Putative candidate genes for blood pressure control in the SHR.BN-RNO8 congenic substrains

The SHR-Lx congenic strain carrying a differential segment of chromosome 8 of BN and PD origin was recently shown to exhibit a significant decrease in blood pressure as compared to the SHR strain. There were two positional candidate genes for blood pressure control mapped to the differential segment: the rat kidney epithelial potassium channel gene (Kcnj1) and brain dopamine receptor 2 gene (Drd2). Bot these genes were separated into SHR.BN-RNO8 congenic substrains. In this communication, we are presenting the assignment of two further putative candidate genes, which might be involved in blood pressure control to the BN/PD differential segment of the SHR-Lx congenic strain. These are: the gene coding for smooth muscle cell specific protein 22 (Sm22) defined by the D8Mcw1 marker and neuronal nicotinic acetylcholine receptor gene cluster, defined by the D8Bord1 marker. Moreover, the glutamate receptor gene Grik4 which also maps to the differential segment of the SHR-Lx should be taken into account. The genetic separation of all these putative candidate genes of blood pressure control is being performed by recombinations and subsequent selection using (SHR×SHR-Lx) intercross population.     

Subcellular localization of the 84,000 dalton heat-shock protein in mouse neuroblastoma cells: evidence for a cytoplasmic and nuclear location

Using affinity-purified antibodies, the 84,000 dalton heat-shock protein (hsp) has been localized in mouse N2A neuroblastoma cells by immunocytochemical techniques. Immunofluorescence microscopy showed that hsp84 was present both in the cytoplasm and in the nucleus. The nucleoli were found to be unlabelled. Immunogold labelling on ultrathin cryosections revealed that hsp84 was evenly distributed throughout the entire cytoplasm. No preferential association of hsp84 with the plasma membrane or with membranes from organelles was observed. In the nucleus the hsp84 was present in both the euchromatin and heterochromatin. In the nucleolus only the fibrillar part was labelled and virtually no gold particles were observed in the granular part. A long-term hyperthermic treatment of 3 h at 42.5 degrees C was found to induce an accumulation of hsp84 inside the nucleus. No alterations in hsp84 distribution were observed during a treatment of the cells with 75 microM sodium arsenite for 3 h. Drastic alterations were observed in the nucleoli after both stress treatments. The granular part had totally disappeared and only remnants of the fibrillar part which contained hsp84, were found. Besides the nuclear accumulations of hsp84 during heat shock, no additional changes in the hsp84 location in stressed cells were observed. During a recovery from the heat shock by replacing the cells at 37 degrees C, a decrease in the nuclear location of hsp84 was observed, indicating the reversibility of this process. The significance of these results for the role of hsp84 in normal and in stressed cells is discussed.     

Physiological activity of immobilized cells of Claviceps fusiformis during long-term semicontinuous cultivation

Summary The 550-day semicontinuous cultivation of Claviceps fusiformis immobilized in calcium alginate is documented. The vegetative mycelium from seed or from early-production submerged culture is the best choice for immobilization. No extracellular glucans are produced by immobilized cells. Immobilized spores give low yields of clavine alkaloids. Alginate concentrations in a range of 2%–4% do not influence yield and spectrum of alkaloids. The cytoplasm of the immobilized cells becomes condensed (after 3 days), polysaccharides disappear, and centres of lipid synthesis are formed in the cytoplasm. After 60 days the cells harbour a great number of lipid particles, mitochondria are diminishing and their cristae partly disappear, indicating a decreased respiration capacity. After 350–500 days the volume of most cells is increased many times and the cells are filled with large oval bodies of electrondense material. Chloramphenicol protects immobilized cultures against bacterial contamination.     

Chydorus arcticus n.sp., a new cladoceran crustacean (Chydoridae: Chydorinae) from the North Atlantic arctic and Subarctic areas

Chydorus arcticus n.sp. (Cladocera: Chydoridae: Chydorinae) is described, figured, and differentiated from the closely relatedC. sphaericus (O.F. Müller, 1785). The known distribution of the species is given, and some aspects of speciation of arctic crustaceans are pointed out.     

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Summary Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both elymoclavine-inhibitionless and clavine alkaloid-decompositionless fermentations if a combination of fermentation and separation units in a closed loop was used.Nomenlacture k ₁ rate constant of agroclavine synthesis (mg Agro · mg Elymo/l·g DW·day for stage 1, mg Agro/g DW·day for stage 2) - k ₂ parameter describing inhibition of agroclavine formation rate by elymoclavine (mg Elymo/l) - k ₃ specific rate of agroclavine decay (l/g DW·day) - k ₄ maximal specific rate of elymoclavine synthesis (stage 1, 1/g DW·day, stage 2, mg Elymo/g DW·day) - k ₄ ^– maximal specific rate of elymoclavine synthesis in stage 1 (inhibition-activation mechanism) (mg Elymo/g DW·day) - k ₅ physiological constant describing the elymoclavine decay rate (l²/g DW·day·mg Elymo) - k ₅ ^– physiological constant describing the activation of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₆ physiological constant describing the repression of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₇ maximal specific growth rate (1/day) - k ₈ specific rate of biomass decay (l/g DW·day) - A agroclavine concentration (mg/l) - E elymoclavine concentration (mg/l) - r _A specific rate of agroclavine biosynthesis (mg Agro/g DW·day) - r _E specific rate of elymoclavine biosynthesis (mg Elymo/g DW·day) - r _i specific rate of alkaloid biosynthesis (mg alkaloid/g DW·day) - X dry biomass concentration (g/l) - specific growth rate (1/day) Abbreviations Agro agroclavine - Elymo elymoclavine - Chano chanoclavine - DW dry weight of biomass     

Saprophytic production of Clavine alkaloids and activity of Hydroxymethylglutaryl-CoA reductase

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.     

Morphological and biochemical evidence for partial nuclear localization of annexin 1 in endothelial cells.

Using immunofluorescence, an affinity-purified anti-annexin-1 polyclonal antibody showed both cytoplasmic and nuclear staining, whereas antibodies against annexins 2, 5 and 6 labelled almost exclusively the cytoplasm of cultured endothelial cells. This was further confirmed by immunogold labelling and electron microscopy using a monoclonal antibody, annexin 1 being detected close to the plasma membrane, in the cytoplasm, as well as inside the nucleus. Finally, using immunoblotting, purified nuclei were shown to contain annexin 1, which was not removed by EDTA treatment. These data open some new perspectives in the understanding of annexin function, including possible involvement in nucleoskeleton dynamics and regulation of proliferation through cell signalling.     

Metabolism of high-density lipoproteins in rainbow trout.

Trout high-density lipoproteins have been labelled with residualizing tracers for the lipid and protein moieties ([3H]cholesteryloleyl ether and 125I-tyramine-cellobiose, respectively). Plasma kinetics and tissue site of catabolism were determined for both tracers. The lipid tracer was cleared about twice as fast from the blood as the protein tracer (half lifes were 63.5 and 125.3 h, respectively). This selective removal of lipid from the lipoprotein was mainly accomplished by the higher liver uptake of the cholesteryl ether. The main catabolic site for HDL protein was kidney tissue. This data established the existence of differential HDL catabolism in a lower vertebrate, in which HDL is the dominant plasma lipoprotein. In addition, the findings confirm the importance of fish kidney as a major site of endocytosis of macromolecules, of both exogenous and endogenous origin.     

The effect of manganese-induced hypercholesterolemia on learning in rats

Since the exact mechanism of manganese (Mn)-induced learning disability is not known, we investigated the role of elevated cholesterol in rats exposed daily to 357 and 714 μg Mn/kg for 30 d. Significant Mn accumulation was accompanied by increased cholesterol content in the hippocampal region of Mn-treated rats. The learning, which is based on the time needed to reach food placed at the exit of a T-maze after a 1-d training period, was significantly slower in exposed rats than in unexposed rats. The rats receiving 357 and 714 μg Mn/kg reached the food in 104.5±13.8 and 113.3±25.7 s, respectively, on d 30, whereas their untreated counterparts reached the food in 28.7±11.4 s. This delay was completely corrected to 29.3±7.8 and 30.7±6.0 s in rats with coadministration of an inhibitor of cholesterol biosynthesis with 357 and 714 μg/kg of Mn. The correction of impaired learning was associated with the normalization of hippocampal cholesterol, but the Mn level in this region of the brain was not influenced in rats treated with a drug that inhibits cholesterol biosynthesis. These results suggested that Mn-induced hypercholesterolemia is involved in Mn-dependent learning disability.