Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

Antimicrobial Resistance, Virulence Genes, and Genetic Lineages of Staphylococcus pseudintermedius in Healthy Dogs in Tunisia

Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec _canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg _v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.     

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC₅₀ values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC₅₀ values.     

Genetic characterization of 12 heterologous microsatellite markers for the giant tropical tree Cariniana legalis (Lecythidaceae)

Twelve microsatellite loci previously developed in the tropical tree Cariniana estrellensis were genetically characterized in Cariniana legalis. Polymorphisms were assessed in 28 C. legalis individuals found between the Pardo and Mogi-Guaçu River basins in the state of São Paulo, Brazil. Of the 12 loci, 10 were polymorphic and exhibited Mendelian inheritance. The allelic richness at each locus ranged from 2-11, with an average of 7 alleles per locus, and the expected heterozygosity ranged from 0.07-0.88. These loci showed a high probability of paternity exclusion. The characteristics of these heterologous microsatellite markers indicate that they are suitable tools for investigating questions concerning population genetics in C. legalis.     

Failure of the gamma-aminobutyric acid (GABA) derivative, baclofen, to stimulate growth hormone secretion in heroin addicts.

In order to establish possible alterations in the gamma aminobutyric acid (GABA)ergic control of growth hormone (GH) secretion in heroin addicts, ten patients (age, 25.8 +/- 1.07 yr (mean +/- SE); duration of heroin addiction, range 3-8 yr; weight, 67.3 +/- 0.87 kg body weight), and ten age (29.1 +/- 0.84 yr)- and weight (69.7 +/- 0.87 kg)-matched normal controls were tested with the GABAergic B-receptor agonist baclofen (10 mg p.o. at 09.00 h) (experimental test) or a placebo (control test). Blood samples for GH assay were taken every 15 min for the next 150 min. Normal controls underwent one control and one experimental test. Heroin addicts were submitted to both baclofen and placebo test twice, once around the time of their admission to a recovery community for drug abusers, when they were still assuming heroin, and again after two months of permanence in the community. From the time of their admission to the community, the patients were forbidden to use heroin. For two weeks after admission they were treated with clonidine and acetylsalicilic acid to attenuate withdrawal symptoms. Thereafter, the patients underwent a period of wash-out of pharmacological treatments for at least 6 weeks before being retested. Basal GH levels were similar in normal controls and heroin addicts in all tests and remained unmodified during control tests in all subjects. The administration of baclofen increased four times the serum GH levels within 120 minutes in the normal controls, whereas it did not modify serum GH concentrations in heroin addicts either during the period of drug abuse or after two months of abstinence. These data show that the control of GH secretion mediated by GABAergic B-receptors is impaired in heroin addicts. It is hypothesized that this neuroendocrine alteration might represent a trait marker of heroin addiction, or more likely, that it was a consequence of a long addiction to heroin persisting after two months of abstinence.     

Exhibition of persistent and drug-tolerant L-form habit of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> during infection in rats

A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.     

Seasonal activity budget of adult baltic ringed seals

Although ringed seals are important components in oceanic and fresh water ecosystems at high latitudes, little is known about how they exploit these harsh environments. Seasonal activity and diving behaviour of 19 adult Baltic ringed seals were studied by satellite telemetry. We elaborated an activity budget for ten months of the year, extending over the period from moult to the breeding season. Seals from three main regions showed explicit site fidelity and the distributions of animals tagged from different areas did not overlap, suggesting separate stocks. Both the mean duration and the mean depth of dives peaked in June and July. Seals spent 70% (females) to 85% (males) of their time diving in June and July which decreased to 50% in late autumn. Less than one percent of dives exceeded 10 min in females, while 10% of male dives lasted longer than 10 min in June to September. Less than one percent of dives lasted for more than 25 min. Both females and males were most active during day time and hauled out predominantly during the night. Activity patterns during the summer are suggested to be correlated to energy accumulation and prey availability. The information on seasonal activity budget is crucial for developing population energetic models where interactions between ringed seals and other trophic levels can be evaluated.     

Consortium diversity of a sulfate‐reducing biofilm developed at acidic pH influent conditions in a down‐flow fluidized bed reactor

Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate‐reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down‐flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate‐reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate‐reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate‐reducers, improving the acetate removal via sulfate reduction in the reactor.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.