Visualization of thrombin receptors on mouse embryo fibroblasts using fluorescein-amine conjugated human α-thrombin

The localization of thrombin receptors on mouse embryo (ME) cells has been examined by direct fluorescence microscopy using a fluorescein aminelabeled thrombin. Two fluorescein amines, 4-(N-6-aminoethyl thioureal)-fluorescein and 4-(N-6-aminohexyl thioureal)-fluorescein, were synthesized and attached to the carbohydrate moiety of highly purified human α-thrombin by periodate oxidation of the carbohydrate and selective reduction of the Schiff's base using sodium cyanoborohydride. Preparations of fluorescent thrombin with from 1 to 4 fluoresceins per molecule of thrombin retained their ability to proteolytically cleave fibrinogin to form fibrin clots, to bind to thrombin receptors on ME cells, and to initiate cell division. After incubating mitogenic concentrations of the fluorescein amine labeled thrombin with ME cells at 4°C, a diffuse fluorescent pattern was observed over the surface of the ME cells. This diffuse pattern was specific: it was not observed on cells from parallel cultures incubated with fluorescent thrombin plus a 20-fold excess of unlabeled thrombin. Thus, thrombin receptors appear to be distributed randomly over the surface of ME cells prior to interaction with thrombin. Increasing the temperature to 37°C following binding at 4° C resulted in a rapid dissociation of the fluorescent pattern from the cells leaving only the autofluorescent vesicles. This result may reflect the unique ability of thrombin to proteolytically cleave its own receptor.     

Relation between plasma hormone profiles,symptoms, and response to oestrogen treatment in women approaching the menopause.

Out of a consecutive series of 300 patients seen at a menopause clinic, 82 complained of symptoms generally associated with the climacteric, although they were still menstruating. Vasomotor disturbances were absent in 42 of these patients (group 1) and present in 40 (group 2). Headaches, insomnia, and dyspareunia were the most common complaints among the women with vasomotor symptoms, whereas loss of libido and depression predominated in those without. Conjugated equine oestrogens (Premarin) 1.25 mg daily given for three weeks out of four relieved nearly all symptoms in group 2, but in group 1 the response was disappointing. The mean plasma oestradiol concentration in women with vasomotor symptoms was significantly lower than that observed during days 1-10 of the menstrual cycle, but plasma testosterone values were not significantly different from those observed in younger women. Plasma follicle-stimulating hormone (FSH) and luteinising hormone (LH) concentrations were similar to those seen after the menopause. Concentrations of these hormones in the women without vasomotor symptoms were similar to those in the younger, regularly menstruating women. After six months of oestrogen treatment patients in group 2 had a 2.1-fold increase in mean plasma oestradiol concentration, and plasma FSH and LH concentrations were reduced to 39% and 66% of their pretreatment values respectively; in group 1, however, no such pronounced changes occurred. High concentrations of FSH were present in patients with oestrogen-responsive symptoms, 15 U/1 being the diagnostic cut-off point. This measurement in the presence of characteristic symptoms therefore constitutes the best method of selecting patients for oestrogen-replacement therapy.     

Chiroptical and stoichiometric evidence of a specific,primary dimerisation process in alginate gelation

The stoichiometry of calcium-ion chelation to alginate chains has been investigated by circular dichroism (c.d.), and by equilibrium dialysis in the presence of various concentrations of sodium chloride. C.d. intensity in the carboxylate π → π * spectral region increases linearly with calcium-ion concentration up to a level equivalent to half the total poly-L-guluronate stoichiometric requirement, and thereafter shows little further change. Similarly, the level of bound calcium resistant to displacement by swamping concentrations of sodium ions is equivalent to half the stoichiometric requirement of poly-L-guluronate chain-sequences alone. In terms of the previously developed “egg-box” model of co-operative junction-zone formation in alginate gelation, these results are interpreted as showing that the primary mechanism of interchain association is by dimerisation of poly-L-guluronate chain-segments in a regular, buckled, two-fold conformation related to that characterized for the free acid in the solid state, with tight interchain chelation of calcium to the carboxylate groups on the interior faces of the dimer (i.e., half the carboxylate residues of the participating chain-sequences). This interpretation is entirely consistent with previous evidence from electron microscopy, and offers a simple rationalisation of experimental results from competitive-ion binding studies.     

SIRT1 Activation by Small Molecules: KINETIC AND BIOPHYSICAL EVIDENCE FOR DIRECT INTERACTION OF ENZYME AND ACTIVATOR?

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340–8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.     

Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens

Seven paralytic peptides were isolated and identified from lepidopteran hemolymph. All of these peptides cause rapid, rigid paralysis when injected into Manduca sexta and some other lepidopteran larvae. Each peptide contains 23 amino acid residues including 2 cysteines and the carboxyl termini are acidic. Synthetic peptides in the disulfide or reduced forms, and as carboxyl-terminal acids or amides were equally paralytic. The most potent paralytic peptide, Mas PP I, has the following sequence: H-Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu- Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The two peptides from M. sexta hemolymph are remarkable in that they are autoparalytic (i.e. factors in collected hemolymph that are paralytic when injected into the same larvae).     

Cultivation and biomass production of the diatom Thalassiosira weissflogii as a live feed for white-leg shrimp in hatcheries and commercial farms in Vietnam

This study investigated the biomass production process from the laboratory to the pilot scale in order to use the nutrient-rich biomass of the diatom Thalassiosira weissflogii as live feed for white-leg shrimp (Litopenaeus vannamei) at larval stages (zoeal, mysis, and postlarval) and in commercial production in hatcheries in Vietnam. Our results showed that T. weissflogii was successfully cultured in 1–2 L Erlenmeyer flasks, 0.2–3.5 m³ composite tanks, and 6.5 m³ tubular photobioreactors, with the highest cell density of 1.6 × 10⁶ cells mL^?1 reached after 6 days of culture. Under optimal culture conditions, the protein, lipid, and carbohydrate contents in this algal biomass were 13.2%, 20.0%, and 10.0% of dry cell weight, respectively. The fatty acid composition contains high amount of palmitic acid (C16:0, 43.11% of total fatty acid), and polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5ω-3), approximated 16.5% of total fatty acid. In a 50 L larval rearing tank, at the optimal stocking density of 125 nauplii L^?1, the survival percentage (75.55%), the total body length (from 5.376 ± 0.007 to 10.860 ± 0.030 mm), and weight (at from PL₁ to PL₁₂ stages) (from 0.145 ± 0.002 to 1.158 ± 0.005 g) of the white-leg shrimp larvae reached the highest values but the metamorphosis time (234 h) was shortest compared with the other stocking densities. Further, adding living T. weissflogii biomass to the diet of white-leg shrimp larvae at the nauplii 6 stage led to an increase in the body length, weight, and survival percentage of white-leg shrimp larvae of 21.17%, 35.7%, and 33% higher compared with those of larvae fed the control diet (without the addition of T. weissflogii), respectively. At the same time, the metamorphosis time of larvae (from Z₁ to PL₁) decreased by 4 h compared to the control group. In intensive ponds (area of 6400 m² pond^?1), using seed stocks at the postlarvae 12 stage that had been fed T. weissflogii, the final weight, yield, and survival percentage of the shrimp were increased by 7.3%, 14.2%, and 16.3%, respectively, compared with those of the control group. There were no statistically significant differences in the protein and carbohydrate contents in the shrimp flesh among the experimental and control group (p > 0.05). The lipid, omega-3, omega-6, and omega-9 fatty acid contents of shrimp flesh in experiment formula (per 100 g shrimp) were 1.21 g, 72.9 mg, 114 mg, and 86.1 mg, 11%, 29%, 21.6%, and 17.7% higher than that those in control, respectively. The obtained results show the great potential of using T. weissflogii as live feed on white-leg shrimp farms in Vietnam.

     

Proteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnaea stagnalis Exposed to Different Endocrine Disrupting Chemicals

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 µg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.     

Racial Discrimination & Cardiovascular Disease Risk: My Body My Story Study of 1005 US-Born Black and White Community Health Center Participants (US)

Objectives

To date, limited and inconsistent evidence exists regarding racial discrimination and risk of cardiovascular disease (CVD).

Methods

Cross-sectional observational study of 1005 US-born non-Hispanic black (n = 504) and white (n = 501) participants age 35–64 randomly selected from community health centers in Boston, MA (2008–2010; 82.4% response rate), using 3 racial discrimination measures: explicit self-report; implicit association test (IAT, a time reaction test for self and group as target vs. perpetrator of discrimination); and structural (Jim Crow status of state of birth, i.e. legal racial discrimination prior 1964).

Results

Black and white participants both had adverse cardiovascular and socioeconomic profiles, with black participants most highly exposed to racial discrimination. Positive crude associations among black participants occurred for Jim Crow birthplace and hypertension (odds ratio (OR) 1.92, 95% confidence interval (CI) 1.28, 2.89) and for explicit self-report and the Framingham 10 year CVD risk score (beta  = 0.04; 95% CI 0.01, 0.07); among white participants, only negative crude associations existed (for IAT for self, for lower systolic blood pressure (SBP; beta  = −4.86; 95% CI −9.08, −0.64) and lower Framingham CVD score (beta  = −0.36, 95% CI −0.63, −0.08)). All of these associations were attenuated and all but the white IAT-Framingham risk score association were rendered null in analyses that controlled for lifetime socioeconomic position and additional covariates. Controlling for racial discrimination, socioeconomic position, and other covariates did not attenuate the crude black excess risk for SBP and hypertension and left unaffected the null excess risk for the Framingham CVD score.

Conclusion

Despite worse exposures among the black participants, racial discrimination and socioeconomic position were not associated, in multivariable analyses, with risk of CVD. We interpret results in relation to constrained variability of exposures and outcomes and discuss implications for valid research on social determinants of health.     

Male-male competition and repeated evolution of terrestrial breeding in Atlantic Coastal Forest frogs*

Terrestrial breeding is a derived condition in frogs, with multiple transitions from an aquatic ancestor. Shifts in reproductive mode often involve changes in habitat use, and these are typically associated with diversification in body plans, with repeated transitions imposing similar selective pressures. We examine the diversification of reproductive modes, male and female body sizes, and sexual size dimorphism (SSD) in the Neotropical frog genera Cycloramphus and Zachaenus, both endemic to the Atlantic rainforest of Brazil. Species in this clade either breed in rocky streams (saxicolous) or in terrestrial environments, allowing us to investigate reproductive habitat shifts. We constructed a multilocus molecular phylogeny and inferred evolutionary histories of reproductive habitats, body sizes, and SSD. The common ancestor was small, saxicolous, and had low SSD. Terrestrial breeding evolved independently three times and we found a significant association between reproductive habitat and SSD, with shifts to terrestrial breeding evolving in correlation with decreases in male body size, but not female body size. Terrestrial breeding increases the availability of breeding sites and results in concealment of amplexus, egg-laying, and parental care, therefore reducing male-male competition at all stages of reproduction. We conclude that correlated evolution of terrestrial reproduction and small males is due to release from intense male-male competition that is typical of exposed saxicolous breeding.