Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity

Anthrax toxin consists of three proteins, protective antigen, lethal factor, and edema factor. Protective antigen translocates lethal factor and edema factor to the cytosol of mammalian cells. The amino-termini of lethal factor and edema factor have several homologous stretches. These regions are presumably involved in binding to protective antigen. In the present study we have determined the role of one such homologous stretch in lethal factor. Residues 187AspLeuLeuPhe190 were replaced by alanine. Asp187Ala and Phe190Ala were found to be non-toxic in combination with protective antigen. Their protective antigen-binding ability was drastically reduced. We propose that Asp187 and Phe190 are crucial for the expression of anthrax lethal toxin activity.     

Squamocin-O(1) and squamocin-O(2), new adjacent bis-tetrahydrofuran acetogenins from the seeds of Annona squamosa

Two bis-tetrahydrofuran acetogenins, squamocin-O(1) (1) and squamocin-O(2) (2), were isolated from a MeOH extract of seeds of Annona squamosa L. Their structures were determined by spectral means including precursor-ion scanning mass spectral analysis for their aminal derivatives. The configurations at the oxymethine chiral centers were assigned as 12R,15R,16R,19R,20R,23R,24S,28S,36S for 1 and 12S,15R,16R,19R,20R,23R,24S, 28S,36S for 2, based on 1H NMR analysis of their Mosher's ester derivatives and CD data.     

Crystal structure of the C. perfringens alpha-toxin with the active site closed by a flexible loop region

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin.     

T-cell receptor:CD3 down-regulation is a selected in vivo function of simian immunodeficiency virus Nef but is not sufficient for effective viral replication in rhesus macaques

We investigated the function of severely truncated simian immunodeficiency virus (SIV) Nef proteins (tNef) in vitro and in vivo. These variants emerged in rhesus monkeys infected with SIVmac239 containing a 152-bp deletion in the nef-unique region and have been suggested to enhance SIV virulence (E. T. Sawai, M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw, J. Virol. 74:2038-2045, 2000). We found that the tNef proteins were unable to down-regulate the cell surface expression of major histocompatibility complex class I proteins, CD4, and CD28 and neither stimulated SIV replication nor enhanced virion infectivity. The tNef proteins did efficiently down-regulate T-cell receptor (TCR):CD3 cell surface expression. Nevertheless, the SIVmac239 tnef variants were strongly attenuated in six infected juvenile rhesus macaques. Thus, while the ability of SIV Nef to down-modulate TCR:CD3 cell surface expression apparently confers a selective advantage in vivo, it is insufficient for efficient viral replication in infected macaques. Additional mutations elsewhere in SIVmac239 tnef genomes are required for a virulent phenotype.     

Induction of apoptosis and inhibition of papilloma formation may signal a new role for okadaic acid

Okadaic acid (OA), a tumor promoter in the mouse skin carcinogenesis model, has been shown to induce apoptosis in tumor cell lines that harbor H-ras mutations. We examined the effects of OA on mouse keratinocytes with (308) and without (C50) H-ras mutation in vitro and in an in vivo system. Following exposure to varying concentrations of OA over time, the effects of OA in vitro were assessed using microscopic, biochemical and flow cytometric techniques. OA effects on the cells included incorporation of propidium iodide, externalization of phosphatidylserine, and development of hypodiploidy. 308 cells demonstrated typical DNA ladder formation, rapid chromatin and nuclear condensation, while C50 cells demonstrated delayed chromatin condensation and nuclear fragmentation, but no DNA ladder formation. In vivo, OA elicited delayed papilloma formation and reduced tumor multiplicity. Though its mechanism of action is not fully known, we found that OA-induced inhibition of the clonal expansion of initiated cells may be related to the presence or absence of H-ras mutation.     

Mining viral protease data to extract cleavage knowledge

MOTIVATION: The motivation is to identify, through machine learning techniques, specific patterns in HIV and HCV viral polyprotein amino acid residues where viral protease cleaves the polyprotein as it leaves the ribosome. An understanding of viral protease specificity may help the development of future anti-viral drugs involving protease inhibitors by identifying specific features of protease activity for further experimental investigation. While viral sequence information is growing at a fast rate, there is still comparatively little understanding of how viral polyproteins are cut into their functional unit lengths. The aim of the work reported here is to investigate whether it is possible to generalise from known cleavage sites to unknown cleavage sites for two specific viruses-HIV and HCV. An understanding of proteolytic activity for specific viruses will contribute to our understanding of viral protease function in general, thereby leading to a greater understanding of protease families and their substrate characteristics. RESULTS: Our results show that artificial neural networks and symbolic learning techniques (See5) capture some fundamental and new substrate attributes, but neural networks outperform their symbolic counterpart.     

RecD plays an essential function during growth at low temperature in the antarctic bacterium Pseudomonas syringae Lz4W

The Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W has been used as a model system to identify genes that are required for growth at low temperature. Transposon mutagenesis was carried out to isolate mutant(s) of the bacterium that are defective for growth at 4 degrees but normal at 22 degrees . In one such cold-sensitive mutant (CS1), the transposon-disrupted gene was identified to be a homolog of the recD gene of several bacteria. Trans-complementation and freshly targeted gene disruption studies reconfirmed that the inactivation of the recD gene leads to a cold-sensitive phenotype. We cloned, sequenced, and analyzed approximately 11.2 kbp of DNA from recD and its flanking region from the bacterium. recD was the last gene of a putative recCBD operon. The RecD ORF was 694 amino acids long and 40% identical (52% similar) to the Escherichia coli protein, and it could complement the E. coli recD mutation. The recD gene of E. coli, however, could not complement the cold-sensitive phenotype of the CS1 mutant. Interestingly, the CS1 strain showed greater sensitivity toward the DNA-damaging agents, mitomycin C and UV. The inactivation of recD in P. syringae also led to cell death and accumulation of DNA fragments of approximately 25-30 kbp in size at low temperature (4 degrees ). We propose that during growth at a very low temperature the Antarctic P. syringae is subjected to DNA damage, which requires direct participation of a unique RecD function. Additional results suggest that a truncated recD encoding the N-terminal segment of (1-576) amino acids is sufficient to support growth of P. syringae at low temperature.