Heterogeneity of ovine prolactin after labeling with iodine 125: value of labeling with lactoperoxidase]

The authors have shown an heterogeneity of the ovine prolactine molecule after labelling with iodine 125. As well with chloramine T as with lactoperoxydase, it appears three molecular species which react with the immune serum antiprolactine (I.S.). The first species is of high molecular weight and is probably constituted of aggregates. Their combination with the I.S. is non specific and give blanks of high value. The second species is a dimere and the third one is the monomere. The two last species react with the I.S. and can give competition curves when they are choosen as tracer. However, if one uses as tracer a product obtained by labelling with chloramine T, the competition appears for high concentrations of native hormone. As if the I.S. recognizes much more the labelled protein than the native one. But if one uses the same species but labelled with lactoperoxydase, the competition appears for concentrations lower than five nanogrammes. In the same time one can see that the specificity toward different I.S. is modified. The authors think that the labelling with lactoperoxydase better preserves the tertiary structure of the native protein than do the labelling with chloramine T.     

Differential distribution of cytokeratins after microinjection of anti-cytokeratin monoclonal antibodies

In order to investigate the relationship of different cytokeratins within one cell, monoclonal antibodies directed against three trophectoderm cytokeratins TROMA 1, 2 and 3 were microinjected into mouse teratocarcinoma-derived trophoblastoma cells and indirect immunofluorescence tests were used to follow the subsequent localization of their respective antigens Endo A, B and C. Microinjection of TROMA 1 or 2 resulted in the perinuclear collapse of Endo A, B and C-containing filaments. Microinjection of TROMA 3 resulted in the perinuclear collapse of filaments containing Endo A and B, whereas Endo C condensed into cytoplasmic aggregates which appear as speckles in the fluorescence microscope. The speckles were electron microscopically located using indirect gold-labeling techniques and had a dense, granulous structure. They were often found to be associated with microtubules, although colchicine treatment before microinjection did not interfere with speckle formation. These experiments demonstrate that cytokeratins can become differentially distributed within the cytoplasm after microinjection of an anti-cytokeratin monoclonal antibody. Since Endo A is a type II cytokeratin and Endo B and C are type I cytokeratins, these results suggest that different members of one cytokeratin subfamily may be associated with cytokeratin filaments which have different functions within the same cell.     

Regulation of sucrose-sucrose-fructosyltransferase in barley leaves

The activity of sucrose-sucrose-fructosyltransferase (SST), a vacuolar enzyme strongly induced by light in excised leaves of barley (Hordeum vulgare L.), rapidly declined even in continuous light upon feeding of cycloheximide (CHI). The rate of decline was similar to that observed in light-treated leaves that were placed into darkness, in the presence or absence of CHI. The protease inhibitor leupeptin totally stopped the decline in SST activity in the dark and caused a substantial increase in the rate of induction of SST activity by light. Feeding of sucrose prevented or even reversed the SST activity decay induced by darkness in the absence of CHI but did not stabilize SST activity in the presence of CHI. The results suggest that SST is continuously subjected to rapid, constant proteolytic degradation in the vacuole, and that the enhancement of SST activity in the light or upon feeding sucrose in the dark is due exclusively to de novo protein synthesis.     

Oviposition deterring pheromone in Rhagoletis cerasi: Behavioral laboratory test to measure pheromone activity

Current investigations concerning the identification of the chemical nature of the oviposition deterring pheromone of the European cherry fruit fly, Rhagoletis cerasi L., are conducted in an interdisciplinary research program by combining chemistry with behavioral evaluation. The methods used to collect and evaluate the pheromone by an improved semi-quantitative behavior test are described.

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Zusammenfassung Nach einer einleitenden Übersicht über den heutigen Stand der Erkenntnisse auf dem Gebiet der eiablage-hemmenden Pheromone (ODP) bei Fruchtfliegen und insbesondere der Kirschenfliege wird der verbesserte Verhaltenstest beschrieben, welcher für den halb-quantitativen Nachweis von ODP-Aktivität entwickelt worden ist. Im laufenden interdisziplinären Forschungsprogramm an der Eidg. Forschungsanstalt Wädenswil wird versucht, durch enge Zusammenarbeit zwischen Biologe, Chemiker und Elektrophysiologe das ODP der Kirschenfliege möglichst mit geringem Anteil an Verunreinigung einzusammeln, zu reinigen und einer chemischen Identifikation der ODP Komponenten zugänglich zu machen.Die Möglichkeiten und Grenzen des Verhaltenstests werden diskutiert und die Empfindlichkeit des Verfahrens mittels einer Konzentrationsreihe von Pheromonextrakt aufgezeigt.

     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3-Glucanase in Pea Pods by Pathogens and Elicitors

Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non-pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and β-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and β-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and β-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and β-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and β-1,3-glucanase.     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Localization of polyphosphate in vacuoles of Saccharomyces cerevisiae

Virtually all of the polyphosphate (PP) present in yeast protoplasts can be recovered in a crude particulate fraction if polybase-induced lysis is used for disrupting the protoplasts. This fraction contains most of the vacuoles, mitochondria and nuclei. Upon the purification of vacuoles the PP is enriched to the same extent as are the vacuolar markers. The amount of PP per vacuole is comparable to the amount of PP per protoplast.The possibility that PP is located in the cell wall is also considered. In the course of the incubation necessary for preparing protoplasts, 20% of the cellular PP is broken down. As this loss of PP occurs to the same extent in the absence of cell wall degrading enzymes, it is inferred that internal PP is metabolically degraded, no PP being located in the cell walls.It is concluded that in Saccharomyces cerevisiae most if not all of the PP is located in the vacuoles, at least under the growth conditions used.Non-Standard Abbreviations PIPES piperazine-N,N-bis-2-ethanolsulfonic acid - DEAE-dextran diethylaminoethyl-dextran     

Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid

A simple and sensitive chemical assay was developed for 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. The assay is based on the liberation of ethylene from ACC at pH 11.5 in the presence of pyridoxal phosphate, MnCl₂ and H₂O₂. This assay was used to detect ACC in extracts of tomato fruits (Lycopersicon esculentum Mill.) and to measure the activity of a soluble enzyme from tomato fruit that converted S-adenosylmethionine (SAM) to ACC. The enzyme had a K_m of 13 M for SAM, and conversion of SAM to ACC was competitively and reversibly inhibited by aminoethoxyvinylglycine (AVG), an analog of rhizobitoxine. The K_i value for AVG was 0.2 M. The level of the ACC-forming enzyme activity was positively correlated with the content of ACC and the rate of ethylene formation in wild-type tomatoes of different developmental stages. Mature fruits of the rin (non-ripening) mutant of tomato, which only produce low levels of ethylene, contained much lower levels of ACC and of the ACC-forming enzyme activity than wild-type tomato fruits of comparable age.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid - SAM S-adenosyl-L-methionine Michigan Agricultural Experiment Station No. 8876