A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of <Emphasis Type="Italic">peste des petits ruminants</Emphasis> virus in the clinical samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T_m of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ～0.0001 cell culture infectious dose 50% units (TCID₅₀). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.     

Computational structural analysis of protein interactions and networks

Protein interactions have been at the focus of computational biology in recent years. In particular, interest has come from two different communities--structural and systems biology. Here, we will discuss key systems and structural biology methods that have been used for analysis and prediction of protein-protein interactions and the insight these approaches have provided on the nature and organization of protein-protein interactions inside cells.     

α-L-rhamnosidase from Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH

An a-L-rhamnosidase secreting fungal strain has been isolated and identified as Aspergillus clavato-nanicus MTCC-9611. The enzyme was purified to homogeneity from the culture filtrate of the fungus using concentration by ultrafiltration membrane and ion-exchange chromatography on CM-cellulose. The native PAGE analysis confirmed the homogeneity of the purified enzyme. The SDS-PAGE analysis of the purified enzyme revealed a single protein band corresponding to the molecular weight 82 kDa. The α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 had optimum at pH 10.0 and 50°C. The K _m values of the enzyme were 0.65 mM and 0.95 mM using p-nitrophenyl α-L-rhamnopyranoside and naringin as a substrates respectively. The enzyme transforms naringin to prunin at pH 10.0 and further hydrolysis of prunin to naringenin does not occur under these reaction conditions that makes α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 promising enzyme to get prunin for pharmaceutical purposes.     

Unexpected high pressure effects on the structural properties of condensed whey protein systems

We show that application of high hydrostatic pressure (600 MPa for 15 min) on condensed whey protein (WP) systems (e.g., 80% w/w solids content) results in unexpected structure–function behavior when compared with conventional thermal treatment. Unraveling the relaxation properties in first‐order thermodynamic transitions, the manifestation of glass transition phenomena and the preservation of native conformation in condensed preparations were recorded using small‐deformation dynamic oscillation in shear, modulated differential scanning calorimetry, and infrared spectroscopy. Informed temperature application results in the formation of continuous networks at the denaturation temperature, which undergo vitrification at subzero temperatures. In contrast, high‐pressure‐treated WPs resist physicochemical denaturation, hence preserving the native conformation of secondary and tertiary structures. This was rationalized on the basis of a critical concentration threshold where transfer of water molecules to nonpolar residues in the protein interior is minimized because of low moisture content and restricted molecular mobility. The physical state and morphology of these high‐solid preparations were further examined by the combined framework of reduced variables and Williams, Landel, and Ferry equation/free volume theory. Theoretical treatment of experimental observations unveils the dynamic range of the mechanical manifestation of the glass transition region in samples subjected to heat or pressure. In addition to preserving native conformation, WPs subjected to high pressure form glassy systems at parity with the structural functionality of the thermally treated counterparts. © 2012 Wiley Periodicals, Inc. Biopolymers 97:963–973, 2012.     

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified. Two MAbs, 2E1 and 3D11, recognized an epitope in the amino-terminal 689 residues of apoB in native apoB-containing lipoproteins (LpBs) from human plasma or from the supernatant of human hepatoma HepG2 cells, but did not react with LpB from HuBTg+/+ mice or LpB secreted by human apoB-100-transfected rat McArdle 7777 hepatoma cells. 2E1 reacted weakly and 3D11 reacted strongly with apoB from HuBTg+/+ mice after SDS-PAGE. The lack of expression of the 2E1 and 3D11 epitopes on native LpB from HuBTg+/+ mice did not solely reflect the abnormal lipid composition of murine LpB. Both epitopes were detected in all human plasma samples tested and in all human plasma LpB classes. Therefore, human apoB expressed by rodent hepatocytes or hepatoma cells appears to adopt a different conformation or undergoes different posttranslational modification than apoB expressed in human hepatocytes or hepatoma cells.     

Decreased plasma DEK Oncogene Levels Correlate with p16-Negative Disease and Advanced Tumor Stage in a Case–Control Study of Patients with Head and Neck Squamous Cell Carcinoma

Head and neck cancer (HNC) remains the sixth most common malignancy worldwide and survival upon recurrence and/or metastasis remains poor. HNSCC has traditionally been associated with alcohol and nicotine use, but more recently the Human Papilloma Virus (HPV) has emerged as a favorable prognostic risk factor for oropharyngeal HNSCC. However, further stratification with additional biomarkers to predict patient outcome continues to be essential. One candidate biomarker is the DEK oncogenic protein, which was previously detected in the urine of patients with bladder cancer and is known to be secreted by immune cells such as macrophages. Here, we investigated if DEK could be detected in human plasma and if DEK levels correlated with clinical and pathological variables of HNSCC. Plasma was separated from the peripheral blood of newly diagnosed, untreated HNSCC patients or age-matched normal healthy controls and analyzed for DEK protein using ELISA. Plasma concentrations of DEK protein were lower in p16-negative tumors compared to both normal controls and patients with p16-positive tumors. Patients with lower plasma concentrations of DEK were also more likely to have late stage tumors and a lower white blood cell count. Contrary to previously published work demonstrating a poor prognosis with high intratumoral DEK levels, we show for the first time that decreased concentrations of DEK in patient plasma correlates with poor prognostic factors, including HPV-negative status as determined by negative p16 expression and advanced tumor stage.     

Proportion and pattern of dystrophin gene deletions in North Indian Duchenne and Becker muscular dystrophy patients

Population-based variations in frequency and distribution of dystrophin gene deletions have been recognized in Duchenne/Becker (DMD/BMD) muscular dystrophy patients. In the present study, DNA samples from 121 unrelated DMD/BMD patients from North India were analyzed for deletional studies with multiplex PCR and Southern hybridization. A total of 88 (73%) patients showed intragenic deletions in the dystrophin gene. The observed proportion of gene deletions is relatively high, particularly compared with that of Asian counterparts. However, the distribution of breakpoints across the gene does not show significant variations. Received: 5 June 1996 / Revised: 4 September 1996