Amino Acid transport into membrane vesicles isolated from zucchini : evidence of a proton-amino Acid symport in the plasmalemma

Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid transport was studied in membrane vesicles isolated from zucchini (Cucurbita pepo L. cv Black Beauty) hypocotyls. An imposed pH gradient (basic interior) was used to energize isolated membrane vesicles and drive amino acid transport. Proton-coupled amino acid accumulation was demonstrated for alanine, glutamate, glutamine, leucine, and tabtoxinine-β-lactam. Alanine transport into the isolated membrane vesicles was studied in detail. Alanine transport was protonophore sensitive and accumulation ratios exceeding 10 times that predicted by diffusion alone were observed. ΔpH-Dependent alanine transport exhibited saturation kinetics, suggesting translocation was mediated via a carrier transport system. In support of that conclusion, 50 micromolar N,N′-dicyclohexylcarbodiimide, a hydrophobic modifier of protein carboxyls, completely inhibited proton-coupled alanine accumulation. Transport activity, equilibrated on a linear sucrose gradient, peaked at 1.16 grams per cubic centimeter and co-migrated with a plasmalemma marker (vanadate-sensitive K⁺-Mg²⁺-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants.     

Reproductive physiology of the clouded leopard: II. A circannual analysis of adrenal-pituitary-testicular relationships during electroejaculation or after an adrenocorticotropin hormone challenge

A circannual analysis was made of serum cortisol, luteinizing hormone (LH), and testosterone concentrations in the male clouded leopard (Neofelis nebulosa). Group I males (n = 4), maintained in a standardized environment, were bled serially during a regimented anesthesia/electroejaculation episode occurring monthly (beginning in January, ending in December). Additional sampling intervals were conducted under anesthesia only (control, n = 8), anesthesia plus a single adrenocorticotropin hormone challenge (ACTH, Cortrosyn, n = 4), or anesthesia plus a single 25 micrograms injection of gonadotropin-releasing hormone (GnRH, Gonadorelin, n = 4). Group II males (n = 6) from various zoological collections were sampled serially under the same semen collection conditions on one random occasion within the year. Serum cortisol levels were 2 times greater than values measured in comparable studies involving other felid species. Cortisol concentrations were similar during electroejaculation and control (anesthesia only) episodes, and mean levels did not rise as a result of semen collection. Adrenocorticotropin caused an immediate rise in cortisol to levels at least 1.5 times greater than electroejaculated or control counterparts. Mean concentrations of basal cortisol in individual males gradually increased as the year progressed, possibly as a consequence of repeated psychogenic stress. Between seasons, there were no differences in mean LH; however, testosterone levels were greater (p less than 0.05) in the winter compared to all other seasons. There were no differences (p greater than 0.05) between individual males in secretory patterns or mean concentrations of cortisol, LH, or testosterone. Within males, distinct temporal fluctuations were observed in both LH and testosterone during the approximately 80-min sampling interval. Neither LH nor testosterone profiles appeared affected by cortisol patterns during electroejaculation or after an ACTH challenge. A bolus of GnRH induced a marked rise in serum LH and testosterone within 15 and 30 min respectively, indicating that these two hormones were coupled. Both LH and testosterone profiles in Group II males mimicked those in Group I; concentrations of cortisol in Group II males immobilized on one occasion were similar to those of Group I animals sampled from January-May but appeared to be less than values measured from June-December. These data demonstrate that the clouded leopard, compared to other felids, produces markedly elevated concentrations of cortisol, which are likely related to an aggressive behavioral temperament.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insect Feeding Deterrents in Endophyte-Infected Tall Fescue

The presence of an endophytic fungus, Acremonium coenophialum, in tall fescue (Festuca arundinacea) deterred aphid feeding by Rhopalosiphum padi and Schizaphis graminum. Both species of aphid were unable to survive when confined to endophyte-infected tall fescue plants. Feeding deterrents and toxic factors to R. padi and Oncopeltus fasciatus, large milkweed bug, were primarily associated with a methanol extract obtained when endophyte-infected tall fescue seed was serially extracted with hexane, ethyl acetate, and methanol. The concentrations of pyrrolizidine alkaloids were determined to be 30 to 100 times greater in the methanol extract than in the hexane and ethyl acetate extracts.     

Centriole as microtubule-organizing centers for marginal bands of molluscan erythrocytes

The erythrocytes of blood clams (arcidae) are flattened, elliptical, and nucleated. They contain elliptical marginal bands (MBs) of microtubules, each physically associated with a pair of centrioles marginal bands (MBs) of microtubles, each physically associated with a pair of centrioles (Cohen, W., and I. Nemhauser, 1980, J. Cell Biol., 86:286-291). The MBs were found to be cold labile in living cells, disappearing within 1-2 h at 0 degrees C. After the cells had been rewarmed for 1-2 h, continuous MBs with associated centrioles were once again present. Time-course studies utilizing phase contrast, antitubulin immunofluorescence, and electron microscopy of cytoskeletons prepared during rewarming revealed structural evidence of centriole participation in MB reassembly. At the earliest stage of reassembly, a continuous MB was not present. Instead, relatively short and straight microtubules focused on a pointed centriolar “pole,” and none were present elsewhere in the cytoskeleton. Thin continuous MBs then formed, still pointed in the centriolar region. Subsequently, the MBs regained ellipticity, with their thickness gradually increasing but not reaching that of controls even after several hours of rewarming. At these later time points, microtubules still radiated from the centrioles and joined the MBs some distance away. In the presence of 0.1 mM colchicines, MB reassembly was arrested at the pointed stage. Electron microscopic observations indicate that pericentriolar material is involved in microtubule nucleation in this system, rather than the centriolar triplets directly. The results suggest a model in which the centrioles and associated material nucleate assembly and growth of microtubules in diverging directions around the cell periphery. Microtubules of opposite polarity would then pass each other at the end of the cell distal to the centrioles, with continued elongation eventually closing the MB ellipse behind the centriole pair.     

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.     

Biochemical and genetic analysis of an antigenic determinant found on N-linked oligosaccharides in Dictyostelium.

Dictyostelium discoideum synthesizes many highly immunogenic carbohydrates of unknown structure and function. We have used monoclonal antibodies prepared against one of these called CA1 to investigate its structure and the consequences of its loss. CA1 is preferentially expressed on lysosomal enzymes as a specific arrangement of mannose-6-SO4 residues on N-linked oligosaccharides. Mutant strains HL241 and HL243 do not express CA1, and synthesize a truncated lipid-linked oligosaccharide (LLO) precursor that lacks the critical mannose residues needed for expression. The lesion appears to result from the loss of mannosyl transferase activity involved in LLO biosynthesis. The truncated LLO is poorly transferred to an artificial peptide acceptor in a cell-free N-glycosylation assay, and this appears to result from improper topological localization of the LLO or to a lower affinity of the LLO for the oligosaccharyl transferase. Although both mutants share these lesions, they are biochemically and genetically distinct. Only HL243 is lower in N-glycosylation in intact cells, and this is not a result of an altered structure of the LLO. There are other differences between the strains. HL241 can form fruiting bodies at a slower rate than normal while HL243 cannot aggregate. Genetic analysis of defects shows that the CA1 lesion in HL241 is recessive, while the lesion in both CA1 and in development are dominant and co-segregate in HL243 and are, therefore, likely to be in the same gene. Lysosomal enzyme targeting is normal but enzyme processing proceeds at a 2-3 fold slower rate in HL241 and HL243 compared to wild-type. Strain HL244 does not express CA1 since it completely lacks protein sulfation, but lysosomal enzyme targeting and processing proceeds at a normal rate, showing that sulfate is not essential for these processes. Alterations in oligosaccharide structure can have individualized effects on the biosynthesis of lysosomal enzymes. The results presented here illustrate how this approach can be used to study both the structure and function of carbohydrate epitopes.