Lipids of the marine bacterium Flexibacter polymorphus

This paper reports on the total fatty acid composition of a marine bacterium representative of the genus Flexibacter. Flexibacter polymorphus is unusual in containing a high proportion of the polunsaturated acid C20:53 whilst the level of branched fatty acids is low. These facts suggest that the membrane flexibility necessary for its gliding motility is a function of the polyunsaturated fatty acid composition. Biosynthetic precursors to the C20:5 acid are present which are characteristic of an oxygen-dependent pathway. The fatty acid composition of the structural lipids is influenced by changes in the culture medium. Na₂S inhibits production of the C20:5 acid at levels much lower than that at which it is known to inhibit growth. The intracellular granules observable in F. polymorphus do not contain elemental sulphur, in contrast to Beggiatoa sps., but instead probably contain lipids.     

Utility of d4-PGE2 as an internal standard to quantify endogenous levels of PGE1, PGE2, 190H PGE1 and 190H PGE2 in human seminal fluid by GC-MS-SIM

Four prostaglandins-PGE₁, PGE₂, 190H PGE₁ and 190H PGE₂-were quantified in human seminal fluid by GC-MS-SIM using only the internal standard, d₄-PGE₂. Methods and calculations were developed to minize errors inherent in using only one internal standard for quantifying four closely related prostaglandins. Preliminary data concerning the statistical significance of the differences found between PGE and 190H PGE levels in fertile, azospermic and oligospermic men are reported.     

Enhanced production of Rauscher leukemia virus after infection of high-passaged JLS-V9 cells.

JLS-V9, a mouse bone marrow cell line infected with Rauscher leukemia virus at high passage level, produced larger amounts of virus than the standard JLS-V10 cells. The enhanced virus production was attributed to the increased saturation density of JLS-V9 cells.     

Isolation, purification, and properties of mouse heavy-chain immunoglobulin mRNAs.

A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.     

Amine dehydrogenase of Pseudomonas putida: properties of the heme-prosthetic group.

There was approximately five times more hemoprotein (amine dehydrogenase) in crude extracts obtained from Pseudomonas putida grown on benzylamine than present in extracts from succinate-grown cells. The difference (reduced minus oxidized) spectrum of the purified enzyme possessed alpha,beta, and gamma bands at 550, 523, and 416 nm, respectively. The difference spectrum of the pyridine hemochrome derivative had absorption maxima at 416, 520, and 550 nm. These results, together with the fact that the heme group was covalently bound to the enzyme, indicated that the amine dehydrogenase from P. putida was a hemoprotein which contained heme c. The heme content was calculated at 2.01 mol/mol of enzyme. The enzyme was composed of two nonidentical subunits, but heme was present solely in the heavier unit. Carbon monoxide did not inhibit enzymatic activity, nor would it combine with the reduced or oxidized form of the enzyme. Amine dehydrogenase activity was inhibited by carbonyl agents with semicarbazide and cuprizone acting noncompetitively, whereas KCN and isoniazid inhibited by competitive and uncompetitive mechanisms, respectively. Spectral observations suggested that inhibition by these reagents was not due to an interaction with the heme moiety.     

Effect of partial hepatectomy on removal of O6-methylguanine from alkylated DNA by rat liver extracts.

1. The activity of an enzyme catalysing the loss of O6-methylguanine from methylated DNA was increasing during liver regeneration after partial hepatectomy. Activity was increased 3-fold by 24h and was maximal (6-fold increase) over the period 48-72h after operation. 2. This activity could also be induced by chronic treatment with dimethylnitrosamine, but the maximal response amounted to a 2-3-fold change (with the greater effect in male rats) after 4-6 weeks of exposure to daily doses of 2 mg of dimethylnitrosamine/kg. 3. Neither partial hepatectomy nor treatment with dimethylnitrosamine increased the activities of two other enzymes repairing alkylated DNA, DNA (7-methylguanine-)glycosylase and DNA (3-methyladenine-)glycosylase. 4. These results therefore indicate that there is a selective induction of the O6-methylguanine removal system during hepatocyte proliferation. Since this product is known to lead to mutations and its persistence in DNA throughout cell replication has been implicated in tumour initiation, this induction may play a role in resistance to carcinogenesis by alkylating agents.     

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.     

Freeze-drying of solutions of serum albumin containing dimethylsulfoxide.

Although several publications have emphasized the inadvisability of drying biologic materials containing dimethylsulfoxide (DMSO) by sublimation of ice in vacuo, our studies showed a relationship to exist between the relative concentrations of serum albumin, human, and dimethylsulfoxide for successful or unsuccessful freeze-drying of albumin-DMSO solutions. The cycle of freeze-drying for the successful drying of an albumin-DMSO solution was a modification of the cycle used for the successful drying of suspensions of measles virus by sublimation of ice in vacuo. Using nuclear magnetic resonance spectroscopy, a strong, sharp signal for DMSO was obtained in preparations of freeze-dried albumin-DMSO solutions rehydrated with deuterium oxide, D₂O.     

Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus)

Summary Exposure of adult brown bullheads Ictalurus nebulosus (120–450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (J _net ^Na+ ) and net chloride flux (J _net ^Cl- ) resulting largely from changes in whole body sodium influx (J _in ^Na+ ) and chloride influx (J _in ^Cl- ). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in J _in ^Cl- during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in J _in ^Na+ did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the post-hypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl^-/HCO ₃ ^- exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.     

Role and specificity of T-cell subsets in spontaneous recovery from Friend virus-induced leukemia in mice.

Spontaneous recovery from Friend virus complex-induced leukemic splenomegaly in H-2Db/b mice correlated with the appearance of Friend virus complex-specific cytotoxic T lymphocytes (CTL) detectable directly in spleen cell populations. By testing CTL on target cells containing expression vectors encoding individual retroviral structural proteins, the main viral protein recognized was shown to be the Friend murine leukemia helper virus envelope glycoprotein. In vivo depletion of CD8-positive T cells drastically reduced the incidence of recovery, providing direct evidence for the role of CD8-positive CTL in the spontaneous recovery process. In vivo depletion of CD4-positive cells had little effect on the early stages of recovery but did cause a marked reduction in the final incidence of recovery at 60 to 90 days. Thus, CD8-positive cells were required for the initiation of the recovery process, whereas CD4-positive cells appeared to be required for maintenance of the recovered status.