Synthesis and biological activity of an azido derivative of paclobutrazol, an inhibitor of gibberellin biosynthesis

A photolabile azido derivative of the kaurene oxidase inhibitor 1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl) pentan-3-ol (paclobutrazol) has been synthesized for use as a photoaffinity labeling agent. The compound was tested as an inhibitor of the oxidation of ent-kaurene catalyzed by cell-free preparations from endosperm of Cucurbita maxima. The I₅₀ of the azido derivative was 9.5 nanomolar, which compares well with that of paclobutrazol (6.3 nanomolar in our measurements). The azido compound bound to Cytochrome P-450 in microsomes from Cucurbita maxima, and induced a Type II spectral change, with an apparent binding constant of 0.24±0.04 micromolar.     

Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati

We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.     

Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.     

Mitochondrial DNA sequence evolution in sharks: rates, patterns, and phylogenetic inferences

Abundant representation of sharks in the fossil record makes this group a superb system in which to investigate rates and patterns of molecular evolution and to explore the strengths and weaknesses of phylogenetic inferences from molecular data. In this report, the molecular evolution of the cytochrome b gene in sharks is described and the information related to results from phylogenetic analysis of the data evaluated in the light of a phylogeny derived independently of the molecular data. Across divergent lineages of sharks there is evidence for significant substitution rate variation, departure from compositional equilibrium, and substantial homoplasy; nevertheless, the signal of evolutionary history is evident in patterns of shared transversions and amino acid replacements.      

Metabolic rate and directional nucleotide substitution in animal mitochondrial DNA

There is marked heterogeneity of nucleotide composition in mitochondrial DNA across divergent animals. Differences in nucleotide composition presumably reflect differences in directional nucleotide substitution for A+T or G+C nucleotides. In mitochondrial DNA, there is A+T directional nucleotide substitution in most (if not all) animals surveyed, and the magnitude of directional A+T nucleotide substitution differs greatly within and among groups. Differences in directional nucleotide substitution among lineages of mammals can be explained by changes in metabolic physiology. This relationship is thought to be mediated by the effect of oxygen radicals because these toxic compounds are by-products of aerobic metabolism and are known mutagens. Association between metabolism and nucleotide composition provides additional evidence in favor of the hypothesis that rates and patterns of nucleotide substitution in mitochondrial DNA can be influenced by factors that impinge on rates of endogenous DNA damage.      

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Evidence for substrate guidance of primordial germ cells

Primordial germ cells (PGCs) have been removed from their normal migratory route in early embryos of Xenopus laevis, and their behaviour studied in vitro. They adhere to, and move over the upper surface of, layers of outgrowing cells from expiants of adult Xenopus mesentery. They move by the extrusion of single filopodia, elongation, forward streaming of the yolky cytoplasm and retraction of their trailing ends. When the underlying cells are polarized in one direction only, PGCs always elongate and move along the same direction. Furthermore, when PGCs elongate and move over less obviously polarized cells, they always do so in the direction of ‘stress fibres’ (actin bundles) in the underlying cells. A substrate-guidance hypothesis for PGC migration is only tenable if there is some orientation in their natural substrate in vivo. Using the scanning electron microscope, we demonstrate that the coelpmic lining cells, beneath which PGCs migrate up the dorsal mesentery of the gut, are orientated in the direction of travel. Furthermore, this orientation changes at the time of gonadal ridge formation. This raises the intriguing possibility that PGCs are guided for at least part of their migration in Xenopus laevis embryos by a substrate-guidance mechanism.     

The Impact of the West Africa Ebola Outbreak on Obstetric Health Care in Sierra Leone

Background

As Sierra Leone celebrates the end of the Ebola Virus Disease (EVD) outbreak, we can begin to fully grasp its impact on already weak health systems. The EVD outbreak in West Africa forced many hospitals to close down or reduce their activity, either to prevent nosocomial transmission or because of staff shortages. The aim of this study is to assess the potential impact of EVD on nationwide access to obstetric care in Sierra Leone.

Methods and Findings

Community health officers collected weekly data between January 2014—May 2015 on in-hospital deliveries and caesarean sections (C-sections) from all open facilities (public, private for-profit and private non-profit sectors) offering emergency obstetrics in Sierra Leone. This was compared to official data of EVD cases per district. Logistic and Poisson regression analyses were used to compute risk and rate estimates. Nationwide, the number of in-hospital deliveries and C-sections decreased by over 20% during the EVD outbreak. The decline occurred early on in the EVD outbreak and was mainly attributable to the closing of private not-for-profit hospitals rather than government facilities. Due to difficulties in collecting data in the midst of an epidemic, limitations of this study include some missing data points.

Conclusions

Both the number of in-hospital deliveries and C-sections substantially declined shortly after the onset of the EVD outbreak. Since access to emergency obstetric care, like C-sections, is associated with decreased maternal mortality, many women are likely to have died due to the reduced access to appropriate care during childbirth. Future research on indirect health effects of health system breakdown should ideally be nationwide and continue also into the recovery phase. It is also important to understand the mechanisms behind the deterioration so that important health services can be reestablished.     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.