Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments.

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time. The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways. Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems.

Pioneering Study on Rhopalurus crassicauda Scorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase.

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Nav1.4, Nav1.6, and BgNav1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.

Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina).

The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.

Purification of hyaluronidase as an anticancer agent inhibiting CD44.

Hyaluronidase (Hyal) can be employed to accomplish a diversity of complications related to hyaluronic acid (HA). Hyal contains some classes of catalysts that cleave HA. This enzyme is detected in several human tissues as well as in animal venoms, pathogenic organisms and cancers. Destructive cancer cells regularly increase the CD44 receptor existing in a cell membrane. This receptor acts as an exact receptor for HA, and HA is recognized to motivate the migration, spread, attack and metastasis of cancer cells. Nearly all of the methods used to purify Hyal are highly costly and not proper for industrial applications. This survey aims to review different methods of Hyal purification, which acts as an anticancer agent by degrading HA in tissues and thus inhibiting the CD44-HA interaction. Hyal can be successfully employed in the management of cancer, which is associated with HA-CD44. This review has described different methods for Hyal purification to prepare an origin to develop a novel purification technique for this highly appreciated protein. Using multiple columns is not applicable for the purification of Hyal and thus cannot be used at the industrial level. It is better to use affinity chromatography of anti-Hyal for Hyal with one-step purification.

Venom characterization of the bark scorpion Centruroides edwardsii (Gervais 1843): Composition, biochemical activities and in vivo toxicity for potential prey.

In this study, we characterize the venom of Centruroides edwardsii, one of the most abundant scorpions in urban and rural areas of Costa Rica, in terms of its biochemical constituents and their biological activities. C. edwardsii venom is rich in peptides but also contains some higher molecular weight protein components. No phospholipase A2, hemolytic or fibrinogenolytic activities were found, but the presence of proteolytic and hyaluronidase enzymes was evidenced by zymography. Venom proteomic analysis indicates the presence of a hyaluronidase, several cysteine-rich secretory proteins, metalloproteinases and a peptidylglycine α-hydroxylating monooxygenase like-enzyme. It also includes peptides similar to the K+-channel blocker margatoxin, a dominant toxin in the venom of the related scorpion C. margaritatus. MS and N-terminal sequencing analysis also reveals the presence of Na+-channel-modulating peptides with sequence similarity to orthologs present in other scorpion species of the genera Centruroides and Tityus. We purified the hyaluronidase (which co-eluted with an allergen 5-like CRiSP) and sequenced ~60% of this enzyme. We also sequenced some venom gland transcripts that include other cysteine-containing peptides and a Non-Disulfide Bridged Peptide (NDBP). Our in vivo experiments characterizing the effects on potential predators and prey show that C. edwardsii venom induces paralysis in several species of arthropods and geckos; crickets being the most sensitive and cockroaches and scorpions the most resistant organisms tested. Envenomation signs were also observed in mice, but no lethality was reached by intraperitoneal administration of this venom up to 120 µg/g body weight.

Development of a New Type of Recombinant Hyaluronidase Using a Hexahistidine; Possibilities and Challenges in Commercialization.

Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The present study aimed to develop a novel, highly active hyaluronidase and assess its function. Therefore, in order to find the most efficient active hyaluronidase, we produced several shortened hyaluronidases with partial removal of the N- or C-terminal regions. Moreover, we created an enzyme that connected six histidines onto the end of the hyaluronidase C-terminus. This simplified subsequent purification using Ni2+ affinity chromatography, making it feasible to industrialize this highly active recombinant hyaluronidase which exhibited catalytic activity equal to that of the commercial enzyme. Therefore, this simple and effective isolation method could increase the availability of recombinant hyaluronidase for research and clinical purposes.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis.

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 µg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

In-Depth Venome of the Brazilian Rattlesnake Crotalus durissus terrificus: An Integrative Approach Combining Its Venom Gland Transcriptome and Venom Proteome.

Snake venoms are complex mixtures mainly composed of proteins and small peptides. Crotoxin is one of the most studied components from Crotalus venoms, but many other components are less known due to their low abundance. The venome of Crotalus durissus terrificus, the most lethal Brazilian snake, was investigated by combining its venom gland transcriptome and proteome to create a holistic database of venom compounds unraveling novel toxins. We constructed a cDNA library from C. d. terrificus venom gland using the Illumina platform and investigated its venom proteome through high resolution liquid chromotography-tandem mass spectrometry. After integrating data from both data sets, more than 30 venom components classes were identified by the transcriptomic analysis and 15 of them were detected in the venom proteome. However, few of them (PLA2, SVMP, SVSP, and VEGF) were relatively abundant. Furthermore, only seven expressed transcripts contributed to ∼82% and ∼73% of the abundance in the transcriptome and proteome, respectively. Additionally, novel venom proteins are reported, and we highlight the importance of using different databases to perform the data integration and discuss the structure of the venom components-related transcripts identified. Concluding, this research paves the way for novel investigations and discovery of future pharmacological agents or targets in the antivenom therapy.

Affinity adsorption of bovine hyaluronidase with ligands targeting to active site.

Four affinity ligands were designed from 6-chloromethyluracil and 2-aminobenzimidazole and simulated for the interaction with bovine hyaluronidase-1. Regarding sequence alignment, bovine hyaluronidase-1 precursor showed circa 83.6% similarity with human hyaluronidase-1. Regarding structural modeling and molecular docking, bovine hyaluronidase-1 interacted with ligands in the active site. Using epichlorohydrin, 1,3-propanediamine and cyanuric chloride as spacers, 6-chloromethyluracil and 2-aminobenzimidazole were composed to Sepharose beads. The modified Sepharose beads were then subjected to adsorption analysis with bovine hyaluronidase. After one step of affinity adsorption, the samples extracted from bovine testes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and activity assay. As calculated, the densities of four ligands on sorbents (entitled as L-1, L-2, L-3 and L-4) were 37.7 ±â€¯2.3, 36.4 ±â€¯3.2, 42.4 ±â€¯4.2 and 33.7 ±â€¯2.3 µmol/g wet gel; the theoretical maximum adsorption (Qmax) of bovine hyaluronidase on the four sorbents were 63.6 ±â€¯1.6, 72.0 ±â€¯0.7, 111.0 ±â€¯4.1 and 121.7 ±â€¯2.3 mg/g wet gel, respectively; the dissociation constants (Kd) of the four sorbents were 18.5 ±â€¯0.8, 48.1 ±â€¯4.3, 35.0 ±â€¯3.0, 40.6 ±â€¯2.7 µg/g wet gel, respectively. After optimization, the proteins captured by sorbents attaching 2-aminobenzimidazole based ligands (L-3 and L-4) revealed the main single band at approximately 50 kDa, and the purities were about 85.2 and 96.4%; the bioactivity recoveries were 83.5 and 89.4%. In addition, the bands on SDS-PAGE gel were also extracted and confirmed with linear trap quadropole mass spectrometry (LTQ-MS) analysis.

Hyaluronidase-like enzymes are a frequent component of venoms from theraphosid spiders.

Theraphosid spider venoms are extremely complex mixtures, composed mainly by low molecular compounds, peptides, and enzymes. The large size of these spiders and their ability to breed in captivity permits access to rather large amounts of venom and an easier venom extraction. In the present study, we conducted a comparative investigation about the content of hyaluronidase-like enzymes in the venoms from several theraphosid spiders, with a special focus on the Poecilotheria species, which are considered as underestimated theraphosids of medical importance. The following species were analyzed: Poecilotheria regalis, Poecilotheria ornata, Poecilotheria rufilata, Poecilotheria vittata, Bonnetina papalutlensis, Aphonopelma sp., Brachypelma smithi, Brachypelma epicureanum, Brachypelma boehmei, Grammostola porteri, Lasiodora klugi, Ceratogyrus darlingi, and Nhandu chromatus. The presence of hyaluronidase-like enzymes was evidenced in all venoms by a turbidimetric method and zymography. Several isoforms of acid-active hyaluronidase-like enzymes were detected in the venoms from Poecilotheria species. These results provide some biochemical characteristics of the high molecular mass proteins of the theraphosid venoms.

Isolation and characterization of Conohyal-P1, a hyaluronidase from the injected venom of Conus purpurascens.

Hyaluronidases are ubiquitous enzymes commonly found in venom and their main function is to degrade hyaluran, which is the major glycosaminoglycan of the extracellular matrix in animal tissues. Here we describe the purification and characterization of a 60kDa hyaluronidase found in the injected venom from Conus purpurascens, Conohyal-P1. Using a combined strategy based on transcriptomic and proteomic analysis, we determined the Conohyal-P1 sequence. Conohyal-P1 has conserved consensus catalytic and positioning domain residues characteristic of hyaluronidases and a C-terminus EGF-like domain. Additionally, the enzyme is expressed as a mixture of glycosylated isoforms at five asparagine sites. The activity of the native Conohyal-P1 was assess MS-based methods and confirmed by classical turbidimetric methods. The MS-based assay is particularly sensitive and provides the first detailed analysis of a venom hyaluronidase activity monitored with this method. The discovery of new hyaluronidases and the development of techniques to evaluate their performance can advance several therapeutic procedures, as these enzymes are widely used for enhanced drug delivery applications. BIOLOGICAL SIGNIFICANCE: Cone snail venom is a remarkable source of therapeutically important molecules, as is the case of conotoxins, which have undergone extensive clinical trials for several applications. In addition to the conotoxins, a large array of proteins have been reported in the venom of several species of cone snails, including enzymes that were found in dissected and injected Conus venom. Here we describe the isolation and characterization of the hyaluronidase Conohyal-P1 from the injected venom of C. purpurascens. We employed a combined transcriptomic and proteomic analysis to obtain the full sequence of this hyaluronidase. The activity of Conohyal-P1 was assessed by a mass spectrometry-based method, which provide the first detailed venom hyaluronidase activity analysis monitored by mass spectrometry allowing the visualization of the substrate degradation by the enzyme.

A simple, rapid and sensitive plate assay for detection of microbial hyaluronidase activity.

Hyaluronidase (hyase) is a glycosidase enzyme that predominantly degrades hyaluronic acid (HA) having important applications in many biotechnological processes and therapeutics. Several assay methods have been proposed to screen hyase producing microorganisms; however, they rely on unique reagents and sophisticated instruments, which are expensive and could be unavailable in general laboratories. In the present studies, a rapid, simple, sensitive, highly reproducible, and cost-effective qualitative plate assay has been developed for the screening of hyase producing microorganisms. The routinely used plate assay method of Richman and Baer requires a special chemical cetylpyridinium chloride and long incubation period of 20 h; but still, the zones of clearance are not very clear and distinct. While, the present method requires an incubation period of only 1 h and the distinct zones of clearance appear with Gram's iodine within 1 min of time. This method does not require any special medium, unlike previously reported methods. Moreover, use of commonly available Gram's iodine makes this method suitable for many researchers. The results of the assay method were validated by TLC, zymographic analysis and determining the growth of isolates in minimal medium containing HA as a sole carbon source.

Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms' Lytic Activity Using Mass Spectrometry and Zymography.

BACKGROUND: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. METHODS: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. RESULTS: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. CONCLUSION: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

Hyaluronidase and Chondroitinase.

Glycosaminoglycans (GAGs) are important constituents of the extracellular matrix that make significant contributions to biological processes and have been implicated in a wide variety of diseases. GAG-degrading enzymes with different activities have been found in various animals and microorganisms, and they play an irreplaceable role in the structure and function studies of GAGs. As two kind of important GAG-degrading enzymes, hyaluronidase (HAase) and chondroitinase (CSase) have been widely studied and increasing evidence has shown that, in most cases, their substrate specificities overlap and thus the "HAase" or "CSase" terms may be improper or even misnomers. Different from previous reviews, this article combines HAase and CSase together to discuss the traditional classification, substrate specificity, degradation pattern, new resources and naming of these enzymes.

Identification of hyaluronidase and phospholipase B in Lachesis muta rhombeata venom.

Hyaluronidases contribute to local and systemic damages after envenoming, since they act as spreading factors cleaving the hyaluronan presents in the connective tissues of the victim, facilitating the diffusion of venom components. Although hyaluronidases are ubiquitous in snake venoms, they still have not been detected in transcriptomic analysis of the Lachesis venom gland and neither in the proteome of its venom performed previously. This work purified a hyaluronidase from Lachesis muta rhombeata venom whose molecular mass was estimated by SDS-PAGE to be 60 kDa. The hyaluronidase was more active at pH 6 and 37 °C when salt concentration was kept constant and more active in the presence of 0.15 M monovalent ions when the pH was kept at 6. Venom was fractionated by reversed-phase liquid chromatography (RPLC). Edman sequencing after RPLC failed to detect hyaluronidase, but identified a new serine proteinase isoform. The hyaluronidase was identified by mass spectrometry analysis of the protein bands in SDS-PAGE. Additionally, phospholipase B was identified for the first time in Lachesis genus venom. The discovery of new bioactive molecules might contribute to the design of novel drugs and biotechnology products as well as to development of more effective treatments against the envenoming.

Comparison of total protein and enzyme levels in successive regenerations of venom from individual coralsnakes.

Coralsnakes produce highly potent neurotoxic venoms, but little is known about variations in specific enzyme components within a species or from one replenishment of venom to the next within the same animal. Since published studies are often conducted using venom pools from multiple snakes, individual differences are masked and variations among individual snakes and between subsequent venom regenerations from the same snake have rarely been documented. This study involves the analysis and comparison of four successive venom collections from each of nine individual coralsnakes in order to detect these differences. Significant variation was found within the successive re-synthesis of venom components. Even greater differences were observed between the venoms from similar individual snakes. Since studies of variation in enzymatic activity would be significant only if they were above these normal variations, it is important to be aware of these differences. These results suggest the importance of understanding the variations present within and between individuals of the same species when interpreting the potential significance of differences found as the result of genetic, environmental or ecological factors.

A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, ß-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.

A novel hyaluronidase produced by Bacillus sp. A50.

Hyaluronidases are a family of enzymes that degrade hyaluronic acid (hyaluronan, HA) and widely used in many fields. A hyaluronidase producing bacteria strain was screened from the air. 16S ribosomal DNA (16S rDNA) analysis indicated that the strain belonged to the genus Bacillus, and the strain was named as Bacillus sp. A50. This is the first report of a hyaluronidase from Bacillus, which yields unsaturated oligosaccharides as product like other microbial hyaluronate lyases. Under optimized conditions, the yield of hyaluronidase from Bacillus sp. A50 could reach up to 1.5×10(4) U/mL, suggesting that strain A50 is a good producer of hyaluronidase. The hyaluronidase (HAase-B) was isolated and purified from the bacterial culture, with a specific activity of 1.02×10(6) U/mg protein and a yield of 25.38%. The optimal temperature and pH of HAase-B were 44°C and pH 6.5, respectively. It was stable at pH 5-6 and at a temperature lower than 45°C. The enzymatic activity could be enhanced by Ca2+, Mg2+, or Ni2+, and inhibited by Zn2+, Cu2+, EDTA, ethylene glycol tetraacetic acid (EGTA), deferoxamine mesylate salt (DFO), triton X-100, Tween 80, or SDS at different levels. Kinetic measurements of HAase-B towards HA gave a Michaelis constant (Km) of 0.02 mg/mL, and a maximum velocity (Vmax) of 0.27 A232/min. HAase-B also showed activity towards chondroitin sulfate A (CSA) with the kinetic parameters, Km and Vmax, 12.30 mg/mL and 0.20 A232/min respectively. Meanwhile, according to the sequences of genomic DNA and HAase-B's part peptides, a 3,324-bp gene encoding HAase-B was obtained.

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins.

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.

[Characteristic of virulence potential of clinical isolates of enterococci].

AIM: Determination of virulence of enterococci strains isolated from clinical material from humans on pheno- and genotype levels. MATERIALS AND METHODS: 30 strains of enterococci isolated from wound exudate, urine, newborn skin lavage were used in the study. Strain identification was carried out by multiplex PCR. Hemolytic activity was determined by dish method, gelatinase - by dissolution of gelatin column, proteolytic--by biuret method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, cylLs, cylL1, ESP, HYL, ASA)--by using PCR. RESULTS: Clinical isolates of enterococci were assigned to E. faecalis and E. faecium species. Virulence factors on phenotype and genotype levels were detected in both species. CONCLUSION: Genetic determinants of virulence are more widespread among clinical isolates of E.faecalis species. Set of genes coding virulence factors in E. faecalis depends on biotope. Gene coding hyaluronidase synthesis is characteristic for E. faecium. A correlation between phenotypic manifestation of features and enterococci genotype was detected.