Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems

The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5 flanking sequences were fused to -glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (–358 to –211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (–211 to –80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.     

Molecular modeling of wild-type and antifolate resistant mutant Plasmodium falciparum DHFR

The development of drug resistance is reducing the efficiency of antifolates as antimalarials. This phenomenon has been linked to the occurrence of mutations in the parasite's dihydrofolate reductase (DHFR). In this way, the resistance to pyrimethamine and cycloguanil, two potent inhibitors of P. falciparum DHFR, is mainly related to mutations (single and crossed) at residues 16, 51, 59, 108 and 164 of the enzyme. In this work, we have refined a recently proposed homology-model of P. falciparum DHFR, and the resulting structure was used to obtain models for 14 mutant enzymes, employing molecular modeling. Ternary complexes of the mutant enzymes with these inhibitors have been superimposed to equivalent ternary complexes of the wild-type enzyme, allowing the proposition of hypotheses for the role of each mutation in drug resistance. Based on these results, possible reasons for antifolate resistance have been proposed.     

The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle

The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing device and estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group (TI; n=40) received a standard superovulation protocol with females of this group being between days 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B program containing 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were at day 0 (TII; n=30), between days 2 and 6 (TIII; n=30), days 7 and 12 (TIV; n=30), days 13 and 16 (TV; n=30) and days 17 and 20 (TVI; n=30) of the estrous cycle. Superovulation was induced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) at intervals of 12h. The donors received PGF2alpha (Cloprostenol) 48 h after beginning the treatment and CIDRs were removed 12h later. Artificial inseminations (AI) were performed 12 and 22 h after the initiation of estrus and embryos were collected 7 days after AI. The mean numbers (+/-S.E.M.) of total ova and embryos, viable (transferable) and degenerated embryos were 14.2+/-11.3, 7.4+/-6.9 and 3.2+/-3.5 (TI), 13.3+/-10.4, 7.1+/-6.2 and 3.3+/-4.3 (TII), 13.5+/-7.0, 8.1+/-6.7 and 2.3+/-3.0 (TIII), 17.4+/-9.9, 9.4+/-6.9 and 4.0+/-4.4 (TIV), 16.9+/-8.8, 9.8+/-8.1 and 2.7+/-2.5 (TV) and 13.0+/-7.8, 7.2+/-6.9 and 2.3+/-2.5 (TVI), with no significant differences (P>/=0.05) among groups. Pregnancy rates of 67.1% (TI; n=86/128), 60.8% (TII; n=59/97), 62.5% (TIII; n=73/115), 64.1% (TIV; n=84/131), 72.3% (TV; n=81/112) and 60.6% (TVI; n=63/104) were obtained with embryos transferred from these collections and did not differ significantly (P>/=0.05) among groups. The results of the present study allow us to conclude that a combination of steroid hormones may be used prior to superovulation in Nelore donors, at any stage of the estrous cycle without affecting the efficiency of embryo transfer programs.     

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of S o Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.     

Redescription of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae)

The phlebotomine sand flies Nyssomyia intermedia and Nyssomyia neivai are the probable vectors of American tegumentary leishmaniasis in the Southern and Southeastern regions of Brazil. These species form a complex, being difficult to separate between either females or males of the two members based on recognized morphological characteristics. Both N. intermedia and N. neivai are redescribed here in the search for characters that facilitate their correct identification. It was possible to differentiate females by means of spermathecal characteristics. Males could be separated with confidence by the tips of the genital filaments, which have the form of a deep spoon, the angle of the concavity being well accentuated in N. intermedia and much shallower in N. neivai.     

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.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

Temperature and meal size effects on the postprandial metabolism and energetics in a boid snake

We investigated the combined effect of meal size and temperature on the aerobic metabolism and energetics of digestion in Boa constrictor amarali. Oxygen uptake rates (Vd2;o2) and the duration of the digestion were determined in snakes fed with meals equaling to 5%, 10%, 20%, and 40% of the snake's body mass at 25 degrees and 30 degrees C. The maximum Vd2;o2 values attained during digestion were greater at 30 degrees C than at 25 degrees C. Both maximal Vd2;o2 values and the duration of the specific dynamic action (SDA) were attained sooner at 30 degrees C than at 25 degrees C. Therefore, the temperature effect on digestion in Boa is characterized by the shortening of the SDA duration at the expense of increased Vd2;o2. Energy allocated to SDA was not affected by meal size but was greater at 25 degrees C compared to 30 degrees C. This indicates that a postprandial thermophilic response can be advantageous not only by decreasing the duration of digestion but also by improving digestive efficiency. Maximal Vd2;o2 and SDA duration increased with meal size at both temperatures.     

Some effects of repeated handling and the use of a mask on stress responses in zebu cattle during restraint

The objective of this investigation was to evaluate the effect of repeated handling and the use of a mask on the order of entry, temperament, cortisol concentration and heart and respiratory rates of Brahman cattle during restraint in a squeeze chute. In the first experiment, 18 cows were restrained in the chute for 10min on 19 test-days and blood samples were collected during five of the trials. Habituation to handling was reflected by a decrease in cortisol concentration as the study advanced (P<0.01). In the second experiment, conducted 6 months later, animals were randomly allotted to two groups, alternating the use of a mask over test-days while restraining the cows in the chute for 3min. Respiratory and heart rates were measured at the beginning and end of each 3min trial. A blood sample was also taken at the end of each trial. Respiratory rates decreased while the animals were restrained and this decrease tended to be greater (P=0.09) when the animals were wearing masks (3.06) than when not (2.2breaths/min). Heart rates also decreased and more so for masked animals (10.6beats/min) than for non-masked (5.0, P<0.01). Cortisol values tended to be higher (9.9 versus 4.7ng/ml, P=0.08) when animals were restrained without a mask. Temperament scores were lower (P>0.01) when cows were masked and have a relative high correlation (r=0.58) with flight distance values, (P<0.05). Finally, the order of entrance was found consistent over test-days, (P<0.01). It is concluded that Brahman cattle: (a) habituate to repeat handling in a squeeze chute; (b) are less emotionally reactive when their eyes were covered during this process and (c) establish a repeatable order of entrance to the chute.