Accurate prediction of the functional significance of single nucleotide polymorphisms and mutations in the ABCA1 gene

The human genome contains an estimated 100,000 to 300,000 DNA variants that alter an amino acid in an encoded protein. However, our ability to predict which of these variants are functionally significant is limited. We used a bioinformatics approach to define the functional significance of genetic variation in the ABCA1 gene, a cholesterol transporter crucial for the metabolism of high density lipoprotein cholesterol. To predict the functional consequence of each coding single nucleotide polymorphism and mutation in this gene, we calculated a substitution position-specific evolutionary conservation score for each variant, which considers site-specific variation among evolutionarily related proteins. To test the bioinformatics predictions experimentally, we evaluated the biochemical consequence of these sequence variants by examining the ability of cell lines stably transfected with the ABCA1 alleles to elicit cholesterol efflux. Our bioinformatics approach correctly predicted the functional impact of greater than 94% of the naturally occurring variants we assessed. The bioinformatics predictions were significantly correlated with the degree of functional impairment of ABCA1 mutations (r² = 0.62, p = 0.0008). These results have allowed us to define the impact of genetic variation on ABCA1 function and to suggest that the in silico evolutionary approach we used may be a useful tool in general for predicting the effects of DNA variation on gene function. In addition, our data suggest that considering patterns of positive selection, along with patterns of negative selection such as evolutionary conservation, may improve our ability to predict the functional effects of amino acid variation.     

Role of protein phosphatase 2A in mGluR5-regulated MEK/ERK phosphorylation in neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.     

First signature of islet beta-cell-derived naturally processed peptides selected by diabetogenic class II MHC molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet beta-cells selected by diabetogenic I-A(g7) molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-A(g7) were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in beta cells from normal islets. Peptides bound to I-A(g7) molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet beta-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.     

High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression

In chickens, high levels of dietary zinc cause molting, and the reproductive system undergoes complete remodeling concomitant to feather replacement. In the present study, the expression profiles of cytokines and chemokines were investigated in the ovary and oviduct of control hens and of hens induced to molt by zinc feeding. The zinc-induced feed-intake suppression, the changes in corticosterone levels, the immune cell populations in the reproductive tract, and the apoptosis of reproductive tissues were analyzed. The expression of mRNAs for interleukin-6 (IL-6), interferon-γ (IFN-γ), the avian ortholog of mammalian IL-8 (chCXCLi2), and a chicken MIP-1β-like chemokine (chCCLi2) in the ovary and of mRNAs for IL-1β, IL-6, IFN-γ, transforming growth factor-β2, chCXCLi2, and chCCLi2 in the oviduct were upregulated significantly during zinc-induced molting. A simultaneous feed-intake reduction was observed with higher expression of cytokines and chemokines. The results of the present investigation also suggested that the upregulation of corticosterone was closely associated with the increased expression of cytokines and chemokines. An increase in apoptosis within reproductive tissue during tissue regression was also noted. We had previously observed the upregulation of these cytokines expression in an earlier study (molting by feed withdrawal). However, the pattern and the level of expression were different among these two methods. These findings indicate that cytokines might be a common mediator of tissue regression during molting induced by diverse methods, although the pattern of induction is different. Thus, a high dose of dietary zinc seems to induce reproductive regression via the upregulation of cytokines and chemokines, the suppression of feed intake, and the increase in serum corticosterone, resulting finally in the apoptosis of reproductive tissues.     

In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.     

Low frequency fatigue in human quadriceps is fatigue dependent and not task dependent.

It is well accepted that a low intensity/long duration isometric contraction induces more low frequency fatigue (LFF) compared to a high-intensity/short-duration contraction. However, previous reports examined the intensity/duration of the contraction but did not control the level of fatigue when concluding fatigue is task dependent. The purpose of this study was to determine whether a long duration/low intensity fatiguing contraction would induce greater LFF than a short duration/high-intensity contraction when the quadriceps muscle was fatigued to similar levels. Eighteen healthy male subjects performed quadriceps contractions sustained at 35% and 65% of maximal voluntary contraction (MVC) on separate days, until the tasks induced a similar amount of fatigue (force generating capacity=45% MVC). Double pulse torque to single pulse torque ratio (D/S ratio) was obtained before, immediately and 5min after fatigue along with the electromyographic (EMG) signal from vastus medialis (VM) and rectus femoris (RF). The D/S ratio significantly (p<0.05) increased by 8.7+/-8.5% (mean+/-SD) and 10.2+/-9.2% after 35% and 65% tasks, respectively, and remained elevated 5min into recovery; however, there was no significant difference in ratio between the two sessions immediately or 5min post-fatigue (p>0.05) even though the endurance time for the 35% fatigue task (124+/-39.68s) was significantly longer (p=0.05) than that of the 65% task (63+/-17.73s). EMG amplitude and median power frequency (MPF) analysis also did not reveal any significant differences between these two sessions after fatigue. These findings indicate that LFF fatigue is fatigue dependent as well as task intensity/duration dependent. These findings assist us in understanding task dependency and muscle fatigue.     

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     

Bacterial genome chimaerism and the origin of mitochondria

Many studies have sought to determine the origin and evolution of mitochondria. Although the Alphaproteobacteria are thought to be the closest relatives of the mitochondrial progenitor, there is dispute as to what its particular sister group is. Some have argued that mitochondria originated from ancestors of the order Rickettsiales, or more specifically of the Rickettsiaceae family, while others believe that ancestors of the family Rhodospirillaceae are also equally likely the progenitors. To resolve some of these disputes, sequence similarity searches and phylogenetic analyses were performed against mitochondria-related proteins in Saccharomyces cerevisiae. The 86 common matches of 5 Alphaproteobacteria (Rickettsia prowazekii, Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Ochrobactrum anthropi) to yeast mitochondrial proteins were distributed fairly evenly among the 5 species when sorted by highest identity or score. Moreover, exploratory phylogenetic analyses revealed that among these common matches, 44.19% (38) had branched most closely with O. anthropi, while only 34.88% (30) corresponded with Rickettsia prowazekii. More detailed phylogenetic analyses with additional Alphaproteobacteria and including genes from the mitochondria of Reclinomonas americana found matches of mitochondrial genes to those of members of the Rickettsiaceae, Anaplasmataceae, and Rhodospirillaceae families. The results support the idea that notable bacterial genome chimaerism has occurred en route to the formation of mitochondria.