Teaching cases: A patient with loss of vision in the right eye and neurofibromatosis type 1

Abstract: Neurofibromatosis type 1 is a common autosomal dominant condition that affects about 1 in 5000 people. We describe a 75-year-old man who, in addition to many classic developmental changes of the disease in his skin, eyes and nervous system, had blindness in his right eye as a complication.Case: A 75-year-old man with long-standing neurofibromatosis type 1 was admitted because the vision in his right eye had decreased progressively over 3 months. Physical examination showed disseminated cutaneous and subcutaneous neurofibromas of varying size (Figure 1) and café-au-lait spots (Figure 2). The patient had a visual acuity of 6/18 (20/60) in his right eye and Lisch nodules (iris hamartomas) (Figure 3). A neurologic examination showed no abnormalities other than his loss of vision. Axial T₁-weighted magnetic resonance imaging of the brain and orbits (Figure 4) showed an isointense mass lateral to the right optic nerve that appeared atrophic and pushed to the left. The mass showed a hyperintense signal on T₂-weighted images with contrast enhancement. These findings are compatible with glioma of the optic nerve.Open in a separate windowFigure 1: Disseminated cutaneous and subcutaneous neurofibromas of varying size on the torso of a patient with neurofibromatosis type 1.Open in a separate windowFigure 2: A café-au-lait spot on the patient''s right knee.Open in a separate windowFigure 3: Lisch nodules on the left iris.Open in a separate windowFigure 4: T₁-weighted axial magnetic resonance imaging of the brain and orbits, showing an isointense mass lateral to the right optic nerve (white arrow) that appears atrophic and pushed to the left (black arrow on inset).Axial and coronal magnetic resonance imaging (Figure 5) showed a mass in the left parietal lobe with hyperintensity on T₂-weighted images and hypointensity on T₁-weighted images. After a contrast medium was administered, the lesion showed a thickened, enhanced wall with a central necrotic area. These findings are compatible with astrocytoma.Open in a separate windowFigure 5: T₂-weighted axial (left) and coronal (right) magnetic resonance imaging showing a mass with hyperintensity (arrow) in the left temporal lobe. After administration of a contrast medium, the lesion is visible with a thickened enhanced wall and a central necrotic area.Because of slight enlargement and increased hardness of the subcutaneous lesions, an excisional biopsy was performed. Histology showed delicate fascicles consisting of cells with oval or spindle-shaped nuclei, scant cytoplasm and round cells with entrapped axons (Figure 6). Only scattered neoplastic Schwann cells were stained during immunostaining for S-100 protein (Figure 7). This pattern is consistent with neurofibroma. The patient chose not to receive further treatment and was discharged.Open in a separate windowFigure 6: Biopsy specimen of a subcutaneous neurofibroma showing spindle-shaped and round cells with entrapped axons (hematoxylin and eosin, original magnification ×10).Open in a separate windowFigure 7: Only scattered neoplastic Schwann cells (arrow) are stained after immunostaining for S-100 protein. Normally, S-100 protein is present in cells derived from the neural crest, such as Schwann cells. It can be found in melanoma cells, in malignant peripheral nerve sheath tumours and in certain types of sarcomas.Neurofibromatosis type 1, also known as von Recklinghausen disease,¹ is characterized by changes in pigmentation and the growth of tumours along nerves in the skin and other parts of the body. It is caused by a defect in a tumour-suppressing gene on chromosome 17q11.2. Normally the gene produces neurofibromin, a protein that regulates cellular proliferation.² With the gene mutation, the lack of neurofibromin results in overgrowth of cells from neural crest areas in both the central nervous system (causing Schwann cell tumours on virtually every nerve) and the skin. All people who inherit a copy of the mutated gene are affected. As the pattern of inheritance is autosomal dominant, only 1 copy of the defective gene is needed to cause the condition. However, it is not necessary to have an affected parent. About 30%–50% of patients have a new mutation.Neurofibromatosis type 2 is a much rarer form of neurofibromatosis caused by mutations in both alleles of a different tumour suppressor gene on chromosome 22q12.1.About 1 in 3000–5000 individuals are affected by neurofibromatosis type 1, without differences related to ethnic background.³ Pigmented small macules and café-au-lait patches are often present shortly after birth, although neurofibromas are rare in early childhood. In later childhood and adolescence, both neurofibromas and pigmented lesions become common. Clinical manifestations are variable (4Table 1Open in a separate windowA diagnosis of neurofibromatosis type 1 is based on clinical findings. The patient should have 2 or more of the following: 6 or more café-au-lait spots of ≥ 1.5 cm in postpubertal individuals or ≥ 0.5 cm in prepubertal individuals; 2 or more neurofibromas of any type or 1 or more plexiform neurofibroma; and freckling in the underarms and groin.¹ The differential diagnosis includes benign café-au-lait pigmentation (present in up to 10% of the general population), multiple lipomas, and sporadic schwannomas, gliomas and meningiomas in the central nervous system.Most people with mild neurofibromatosis have little disability. People affected by more severe variants have a shortened life expectancy, especially if tumours of the central nervous system or other malignant neoplasms arise during the course of illness.^1,3 The condition can have a serious psychological impact because the accumulation of skin nodules can be quite disfiguring.⁵ Surgical excision and laser treatment of the neurofibromas are possible, but neither treatment is universally effective.⁶ Transplantation with an allograft of composite tissue on the lower and middle parts of a patient''s face was recently reported.⁷Gliomas of the optic nerve are found in up to 15% of pediatric patients with neurofibromatosis type 1. Best detected using magnetic resonance imaging, these gliomas are symptomatic in about 50% of patients at diagnosis. A minority will progress to vision loss.⁸ The high prevalence of gliomas of the optic nerve that are asymptomatic may, however, be biased by referral patterns, Indeed, in patients with neurofibromatosis type 1, the threshold of risk for optic nerve glioma is low.⁹Guidelines are available for the diagnosis and management of neurofibromatosis type 1.^10,11 Physicians who identify patients with neurofibromatosis type 1 should refer them early to facilities where appropriate evaluation and monitoring of lesions can be carried out. Early detection and monitoring may help to prevent disability and death.     

Teaching cases: Pulmonary lymphangitic carcinomatosis as a primary manifestation of colon cancer in a young adult

Abstract: Pulmonary lymphangitic carcinomatosis is a metastatic lung disease characterized by diffuse spread of the tumour to the pulmonary lymphatic system. We describe the case of a 31-year-old woman who initially received a diagnosis of sarcoidosis based on the results of imaging studies. However, results of a transbronchial biopsy led to the diagnosis of pulmonary lymphangitic carcinomatosis from metastatic colon cancer.The case: A 31-year-old woman presented to her primary care provider with a nonproductive cough and shortness of breath, which she had experienced intermittently for 1 month. She had no fever, chills or night sweats. She had type 1 diabetes mellitus and hypertension. The patient had experienced multiple miscarriages and was 1 month post partum. She had a 10-pack-year history of smoking. Her father and maternal grandfather both had colon cancer, which, in her father, had been diagnosed after his death at the age of 45. The patient received a diagnosis of exacerbated chronic obstructive pulmonary disease, which was treated with a short course of corticosteroids and broad-spectrum antibiotics. Her condition did not improve.Two months later, the patient presented to the emergency department with dyspnea. A radiograph of her chest showed interstitial lung disease, thickening of the right paratracheal region and hilar prominence. A computed tomography (CT) scan of her chest revealed extensive paratracheal, subcarinal and para-aortic lymphadenopathy. It also showed diffuse consolidation with a nodular pattern throughout the lungs, particularly in the upper lobes and the lower left lobe. She received a diagnosis of sarcoidosis, which her primary care physician treated with corticosteroids. Her symptoms abated, but less than 1 week later, her condition worsened. Oxygen therapy was started at home.A respirologist saw the patient 6 months after the onset of her symptoms. She still had exertional dyspnea and a nonproductive cough without hemoptysis. She had lost 20 pounds in the 5 months preceding the visit. She had tachypnea and tachycardia, and was normotensive and afebrile. She did not have enlarged lymph nodes, skin changes or peripheral edema.Examinations of the patient''s abdomen and heart sounds were unremarkable. We heard rhonchi and crackles in the base of both lungs. A radiograph of her chest showed diffuse interstitial and septal thickening in both lungs (Figure 1). A CT scan showed large nodular areas, ground-glass opacities and thickened interlobular septa with interstitial lung disease (Figure 2).Open in a separate windowFigure 1: Chest radiograph of a 31-year-old woman showing diffuse interstitial and septal thickening (arrow) in both lungs.Open in a separate windowFigure 2: Computed tomography scan showing nodular thickening of interlobular septa (white arrow), seen as polygonal arcades with thickened and nodular limbs, and ground-glass opacities (black arrows).Results of a transbronchial biopsy showed a moderate to poorly differentiated adenocarcinoma. Immunohistochemical studies of the biopsy specimen showed a pattern of staining consistent with metastatic adenocarcinoma most suggestive of a primary origin in the colon. A colonoscopy showed a mass in her proximal sigmoid colon, and biopsy results confirmed the mass to be a moderately differentiated adenocarcinoma. A bone scan showed multiple tomours in her thorax and lumbar spine.The patient was admitted to hospital for chemotherapy (FOLFOX4, a regimen of oxaliplatin, leucovorin and 5-fluorouracil). She was transferred the next day to the intensive care unit because of worsening respiratory distress, necessitating mechanical ventilation. The chemotherapy was continued; however, the patient died of respiratory failure 11 days after admission to hospital.Pulmonary lymphangitic carcinomatosis occurs in 6%–8% of patients with pulmonary metastases.¹ The spread of tumour cells to the pulmonary lymphatic system or the adjacent interstitial tissue results in thickening of the bronchovascular bundles and septa. Desmoplastic reaction due to proliferation of neoplastic cells, and lymphatic dilation by edema fluid or tumour secretions contribute to this interstitial thickening. Spread of the neoplasm outside the interstitium and lymphatic spaces into the adjacent parenchyma can result in a nodular pattern.² Although virtually any metastatic neoplasm can cause pulmonary lymphangitic carcinomatosis, the common locations of the primary tumour are the breasts, stomach, lungs, pancreas and prostate^1,3 (Open in a separate windowPatients with pulmonary lymphangitic carcinomatosis often present with breathlessness and a nonproductive cough. As with our patient, the onset of pulmonary symptoms may precede diagnosis of the primary tumour; however, the frequency of this presentation is unknown. Although chest radiographs appear normal for 30%–50% of patients with histologically proven disease,² pulmonary lymphangitic carcinomatosis has several characteristic changes that can be observed on radiographs (Box 1). Transbronchial biopsy is required for a definitive diagnosis.Open in a separate windowBox 1Pulmonary lymphangitic carcinomatosis can mimic sarcoidosis radiologically. Nodular thickening and ground-glass attenuation are seen in 30%–60% of patients with sarcoidosis. The nodules in sarcoidosis mainly involve central regions of the middle and upper lobes of the lungs. In contrast, changes usually occur in the lower lobes in pulmonary lymphangitic carcinomatosis. Although imaging studies may suggest sarcoidosis, the diagnosis should be confirmed by biopsy results indicating noncaseating granulomas and by the exclusion of other causes of granulomatous disease. Rapid onset and progression of symptoms, asymmetrically enlarged lymph nodes, predominant disease in the lower lobes of the lungs and lack of response to steroids within 2–4 weeks also should alert clinicians to a diagnosis other than sarcoidosis. Thickening of the interlobular septa and peribronchovascular interstitium without a nodular pattern may be seen in other conditions, such as pulmonary edema and idiopathic pulmonary fibrosis.²Although the diagnosis was delayed for our patient, an earlier diagnosis may not have altered the outcome because of the condition''s extremely poor prognosis in most cases. Less than half of patients with pulmonary lymphangitic carcinomatosis who present with respiratory symptoms survive for 3 months.¹ However, platinum-based chemotherapy has led to transient remissions in some cases.⁴Although our patient did not undergo genetic testing, her young age at presentation and strong family history of colon cancer led to a diagnosis of hereditary nonpolyposis colorectal cancer. This condition accounts for 2%–3% of colorectal cancers and is the most common form of hereditary colorectal cancer.⁵ It is characterized by an early onset, with an average age at diagnosis of about 45 years and the presence of tumours predominantly on the right side of the colon. Patients with this condition are also at increased risk of having more than 1 primary colorectal tumour at the time of diagnosis (synchronous neoplasms) and of having another primary tumour after successful treatment of the index tumour (metachronous carcinoma). Patients with this condition are more likely than others of the same age and sex to experience tumours of the endometrium, small bowel, stomach, renal pelvis ureter and ovaries. Skin lesions, including carcinomas and nonmalignant lesions such as sebaceous adenomas and keratoacanthomas, are also more common among these patients.⁶Our patient had unusual features of pulmonary lymphangitic carcinomatosis, including involvement of the sigmoid colon and metastatis to the lungs and spine without liver involvement. This presentation is seen in only about 9% of rectal cancers.⁷ If hereditary nonpolyposis colorectal cancer is suspected, a biopsy of the tumour and genetic testing should be carried out to identify mutations. Once identified, relatives at high risk for colorectal cancer should be counselled and screened for these mutations. Those who carry the mutations should have a colonoscopy every 2 years starting at age 20–25.⁶Anish Thomas MD Robert Lenox MD Department of Medicine State University of New York Syracuse, New York     

Teaching cases: A 1-week-old newborn with hypercalcemia and palpable nodules: subcutaneous fat necrosis

Abstract: We present a 1-week-old newborn with subcutaneous fat necrosis complicated by hypercalcemia. She received conservative treatment of adequate hydration and restricted supplementary vitamin D.The case: A 1-week-old term newborn girl was brought to her physician with a 2-day history of subcutaneous masses. The girl had been born by vacuum-assisted vaginal delivery with a birth weight of 3.5 kg. She did not require resuscitation but was observed for 24 hours in a special care nursery because of tachypnea. The patient was discharged home after 48 hours, and her course over the next 5 days was unremarkable.On physical examination, the newborn was afebrile and in no obvious distress. She had multiple firm, mobile, mildly tender subcutaneous nodules with overlying erythema (Figure 1). The largest mass on palpation was located in the left deltoid area and measured 2 × 2.5 cm (Figure 1). Two smaller lesions were located in the left posterior axillary area (Figure 1) and a fourth lesion was in the right posterior auricular region. The remainder of the physical examination and the results of a complete blood count were normal. The newborn''s total serum calcium level was elevated (2.94 [normal 1.96–2.66] mmol/L) as was her ionized calcium level (1.36 [normal 1.14–1.29] mmol/L. A biopsy was not performed because the infant was well and the results of clinical investigations were consistent with subcutaneous fat necrosis.Open in a separate windowFigure 1: A 5-day-old infant with lesions of subcutaneous fat necrosis. The largest mass (black arrow) measured 2 × 2.5 cm. Two smaller lesions (white arrows) were located in the left posterior axillary area.Subcutaneous fat necrosis of the newborn is a relatively uncommon condition that occurs in the first several weeks after birth. The incidence is unknown; however, it is more frequently reported after perinatal distress than after uncomplicated deliveries, and maternal risk factors include gestational diabetes and preeclampsia.¹ Skin lesions are characterized by indurated nodules that range from flesh-coloured to blue and by plaques on the face, trunk and buttocks as well as on the arms and legs near the trunk. Figure 2 is a representative microscopic image of this condition.² The differential diagnosis is bacterial cellulitis, erysipelas and sclerema neonatorum.³Open in a separate windowFigure 2: Left: A typical photomicrograph from a different patient showing lobular panniculitis with sparing of the dermis and epidermis (original magnification × 20). Right: A high-power view shows that the inflammatory infiltrate is mixed and composed of histiocytes, lymphocytes, neutrophils and eosinophils. Cleft-like spaces (arrow) suggestive of dissolved crystals can be seen at the periphery of some of the fat cysts (original magnification × 400). Reproduced with permission from Macmillan Publishers Ltd: Journal of Perinatology (Diamantis et al.²) © 2006.Although subcutaneous fat necrosis of the newborn is often benign and self-limited, the most important concern is hypercalcemia, which can lead to neurologic or cardiac problems, nephrocalcinosis and nephrolithiasis. Clinical signs of newborn hypercalcemia include irritability, poor feeding and vomiting. Skin lesions typically resolve over a period of weeks to several months; however, hypercalcemia can persist longer and requires serial monitoring. The treatment of hypercalcemia ranges from conservative measures such as hydration and restriction of vitamin D and calcium to more aggressive interventions such as furosemide, glucocorticoid or bisphosphonate therapy in severe cases.⁴In our patient, mild hypercalcemia was accompanied by mild elevations in the ratio of calcium to creatinine in the urine and a normal 1,25-dihydroxyvitamin D level. Because our patient was otherwise well, we opted for conservative management. In 2 months, her calcium level had normalized and the lesions completely regressed.Fahed Aljaser MD Michael Weinstein MD Division of Pediatric Medicine Hospital for Sick Children University of Toronto Toronto, Ont.     

Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

Spotlight on Zebrafish: Translational Impact

In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications.Open in a separate windowE. Elizabeth PattonOpen in a separate windowJames F. AmatrudaOpen in a separate windowLalita Ramakrishnan     

Association of Rice Gall Dwarf Virus with Microtubules Is Necessary for Viral Release from Cultured Insect Vector Cells

Vector insect cells infected with Rice gall dwarf virus, a member of the family Reoviridae, contained the virus-associated microtubules adjacent to the viroplasms, as revealed by transmission electron, electron tomographic, and confocal microscopy. The viroplasms, putative sites of viral replication, contained the nonstructural viral proteins Pns7 and Pns12, as well as core protein P5, of the virus. Microtubule-depolymerizing drugs suppressed the association of viral particles with microtubules and prevented the release of viruses from cells without significantly affecting viral multiplication. Thus, microtubules appear to mediate viral transport within and release of viruses from infected vector cells.Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV), and Wound tumor virus, members of the genus Phytoreovirus in the family Reoviridae, multiply both in plants and in invertebrate insect vectors. Each virus exists as icosahedral particles of approximately 65 to 70 nm in diameter, with two concentric layers (shells) of proteins that enclose a core (1, 13). The viral genome of RGDV consists of 12 segmented double-stranded RNAs that encode six structural (P1, P2, P3, P5, P6, and P8) and six nonstructural (Pns4, Pns7, Pns9, Pns10, Pns11, and Pns12) proteins (reference 21 and references therein). The core capsid is composed of P3, the major protein, which encloses P1, P5, and P6 (12). The outer layer consists of two proteins, namely, P2 and P8 (10, 12).Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of viruses in the family Reoviridae. After infecting insect vector cell monolayers (VCMs) in culture with RDV, Wei et al. (19) examined the generation of RDV particles in and at the periphery of such viroplasms. VCMs are also useful for studies of RGDV, allowing detailed analysis of the synchronous replication and multiplication of this virus (14). In order to identify the viroplasms in RGDV-infected VCMs, we examined the subcellular localization of Pns7, Pns12, P5, and RGDV particles by confocal immunofluorescence microscopy. Pns7 and Pns12 of RGDV correspond to Pns6 and Pns11, respectively, which are components of the viroplasm of RDV (12, 19). RGDV P5 is a counterpart of RDV P5, a core protein that locates inside the viroplasm in RDV-infected cells. We inoculated VCMs with RGDV, purified by the method reported in reference 15, at a multiplicity of infection (MOI) of 1; fixed them 48 h postinfection (p.i.); probed the cells with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (11, 12) that had been conjugated to fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO) or rhodamine (Sigma); and examined them by confocal microscopy, as described previously (19). In RGDV-infected cells, Pns7, Pns12, and P5 were detected as punctate inclusions (Fig. ​(Fig.1).1). Immunostained viral antigens formed ringlike structures around the punctate inclusions. When the images were merged, Pns7, Pns12, and P5 were colocalized in the punctate inclusions, indicating that these proteins were constituents of the viral inclusions (Fig. ​(Fig.1).1). Our observations revealed the similar respective localizations of the corresponding nonstructural proteins, core proteins, and viral particles of two phytoreoviruses, RGDV and RDV, in infected cells. Thus, Pns7 and Pns12 of RGDV had attributes common to their functional counterparts—Pns6 and Pns11, respectively—of RDV (19). The core protein P5 was located inside the viroplasms, and the viral antigens were distributed at the periphery of the viroplasms. The results, together, suggest that RGDV and RDV exploit similar replication strategies. Specific fluorescence was not detected in noninfected cells after incubation with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (data not shown).Open in a separate windowFIG. 1.Subcellular localization of Pns7, Pns12, and P5 of RGDV and viral antigens in RGDV-infected VCMs 48 h p.i. Arrowheads show ringlike profiles of viral antigens that surround viral inclusions, which have been immunostained with the Pns12-specific antibodies. Arrows show the fibrillar profiles of immunostained viral antigens. Bars, 5 μm.In addition to the viral location at the periphery of the viral inclusions visualized as immunostained Pns12 (Fig. ​(Fig.1),1), the antigens were distributed as bundles of fibrillar structures, a form not observed in RDV-infected cells. To analyze the entity of the bundles of fibrillar structures, VCMs on coverslips were inoculated with RGDV at an MOI of 1, fixed at 48 h p.i., and examined by electron microscopy (EM), as described previously (19). We observed viral particles of approximately 70 nm in diameter in close association with the free ends, as well as along the edges, of tubules of approximately 25 nm in diameter (Fig. 2A to D). The abundant bundles of tubules with closely associated viral particles were clearly in contact with the periphery of granular, electron-dense inclusions of 800 to 1,200 nm in diameter (Fig. ​(Fig.2B),2B), namely, viroplasms. The dimensions and appearance of the tubular structures resembled those of microtubules (Fig. ​(Fig.2C)2C) (17). Transverse sections of tubules revealed arrays of closed circles of approximately 25 nm in diameter, with viral particles attached directly or via a filament to the circumference (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.Association of RGDV particles with microtubules. (A) Electron micrograph showing RGDV particles associated with microtubules in virus-infected VCMs 48 h p.i. Bar, 300 nm. VP, electron-dense inclusion. (B) Virus-associated microtubules in contact with the periphery of the electron-dense inclusion indicated by a white rectangle in panel A. Bar, 300 nm. (C) Viral particles along the edges of tubules of approximately 25 nm in diameter. Bar, 150 nm. (D) Transverse sections of arrays of closed circles of approximately 25 nm in diameter with viral particles attached to their circumference directly (arrow) or via a filament (arrowhead). Bar, 150 nm. (E) Confocal micrograph showing the association of viral particles with microtubules in virus-infected VCMs 48 h p.i. Microtubules were stained with α-tubulin-specific antibodies conjugated to FITC; viral particles were stained with viral-antigen-specific antibodies conjugated to rhodamine. Arrowheads indicate the ringlike organization of viral antigens. Arrows show the colocalization of fibrillar profiles of viral antigens with microtubules. The insets show ringlike and fibrillar profiles of immunostained viral antigens. The circular areas inside the ringlike structures are viroplasms. Bar, 5 μm.Our observations suggested that RGDV particles might attach to microtubules in infected cells. To examine this possibility, we inoculated VCMs with RGDV at an MOI of 1, fixed the cells 48 h p.i., immunostained them with α-tubulin-specific antibodies conjugated to FITC and with viral-antigen-specific antibodies conjugated to rhodamine, and analyzed them by confocal microscopy, as described previously (19). Viral antigens were visualized as ringlike and fibrillar structures (Fig. ​(Fig.2E).2E). Double immunostaining of the infected cells revealed that a network of microtubule-based filaments colocalized with most of the fibrillar structures that represented viral antigens, confirming the association of viral particles with the microtubule-like inclusions visualized by EM (Fig. ​(Fig.2A).2A). Nonspecific reactions were not detected with either of the stainings (data not shown). Our results suggested that RGDV particles, which assembled at the periphery of viroplasms, might be transported along microtubules. Due to the lack of RGDV infectious clones fused with green fluorescent protein and the effective gene transfection system for VCMs, we could not observe the trafficking of RGDV particles along microtubules in living cells.We then used three-dimensional (3-D) electron tomographic microscopy (ET) to reveal a new level of morphological detail about the association of RGDV with microtubules. To produce 3-D reconstructions of RGDV-infected cells, we fixed, embedded, and sectioned infected leafhopper cells as described previously (5). We chose a representative region that showed numerous RGDV particles close to bundles of microtubules for this novel tomographic analysis. A single-axis tilt series was collected manually from −60° to 60° with 2° increments using an H9500SD EM (Hitachi, Tokyo) operated at 200 kV. These tomographic data were recorded at a defocus of 3.6 μm on the TVIPS 2k × 2k charge-coupled-device camera (TVIPS, Gauting, Germany). Microscopic magnification of ×15,000, providing 1.28 nm/pixel, was enough to view the microtubules and virus particles following tomographic reconstruction of the tilt series using IMOD (7). As shown in the 3-D tomogram in Fig. ​Fig.3,3, most of the RGDV particles were bound to the edges of bundles of microtubules. The RGDV particles along the edges of microtubules were arrayed in an orderly but uncrowded manner (Fig. ​(Fig.3).3). Our ET analysis also revealed that some viral particles were linked to filaments of approximately 10 nm in diameter (Fig. ​(Fig.3B).3B). Morphologically, these filaments resembled vimentin intermediate filaments (4). In many lines of cultured cells, vimentin intermediate filaments partially overlap the microtubules, and there is evidence that the two filament systems interact (3, 9, 20). Unfortunately, vimentin-specific monoclonal antibodies from mouse and rabbit did not react specifically with our leafhopper cells (data not shown), but the nature of the intermediate filaments was apparent from their dimensions, intracellular location, and organization. Thus, our ET analysis indicated that RGDV particles were able to associate directly and/or via intermediate filaments with microtubules.Open in a separate windowFIG. 3.ET analysis showing the association of RGDV particles with microtubules either directly or via intermediate filaments. (A) Translucent representation of the reconstructed viruses lining up with microtubules. (B) Slice of the reconstructed volumes from the inset of A to show the association of RGDV particles with intermediate filaments (arrows). Bars, 150 nm.To examine the role of the microtubules for RGDV activity, we added a microtubule-disrupting agent, either nocodazole (Sigma) or colchicine (Sigma), 2 hours after inoculation of VCMs with RGDV at an MOI of 1 and then continued the incubation for a further 46 h. Cells were fixed 48 h p.i. and stained with α-tubulin-specific antibodies conjugated to FITC (Sigma) and viral-particle-specific antibodies conjugated to rhodamine, with subsequent confocal fluorescence microscopy, as described previously (19). We tested a range of drug concentrations in preliminary experiments (data not shown) and determined optimal concentrations. Treatment of infected cells with 10 μM nocodazole or 5 μg/ml colchicine resulted in the complete disassembly of microtubules, with the accumulation of ringlike structures exclusively and no fibrillar structures representative of viral antigens in the cytoplasm (Fig. ​(Fig.4A).4A). These ringlike aggregates of viral antigens were confirmed to surround viroplasms when the latter were immunostained for Pns12, as described above and shown in Fig. ​Fig.1.1. Nonspecific reactions were not detected with either staining (data not shown). These results suggest that RGDV particles multiply around the viroplasm but are unable to distribute along the microtubules in the presence of the chemicals.Open in a separate windowFIG. 4.(A) Effects of microtubule-disrupting agents on the formation of microtubules and fibrillar profiles of immunostained viral antigens. Bars, 5 μm. The insets show the ringlike profiles of immunostained viral antigens after treatment with inhibitors, suggesting that viral replication occurs in the presence of each inhibitor. (B) Effects of drugs on the production of cell-associated (gray bars) and extracellular (black bars) viruses in VCMs infected with RGDV. The error bars indicate standard deviations.During the process of infection, microtubules play important roles in viral entry, intracellular trafficking, and extracellular release (2, 8, 16). We next investigated the effects of the microtubule-disrupting agents on the production in and release of viruses from virus-infected cells by the method described previously (18). Nocodazole or colchicine was added 2 h after inoculation of VCMs with RGDV at an MOI of 1, and incubation was continued for a further 46 h. The extracellular medium and the cells were collected separately. The medium was centrifuged for 30 min at 15,000 × g, and the supernatant was collected. The cells were subjected to three cycles of freezing and thawing to release viral particles. The viral titer of each sample was determined, in duplicate, by the fluorescent focus assay as described previously (6), with VCMs and a magnification of ×10. As shown in Fig. ​Fig.4B,4B, nocodazole (20 μM) and colchicine (10 μg/ml) caused a fivefold reduction in the number of released viruses, compared to that from untreated control infected cells. In contrast, each inhibitor at the selected dose failed to significantly reduce the titer of cell-associated viruses (less then 5% compared to that from untreated control). These results suggest that the inhibitors impeded the release of viruses into the medium without affecting viral production in infected cells. We do not yet understand why the viral titer was not elevated in drug-treated cells from which viral release was inhibited. However, our data show clearly that disruption of microtubules directly inhibited the release of mature viral particles from infected cells.In conclusion, EM, ET, immunofluorescence staining, and experiments with two inhibitors support the hypothesis that the transport of RGDV from viroplasms to the plasma membrane and into the medium is dependent on microtubules. In the case of RDV, vesicular compartment-containing viral particles that locate adjacent to the viroplasms were considered to play an important role in the transport and release of the virus from the viroplasm to the culture medium in infected VCMs (18). On the other hand, a fibrillar structure (Fig. ​(Fig.11 and ​and2),2), not observed in RDV-infected cells, was considered to function in the trafficking of RGDV from viroplasm into the culture medium (Fig. ​(Fig.4)4) in the present study. RGDV and RDV, both members of the Phytoreovirus genus, have some common biological and biochemical properties but are distinct from each other (13). For example, viruses are restricted to phloem-related cells in RGDV-infected plants but distributed in many types of cells in RDV-infected plants, and a P2 protein with a function to adsorb to and/or penetrate into insect vector cells is present in RGDV and absent in RDV in particles purified using carbon tetrachloride. The present molecular cytopathological study revealed one more difference between the viruses: they have different means for transporting and releasing infectious particles to the cell exterior. The presence of such a molecular mechanism may accelerate the secondary infections by the viruses in infected vector insects, and the high propagation speed would allow the viruses to complete infection cycles through insects and plants.     

Genetic Basis for Rhizobium etli CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis.O antigens typically constitute the distal portions of lipopolysaccharides (LPS) and help determine the diverse surface characteristics of Gram-negative bacteria. These repeat unit carbohydrate polymers vary tremendously in structure and, as a family, they exhibit all known sugars and sugar modifications, linked in myriad ways forming homopolymers and heteropolymers. Control of polymer length also varies, allowing highly uniform to completely random lengths. Great diversity of O-antigen structures even within a species is well known. Moreover, O antigens of a single strain can vary according to growth and environmental conditions. One such condition is the presence of a multicellular host (5, 18, 36, 40, 42, 44).Rhizobium etli CE3 fixes nitrogen inside root nodules it incites on the common bean Phaseolus vulgaris. The O antigen of its LPS (Fig. ​(Fig.1)1) is essential for bacterial infection during development of this symbiosis (41). In addition, at least two alterations occur in the O antigen when R. etli CE3 is grown in the presence of either the host plant or plant exudates. The content of the multiply O-methylated terminal fucose is decreased (19, 44), whereas the 2-O methylation of internal fucoses (2OMeFuc) increases twofold (Fig. ​(Fig.1)1) (15, 44). In addition to the multiply O-methylated terminal fucose and 2OMeFuc, methylation occurs always on 6-deoxytalose and likely on glucuronic acid to yield 3-O-methyl-6-deoxytalose (3OMe6dTal) and methyl-esterified glucuronyl (MeGlcA) residues (Fig. ​(Fig.1)1) (22); however, the incidence of these methylations is not known to vary with growth condition. The genetics responsible for the variable O methylations and the additions of the residues they modify have not been elucidated.Open in a separate windowFIG. 1.R. etli CE3 O-antigen structure (22). The portion of the LPS conceptually defined as O antigen begins with N-acetyl-quinovosamine (QuiNAc) at the reducing end followed by a mannose (Man) residue and a fucose (Fuc) residue. Attached to this fucose is the repeating unit consisting of one fucose residue, one 3-O-methyl-6-deoxytalose residue (3OMe6dTal), and one glucuronyl methyl ester residue (MeGlcA). The sugars of the repeating unit are added sequentially exactly five times (in most molecules). An O-acetyl group is present in each of the repeating units, but its location is unknown at this time. Growth in TY culture results in one 2-O-methylfucose (2OMeFuc) per O antigen on average (22). The O-antigen backbone is capped with a 2,3-di-O-methylfucose (referred to as the terminal residue in this report) on which additional O methylation at the 4-position is variable as indicated by parentheses. Growth of the bacteria in the presence of the host plant or plant exudates induces the increase of 2-O methylation of internal fucose (2OMeFuc) residues and decreased relative amount of the terminal residue (44).Most mutations affecting the known R. etli CE3 O-antigen structure map to a 28-kb genetic cluster on the chromosome (Fig. ​(Fig.2)2) (previously referred to as lps region α [8, 19, 37, 40, 45]). Genes and mutations within this cluster previously have been given the designations lps (9) and lpe (19). Recently, the new designation wre has been sanctioned by the Bacterial Polysaccharide Gene Database for this genetic cluster and other genes specifically devoted to the R. etli CE3 O antigen, in keeping with the system of nomenclature for bacterial polysaccharide genes (47).Open in a separate windowFIG. 2.R. etli CE3 O-antigen genetic cluster. (A) The R. etli CE3 chromosomal O antigen genetic cluster spans nucleotides 784527 to 812262 of the genome sequence (28) and consists of 25 putative ORFs. ORFs relevant to the present study are enlarged, and the relative locations of mutations are indicated. White triangles indicate mutations created by insertion of antibiotic cassettes, and black triangles indicate mutations created by Tn5 mutagenesis. The strain numbers carrying these mutations are indicated above the triangles. (B) The solid bars represent the extents of R. etli CE3 DNA cloned for complementation analysis. The scale and positions match those of the lower map in panel A.Duelli et al. (19) identified a 3-kb genetic locus that is required for the presence of the 2,3-di-O-methylfucose or 2,3,4-tri-O-methylfucose at the terminus of the O antigen. Now known to be near one end of the O-antigen genetic cluster (Fig. ​(Fig.2),2), the DNA sequence reported by Duelli et al. encompasses nucleotides 807701 to 810147 of the subsequently determined genome sequence (28). Sequence and annotation of the 3-kb locus have since been revised. In place of the four open reading frames (ORFs) suggested previously (19), the current annotation predicts two ORFs: wreA and wreC (Fig. ​(Fig.2).2). The wreA ORF is predicted to encode a methyltransferase (19), but the predicted WreC polypeptide sequence matches no known methyltransferase or glycosyltransferase or any other polypeptide sequence in the database (Fig. ​(Fig.3).3). When it became clear that this locus was part of the larger O-antigen genetic cluster, the nucleotide sequence suggested that three genes contiguous to wreA also might encode functions needed for synthesis and addition of the terminal fucose. The results to be shown bore out predictions of this hypothesis.Open in a separate windowFIG. 3.Conserved domain predictions. Spanning nucleotides 804817 to 810147 of the genome sequence (28), ORFs RHE_CH00766, RHE_CH00767, RHE_CH00768, RHE_CH00769, and RHE_CH00770 were named wreB, wreD, wreF, wreA, and wreC, respectively. Previously, wreF, wreA, and wreC were referred to as nlpe2, lpeA, and nlpe1, respectively (19). ORF RHE_CH00755, spanning nucleotides 791286 to 794093, was named wreM. Predicted positions of conserved domains are indicated by amino acid positions. Abbreviations: GT, conserved glycosyltransferase domain; MT, conserved methyltransferase domain. Gray boxes indicate the predicted transmembrane domains.The gene responsible for the other conditionally variable O-antigen methylation, the 2-O methylation of internal fucose residues (2OMeFuc), had not been identified in prior published work. However, among mutants isolated by random Tn5 mutagenesis, a few had been shown to lack 2OMeFuc entirely (44). We show here that the transposon insertions were located in the bifunctional gene wreM. Furthermore, results of directed insertion mutagenesis confirm two separate enzymatic domains encoded by this gene, with the α domain being required for the 2-O methylation activity and mutation of the other domain resulting in a truncated O antigen. Mutants from the directed mutagenesis that appeared to have no LPS defects other than the lack of 2OMeFuc served as tools to assess the importance of just this structural feature in the symbiosis with P. vulgaris.     

Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.     

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.Open in a separate windowClick here to view.^{(52M, flv)}     

Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes

Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2′ loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive ∼6000-fold reduction in catalytic efficiency. Mutations in the Arg-187–Tyr-223 couple have a far less dramatic effect (3–20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.Caspases are a family of dimeric cysteine proteases whose members control the ultimate steps for apoptosis (programmed cell death) or innate inflammation among others (for reviews, see Refs. 1 and 2). During apoptosis, the upstream initiator caspases (caspase-8 and -9) activate the downstream executioner caspases (caspase-3, -6, and-7) via zymogen maturation (3). The activated executioner caspases then cleave upwards of 500 key proteins (4–6) and DNA, leading to cell death. Due to their pivotal role in apoptosis, the caspases are involved both in embryonic development and in dysfunction in diseases including cancer and stroke (7). The 11 human caspases share a common active site cysteine-histidine dyad (8), and derive their name, cysteine aspartate proteases, from their exquisite specificity for cleaving substrate proteins after specific aspartate residues (9–13). Thus, it has been difficult to develop active site-directed inhibitors with significant specificity for one caspase over the others (14). Despite difficulties in obtaining specificity, there has been a long-standing correlation between efficacy of caspase inhibitors in vitro and their ability to inhibit caspases and apoptosis in vivo (for review, see Ref. 31). Thus, a clear understanding of in vitro inhibitor function is central to the ability control caspase function in vivo.Caspase-7 has been a paradigm for understanding the structure and dynamics of the executioner caspases (15–21). The substrate-binding site is composed of four loops; L2, L3, and L4 are contributed from one-half of the caspase dimer, and L2′ is contributed from the other half of the caspase dimer (Fig. 1). These loops appear highly dynamic as they are only observed in x-ray structures when bound to substrate or substrate analogs in the catalytically competent conformation (17–19, 22) (Fig. 1B).Open in a separate windowFIGURE 1.Allosteric site and dimeric structure in caspase-7. A, the surface of active site-bound caspase-7 shows a large open allosteric (yellow) site at the dimer interface. This cavity is distinct from the active sites, which are bound with the active site inhibitor DEVD (green sticks). B, large subunits of caspase-7 dimers (dark green and dark purple) contain the active site cysteine-histidine dyad. The small subunits (light green and light purple) contain the allosteric site cysteine 290. The conformation of the substrate-binding loops (L2, L2′, L3, and L4) in active caspase-7 (Protein Data Bank (PDB) number 1f1j) is depicted. The L2′ loop (spheres) from one-half of the dimer interacts with the L2 loop from the other half of the dimer. C, binding of allosteric inhibitors influences the conformation of the L2′ loop (spheres), which folds over the allosteric cavity (PDB number 1shj). Subunit rendering is as in panel A. Panels A, B, and C are in the same orientation.A potential alternative to active site inhibitors are allosteric inhibitors that have been seeded by the discovery of selective cysteine-tethered allosteric inhibitors for either apoptotic executioner caspase-3 or apoptotic executioner caspase-7 (23) as well as the inflammatory caspase-1 (24). These thiol-containing compounds bind to a putative allosteric site through disulfide bond formation with a thiol in the cavity at the dimer interface (Fig. 1A) (23, 24). X-ray structures of caspase-7 bound to allosteric inhibitors FICA³ and DICA (Fig. 2) show that these compounds trigger conformational rearrangements that stabilize the inactive zymogen-like conformation over the substrate-bound, active conformation. The ability of small molecules to hold mature caspase-7 in a conformation that mimics the naturally occurring, inactive zymogen state underscores the utility and biological relevance of the allosteric mechanism of inhibition. Several structural changes are evident between these allosterically inhibited and active states. (i) The allosteric inhibitors directly disrupt an interaction between Arg-187 (next to the catalytic Cys-186) and Tyr-223 that springs the Arg-187 into the active site (Fig. 3), (ii) this conformational change appears to be facilitated by a hinge-like movement about Gly-188, and (iii) the L2′ loop folds down to cover the allosteric inhibitor and assumes a zymogen-like conformation (Fig. 1C) (23).Open in a separate windowFIGURE 2.Structure of allosteric inhibitors DICA and FICA. DICA and FICA are hydrophobic small molecules that bind to an allosteric site at the dimer interface of caspase-7. Binding of DICA/FICA is mediated by a disulfide between the compound thiol and Cys-290 in caspase-7.Open in a separate windowFIGURE 3.Movement of L2′ blocking arm. The region of caspase-7 encompassing the allosteric couple Arg-187 and Tyr-223 is boxed. The inset shows the down orientation of Arg-187 and Tyr-223 in the active conformation with DEVD substrate mimic (orange spheres) in the active site. In the allosteric/zymogen conformation, Arg-187 and Tyr-223 are pushed up by DICA (blue spheres).Here, using mutational analysis and small molecule inhibitors, we assess the importance of these three structural units to modulate both the inhibition of the enzyme and the coupling between allosteric and active site labeling. Our data suggest that the hinge movement and pinning of the L2-L2′ are most critical for transitioning between the active and inactive forms of the enzyme.     

Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.Open in a separate windowClick here to view.^{(19M, flv)}     

Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.We have previously shown that 2-keto acids generated from amino acid biosynthesis can serve as precursors for the Ehrlich degradation pathway (15) to higher alcohols (3). In order to produce isobutanol, the valine biosynthesis pathway was used to generate 2-ketoisovalerate (KIV), the precursor to valine, which was then converted to isobutanol via a decarboxylation and reduction step (Fig. ​(Fig.1A).1A). The entire pathway to isobutanol from glucose is shown in Fig. ​Fig.1A.1A. To produce isobutanol, we overexpressed five genes, alsS (Bacillus subtilis), ilvC (Escherichia coli), ilvD (E. coli), kdc (Lactococcus lactis), and ADH2 (Saccharomyces cerevisiae) (Fig. ​(Fig.1A).1A). This E. coli strain produced 6.8 g/liter isobutanol in 24 h (Fig. ​(Fig.1B)1B) and more than 20 g/liter in 112 h (3). More recently, we have found that an alcohol dehydrogenase (Adh) encoded by yqhD on the E. coli genome can convert isobutyraldehyde to isobutanol efficiently (5) (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.Schematic representation of the pathway for isobutanol production. (A) The Kdc-dependent synthetic pathway for isobutanol production. (B) Isobutanol production with the Kdc-dependent and -independent synthetic pathways. IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy acid dehydratase. (C) Enzymatic reaction of Als, Ahbs, and Kdc activities.One key reaction in the production of isobutanol is the conversion of KIV to isobutyraldehyde catalyzed by 2-ketoacid decarboxylase (Kdc) (Fig. ​(Fig.1C).1C). Since E. coli does not have Kdc, kdc from L. lactis was overexpressed. Kdc is a nonoxidative thiamine PP_i (TPP)-dependent enzyme and is relatively rare in bacteria, being more frequently found in plants, yeasts, and fungi (8, 19). Several enzymes with Kdc activity have been found, including pyruvate decarboxylase, phenylpyruvate decarboxylase (18), branched-chain Kdc (8, 19), 2-ketoglutarate decarboxylase (10, 17, 20), and indole-3-pyruvate decarboxylase (13).In this work, unexpectedly, we find that Kdc is nonessential for E. coli to produce isobutanol (Fig. ​(Fig.1).1). An E. coli strain overexpressing only alsS (from B. subtilis), ilvC, and ilvD (both from E. coli) is still able to produce isobutanol. Since E. coli is not a natural producer of isobutanol, it cannot be detected from the culture media in any unmodified strain. We identify that AlsS from B. subtilis, which was introduced in E. coli for acetolactate synthesis (Als), catalyzes the decarboxylation of 2-ketoisovalerate like Kdc both in vivo and in vitro. AlsS is part of the acetoin synthesis pathway and catalyzes the aldo condensation of two molecules of pyruvate to 2-acetolactate (Als activity) (Fig. ​(Fig.1C)1C) (11). The overall reaction catalyzed by AlsS is irreversible because of CO₂ evolution. The first step in catalysis is the ionized thiazolium ring of TPP reacting with the first pyruvate, followed by decarboxylation. This intermediate then reacts with the second pyruvate. Deprotonation followed by C-C bond breakage produces 2-acetolactate. In this work, mutational approaches were used to assess the importance of Q487 in the Kdc activity of AlsS.     

The H+-ATPase in the Plasma Membrane of Saccharomyces cerevisiae Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability

Saccharomyces cerevisiae plasma membrane H⁺-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pH_i). The cell population used as inoculum exhibited a significant heterogeneity of pH_i, and the fall of pH_i correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.The biological target sites of octanoic acid in Saccharomyces cerevisiae may be related to processes of transport across membranes, particularly the plasma membrane (21). Like other weak acids at low pH, octanoic acid, a highly toxic by-product of yeast alcoholic fermentation (23) and an antimicrobial food additive (6), leads to the reduction of cytosolic pH (pH_i) due to its dissociation in the approximately neutral cytoplasm following the entrance of the undissociated toxic form into the cell by passive diffusion (5, 20, 23). It is likely that this highly liposoluble weak acid significantly affects the spatial organization of the plasma membrane, affecting its function as a matrix for enzymes and as a selective barrier, thereby leading to the dissipation of the proton motive force across the plasma membrane and to intracellular acidification (16, 18). Significantly, the H⁺-ATPase in the plasma membrane in yeast, which creates the electrochemical proton gradient that drives the secondary transport of solutes and is implicated in the maintenance of pH_i around neutrality, has been pointed out as a critical component of yeast adaptation to weak acids (8, 19, 24). Indeed, yeast plasma membrane H⁺-ATPase is activated during exponential growth with octanoic acid (19, 24), and the duration of lag phase before yeast cells enter exponential growth in the presence of sorbic acid is significantly extended in a mutant with reduced levels of plasma membrane ATPase activity (8). The activation of the H⁺-ATPase in the plasma membrane in yeast cells exposed to other stresses that also lead to the dissipation of the H⁺ gradient and intracellular acidification (such as subcritical inhibitory concentrations of ethanol [12, 14, 15], supraoptimal temperatures below 40°C [25], presence of other organic acids at low pH [1, 5, 8], and deprivation of nitrogen source [2]) have also been observed. Several lines of evidence indicate that ATPase activation is due to posttranslational modifications of the PMA1 ATPase (2, 12, 24, 25). Considerable information has been obtained on the variation of plasma membrane ATPase activity during exponential growth and early stationary phase of yeast cells cultivated in media, at low pH, supplemented or not with octanoic acid (24). However, this is not the case during the period of latency preceding exponential growth at concentrations of octanoic acid close to the maximal concentration allowing growth. The main objective of the present work was to obtain information about the pattern of ATPase activity and the changes in pH_i and cell viability during the lag phase necessary for yeast adaptation to the physiological effects of octanoic acid before exponential growth.

Duration of yeast growth latency in octanoic acid-supplemented media.

When cells of S. cerevisiae IGC3507III grown, at 30°C, in medium that had not been supplemented with octanoic acid were used to inoculate buffered YG media (30 g of glucose liter⁻¹, 6.7 g of Yeast Nitrogen Base [Difco] liter⁻¹) (pH 4.0) supplemented with increasing concentrations of this toxic acid up to around 0.35 mM total acid (19, 23), exponential growth was initiated without significant delay (Fig. ​(Fig.1a),1a), although a dose-dependent decrease in specific growth rate was observed (Fig. ​(Fig.1b).1b). However, for higher concentrations up to the maximal that allowed growth (0.42 mM), a lag phase was observed and its duration strongly increased with the severity of octanoic acid stress (Fig. ​(Fig.1a).1a). The duration of latency was drastically reduced when exponential cells used as inoculum were grown in medium with an identical concentration of octanoic acid (Fig. ​(Fig.1a),1a), but the specific growth rate was not modified (Fig. ​(Fig.1b).1b). At a concentration of total octanoic acid of 0.39 mM, a lag phase of around 55 h was necessary for yeast cells, which had been cultivated under nonstressing conditions, to adapt to the deleterious effects of octanoic acid and to initiate inhibited exponential growth (Fig. ​(Fig.2).2). Open in a separate windowFIG. 1Effect of the addition to the growth medium of increasing concentrations of octanoic acid on the duration of lag phase (a) and the specific growth rate of S. cerevisiae IGC 3507III (b) for exponentially growing cells (used as inoculum) cultivated at 30°C at pH 4.0 in the absence (○) or presence (•) of concentrations of toxic lipophilic acid identical to those present in the growth medium. Results are representative of the many growth experiments carried out.Open in a separate windowFIG. 2Specific activity of plasma membrane H⁺-ATPase (filled symbols) and growth curve (open symbols) of S. cerevisiae IGC 3507III during cultivation in the presence (a) or absence (b) of 0.39 mM total octanoic acid (at pH 4.0, 30°C). The data are averages with standard deviations for at least three enzyme assays using cells from at least two independent growth experiments. OD, optical density.

Activation of plasma membrane ATPase during octanoic acid-induced latency.

The specific activity of plasma membrane ATPase assayed in crude membrane suspensions prepared from nonadapted cells, as previously reported (19, 25), during cultivation in buffered medium (at pH 4.0) supplemented with 0.39 mM octanoic acid, increased during the 55 h of latency (Fig. ​(Fig.2a).2a). A peak of activity was reached during the early stages of exponential growth and values of ATPase activity were consistently higher (twofold) in cells grown under octanoic acid stress (Fig. ​(Fig.2),2), as described by Viegas et al. (24). Yeast cells must adapt to the physiological effects of octanoic acid during an extended lag period, the length of which depended on the severity of acid stress (Fig. ​(Fig.1a),1a), before eventually recovering and entering exponential growth; the activation of plasma membrane H⁺-ATPase observed during this period of latency reinforces the idea that this proton pump is an important component of this adaptative response (5, 8, 19, 24). In fact, the ability of yeast cells to grow in the presence of lipophilic acids at a low pH reflects their capacity to maintain control over their internal pH by excluding protons. This adaptative phenomenon, reported for the first time in the present work, complements the observation of Holyoak et al. (8) that a strain with reduced plasma membrane H⁺-ATPase activity displayed increased lag phase in the presence of the weak-acid preservative sorbic acid. Significantly, plasma membrane H⁺-ATPase activity was also pointed out to play a critical role in yeast tolerance of ethanol (15) or supraoptimal temperatures (13, 25). The mechanism underlying plasma membrane ATPase activation during octanoic acid-induced latency remains obscure at the present time, but it is likely that this is due to a posttranslational modification of ATPase, as proposed for ATPase activation during octanoic acid-stressed exponential growth (24). It is likely that during lag phase the amount of H⁺-ATPase in the plasma membrane slightly decreases, as found by Benito et al. (2) in yeast cells deprived of nitrogen source where ATPase activation also occurred (2), as the estimated half-life of the enzyme is about 11 h (2). ATPase activation during latency can hardly be attributed to the adaptative modification of the ATPase lipid environment in cells grown under lipophilic acid stress, as suggested by Alexandre et al. (1).

Changes in yeast pH_i and viability during octanoic acid-stressed cultivation.

The change in pH_i during cultivation of nonadapted cells with 0.39 mM octanoic acid was monitored by using an adaptation of the fluorescence microscopic image processing technique developed by Imai and Ohno (9); 5- (and 6)-carboxyfluorescein (cF) was used as the internal pH-dependent fluoroprobe. Cells washed and resuspended in cold CF buffer (citrate-phosphate buffer [at pH 4.0] with 50 mM glycine [Sigma], 110 mM NaCl, 5 mM KCl, and 1 mM MgCl₂) to a cellular density of 2 × 10⁸ ml⁻¹ were loaded with cF by adding 20 μM of 5 (and 6)-carboxyfluorescein-diacetate (Sigma) and vortexing in two bursts of 1 min each, interspersed with 15 min on ice (9). After being washed twice with cold CF buffer, cF-loaded cells were immediately examined with a Zeiss Axioplan microscope equipped with adequate epifluorescence interference filters (Zeiss BP450-490 and Zeiss LP520) and connected to a video camera and to a computer with an image- analysis program (gel documentation system SW2000; UVP, San Gabriel, Calif.). Following a cell-by-cell analysis, the value of fluorescence intensity (fI) emitted by each cell, measured by direct densitometry, corresponded to the arithmetical mean value of fI measured in two or three different regions in the cytoplasm of the same cell, with the less fluorescent vacuole excluded. To estimate average pH_i, an in vivo calibration curve was prepared (Fig. ​(Fig.3)3) by using cell suspensions grown in the absence of toxics which were loaded with cF as described above and incubated, at 30°C, with 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to dissipate the plasma membrane pH gradient (4), before adjustment of external pH (in the range 3.5 to 7.5) by the addition of HCl or NaOH at 2 M. Fluorescence images were fixed 15 s after the occurrence of the excitation radiation in order to minimize interferences due to leakage of cF as well as fluorescence quenching (3, 7). Cells were kept on ice throughout the procedure, and CF buffer lacked glucose; therefore, the active efflux of cF (3) was minimized as confirmed by measuring the fluorescence in the medium surrounding the cells, which was negligible. Under the experimental conditions used and for the purpose of the study, this technique proved to be highly useful and suitable despite the limitations that might be raised (3, 7). It allowed a clear-cut picture of the pH_i of individual cells, giving information about the distribution of pH_i values of a yeast population (Fig. ​(Fig.44 and ​and5a5a to c), instead of solely an estimation of the average value of the whole population, as is the case with techniques based on the distribution of radioactive propionic acid (20) or on the in vivo ³¹P nuclear magnetic resonance (5). Moreover, values calculated for the average pH_i of the whole yeast population during latency and exponential growth in medium with octanoic acid (Fig. ​(Fig.5d)5d) were close to, although slightly lower than, the values previously obtained based on the distribution of [¹⁴C]propionic acid (20, 22). Results revealed that the cell population used to inoculate octanoic acid-supplemented medium exhibited a significant heterogeneity (Fig. ​(Fig.4);4); around 31% showed a pH_i in the optimal range (above 6.5) (Fig. ​(Fig.4),4), with the average pH_i value of the whole population estimated to be approximately 6.0. This low pH_i value results from cell cultivation in a rich medium with high production of organic acids (11) (external pH, 3.6), followed by washing of the cells with YG medium buffered at pH 4.0 (17). The introduction of the inoculum in octanoic acid-supplemented medium led to the very rapid (5-min) increase in the percentage of the cell population with pH_i below 5.5, consistent with the rapid kinetics of cytosol acidification when yeast cells are exposed to weak acids (5). During extended incubation with octanoic acid and until the end of latency, the percentage of the population with a very low pH_i (below 5.5) continued to increase, reaching 80% of the cell population, while the percentage of cell population with a pH_i above 6.0 suffered a corresponding decrease (Fig. ​(Fig.5).5). During exponential growth, the opposite pH_i modification was observed, consistent with a recovery of pH_i to physiological levels (Fig. ​(Fig.5).5). The time-dependent pattern of internal acidification during lag phase correlated with plasma membrane ATPase activation (Fig. ​(Fig.2a2a and ​and5),5), suggesting that this activation was triggered by intracellular acidification, as proposed for acetic acid (5)- or nitrogen starvation (2)-induced activation. Immediately before yeast cells entered exponential growth, 80% of the initial viable population had lost viability, as assessed by the number of CFU (21) (Fig. ​(Fig.6),6), suggesting that octanoic acid-induced death during latency is related to internal acidification down to critical values (Fig. ​(Fig.55 and ​and6),6), in agreement with the relationship established by Imai and Ohno (10) between yeast viability and intracellular pH. Only about 20% of the initial population was able to start cell division in octanoic acid-supplemented medium, presumably those cells that in the inoculum exhibited pH_i values around neutrality (Fig. ​(Fig.55 and ​and6).6). These results suggest that despite plasma membrane H⁺- ATPase activation, this system of pH homeostasis may not be able to fully counteract the physiological effects of increasing octanoic acid concentrations and eventually fails at very severe acid stress. Open in a separate windowFIG. 3In vivo calibration curve, showing the pH dependence of the fI of cF-loaded-cells of S. cerevisiae IGC 3507III. Intracellular and extracellular pHs were equilibrated by incubation of cF-loaded cells, for 10 min at 30°C, with 0.5 mM CCCP. At each pH, values of fI correspond to the average fI of about 20 cells. The data are averages with standard deviations for three independent experiments.Open in a separate windowFIG. 4Distribution of cells with different pH_i values present in the inoculum of S. cerevisiae IGC 3507III prepared in growth medium without octanoic acid supplementation.Open in a separate windowFIG. 5Percentage of yeast cells with pH_i below 5.5 (a), between 5.5 and 6.0 (b), or above 6.0 (c); average pH_i of the whole cell population (▴) during S. cerevisiae IGC3507III cultivation in medium supplemented with 0.39 mM total octanoic acid (pH 4.0, 30°C); and the optical density (OD) of the culture at 600 nm (▪). The average pH_i values estimated for the whole cell population are the arithmetical mean values of the various average pH_i values calculated for individual cells. The percentage of cells present in the inoculum with pH_i values within the three ranges (○) and the average pH_i of the inoculum cell population (▵) are indicated.Open in a separate windowFIG. 6Concentration of viable cells (▴) and culture optical density (O.D.) at 600 nm (□) during lag and exponential phases of S. cerevisiae IGC 3507III growth in medium supplemented with 0.39 mM octanoic acid, at pH 4.0 and 30°C.

Adaptative response to octanoic acid.

The adaptation of yeast cells to octanoic acid at a low pH appears to depend on their H⁺-exporting ability, but this requires not only a highly active H⁺-ATPase in the plasma membrane but the provision of sufficient ATP to drive this energy-demanding process as indicated by the results of Holyoak et al. (8). It is likely that increased ATPase activity under octanoic acid stress may reduce cellular ATP levels and that ATP depletion contributes to the failure of the maintenance of pH_i homeostasis, particularly among the subpopulation that in the inoculum exhibited the lowest pH_i values. The loss of viability might occur for those cells where pH_i decreased down to nonphysiological values. The eventual recovery of growth therefore depends on the remaining viable population, in agreement with the well-known critical role played by the physiology and number of viable cells in the inoculum in the duration of latency under acid stress. The observation that octanoic acid-adapted cells reinoculated into the same fresh medium can resume growth after a much shorter latency (Fig. ​(Fig.1a)1a) is a good example of the importance of the physiology of the inoculum cells. Besides the increased plasma membrane H⁺-ATPase activity of octanoic acid-adapted cells, other mechanisms may underlie the adaptation to acid stress, such as the increased cellular buffering capacity of octanoic acid-grown cells due to their lower intracellular volume (20), the more favorable plasma membrane lipid composition (1), and the possible induction of the active efflux of the anion (26).     

Computational Analysis of Human Adenovirus Type 22 Provides Evidence for Recombination among Species D Human Adenoviruses in the Penton Base Gene

Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.Adenoviruses were first isolated in 1953 (18, 31) and currently cause an array of human diseases. These include respiratory, gastrointestinal, and ocular surface infections, opportunistic infections in immune-deficient individuals, and possibly, obesity (9, 13, 14, 20, 40). Adenoviruses have also recently been used as gene therapy vectors (19, 35). Thus, while adenoviruses continue to cause significant morbidity and mortality in the human population, their existence also provides a potential benefit for the treatment of patients with an even broader range of ailments.Since the characterization of the first human adenovirus (HAdV), 53 types have been identified and subsequently classified into seven species (A to G) on the basis of serology, restriction endonuclease digestion patterns, and to a lesser degree, genotyping. Recently, high-throughput sequencing technology has made whole-genome sequencing both rapid and affordable (27). However, the genomic sequences of 23 out of 53 HAdV types remain to be determined, with most of those within species D.Species D HAdV type 22 (HAdV-D22) was originally isolated from a child in 1956 (3). While it is not clear what role HAdV-D22 plays in human disease, one report revealed a possible tropism for the eye (32). Recently, recombination with HAdV-D22 has been identified as the source of a novel HAdV, HAdV-D53, causing an outbreak of keratoconjunctivitis in Germany (37). HAdV-D22 recombination was also identified in a possible variant of HAdV-D53 that was isolated from a patient in Japan (2). Therefore, HAdV-D22 has shown the propensity to recombine with other viruses, with clinically important consequences. The emergence of new pathogenic HAdV genotypes, along with continued interest in HAdVs as vectors for human gene therapy, make adenovirus recombination a critically important issue.HAdV-D22 was acquired from the American Type Culture Collection (Manassas, VA). The complete genome of the prototype strain AV-2711 (ATCC VR-1100) was sequenced on an Applied Biosystems (Foster City, CA) 3730 XL DNA sequencer in the Laboratory for Genomics and Bioinformatics at the University of Oklahoma Health Sciences Center using a previously described protocol (29). The sequence was validated by sequencing on an ABI SOLiD DNA sequencer. Sequences from both methodologies were 100% identical and provided 7,727-fold coverage for the genome.The mVISTA Limited Area Global Alignment of Nucleotides (LAGAN) tool (http://genome.lbl.gov/vista/index.shtml) was used to align and compare the whole HAdV genomes (6) of HAdV-D22 and each of the other nine completely sequenced HAdV-Ds. Analysis revealed sequence diversity in the penton base, hexon, E3, and fiber open reading frames (Fig. ​(Fig.1).1). Surprisingly, comparison of HAdV-D22 to HAdV-D19 strain C (30) and HAdV-D37 (29) revealed considerable sequence conservation in the penton base gene (Fig. ​(Fig.11 and 2A and B).Open in a separate windowFIG. 1.Global pairwise sequence alignment of HAdV-D22 with nine other HAdV-D types. The percent sequence conservation is reflected in the height of each data point along the y axis. A conserved sequence in the penton base open reading frame is designated by an asterisk. GenBank accession numbers are as follows: HAdV-D8, AB448768; HAdV-D9, AJ854486; HAdV-D19, EF121005; HAdV-D26, EF153474; HAdV-D37, DQ900900; HAdV-D46, AY875648; HAdV-D48, EF153473; HAdV-D49, DQ393829; and HAdV-D53, FJ169625.Open in a separate windowFIG. 2.Multisequence alignment of HAdV-D penton base genes. Nucleotide alignment of the bp 397 to 594 (A) and bp 694 to 1089 (B) regions of the penton base gene.Based on the sequence conservation seen in the penton base, Simplot 3.5.1 (http://sray.med.som.jhmi.edu/SCRoftware/simplot/) and Recombination Detection Program (RDP) version 3.34 (http://darwin.uvigo.es/rdp/rdp.html) were used to identify possible recombination sites (21, 24). Bootscan analysis identified two possible recombination loci in the HAdV-D22 penton base, the first in HAdV-D37, encompassing nucleotides 400 to 600, and the second in HAdV-D19 at nucleotides 750 to 1350 (Fig. ​(Fig.3A).3A). In silico amino acid analysis showed that these two probable recombination areas code for the two variable loops in the penton base protein (Fig. ​(Fig.3A),3A), located on the exterior of the viral capsid (16, 44).Open in a separate windowFIG. 3.Bootscan analysis of HAdV-D penton base genes. Comparison of HAdV-D22 (A) with completely sequenced HAdV types. Similar comparisons of the same region were performed with HAdV-D9 (B), HAdV-D49 (C), HAdV-A12 (D), HAdV-B3 (E), HAdV-C2 (F), or HAdV-F41 (G) as the reference type. HAdV-D53 was left out of the analysis due to the 100% identity of its penton base to that of HAdV-D37. The axes of all panels are as labeled in panel A. GenBank accession numbers are as follows: HAdV-A12, NC_001460; HAdV-B3, DQ086466; HAdV-C2, AC_000007; and HAdV-F41, DQ315364.We extended our investigation to determine if these recombination sites were common to penton base genes of other HAdV genomes. We found evidence for recombination between HAdV-D9 and HAdV-D26 in both the nucleotide 400 to 600 and 750 to 1350 regions (Fig. ​(Fig.3B),3B), between HAdV-D49 and HAdV-D48 in the nucleotide 400 to 600 region, and between HAdV-D49 and HAdV-D8 in the nucleotide 750 to 1350 region (Fig. ​(Fig.3C).3C). A similar pattern was observed in one or both of these nucleotide regions for HAdV-D8, -17, -19, -26, -37, and -48 (data not shown). Remarkably, this pattern of recombination in the penton base gene was unique to HAdV species D (Fig. 3D, E, F, and G).Bootstrap-confirmed neighbor-joining phylogenetic trees of HAdV-D penton base genes were constructed with Molecular Evolutionary Genetics Analysis (MEGA) 4.0.2 (http://www.megasoftware.net/index.html) to examine viral evolution in HAdV-D (34). Analysis of the entire penton gene revealed a close relationship of HAdV-D22 and HAdV-D19 strain C (Fig. ​(Fig.4A)4A) (30). Additional phylogenetic trees were constructed to encompass the two proposed recombination sites within this gene. Phylogenetic analysis of the nucleotide 400 to 600 region revealed a close identity among HAdV-D22, HAdV-D37, and HAdV-D53 (Fig. ​(Fig.4B).4B). Analysis of the nucleotide 750 to 1350 region of the penton base gene revealed a close identity of HAdV-D22 and HAdV-D19 (Fig. ​(Fig.4C4C).Open in a separate windowFIG. 4.Phylogenetic analysis of HAdV-D penton base genes. Bootstrap neighbor-joining tree designed using MEGA 4.0.2. The Gonnet protein weight matrix in ClustalX alignment was used, along with complete deletion options. Percent bootstrap confidence levels (1,000 replicates) are shown on the relevant branches. (A) Analysis of sequenced HAdV-D penton base genes. (B) Analysis of the nucleotide 400 to 600 region of sequenced HAdV-D penton base genes. (C) Analysis of the nucleotide 750 to 1350 region of sequenced HAdV-D penton base genes.In summary, comparison of the HAdV-D22 genome to other sequenced HAdV-D genomes identified substantial sequence divergence in the penton, hexon, E3, and fiber open reading frames. Differences between HAdV-D genomes in these areas have previously been described by us in reports on HAdV-D19 strain C and HAdV-D37 genomes (29, 30). However, the sequence conservation among HAdV-D22, HAdV-D19, and HAdV-D37 in the penton base gene was unexpected and suggests recombination among these viruses. Bootscan analysis identified recombination events at two regions within the penton base gene, encompassing nucleotides 400 to 600 and 750 to 1350. Further analysis of other HAdV-D penton genes suggests that these areas represent hot spots for recombination.The propensity for recombination among different HAdV-Ds in the penton base gene can be understood in the context of penton base function. The adenovirus penton base acts as the ligand for a secondary attachment event that is critical to host cell internalization (38) and thus is critical to infection. The penton base protein contains two hypervariable loops located on the surface of the viral capsid (16, 44). One loop contains the host cell integrin binding Arg-Gly-Asp (RGD) motif mediating attachment to host cell integrins (8, 38). The RGD motif is conserved in almost every HAdV penton base protein, with the exception of HAdV-F40 and HAdV-F41, which do not use host cell integrins for internalization (1, 11). The second exterior loop, known as the variable loop (HVL1), has no known function. Both of these regions are highly variable among different HAdV types. We identified recombination around both of these loops for multiple viruses within HAdV-D (Fig. ​(Fig.3A3A).In recent epidemiological studies, patients were identified with coincident clinical infections with two or more adenovirus types (17, 36). Simultaneous infection by more than a single HAdV type is possible because of conserved host receptor affinity, common tissue tropisms, and an absence of immunity across serotypes. Our data, along with evidence from previous studies (22, 23, 37, 43), identified recombination events among viruses with similar tissue tropisms, providing evidence that the restriction of tissue tropism might determine in part the observed recombination within adenoviruses of the same species. Recently, a novel HAdV, HAdV-D53, was isolated from an outbreak of keratoconjunctivitis. Subsequent analysis revealed recombination among HAdV-D22, HAdV-D37, HAdV-D8, and a previously unknown adenoviral sequence, suggesting the potential for the emergence of new pathogens, with important ramifications for human disease.Previous work provides evidence for recombination in HAdVs (10, 12, 22, 25, 26, 37, 39, 41-43), but the mechanisms of recombination have yet to be identified. Recombination may result from selective pressure from the host immune system relative to surface capsid proteins, a nucleotide motif that directs cellular recombination machinery to the local sites on the viral DNA, or a combination of both. Two eukaryotic recombinases, RAD51 and Dmc1, both homologues of the bacterial recombinase RecA, act in host cell DNA recombination (5, 33). RAD51 mediates recombination during mitosis, while Dmc1 acts during meiosis. RAD51 is of potential interest because it colocalizes with promyelocytic leukemia nuclear bodies in the nucleus (4). Adenoviral proteins E1A and E4 Orf3 have been shown to interact with promyelocytic leukemia nuclear bodies, which play an important role in adenoviral replication (7, 15). An interaction between RAD51 and adenoviral DNA has not been studied. A proposed motif consisting of CCNCCNTNNCCNC was recently identified as being associated with loci of recombination and genome instability in humans (28). Although not present in the viruses we studied, sequencing of other viruses within HAdV-D may yet reveal a consensus site for recombination. The elucidation of recombination mechanisms for HAdVs should allow a better understanding of adenoviral evolution.In conclusion, our analysis of the penton base gene of HAdV-D identified a potential paradigm for adenovirus recombination and the emergence of pathogenic strains. An in depth understanding of adenovirus recombination and evolution is critical to ensure the safety of adenoviral gene therapy.