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.     

In-vivo studies of mammary development in the goat using magnetic resonance imaging (MRI)

Mammary development and regression were measured in goats in vivo using magnetic resonance imaging (MRI). Measurements were made during the first and second cycles of pregnancy, lactation and involution. In primiparous goats, and exponential pattern of growth was evident during gestation and for the first 2 weeks of lactation. Parenchyma volume correlated significantly with milk yield across goats during early lactation, and across stage of lactation within goats. Milking was discontinued in Week 26 of the first lactation. Involution was characterized by an initial accumulation of fluid (over 2 days) followed by reabsorption; parenchyma volume did not decrease significantly until the 3rd week of involution, which was also the time at which these goats were mated to start their second gestation. Their udders still contained significant quantities of fluid (40-60% of the gross volume), but parenchyma volume was also greater (by 4.7-fold) than in goats beginning their first gestation. By Week 15 of gestation there was no longer a parity difference in parenchyma; the udders of first-gestation goats had grown significantly, but those of second-gestation goats had not. Conversely, between gestation Week 15 and lactation Week 2 mammary growth was significantly more rapid in the second cycle, such that the udder was larger at the start of the second lactation.     

Metabolism of larger high density lipoproteins accumulating in some families of baboons fed a high cholesterol and high saturated fat diet

Progeny of certain baboon sires accumulate lipoproteins in high density lipoprotein-1 (HDL1) when challenged with a high cholesterol, high saturated fat diet. These studies were conducted to determine the apoprotein composition and metabolic fate of HDL1 in the plasma. HDL1 particles containing apoA-I with and without apoE were detected. The majority of particles, however, contained apoA-I without any detectable apoE. To determine the metabolic fate of HDL1 in plasma, HDL1 labeled with iodinated apoA-I from animals with high levels of HDL1 and iodinated apoA-I-labeled autologous HDL were coinjected into both high and low HDL1 animals. The data for the decay of radioactivity in HDL1 and HDL were analyzed by multicompartment modelling. The radioactivity from HDL1 was cleared from the plasma either via direct removal (9.1 +/- 4.7% in low and 21.7 +/- 8.3% in high HDL1 animals) or via its conversion to HDL. A large proportion of radioactivity from HDL1 was rapidly transferred to HDL directly or metabolized via an intermediate compartment. Most of the radioactivity from apoE-poor HDL1, however, was transferred to HDL. Both high and low HDL1 animals catabolized HDL1 and HDL similarly. Low HDL1 animals transferred HDL1 radioactivity to HDL much faster. No detectable radioactivity from HDL was transferred to HDL1. Thus, HDL1 that accumulates in high HDL1 animals is mainly a precursor for HDL. Our hypothesis is that this accumulation of HDL1 is due to the slower cholesteryl ester transfer from HDL to lower density lipoproteins, thus affecting reverse cholesterol transport in high HDL1 baboons.     

Metabolism of 125I-atrial natriuretic factor by vascular smooth muscle cells. Evidence for a peptidase that specifically removes the COOH-terminal tripeptide

The addition of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) to cultured bovine aortic smooth muscle cells at 37 degrees C resulted in a rapid clearance from the medium (t1/2 approximately 7.5 min). Within 5 min, [125I]iodotyrosine126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg-[125]iodotyrosine126 (125I-FRY) appeared in the medium. The identities of these degradation products were confirmed by 1) retention time on high performance liquid chromatography (HPLC) relative to standards, 2) products generated by digestion with aminopeptidase M, and 3) the absence of the Met110. Preincubation of the cells with ammonium chloride or chloroquine resulted in a significant increase in the intracellular accumulation of radiolabel, indicative of endocytosis and rapid delivery of 125I-hANF to an acidic intracellular compartment (endosome and/or lysosome). Neither ammonium chloride, chloroquine, nor excess unlabeled hANF blocked the rapid appearance in the medium of 125I-RY or 125I-FRY. Bestatin inhibited the generation of 125I-RY, with a concomitant increase in 125I-FRY, suggesting that the 125I-RY is produced by aminopeptidase action on 125I-FRY. The endopeptidase 24.11 (enkephalinase) inhibitor, SCH 39370, did not inhibit the formation of 125I-FRY. These results provide evidence of a peptidase capable of specifically removing the COOH-terminal tripeptide from 125I-hANF. The COOH-terminal tripeptide, Phe124-Arg-Tyr126, was also isolated from cell digests of hANF by HPLC and its identity confirmed by amino acid analysis. Since it is generally believed that the COOH-terminal tripeptide is critical to many of atrial natriuretic factor-(99-126)'s bioactivities, this enzyme may be involved in the inactivation of atrial natriuretic factor-(99-126) in target tissues.     

Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4

The gene coding for protein A (spa) has been mapped close to nov on the genetic map of the chromosome of Staphylococcus aureus 8325-4. A rapid mapping procedure has been developed which first allowed the region of the chromosome carrying the spa gene to be identified by blot +hybridization of large DNA fragments which had been separated by pulsed-field gel electrophoresis. Restriction endonuclease SmaI fragment G was shown to carry the spa gene. An insertion mutation in spa was constructed by in vitro insertion of a fragment of DNA expressing resistance to kanamycin and neomycin. A spa::Kan(r)Neo(r) mutation was isolated in S. aureus 8325-4 by allele replacement. This provided a selectable marker which allowed the spa gene to be mapped by transformation analysis.