Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

Harijans and Girijans of Andhra Pradesh: an application of analysis of variance in the study of fertility,mortality and family planning

Research on the Bagatha tribe and the Malas and Madigas in India has been done for economic and social planning purposes in regard to family planning. Bagatha are mostly agricultural people where the nuclear family is prevalent and polygamy is popular as well as cousin marriage. The Madigas and Males (Harijans) are lower caste with the 1st being leather workers and the latter being agricultural helpers. The data was collected by direct interview of 202 tribesmen and 202 caste households with women from 15-49 years of age. The data collected on fertility include live births, child survival rate, fetal wastage, husband and wives education, income, and occupations. On mortality, the number of deaths, age at marriage, number of and intervals of pregnancies. As expected, educated and employed families show healthier and higher levels of fertility especially if the wife is educated. The wife shows more of the responsibility for family planning. The age at marriage and the number of pregnancies appears to have little effect on mortality. In the caste group the education level of the husband has little effect on fertility and again the wife has the primary responsibility in using family planning techniques.     

Pentacyclic steroids,part XVI,studies on the total syntheses of racemic 1,6-dithiabenz[3,4]estra-3, 5(10),8,14-tetraen-17-one and its D-homo analogue

The total syntheses of racemic 1,6-dithiabenz[3,4]-estra-3,5(10), 8,14-tetraen-17-one [VII]and 1,6-dithiabenz-[3,4]-D-homoestra-3,5(10),8,14-tetraen-17a-one [IX]starting fron isothiochroman-4-one [I]are described.     

Motifs Q and I are required for ATP hydrolysis but not for ATP binding in SWI2/SNF2 proteins

Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases.     

Biodegradation of pyridine by an isolated bacterial consortium/strain and bio-augmentation of strain into activated sludge to enhance pyridine biodegradation

Pyridine and pyridine based products are of major concern as environmental pollutants due to their recalcitrant, persistent, toxic and teratogenic nature. In this study, we describe biodegradation of pyridine by an isolated consortium/strain under aerobic condition. Batch experiment results reveal that at lower initial pyridine concentrations (1-20 mg l(-1)), almost complete degradation was observed whereas at higher concentration (30-50 mg l(-1)), the degradation efficiency was dropped significantly. This may be due to inhibitory effect of pyridine at higher concentrations. The value of decay and yield coefficient was also determined. Furthermore, the bio-augmentation of isolated consortium/strain into the activated sludge consortium in different quantity has been also done and the effect of bio-augmentation on degradation has been studied. The results reveal that as the quantity of bio-augmentation increases, the degradation of pyridine increases. At 25% bio-augmentation, complete degradation of 20 mg l(-1) of pyridine can be achieved within 96 h of incubation. Thus, the study concluded that the bio-augmentation of the isolated consortium/strain into the sludge enhances the pyridine degradation efficiency of the biomass.     

Nanotechnology a novel approach to enhance crop productivity

Nanobiotechnology provides novel set of tools to manipulate and enhance crop production using nanoparticles, nanofibres, nanoemulsions, and nanocapsules. Nanomaterials provide a platform to deliver agrochemicals and various macromolecules needed for plant growth enhancement and resistance to stresses. Smart delivery of agrochemicals increases the yield by optimizing water and nutrient conditions. Another added advantage is controlled release and site-directed delivery of agrochemicals. Further enhancement in quality and quantity in agriculture can be achieved by nanoparticle-mediated gene transformation and delivery of macromolecules that induces gene expression in plants. Various types of nanomaterials have been tested so far and the results have been promising in terms of productivity and quality enhancement.     

Utility of Host Delivered RNAi of Two FMRF Amide Like Peptides,flp-14 and flp-18, for the Management of Root Knot Nematode,Meloidogyne incognita

Root knot nematode, Meloidogyne incognita, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (flp-14 and flp-18) for infection and development of resistance to M. incognita through host-derived RNAi. The study demonstrated both in vitro and in planta validation of RNAi-induced silencing of the two genes cloned from J2 stage of M. incognita. In vitro silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of flp-14 and flp-18 and evaluated against M. incognita. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of M. incognita resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for in vitro and in planta silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of M. incognita. This is the first ever report that has revealed the utility of host delivered RNAi of flps to control M. incognita. The strategy can also be extended to other crops and nematodes.     

Assessment of SLX4 Mutations in Hereditary Breast Cancers

Background

SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.

Methods and Results

To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65–1). We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare) cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* SLX4 mutant failed to do so.

Conclusion

Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.     

Palmitate and Lipopolysaccharide Trigger Synergistic Ceramide Production in Primary Macrophages

Macrophages play a key role in host defense and in tissue repair after injury. Emerging evidence suggests that macrophage dysfunction in states of lipid excess can contribute to the development of insulin resistance and may underlie inflammatory complications of diabetes. Ceramides are sphingolipids that modulate a variety of cellular responses including cell death, autophagy, insulin signaling, and inflammation. In this study we investigated the intersection between TLR4-mediated inflammatory signaling and saturated fatty acids with regard to ceramide generation. Primary macrophages treated with lipopolysaccharide (LPS) did not produce C16 ceramide, whereas palmitate exposure led to a modest increase in this sphingolipid. Strikingly, the combination of LPS and palmitate led to a synergistic increase in C16 ceramide. This response occurred via cross-talk at the level of de novo ceramide synthesis in the ER. The synergistic response required TLR4 signaling via MyD88 and TIR-domain-containing adaptor-inducing interferon beta (TRIF), whereas palmitate-induced ceramide production occurred independent of these inflammatory molecules. This ceramide response augmented IL-1β and TNFα release, a process that may contribute to the enhanced inflammatory response in metabolic diseases characterized by dyslipidemia.