Sterol-phospholipid interaction in model membranes: role of C5-C6 double bond in cholesterol

Several double-bond isomers of cholesterol where the normal C5-C6 double bond (delta 5) has been moved to different positions in the ring skeleton, i.e., delta 1, delta 4, delta 7, delta 8(9), delta 8(14), and delta 14, have been synthesized and incorporated in phosphotidylcholine vesicles. In addition, dienes like delta 5,7, delta 7,14, and delta 8,14 have also been studied. Many of these cholesterol analogues are intermediates in the sterol biosynthesis in different organisms. The incorporation studied indicated that more than 90% of the sterol was present in the vesicles. The effect of these cholesterol analogues was studied by glucose permeability, electron spin resonance, and fluorescence polarization spectroscopy. These studies indicated that delta 14-cholesten-3 beta-ol was most effective in restricting glucose permeability or in increasing the order parameter but was still not as effective as cholesterol. This was followed by delta 8(14)- and delta 8(9)-cholesten-3 beta-ol. The delta 1, delta 4, and delta 7 analogues and the dienols were relatively less effective in condensing the membrane. These studies indicate that the double bond at C5-C6 in cholesterol is most effective for optimal sterol-phospholipid interaction and may have formed the basis of the migration of the double bond from rings C and D in sterols to C5-C6 during the evolution of cholesterol.     

Metabolism of high-density lipoproteins in cultured rat luteal cells

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.     

Mechanism of down regulation of luteinizing hormone receptors and steroidogenesis in corpora lutea

The mechanism of ‘down regulation’ of luteinizing hormone receptors was investigated in pseudopregnant rats using a modified radioimmunoassay capable of measuring endogenous tissue-bound hormone. Treatment of pseudopregnant animals with a desensitizing dose (desensitization treatment) of human chorionic gonadotropin resulted in a decrease in receptor concentration. This decrease was prevented if the animals were treated prior to the desensitization treatment with indomethacin, an inhibitor of prostaglandin biosynthesis, suggesting a role for prostaglandins in down regulation. The desensitization treatment resulted in a time-dependent decrease in subsequent responsiveness of the tissue to luteinizing hormone. Basal progesterone production rate was also decreased following desensitization. Total tissue cholesterol was found to be decreased following desensitization treatment, without any change in the ratio of free to esterified cholesterol. Mitochondrial cholesterol was significantly reduced and pregnenolone production by the mitochondria of desensitized corpora lutea was also markedly reduced. However, when cholesterol was added to the mitochondria of desensitized corpora lutea, pregnenolone production was increased, reaching values almost equal to that shown by the control mitochondria. These results show that decrease in the responsiveness following desensitization treatment is due to, besides receptor loss, decrease in tissue cholesterol, in particular mitochondrial cholesterol. The cholesterol side chain cleavage activity, although low, appears to be functionally intact; the low activity could be attributed to low levels of mitochondrial cholesterol.     

Decidual cell-specific surface antigen(s) recognized by monoclonal antibodies: tissue and species distribution

Decidual cells are direct descendants of endometrial stromal cells and the ultimate progeny of bone marrow-derived precursors. In view of their bone marrow genealogy and demonstrated immunoregulatory role during pregnancy, this study attempted to identify a lineage-specific differentiation marker(s) on murine decidual cells with the hope of tracing their developmental pathway and exploring their familial relationship to other lymphomyeloid cells. Two protein A-binding, IgG2b isotype monoclonal antibodies (secreted by clones 16F12 and 2G4F8) were raised by immunizing virgin CBA mice with syngeneic decidual cells. The presence and the density of the antigenic marker(s) recognized by these antibodies were examined by radioautography on various cell types in single cell suspensions of the decidua, placenta, and lymphomyeloid organs after a sandwich labeling with hybridoma supernatants followed by 125I-protein A. Both antibodies appeared to recognize antigen(s) unique for the decidual cell lineage in mice, humans, and rats. The incidence of antigen-bearing decidual cells increased with gestational age in CBA, C3H, and CD1 mice between days 8 and 14, and in humans between 6 and 10.5 wk; in rats, however, some decline was noted between days 8 and 14. The binding was always higher with 16F12 than with 2G4F8 supernatants. No significant binding of either antibody to trophoblast cells of the placenta or leukocytes within the decidua was noted in any of the above mouse strains or species. Little or no labeling of any cell type was seen on lymphomyeloid cells of the virgin or pregnant CBA mice, but a consistent labeling of a rare blast-type cell in the blood was observed with both antibodies, raising the possibility that this cell may represent the circulating precursor of the decidual cell lineage. It remains to be investigated whether these antibodies are recognizing the same or different differentiation antigen(s) on the decidual cells, and whether a conservation of this antigen(s) during speciation signifies its functional importance.     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Three methods for producing fertile hemopoietic chimeras in mice

Three methods for producing semiallogeneic (F1----parental) hemopoietic chimeras with retained or regained fertility are detailed here. Prenatal (PN) chimeras were produced by injecting F1 ([BALB/c female x C3H/HeJ male] or [CBA/J female x C57BL/6 male]) fetal liver (days 13-18) or adult bone marrow cells (10(6)-10(7) cells/20 microliters/embryo) into the yolk-sac cavities of days 13-17 gestation BALB/c or CBA/J embryos, respectively, and allowing them to be born naturally. Neonatal (NN) chimeras were made by introducing F1 bone marrow cells (1-2 x 10(7) cells/0.25 ml) into newborn (less than 24 hr old) female mice through the anterior facial vein. Female mice were raised to maturity in both cases. Ovary-transplanted (OT) chimeras were made by first irradiating (9.5 Gy) and repopulating young female adult mice with 10(7) F1 bone marrow cells, followed by bilateral orthotopic transplantation of syngeneic ovarian tissue six weeks later. Females reconstituted with the above three methods were mated with normal syngeneic males and sacrificed at 11-16 days of pregnancy to evaluate hemopoietic chimerism. This was determined in all cases by a radioautographic evaluation of the extent of donor H-2 phenotype marker expression on splenic small lymphocytes, after an indirect labelling of single-cell suspensions with monospecific antibody and [125I]protein-A. Results indicate that hemopoietic chimerism was best in the PN group (0.3-78.1%, mean = 27.1); intermediate in the OT group (5.8-38.2%, mean = 18.1); and low in the NN group (0-14%, with one exception, which was 83.6%). Observed fertility was best for BALB/c host PN chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of Selenate and Selenite to Elemental Selenium by a Pseudomonas stutzeri Isolate

A Pseudomonas stutzeri isolate rapidly reduced both selenite and selenate ions to elemental selenium at initial concentrations of both anions of up to 48.1 mM. Optimal selenium reduction occurred under aerobic conditions between pH 7.0 and 9.0 and at temperatures of 25 to 35°C. Reduction of both selenite and selenate was unaffected by a number of anions except for sulfite, chromate, and tungstate ions, which inhibited both growth and reduction.