α-MSH Stimulates Glucose Uptake in Mouse Muscle and Phosphorylates Rab-GTPase-Activating Protein TBC1D1 Independently of AMPK

The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate α-MSH stimulation in both wild type and AMPK deficient mice. We found that α-MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that α-MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation.     

The deviation from linkage equilibrium with multiple loci varying in a stepping-stone cline

The balance between the creation of associations between alleles at different loci by immigration and the convergence to linkage equilibrium due to the recombination process is studied in a theoretical model. The geographical structure of the model is a stepping-stone chain of populations linking two genetically constant source populations. The model assumes an arbitrary number of autosomal loci and considers genetic variation (two alleles at each locus) that is not subject to natural selection. The gene frequencies at each locus will then show a linear cline through the stepping-stone chain of populations. The deviation from linkage equilibrium through the stepping-stone cline is characterized by an equation for linear measures that provide the linkage disequilibrium measures for a given set of loci in terms of the gene frequencies and the linkage disequilibria in the source populations and in terms of the linkage disequilibrium measures through the cline for lower numbers of loci. Numerical examples of this iterative solution are given, and it is shown that the build-up of the higher order Bennett-disequilibria through the cline is considerably more pronounced than the build-up of two-locus disequilibria.     

Kinetics of Butyrate, Acetate, and Hydrogen Metabolism in a Thermophilic, Anaerobic, Butyrate-Degrading Triculture

Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, K_m, for butyrate, acetate, and dissolved hydrogen were 76 μM, 0.4 mM, and 8.5 μM, respectively. Butyrate and hydrogen were metabolized to a concentration of less than 1 μM, whereas acetate uptake usually ceased at a concentration of 25 to 75 μM, indicating a threshold level for acetate uptake. No significant differences in K_m values for butyrate degradation were found between chemostat- and batch-grown tricultures, although the maximum growth rate was somewhat higher in the batch cultures in which the medium was supplemented with yeast extract. Acetate utilization was found to be the rate-limiting reaction for complete degradation of butyrate to methane and carbon dioxide in continuous culture. Increasing the dilution rate resulted in a gradual accumulation of acetate. The results explain the low concentrations of butyrate and hydrogen normally found during anaerobic digestion and the observation that acetate is the first volatile fatty acid to accumulate upon a decrease in retention time or increase in organic loading of a digestor.     

Ontogeny of hormone-secreting cells of the rat pituitary gland: An immunocytochemical study on dissociated cells

Summary An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18–21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80–95% of the cells were immunoreactive.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

The structures of oligosaccharides excreted by sheep with swainsonine toxicosis

Eleven oligosaccharides were purified form the urine of sheep with swainsonine toxicosis induced by the feeding of Astragalus lentiginosus. Oligosaccharides were extracted by charcoal adsorption, chromatographed on Bio-Gel P-2, and partially fractionated by preparative-layer chromatography. Separation into individual compounds was completed by semi-preparative high pressure liquid chromatography. Structures were determined by a combination of high pressure liquid chromatography and exo- and endo- glycosidase action, methanolysis followed by gas-liquid chromatography, methylation analysis, and high resolution nuclear magnetic resonance spectroscopy. Two homologous series of oligosaccharides were identified: (a) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----2)-alpha-D-Manp(1----3)-[alpha-D-Manp+ ++-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)-[alpha- D-Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc (minor series); (b) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-alpha-D-Manp-(1----6)-beta-D-Manp -(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp++ +-(1----4)-beta-D-GlcpNAc - (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)- [alpha-D-Manp-(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D- GlcpNAc (major series).     

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.