HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.     

Production of bio-ethanol from pretreated agricultural byproduct using enzymatic hydrolysis and simultaneous saccharification

Global warming alerts and threats are on the rise due to the utilization of fossil fuels. Alternative fuel sources like bio-ethanol and biodiesel are being produced to combat against these threats. Bio-ethanol can be produced from a range of substrate. The present study is aimed at the Production of bioethanol from pretreated agricultural substrate using enzymatic hydrolysis and simultaneous saccharification with the addition of purified fungal enzyme. Most cellulosic biomass is not fermentable without appropriate pretreatment methods and so dilute sulfuric acid pretreatment was applied to make the cellulose contained in the waste susceptible to endoglucanase enzyme. A range of acid pretreatment of wheat bran was made in which the sample that was pretreated with 1% dilute sulfuric acid gave maximum yield of ethanol in both methods such as 5.83 g L(-1) and 5.27 g L(-1), respectively. Ethanol produced from renewable and cheap agricultural products (wheat bran) provides reduction in green house gas emission, carbon monoxide, sulfur, and helps to eliminate smog from the environment.     

Pre-steady state kinetic analysis of cyclobutyl derivatives of 2'-deoxyadenosine 5'-triphosphate as inhibitors of HIV-1 reverse transcriptase

Pre-steady state kinetic analysis was utilized for biochemical evaluation of a series of cyclobutyl adenosine nucleotide analogs with HIV-1 RT(WT). The phosphonyl-diphosphate form of the cyclobutyl nucleotide, 5, was the most efficiently incorporated of the series. Nucleotide 5 was fourfold more efficiently incorporated than the FDA approved TFV-DP by RT(WT). The kinetics of incorporation for 5 using the drug resistant mutant enzyme K65R was also determined. Compound 5 was threefold more efficiently incorporated compared to TFV-DP with RT(K65R). These results demonstrate cyclobutyl adenosine analogs can act as substrates for incorporation by HIV-1 RT and be a potential scaffold for HIV inhibitors.     

Catalysis by molecular iodine: a rapid synthesis of 1,8-dioxo-octahydroxanthenes and their evaluation as potential anticancer agents

Molecular iodine facilitated the reaction of 5,5-dimethyl-1,3-cyclohexanedione with aromatic aldehydes in iso-propanol affording a variety of 1,8-dioxo-octahydroxanthenes in high yields. Most of the compounds synthesized showed good anti-proliferative properties in vitro against three cancer cell lines and 9-(2-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione possessing a 2-hydroxy phenyl group at C-9 position was found to be promising. Further structure elaboration of the same compound and the crystal structure analysis and hydrogen bonding patterns of another compound that is, 9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione prepared by using this methodology is presented.     

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family.     

Cloning and characterization of two neuropeptide genes from cereal cyst nematode, Heterodera avanae from India

The cereal cyst nematode, Heterodera avenae (Wollenweber, 1924) is one of the most important plant parasitic nematodes of cereals. It is an obligate sedentary endo parasite causing considerable crop losses in wheat, barley and oats worldwide. FMRFamide-like peptides (FLPs) play critical role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time we have cloned and characterized two neuropeptide genes (flp-12 and flp-16) from the cDNA library of feeding female of H. avenae. Sequence analysis of FLPs revealed that both the neuropeptides are closely related with the parasitic as well as free-living nematodes. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting its association with extra-cellular functions, while flp-16 does not contain signal peptide. Besides this, we have found highly conserved motif KFEFIRF in flp-12 and RFGK motif in flp-16. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.     

A key structural domain of the Candida albicans Mdr1 protein

A major multidrug transporter, MDR1 (multidrug resistance 1), a member of the MFS (major facilitator superfamily), invariably contributes to an increased efflux of commonly used azoles and thus corroborates their direct involvement in MDR in Candida albicans. The Mdr1 protein has two transmembrane domains, each comprising six transmembrane helices, interconnected with extracellular loops and ICLs (intracellular loops). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest interdomain ICL3 of the MDR1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletion variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface-charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the CCL (central cytoplasmic loop) of the fungal MFS transporter of the DHA1 (drug/proton antiporter) family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.