Modulation of adenylate cyclase by guanine nucleotides and Kirsten sarcoma virus mediated transformation

Certain tumour cells contain activated ras genes that code for 21 000 dalton proteins (p21). These proteins associate with the inner face of the plasma membrane and bind guanine nucleotides specifically. In order to determine whether p21s have functions similar to other GTP binding proteins, we investigated the regulation, by guanine nucleotides, of adenylate cyclase (AC) activity in membrane preparations isolated from fibroblasts (C127) transformed by a temperature sensitive mutant of Kirsten sarcoma virus (Ts 371). The degree of AC stimulation by GMP P(NH)P increased when these cells were shifted from the permissive temperature (33 degrees C) to the non-permissive temperature (39 degrees C). This effect was more pronounced at low Mg++ and low GMP P(NH)P concentrations. AC stimulation remained unchanged in rat fibroblasts infected with a temperature sensitive mutant of Rous Sarcoma virus. AC activity was depressed in C127 cells infected with wild type KiMSV. Our data illustrate the feasibility of correlating alterations in the AC system with ras gene expression and using such experimental approaches to elucidate the physiological functions of the p21 proteins.     

Sterol and squalene carrier protein interactions with fluorescent delta 5,7,9(11)-cholestatrien-3 beta-ol

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Serine-specific tRNAs in Escherichia coli: relative abundance and sequence

Serine isoaccepting tRNAs were isolated from bulk tRNA of Escherichia coli by affinity chromatography on immobilized bacterial elongation factor Tu and their relative abundance was determined. The three major species, which are sufficient to read all six serine codons, were identified by sequencing. The sequence of a novel tRNASer with the anticodon GGA was elucidated.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

Synthesis and phosphorylation of cytoskeleton components in foetal,regenerating and adult normal rat hepatocytes during culture

Summary Detergent insoluble material (DIM) was prepared by gentle treatment with detergent from foetal, regenerating and adult normal rat hepatocytes cultured for various times. It retained to some degree the morphology of the cells. After incubation of intact cells with ³⁵S-methionine, most of the labelled DIM proteins were found to be components of the cytoskeleton. They included several cytokeratins, vimentin and actin. The synthesis rate varied with the age of animals and culture conditions. The high synthetic rate of vimentin in foetal and regenerating hepatocytes could be associated with cell proliferation. No correlation was found between cytokeratin synthesis and hepatocyte growth. Most of the cytoskeleton proteins could be phosphorylated in intact cells and in DIM from cultured hepatocytes. However the degree of phosphorylation of these proteins was not related to their synthetic rate. The decreased phosphorylation level in cultured adult rat hepatocytes could be related to the rapid loss of specific functions.     

Production and release of peptidoglycan and wall teichoic acid polymers in pneumococci treated with beta-lactam antibiotics

Autolysin-defective pneumococci treated with inhibitory concentrations of penicillin and other beta-lactam antibiotics continued to produce non-cross-linked peptidoglycan and cell wall teichoic acid polymers, the majority of which were released into the surrounding medium. The released cell wall polymers were those synthesized by the pneumococci after the addition of the antibiotics. The peptidoglycan and wall teichoic acid chains released were not linked to one another; they could be separated by affinity chromatography on an agarose-linked phosphorylcholine-specific myeloma protein column. Omission of choline, a nutritional requirement and component of the pneumococcal teichoic acid, from the medium inhibited both teichoic acid and peptidoglycan synthesis and release. These observations are discussed in terms of plausible mechanisms for the coordination between the biosynthesis of peptidoglycan and cell wall teichoic acids.     

Investigations on karyotype evolution in patients with chronic myeloid leukemia (CML)

In an attempt to relate karyotype evolution to clinical and hematological data serial chromosomal analyses were performed in 31 patients with chronic myeloid leukemia (CML), both in chronic and acute phases. Our results in Philadelphia chromosome (Ph1)-positive CML are in line with karyotype profiles described in the literature. In addition, we report on chromosomal findings in 4 cases of Ph1-negative disease, one presenting with an iso17q chromosome in the positive CML. The same chromosomal abnormality was observed in a small population of Ph1-negative cells present in one of two patients with mixed Ph1-positive/Ph1-negative CML. The first case of a female patient with the loss of a sex chromosome in Ph1-positive cells is reported. Two patients with unusually long and mild chronic phases despite the presence of trisomy 8 in their karyotypes are described. Our findings suggest that the order of appearance of additional chromosomal changes of CML is of prognostic significance for the progression and the clinical picture of the disease.