The asymmetric distribution of enzymic activity between the six subunits of bovine liver glutamate dehydrogenase. Use of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid.

A method for the preparation of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid) is described. These chloromethyl ketones irreversibly inactivated bovine glutamate dehydrogenase, whereas several other related compounds had no adverse effect on the activity of the enzyme. The inactivation process was shown to be due to the modification of lysine-126. The time-courses for the inactivation and the incorporation of radioactivity from tritiated L-glutamyl alpha-chloromethyl ketone into the glutamate dehydrogenase were biphasic. The results were interpreted to suggest the involvement of 'negative co-operative' interactions in the reactivity of lysine-126. From the cumulative evidence it is argued that the first subunit of the enzyme, which takes part in catalysis, makes the largest, and the last the smallest, contribution to the overall catalysis. It is emphasized that three of the six subunits of the enzyme may possess as much as 80% of the total activity of bovine glutamate dehydrogenase.     

Studies on N-acetylneuraminic acid aldolase

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.     

Vaccination with a non-human random sequence amyloid oligomer mimic results in improved cognitive function and reduced plaque deposition and micro hemorrhage in Tg2576 mice

ABSTRACT: BACKGROUND: It is well established that vaccination of humans and transgenic animals against fibrillar amyloid beta (Abeta) prevents amyloid accumulation in plaques and preserves cognitive function in transgenic mouse models. However, autoimmune side effects have halted the development of vaccines based on full length human Abeta. Further development of an effective vaccine depends on overcoming these side effects while maintaining an effective immune response. RESULTS: We have previously reported that the immune response to amyloid oligomers is largely directed against generic epitopes that are common to amyloid oligomers of many different proteins and independent of a specific amino acid sequence. Here we have examined whether we can exploit this generic immune response to develop a vaccine that targets amyloid oligomers using a non-human random sequence amyloid oligomer. In order to study the effect of vaccination against generic oligomer epitopes, a random sequence oligomer (3A) was selected as it forms oligomers that react with the oligomer specific A11 antibody. Oligomer mimics from 3A peptide, Abeta, islet amyloid polypeptide (IAPP), and Abeta fibrils were used to vaccinate Tg2576 mice, which develop a progressive accumulation of plaques and cognitive impairment. Vaccination with the 3A random sequence antigen was just as effective as vaccination with the other antigens in improving cognitive function and reducing total plaque load (Abeta burden) in the Tg2576 mouse brains. CONCLUSION: These results show that the amyloid Abeta sequence is not necessary to produce a protective immune response that specifically targets generic amyloid oligomers. Using a non-human, random sequence antigen may facilitate the development of a vaccine that avoids autoimmune side effects.     

Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU)

The aim of this study was to investigate the prevalence of ESBL and MBL encoding genes among A. baumannii isolates. In this cross sectional study, 100 A. baumannii strains were isolated from ICU wards of 3 educational hospitals of Hamadan City, Iran in 2011. Phenotypic identification of the production of ESBLs and MBLs has been carried out by using E-test and DDST methods, respectively. PCR technique was used for amplification of the ESBL and MBL encoding genes, namely: CTX-M, SHV, TEM, OXA-51, VIM-Family, IMP-Family, SPM-1, SIM-1, and GIM-1. Eighty seven (87%), 95 (95%), 98 (98%) and 95 (95%) out of 100 A. baumannii isolates were resistant to imipenem, meropenem, ceftazidime and cefotaxime, respectively. Also, 99% and 7% of the isolates were MBLs and ESBLs produced phenotypically. Thirty (30%), 20 (20%) and 58 (58%) out of 100 A. baumannii isolates have been confirmed to harbor the bla_VIM-family, TEM and SHV genes, respectively. Our results show no significant relationship between the detected gens with production of MBLs and ESBLs in spite of high prevalence of MBL encoding and drug resistant A. baumannii. Probably some other genes rather than what we studied are involved in phenotypic production of MBLs and ESBLs and subsequent drug resistance in Hamadan area, Iran.     

Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding.

Conflicting results have been reported regarding the role of carbohydrate on human immunodeficiency virus (HIV) envelope glycoprotein gp120 in CD4 receptor binding. Glycosylated, deglycosylated, and nonglycosylated forms of HIV type 1 (HIV-1) and HIV-2 gp120s were used to examine CD4 receptor-binding activity. Nonglycosylated forms of gp120 generated either by deletion of the signal sequence of HIV-1 gp120 or by synthesis in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated gp120 bound to soluble CD4 molecules well. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H or an endoglycosidase F/N glycanase mixture had no effect on the ability of gp120 to bind CD4. An experiment which measured the ability of gp120 to bind to CD4 as an assay of the proper conformation of gp120 showed that carbohydrate chains on gp120 are not required for the interaction between gp120 and CD4 but that N-linked glycosylation is essential for generation of the proper conformation of gp120 to provide a CD4-binding site.     

Contribution of TGFβ1 Codon 10 Polymorphism to High Myopia in an Ethnic Kashmiri Population from India

This study looks at novel variants of the TGFβ1 gene and their potential association with high myopia in an ethnic population from Kashmir, India. Allele frequencies of 247 Kashmiri subjects (from India) with high myopia and 176 ethnically matched healthy controls were tested for Hardy–Weinberg disequilibrium. The genotype and allele frequencies were evaluated using chi-square or Fisher’s exact tests. One of the three SNPs in codon 10 showed a significant difference between patients and control subjects (rs1982073: p genotype = 0.003, p allele = 0.001). There were no statistically significant differences between patients and control subjects for the other two SNPs, rs1800471 at codon 25 and a novel variant at codon 52. SNP rs1982073, substituting proline with leucine, appeared to be significantly associated with high myopia (p < 0.05). In silico predictions show that substitutions are likely to have an impact on the structure and functional properties of the protein, making it imperative to understand their functional consequences in relation to high myopia.     

Diversity of Arsenate Reductase Genes (<Emphasis Type="Italic">arsC</Emphasis> Genes) from Arsenic-Resistant Environmental Isolates of <Emphasis Type="Italic">E. coli</Emphasis>

A polymerase chain reaction (PCR) approach was used to assess the occurrence and diversity of arsenate reductase gene (arsC gene) in arsenic-resistant environmental E. coli strains. For this purpose, two different sets of primers were designed for the specific amplification of approximately 370-bp fragments from the arsC gene. These primers were used to screen a collection of 25 environmental arsenic-resistant strains isolated from different geographical regions of India, as well as Bangladesh. The PCR results showed that 17 out of the 25 environmental isolates (68%) contained a gene related to the arsC family. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsC genes in the isolated strains. A significant divergence in the DNA sequence was found in the arsC genes among As-resistant environmental E. coli strains from this study, and arsenic resistance, a genetic character, arose from a common ancestral background.     

Synthesis and pharmacological screening: Sulfa derivatives of 2-pipecoline-bearing 1,3,4-oxadiazole core

An electrophile, 1-(4-(bromomethylbenzenesulfonyl)-2-methylpiperidine, was synthesized by the reaction of 2-methylpiperidine (2-pipecoline) and 4-bromomethylbenzenesulfonyl chloride in a weak basic medium under pH control. A series of nucleophiles, 5-aryl/aralkyl-1,3,4-oxadiazol-2-thiols, were synthesized from corresponding carboxylic acids in three steps. The title molecules were synthesized by coupling the electrophile to nucleophiles in an aprotic medium using LiH as an activator. The structures of all synthesized compounds were corroborated through IR, ¹H NMR, and EI-MS techniques. All the compounds were screened for their pharmacological behavior, particularly, antibacterial and enzyme inhibitory activities. Notably efficient results were obtained against both gram-positive and gram-negative bacterial strains. Regarding enzyme inhibition, compounds were efficient against acetylcholinesterase and butyrylcholinesterase.     

Photophysical and biological aspects of α, β-unsaturated ketones: Experimental and in silico approach

In this work, four fluorinated α, β-unsaturated ketones named as 3-(3-bromophenyl)−1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (1), 3-(4-methoxyphenyl)−1-(3-(trifluoromethyl)phenyl) prop-2-en-1-one (2), 3-(3-bromo-5-chloro-2-hydroxyphenyl)-1-(3-(trifluoromethyl)phenyl) prop-2-en-1-one (3) and 3-(2-hydroxy-5-methylphenyl)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (4) were synthesized by Claisen–Schmidt reaction. The synthesized molecules were then characterized through ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared (FTIR), ¹H-NMR, ¹³C-NMR, and mass spectrometry. The antioxidant potential, Urease inhibition, and interaction of compounds 1 – 4 with Salmon sperm DNA were experimentally explored and supported by molecular docking studies. The synthesized compounds strongly interact with SS-DNA through intercalative mode. It was noticed that compound 1 served as potent Urease inhibitor while compound 4 as better antioxidant among synthesized compounds. Moreover, frontier molecular orbitals, nonlinear optical (NLO) properties, natural bond orbitals, molecular electrostatic potential, natural population analysis, and photophysical properties of synthesized compounds were accomplished through density functional theory and time-dependent density functional theory. The band gap of all the compounds have been worked out using Taucs method. In addition to that, a precise comparative account of UV and IR data obtained from theoretical and experimental findings showed good agreement between theoretical and experimental data. The findings of our studies reflected that compounds  1 – 4 possess better NLO properties than Urea standard and the band gap data also reflected their prospective use towards optoelectronic materials. The better NLO behavior of compounds was attributed to the noncentrosymmetric structure of synthesized compounds.