Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Three new HLA-G alleles and their linkage disequilibria with HLA-A

Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A T), but conservative, (Thr Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A T) at the third base of codon 107 and (G A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.The nucleotide sequence data reported in this paper have been submitted to the GenBank and EMBL nucleotide sequence databases and have been assigned the following accession numbers: EMBL-X60983 (HLA-G*II), GenBank-M99048 (HLA-G*III), and GenBank-L07784 (HLA-G*IV).The contribution to this paper by P. Morales and A. Corell is equal, and the order of authorship is arbitrary. Correspondence to: A. Arnaiz-Villena.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.     

The stimulatory effect of human chorionic gonadotropin on amino acid uptake by amphibian follicles.

Human chorionic gonadotropin (hCG) stimulates the uptake of eight different amino acids and four nucleosides by Xenopus laevis ovarian follicles. This hormone also stimulates amino acid uptake in the follicles of another amphibian, Callyptocephallela caudiverbera. The stimulation of uptake is due to a reduction in the amino acid concentration required for half-maximal uptake velocity and not to an increment in V_max. The effect of hCG does not require protein synthesis but requires physiological conditions of temperature and pH. Incorporation of radioactive exogenous amino acid into proteins is also stimulated by the hormone, but high-resolution electrophoresis shows that there are no drastic qualitative changes in the pattern of proteins synthesized at early times after hCG treatment. The effect of hCG on the uptake of exogenous amino acids does not appear to be required for oocyte maturation because other hormones such as progesterone and testosterone which induce maturation do not increase amino acid uptake. Also the concentration of hCG required for oocyte maturation is significantly lower than that required for an effect on amino acid transport. Inhibitors of oocyte maturation such as theophylline and cycloheximide do not inhibit the action of hCG on amino acid uptake by the amphibian follicles.     

Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions.

The maturation of Xenopus laevis oocytes was studied in media free of added potassium salts. Under these conditions maturation could be triggered by 1 mM Mn²⁺ and La³⁺ and, to a lesser extent, by 2–4 mM Ca²⁺ and Mg²⁺. Maturation induced by 1.5 mM Mn²⁺ was inhibited by K⁺ concentrations above 0.25 mM. Potassium was inhibitory when added up to 2 hr before germinal vesicle breakdown occurred. In potassium-free media, maturation could be induced by incubation of oocytes under mild alkaline media (pH 8.5–9). A high percentage of medium-sized oocytes (stage IV according to Dumont) was induced to mature by progesterone in the absence of potassium. Maturation of oocytes in potassium-free media was normal by the criteria of germinal vesicle breakdown, production of maturation promoting factor, vitelline membrane activation, and inhibition by known maturation inhibitors.     

Short-term analysis of the phytoplankton structure and dynamics in two ponds with distinct trophic states from Cierva Point (maritime Antarctica)

Phytoplankton communities dominating Musgos and Papúa ponds with differing trophic states were sampled over 3 days enabling the detection of the physiological and population responses of microalgae to short-scale changes in biotic and abiotic factors, rather than frequently analyzed changes in community composition responses to long-scale environmental changes. We hypothesized that both environments undergoing diel changes would be dominated by phytoplankton with generalist strategies, while community structure would be mostly dictated by the trophic state of each water body. The phytoplankton biovolumes of both ponds were strongly dominated by euplanktonic nanoflagellated Chlorophyta, while phycocyanin-rich picocyanobacteria dominated the picophytoplankton. Parallel diel cycles of air and water temperatures were more pronounced on a sunny, warm day which prompted algal photosynthesis, revealed by strong increases in dissolved oxygen and pH. Nutrient and phytoplanktonic chlorophyll a confirmed the hypertrophic condition of Papúa pond. This accounted for the distinct community composition encountered in each pond, which remained stable throughout the study, as revealed by the SIMI index. The inverse relationship between the chl a/abundance ratio and the abundances of dominant species together with varying net growth rates (k′) showed algal reproduction, yet densities remained rather stable in both cases. In Musgos pond, fluctuations in k′ for small and median ciliates shadowed those of pico- and nanophytoplankton, respectively, strongly suggesting that they can control algal growth in these 2-level trophic chains.     

Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis

Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1^−/− mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1^−/− pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1^−/− mice. Keratinocytes isolated from the skin of Sgpp1^−/− pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.