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The assay of mammalian hyaluronidases (HAases) is important in understanding the role of the hyaluronan-hyaluronidase (HA-HAase) system in various pathophysiological processes. Despite several quantitative assay method options, the Morgan-Elson colorimetric method modified by Reissig et al is considered the best for determining the activity in clinical samples. However, the sensitivity of the method was greatly limited by presence of protein above 400 microg due to turbidity interference that led to chromogen quenching. Therefore, an effort has been made to reinvestigate the Reissig et al method. In the reinvestigated method, a standardized optimal 0.32 M potassium tetraborate (PTB) was used against 0.13 M (native) to overcome the turbidity interference. The estimated mean OD at 585 nm of serum for native method was 0.043 (95% CI: 0.040 to 0.045), while that for the re-investigated method was 0.138 (95% CI: 0.133 to 0.143, p < 0.0001). The mean OD at 585 nm of serum of native method was significantly lower than that of re-investigated method (p < 0.05) at all protein levels. This was also true for estimated mean OD at 585 nm of plasma. The mean intrasample CVs for native and re-investigated methods were 0.9% and 0.5%, respectively, for normal serum. Furthermore, the repeatability coefficient of normal serum for native was 0.003 IU, while re-investigated method experienced that of 0.002 IU.

Despite a long history on treatment and management of snakebite, as of now, no satisfactory cure exists to treat local toxicity, including anti-venom therapy. Several natural compounds from plants and their synthetic analogs have shown to be protective. In this study 3, 4, 5-tri-hydroxy benzoic acid, the gallic acid (GA) was tested against the local toxicity of Daboia russelli (DR) venom and its purified hemorrhagic complex (HC). GA inhibited in vitro proteolytic activity of both DR venom and HC but, it did not inhibit phospholipase activity of DR venom. GA inhibited hemorrhage, edema forming, dermo- and myonecrotic activities of both HC and DR venom in in vivo experiments. GA was particularly effective against hemorrhagic activity but, GA inhibition had a greater effect on HC when compared to DR venom. The inhibition was likely due to GA induced structural changes in HC as revealed by alterations in fluorescence emission and CD spectral properties. However, the inhibition was not due to chelating property of GA as suggested by UV-visible spectral studies. Inhibition of collagen type IV, laminin and fibronectin degradation essentially provided the biochemical basis for GA which inhibited local effects of HC as well as DR venom. Thus, the study appears highly promising to explore GA and its generics against ruthless local effects and perhaps systemic hemorrhage of DR and other snake bites as well. Further, these agents will possibly find an immense value in the regulation of matrix metalloproteases (MMPs) in processes such as wound healing, inflammation and in the treatment of cancer.

Antivenom therapy is the current best therapy available for the treatment of fatal snake envenomation. However, the antivenom offers less or no protection against local effects such as extensive edema, hemorrhage, dermo-, myonecrosis and inflammation at the envenomed region. Viperidae snakes are highly known for their violent local effects and such effects have been commonly treated with plant extracts without any scientific validation in rural India. In this investigation Morus alba plant leaf extract has been studied against the Indian Vipera/Daboia russelii venom induced local and systemic effects. The extract completely abolished the in vitro proteolytic and hyaluronolytic activities of the venom. Edema, hemorrhage and myonecrotic activities were also neutralized efficiently. In addition, the extract partially inhibited the pro-coagulant activity and completely abolished the degradation of Aalpha chain of human fibrinogen. Thus, the extract processes potent antisnake venom property, especially against the local and systemic effects of Daboia russelii venom., (Copyright 2009 John Wiley & Sons, Ltd.)

Snakebites in rural areas of tropical and subtropical regions are commonly treated with medicinal plants. In this report, we have studied the ability of Anacardium occidentale bark extract to neutralize enzymatic as well as pharmacological effects induced by Vipera russelii venom. The extract neutralized the viper venom hydrolytic enzymes such as phospholipase, protease, and hyaluronidase in a dose dependent manner. These enzymes are responsible for both local effects of envenomation such as local tissue damage, inflammation and myonecrosis, and systemic effects including dysfunction of vital organs and alteration in the coagulation components. In addition, extract neutralized the pharmacological effects such as edema, hemorrhage, and myotoxic effects including lethality, induced by venom. Since, it inhibits both hydrolytic enzymes and pharmacological effects; it may be used as an alternative treatment to serum therapy and, in addition, as a rich source of potential inhibitors of hydrolytic enzymes involved in several physiopathological diseases.

Despite the long history [Kaiser, E., 1956. Enzymatic activity of spider venoms. In: Buckley, E.E., Porges, N. (Eds.), Venoms. American Association for the Advancement of Science, Washington, DC, pp. 91-93] on proteolytic activity, no study so far claims the isolation of a serine protease from the spider venom/venom gland extract. Therefore, the present study describes the isolation and characterization of a low molecular weight serine protease from Hippasa agelenoides venom gland extract. The protease (Hag-protease) was purified to homogeneity using the combination of gel-permeation and ion-exchange chromatography. The molecular mass was found to be 16.350 kDa by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Hag-protease was optimally active at pH 7.5 and temperature of 37 degrees C. PMSF abolished the enzyme activity while EDTA, EGTA, IAA, 1, 10-phenanthrolene did not. It hydrolyzed proteins such as casein, fibronectin and collagen type-I dose dependently but did not degrade gelatin and collagen type-IV. The isolated protease was non-lethal and devoid of hemorrhagic, myotoxic and edema forming activities. The light microscopy of Hag-protease treated skin tissue sections at the site of injection showed extensive damage of extracellular matrix (ECM) of hypodermis without causing any damage to blood vessels and capillaries. Similar damage of ECM of muscle tissue sections without affecting myocytes was noticed. Hag-protease was found to be procoagulant in property when studied plasma recalcification time.

Spider venom is a complex mixture of protein and peptide toxins. Hyaluronidase a 'spreading factor' has not been studied extensively in spider venom. In this paper, we describe the purification and characterization of a hyaluronidase from Hippasa partita venom gland extract. Hyaluronidase (HPHyal) has been purified by the successive chromatography on a Sephadex G-100 and on CM-Sephadex C-25 columns. HPHyal has been purified to an extent of about approximately 20-folds. The molecular mass was found to be 42.26 kDa by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. HPHyal was optimally active at pH 5.8 at 37 degrees C and in the presence of 300 mM NaCl in the reaction mixture. HPHyal showed absolute specificity for hyaluronan and belongs to neutral active group of enzymes. HPHyal revealed single-precipitin line, while venom gland extract revealed multiple bands in Western blotting with the antiserum prepared against venom gland extract. HPHyal indirectly potentiates the myotoxicity of VRV-PL-VIII myotoxin and also the hemorrhagic potency of hemorrhagic complex-I. Cations, Na(+) and K(+) enhanced the activity and chloride ions do not have any effect while, divalent cations, inhibited the enzyme activity.

In Indian traditional medicine, various plants have been used widely as a remedy for treating snake bites. The aim of this study was to evaluate the effect of Tamarindus indica seed extract on the pharmacological as well as the enzymatic effects induced by V. russelli venom. Tamarind seed extract inhibited the PLA(2), protease, hyaluronidase, l-amino acid oxidase and 5'-nucleotidase enzyme activities of venom in a dose-dependent manner. These are the major hydrolytic enzymes responsible for the early effects of envenomation, such as local tissue damage, inflammation and hypotension. Furthermore, the extract neutralized the degradation of the Bbeta chain of human fibrinogen and indirect hemolysis caused by venom. It was also observed that the extract exerted a moderate effect on the clotting time, prolonging it only to a small extent. Edema, hemorrhage and myotoxic effects including lethality, induced by venom were neutralized significantly when different doses of the extract were preincubated with venom before the assays. On the other hand, animals that received extract 10 min after the injection of venom were protected from venom induced toxicity. Since it inhibits hydrolytic enzymes and pharmacological effects, it may be used as an alternative treatment to serum therapy and, in addition, as a rich source of potential inhibitors of PLA(2), metalloproteinases, serine proteases, hyaluronidases and 5 cent-nucleotidases, the enzymes involved in several physiopathological human and animal diseases., (Copyright 2006 John Wiley & Sons, Ltd.)

We present the first analysis of the human proteome with regard to interactions between proteins. We also compare the human interactome with the available interaction datasets from yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans) and fly (Drosophila melanogaster). Of >70,000 binary interactions, only 42 were common to human, worm and fly, and only 16 were common to all four datasets. An additional 36 interactions were common to fly and worm but were not observed in humans, although a coimmunoprecipitation assay showed that 9 of the interactions do occur in humans. A re-examination of the connectivity of essential genes in yeast and humans indicated that the available data do not support the presumption that the number of interaction partners can accurately predict whether a gene is essential. Finally, we found that proteins encoded by genes mutated in inherited genetic disorders are likely to interact with proteins known to cause similar disorders, suggesting the existence of disease subnetworks. The human interaction map constructed from our analysis should facilitate an integrative systems biology approach to elucidating the cellular networks that contribute to health and disease states.