Topographic distribution of dopamine uptake,choline uptake,choline acetyltransferase,and GABA uptake in the striata of weaver mutant mice

The topographic distribution of dopamine (DA) uptake, choline uptake, choline acetyltransferase (ChAT) activity and GABA uptake within the striata of weaver mutant mice and control mice was determined. Uptake of [³H]dopamine, [³H]choline and [¹⁴C]GABA, as well as ChAT activity were determined in samples prepared from the dorsolateral, dorsomedial, ventrolateral and ventromedial portions of the striatum. In 45–60 day old control mice, dopamine uptake was homogeneously distributed throughout the striatum. On the other hand, striata from weaver mice exhibited an uneven distribution with the ventral aspects having greater uptake activity than the dorsal regions. Thus, although the ventral portion of the striatum is less severely affected than the dorsal portion, all areas of the striatum exhibited significantly reduced uptake rates. In 9 and 12 month old mice, choline uptake was higher in lateral than medial zones of the striatum of both genotypes and no differences were observed between genotypes. GABA uptake was higher in the ventral striatum than in the dorsal striatum but again no differences were found between weaver and control mice. The results of this study indicate that the entire weaver striatum is severely deficient in its ability to recapture dopamine and thus is functionally compromised. The results also indicate that the striatal cholinergic and GABAergic interneurons are not directly or indirectly affected by the weaver gene.Special ïssue dedicated to Dr. Morris H. Aprison     

Dolichol and N-linked oligosaccharide synthesis in the rat testis: interaction between Sertoli and spermatogenic cells, evidence for paracrine effects.

The relative contribution of the Sertoli cell and the pachytene spermatocyte to dolichol and N-linked oligosaccharide biosynthesis within the seminiferous tubule was investigated. Evidence is presented to show that the interaction between these two cell types affects dolichol and N-linked oligosaccharide biosynthesis. Analysis of the dolichol content of Sertoli cultures confirms earlier data suggesting that the Sertoli cell constitutes the major pool of dolichols within the seminiferous tubule. [14C]Acetate incorporation studies suggest that the Sertoli cell in culture synthesizes dolichol much more rapidly than does the isolated pachytene spermatocyte. This information, in addition to previous data in the literature, infers an interactive effect whereby the presence of the spermatogenic cell in the tubule stimulates dolichol synthesis in the Sertoli cell. The absence of normal Sertoli-spermatocyte interactions in in vitro incubations may also limit dolichol synthesis in the pachytene spermatocyte. The distribution of dolichol kinase between the Sertoli and the pachytene spermatocyte was also examined. The concentration of this enzyme in the Sertoli cell suggests the presence of an active salvage pathway within that cell. The correlation between the appearance of the pachytene spermatocyte and the previously described peak of dolichol kinase activity in the seminiferous tubules of the prepubertal animal implies cell-cell interactions. Radiolabelling studies of N-linked oligosaccharides were conducted using [3H]mannose and concanavalin A affinity chromatography to identify multiantennary, biantennary, and high-mannose oligosaccharide pools. An in vitro bicameral coculture system was used to demonstrate that pachytene spermatocytes stimulate incorporation of [3H]mannose into Sertoli cell oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of radical adduct reduction and reoxidation of the corresponding hydroxylamines in in vivo spin trapping of carbon tetrachloride-derived radicals.

In vivo spin trapping of radical metabolites has become a promising tool in understanding and predicting toxicities caused by different xenobiotics. However, in biological systems radical adducts can be reduced to electron paramagnetic resonance (EPR)-silent hydroxylamines. To overcome this difficulty, different procedures for reoxidation of the reduced radical adducts were systematically investigated and some metabolic inhibitors of nitroxide reduction were tested. As a test system, carbon tetrachloride (CCl4), a known hepatotoxic substance, was used. CCl4 is metabolized by liver to .CCl3 and, in the presence of the spin trap phenyl N-t-butylnitrone (PBN), forms the PBN/.CCl3 and PBN/.CO2- radical adducts. These radical adducts were measured in the bile using electron paramagnetic resonance after administration of CCl4 and PBN to the rat. We have shown that these radical adducts were reduced to the corresponding hydroxylamines in vivo, since immediately after the collection of bile only traces of the radical adducts could be detected, but after oxidation by different procedures such as bubbling with oxygen, addition of mild oxidant potassium ferricyanide or autoxidation the EPR spectra intensity increases, indicating that the hydroxylamines had been re-oxidized back to nitroxides. The collection of bile into plastic Eppendorf tubes containing the sulfhydryl reagent N-ethylmaleimide (NEM) or the enzyme ascorbate oxidase did not increase the intensity of the spectra significantly, demonstrating that neither reduction by reduced glutathione (GSH) nor ascorbic acid occurred ex vivo. However in the presence of NEM faster re-oxidation was observed. A new radical adduct that was not observed previously in any in vivo experiment and which exhibited 13C hyperfine coupling was detected when the rats were injected with 13CCl4. We have proven that this is the same adduct detected previously in vitro in microsomal incubations of CCl4, PBN, GSH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). As a general rule, we have shown that a variety of oxidation procedures should be tried to detect the different radical adducts which are otherwise not observable due to the in vivo reduction of radical adducts.     

Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis.

Previous work demonstrating the presence and differential distribution of Ca(2+)-binding proteins in the CNS has led to the proposal that cytosolic proteins, such as calbindin-D28k (CB), may play a pivotal role in neurons. We have used a retrovirus containing the full-length cDNA for CB to transfect the pituitary tumor cell line GH3, to generate CB-expressing GH3 cells and to investigate whether ionic channel activities as well as the concentration of intracellular free Ca2+ ([Ca2+]i) homeostasis could be altered by the presence of this Ca(2+)-binding protein. We show that CB-transfected GH3 cells exhibited lower Ca2+ entry through voltage-dependent Ca2+ channels and were better able to reduce [Ca2+]i transients evoked by voltage depolarizations than the wild-type parent cell line. These observations provide a mechanism by which CB may protect tissues against Ca(2+)-mediated excitotoxicity.     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes

Summary Hybrid (1-3,1-4)--glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)--glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)--glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)--glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)--glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.Abbreviations AMY mature form of Bacillus amyloliquefaciens (1-3,1-4)--glucanase; - MAC mature form of B. macerans (1-3,1-4)--glucanase - SUB mature form of B. subtilis (1-3,1-4)--glucanase - H(A16-M), H(A36-M), H(A78-M), H(A107-M), H(A152-M) mature forms of hybrid enzymes having 16, 36, 78, 107, 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC     

Interactions between freshwater snails and tadpoles: competition and facilitation

Summary Freshwater snails and anuran tadpoles have been suggested to have their highest population densities in ponds of intermediate size where abiotic disturbance (e.g. desiccation) is low and large predators absent. Both snails and tadpoles feed on periphytic algae and, thus, there should be a large potential for competitive interactions to occur between these two distantly related taxa. In a field experiment we examined the relative strength of competition between two closely related snail species, Lymnaea stagnalis and L. peregra, and between L. stagnalis and tadpoles of the common frog, Rana temporaria. Snail growth and egg production and tadpole size at and time to metamorphosis were determined. Effects on the common food source, periphyton, were monitored with the aid of artificial substrates. Periphyton dry weight was dramatically reduced in the presence of snails and/or tadpoles. There were no competitive effects on growth or egg production of the two snail species when they were coexisting. Mortality of L. peregra was high (95%) after reproduction, but independent of treatment. Growth of L. stagnalis was reduced only at the highest tadpole densities, whereas egg production was reduced both by intraspecific competition and by competition with tadpoles. Differences in egg production were retained after tadpole metamorphosis. Tadpole larval period increased, weight of metamorphosing frogs decreased and growth rate was reduced as a function of increasing tadpole density. However, contrary to expectation, snails had a positive effect on tadpole larval period, weight and growth rate. Further, in experimental containers without snails there was a dense growth of the filamentous green alga Cladophora sp. We suggest that the facilitative effects of snails on tadpoles are due to an indirect mutualistic mechanism, involving competition between food sources of different quality (microalgae and Cladophora sp.) and tadpoles being competitively dominant over snails for the preferred food source (microalgae). In the presence of tadpoles snails will be forced to feed on low-quality Cladophora, increasing nutrient turnover rates, which results in enhanced productivity of microalgae, increasing tadpole food resources. Thus, tadpoles have a negative effect on snails through resource depression, while snails facilitate tadpole growth through an indirect enhancement of food availability.     

Root Flooding of Muskmelon (Cucumis melo L.) Affects Fruit Sugar Concentration But Not Leaf Carbon Exchange Rate

Flooding the roots of greenhouse-grown muskmelon (Cucumis meloL. cv. Noy Yizreel) plants for 4 days reduced sucrose accumulation36% in the inner mesocarp and 88% in the outer mesocarp of developingfruit. Concentration of the translocated sugars raffinose andstachyose were also lower in fruit on flooded plants than inthose from nonflooded plants. In contrast, fruit hexose concentrationwas similar in both flooded and nonflooded plants. There wasno alteration in activities of enzymes associated with sucrosemetabolism in the fruit which could explain the decreased sucroseconcentration. Four days of root flooding caused no reductionin leaf carbon exchange rate or assimilate export rate, indicatingthat the reduction in fruit sucrose accumulation was not dueto source limitation. Root respiration, measured as CO₂ evolution,was approximately 30% lower in anaerobic roots than in aerobicroots. When viewed as carbohydrate consumed, a doubling of glycolyticactivity occurred in the anaerobic root mass. Increased demandfor carbohydrates by anaerobic roots may lead to a reductionin translocated carbohydrates available for sucrose biosynthesisin the developing fruit. (Received August 29, 1990; Accepted February 21, 1991)