Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

Changes in large herbivore populations across large areas of Tanzania

We collated aerial census data collected during the late 1980s to early 2000s for large herbivore populations in eight large census zones in Tanzania. Of the ungulate populations that showed significant changes in densities at the start versus end of this decade, most declined; very few populations increased significantly. Thomson's gazelle, Grant's gazelle, hartebeest, reedbuck, roan antelope, sable antelope, warthog and zebra, for example, declined in over 50% of the zones where they were surveyed. Interestingly, small‐bodied species fared particularly poorly in many census zones, whereas elephant and giraffe generally fared well across the country. Most populations of all herbivores declined in some portions of the country (e.g. Burigi‐Biharamulo, Katavi, Greater Ruaha and Tarangire census zones). These surveys suggest that, even in a country renowned for its protected areas and conservation commitment, some large herbivore populations need more conservation attention in order to remain stable.     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family.     

Origin and sequence of chromosome replication in Escherichia coli

Two methods have been used to determine the origin and direction of chromosome replication in Escherichia coli: gradient of marker frequency and sequence of replication in synchronized cultures. In both cases, DNA-DNA hybridization was used to assay for gene dosage. A series of isogenic strains were made lysogenic for phage λ and for phage Mu-1, with phage Mu-1 in a different chromosomal location in each strain. In a first group of experiments, DNA from exponential cultures of the various strains was extracted, denatured, immobilized on filters and hybridized against a mixture of differentially labeled phage λ and phage Mu-1 DNA. This was done for several culture conditions. The ratio of hybridization Mu-1/λ gives a measurement of the dosage of the chromosome region where phage Mu-1 is integrated. A plot of this ratio versus map position reflects the marker frequency distribution.     

Viability and Virulence of Experimentally Stressed Nonculturable Salmonella typhimurium

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities.     

How the ovarian follicle of Podarcis sicula recycles the DNA of its nurse,regressing follicle cells

In Podarcis sicula specialized follicle cells send reserve materials to the previtellogenic oocyte via intercellular bridges. Immediately before the onset of vitellogenesis this transferring becomes particularly massive so that the cell volume significantly reduces, meanwhile in the nucleus the morphological alterations typical of apoptosis appear. To clarify why these follicle cells are not simply fully resorbed by the oocyte and to determine whether their DNA is discarded or recycled, we carried out a series of morphological and biochemical investigations. The finding that large macromolecular scaffolds are formed and that these are able to retain the DNA until it is extensively cut by two different endonucleases suggests that regression of the follicle cells is programmed and that the fate of their DNA is strictly controlled. Following its genetical neutralization via fragmentation, the DNA is apparently recycled by being transferred into the oocyte via the intercellular bridges, that, in fact, remain open until the very late stages of cell regression. The small DNA fragments reaching the oocyte cytoplasm would not interfere with meiosis completion but could significantly contribute to the stock of reserve materials to the advantage of the growing oocyte and/or developing embryo. Mol. Reprod. Dev. 51:421–429, 1998. © 1998 Wiley-Liss, Inc.     

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

Fertilization is a complex and multiphasic process, consisting of several steps, where egg‐coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm‐associated β‐N‐acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid‐phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well‐defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β‐N‐acetylglucosamine, but not core β‐N‐acetylglucosamine residues. The capacity of sperm β‐N‐acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β‐N‐acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β‐N‐acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm–egg interactions in Drosophila.     

Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis,Lucinidae) and Its Symbiont Population

The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.The entire marine Lucinidae family, found in a wide range of sulfidic habitats, lives in association with chemoautotrophic sulfide-oxidizing bacterial symbionts, generally hosted in the gills of the bivalve. Lucinids are usually found in shallow water, such as intertidal mud or seagrasses (4, 53), in deeper water, e.g., Bathyaustriella thionipta (30), and in deep oceans at a 2,000-m depth, i.e., Lucinoma kazani (21, 55). The chemoautotrophic endosymbionts involved in such relationships are always localized inside specialized cells called bacteriocytes, and they have been found in several genera of the Lucinidae family, such as Codakia (4, 28), Loripes (39, 43), Lucina, and Lucinoma (17). Sulfur granules inside the symbiont cytoplasm have been demonstrated in most of the investigated species. The intracellular symbionts take energy from the oxidation of reduced sulfur compounds (27, 56, 59) and synthesize organic molecules by CO₂ fixation in a Calvin-Benson cycle, translocated to the host (18). This relationship between the host and its symbionts represents the autotrophic pathway for host nutrition (27). It has also been suggested that in symbiotic bivalves, intracellular digestion of the symbionts may be a nutrient source for the host, based on studies of hydrothermal vent and shallow water bivalves (6, 26, 39).The relative importance of the autotrophic versus heterotrophic nutritional pathway can be estimated by measuring the carbon isotope (δ-¹³C) ratios in the host tissue. Measuring this δ-¹³C ratio on a wide range of invertebrates suggested that bivalves, including members of the Lucinidae, that live in reduced sediment may obtain a significant proportion of their organic carbon from chemoautotrophic endosymbionts (4, 10, 51, 52, 57). This suggestion was in agreement with the reduced functional digestive system previously described for the Lucinidae family (2, 53). Structural and morphological studies of gills of a few lucinids (belonging to the genera Lucina and Lucinoma) strongly suggested that symbionts play an important role in host nutrition since they occupy about 30% of the gill tissue and produce most of the host energy (17). Nevertheless, an alternative pathway for feeding, i.e., heterotrophic particulate feeding, could occur in some of the lucinid bivalves, since diatoms were found in the stomach of some lucinids (57). Duplessis et al. (22) showed that particulate feeding could be an important part of the nutritional strategy in symbiont-bearing Lucinoma, as opposed to the anatomical features that gave the impression that this bivalve relied only on symbiont nutrition.In natural habitats, chemoautotrophic bivalves live at the interface between an anoxic sulfide-generating zone and water column oxygenated sediment. However, even if they are not close to a vent, these symbiotic organisms often have to deal with environments that are periodically depleted of oxygen (5, 12) and with extremely low sulfide concentration (13, 15, 16). These natural environmental variations lead to annual and seasonal changes in the δ-¹³C ratio, as observed for some thyasirid species (13, 14). This δ-¹³C ratio variation may be assumed to correspond to the capability of the host to rely on both autotrophic and heterotrophic pathways, and the preponderance of one pathway versus the other in the mixotrophic diet has been considered to be the way in which these organisms deal with changes in the chemical composition of their environment.Apart from the decrease in symbiont abundance suggested by transmission electron microscopy (TEM) analysis and a decrease in sulfur and protein content in the gill tissue of thyasirids (20, 38, 40), little is known about the physiological status of these symbionts and the changes undergone by the symbiotic population of starved bivalves. A previous study of the population was carried out under natural conditions with Codakia orbicularis, a chemoautotrophic bivalve. This tropical bivalve lives in shallow-water sediment among the roots of seagrasses (Thalassia testudinum) (1). Like all lucinids studied so far, it is associated with sulfur-oxidizing symbionts (4, 27, 28) containing elemental sulfur in their cytoplasm (42). The bacterial symbiont of C. orbicularis, environmentally transmitted to the host (31), belongs to a single taxonomic group (Gammaproteobacteria) (25) and is shared by several other tropical lucinids (24, 25, 34, 35). Only a few data are available on the physiology of this symbiont. It was characterized by the presence of Rubisco and ATP sulfurylase enzymes and a δ-¹³C ratio typical of chemoautotrophic bivalves (4). Unlike other related thyasirids tested for nitrate respiration even under oxygenated conditions (37), the symbionts of C. orbicularis use oxygen as the primary electron acceptor (23). Initial investigations of the population of Codakia orbicularis'' symbiont revealed that the symbiotic population hosted by freshly collected individuals contained a high proportion of large bacterial cells containing multiple copies of their genome, typical of actively growing cells, despite the absence of dividing cells (11). It was assumed that the host could maintain a pure culture of the symbiont inside the bacteriocytes by regulating the entry and growth of newly recruited symbionts from sediment and probably regulating symbiont densities by host digestion (11).This study was undertaken to investigate the dynamics of the symbiotic population hosted by C. orbicularis under experimental conditions based on long-term starvation of bivalves, i.e., incubated without planktonic food. We set out the ultrastructural, structural, and physiological changes that occurred in the symbiont population by examining the host gill sections using TEM and fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH). A purified fraction of gill endosymbionts was analyzed by flow cytometry (FCM) to investigate the nucleic acid content and cell size of symbionts and by using the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) method and epifluorescence microscopy to detect the respiratory activity of symbionts. The modifications induced by host starvation in the symbiotic population are described for the period of long-term starvation.