Conservation genetics of the European brown bear - a study using excremental PCR of nuclear and mitochondrial sequences

In the Brenta area of northern Italy, a brown bear Ursus arctos population is rapidly going extinct. Restocking of the population is planned. In order to study the genetics of this highly vulnerable population with a minimum of stress to the animals we have developed a PCR-based method that allows the study of mitochondrial and nuclear gene sequences from droppings collected in the field. This method is generally applicable to animals in the wild. Using excremental as well as hair samples, we show that the Brenta population is monomorphic for one mitochondrial lineage and that female as well as male bears exist in the area. In addition, 70 samples from other parts of Europe were studied. As others have previously reported, the mitochondrial gene pool of European bears is divided into two major clades, one with a western and the other with an eastern distribution. Whereas populations generally belong to either one or the other mitochondrial clade, the Romanian population contains both clades. The bears in the Brenta belong to the western clade. The implications for the management of brown bears in the Brenta and elsewhere in Europe are discussed.     

ADSORPTION AND UPTAKE OF COPPER BY THE GREEN ALGA SCENEDESMUS SUBSPICATUS (CHLOROPHYTA)1

Copper (II) accumulation has been investigated in the green alga Scenedesmus subspicatus G. Brinkmann considering both adsorption and uptake kinetics. Experiments were conducted in a Cu- and PH-buffered medium at different free Cu²⁺ concentrations that were neither growth limiting nor toxic. We distinguished between adsorption on the cell surface and intracellular uptake by extracting copper from the cells with EDTA. Data from short-term experiments were compared with data obtained from experiments under steady state conditions. The accumulation of Cu can be described by two processes, an initial fast adsorption occurring within a minute followed by a slower intracellular uptake. Metal uptake followed Michaelis-Menten kinetics and is mediated by two systems, one with low and the other with high affinity. The maximum uptake rates (1.30 × 10^?-¹⁰ mol·[g dry wt algae]^?1· min^?1, 3.67 × 10^?-⁹ mol·[g dry wt algae]^?1·min^?1), and the half-saturation constants (6.84 × 10^?-¹⁴ M, 2.82 × 10^?-¹² M) for the two uptake systems were determined using the Lineweaver-Burk plot. The calculated maximum concentration of binding sites on the surface of the algae is initially higher (9.0 × 10^?-⁶ mol Cu.[g dry wt algae]^?1) than under steady state conditions (2.9 × 10^?-⁶ mol Cu·[g dry wt algae]^?1). This suggests that the initial binding to the algal surface comprises the binding to specific transport ligands as well as to inert adsorption sites. The conditional stability constant of the Cu binding to surface ligands was calculated as log K_Cu= 11.0 at pH 7.9. This freshwater alga has a high ability to accumulate Cu, reflecting its adaptation to the bioavailable concentration of copper.     

Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.

The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.     

A comparison of in situ and simulated in situ methods for estimating oceanic primary production

Primary production data measured by in situ (IS) and ‘simulated’in situ (SIS) incubations were compared. To minimize differencesbetween the two types of incubations, SIS experiments were conductedin temperature-controlled incubators in which the spectral distributionand irradiance were adjusted to approximate IS conditions. ISavailable irradiance (I_Is) was computed from vertical attenuationof integrated surface irradiance. Vertical attenuation was estimatedusing a spectral irradiance model, validated by measured profilesof the vertical attenuation coefficient. IS incubations werecarried out using two methods. The first involved deploymentof bottles on a drifting array for whole-day (dawn to dusk)incubations. The second method employed an autonomous submersibleincubation device that performed short term (<1 h) incubationsat multiple depths. Differences between whole-day IS and SISincubation estimates were attributed partially to differencesbetween I_IS and SIS-available irradiance (I_SIS). Photosynthesis-irradiance(P-I) properties of IS and SIS populations from the whole-dayincubations were not significantly different. P-I propertiesof the short-term IS and SIS populations were significantlydifferent, although estimates of P^B (mg C mg Chl^–1 h^–1)from contemporaneous IS and SIS incubations did not differ by>40%. Integrated water-column primary production (IPP) estimatedusing P-I models derived from SIS data were within 15% of ISestimates of IPP.     

ADSORPTION OF FULVIC ACID ON ALGAL SURFACES AND ITS EFFECT ON CARBON UPTAKE

Adsorption of Suwannee River fulvic acid (SRFA) to algal surfaces of three green algae was studied at environmentally relevant pH values (4 –7) and SRFA concentrations (5–100 mg·L ^{− 1}). The influence of adsorbed SRFA on carbon uptake of Scenedesmus subspicatus Chodat was also examined. Although no adsorption was observed at neutral pH values (pH 6 and 7), at pH 4 up to 31 mg SRFA·m ^{− 2} and at pH 5 up to 4 mg SRFA·m ^{− 2} was adsorbed to the algal surfaces. Electrophoretic mobility measurements of S. subspicatus demonstrated an increase in the negative surface charge of the alga in the presence of SRFA at pH 4. The adsorbed SRFA also influenced ¹⁴C uptake in S. subspicatus; in this case, enhanced carbon uptake could be related to the amount of adsorbed SRFA. The binding of humic substances by algal surfaces was interpreted as the result of hydrogen bonding and hydrophobic interactions.     

Intronic gene conversion in the evolution of human X-linked color vision genes

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.      

Evidence for increased formation of preprothrombin and the noninvolvement of vitamin K-dependent reactions in sex-linked hyperprothrombinemia in the rat.

The rate of appearance of plasma prothrombin was measured in vitamin K-deficient male and female rats after the administration of vitamin K₁, and the disappearance of prothrombin was measured in normal rats after injection of cycloheximide. The results suggest that hyperprothrombinemia in female rats is due to a faster rate of formation of the clotting protein rather than to a slower rate of its degradation. Preprothrombin activity in liver microsomes was higher in warfarin-treated female rats than in warfarintreated male rats; but the activity of preprothrombin in liver disappeared at approximately the same rate in both sexes after administration of vitamin K. The rate and extent of vitamin K-dependent formation of γ-carboxyglutamic acid and the appearance of prothrombin activity in vitro were not significantly different between the sexes. These results suggest that elevated levels of plasma prothrombin in female rats are probably due to a higher rate of synthesis of preprothrombin and not to any difference in the vitamin K-dependent step. A difference was observed in the amount of cycloheximide required to inhibit synthesis of liver microsomal protein in the two sexes.     

ACCUMULATION AND REDUCTION OF ARSENATE BY THE FRESHWATER GREEN ALGA CHLORELLA SP. (CHLOROPHYTA)

Arsenate accumulation and reduction kinetics at both high and low phosphate concentrations were investigated in the green alga Chlorella sp, isolated from the arsenic-contaminated Upper Mystic Lake near Boston, MA. Growth rate, accumulated cellular arsenic, and release of As(III) were determined over a range of arsenate concentrations. Arsenate inhibited growth and reduced final cell yield at high phosphate concentration. However, growth rate, final cell yield, and cellular arsenic content were all enhanced by higher arsenate concentrations in cultures grown at a low concentration of phosphate. The traditional view that phosphate-limited cells are necessarily more sensitive to As(V) toxicity may not be correct. The reduction rates of As(V) by Chlorella sp. obtained in our laboratory were similar to net reduction rates measured in epilimnetic water from the Upper Mystic Lake, demonstrating the importance of phytoplankton in arsenic reduction in freshwater.     

Differential phosphoproteome profiling reveals a functional role for VASP in Helicobacter pylori-induced cytoskeleton turnover in gastric epithelial cells

Infection with Helicobacter pylori induces various gastric diseases, including ulceration, gastritis and neoplasia. As H. pylori-induced cellular mechanisms leading to these disease states are widely unclear, we analysed the phosphoproteome of H. pylori-infected gastric epithelial cells. Phosphoproteins from infected cells were enriched using affinity columns and analysed by two-dimensional gel electrophoresis and mass spectrometry. Eleven novel phosphoproteins that showed differentially regulated phosphorylation levels during H. pylori infection were identified. Interestingly, the identified proteins were actin-binding, transport and folding, RNA/DNA-binding or cancer-associated proteins. We analysed functions of one identified H. pylori-regulated candidate, the vasodilator-stimulated phosphoprotein (VASP). H. pylori induced VASP phosphorylation at residues Ser157, Ser239 and Thr278, which was enhanced by the bacterial oncogene cytotoxin-associated gene A. Overexpression of a phosphorylation-resistant VASP mutant efficiently blocked host cell elongation. We identified cGMP-dependent protein kinase G-mediated Ser239 and Thr278 phosphorylation of VASP as a crucial event in H. pylori-dependent host cell elongation. These results suggest that phosphorylated VASP could be a novel target candidate for therapeutic intervention in H. pylori-related gastric diseases.     

Peptidic nanoparticles: for cancer diagnosis and therapy

A challenging topic in cancer research is to create drug delivery system that can bring in a specific and noncytotoxic manner a therapeutic compound. Usually, tumor targeting requires very specific compounds. Currently, peptide analogues like somatostatin, neurotensin, or bombesin are used to target G-coupled receptors, which are overexpressed on tumor cells. However, many of those analogues are rapidly degraded in the plasma and are cytotoxic [1–2]. Due to the limited efficiency and high toxicity of conventional chemotherapy different strategies have been developed for non-cytotoxic cancer treatment and cancer localization [3–5]. The recent development in bio-nanotechnology offers new avenues for cancer therapy. A lot of studies have been devoted to nanoparticulate delivery systems (10–100nm) like lipid or polymer particles [6–8]. Due to the nanometer sized of such cargos, the transportation of therapeutic compounds in the blood stream is increased in terms of time circulation. But their surface functionalization to improve drug-targeting properties is usually complicated and rather uneffective. We have recently designed a novel type of functional nanoparticles with regular icosahedral symmetry, mimicking small, rigid viral capsids (Fig. 1 (A)) and a diameter of about 17 nm (Fig. 1 (C)) which self-assemble from single polypeptide chains (Fig. 1 (B)).