L-amino Oxidase Research Articles

Exploring venom diversity in Mixcoatlus browni and Mixcoatlus barbouri: A comparative analysis of two rare Mexican snake species with crotoxin-like presence

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri, M. browni, and M. melanurus, of which the venom composition of M. melanurus, the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni, and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri, and 13 for M. browni. The latter venom was further characterized by using a quantitative ‘venomics’ protocol, which revealed that it is mainly composed of 51.1 % phospholipases A2 (PLA2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l-amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri, on the other hand, remains necessary.

Arginine as the sole nitrogen source for Ostreococcus tauri growth: Insights on nitric oxide synthase enzyme

IntroductionPhotosynthetic organisms respond to nitrogen (N) deprivation with the slowdown of photosynthesis and electron transport resulting in the balance the carbon (C)/N ratio. Under this extreme condition, organisms trigger complex mechanisms to keep growing using different N sources and recycling N containing molecules. In particular, phytoplankton are able to uptake L-arginine (L-Arg) as an organic N source. L-Arg can be assimilated mainly by the arginase, arginine deimidase, arginine decarboxylase or L-amino oxidase pathways.ResultsWe analyzed the effect of different N sources on the growth of the green algae Ostreococcus tauri. N starvation caused an inhibition of culture growth and a decrease in chlorophyll content. The addition of L-Arg to an N-deprived medium promotes a sustained growth rate of O. tauri culture and the increase of chlorophyll levels. The transcript level of genes involved in N uptake and metabolism were increased in N-starved condition while the addition of L-Arg as the sole N source reduced their induction. Since the O. tauri genome lacks the classical pathways to metabolize L-Arg, another enzyme/s may be responsible for L-Arg catabolism. Previously, we characterized the nitric oxide synthase (NOS) enzyme from O. tauri (OtNOS) which oxidizes L-Arg producing nitric oxide (NO) and citrulline. The NOS inhibitor L-NAME blocks the effect promoted by L-Arg on N-deprived O. tauri growth. Besides, NO level increased in O. tauri cells growing in L-Arg containing medium, suggesting the participation of OtNOS enzyme in L-Arg metabolism during N starvation.DiscussionOur hypothesis suggests that, after NOS-dependent Arg degradation, non-enzymatic oxidation of NO produces N oxides (mainly NO2-) that are re-incorporated to the N primary metabolism. As expected, N deprivation increases the lipid content in Ostreococcus. The addition of L-Arg or NO2- as the sole N sources showed a similar increase in lipid content to N deprivation. In summary, our results demonstrate that L-Arg is able to function as N source in Ostreococcus. The evidences on an alternative pathway of N supply and metabolism in a photosynthetic microorganism are discussed. These results could also allow the development of biotechnological tools for increasing lipid production for industry.

Venom gland transcriptome from Heloderma horridum horridum by high-throughput sequencing

Lizards of the Helodermatidae (Anguimorpha) family consist of at least two well recognized species: Heloderma horridum horridum and Heloderma suspectum suspectum. They contain specialized glands in their jaws that produce venomous secretions that causes envenoming symptoms to bitten animals. One way to study proteins from such secretions is by RNA-seq; a powerful molecular tool to characterize the transcriptome of such specialized gland, and its protein secretions. The total RNA from venom gland tissues of H. horridum horridum was extracted and a cDNA library was constructed and sequenced. Overall, 114,172 transcripts were found, and 199 were annotated based on sequence similarities to previously described peptides/proteins. Transcripts coding for putative exendins, defensins, natriuretics and serine protease inhibitors were the most highly expressed. Transcripts that code for several putative serine proteases, phospholipases, metalloproteases, lipases, L-amino oxidase and nucleases were also found. Some of the novel identified transcripts were translationally controlled tumor proteins, venom factors, vespryns, waprins, lectins, cystatins and serine protease inhibitors. All these new protein structures may contribute to a better understanding of the venomous secretions of the Helodermatidae family.

Nitrogen scavenging from amino acids and peptides in the model alga Chlamydomonas reinhardtii. The role of extracellular l-amino oxidase

Phytoplankton live under constantly changing environments in which concentrations of inorganic nitrogen can be limiting but organic nitrogen sources (urea, amino acids or peptides) are available. Understanding how algae, as primary producers, assimilate organic nitrogen is of eco-physiological importance although such studies are rare. Given genetic tractability, the green microalga Chlamydomonas reinhardtii is an excellent model system for elucidating amino acid assimilation by algal species. This alga can extracellularly deaminate most amino acids using a l-Amino acid Oxidase (LAO1), which generates ammonium that can then be taken up as a source of nitrogen. In this work, we have used lao1 mutant strains to investigate the impact of the absence of this enzyme on C. reinhardtii growth using amino acids and di-/tri-peptides as sole nitrogen sources. Our results show that LAO1 enzyme is crucial for growth on most proteinogenic amino acids and peptides by this alga. We present findings from an analysis of algal genomes that reveal a new evolutionary branch for algal l-amino acid oxidase genes (ALAAO) that includes Rhodophyta, Alveolata, Heterokonta, Haptophyta, and Dinophyta species. Interestingly, C. reinhardtii appears to be the only green algal that contains an ALAAO homolog, and we present a hypothesis about the possible origins of ALAAO genes in algae based on a comparative analysis of currently available and assembled algal genomes.

Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family

The objective of this study was to characterize six different scorpion venoms using biological and biochemical methods, including a preliminary MS/MS and a post-translational modifications analysis. Despite the diversity of scorpion species of medical importance in Africa and Colombia, the venoms of these arachnids have been poorly studied in these two regions. We report the biochemical, electrophoretic, chromatographic profile, internal peptide sequences with a post-translational modification report, and a preliminary antitumor activity of five different scorpions of the Buthidae family, Androctonus amoreuxi, Babycurus jacksoni, Grosphus grandidieri, Hottentotta gentili and Tityus fuhrmanni, and one of the Scorpionidae family Pandinus imperator. No L-amino oxidase activity was detected in the evaluated venoms. Proteolytic activity using azocasein was detected only in G. grandidieri and P. imperator, indicating the possible presence of metalloproteinases in these two venoms. Proteolytic activity using NOBA was detected in all venoms indicating the possible presence of serine-proteinases. Phospholipase A2 activity was detected in the venoms of P. imperator, G. grandidieri, H. gentili and A. amoreuxi, with P. imperator venom being the most active. All venoms analyzed contained defensin-like proteins, alpha toxins, metalloproteinases, neuropeptides, DBP affecting ion channels, DBP with antimicrobial activity, among others. Venoms from P. imperator, G. grandidieri and T. fuhrmanni showed a dose-dependent cytotoxic activity over MCF-7 cells. Only two isolated RP-HPLC fractions from P. imperator and T. fuhrmanni showed cytotoxic activity over MCF-7. No cytotoxic activity was found in the venoms from A. amoreuxi, B. jacksoni, and H. gentili.

An Alternative Inactivant for Rift Valley Fever Virus using Cobra Venom-derived L-Amino Oxidase, which is Related to its Immune Potential

Vaccine improvement depends on the formulation, adjuvant type and inactivant used. The type of formulation may interfere with immunogenicity. The present work aimed to evaluate the inactivation activity and related immune potential of the Cobra venom-derived LAO enzyme compared to the currently used inactivants (BPL and formalin) for both animal and human vaccines. The RVF virus was completely inactivated within 6 hrs, 4 hrs and 2 hrs after treatment with Formalin, LAO and BPL, respectively. The vaccine potency [ED50] was arranged in a descending order from formalin (0.016) to BPL (0.005) and LAO (0.002). The total IgG levels, Neutralizing Index (NI) and Interferon levels were significantly increased compared to those detected after immunization with the BPL- and Formalin-inactivated vaccine candidates.

Evaluation of an Antimicrobial L-Amino Acid Oxidase and Peptide Derivatives from Bothropoides mattogrosensis Pitviper Venom

Healthcare-associated infections (HAIs) are causes of mortality and morbidity worldwide. The prevalence of bacterial resistance to common antibiotics has increased in recent years, highlighting the need to develop novel alternatives for controlling these pathogens. Pitviper venoms are composed of a multifaceted mixture of peptides, proteins and inorganic components. L-amino oxidase (LAO) is a multifunctional enzyme that is able to develop different activities including antibacterial activity. In this study a novel LAO from Bothrops mattogrosensis (BmLAO) was isolated and biochemically characterized. Partial enzyme sequence showed full identity to Bothrops pauloensis LAO. Moreover, LAO here isolated showed remarkable antibacterial activity against Gram-positive and -negative bacteria, clearly suggesting a secondary protective function. Otherwise, no cytotoxic activities against macrophages and erythrocytes were observed. Finally, some LAO fragments (BmLAO-f1, BmLAO-f2 and BmLAO-f3) were synthesized and further evaluated, also showing enhanced antimicrobial activity. Peptide fragments, which are the key residues involved in antimicrobial activity, were also structurally studied by using theoretical models. The fragments reported here may be promising candidates in the rational design of new antibiotics that could be used to control resistant microorganisms.

Antimicrobial Profile of Selected Snake Venoms and Their Associated Enzymatic Activities

Aims: To investigate the antiviral and antibacterial profile of several crude snake venoms and to assess some of their enzymatic activities. Methodology: The antiviral activities of Naja haje, Bitis arietans, Naja nigricollis and Echis carinatus snake venoms were investigated against Herpes simplex virus type1, Rift valley fever virus and Vesicular stomatitis virus using the end point of cytopathic effect method. Antibacterial activities of Bitis arietans, Cerastes cerastes, Echis carinatus, Vipera lebetina, Naja naja, Pseudechis australis, Naja nigricollis and Naja haje venoms were examined against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa using disc diffusion method. Microdilution method was used to determine the venom's minimum inhibitory concentration. L-amino acid oxidase and phospholipase A2 activities of crude venoms were evaluated using enzymatic assays. Results: Naja nigricollis, Bitis arietans and Echis carinatus snake venoms exhibited significant antiviral activities against all test viruses, except for N. haje treated cells. The mean depletion of viral infectivity titer of venom pretreated cells was higher than its depletion post viral infection for all three venoms showing antiviral activities. Naja nigricollis exhibited the highest antiviral activity against test viruses and recorded a mean depletion of viral infectivity titer in venom pretreated cells of 3.8 log (10) / ml , 3.2 log (10) / ml and 2.5 Research Article British Microbiology Research Journal, 2(4): 251-263, 2012 252 log (10) / ml for HSV-1, RVFV and VSV, respectively. Pseudechis australis, followed by Naja naja and Naja nigricollis venoms, showed the highest inhibitory activity against test bacteria with inhibition zones ranging from 11-17 mm, 8-14 mm and 8-13 mm, respectively. Minimum inhibitory concentrations of test venoms against different bacterial strains ranged from 156 μg / ml to 1.25 mg / ml. Maximum Lamino oxidase activity was detected in Naja naja, Cerastes cerastes and Pseudechis australis. The highest Phospholipase A2 activity was identified in Bitis arietans, Pseudechis australis, Naja naja and Naja nigricollis. Conclusion: It can be concluded that snake venoms or their bioactive derivatives can be promising therapeutic agents against some microbial infections. Further investigations will be carried out for purification and more characterization of the biologically active components in snake venoms.

Identification of antibacterial mechanism of l‐amino acid oxidase derived from Trichoderma harzianum ETS 323

Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.

A Novel l-Amino Acid Oxidase from Trichoderma harzianum ETS 323 Associated with Antagonism of Rhizoctonia solani

Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.

Origin and diversification of the L-amino oxidase family in innate immune defenses of animals

L-amino acid oxidases (LAOs), because they produce hydrogen peroxide as a by-product, function in innate immune defenses of both vertebrates and mollusks. Phylogenetic analysis revealed two major subfamilies of LAOs: (1) a subfamily including LAOs from vertebrates and mainly from Terrabacteria and (2) a subfamily including LAOs from mollusks and Hydrobacteria. These subfamilies thus originated early in the history of life, implying that their innate immune functions in vertebrates and mollusks have evolved separately. Mammalian LAOs were found to belong to three separate clades: (1) LAO1, (2) LAO2, and (3) IL4I1. Phylogenetic analysis supported the hypothesis that LAO1 and LAO2 arose by a gene duplication prior to the divergence of marsupials from placental mammals, while IL4I1 duplicated from the ancestor of the LAO1 and LAO2 prior to the divergence of tetrapods from bony fishes. Mammalian IL4I1 clustered with LAOs from bony fishes, and these molecules shared a number of unique sequence features, including both amino acid replacements and a unique two-codon deletion. It is certain such unique features may be functionally important, especially three unique amino acid replacements in close proximity to the putative active site.

Comparative analysis of newborn and adult Bothrops jararaca snake venoms

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5′-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.

Cytogenetic and radiation hybrid mapping of the bovine <i>interleukin 4 induced 1 (IL4I1)</i> gene to cattle chromosome 18 (Brief Report)

Abstract. The interleukin 4 induced 1 (IL4I1) gene, also known as FIG1, encodes a lysosomal protein in B cells with L-amino oxidase (EC 1.4.3.2) property and a high preference for aromatic amino acid substrates implicating a fundamental role in antigene processing and presentation (MANSON et al. 2004). In human, IL4I1 has been assigned to chromosome 19q13.3-19q13.4, a hot spot for susceptibility to several autoimmune diseases (CHAVAN et al. 2002). Based on comparative genome data between human and cattle (GOLDAMMER et al. 2002), an assignment of IL4I1 to Bos taurus chromosome 18 (BTA18) has been predicted within a quantitative trait locus for somatic cell score (KÜHN et al. 2003). This prediction is supported by the putative bovine sequence for IL4I1, LOC520692, which has been assigned to BTA18 close to 56.1 megabases within the annotated bovine sequence (NCBI build Btau-4.0). Therefore, we started positional cloning of bovine IL4I1, as a candidate gene related to adaptive immunity, i.e. during udder infections in cattle. Here we present the physical assignment of the bovine IL4I1 gene by fluorescence in situ hybridization (FISH) and radiation hybrid (RH) mapping.

Comparative venom gland transcriptome surveys of the saw-scaled vipers (Viperidae: Echis) reveal substantial intra-family gene diversity and novel venom transcripts

BackgroundVenom variation occurs at all taxonomical levels and can impact significantly upon the clinical manifestations and efficacy of antivenom therapy following snakebite. Variation in snake venom composition is thought to be subject to strong natural selection as a result of adaptation towards specific diets. Members of the medically important genus Echis exhibit considerable variation in venom composition, which has been demonstrated to co-evolve with evolutionary shifts in diet. We adopt a venom gland transcriptome approach in order to investigate the diversity of toxins in the genus and elucidate the mechanisms which result in prey-specific adaptations of venom composition.ResultsVenom gland transcriptomes were created for E. pyramidum leakeyi, E. coloratus and E. carinatus sochureki by sequencing ~1000 expressed sequence tags from venom gland cDNA libraries. A standardised methodology allowed a comprehensive intra-genus comparison of the venom gland profiles to be undertaken, including the previously described E. ocellatus transcriptome. Blast annotation revealed the presence of snake venom metalloproteinases, C-type lectins, group II phopholipases A2, serine proteases, L-amino oxidases and growth factors in all transcriptomes throughout the genus. Transcripts encoding disintegrins, cysteine-rich secretory proteins and hyaluronidases were obtained from at least one, but not all, species. A representative group of novel venom transcripts exhibiting similarity to lysosomal acid lipase were identified from the E. coloratus transcriptome, whilst novel metallopeptidases exhibiting similarity to neprilysin and dipeptidyl peptidase III were identified from E. p. leakeyi and E. coloratus respectively.ConclusionThe comparison of Echis venom gland transcriptomes revealed substantial intrageneric venom variation in representations and cluster numbers of the most abundant venom toxin families. The expression profiles of established toxin groups exhibit little obvious association with venom-related adaptations to diet described from this genus. We suggest therefore that alterations in isoform diversity or transcript expression levels within the major venom protein families are likely to be responsible for prey specificity, rather than differences in the representation of entire toxin families or the recruitment of novel toxin families, although the recruitment of lysosomal acid lipase as a response to vertebrate feeding cannot be excluded. Evidence of marked intrageneric venom variation within the medically important genus Echis strongly advocates further investigations into the medical significance of venom variation in this genus and its impact upon antivenom therapy.

INTERACTION OF SNAKE-VENOM PROTEINS WITH BLOOD COAGULATION FACTORS: MECHANISMS OF ANTICOAGULANT ACTIVITY

Snake venoms are rich in a large variety of proteins and peptides that can interfere with the hemostatic system. This review focuses on snake-venom proteins, with or without enzymatic activity, that bind to blood coagulation factors and exhibit anticoagulant effects. These proteins include (1) anticoagulant phospholipases A2, which bind to factor X and impair the formation of prothrombinase complex independently from their enzymatic activity; (2) factor IX- and X-binding proteins, which belong to the family of C-type lectin-like proteins and interact with the Gla domain of factor X/Xa and/or IX/IXa; (3) prothrombin- and thrombin-binding proteins, represented by bothrojaracin and salmorin, which are also C-type lectin-like molecules and either bind to prothrombin, impairing thrombin formation, or to thrombin, inhibiting its biological activities, such as clotting of fibrinogen, platelet activation, and so forth; and (4) L-amino oxidases that have recently been described to have anticoagulant activity. The general structural characteristics of these proteins and current knowledge regarding their mechanisms of action are discussed. These proteins have helped in our understanding of some molecular aspects of the hemostatic process. A more detailed analysis of the structure–function relationship of these molecules will certainly result in new medical and pharmacological applications. Furthermore, venom components offer attractive templates for the development of rationally designed therapeutic agents.

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox

BackgroundBothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.ResultsOptimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3–10 pH range during the isoelectric focusing step and 10–20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins.ConclusionThe analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel α 9β 1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases

Echis ocellatus is the most medically important snake in West Africa. However, the composition of its venom and the differential contribution of these venom components to the severe haemorrhagic and coagulopathic pathology of envenoming are poorly understood. To address this situation we assembled a toxin transcriptome based upon 1000 expressed sequence tags (EST) from a cDNA library constructed from pooled venom glands of 10 individual E. ocellatus. We used a variety of bioinformatic tools to construct a fully annotated venom-toxin transcriptome that was interrogated with a combination of BLAST annotation, gene ontology cataloguing and disintegrin-motif searching. The results of these analyses revealed an unusually abundant and diverse expression of snake venom metalloproteinases (SVMP) and a broad toxin-expression profile including several distinct isoforms of bradykinin-potentiating peptides, phospholipase A 2, C-type lectins, serine proteinases and l-amino oxidases. Most significantly, we identified for the first time a conserved α 9β 1 integrin-binding motif in several SVMPs, and a new group of putative venom toxins, renin-like aspartic proteases.

From genome to “venome”: Molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins

This study analyzed the origin and evolution of snake venom proteome by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. The snake toxins were shown to have arisen from recruitment events of genes from within the following protein families: acetylcholinesterase, ADAM (disintegrin/metalloproteinase), AVIT, complement C3, crotasin/beta defensin, cystatin, endothelin, factor V, factor X, kallikrein, kunitz-type proteinase inhibitor, LYNX/SLUR, L-amino oxidase, lectin, natriuretic peptide, betanerve growth factor, phospholipase A(2), SPla/Ryanodine, vascular endothelial growth factor, and whey acidic protein/secretory leukoproteinase inhibitor. Toxin recruitment events were found to have occurred at least 24 times in the evolution of snake venom. Two of these toxin derivations (CRISP and kallikrein toxins) appear to have been actually the result of modifications of existing salivary proteins rather than gene recruitment events. One snake toxin type, the waglerin peptides from Tropidolaemus wagleri (Wagler's Viper), did not have a match with known proteins and may be derived from a uniquely reptilian peptide. All of the snake toxin types still possess the bioactivity of the ancestral proteins in at least some of the toxin isoforms. However, this study revealed that the toxin types, where the ancestral protein was extensively cysteine cross-linked, were the ones that flourished into functionally diverse, novel toxin multigene families.

Partial purification of the aminopeptidase from the midgut of the human body louse, Pediculus humanus humanus

Summary The midgut of the human body louse Pediculus humanus humanus contains a thermally stable leucine aminopeptidase, which was detected by agarose gel electrophoresis using l-amino oxidase. Midgut extracts were homogenized in saline or in 1% Triton X-100 and the aminopeptidase was purified by Superose 6 gel filtration chromatography. A peak with enzyme activity that was extracted with or without Triton X-100 was eluted at a molecular weight 67–69 kDa. Non-denaturing polyacrylamide gel electrophoresis resolved one band of molecular weight of 69 kDa for samples that were extracted in a saline buffer. Two closely linked bands of molecular weight 67 kDa and 69 kDa were observed in samples that were extracted in 1% Triton X-100.

A Neurospora crassa mutant altered in the regulation of L-amino acid oxidase.

The isolation and characterization of a Neurospora crassa mutant altered in L-amino oxidase regulation is reported. The previously isolated gln-1bR8 strain, which only synthesizes the glutamine synthetase alpha monomer and lacks the beta monomer, was used as parental strain. A mutant derivative of strain was selected for its ability to grow on minimal medium in the presence of DL-methionine-SR-sulfoximine (MSO), an inhibitor of glutamine synthetase activity. This gln-1bR8;MSOR mutant overcame the inhibitory effect of MSO by increasing the activity of L-amino acid oxidase, an enzyme capable of degrading this compound. In contrast with the wild-type strain, the L-amino acid oxidase of the MSOR mutant was resistant to glutamine repression; in fact, it was induced by this amino acid but repressed by ammonium. This mutant is different from other nitrogen regulatory N. crassa mutants reported and is only altered in the regulation of L-amino acid oxidase. The MSOR mutation is epistatic to nit-2 since the nit2;MSOR double mutant regulated the L-amino acid oxidase in the same way as the MSOR single mutant.