Construction of Improved Tools for Protein Localization Studies in Streptococcus pneumoniae

We have constructed a set of plasmids that allow efficient expression of both N- and C-terminal fusions of proteins of interest to fluorescent proteins mCherry, Citrine, CFP and GFP in the Gram-positive pathogen Streptococcus pneumoniae. In order to improve expression of the fluorescent fusions to levels that allow their detection by fluorescence microscopy, we have introduced a 10 amino acid tag, named i-tag, at the N-terminal end of the fluorescent proteins. This caused increased expression due to improved translation efficiency and did not interfere with the protein localization in pneumococcal bacteria. Localizing fluorescent derivatives of FtsZ, Wzd and Wze in dividing bacteria validated the developed tools. The availability of the new plasmids described in this work should greatly facilitate studies of protein localization in an important clinical pathogen.     

Acute Hypoxia Differentially Affects the NMDA Receptor NR1, NR2A and NR2B Subunit mRNA Levels in the Developing Chick Optic Tectum: Stage-Dependent Plasticity in the 2B–2A Ratio

It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1–NR2A and NR1–NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis.     

Dissolved carbon in an urban area of a river in the Brazilian Amazon

The main objective of this study was to evaluate dissolved organic and inorganic carbon dynamics along upstream and downstream reaches of the Acre River draining the city of Rio Branco, in the state of Acre, Brazil. Dissolved organic carbon (DOC) concentrations in the Acre River were significantly higher during the wet season, ranging from 385 ± 160 to 430 ± 131 ??M among the stations, with no difference in upstream and downstream concentrations. Dissolved inorganic carbon (DIC) showed an inverse pattern, with higher concentrations in the dry season, ranging from 816 ± 215 to 998 ± 754 ??M among the stations, as well as no difference in upstream and downstream DIC concentrations. Bicarbonate was the dominant DIC fraction and was mainly observed during the dry season. Due to lower pH values during the wet season, CO₂ partial pressure (pCO₂) in the Acre River was higher in the wet season, with values ranging from 4,567 ± 1,813 to 4,893 ± 837 ppm among the stations. Our results indicate that, although the Acre River drains a large city with significant sewage disposal into the river, seasonal hydrological processes are the main driver of dissolved carbon dynamics, even in the downstream study reach directly influenced by urbanization.     

Determinants of human immunodeficiency virus (HIV) prevalence in homosexual and bisexual men screened for admission to a cohort study of HIV negatives in Belo Horizonte,Brazil: Project Horizonte

Project Horizonte, an open cohort of homosexual and bisexual human immunodeficiency virus (HIV-1) negative men, is a component of the AIDS Vaccine Program, in Belo Horizonte, Minas Gerais, Brazil. The objective of this study was to compare volunteers testing HIV positive at cohort entry with a sample of those who tested HIV negative in order to identify risk factors for prevalent HIV infection, in a population being screened for enrollment at Project Horizonte. A nested case-control study was conducted. HIV positive volunteers at entry (cases) were matched by age and admission date to three HIV negative controls each. Selected variables used for the current analysis included demographic factors, sexual behavior and other risk factors for HIV infection. During the study period (1994-2001), among the 621 volunteers screened, 61 tested positive for HIV. Cases were matched to 183 HIV negative control subjects. After adjustments, the main risk factors associated with HIV infection were unprotected sex with an occasional partners, OR = 3.7 (CI 95% 1.3-10.6), receptive anal intercourse with an occasional partner, OR = 2.8 (95% CI 0.9-8.9) and belonging to the negro racial group, OR = 3.4 (CI 95% 1.1-11.9). These variables were associated with an increase in the risk of HIV infection among men who have sex with men at the screening for admission to an open HIV negative cohort.     

The Scottish Structural Proteomics Facility: targets, methods and outputs

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.     

Alteration in the endogenous intestinal flora of Swiss Webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.     

Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles

The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P < 0.06) and cultured without FSH (P < 0.1). There was a higher (P < 0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P < 0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P < 0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P < 0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.     

Characterization of pregnancy-associated glycoproteins extracted from zebu (Bos indicus) placentas removed at different gestational periods

In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.     

Immune depression in Rhodnius prolixus by seco-steroids, physalins

A comparative study of the effects of physalins, seco-steroidal substances of Physalis angulata (Solanaceae), on the immune reactions of R. prolixus was carried out. Ecdysis and mortality were not affected by treatment with physalins B, D, F or G (1-10 microg/ml of blood meal). R. prolixus larvae fed with blood containing physalins and inoculated with 1 microl of Enterobacter cloacae beta12 (5 x 10(3)/insect) exhibited mortality rates three times higher than controls. The insects treated with physalin B, and F (1 microg/ml) and inoculated with E. cloacae beta12 showed significant differences on lysozyme activity in the hemolymph compared to untreated insects. Furthermore, physalin D (1 microg/ml) significantly reduced the antibacterial activity. Concerning cellular immune reactions, all insects treated with physalins (1 microg/ml), exhibited drastic reductions in the quantity of yeast cell-hemocyte binding and subsequent internalization. Insects inoculated with bacteria and treated with physalins B, F and G showed reductions of microaggregate formation but physalin D did not. Physalins B and F also reduced total hemocyte count in the hemolymph. These results suggest that, in different ways, probably due to their different chemical structures, physalin B, D, F and G are immunomodulatory substances for the bloodsucking insect, R. prolixus.     

Changes in leaf epicuticular wax,gas exchange and biochemistry metabolism between <Emphasis Type="Italic">Jatropha mollissima</Emphasis> and <Emphasis Type="Italic">Jatropha curcas</Emphasis> under semi-arid conditions

Jatropha curcas and Jatropha mollissima plants were evaluated under conditions of high (HSM) and low (LSM) soil moisture in a semi-arid environment, as changes in the content and concentration of epicuticular wax and the leaf metabolism which could have a relationship with drought tolerance. Besides epicuticular wax, gas exchange, antioxidant system and biochemical parameters of the photosynthetic metabolism were measured. The epicuticular wax content increased only in J. mollissima leaves 95 % under LSM, when compared with HSM conditions. Therefore, J. curcas invested less in the production of long-chain n-alkanes than did J. mollissima under LSM conditions. J. mollissima plants showed the highest CO₂ assimilation rate during the HSM period compared to J. curcas. Both species showed high stability in some leaf biochemistry products, highlighting the highest sugar content, free amino acids, total soluble protein, and photosynthetic pigments in the leaves of J. mollissima plants under both of the soil moisture conditions. Moreover, the stability and performance of the different parameters, such as morphologic variables, seem to allow J. mollissima plants to tolerate semi-arid conditions.