The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95–Discs Large–zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity

Multiplex PCR for rapid diagnosis of gastrointestinal tuberculosis

Background: Rapid and specific diagnosis of gastrointestinal tuberculosis (GITB) is of utmost importance. Aim: To evaluate Multiplex PCR (MPCR) using MPB64 and IS6110 primers specific for M. tuberculosis for rapid diagnosis of GITB. Materials and Methods: MPCR was performed on colonoscopy biopsy specimens on 11 GITB confirmed (culture/AFB/histopathology was positive), 29 GITB suspected and 30 Non GITB (control group) patients. Results: MPB64 PCR had sensitivity and specificity of 90% and 100% for confirmed GITB cases. In 29 clinically diagnosed but unconfirmed GITB cases, MPCR was positive in 72.41%. MPCR was negative in all control group patients. The overall sensitivity and specificity of microscopy, culture, histopathology and MPCR was 5%, 2% 20% and 77.5% and 100%, 100%, 100% and 100% respectively. Conclusion: MPCR has good sensitivity and specificity in diagnosing gastrointestinal tuberculosis

Evaluation of PCR using MPB64 Primers for Rapid Diagnosis of Tuberculous Meningitis

TBM control group and its comparison with conventional techniques like microscopy and culture. Materials and Methods A total of 130 CSF samples received for AFB smear and culture in laboratory of tertiary care hospital of India, between September 2008 and December 2009 were evaluated. Patient's age ranged from 12-90 years. The relevant history and other details of the patients were noted from the case records. The patients were divided into 3 groups: Group I: TBM (n=90): (a) confirmed TBM-culture/smear positive (n=9), and (b) suspected TBM: smear/culture negative, clinical and laboratory features suggestive of TBM and response to anti-tuberculosis therapy Processing of CSF sample All the 130 CSF samples were subjected to three microbiological tests: Ziehl-Neelsen staining (ZN), culture on Lowenstein-Jensen medium and PCR with MPB-64. The CSF samples of the subjects were Keywords: PCR; Tubercular meningitis Introduction Tuberculosis is a major cause of morbidity and mortality worldwide. The World Health Organization has noted that the global incidence of TB is increasing by 0.4% per annum The development of rapid, sensitive and specific test for detection of mycobacterium has been a long standing need. A number of mycobacterial antigen Nucleic Acid Amplification Techniques (NAAT) such as Polymerase Chain Reaction (PCR) has been reported to be more sensitive and specific. Several Mycobacterium tuberculosis specific sequences like IS6110, Protein antigen b [8], MPB64 and 65 kDa have been evaluated Abstract Purpose: Diagnosis of Tuberculosis (TB) is largely based on microscopy and culture, which either lack of sensitivity or time consuming. In the present study a PCR test based on DNA sequence coding for MPB64, specific for Mycobacterium tuberculosis was compared with Ziehl-Neelson (ZN) stained AFB smear examination, culture based on conventional LJ medium for diagnosis of tuberculosis using clinical samples obtained from Cerebro-Spinal Fluid (CSF) samples from TBM patients. Methods: PCR using MPB64 primers was performed on 9 TBM confirmed (culture was positive), 81TBM suspected and 40 Non TBM (control group) patients. Results: MPB64 PCR had sensitivity of 88.8% and specificity of 100% for confirmed TBM cases, where as in 81 clinically diagnosed but unconfirmed TBM cases MPB64 PCR was positive in 81.48% cases respectively. The overall sensitivity of microscopy, culture and PCR using MPB64 were 1.11%, 10%, 82.22% and specificity was 100%, 100% and 100% respectively

NFκB Promotes Inflammation, Coagulation, and Fibrosis in the Aging Glomerulus

The peak prevalence of ESRD from glomerulosclerosis occurs at 70 to 79 years. To understand why old glomeruli are prone to failure, we analyzed the Fischer 344 rat model of aging under ad libitum-fed (rapid aging) and calorie-restricted (slowed aging) conditions. All glomerular cells contained genes whose expression changed “linearly” during adult life from 2 to 24 months: mesangial cells (e.g., MMP9), endothelial cells (e.g., ICAM and VCAM), parietal epithelial cells (e.g., ceruloplasmin), and podocytes (e.g., nephrin and prepronociceptin). Patterns of aging glomerular gene expression closely resembled atherosclerosis, including activation of endothelial cells, epithelial cells, and macrophages, as well as proinflammatory pathways related to cell adhesion, chemotaxis, blood coagulation, oxidoreductases, matrix metalloproteinases, and TGF-β activation. We used a nonbiased data-mining approach to identify NFκB as the likely transcriptional regulator of these events. We confirmed NFκB activation by two independent methods: translocation of NFκB p50 to glomerular nuclei and ChIP assays demonstrating NFκB p50 binding to the κB motif of target genes in old versus young glomeruli. These data suggest that old glomeruli exhibit NFκB-associated up-regulation of a proinflammatory, procoagulable, and profibrotic phenotype compared with young glomeruli; these distinctions could explain their enhanced susceptibility to failure. Furthermore, these results provide a potential mechanistic explanation for the close relationship between ESRD and atherosclerotic organ failure as two parallel arms of age-associated NFκB-driven processes

Urine Podocyte mRNAs Mark Progression of Renal Disease

Because loss of podocytes associates with glomerulosclerosis, monitoring podocyte loss by measuring podocyte products in urine may be clinically useful. To determine whether a single episode of podocyte injury would cause persistent podocyte loss, we induced limited podocyte depletion using a diphtheria toxin receptor (hDTR) transgenic rat. We monitored podocyte loss by detecting nephrin and podocin mRNA in urine particulates with quantitative reverse transcriptase–PCR. Aquaporin 2 mRNA served as a kidney reference gene to account for variable kidney contribution to RNA amount and quality. We found that a single injection of diphtheria toxin resulted in an initial peak of proteinuria and podocyte mRNAs (podocin and nephrin) followed 8 d later by a second peak of proteinuria and podocyte mRNAs that were podocin positive but nephrin negative. Proteinuria that persisted for months correlated with podocin-positive, nephrin-negative mRNAs in urine. Animals with persistent podocyte mRNA in urine progressed to ESRD with global podocyte depletion and interstitial scarring. Podocytes in ectatic tubules expressed podocalyxin and podocin proteins but not nephrin, compatible with detached podocytes’ having an altered phenotype. Parallel human studies showed that biopsy-proven glomerular injury associated with increased urinary podocin:aquaporin 2 and nephrin:aquaporin 2 molar ratios. We conclude that a single episode of podocyte injury can trigger glomerular destabilization, resulting in persistent podocyte loss and an altered phenotype of podocytes recovered from urine. Podocyte mRNAs in urine may be a useful clinical tool for the diagnosis and monitoring of glomerular diseases