Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.     

The genetic fine structure of the complex locus aro3 involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe.

Summary The complex locus aro3 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis. By comparing the complementation map and the meiotic recombination map, the aro3 locus could be subdivided into the five adjacent subregions A, B, C, D and E. Out of 115 aro3 alleles, 26 nonsense alleles and 30 missense alleles could be identified by the criteria of nonsense suppressor sensitivity and leakiness, respectively. Most alleles with a pleiotropic complementation pattern are of the nonsense type. We conclude from the polarity of the complementation patterns characterising the nonsense alleles that the translation direction proceeds from subregion A to subregion E. Antipolar effects in complementation are more frequent than in the analogous system of the arom gene cluster of Neurospora crassa.This work formed part of a Ph.D. thesis submitted to the University of Bern     

Microbial Oxidation of Methane and Methanol: Crystallization of Methanol Dehydrogenase and Properties of Holo- and Apo-Methanol Dehydrogenase from Methylomonas methanica

Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s(20,w)) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful.     

Autolytic Activity and Butanol Tolerance of Clostridium acetobutylicum

The effects of acetone and butanol on the growth of vegetative cells and the stability of swollen-phase bright-stationary-phase cells (clostridial forms) of Clostridium acetobutylicum P262 and an autolytic deficient mutant (lyt-1) were investigated. There was little difference in the sensitivity of strain P262 and the lyt-1 mutant vegetative cells and clostridial forms to acetone. The stability of the different morphological stages was unaffected by acetone concentrations far in excess of those encountered in factory fermentations. Butanol concentrations between 7 and 16 g/liter, which are within the range obtained in industrial fermentations, increased the degeneration of strain P262 clostridial forms but had no effect on the stability of lyt-1 clostridial forms which never underwent autolysis. Vegetative cells of the lyt-1 mutant were able to grow in higher concentrations of butanol than strain P262 vegetative cells. It was concluded that there is a relationship between butanol tolerance and autolytic activity.     

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.     

Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.

A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).     

Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding

The nerve growth factor (NGF) receptor is a glycosylated transmembrane protein present on the cell surface as both high and low affinity forms, but biological responsiveness requires interactions of NGF with the high affinity site. We have tested the effects of mutations in the intracellular domain of the receptor upon its cell surface expression and equilibrium binding of 125I-NGF. Although mutant receptors lacking the entire cytoplasmic domain are processed and expressed at the cell surface and are capable of binding to NGF, the absence of cytoplasmic sequences leads to a loss of high affinity binding and to a lack of an appropriate cross-linking pattern as assessed by N-hydroxysuccinimidyl 4-azidobenzoate photoaffinity cross-linking. These results, taken together with the highly conserved nature of these cytoplasmic sequences, implies that the interaction of the receptor with an accessory molecule is necessary to form the high affinity receptor.