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.     

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.     

Multiple Forms of Polyglucoside-Branching Enzyme in the Algae

Three isozymes specifically concerned with the “branching” of linear polyglucosides have been delected in algae. These enzymes were detected using two-dimensional polyacrylamide gel electrophoresis, and were found to be present in blue-green, red and in green algae. Two isozynies were found in Oscillatoria princeps; three enzymes were present in Spirogyra setiformis, and two and three such enzymes were detected in red algae of the Rhodymenia type. The significance of the multiple forms of this branching enzyme was assessed in light of the type of storage poly-glucosides formed by these plants. The “degree of branching” of the storage sugar appeared to be related to the evolutionary status of these algae.     

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)     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

On the mechanism of a 60-Hz electric field induced growth reduction of mammalian cellsin vitro

Data on 60-Hz electric field (EF) induced reduction in growth rate of plant roots have strongly supported the hypothesis that the effect is related to an EF-induced transmembrane potential (V ⁱ _m). An investigation was undertaken to determine if this hypothesis is also applicable to 60-Hz EF-induced reductions in growth rate of mammalian cells in vitro. Human lymphoblastic (RPMI 1788) and human carcinoma (HeLa) cells were selected for study, the former having a relatively small diameter (11.2 m), and the latter having a relatively large diameter (15.4 tm). The 60-Hz EFs ranged from 430–1200 V/m in the culture medium. The growth rate of RPMI 1788 cells after 4-days was depressed by about 42% at a 60-Hz EF of 1000–1200 V/m with a response threshold occurring at 950 V/m; theV ⁱ _m at the response threshold was 8 mV There was no 60-Hz EF-induced effect on HeLa cell growth rate of aV ⁱ _m of 8 mV (60-Hz EF=700 V/m); a statistically significant effect was achieved atV ⁱ _m of 11 mV (950 V/m). The data support the hypothesis that above a threshold 60-Hz EF,V ⁱ _m acts as the initial signal leading to growth rate reductions.     

Seasonality in the equatorial tropics: Flower,fruit, and leaf phenology of montane trees in the highlands of Southwest Uganda

Phenology influences many forest functions and can inform forest conservation and management, yet representative phenological data for most common tropical forest tree species remain sparse or absent. Between June 2011 and December 2013, we investigated flowering, fruiting, and leafing patterns in the Bwindi Impenetrable National Park, a montane forest located near the equator in Uganda, drawing on 16,410 observations of 530 trees of 54 species located between 2066 and 2527 m in elevation. The park's climate is equatorial with two wet and dry seasons each year. Flowering and fruiting were strongly seasonal while patterns in leafing were less pronounced. Flower occurrence peaked at the beginning of the short dry season followed by a pronounced trough during the beginning and the middle of the short wet season. Fruit occurrence had a pronounced peak during high rainfall months in March through April with most fruits ripening during drier months in May through July. Fruit scarcity was observed for a 4-month period spanning September to December and most flushing of leaves noted at the end of the wet season in November and December. Our binomial generalized linear mixed models indicated that flowering and fruiting were negatively associated with temperature and that leafing activity was positively associated with rainfall and temperature. These findings are consistent with the insolation- and water-limitation hypotheses suggesting that the seasonally varying availability of resources such as light, water, and nutrients determines these phenological patterns. Ideally, prolonged, multi-year community-level studies would be supported so as to better characterize the influence of climate and of climate variability.     

Role of gonadal hormones in development of the sexual phenotypes

Summary Male and female embryos develop in an identical fashion during the initial portion of gestation. If the indifferent gonad differentiates into an ovary (or if no gonad is present), a female phenotype is formed. Male phenotypic differentiation, however, requires the presence of an endocrinologically active testis. Two secretion of the fetal testis, Müllerian inhibiting substance and testosterone, are responsible for male development. Studies of single gene mutations that interfere with androgen action indicate that testosterone itself is responsible for virilization of the Wolffian duct system into the epididymis, vas deferens, and seminal vesicle, whereas the testosterone metabolite dihydrotestosterone induces development of the prostate and male external genitalia. Thus, impairment of dihydrotestosterone formation results in a characteristic phenotype consisting of predominantly female external genitalia but normally virilized Wolffian ducts. The molecular mechanisms by which testosterone and dihydrotestosterone act during fetal development appear to involve the same high affinity receptor, a protein that transports both testosterone and dihydrotestosterone to the nucleus of target cells. When this receptor is either absent, deficient, or structurally abnormal, the actions of both testosterone and dihydrotestosterone are impaired, and the resulting developmental anomalies involve both internal and external genital structures.The original work described in this review was supported by grant AM 03892 from the National Institutes of Health