Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Two new species and new records of biting midges of the genus Dasyhelea Kieffer (Diptera: Ceratopogonidae) from India

Pupa, male and female adults of Dasyhelea (Prokempia) flava Carter, Ingram & Macfie, pupa and male adult of D. (Pseudoculicoides) acuta n. sp., and male adult of D. (Pseudoculicoides) comosa n. sp. are described and illustrated.     

Clinical Significance of Circulating Serum Levels of sCD95 and TNF-α in Cytoprotection of Cervical Cancer

Background:This study correlates the serum levels of sCD95 & TNF-α with a simple cell-based assay to evaluate the capacity of the serum sample to induce apoptosis in Jurkat cells. Interlinking of these parameters can be explored to design a minimum invasive diagnostic strategy for cervical cancer (CC).Methods:Sera samples were assessed to induce apoptosis in Jurkat cells through FACS. Serum levels of sCD95 and TNF-α were measured by ELISA. JNK phosphorylation was evaluated in sera incubated Jurkat cells. Data was scrutinized through statistical analysis.Results:Significantly higher serum levels of sCD95 and lower TNF-α levels were observed in CC patients; their sera samples inhibited induction of apoptosis in Jurkat cells through reduced JNK phosphorylation. Statistical analysis linked these three parameters for the early screening of CC.Conclusion:Distinct sera levels of sCD95 & TNF-α in CC patients showed an anti-apoptotic effect, which can be considered for early detection of CC.Key Words: Apoptosis, sCD95, Jurkat Cells, Tumor Necrosis Factor-alpha, Uterine Cervical Neoplasms     

Differential antioxidant enzyme and thiol responses of tolerant and non-tolerant clones of <Emphasis Type="Italic">Chloris barbata</Emphasis> to cadmium-stress

Tolerant and non-tolerant clones of Chloris barbata Sw. obtained, respectively, from an erstwhile mercury contaminated solid waste dump site near a chloralkali plant and a non-contaminated (control) site were subjected to cadmium-stress by growing the rooted cuttings in water containing CdSO₄, 13 and 130 μM. Differences between the two clones in their response to cadmium-stress were noted in root growth, and also with respect to certain biochemical parameters. Whereas catalase activity decreased and non protein-thiol levels increased in the non-tolerant clone, the level of protein-thiol alone increased significantly in the tolerant clone in response to cadmium-stress. No remarkable differences between the clones, however, were noted with respect to total soluble protein, peroxidase activity and lipid peroxidation. Remarkably the two clones responded differently to buthionine sulfoximine, an inhibitor of glutathione and/or phytochelatin synthesis, which inhibited root growth significantly in non-tolerant clone but not in the tolerant clone. Buthionine sulfoximine, nonetheless, could potentate cadmium toxicity in either of the clones, but more effectively in the tolerant clone. The high sensitivity of tolerant-clone to the combined treatment of BSO and Cd in the present study could, therefore, be attributed to the cumulative oxidative stress generated synergistically by BSO and Cd.     

Ascorbate inhibits edema in brain slices

Ascorbate is an essential antioxidant in the CNS, localized predominantly in neuronal cytosol. Slices of mammalian brain rapidly lose ascorbate, however, when incubated in ascorbate-free media; brain slices also take up water and swell. Here we investigated water gain in coronal slices of rat forebrain incubated with and without ascorbate for 1-3 h at 34 degrees C. Slices progressively gained water in ascorbate-free media, with a significant 12% water increase after 3 h at 34 degrees C, compared with the water content of slices after a 1-h recovery period at 24 degrees C, immediately following slice preparation. Inclusion of 400 micro M ascorbate in the medium led to an increase in tissue ascorbate content and prevented water gain at 34 degrees C. By contrast, water gain was not inhibited by isoascorbate or thiourea, which are antioxidants that are not accumulated in brain cells. The oxidant H2O2 enhanced water gain, whereas a cocktail of NMDA and non-NMDA receptor blockers inhibited edema formation to the same extent as ascorbate. These data demonstrate that brain edema, linked to glutamate-receptor activation, can result from intracellular oxidative stress and that this can be prevented by ascorbate.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

Human DNA polymerase kappa encircles DNA: implications for mismatch extension and lesion bypass

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: analysis of its hysteretic behavior during catalysis

UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor. This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP. The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared. It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.     

Ragweed Subpollen Particles of Respirable Size Activate Human Dendritic Cells

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3⁺ pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs'' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.