Mutational analysis of the Caenorhabditis elegans cell-death gene ced-3

Mutations in the gene ced-3, which encodes a protease similar to interleukin-1beta converting enzyme and related proteins termed caspases, prevent programmed cell death in the nematode Caenorhabditis elegans. We used site-directed mutagenesis to demonstrate that both the presumptive active-site cysteine of the CED-3 protease and the aspartate residues at sites of processing of the CED-3 proprotein are required for programmed cell death in vivo. We characterized the phenotypes caused by and the molecular lesions of 52 ced-3 alleles. These alleles can be ordered in a graded phenotypic series. Of the 30 amino acid sites altered by ced-3 missense mutations, 29 are conserved with at least one other caspase, suggesting that these residues define sites important for the functions of all caspases. Animals homozygous for the ced-3(n2452) allele, which is deleted for the region of the ced-3 gene that encodes the protease domain, seemed to be incompletely blocked in programmed cell death, suggesting that some programmed cell death can occur independently of CED-3 protease activity.     

Molecular neuroadaptations in the accumbens and ventral tegmental area during the first 90 days of forced abstinence from cocaine self-administration in rats

Cocaine self-administration is associated with a propensity to relapse in humans and reinstatement of drug seeking in rats after prolonged withdrawal periods. These behaviors are hypothesized to be mediated by molecular neuroadaptations within the mesolimbic dopamine system. However, in most studies of drug-induced neuroadaptations, cocaine was experimenter-delivered and molecular measurements were performed after short withdrawal periods. In the present study, rats were trained to self-administer intravenous cocaine or oral sucrose (a control non-drug reward) for 10 days (6-h/day) and were killed following 1, 30, or 90 days of reward withdrawal. Tissues from the accumbens and ventral tegmental area (VTA) were assayed for candidate molecular neuroadaptations, including enzyme activities of cAMP-dependent protein kinase (PKA) and adenylate cyclase (AC), and protein expression of cyclin-dependent kinase 5 (cdk5), tyrosine hydroxylase (TH) and glutamate receptor subunits (GluR1, GluR2 and NMDAR1). In the accumbens of cocaine-trained rats, GluR1 and NMDAR1 levels were increased on days 1 and 90, while GluR2 levels were increased on days 1 and 30, but not day 90; PKA activity levels were increased on days 1 and 30, but not day 90, while AC activity, TH and cdk5 levels were unaltered. In the VTA of cocaine-trained rats, NMDAR1 levels were increased for up to 90 days, while GluR2 levels were increased only on day 1; TH and Cdk5 levels were increased only on day 1, while PKA and AC activity levels were unaltered. Cocaine self-administration produces long-lasting molecular neuroadaptations in the VTA and accumbens that may underlie cocaine relapse during periods of abstinence.     

Regulation of developing B cell survival by RelA-containing NF-kappa B complexes

Mice deficient in the RelA (p65) subunit of NF-kappaB die during embryonic development. Fetal liver (FL) hemopoietic precursors from these mice were used to generate RelA-deficient lymphocytes by adoptive transfer into lethally irradiated mature lymphocyte-deficient recombination-activating gene-1(-/-) mice. Strikingly, RelA(-/-) lymphocyte generation was greatly diminished compared with that of RelA(+/+) lymphocytes. The most dramatic reduction was noticed in the numbers of developing B cells, which were considerably increased when RelA(-/-) FL cells that were also TNFR1 deficient were used. The role of RelA was further investigated in FL-derived developing B cells in vitro. Our results show that RelA is a major component of constitutive and TNF-alpha-induced kappaB site-binding activity in developing B cells, and provide evidence for a direct role of TNF-alpha in killing RelA(-/-) B cells. The absence of RelA significantly reduced mRNA expression of the antiapoptotic genes cellular FLICE-inhibitory protein and Bcl-2. Retroviral transduction of RelA(-/-) B cells with either cFLIP or Bcl-2 significantly reduced TNF-alpha killing. Together, these results indicate that RelA plays a crucial role in regulating developing B cell survival by inhibiting TNF-alpha cytotoxicity.     

An overview on fermentation,downstream processing and properties of microbial alkaline proteases

Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8-12 and at temperatures between 50 and 70 degrees C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80-90 degrees C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8-11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.     

Sister chromatid exchange in pathology staff occupationally exposed to formaldehyde

Sister chromatid exchange (SCE) was measured in peripheral lymphocytes of 90 workers from 14 hospital pathology departments in Israel who were occupationally exposed to formaldehyde (FA) and of 52 unexposed workers from the administrative section of the same hospitals. The mean exposure period to FA was 15.4 years (range 1-39). The results of SCEs are expressed in two variables: (a) mean number of SCEs per chromosome and (b) proportion of high frequency cells (cells with more than eight SCEs). A high correlation was found between these two variables. The adjusted means of both SCEs variables were significantly higher among the exposed compared with that of the unexposed group (P<0.01). Adjustment was made for age, sex, smoking habits, education workers and origin. Evaluation of the influence of years of exposure on the frequency of SCEs showed that the two variables of SCEs were higher among those who were exposed to FA for 15 or more than among those with less than 15 years of exposure. Concerning levels of exposure, both variables of SCEs were the same in the low and in the high levels of exposure sub-groups. However, among the smokers, both variables of SCEs were higher in the high exposure sub-group than in the low exposure sub-group.Our finding of a significant increase of SCEs frequency in peripheral lymphocytes in pathology staff indicates a potential cytogenetic hazard due to FA exposure. We conclude that our data indicate that FA is mutagenic to humans.     

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).     

Analysis of Heparin-Binding Sites in Human Lipoprotein Lipase Using Synthetic Peptides

Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388–395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261–287) and cluster 3 (residues 147–151) peptides followed by cluster 2 (residues 290–302) peptide. A peptide corresponding to LPL cluster 4 (residues 405–414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications.     

Isolation and immunological characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been isolated from human liver utilizing HMG-CoA affinity chromatography. The apparent monomer molecular weight of purified human HMG-CoA reductase by SDS-gel electrophoresis was 53,000, and the oligomeric molecular weight determined by sucrose density centrifugation was 104,000. A monospecific antibody prepared against rat liver HMG-CoA reductase inhibited the enzymic activity of microsomal and purified human liver enzyme and formed a single immunoprecipitin line by radial immunodiffusion. These results represent the initial isolation and characterization of human liver HMG-CoA reductase.     

The fuel properties of pyrolysis liquid derived from urban solid wastes in Bangladesh

Urban solid wastes in the form of scrap tyre, waste plastic and waste paper, were pyrolyzed in an externally heated fixed bed reactor. The condensed liquids were analyzed for their properties as fuels and compared with petroleum-derived products. The properties were physical properties, higher heating value, elemental (CHNOS) analysis and chemical composition using Fourier transform infra-red (FTIR) spectroscopy. The results showed that the pyrolysis of urban solid wastes may be a potential source of liquid hydrocarbon fuel.