Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.     

Freezing induces biased results in the molecular detection of Flavobacterium columnare

Specific PCR detection and electron microscopy of Flavobacterium columnare revealed the risk of false-negative results in molecular detection of this fish pathogen. Freezing and thawing destroyed the cells so that DNA was for the most part undetectable by PCR. The detection of bacteria was also weakened after prolonged enrichment cultivation of samples from infected fish.     

Mycobacteria and Fungi in Moisture-Damaged Building Materials

In contrast to the growth of fungi, the growth of mycobacteria in moisture-damaged building materials has rarely been studied. Environmental mycobacteria were isolated from 23% of samples of moisture-damaged materials (n = 88). The occurrence of mycobacteria increased with increasing concentrations of fungi. Mycobacteria may contribute to indoor exposure and associated adverse health effects.     

Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate

Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.     

SIRT1 longevity factor suppresses NF‐κB ‐driven immune responses: regulation of aging via NF‐κB acetylation?

The aging process involves changes in immune regulation, i.e. adaptive immunity declines whereas innate immunity becomes activated. NF-kappaB signaling is the master regulator of the both immune systems. Two recent articles highlight the role of the NF-kappaB system in aging and immune responses. Adler et al showed that the NF-kappaB binding domain is the genetic regulatory motif which is most strongly associated with the aging process. Kwon et al studying HIV-1 infection and subsequent immune deficiency process demonstrated that HIV-1 Tat protein binds to SIRT1 protein, a well-known longevity factor, and inhibits the SIRT1-mediated deacetylation of the p65 component of the NF-kappaB complex. As a consequence, the transactivation efficiency of the NF-kappaB factor was greatly potentiated, leading to the activation of immune system and later to the decline of adaptive immunity. These observations support the scenario where immune responses and aging process can be enforced by the potentiation of NF-kappaB transactivation efficiency. Longevity factors, such as SIRT1 and its activators, might regulate the efficiency of the NF-kappaB signaling, the major outcome of which is inflamm-aging via proinflammatory responses.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.     

Chemical interplay in the mechanism of partial agonist activation in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, one subtype in the family of ionotropic glutamate receptors, are the main receptors responsible for excitatory signaling in the mammalian central nervous system. Previous studies utilitizing the isolated ligand binding domain of these receptors have provided insight into the role of specific ligand-protein interactions in mediating receptor activation. However, these studies relied heavily on the partial agonist kainate, in which the alpha-amine group is constrained in a pyrrolidine ring. Here we have studied a series of substituted and unsubstituted willardiines with primary alpha-amine groups similar to that of the full agonist glutamate whose activation can be varied depending on the size of the substituent. The specific ligand-protein interactions in the mechanism of partial agonism in this subtype were investigated using vibrational spectroscopy, and the large-scale conformational changes in the ligand binding domain were studied with fluorescence resonance energy transfer (FRET). These investigations show that the strength of the interaction at the alpha-amine group correlates with the extent of cleft closure and extent of activation, with the agonist of higher efficacy showing larger cleft closure and stronger interactions at this group, suggesting that this is one of the mechanisms by which the agonist controls receptor activation.     

The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A.

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5-5.6 (Tm, 62 +/- 2 degrees C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstr?m, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.