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.     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

A new species of Stenoloba Staudinger, 1892 from China (Lepidoptera,Noctuidae, Bryophilinae)

A new species of Stenoloba from the olivacea species group, Stenoloba solaris, sp. n. (Lepidoptera, Noctuidae), is described from Yunnan, China. Illustrations of the male holotype and its genitalia are provided. A diagnostic comparison is made with Stenoloba albistriata Kononenko & Ronkay, 2000, Stenoloba olivacea (Wileman, 1914), and Stenoloba benedeki Ronkay, 2001 (Fig. 4).Open in a separate windowFigures 1–5.Stenoloba spp. adults and biotope. 1 Stenoloba solaris, sp. n., male, holotypus, Yunnan, China (GBG/ZSM) 2 Stenoloba albistriata, male, paratypus, N. Vietnam (ZFMK) 3 Stenoloba olivacea, male, Taiwan (HNHM) 4 Stenoloba benedeki, male, paratypus, N. Vietnam (HNHM) 5 Type locality of Stenoloba solaris, sp. n. China, NW Yunnan, Lijiang/Zhongdian near Tuguancum, 27°29''700"N, 99°53''700"E.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.     

Conserved residues in the ankyrin domain of VAPYRIN indicate potential protein-protein interaction surfaces

Plant VAPYRINs are required for the establishment of arbuscular mycorrhiza (AM) and root nodule symbiosis (RNS). In vapyrin mutants, the intracellular accommodation of AM fungi and rhizobia is blocked, and in the case of AM, the fungal endosymbiont cannot develop arbuscules which serve for nutrient exchange. VAPYRINs are plant-specific proteins that consists of a major sperm protein (MSP) domain and an ankyrin domain. Comparison of VAPYRINs of dicots, monocots and the moss Physcomitrella patens reveals a highly conserved domain structure. We focused our attention on the ankyrin domain, which closely resembles the D34 domain of human ankyrin R. Conserved residues within the petunia VAPYRIN cluster to a surface patch on the concave side of the crescent-shaped ankyrin domain, suggesting that this region may represent a conserved binding site involved in the formation of a protein complex with an essential function in intracellular accommodation of microbial endosymbionts.Key words: VAPYRIN, arbuscular mycorrhiza, petunia, symbiosis, glomus, ankyrin, major sperm protein, VAPPlants engage in mutualistic interactions such as root nodule symbiosis (RNS) with rhizobia and arbuscular mycorrhiza (AM) with Glomeromycotan fungi. These associations are referred to as endosymbioses because they involve transcellular passage through the epidermis and intracellular accommodation of the microbial partner within root cortical cells of the host.^1,2 Infection by AM fungi and rhizobia is actively promoted by the plant and requires the establishment of infection structures namely the prepenetration apparatus (PPA) in AM and a preinfection thread in RNS, respectively.^3–5 In both symbioses the intracellular microbial accommodation in epidermal and root cortical cells involves rebuilding of the cytoskeleton and of the entire membrane system.^6–8 Recently, intracellular accommodation of rhizobia and AM fungi, and in particular morphogenesis of the AM fungal feeding structures, the arbuscules, was shown to depend on the novel VAPYRIN protein.^9–11VAPYRINs are plant-specific proteins consisting of two protein-protein interaction domains, an N-terminal major sperm protein (MSP) domain and a C-terminal ankyrin (ANK) domain. MSP of C. elegans forms a cytoskeletal network required for the motility of the ameboidal sperm.¹² MSP domains also occur in VAP proteins that are involved in membrane fusion processes in various eukaryotes.¹³ The ANK domain, on the other hand, closely resembles animal ankyrins which serve to connect integral membrane proteins to elements of the spectrin cytoskeleton,¹⁴ thereby facilitating the assembly of functional membrane microdomains in diverse animal cells.¹⁵ Ankyrin repeats exhibit features of nano-springs, opening the possibility that ankyrin domains may be involved in mechanosensing.¹⁶ Based on these structural similarities, VAPYRIN may promote intracellular accommodation of endosymbionts by interacting with membranes and/or with the cytoskeleton. Indeed, VAPYRIN protein associates with small subcellular compartments in petunia and in Medicago truncatula.^9,10Ankyrin repeats typically consist of 33 amino acids, of which 30–40% are highly conserved across most taxa. These residues confer to the repeats their basic helix-turn-helix structure.¹⁷ Ankyrin domains often consist of arrays of several repeats that form a solenoid with a characteristic crescent shape.¹⁷ Besides the ankyrin-specific motiv-associated amino acids there is little conservation between the ankyrin domains of different proteins, or between the individual repeats of a given ankyrin domain,¹⁷ a feature that was also observed in petunia VAPYRIN (Fig. 1A).⁹ However, sequence comparison of VAPYRINs from eight dicots, three monocots and the moss Physcomitrella patens revealed a high degree of sequence conservation beyond the ankyrin-specific residues (Fig. 1B and Sup. Fig. S1). When the degree of conservation was determined for the individual ankyrin repeats among all the 12 species, it appeared that repeats 7, 9 and 10 exhibited particularly high conservation (Fig. 1C).Open in a separate windowFigure 1Sequence analysis and phylogeny of VAPYRIN from diverse plants. (A) Predicted amino acid sequence of the petunia VAPYRIN protein PAM1. The 11 repeats of the ankyrin domain are aligned, and the ankyrin consensus sequence is shown below the eleventh ankyrin repeat (line c). Conserved residues that are characteristic for ankyrin repeats (Mosavi et al. 2004)¹⁷ are depicted in bold face. (B) Unrooted phylogenetic tree representing the VAPYRINs of eight dicot species (Petunia hybrida, Solanum lycopersicon, Solanum tuberosum, Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula and Glycine max) three monocot species (Sorghum bicolor, Zea mays and Oryza sativa), and the moss Physcomitrella patens. (C) Degree of conservation of the individual ankyrin repeats of VAPYRIN. Schematic representation of the MSP domain as N-terminal barrel-shaped structure, and of the individual ankyrin repeats as pairs of alpha-helices. An additional loop occurring only in monocots (grass-loop) is inserted above repeat 4, and the deletion between repeat 7 and 8 is indicated (gap). This latter feature is common to all VAPYRIN proteins. The percentage of amino acid residues that are identical in at least 11 of the 12 VAPYRINS is given below the MSP domain and the eleven ankyrin repeats. The box highlights repeats 7–10 which contribute to the predicted binding site (compare with Figs. 3 and ​and44).Sequence comparison of the eleven repeats of all the twelve plant species revealed that the individual repeats clustered according to their position in the domain, rather than according to their origin (plant species) (Fig. 2). This shows that the repeats each are well conserved across species, but show little similarity among each other within a given VAPYRIN protein. The higher conservation of repeats 9 and 10 was reflected by the compact appearance of the respective branches, in which the monocot and moss sequences were nested closely with the dicot sequences, compared to other repeats, where the branches appeared fragmented between monocots and dicots, and where the P. patens sequence fell out of the branch as in the case of repeats 4–6 (Fig. 2). Taken together, this points to an old evolutionary origin of the entire ankyrin domain in lower land plants, with no subsequent rearrangement of ankyrin repeats.Open in a separate windowFigure 2Phylogenetic analysis of the individual ankyrin repeats of VAPYRIN. Phylogenetic representation of an alignment of all the 11 repeats of the 12 VAPYRINs compared in Figure 1B and C. The repeats cluster according to their position within the domain, rather than to their origin (plant species). Numbers indicate the position of the repeats within the domain (compare with Fig. 1C). P. patens repeats are highlighted (small circles) for clarity. The monocot repeat 4 sequences (boxed) are remote from the remaining repeat 4 sequences because of the grass loop (compare with Fig. 1C).Ankyrin domains function as protein-protein interaction domains,¹⁷ in which the residues on the surface are involved in the binding of their protein partners.¹⁴ The fact that repeats 9 and 10 exhibited particularly high levels of conservation across species from moss to angiosperms indicated that this region may contain functionally important residues. Within repeat 10, sixteen amino acid positions were identical in >90% of the analyzed species (Fig. 3A and grey bars). Nine of those represent residues that are characteristic for ankyrin repeats (red letters) and determine their typical 3D shape.¹⁷ These residues are considered ankyrin-specific, and are unlikely to be involved in a VAPYRIN-specific function. The remaining seven highly conserved residues in repeat 10, however, are VAPYRIN-specific, since they have been under positive selection, without being essential for the basic structure of the ankyrin repeat. Ankyrin-specific and VAPYRIN-specific residues where identified throughout the entire ankyrin domain (Sup. Fig. 1), and subsequently mapped on a 3-dimensional model of petunia VAPYRIN to reveal their position in the protein (Fig. 3B–G). The ankyrin-specific residues were found to be localized primarily to the interior of the ankyrin domain, with the characteristic glycines (brown) marking the turns between helices and loops (Fig. 3B, D and F, compare with A). In contrast, the VAPYRIN-specific residues were localized primarily on the surface of the ankyrin domain (Fig. 3C, E and G). A prominent clustering of VAPYRIN-specific residues was identified on the concave side of the crescent-shaped ankyrin domain comprising repeats 7–10 close to the gap (Figs. 3G and ​and44). This highly conserved VAPYRIN-specific region contains several negatively and positively charged residues (D, E and K, R, respectively) and aromatic residues (W, Y, F), which may together form a conserved binding site for an interacting protein.Open in a separate windowFigure 33D-Mapping of conserved positions within the ankyrin domain of VAPYRIN. (A) Conserved amino acid residues were evaluated for ankyrin repeat 10 of petunia VAPYRIN as an example. The degree of conservation between the 12 VAPYRINs analyzed in Figures 1B and ​and22 is depicted with grey bars. Average conservation between all the 132 ankyrin repeats of the 12 VAPYRIN sequences is shown with black bars. Residues that are conserved in all 132 repeats (red letters) define the ankyrin consensus sequence, which confers to the repeats their characteristic basic structure.¹⁷ Residues that are >90% conserved but are not part of the basic ankyrin sequence (highlighted with asterisks) are VAPYRIN-specific and may therefore have been conserved because of their specific function in VAPYRIN. Arrows indicate the characteristic antiparallel helices, the turns are marked by conserved glycine residues (underlined; compare with B, D and F). (B–G) 3D-models of the petunia VAPYRIN PAM1. Conserved amino acid residues were color-coded according to their physico-chemical properties (http://life.nthu.edu.tw/∼fmhsu/rasframe/SHAPELY.HTM) with minor modification (see below). In (B, D and F) the ankyrin-specific residues are highlighted (corresponding to the bold letters in Fig. 1A). In (C, E and G), the VAPYRIN-specific residues are highlighted. Note the patch of high conservation on the concave side of the crescent-shaped ankyrin domain between repeats 7–10 next to the gap. (B–E) represent respective side views of the ankyrin domain, (F and G) exhibit the concave inner side of the domain. Color code: Bright red: aspartic acid (D), glutamic acid (E); Yellow: cysteine (C); Blue: lysine (K), arginine (R); Orange: serine (S), threonine (T); Dark blue: phenylalanine (F), tyrosin (Y); Brown: glycine (G); Green: leucin (L), valine (V), isoleucin (I), alanine (A); Lilac: tryptophane (W); Purple: histidine (H); Pink: proline (P).Open in a separate windowFigure 4The highly conserved surface area in domain 8–10 of the ankyrin domain of petunia VAPYRIN. Close-up of the highly conserved region of petunia PAM1 as shown in Figure 3G. Amino acids were color-coded as in Figure 3 and their position in the amino acid sequence is indicated (compare with Sup. Fig. 1).In this context, it is interesting to note that human ankyrin R also contains a binding surface on the concave side of the D34 domain for the interaction with the CBD3 protein.¹⁴ Consistent with an essential function of the C-terminal third of the ankyrin domain, mutations that abolish this relatively short portion of VAPYRIN, have a strong phenotype, indicating that they may represent null alleles.⁹ Based on this collective evidence, we hypothesize that repeats 7–10 are involved in the formation of a protein complex that is essential for intracellular accommodation of rhizobia and AM fungi. Biochemical and genetic studies are now required to identify the binding partners of VAPYRINs, and to elucidate their role in plant endosymbioses.     

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     

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.     

A photoreceptor going nowhere but the nucleus

Cryptochrome 2 (CRY2) is a blue/UV-A light receptor that regulates light inhibition of cell elongation and photoperiodic promotion of floral initiation in Arabidopsis. We and others have previously shown that CRY2 is a nuclear protein that regulates gene expression to affect plant development. We also showed that CRY2 is phosphorylated in response to blue light and the phosphorylated CRY2 is most likely active and degraded in blue light. Given that protein translation (and probably chromophore attachment) takes place in the cytosol and that a photoreceptor would absorb photon instantaneously, it would be interesting to know where those inter-connected events occur in the cell. Our results showed that freshly synthesized CRY2 photoreceptor is inactive in the cytosol although it may be photon-excited, it is imported into the nucleus where the photoreceptor is phosphorylated, performs its function, becomes ubiquitinated, and eventually gets degraded (Fig. 1).¹ To our knowledge, this is the first example in any organism that a photoreceptor is shown to complete its post-translational life cycle in a single subcellular compartment.Open in a separate windowFigure 1A model depicting the post-translational life cycle of CRY2. Pi, phosphate group; Ubq, ubiquitin.Key words: blue light, cryptochrome, ubiquitination, phosphorylation, Arabidopsis