Snake venom toxins. The amino acid sequence of three toxins (CM-2e, CM-4a and CM-) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-2e, CM-4a and CM-7) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex and by ion-exchange chromatography on CM-cellulose. They comprise 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of the three toxins have been elucidated. The toxicities, the serological properties, the sequences and the invariant amino acid residues of toxin CM-2e, CM-4a and CM-7 resemble the corresponding properties of the cytotoxin group.     

Acid Phosphatase in the Digestive Vacuoles and Lysosomes of Paramecium caudatum: A Timed Study1

Enzyme cytochemistry was used to determine when acid phosphatase (AcPase) becomes associated with the digestive vacuoles (DVs) of axenically grown Paramecium caudatum that were pulsed with latex beads for 2–3 min. When cells were incubated in the Gomori medium, AcPase was not observed in the discoidal vesicles, the acidosomes, and the newly released DVs up to 3 min old or in most DVs 3–6 min old. The number of AcPase-positive DVs increased to 56% when DVs were 12–18 min old. Similar results were obtained using the napthol AS-TR phosphate-hexaotized rosanilin method at the light microscopic level where hundreds of DVs were scored though the maximal level of positive DVs obtained by this method was lower. In addition to DVs of specific ages, AcPase was found in ER, in some Golgi vesicles, and small vesicles similar in diameter to Golgi vesicles which may represent primary lysosomes in this ciliate. Larger vesicles abundant near the DV-II were only partially filled with reaction product. These vesicles, which could be identified by their paracrystalline sheets and a prominent glycocalyx lining the luminal surface of their membranes, fit the definition for secondary lysosomes. These results, which indicate that lysosomes fuse with DVs only after they have attained a certain age, suggest the existence of specific recognition factors on the membranes of secondary lysosomes as well as DV-II.     

Ultrastructural Organization of Bovine Chromaffin Cell Cortex—Analysis by Cryofixation and Morphometry of Aspects Pertinent to Exocytosis

We have analyzed ultrathin sections from isolated bovine chromaffin cells grown on plastic support, after fast freezing, by quantitative electron microscopy. We determined the size and intracellular distribution of dense core vesicles (DVs or chromaffin granules) and of clear vesicles (CVs). The average diameter of DVs is 356 nm, and that of CVs varies between 35–195 nm (average 90 nm). DVs appear randomly packed inside cells. When the distance of the center of DVs to the cell membrane (CM) is analyzed, DV density is found to decrease as the CM is approached. According to Monte Carlo simulations performed on the basis of the measured size distribution of DVs, this decay can be assigned to a “wall effect.” Any cortical barrier, regardless of its function, seems to not impose a restriction to a random cortical DV packing pattern. The number of DVs closely approaching the CM (docked DVs) is estimated to be between 364 and 629 (average 496), i.e., 0.45 to 0.78 DVs/μm² CM. Deprivation of Ca²⁺, priming by increasing [Ca²⁺]_i, or depolarization by high [K⁺]_e for 10 s (the effect of which was controlled electrophysiologically and predicted to change the number of readily releasable granules [RRGs]) does not significantly change the number of peripheral DVs. The reason may be that (a) structural docking implies only in part functional docking (capability of immediate release), and (b) exocytosis is rapidly followed by endocytosis and replenishment of the pool of docked DVs. Whereas the potential contribution of DVs to CM area increase by immediate release can be estimated at 19–33%, that of CVs is expected to be in the range of 5.6–8.0%.     

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding.     

Processing of digestive vacuoles in Tetrahymena and the effects of dichloroisoproterenol

The digestive-lysosomal system in Tetrahymena has been extensively studied; however, the various vacuole stages and the existence of a required processing period prior to defecation have not been clearly defined. In this study the presence of such a required processing period and the rate of DV defecation in Tetrahymena thermophila were determined. Like the cycle in Paramecium, a digestive cycle in Tetrahymena consisted of two periods: the processing period was 45 min and the defecation period was approximately 2 h, making the complete cycle approximately 3 h. During the defecation period vacuole egestion followed the kinetics of a first-order rate reaction and had a rate constant of 0.0187/min and a t1/2 of 37 min (82 min into the cycle). Using the naphthol AS-TR phosphate-hexazotized rosanilin method to visualize acid phosphatase activity at the light microscopic level, DVs became positive beginning at 10 min. The number of positive DVs increased to a maximum of 13% when DVs were 20-min old and declined to 5-7% beyond 30 min. Although dichloroisoproterenol (DCI) has been reported by others to stimulate vacuole defecation, we found it inhibited the defecation rate. The extent of inhibition depended on the age of the DVs when exposed to DCI. Vacuole formation was completely blocked in cells preexposed to 40 microM DCI for only 10 min; however, upon further exposure, cells could recover from this inhibition. The time required for complete recovery increased with increasing DCI concentrations. If DCI was given to cells simultaneously with latex beads, it was found to exert a dose-dependent inhibitory effect on DV formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

Summary Although it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t_1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.Abbreviations BS-FITC bovine serum albumin-fluorescein isothiocyanate - CVC contractile vacuole complex - DV-I to DV-IV digestive vacuole stages 1 to 4 - HRP horseradish peroxidase - V-ATPase vacuolar proton pump     

Effects of beta-chlornaltrexamine on separation distress in chicks

Intraventricular B-chlornaltrexamine (2 micrograms) increased distress vocalizations (DVs) in chicks, and reduced the ability of intraventricular morphine (.1-.5 micrograms), to inhibit DVs. Object imprinting was not blocked by central CNA, but systemic naloxone (10 mg/kg) did attenuate imprinting to a green but not a red object.     

Immunohistochemical expression of p53 in animal tumors: a methodological study using four anti-human p53 antibodies

Mutations in the p53 tumor suppressor gene are the most common genetic alterations in human cancers. These mutations usually lead to strongly enhanced protein stabilization and allow detection by immunohistochemistry. Two monoclonal (DO-7 and PAb-240) and two polyclonal (Ab-7 and CM-1) antibodies were evaluated by standard immunoperoxidase method in domestic animal tumors, chiefly squamous cell carcinomas (SCC), and osteosarcomas as positive controls. Immunoreactivity was detected in SCC of cattle, sheep, horse and cat as well as in feline actinic keratosis, with PAb-240 and CM-1 antibodies. One polyclonal antibody (Ab-7) did not give positive result at all, whereas DO-7 monoclonal antibody did not react in dogs and cats. Immunodetection of p53 protein is thus possible in all domestic species tested, especially with CM-1 and PAb-240 antibodies, and p53 alterations seem to occur early in carcinogenesis of feline SCC as in comparable human lesions.     

Modulation of separation distress by alpha-MSH

The effects of centrally administered alpha-MSH on separation-induced distress vocalizations (DVs) and squatting were evaluated in domestic chicks for dose-response, time course, and interactions with peripheral naloxone and both peripheral and central morphine. Some of the tests were conducted in both the presence and absence of social stimuli (mirrors or a conspecific). Doses of 0.04 microgram of alpha-MSH or greater eliminated the usual suppression of DVs produced by mirrors or conspecifics. This effect lasted 10-15 minutes and was followed by inhibition of DVs, accompanied by a dose-dependent vigilant squatting posture, that lasted about one hour. These effects showed no development of tolerance to repeated alpha-MSH injections over a six-day period, and no apparent interaction with the effects of peripherally injected naloxone or either peripherally or centrally injected morphine. It is suggested that, in keeping with its role in defensive camouflage in amphibians, alpha-MSH in chicks may activate a central state akin to fear to adaptively modulate DVs and defensive hiding.     

Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses

Dengue virus (DV) is a mosquito-borne flavivirus that causes haemorrhagic fever in humans. DV primarily targets immature dendritic cells (DCs) after a bite by an infected mosquito vector. Here, we analysed the interactions between DV and human-monocyte-derived DCs at the level of virus entry. We show that the DC-specific ICAM3-grabbing non-integrin (DC-SIGN) molecule, a cell-surface, mannose-specific, C-type lectin, binds mosquito-cell-derived DVs and allows viral replication. Conclusive evidence for the involvement of DC-SIGN in DV infection was obtained by the inhibition of viral infection by anti-DC-SIGN antibodies and by the soluble tetrameric ectodomain of DC-SIGN. Our data show that DC-SIGN functions as a DV-binding lectin by interacting with the DV envelope glycoprotein. Mosquito-cell-derived DVs may have differential infectivity for DC-SIGN-expressing cells. We suggest that the differential use of DC-SIGN by viral envelope glycoproteins may account for the immunopathogenesis of DVs.     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L

Chromatography on DEAE cellulose equilibrated with Pipes buffer resolved three forms of chorismate mutase (CM) from tubers and leaves of Solanum tuberosum: CM-1A and CM-1B were activated by tryptophan and inhibited by phenylalanine and tyrosine; CM-2 was unaffected by these aromatic amino acids. When compared to freshly excised discs, 3 day old tuber discs demonstrated a 4.5-fold increase in CM-1 activity following wounding. By contrast, CM-2 activity levels were not affected by this treatment. In aged tuber discs the CM-1:CM-2 activity ratio was 9:1. However, in green leaves the CM-1:CM-2 activity ratio was 1:4 suggesting organ specific regulation for the expression of these isozymes. The CM-1 isozymes isolated from both tubers and leaves shared similar native molecular weight values of 55,000, K_m values of 40 to 56 micromolar, and inhibition by phenylalanine (110-145 micromolar concentrations required for 50% inhibition) and tyrosine (50-70 micromolar concentrations required for 50% inhibition). The resolution of CM-1 into two forms occurred only in the presence of Pipes buffer. When this buffer was replaced with Aces, Bes, imidazole or Tris, only a single peak of CM-1 activity was observed. In these buffers CM-2 eluted as a shoulder on the CM-1 peak. Analytical isoelectric focusing of the CM-1 fraction followed by assay of the gel yielded only one form of CM-1 with an isoelectric point of 5.0. Gel filtration studies with Pipes buffer yielded molecular weights of 60,000 for both CM-1A and CM-1B indicating these forms are not the result of aggregation. The two forms of CM-1 may be artifacts generated by Pipes buffer.     

Chorismate mutase isoenzymes from Sorghum bicolor: purification and properties

Two forms of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, have been detected in etiolated seedlings of Sorghum bicolor after DEAE-cellulose chromatography. CM-1 and CM-2 contained 44 and 56%, respectively, of the total activity measured after DEAE-cellulose chromatography. CM-1 was activated by tryptophan and inhibited by phenylalanine and tyrosine. In contrast, CM-2 was insensitive to all three aromatic amino acids. CM-1 and CM-2 were purified 1389- and 1018-fold, respectively, by anion exchange, hydrophobic, and dye matrix chromatography. The molecular weights estimated by gel filtration on Sephacryl S-200 were 56,000 for CM-1 and 48,000 for CM-2. Subunit molecular weights of the two forms were estimated by sodium dodecyl sulfate-gel electrophoresis at 36,000 and 51,000 for CM-1 and CM-2, respectively. Tryptophan was required for the stability of CM-1 at all stages of purification. Both isoenzymes were stable at 0 or -20 degrees C and had broad pH optima (6-10 for CM-1 and 7.5-9.5 for CM-2).     

Chorismate Mutase Isoenzymes from Selected Plants and Their Immunological Comparison with the Isoenzymes from Sorghum bicolor

The isoenzyme pattern of chorismate mutase (EC 5.4.99.5) was examined by diethylaminoethyl-cellulose chromatography in a wide variety of plants. All plants contained a regulated form of chorismate mutase (CM-1), and most contained an additional, unregulated form (CM-2). The regulatory properties of CM-1 differed significantly between plants. Antisera prepared against CM-1 and CM-2 from Sorghum bicolor were used to test immunological cross reaction of chorismate mutases from other plants. There was a high degree of similarity between chorismate mutase isoenzymes from Sorghum bicolor and Zea mays and some with Hordeum vulgare, but all other species studied were antigenically distinct from sorghum. No homology between the structure of CM-1 and CM-2 was detected within any species.     

Regulation of aromatic amino Acid biosynthesis in higher plants: I. Evidence for a regulatory form of chorismate mutase in etiolated mung bean seedlings

Etiolated mung bean seedlings were examined for chorismate mutase activity. Evidence for the occurrence of two forms of the enzyme (designated CM-1 and CM-2) was obtained by ammonium sulfate fractionation, anion exchange cellulose chromatography, and isoelectric focusing. The two forms showed distinctly different properties, as CM-1 was inhibited by phenylalanine and tyrosine and activated by tryptophan, but inhibition by phenylalanine and tyrosine was reversed by tryptophan. The other form, CM-2, was unaffected by any of the three aromatic amino acids. Isoelectric points of the two forms were CM-1, pH 4.6, and CM-2, pH 5.6. The molecular weights estimated by molecular sieving on Sephadex G-200 were CM-1, 50,000, and CM-2, 36,000.     

Differential expression of DAHP synthase and chorismate mutase isozymes during rhizogenesis of Brassica juncea cultured in vitro

The regulatory patterns of two of the enzymes of the shikimate pathway. 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase or DS. EC 4. 1. 2. 15) and chorismate motase (CM, EC 5. 4. 99. 5), were investigated using in vitro cultures of Brassica juncea at two stages, viz. undifferentiated, proliferating callus and the root-forming callus. Our studies revealed the presence of the two isozymes of DAHP synthase, DS-Mn and DS-Co. in undifferentiated callus. However, during the rhizogenesis of the callus DS-Mn was absent. Similarly, for chorismate mutase, whereas both the isozymes CM-1 and CM-2 were present in undifferentiated callus only CM-2 was detected at rhizogenesis. The possible involvement of these isozymes in callus growth and rhizogenesis is discussed.     

Phagosomal acidification in Paramecium: effects on lysosomal fusion

Phagosomes of paramecia and amoeba and endosomes of fibroblasts and other mammalian cells are acidified prior to lysosomal fusion. The question, whether the phagosomal acidification process in paramecia is required for phagosome-lysosome fusion, was studied using ionophores, weak bases, and cytochalasin B (CB) in combination with monoclonal antibodies, acid phosphatase (AcPase) cytochemistry, and lysosome morphometry. Digestive vacuoles (DVs) of known ages were treated and examined. In untreated cells, lysosome binding to the membrane of the acidified DV increased linearly with age and reached a maximum before lysosome-DV fusion. When the fusion of the acidosomes with the very young DVs was prevented by CB, causing a block in the normal vacuole-pH drop, lysosome binding to the DVs as well as the rate and extent of lysosome-DV fusion were all greatly reduced. These effects of CB were reversible. When present prior to acidification, three ionophores and two weak bases did not inhibit the acidosome-DV fusion but raised the phagosomal pH and reduced both the rates of DV acidification and of lysosome-DV fusion. However, when added after acidification but prior to lysosome-DV fusion, five of the six perturbants studied did not inhibit this fusion but prolonged the period when DVs remained AcPase positive. Lastly, lysosome-DV fusion rates were found to be related to acidification rates. We conclude that an inhibition in acidosome-DV fusion or a reduction in both the acidification rate and vacuolar-pH drop would inhibit lysosome-DV fusion.     

Purification and characterization of chorismate mutase isoenzymes from ruta graveolens l.

Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K₁ = 4 · 10^?6 M) and tyrosine (K₁ = 8. 10^?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The K_m-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens.     

Multivesicular bodies in developing tobacco seed and mung bean are functionally equivalent

Protein storage vacuoles (PSVs) are the primarily storage organelles in cotyledon cells for protein preservation in seeds. Storage proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus for subsequent delivery to PSVs via presumably Golgi-derived dense vesicles (DVs). However, recent studies demonstrated that storage proteins in early stage of developing cotyledon of mung beans reached the multivesicular bodies (MVBs) prior to the detection of DVs, indicating the possible involvement of MVBs in mediating transport of storage proteins during the early stage of seed development. Here, we further show that the MVBs in developing tobacco seeds are functionally and biochemically equivalent to those in developing mung beans. Thus, MVBs in developing tobacco seeds are structurally distinct from DVs, contain both vacuolar sorting receptors (VSRs) and storage proteins, and they are insensitive to treatments of wortmannin and brefeldin A (BFA).