Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.     

In silico approach to inhibition of signaling pathways of Toll-like receptors 2 and 4 by ST2L

Toll-like receptors (TLRs) activate a potent immunostimulatory response. There is clear evidence that overactivation of TLRs leads to infectious and inflammatory diseases. Recent biochemical studies have shown that the membrane-bound form of ST2 (ST2L), a member of the Toll-like/IL-1 receptor superfamily, negatively regulates MyD88-dependent TLR signaling pathways by sequestrating the adapters MyD88 and Mal (TIRAP). Specifically, ST2L attenuates the recruitment of Mal and MyD88 adapters to their receptors through its intracellular TIR domain. Thus, ST2L is a potent molecule that acts as a key regulator of endotoxin tolerance and modulates innate immunity. So far, the inhibitory mechanism of ST2L at the molecular level remains elusive. To develop a working hypothesis for the interactions between ST2L, TLRs (TLR1, 2, 4, and 6), and adapter molecules (MyD88 and Mal), we constructed three-dimensional models of the TIR domains of TLR4, 6, Mal, and ST2L based on homology modeling. Since the crystal structures of the TIR domains of TLR1, 2 as well as the NMR solution structure of MyD88 are known, we utilized these structures in our analysis. The TIR domains of TLR1, 2, 4, 6, MyD88, Mal and ST2L were subjected to molecular dynamics (MD) simulations in an explicit solvent environment. The refined structures obtained from the MD simulations were subsequently used in molecular docking studies to probe for potential sites of interactions. Through protein-protein docking analysis, models of the essential complexes involved in TLR2 and 4 signaling and ST2L inhibiting processes were developed. Our results suggest that ST2L may exert its inhibitory effect by blocking the molecular interface of Mal and MyD88 adapters mainly through its BB-loop region. Our predicted oligomeric signaling models may provide a basis for the understanding of the assembly process of TIR domain interactions, which has thus far proven to be difficult via in vivo studies.     

Protection of DNA and membrane from gamma radiation induced damage by gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a naturally occurring plant phenol. In vitro and in vivo studies have shown that this phytochemical protected DNA and membranes against ionizing radiation. Rat liver microsomes and plasmid pBR322 DNA were exposed to various doses of gamma radiation in presence and absence of GA. Exposure of the microsomes to gamma radiation resulted in the formation of peroxides of membrane lipids measured as thiobarbituric acid reactive substances and presence of GA during irradiation prevented the formation of lipid peroxidation. Gamma irradiation of plasmid DNA resulted in induction of strand breaks in DNA resulting in disappearance of the supercoiled (ccc) form. Presence of GA during irradiation protected the DNA from undergoing the strand breaks. In in vivo studies it was found that whole body exposure of mice to gamma radiation (4 Gy) increased the formation of lipid peroxides in various tissues and damage to cellular DNA (as measured by alkaline comet assay) in peripheral blood leucocytes. Administration of GA to mice prior to whole body radiation exposure reduced the peroxidation of lipids and the damage to the cellular DNA indicating in vivo radiation protection of membranes and DNA by GA. (Mol Cell Biochem 278: 111–117, 2005)     

Chloroplast and nuclear microsatellite analysis of Aegilops cylindrica

Aegilops cylindrica Host (2n=4x=28, genome CCDD) is an allotetraploid formed by hybridization between the diploid species Ae. tauschii Coss. (2n=2x=14, genome DD) and Ae. markgrafii (Greuter) Hammer (2n=2x=14, genome CC). Previous research has shown that Ae. tauschii contributed its cytoplasm to Ae. cylindrica. However, our analysis with chloroplast microsatellite markers showed that 1 of the 36 Ae. cylindrica accessions studied, TK 116 (PI 486249), had a plastome derived from Ae. markgrafii rather than Ae. tauschii. Thus, Ae. markgrafii has also contributed its cytoplasm to Ae. cylindrica. Our analysis of chloroplast and nuclear microsatellite markers also suggests that D-type plastome and the D genome in Ae. cylindrica were closely related to, and were probably derived from, the tauschii gene pool of Ae. tauschii. A determination of the likely source of the C genome and the C-type plastome in Ae. cylindrica was not possible.     

Community-Level Physiological Profiles of Cloacal Microbes in Songbirds (Order: Passeriformes): Variation Due to Host Species, Host Diet, and Habitat

The relationship between microorganisms and birds has received increased attention recently. The state of knowledge of this relationship, however, is based largely on examination of sick or dead birds, and knowledge of the prevalence and community structure and function of microbes in healthy wild populations is limited. Using carbon substrate utilization profiles, microbial communities were examined in 91 cloacal samples from 14 species within apparently healthy summer and winter passerine populations. Within each season, gradient lengths and eigenvalues from ordination analyses suggested that many samples differed in their carbon substrate utilization and several had very different communities. Cloacal microbe carbon utilization profiles were distinguishable among host species, season-specific diet, and study site in the ordination analyses. However, these patterns were only observed for the analysis of the summer data set. The results of this study support the idea that the avian host’s microbial community, relative to carbon substrate utilization, is related to host diet. Previously, this pattern had only been reported for potential pathogens isolated from the avian cloaca. Study site–specific patterns in the ordination analysis suggest that environmental conditions at a particular study site may influence cloacal microbial communities in birds. Results of this study indicate that examination of community-level physiological profiles may be a useful technique for distinguishing among avian cloacal samples, similar to that already established for discriminating aqueous and soil samples. Future studies that correlate microbe physiological profiles to condition-based indices of avian hosts may be most useful for eventually using the profile as an indicator of environmental conditions experienced by hosts.     

Population genetic approaches to neurological disease: Parkinson's disease as an example

Parkinson's disease (PD) is a common, progressive, incurable disabling condition. The cause is unknown but over the past few years tremendous progress in our understanding of the genetic bases of this condition has been made. To date, this has almost exclusively come from the study of relatively rare Mendelian forms of the disease and there are no currently, widely accepted common variants known to increase susceptibility.The role that the "Mendelian" genes play in common sporadic forms of PD is unknown. Moreover, most studies in PD can really be described as candidate polymorphism studies rather than true and complete assessments of the genes themselves. We provide a model of how one might tackle some of these issues using Parkinson's disease as an illustration. One of the emerging hypotheses of gene environment interaction in Parkinson's disease is based on drug metabolizing (or xenobiotic) enzymes and their interaction with putative environmental toxins. This motivated us to describe a tagging approach for an extensive but not exhaustive list of 55 drug metabolizing enzyme genes. We use these data to illustrate the power, and some of the limitations of a haplotype tagging approach. We show that haplotype tagging is extremely efficient and works well with only a modest increase in effort through different populations. The tagging approach works much less well if the minor allele frequency is below 5%. However, it will now be possible using these tags to evaluate these genes comprehensively in PD and other neurodegenerative conditions.     

Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes

Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC-conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC-conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1-1,000 ng/ml)-conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS-binding protein (LBP) although CD14 (a receptor for LPS-LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF-alpha, interleukin (IL)-6, and IL-1beta but not of TGF-beta in qHSCs. Individually or together, L-N(G)-monomethylarginine and antibodies to IL-1beta, IL-6, and TNF-alpha only partly reversed qHSC + LPS-conditioned medium-induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF-beta, TNF-alpha, IL-1beta, and IL-6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.     

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.     

Lingual incisor traits in modern hominoids and an assessment of their utility for fossil hominoid taxonomy

The morphology of the anterior dentition has received scant attention for purposes of taxonomic discrimination. Recently, however, lingual incisor morphology was used in differentiating several Miocene ape species and genera. This paper assesses the utility of this morphology for taxonomic discrimination by examining the nature and patterns of variation in lingual incisor morphology in extensive samples of modern chimpanzees, gorillas, orangutans, and gibbons. This paper documents discrete morphological traits on the lingual side of incisors. Trait frequencies are used in univariate and multivariate analyses to examine the apportionment of variation in species, subspecies, and populations. A correlation between lingual incisor traits, tooth dimensions, and sex attempts to determine if such factors affect the manifestation of traits. Finally, the findings are applied to understanding patterns of variation in the Miocene hominids. The study demonstrates that: 1) lingual incisor morphology differs substantially between the hylobatids and great apes; 2) variation in incisor traits is high within species, and most of it is found within local populations; and 3) incisor traits do not correlate significantly with incisor dimensions or sex. Species and to some extent subspecies of extant hominoids can be differentiated statistically using lingual incisor traits, but the frequency of traits such as continuous or discontinuous cingulum, or the presence or absence of pillars, differentiates them. Given this pattern of variation, I argue that it is necessary to assume and document similar patterns of variation in Miocene apes before incisor morphology is used for differentiating taxa.