Effect of long-term n-butanol ingestion on rat brain polypeptide synthesis directed by endogenous messengers.

Long-term ingestion of sublethal n-butanol doses by rats led to a noteworthy increase in the resistance of in vitro brain ribosomal function to the acute inhibitory action of ethanol and isopropanol. Withdrawal of n-butanol did not change this adaptation process immediately. The step affected seems to be the elongation of polypeptide chains. The dependence of in vitro translation on incubation temperature was affected by the adaptation process, the translation system of chronic animals being less stimulatable than that of control animals at low temperature.     

Improved procedure for the purification of hepatic lipase from rat liver homogenate

A procedure is described for the purification of hepatic lipase (HL)4 from rat liver homogenate which results in a high yield (41%) of electrophoretically homogeneous enzyme. The method is based on that of Twu et al. (Biochim. Biophys. Acta 1984: 792, 330), but it is more efficient with respect to yield (about 4-fold) and purity (1.6-fold). It includes the preparation of a high-speed supernatant, chromatography in series on octyl-, heparin- and concanavalin A-Sepharose, and finally gel filtration. On SDS-PAGE analysis, the purified enzyme exhibited an apparent molecular mass of 63.6 +/- 3.2 kDa. Heterogeneity was observed, when purified HL was subjected to isoelectric focussing. The enzyme displayed a specific catalytic activity of 23,000 U* (mumol fatty acid released per h at 37 degrees C) per mg protein, when assayed with trioleoyl glycerol suspensions in arabic gum. A highly specific antiserum against rat liver HL, capable of inhibiting 817 mU* HL per microliter antiserum, was raised in rabbits.     

Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.

A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.     

Thermogenesis of white adipocytes: a novel method allowing long-term microcalorimetric investigations

A microcalorimetric method was developed to facilitate long-term assessment of energy balance in isolated fat cells. White rat adipocytes were primary cultured in glass ampoules with a matrix of agar-gel. Heat production was measured continuously over 3 days with an LKB BioActivityMonitor. In order to assist interpretation of the microcalorimetric measurements, glucose consumption and lactate and pyruvate production were determined in parallel cultures. Heat production, glucose consumption and lactate production were in an apparent steady state throughout the study whether employing aerobic (94 pW, 0.50 and 0.44 pmol/cell. h, respectively) or partial anaerobic experimental conditions (39 pW, 0.41 and 0.57 pmol/cell.h, respectively). However, oxygen availability influenced the apparent heat production and glucose and lactate metabolism. With partial anaerobiosis a 59% lower heat production, an 18% lower glucose consumption and a 30% higher lactate production than by employing aerobic experimental conditions were observed.     

PACAP, a VIP-like peptide, in neurons of the esophagus.

The lower esophagus of guinea-pig, cat, sheep and man was analyzed for pituitary adenylate cyclase activating peptide (PACAP), a novel vasoactive intestinal peptide (VIP)-like peptide, using immunocytochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were numerous in the longitudinal and circular muscle layers of sheep and man, moderate in numbers in cat, while being few in the esophagus of guinea-pig. A few PACAP-immunoreactive nerve cell bodies and numerous nerve fibers were seen in the myenteric ganglia of the esophagus of cat, sheep and man. In the lower esophagus of cat, sheep and man all PACAP-containing nerve cell bodies and nerve fibers stored VIP. The results of radioimmunoassay of PACAP in extracts of specimens from man were in good agreement with the immunocytochemical findings. High performance liquid chromatography revealed one major peak of PACAP-like immunoreactivity in extracts of human esophagus. We suggest that neuronal PACAP may serve to modulate motor activity and secretion in the lower esophageal sphincter region.     

Photosynthesis: Molecular biology and bioenergetics

Book Review

Photosynthesis: Molecular biology and bioenergeticsG.S. Singhal, J. Barber, R.A. Dilley, Govindjee, R. Haselkorn and P. Mohanty (Eds), Proceedings of the International Workshop on Application of Molecular Biology and Bioenergetics of Photosynthesis. Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong and Narosa: Springer-Verlag, 1989. xiv+441 pages. DM 148,00. ISBN 3-540-50451-6     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Plant protoplasts and genetic engineering II

Book Review

Plant protoplasts and genetic engineering IIY.P.S. Bajaj (Ed.), (Biotechnology in Agriculture and Forestry, Vol. 9). Berlin: Springer-Verlag, 1989. 510 pages. DM398.00. ISBN 3-540-50789-2     

Dye-promoted precipitation of serum proteins. Mechanism and application.

Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions.     

Nuclear location of phosphoglycerate mutase BB isozyme in rat tissues

Summary We have previously reported (Ureña et al. Eur. J. Cell Biol. 1990) that in skeletal muscle, type MM phosphoglycerate mutase isozyme is present in the nucleus as well as in the cytosol. To determine whether type BB phosphoglycerate mutase isozyme is also present in nucleus, the subcellular location of this isozyme was studied in different rat tissues by cell fractionation and immunogold techniques. With the aid of high affinity-purified anti-phosphoglycerate mutase BB isozyme antibodies, the isozyme was located in the nucleus of neuronal, astroglial and liver cells but not in the nucleus of oligodendroglial and endothelial cells. Biochemical studies on purified nuclear fractions also demonstrated the presence of phosphoglycerate mutase activity in the nucleus. Both immunocytochemical and biochemical techniques showed that nuclear phosphoglycerate mutase-specific activity depended on the type of cell.Abbreviations PGAM phosphoglycerate mutase - PGAM-M(M) muscle specific subunit (isozyme) of PGAM - PGAM-B(B) brain type subunit (isozyme) of PGAM - ssDNA single stranded DNA - PBS 0.001 M phosphate buffer, pH 7.4, containing 0.15 M NaCl - kDa kilodalton