Effect of a cold stimulus on the synthesis of uncoupling protein in brown adipose tissue of rats and newborn rabbits

Rats are known to respond to a cold stimulus by increasing the activity and amount of the uncoupling protein in brown adipose tissue. A 48 h cold stimulus was found to increase the synthesis of uncoupling protein 3.g-fold in 4–5 week old rats whereas no change was observed with newborn rabbits. The lack of response in the latter case may reflect a difference between rabbits and rats or that synthesis is already maximal in newborn rabbits.     

Thermophilic anaerobic spirochetes in New Zealand hot springs

Abstract Electron and light microscopy revealed the presence of spirochetes in New Zealand thermal springs. The spirochete population in one spring studied (Kuirau Lake) was affected by fluctuations in temperature and/or pool level. A pure culture of the strictly anaerobic bacterium revealed that it grew optimally at a temperature of 45–50°C, with no growth occurring above 60°C, and a pH of 7.0–7.5 with no growth occurring at pH 5.5 or 8.5. Growth was inhibited by chloramphenicol, penicillin, streptomycin, tetracycline and neomycin but not by d -cycloserine, novobiocin or phosphomycin at 10 μg/ml. A wide range of carbohydrates were utilized but not organic acids. Acetate was the major end product of glucose fermentation with substantial amounts of ethanol and traces of lactate being produced.     

Comparison of a Thermoanaerobium sp. from a New Zealand hot spring with Thermoanaerobium brockii

Abstract An anaerobic ethanologenic strain of extremely thermophilic bacteria isolated from a New Zealand hot spring resembled Thermoanaerobium brockii in morphology and cell-wall ultrastructure. However, antibodies produced against the New Zealand isolate did not crossreact with the type strain of T. brockii . The New Zealand isolate strain Tok6-B1 fermented a wider range of carbohydrate substrates, including pentoses, and was less inhibited by a hydrogen atmosphere. Ethanol and acetate were major end-products and lactate a minor product of glucose fermentation. Under a hydrogen atmosphere, these 3 end-products were formed in approximately equal amounts.     

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.     

Rapid recovery from K current inactivation on membrane hyperpolarization in molluscan neurons

Recovery from K current inactivation was studied in molluscan neurons using two-microelectrode and internal perfusion voltage clamps. Experiments were designed to study the voltage-dependent delayed outward current (IK) without contamination from other K currents. The amount of recovery from inactivation and the rate of recovery increase dramatically when the membrane potential is made more negative. The time course of recovery at the resting potential, -40 mV, is well fit by a single exponential with a time constant of 24.5 s (n = 7). At more negative voltages, the time course is best fit by the sum of two exponentials with time constants at -90 mV of 1.7 and 9.8 s (n = 7). In unclamped cells, a short hyperpolarization can cause rapid recovery from inactivation that results in a shortening of the action potential duration. We conclude that there are two inactivated states of the channel and that the time constants for recovery from both states are voltage dependent. The results are discussed in terms of the multistate model for K channel gating that was developed by R. N. Aldrich (1981, Biophys. J., 36:519-532).     

Regulation of carbonic-anhydrase activity,inorganic-carbon uptake and photosynthetic biomass yield inChlamydomonas reinhardtii

The regulation of carbonic anhydrase by environmental conditions was determined forChlamydomonas reinhardtii. The depression of carbonic anhydrase in air-grown cells was pH-dependent. Growth of cells on air at acid pH, corresponding to 10 m CO₂ in solution, resulted in complete repression of carbonic-anhydrase activity. At pH 6.9, increasing the CO₂ concentration to 0.15% (v/v) in the gas phase, corresponding to 11 M in solution, was sufficient to completely repress carbonic-anhydrase activity. Photosynthesis and intracellular inorganic carbon were measured in air-grown and high-CO₂-grown cells using a silicone-oil centrifugation technique. With carbonic anhydrase repressed cells limited inorganic-carbon accumulation resulted from non-specific binding of CO₂. With air-grown cells, inorganic-carbon uptake at acid pH, i.e. 5.5, was linear up to 0.5 mM external inorganic-carbon concentration whereas at alkaline pH, i.e. 7.5, the accumulation ratio decreased with increase in external inorganic-carbon concentration. It is suggested that in air-grown cells at acid pH, CO₂ is the inorganic carbon species that crosses the plasmalemma. The conversion of CO₂ to HCO ₃ ^- by carbonic anhydrase in the cytosol results in inorganic-carbon accumulation and maintains the diffusion gradient for carbon dioxide across the cell boundary. However, this mechanism will not account for energy-dependent accumulation of inorganic carbon when there is little difference in pH between the exterior and cytosol.     

GABA neurones in retinas of different species and their postnatal development in situ and in culture in the rabbit retina

Summary The localisation of GABA immunoreactive neurones in retinas of a variety of animals was examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz. rat, rabbit, goldfish, frog, pigeon and guinea-pig. All species, with the exception of the frog, possessed immunoreactive perikarya in their retinal ganglion cell layers. These perikarya are probably displaced amacrine cells because GABA immunoreactivity was absent from the optic nerves and destruction of the rat optic nerve did not result in degeneration of these cells. GABA immunoreactivity was also associated with the outer plexiform layers of all the retinas studied; these processes are derived from GABA-positive horizontal cells in rat, rabbit, frog, pigeon and goldfish retinas, from bipolar-like cells in the frog, and probably from interplexiform cells in the guinea-pig retina.The development of GABA-positive neurones in the rabbit retina was also analysed. Immunoreactivity was clearly associated with subpopulations of amacrine and horizontal cells on the second postnatal day. The immunoreactivity at this stage is strong, and fairly well developed processes are apparent. The intensity of the immunoreactivity increases with development in the case of the amacrine cells. The immunoreactive neurones appear fully developed at about the 8th postnatal day, although the immunoreactivity in the inner plexiform layer becomes more dispersed as development proceeds. The immunoreactive horizontal cells become less apparent as development proceeds, but they can still be seen in the adult retina.The GABA immunoreactive cells in rabbit retinas can be maintained in culture. Cultures of retinal cells derived from 2-day-old animals can be maintained for up to 20 days and show the presence of GABA-positive cells at all stages. In one-day-old cultures the GABA immunoreactive cells lacked processes but within three days had clearly defined processes. After maintenance for 10 days a meshwork of GABA-positive fibres could also be seen in the cultures.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein.     

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.