Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease

The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the O_b atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.     

Carnitine,carnitine acetyltransferase,and glutathione in Alzheimer brain

Glutathione and total carnitine (i.e., free carnitine plus acid-soluble carnitine esters) were measured in an affected (superior frontal gyrus; SFG) and unaffected (cerebellum: CBL) region of Alzheimer disease (AD) and control brains. Average glutathione content in AD SFG (n=13) and AD CBL (n=7) (7.9±2.1 and 11.9±4.0 nmol/mg protein, respectively (mean ±S.D.)) was similar to that in control SFG (n=13) and CBL (n=6) (7.7±2.0 and 11.6±2.6 nmol/mg protein, respectively). However, glutathione increased significantly with age in AD brain (p=0.003) but not in control brain. Average total carnitine in AD SFG (84±47 pmol/mg protein; n=10) and AD CBL (108±86 pmol/mg protein; n=7) was not significantly different from that in the corresponding regions of control brain (148±97 (n=10) and 144±107 (n=6) pmol/mg protein, respectively). However, a significant decline of total carnitine with age in both regions was noted for AD brain, but not for control brain. Carnitine acetyltransferase activity in the AD SFG (n=13) was not significantly different from that of control SFG (n=13) (1.83±1.05 and 2.04±0.82 nmol/min/mg protein, respectively). However, carnitine acetyltransferase activity of AD CBL (n=7) was significantly lower than that of control CBL (n=6) (1.33±0.88 versus 2.26±0.66 nmol/min/mg protein; p=0.05).     

Mitochondrial haplogroup N1a phylogeography,with implication to the origin of European farmers

Background  

Tracing the genetic origin of central European farmer N1a lineages can provide a unique opportunity to assess the patterns of the farming technology spread into central Europe in the human prehistory. Here, we have chosen twelve N1a samples from modern populations which are most similar with the farmer N1a types and performed the complete mitochondrial DNA genome sequencing analysis. To assess the genetic and phylogeographic relationship, we performed a detailed survey of modern published N1a types from Eurasian and African populations.     

Risk of Becoming Schizophrenic: Birth Order and HLA Profile

The present study was carried out to ascertain the birth order as a risk factor for schizophrenia on the basis of HLA genetics. India born schizophrenic patients of Siliguri, West Bengal who attended Outpatient Department of Psychiatry, North Bengal Medical College and Hospital were included in the study. After longitudinal follow up of 136 patients and 150 controls matched in age, sex and ethnicity were screened for the study. The typing of HLA was done by PCR-SSP method. The results showed a significantly increased frequency of HLA*A3 among the patients. Further, birth order was studied among HLA*A3 positive 108 patients and 41 controls. Although, results indicated higher incidence of schizophrenia among second children irrespective of sex but it was not found to be statistically significant. However, when the birth order of male and female patients was analyzed separately, a significant decreased incidence of schizophrenia was observed among the third female child. The findings do not corroborate with the earlier findings of association of birth order with schizophrenia.     

Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light of variable photoperiods

Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration in human. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Although birds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences a cone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina of white Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7 days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 lux at 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed for transmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synaptic proteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in 24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantly decreased expressions of opsins and synaptic proteins, compared to those seen in 12L:12D and 18L:6D conditions. Also, there were ultrastructural changes in outer and inner plexiform layer (OPL, IPL) of the retinas exposed to 24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, with changes (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic protein expressions in OPL and IPL and death of degenerated axonal processes in IPL.     

Murine splenocyte proliferation by porin of Shigella dysenteriae type 1 and inhibition of bacterial invasion of HeLa cell by anti-porin antibody

Abstract A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.     

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)₂₂ forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)₉AGG (CGG)₁₂AGG(CGG)₉₇, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3′ part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.