Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum.

Studies on the subcellular distribution of rat liver nucleotide pyrophosphatase activity revealed its presence in the plasma membrane and the endoplasmic reticulum only. The enzymes from either source were solubilized specifically with trypsin without an apparent change of their catalytic properties. A 200-fold and 1600-fold purification, respectively, was achieved by a procedure including DEAE-cellulose and affinity-chromatography with AMP as ligand, gel filtration on Sephadex G-200 and gel electrophoresis. Both nucleotide pyrophosphatases were isolated as electrophoretically homogeneous soluble proteins. They were shown to contain carbohydrate moieties. The electrophoretic mobility of both enzymes in polyacrylamide gels was identical at three pH values. Dodecylsulfate gel electrophoresis indicated a molecular weight of 137 000 for both glycoproteins. The enzymes hydrolyze a variety of purine and pyrimidine nucleotides yielding a 5'-nucleoside monophosphate. Adenosine 3':5'-monophosphate, nucleic acids and phosphate monoesters are not cleaved, but p-nitrophenyl-thymidine5'-monophosphate is readily hydrolyzed. In view of their substrate and inhibitor specificities the enzymes are considered nucleotide pyrophosphatases rather than phosphodiesterases.     

Induction, by adriamycin and mitomycin C, of modifications in lipid composition, size distribution, membrane fluidity and permeability of cultured RDM4 lymphoma cells

Adriamycin and mitomycin C were previously found to modulate the sensitivity of lymphoma cells to lysis by certain effectors of immunity and this modulation was dependent on drug concentration. In the present studies, RDM4 lymphoma cells were treated with different concentrations of the two drugs for 24 h in culture. These treatments resulted in changes in the lipid composition, membrane fluidity, cell size distribution, and permeability to 51CrO4, Trypan blue, Acridine orange and trimethylaminodiphenylhexatriene (TMA-DPH) of the cells. Changes in some of these parameters, as a function of drug concentration, resulted in dose-response curves which were bell-like shaped, hence paradoxical similarities between non-drug-treated cells and cells treated with higher drug concentrations were observed.     

Ultrastructural differences between the myelin sheaths of peripheral nerve fibres and CNS white matter

Summary Electron microscopic studies of peripheral nerves and spinal cord white matter of adult mice, as prepared by the freeze-etching method, show distinct differences in the periodicity of the myelin lamellae as well as of the ultrastructural lamellar pattern. A periodicity of 185 Å is to be determined for peripheral myelin whereas it is 160 Å in the myelin of central origin.In contrast to the appearance in the peripheral myelin the lamellar structure in the central myelin sheath is less pronounced and tends towards a fundamental repeating unit of 80 Å.This work was supported by the Swiss National Foundation (Nr. 4065).     

Reactive extraction of penicillin G in a pilot plant Karr-column

Summary Penicillin G was extracted from a model medium with a secondary amine (Amberlite LA-2) as carrier in n-butylacetate as solvent in a 7.6 m high pilot plant Karr-column at different stroke frequencies, throughput of the phases, concentrations of Penicillin G and carrier and ratios of the throughputs of the aqueous and organic phases. Up to penicillin concentrations of 30 gl^–1, throughputs of the aqueous phase of 100 lh^–1 and throughput ratios of the aqueous phase-to-organic phase of 3, very high degrees of extraction (99%) can be achieved with a penicillin loss below 1%.Symbols a specific interfacial area with regard to the volume of the continuous phase - C partition coefficient - cA, cA, i concentration of carrier (sec. amine) in the bulk at the interface - cAHP, cAHP, i concentration of complex in the bulk at the interface - cH proton concentration - cHP_a, cHP_a,i concentration of free acid in the bulk of the aqueous phase at the interface - cHP_o, cHP_{o, i} concentration of free acid in the bulk of the organic phase, at the interface - cP, cP, i concentration of acid anions in the bulk of the aqueous phase, at the interface - d₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - KG reaction equilibrium constant - K_phys distribution coefficient - N number of stages in cascade - t mean residence time of the aqueous phase - _aq throughput of the aqueous phase - _o throughput of the organic phase - Z dimensionless longitudinal coordinate of the column with regard to its active length (4 m) - holdup of the organic phase     

Reactive extraction of penicillin G in a pilot plant Karr-column II. Fermentation broths

Summary Penicillin G was extracted from mycelfree fermentation broths by means of the carrier (Amberlite LA-2) in n-butylacetate at pH 5 in a 7.6 m high pilot plant Karr-column with degrees of extraction E=98–99% and penicillin enrichments up to 3. The reextraction was carried out with phosphate buffer at pH-values above 7.5 with degree of extractions E=86–88% and penicillin enrichments up to 3. The penicillin and carrier losses were negligible. The influence of the process variables on the extraction degree was investigated. The penicillin extraction of the model medium and the fermentation broths were compared. Recommendations are given for the optimal penicillin recovery with reactive extraction.Symbols a specific interfacial area with regard to the volume of the continuous phase - cA concentration of carrier - cAHP,O concentration of complex in feed - cP,cP,O concentration of penicillin acid anion in theaqueous phase, in the feed - d ₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - V _aq throughput of the aqueous phase - V ₀ throughput of the organic phase - Z dimensionsless longitudinal coordinate of the column with regard to its active length (4m) - holdup of the organic phase     

Introduction of 5'-terminal functional groups into synthetic oligonucleotides for selective immobilization

Oligodeoxyribonucleotides terminating in a 5'-primary amine group are synthesized using solid-phase supported phosphoramidite chemistry. The 5'-terminal amine group in the deprotected oligomers is further derivatized with either succinic anhydride to give 5'-carboxylic acid or with dithiobis(succinimidylpropionate) followed by treatment with dithioerythritol to produce 5'-thiol-terminated oligonucleotides. The 5'-thiol-terminated oligonucleotides are selectively immobilized on solid supports containing either p-chloromercuribenzoate or 2,2'-dithiobis(5-nitropyridine) activated thiol groups.     

Reactive extraction of penicillin G from mycel-containing broth in a countercurrent extraction decanter

Penicillin was recovered from mycel-containing fermentation broth by direct reactive extraction into a counter-current extraction decanter, Type CA 226-290 of the Westfalia Separator Co., at room temperature via steady state operation. Penicillin concentrations in the feed varied from 3 to 41 g L(-1), Amberlite LA-2 carrier concentrations from 7 to 20 g L(-1)and/or DITDA carrier concentrations from 7.2 to 84 g L(-1), the LA-2-to-penicillin mole concentration ratio from 4 to 6.4, and/or the DITDA-to-penicillin mole concentration ratio was maintained at 2. The throughputs of the fermentation broth (520 to 880 L h(-1)) of the solvent phase (200 to 860 L h(-1)) and the over all throughput (800 to 1750 L h(-1)) were high. Extraction degrees of 72 to 96% were achieved between pH 4.6 and 5.1. Without carriers in the same pH range, extraction degrees of only 17 to 19% were attained. By reducing the pH to 2.3 and in the absence of carriers, the degree of extraction was increased to 61%. However, during the extraction, 6.5% of the penicillin decomposed. At these high throughputs, the steady state was attained within 1 to 4 min. Through the mechanical stress, the length of the hyphae was reduced and the protein content of the broth was increased by 50 to 100%. However, this protein content had no appreciable influence on the phase separation.     

Human p53 inhibits growth in Schizosaccharomyces pombe.

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.     

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.     

Isolation of specific tRNAs using an ionic-hydrophobic mixed-mode chromatographic matrix

The coating of a C18-reversed-phase high performance liquid chromatography support (octadecylsilyl-Hypersil) with a tetraalkylammonium salt (methyltrioctylammonium chloride) produces a chromatographic matrix with both ionic and hydrophobic character. Using this material oligonucleotides and tRNAs can be separated with high resolution. The observed resolution is in part due to the apparent lack of diffusion processes occurring during chromatography with this matrix. Some tRNAs can be obtained in high purity from a bulk tRNA mixture after a single chromatographic step. In general it is more efficient to use the matrix as the last step of a purification procedure for a particular tRNA. A two-step procedure is described which allows, in some cases, the isolation of small quantities of specific tRNA isoacceptors.