Colony stimulating activity of serum from germfree normal and leukemic mice

Serum from germfree Swiss/HaM mice exhibited a reduced capacity to stimulate granulocytic and mononuclear cell colony formation by DBA/1 bone marrow cells in vitro when compared with serum from conventional Swiss/HaM mice. Sera from germfree preleukemic and leukemic AKR mice exhibited strong colony stimulating activity, indicating that the increased colony stimulating activity previously observed in the serum of conventional leukemic mice is not the consequence of bacterial or fungal infections supervening in leukemic animals with deficient immune responses.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

Bacterial oxidation of cycloparaffinic hydrocarbons

A series of 10 branched-chain alkanes and 4 cycloalkanes were employed individually as elective culture substrates for bacteria in soil. Only 2-methylbutane and 2-methylpentane yielded bacteria, one each. Both bacteria grew at the expense of eachn-alkane from C₁ to C₂₂ but they were very selective for branched-chain substrates. Compounds with less branching were most readily utilized. Neither organism grew at the expense of various cycloalkanes as sole sources of carbon and energy. The 2-methylbutane isolate was studied in detail. Resting cell suspensions were able to produce α-ketoglutaric acid from each of the compounds the bacterium was able to utilize for growth. “Non-growth hydrocarbons” were also oxidized; in each case only neutral ketonic substances were detected. A series of cycloparaffins, from C₃- to C₈-membered rings, was oxidized to the corresponding cyclomonoketones. No oxidation products of cyclododecane (C₁₂), 1,4-cyclohexadiene (C₆) or benzene could be detected. The metabolic products identified are consistent with the formation of a cyclomonoalcohol as the immediate precursor of the ketone. The alcohol is formed from cycloalkanes, the cycloalkenes, and cycloalkene oxide as substrates. Alcohol formation from the first two probably takes place by independent parallel, rather than sequential, reaction pathways. The epoxide may be a non-obligate intermediate in the cyclomonoolefin conversion to the alcohol. Significant aspects of these conversions are discussed.     

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.     

Influence of exchangeable ions on germinability of bacterial spores

Rode, L. J. (The University of Texas, Austin), and J. W. Foster. Influence of exchangeable ions on germinability of bacterial spores. J. Bacteriol. 91:1582-1588. 1966.-Native spores of Bacillus megaterium Texas, and H-spores produced by titration of native spores to pH 4 with mineral acid, did not germinate in a solution of alanine and inosine unless a strong electrolyte was present. Ca-spores prepared from either H-spores or native spores did germinate efficiently in the same solution without a strong electrolyte. Of several other bivalent cations tested, only strontium and barium could substitute for calcium in conditioning spores for subsequent germination in the absence of an electrolyte. Variable responses were obtained with different metal ion forms of 62 unidentified soil isolates and several stock species of Bacillus. Although the pattern of response was not uniform in all organisms, ions played a crucial role in the germinability of the great majority of strains tested.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.     

Effects of fish oil on VLDL triglyceride kinetics in humans

Dietary n-3 fatty acids (FAs) found in fish oils markedly lower plasma triglyceride (TG) and very low density lipoprotein (VLDL) levels in both normal and hypertriglyceridemic subjects. The present study examined the mechanism of this effect. Ten subjects with widely different plasma triglyceride levels (82 to 1002 mg/dl) were fed metabolically controlled diets containing 20% fat. The control diet contained a blend of cocoa butter and peanut oil (P/S = 0.8). The test diet contained fish oil (P/S = 1.1) and provided 10-17 g of n-3 FAs per day (depending on calorie intake). After 3 to 5 weeks of each diet, the kinetics of VLDL-TG were determined over a 48-h period after the injection of [3H]glycerol. The fish oil diet reduced the VLDL-TG synthetic rate from 23 +/- 14.3 (mean +/- SD) to 12.6 +/- 7.5 mg/h per kg ideal weight (P less than 0.005) and increased the fractional catabolic rate (FCR) for VLDL-TG from 0.23 +/- 0.12 to 0.38 +/- 0.16 h -1 (P less than 0.005). At the same time, there was a 66% reduction of plasma triglyceride levels, resulting largely from a 78% decrease in VLDL-TG levels (398 +/- 317 to 87 +/- 77 mg/dl; P less than 0.005). There was a strong correlation (r = 0.83; P less than 0.01) between the change in synthetic rates and pool sizes, but there was no correlation (r = 0.24; NS) between changes in FCRs and pool sizes. The VLDL cholesterol: triglyceride ratio increased during the n-3 diet suggesting that smaller VLDL particles were present. These particles would be expected to leave the VLDL fraction more rapidly than larger particles producing a higher FCR. We conclude that the hypotriglyceridemic effect of fish oil appears to be caused primarily by an inhibition of very low density lipoprotein-triglyceride synthesis, but an additional, independent effect upon VLDL catabolism cannot be ruled out.