蛇毒的毒性成分及其应用研究

蛇毒是从毒蛇的毒腺中分泌出来的一种毒液,属于生物毒素。一般蛇毒的新鲜毒液呈蛋清样粘稠液体,振摇时易起泡沫,呈弱酸性,有特殊腥味,含水量约为65％～80％,比重为1．03～1．06,常温下易失活,置冰箱中1周后有部分失去活力。蛇毒中的蛋白类物质是蛇毒主要毒性成分,包括蛇毒     

Subcellular fractionation of tissue culture cells

Subcellular fractionation has two major steps, (1) the homogenization of the cells and (2) the subsequent separation of the organelles. The homogenization step is discussed with reference to the problems encountered using tissue culture cells. Promising techniques for the isolation of specific compartments are illustrated using the isolation of the endosomal compartment as the example.     

The complex toxic components of the Russell's viper venom

Russell's viper venom has been fractionated on CM-cellulose, CM-Sephadex and Sephadex gel filtration and disc electrophoresis to obtain 4 toxins one of which is a glycoprotein with 2 subunits, one with 4 cationic subunits one of the subunits being dialysable, one with 3 cationic subunits and a dialysable low molecular weight minor toxin. The specific subunits of individual toxins were found to be necessary for their biological activities.     

Isolation and characterization of a protein C activator from the moccasin snake venom

The protein C activator detectable in the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) was isolated by a combination of chromatofocusing on PBE-94 in the range pH9-7 and gel-filtration on Sephadex G-100 column. The peak protein C activator from Sephadex G-100 column appeared as double diffuse bands with apparent molecular weight of 37,700 and 31,400 after electrophoresis in the presence of sodium dodecylsulfate and 2-mercaptoethanol. The isolated enzyme does not clot human fibrinogen and when mixed with normal plasma generates activity of Protein C. It can be used for the measurement of protein C functional activity.     

Lysophosphatidylserine-dependent interaction between rat leukocytes and mast cells

The production of lysophosphatidylserine has been studied in a population of rat peritoneal cells; 67% polymorphonuclear and 33% mononuclear leukocytes. Pulse-chase experiments with L-[U-14C]serine reveal a net lysophosphatidylserine production of 0.33 nmol/mg protein in 2 h of incubation. The source of lysophosphatidylserine is probably the phosphatidylserine of cells damaged during the incubation, since plasma membrane fragments obtained from the leukocytes yield higher lysophosphatidylserine production (1.9 nmol/mg protein in 1 h of incubation). Both leukocytes and plasma membranes show phosphatidylserine splitting activity when tested with vesicles of this phospholipid. In the presence of albumin a fraction of produced lysophosphatidylserine is recovered in the incubation medium. Under these conditions efficient incorporation of lysoderivative into surrounding leukocytes and conversion to phosphatidylserine requires cell activation by tetradecanoylphorbol acetate. In agreement with radiochemical data it is found that a suspension of leukocytes elicits histamine release when rat peritoneal mast cells and nerve growth factor are subsequently added. This typical, lysophosphatidylserine-dependent mast cell response is retained when leukocyte plasma membranes substitute the whole cells. These results suggest that leukocyte lysis at sites of tissue injury results in the production of a sufficient amount of lysophosphatidylserine to reach and activate surrounding mast cells.     

Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.     

Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells

The beige mouse, a homologue of the Chediak-Higashi syndrome in man, possesses abnormally large granules in many tissue cells. The granules in the mucosal mast cells (MMC) of the small intestine of beige and littermate C57BL/6J mice were examined after infecting the mice with the intestinal parasite, Nippostrongylus brasiliensis. MMC in both beige and littermate mice had irregular granules which contained paracrystalline substructures embedded in an amorphous matrix. Granules were not observed in fusion with the cell membrane. Instead, in late-stage mast cells, the granule membrane broke down, the granule contents were spread throughout the cytoplasm, and the cell organelles disintegrated. Unlike connective tissue mast cells, MMC were poorly demonstrated with formalin fixation and toluidine blue staining.     

Identification of toxic components from the venom of Cerastes cerastes and Vipera lebetina vipers; purification of phospholipases

Cerastes cerastes and Vipera lebetina venoms have been fractionated and the different components analysed by electrophoresis on polyacrylamid gels. Phospholipases A2 contained in these two venoms have been purified and their electrophoretic properties compared.     

Regulatory effects of mast cells on lymphoid cells: the role of histamine type 1 receptors in the interaction between mast cells, helper T cells and natural suppressor cells

We have investigated the role of mast cells as modulators of lymphocyte function because the mast cells are concentrated in the areas of lymphoid storage; they are dependent upon T-cell growth factor for their proliferation; and they appear to be the principle if not sole storage site for histamine. We have tested the influence of mast cells on the proliferation of alloreactive cloned helper T cells, mixed leukocyte reactions, and the suppressive capacity of natural suppressor cells. We used an IL-3-dependent mast cell line that at high numbers (greater than 10(5)) suppressed and at low numbers (10(3) to 6 X 10(4)) augmented the proliferation of TH cells. Addition of histamine to cocultures enhanced the mast cell mediated proliferation of TH cells without directly affecting the helper cells. The action of histamine appeared to be mediated with H1 type receptors on these mast cells. Pretreatment of natural suppressor cells with supernatants from mast cell enhanced their suppressive capability. Here too, histamines enhanced suppression by the NS cell via histamine type 1 receptors on the natural suppressor cells. Our data suggest that mast cells may be a major modulator of the lymphoid cell immune function and demonstrate a role of histamine type 1 receptors in the interaction between mast cells, helper T cells, and natural suppressor cells.     

Histamine content of peritoneal and tissue mast cells of growing rats

Summary p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.Supported by NIH Grants DE 04730, DE 02668 and DE 00288 from the National Institute for Dental Research, NIH Grant RR 0533 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation