Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Biosynthetic and genetic relationships of B-series fumonisins produced by Gibberella fujikuroi mating population A

Fumonisins are mycotoxins produced by the maize pathogen Gibberella fujikuroi mating population A and frequently contaminate maize. Wild-type G. fujikuroi produces four B-series fumonisins, FB1, FB2, FR3 and FB4. These toxins are identical in structure except for the number and positions of hydroxyls along their linear carbon backbone. To elucidate the genetic and biosynthetic relationships among these fumonisins, we conducted meiotic and biochemical analyses of G. fujikuroi mutants with altered fumonisin production that resulted from defective alleles at three loci, Fum1, Fum2 and Fum3. These mutants produced either no fumonisins, only FR2 and FB4, or only FR3 and FR4. Genetic analyses revealed the orientation of the Fum loci along linkage group 1 of the fungus. The mutants were grown together in pair-wise combinations to determine if their fumonisin production phenotypes could be complemented. When FR3- and FB2-producing mutants were grown together, complementation occurred. However, when a nonproducing mutant was grown with a FR2- or FB3-producing mutant, complementation did not occur or was incomplete. When purified FR2, FR3, or FB4 was fed to mutant cultures, FR4 was converted primarily to FR2, FR3 was converted to FB1 and FB2 was not converted. The results from these assays suggest a previously unrecognized branch in the fumonisin biosynthetic pathway.     

Fumonisin blunts nitric oxide-induced and nitroprusside-induced cardiomyocyte death.

The objective of this study was to determine whether nitric oxide (NO)-induced cell death in cardiomyocytes was operative through de novo synthesis of ceramide by determining whether the ceramide synthase inhibitor fumonisin blocked NO-mediated cell death. Neonatal mouse cardiomyocytes in culture were pretreated with fumonisin B1 (FB1). FB1 is a competitive inhibitor of sphinganine N-acyl transferase, also known as ceramide synthase (EC 2.3.1.24). Cell viability was assessed by the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on the ability of viable cells to reduce MTT. Treatment with the NO donor nitroso-glutathione (NO-GSH) for 24h produced a significant (p<0.05) concentration-dependent reduction in OD(570) or an increase in cell death. Sodium nitroprusside (SNP) treatment for 24h produced a significant (p<0.001) concentration-dependent reduction in OD(570) and an increase in cardiomyocyte cell death but the effects of SNP were greater than those of NO-GSH. FB1 significantly (p<0.05) reduced cell death induced by either SNP or NO-GSH. The SNP (0.1mM) increase in cell death of 36.9+/-2.8% was significantly (p<0.05) reduced to 24.7+/-1.8% by FB1 (10 microM). The effect of FB1 was not mediated through inhibition of the cell death effects of H(2)O(2), which is produced by SNP, as FB1 did not prevent H(2)O(2)-induced cell death. Confirmation of the ability of ceramide to produce cell death was demonstrated by the cell-permeable ceramide analogue, C(2)-ceramide (100 and 200 microM), which induced, respectively, 23.4+/-11.3 and 78.0+/-3.7% increases in cell death. The cell death effects of SNP and NO-GSH are likely independent of cGMP signal transduction pathways, which are activated by either SNP or NO-GSH, as there was no significant concentration-dependent change in cardiomyocyte viability after treatment with the cell-permeable analogue dibutyryl-GMP. These data show that FB1 blunts SNP- and NO-induced cardiomyocyte death and raise the novel possibility of preventing some of SNP- or NO-induced cardiomyocyte cell death by ceramide synthase inhibition.     

On the functional role of coupling factor B in the mitochondrial H+ -ATPase

Evidence for the presence of a functionally important vicinal dithiol in mitochondrial coupling factor B (FB) has been presented earlier (Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56). FB was completely inactivated by 38 micron of copper o-phenanthroline or 0.63 mM iodosobenzoate, and the kinetics were consistent with intramolecular disulfide formation as were polyacrylamide gel patterns which showed that FB which had been treated with copper o-phenanthroline had a different mobility from that of untreated FB. ATP-Pi exchange activity and ATP-induced binding of bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol (oxonol VI) to H+ -ATPase were also inhibited by the thiol oxidizing reagents, although oligomycin-sensitive ATPase activity was unaffected. F0 isolated from H+ -ATPase rebinds purified F1 with the restoration of ATP-induced oxonol-binding activity. Prior treatment of F0 (but not of F1) with copper o-phenanthroline abolished the oxonol-binding activity of reconstituted F0-F1. 115Cd binds tightly to H+ -ATPase and the bound protein can be recovered by gel electrophoresis in phosphate buffer in the presence of sodium dodecyl sulfate at a position corresponding to FB. Prior treatment of the H+ -ATPase with copper o-phenanthroline abolished 115Cd binding. The results indicate that the major effect of these inhibitors is on FB dithiol and leave little doubt that Cd2+ is indeed bound to a vicinal dithiol group.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific Vinca alkaloid-binding polypeptides identified in calf brain by photoaffinity labeling

A radioactive, photoactive Vinca alkaloid, N-(p-azido-[3,5-3H]-benzoyl)-N'-beta-aminoethylvindesine [( 3H]NABV) with pharmacological and biological activities similar to vinblastine was synthesized and used to identify specific Vinca alkaloid macromolecular interactions in calf brain homogenate by photoaffinity labeling. The most prominent photolabeled species were 54.3- and 21.5-kDa polypeptides. The Vinca alkaloid-binding specificity of these polypeptides was confirmed by competitive blocking of specific photolabeling by vinblastine but not by colchicine or daunorubicin. The 54.3- and 21.5-kDa polypeptides exhibited specific half-maximum saturable photolabeling at 2.1 and 0.95 X 10(-7) M [3H]NABV, respectively. Relative vinblastine and NABV association constants (Ka vinblastine/Ka NABV) for the 54.3- and 21.5-kDa polypeptides were estimated to be 0.86 and 1.4, respectively. The 54.3-kDa component was found in both high speed (100,000 X g; 1 h) pellet and supernatant fractions, whereas the 21.5-kDa component was located primarily in the high speed pellet. Photolabeling of both components was maximal after 12-min UV light exposure, linear up to 120 micrograms of homogenate protein and only slightly affected by the nitrene scavenger p-aminobenzoic acid. The 54.3-kDa polypeptides of [3H]NABV-photolabeled calf brain high speed supernatant and detergent-solubilized high speed pellet fractions were identified as tubulin subunits by immunoprecipitation with monoclonal antibodies to alpha- or beta-tubulin subunits. Although the identity and function of the 21.5-kDa polypeptide is not known, this polypeptide may have a role in membrane-related effects of the Vinca alkaloids. These results demonstrate that [3H]NABV is an attractive tool for identifying and characterizing specific high affinity vinblastine cellular polypeptide acceptors which may initiate or mediate known and unknown mechanisms of Vinca alkaloid action.     

Microbial reclamation of shellfish wastes for the production of chitinases

Shrimp and crab shell powder (SCSP), prepared by treating shellfish processing waste with boiling and crushing, was used as a substrate for isolating chitinolytic microorganisms. Three potential strains (E1, J1, and J1-1) were isolated and identified as Bacillus cereus, B. alvei, and B. sphaericus, respectively. Three extracellular chitinases (FB1, FB2, and FB3) were purified from the culture supernatants of Bacillus cereus E1, B. alvei J1, and B. sphaericus J1-1, respectively. The molecular weights of FB1, FB2, and FB3 were 71,000, 71,000, and 65,000, respectively, by SDS-PAGE. The pIs for FB1, FB2, and FB3 were 7.1, 7.2, and 7.4, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FB1 were pH 9, 50 degrees C, pH 7 to 10, and 70 degrees C; those of FB2 were pH 9, 60 degrees C, pH 5 to 9, and 70 degrees C; and those of FB3 were pH 7, 50 degrees C, pH 5 to 9, and 60 degrees C. The activities of all enzymes were strongly inhibited by Hg(2+) and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol.     

Genetic polymorphism of erythrocyte histone H1 in Japanese quail

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.     

Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.     

Dissection of defective antigen presentation by interferon-gamma-treated fibroblasts

The capacity of interferon-gamma (IFN-gamma)-treated HLA-DR expressing human dermal fibroblasts (FB) to function as antigen-presenting cells (APC) was examined. FB were cultured with 250 U/ml IFN-gamma for 4 days to induce HLA-DR expression. Peripheral blood monocytes (M phi), FB, or IFN-gamma-treated FB from the same donor were then cultured overnight with or without the recall antigen streptokinase streptodornase (SKSD), and their capacity to stimulate autologous T4 cell DNA synthesis was examined. SKSD-bearing M phi stimulated T4 cell proliferation, whereas antigen-bearing HLA-DR (+) FB did not. Even after fixation with paraformaldehyde to eliminate metabolic activity, SKSD-bearing M phi, but not FB, were able to function as APC. However, when HLA-DR (-) endothelial cell (EC) or autologous or HLA-D-mismatched M phi were added to the cultures, antigen-pulsed IFN-gamma-treated FB and M phi were comparably effective stimulators of autologous T4 cell DNA synthesis. Antigen recognition by the T4 cell was restricted by the class II major histocompatibility complex (MHC)-encoded gene products expressed by the IFN-gamma-treated FB and was unrelated to the class I or II MHC-encoded gene products expressed by the additional cell type. EC-promoted T4 cell DNA synthesis induced by antigen-bearing IFN-gamma-treated FB was inhibited by 60.3, a monoclonal antibody directed at an epitope common to LFA-1, CR3, and the p150,95 molecule. Inhibition caused by 60.3 was completely reversed by the addition of IL 2 to the cultures. Antigen presentation by IFN-gamma-treated FB was also enhanced somewhat by IL 1, IL 2, or monoclonal antibody directed at Tp44 (9.3). However, each of these additions alone promoted T cell proliferation less effectively than EC and resulted in responses that were smaller than those triggered by antigen-bearing M phi. The data suggest that IFN-gamma-treated FB take up and process antigen effectively, but lack an accessory cell property necessary for antigen-induced T4 cell IL 2 production and proliferation.     

Atmospheric deposition of nitrogen and sulphur compounds in the Czech Republic

Estimates of dry and wet deposition of nitrogen and sulphur compounds in the Czech Republic for the years 1994 and 1998 are presented. Deposition has been estimated from monitored and modeled concentrations in the atmosphere and in precipitation, where the most important acidifying compounds are sulphur dioxide, nitrogen oxides, ammonia, and their reaction products. Measured atmospheric concentrations of SO2, NOx, NH3, and aerosol particles (SO4(2-), NO3-, and NH4+), along with measured concentrations of SO4(2-), NO3-, and NH4+ in precipitation, weighted by precipitation amounts, were interpolated with Kriging technique on a 10- x 10-km grid covering the whole Czech Republic. Wet deposition was derived from concentration values for SO4(2-), NO3-, and NH4+ in precipitation and from precipitation amounts. Dry deposition was derived from concentrations of gaseous components and aerosol in the air, and from their deposition velocities. A multiple resistance model was used for calculation of SO2, NOx, and NH3 deposition velocities. Deposition velocities of particles were parameterized. It was estimated that the annual average deposition of SOx in the Czech Republic decreased from 1384 to 1027 mol H + ha(-1) a(-1) between 1994 and 1998. The annual average NOy deposition was estimated to be 972 and 919 mol H + ha(-1) a(-1) in 1994 and 1998, respectively. The annual average NHx deposition was estimated to be 887 mol H+ ha(-1) a(-1) and 779 mol H + ha(-1) a(-1) in 1994 and 1998, respectively. It was estimated that the annual average of the total potential acid deposition decreased from 3243 to 2725 mol H + ha(-1) a(-1) between 1994 and 1998. Sulphur compounds (SOx) contributed about 38%, oxidized nitrogen species (NOy) 34%, and reduced nitrogen species (NHx) 28% to the total potential acid deposition in 1998. The wet deposition contributed 42% to the total potential acid deposition in 1998.     

Changes in the composition of the hamster zona pellucida after fertilization in vivo but not in vitro

Hamster zonae pellucidae were obtained from follicular oocytes, superovulated eggs, and eggs fertilized in vivo or in vitro. Zonae were labelled with N-succinimidyl-3(4-hydroxy,5-[125I]iodophenyl)propionate, and compared on single- and two-dimensional SDS-PAGE. Single-dimensional electrophoresis showed considerable differences between zona categories in the amount of label that they incorporated; follicular zonae incorporated the least label and zonae from eggs fertilized in vivo the most. On two-dimensional electrophoresis, polypeptides from 3 of the 4 zona categories migrated into 4 major groups: two of these groups each with Mr 150,000-250,000 were within the Mr range of ZP1, and two others, at Mr 90,000 and 55,000, appeared to be analogous to ZP2 and ZP3, respectively. The fourth zona category (zonae from eggs fertilized in vivo) showed a changed polypeptide profile as well as incorporating the most label; one of the polypeptides, Mr 150,000-250,000, was undetectable, but a train of Mr 70,000-90,000 polypeptides and a discrete polypeptide at Mr 20,000 were new. Since this changed profile did not occur in zonae from superovulated eggs, or in zonae from eggs fertilized in vitro, a synergism between oviductal factors and factors from the spermatozoon or egg, or both, towards the zona in vivo is indicated.     

Effect of fumonisin B1 on inducible nitric oxide synthase and cyclooxygenase-2 in LPS-stimulated J774A.1 cells

Fumonisin B1 (FB1) is a water-soluble fungal metabolite that elicits a wide spectrum of toxicological effects. Cellular targets of FB1 include immune cells and in particular macrophages. In the present study the cytotoxic effect of FB1 (1-100 microM) was evaluated using a murine macrophage cell line (J774A.1) as model system. The effect of FB1 on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS, 10 and 100 ng/ml) was also investigated. Macrophages were pretreated with FB1 for 72 h and then stimulated with LPS for 24 h. The increase of LPS-induced production of these inflammatory mediators was observed at increasing concentrations of FB1 (0.1-10 microM) and was found to be concentration dependent. By western blot analysis we demonstrated that the observed increase of NO and PGE2 production by FB1 was related to an enhancement of iNOS and COX-2 expression.     

Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium

Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell sig-nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav-1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1(-/-) LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav.     

Effect of Aerobic Priming on the Response of Echinochloa crus-pavonis to Anaerobic Stress (Protein Synthesis and Phosphorylation)

Echinochloa species differ in their ability to germinate and grow in the absence of oxygen. Seeds of Echinochloa crus-pavonis (H.B.K.) Schult do not germinate under anoxia but remain viable for extended periods (at least 30 d) when incubated in an anaerobic environment. E. crus-pavonis can be induced to germinate and grow in an anaerobic environment if the seeds are first subjected to a short (1-18 h) exposure to aerobic conditions (aerobic priming). Changes in polypeptide patterns (constitutive and de novo synthesized) and protein phosphorylation induced by aerobic priming were investigated. In the absence of aerobic priming protein degradation was not evident under anaerobic conditions, although synthesis of a 20-kD polypeptide was induced. During aerobic priming, however, synthesis of 37- and 55-kD polypeptides was induced and persisted upon return of the seeds to anoxia. Furthermore, phosphorylation of two 18-kD polypeptides was observed only in those seeds that were labeled with 32PO4 during the aerobic priming period. Subsequent chasing in an anaerobic environment resulted in a decrease in phosphorylation of these polypeptides. Likewise, phosphorylation of the 18-kD polypeptides was not observed if the seeds were labeled in an anaerobic atmosphere. These results suggest that the regulated induction of the 20-, 37-, and 55- kD polypeptides may be important for anaerobic germination and growth of E. crus-pavonis and that the specific phosphorylation of the 18-kD polypeptides may be a factor in regulating this induction.     

The exoskeleton collagens in Caenorhabditis elegans are modified by prolyl 4-hydroxylases with unique combinations of subunits

The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly.     

Two polypeptides from Dendroaspis angusticeps venom selectively inhibit the binding of central muscarinic cholinergic receptor ligands.

Two new polypeptides were isolated and purified from the venom of the snake Dendroaspis angusticeps, which also contains other neuroactive peptides such as Dendrotoxins and Fasciculins. The amino acid composition of the peptides was determined and the first 10 amino acids from the MTX2 N-terminal fragment were sequenced. The so-called muscarinic toxins (MTX1 and MTX2) have been shown to inhibit the specific binding of [3H]QNB (0.15 nM), [3H]PZ (2.5 nM) and [3H]oxoM (2 nM) to bovine cerebral cortex membranes by 60, 88 and 82% respectively. In contrast, they caused only a 30% blockade of the [3H]QNB specific binding to similar membrane preparations from the brainstem. The Hill number for the [3H]PZ binding inhibition by the putative muscarinic toxin MTX2 was 0.95 suggesting homogeneity in the behaviour of the sites involved. The data from [3H]oxoM binding gave a Hill number of 0.83. The decreases in the specific binding involved increases in KD for the three different ligands (8-fold for [3H]QNB, 4-fold for [3H]PZ and 3.5-fold for [3H]oxoM) without significant changes in Bmax, except for a slight decrease in the [3H]oxoM binding sites (-19%); such results suggest that there may be a competitive inhibition between the MTXs and these ligands. The Ki for MTX2/[3H]PZ was 22.58 +/- 3.52 nM; for MTX2/[3H]oxoM, 144.9 +/- 21.07 nM and for MTX2/[3H]QNB, 134.98 +/- 18.35 nM. The labelling of MTX2 with 125I allowed direct demonstration of specific and saturable binding to bovine cerebral cortex synaptosomal membranes. In conclusion, the results reported in this study strongly support the hypotheses that the two polypeptides isolated from D. angusticeps venom selectively inhibit specific ligand binding to central muscarinic receptors, in a competitive manner at least for the antagonist [3H]PZ and that the MTX2 specifically binds to a central site that is suggested to be a muscarinic receptor of the M1 subtype.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.