Violacein antimicrobial activity on Staphylococcus epidermidis and synergistic effect on commercially available antibiotics.

AIMS: The study aimed to assess whether violacein has antimicrobial activity on Staphylococcus epidermidis and synergistically modulates the action of commercially available antimicrobial drugs. METHODS AND RESULTS: Violacein showed excellent antimicrobial activity on biofilm-forming and nonbiofilm-forming S. epidermidis strains (ATCC 35984) (ATCC 12228), with bacteriostatic (MIC = 20 µg ml-1 and 10 µg ml-1 respectively) and bactericidal effects (MBC = 20 µg ml-1 for both strains), observed in short periods of exposure. The violacein bactericidal concentration led to S. epidermidis death after 2-3 h of exposure. Additionally, violacein synergistically modulated the activity of different antimicrobial classes on S. epidermidis ATCC 12228 (81·8%; n = 9) and on S. epidermidis ATCC 35984 (54·5%; n = 6), reducing the MIC of these antibiotics by up to 16-fold. CONCLUSION: Violacein shows excellent antimicrobial activity on S. epidermidis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Violacein shows the potential for the development of a new drug for the treatment of infections caused by S. epidermidis.

Biased sex ratio and niche restriction in Baruscapillaria obsignata (Madsen 1945) (Nematoda, Capillariidae) from Columba livia (Aves, Columbidae).

In the present study populations of the avian nematode species Baruscapillaria obsignata are described from Columba livia. Male and female individuals were obtained from 27 birds, fixed in alcohol/formalin/acetic acid (AFA) and preserved in 70% ethanol. Nematodes were identified and then counted under a stereoscopic microscope. Baruscapillaria obsignata were much more frequent in the anterior third of the small intestine, and females were more abundant than males in all infra populations. The prevalence was 55.6%, mean intensity was 11.8 (median 11.0; range 1-31) and abundance 6.56. In the present study, we observed an aggregated distribution of parasite infrapopulations, as demonstrated by the value of the exponent of the negative binomial distribution, K = 0.2773; by the discrepancy index, D = 0.656 and by the variance/mean ratio, 12.44. The female/male sex ratios found in all infrapopulations were always greater than 1, showing a bias in favour of female abundance. This tendency was especially marked in infrapopulations containing fewer individuals. The sizes of infrapopulations ranged from 5 to 31 individuals. The mean sex ratio observed was 2.69 ± 3.28 (median 1.83; range 0-11). In infrapopulations with 5-15 individuals, the sex ratios observed varied from 2.6 to 11, while in those with 17-31 individuals, the sex ratios were lower, ranging from 1.7 to 2.4. There was a negative correlation between the intensity of infection and the sex ratio of infrapopulations. Results are discussed in terms of possible factors influencing the processes that lead to niche restriction and biased sex ratios in parasite infrapopulations.

Spatio-temporal dynamics of phytoplankton community in a well-mixed temperate estuary (Sado Estuary, Portugal).

Estuaries are highly productive ecosystems, which are strongly affected by several anthropogenic pressures. Phytoplankton is a key element for assessing the ecological quality status in these transitional waters. Moreover, understanding physico-chemical and biological drivers is crucial to disentangle their effect on the structure of phytoplankton community. The present work aims to study the effect of the main physico-chemical drivers on the phytoplankton community structure and dynamics in a temperate well-mixed estuary (Sado Estuary). Four sampling stations were analyzed monthly in three regions of the estuary, from 2018 to 2019. Surface water samples were collected to analyze the phytoplankton community and several concomitant physico-chemical parameters. Temperature, turbidity, salinity, and nutrients availability were the drivers that best explained the spatio-temporal patterns observed in the phytoplankton community. The upper estuary was characterized by higher phytoplankton cell abundances and biomass. Three phytoplankton groups stood out in the characterization of the estuarine assemblages: diatoms, cryptophytes, and dinoflagellates. Diatoms were the dominant group most of the year, being dominated by small cell species (single and chain-forming) upstream, and by larger chain-forming species downstream. Cryptophytes had a high contribution to the community in the inner regions of the estuary, while dinoflagellates contributed more for the community composition downstream, where high abundances of harmful algal species were sporadically found. Previous studies on the phytoplankton community dynamics in this estuary are limited to the 1990s. Thus, the present study provides insight into changes in the dominant phytoplankton groups of the Sado Estuary in the last 25 years, namely an increase in cryptophytes over diatoms in the inner estuarine regions, and an increase in dinoflagellates near the estuary mouth.

Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene.

The HER2 protooncogene encodes a 185-kD transmembrane phosphoglycoproteins, human epidermal growth factor receptor 2 (p185HER2), whose amplified expression on the cell surface can lead to malignant transformation. Overexpression of HER2/p185HER2 is strongly correlated with progression of human ovarian and breast carcinomas. Recent studies have shown that human T cells can be targeted with bispecific antibody to react against human tumor cells in vitro. We have developed a bispecific F(ab')2 antibody molecule consisting of a humanized arm with a specificity to p185HER2 linked to another arm derived from a murine anti-CD3 monoclonal antibody that we have cloned from UCHT1 hybridoma. The antigen-binding loops for the anti-CD3 were installed in the context of human variable region framework residues, thus forming a fully humanized BsF(ab')2 fragment. Additional variants were produced by replacement of amino acid residues located in light chain complementarity determining region 2 and heavy chain framework region 3 of the humanized anti-CD3 arm. Flow cytometry analysis showed that the bispecific F(ab')2 molecules can bind specifically to cells overexpressing p185HER2 and to normal human peripheral blood mononuclear cells bearing the CD3 surface marker. In additional experiments, the presence of bispecific F(ab')2 caused up to fourfold enhancement in the cytotoxic activities of human T cells against tumor cells overexpressing p185HER2 as determined by a 51Cr release assay. These bispecific molecules have a potential use as therapeutic agents for the treatment of cancer.

Grapevine under deficit irrigation: hints from physiological and molecular data.

BACKGROUND: A large proportion of vineyards are located in regions with seasonal drought (e.g. Mediterranean-type climates) where soil and atmospheric water deficits, together with high temperatures, exert large constraints on yield and quality. The increasing demand for vineyard irrigation requires an improvement in the efficiency of water use. Deficit irrigation has emerged as a potential strategy to allow crops to withstand mild water stress with little or no decreases of yield, and potentially a positive impact on fruit quality. Understanding the physiological and molecular bases of grapevine responses to mild to moderate water deficits is fundamental to optimize deficit irrigation management and identify the most suitable varieties to those conditions. SCOPE: How the whole plant acclimatizes to water scarcity and how short- and long-distance chemical and hydraulic signals intervene are reviewed. Chemical compounds synthesized in drying roots are shown to act as long-distance signals inducing leaf stomatal closure and/or restricting leaf growth. This explains why some plants endure soil drying without significant changes in shoot water status. The control of plant water potential by stomatal aperture via feed-forward mechanisms is associated with 'isohydric' behaviour in contrast to 'anysohydric' behaviour in which lower plant water potentials are attained. This review discusses differences in this respect between grapevines varieties and experimental conditions. Mild water deficits also exert direct and/or indirect (via the light environment around grape clusters) effects on berry development and composition; a higher content of skin-based constituents (e.g. tannins and anthocyanins) has generally being reported. Regulation under water deficit of genes and proteins of the various metabolic pathways responsible for berry composition and therefore wine quality are reviewed.

Violacein antimicrobial activity on Staphylococcus epidermidis biofilm.

The aim of this study was to evaluate the antimicrobial potential of violacein (VIO) on Staphylococcus epidermidis biofilm. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined, as well as the effect of VIO exposure time on microbial viability in mature biofilm. Violacein showed good antibiofilm action, inhibiting biofilm formation and eradicating mature biofilm of S. epidermidis at concentrations of 20 µg.mL-1 and 160 µg.mL-1, respectively. At concentrations equal to MBEC and 2x MBEC, the biofilm was eradicated in 3 h and 2h30min of incubation, respectively.When evaluating VIO modulating effect on the action of clinically-used drugs (vancomycin, cefepime, ciprofloxacin and meropenem), especial synergism was observed in the violacein-ciprofloxacin association, it can completely erradicated the mature biofilm at the concentration of 1/2xMBEC and 1/4xMBEC, respectively. VIO shows good antimicrobial action on S. epidermidis biofilm and has the potential to synergistically modulate the activity of clinically-used antimicrobials.

Acclimation to short-term low temperatures in two Eucalyptus globulus clones with contrasting drought resistance.

We tested the hypothesis that Eucalyptus globulus Labill. genotypes that are more resistant to dry environments might also exhibit higher cold tolerances than drought-sensitive plants. The effect of low temperatures was evaluated in acclimated and unacclimated ramets of a drought-resistant clone (CN5) and a drought-sensitive clone (ST51) of E. globulus. We studied the plants' response via leaf gas exchanges, leaf water and osmotic potentials, concentrations of soluble sugars, several antioxidant enzymes and leaf electrolyte leakage. Progressively lowering air temperatures (from 24/16 to 10/-2 degrees C, day/night) led to acclimation of both clones. Acclimated ramets exhibited higher photosynthetic rates, stomatal conductances and lower membrane relative injuries when compared to unacclimated ramets. Moreover, low temperatures led to significant increases of soluble sugars and antioxidant enzymes activity (glutathione reductase, ascorbate peroxidase and superoxide dismutases) of both clones in comparison to plants grown at control temperature (24/16 degrees C). On the other hand, none of the clones, either acclimated or not, exhibited signs of photoinhibition under low temperatures and moderate light. The main differences in the responses to low temperatures between the two clones resulted mainly from differences in carbon metabolism, including a higher accumulation of soluble sugars in the drought-resistant clone CN5 as well as a higher capacity for osmotic regulation, as compared to the drought-sensitive clone ST51. Although membrane injury data suggested that both clones had the same inherent freezing tolerance before and after cold acclimation, the results also support the hypothesis that the drought-resistant clone had a greater cold tolerance at intermediate levels of acclimation than the drought-sensitive clone. A higher capacity to acclimate in a short period can allow a clone to maintain an undamaged leaf surface area along sudden frost events, increasing growth capacity. Moreover, it can enhance survival chances in frost-prone sites expanding the plantation range with more adaptive clones.

Fungal extracellular vesicles: modulating host-pathogen interactions by both the fungus and the host.

The secretion of biomolecules by fungal cells occurs via the conventional export of signal peptide-coupled soluble molecules, but it also results from transport within extracellular vesicles (EV). During the last ten years since the description of this non-conventional secretion pathway, varied, interesting biological roles have been associated with EV release by fungi. The various organic molecules carried by these structures are involved in pathogenesis and immune evasion, and may be associated with cell-cell communication. In regards to host-pathogen interactions, EV roles are diverse and organism-specific, although some features seem to be conserved among the pathogenic fungal organisms studied to date. This review aims to highlight our current understanding of the biologically relevant findings regarding EV released by the pathogenic fungal organisms and describes our knowledge of the roles of EV in host-pathogen interactions.

Plant antiherbivore defenses in Fabaceae species of the Chaco.

The establishment and maintenance of plant species in the Chaco, one of the widest continuous areas of forests in the South American with sharp climatic variations, are possibly related to biological features favoring plants with particular defenses. This study assesses the physical and chemical defenses mechanisms against herbivores of vegetative and reproductive organs. Its analyses of 12 species of Fabaceae (Leguminosae) collected in remnants of Brazilian Chaco shows that 75% present structural defense characters and 50% have chemical defense - defense proteins in their seeds, like protease inhibitors and lectins. Physical defenses occur mainly on branches (78% of the species), leaves (67%), and reproductive organs (56%). The most common physical characters are trichomes and thorns, whose color represents a cryptic character since it does not contrast with the other plant structures. Defense proteins occur in different concentrations and molecular weight classes in the seeds of most species. Protease inhibitors are reported for the first time in seeds of: Albizia niopoides, Anadenanthera colubrina, Mimosa glutinosa, Prosopis rubriflora, and Poincianella pluviosa. The occurrence of physical and chemical defenses in members of Fabaceae indicate no associations between defense characters in these plant species of the Chaco.

Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough.

The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 A resolution. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 A. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

Metabolic responses to water deficit in two Eucalyptus globulus clones with contrasting drought sensitivity.

We compared the metabolic responses of leaves and roots of two Eucalyptus globulus Labill. clones differing in drought sensitivity to a slowly imposed water deficit. Responses measured included changes in concentrations of soluble and insoluble sugars, proline, total protein and several antioxidant enzymes. In addition to the general decrease in growth caused by water deficit, we observed a decrease in osmotic potential when drought stress became severe. In both clones, the decrease was greater in roots than in leaves, consistent with the observed increases in concentrations of soluble sugars and proline in these organs. In roots of both clones, glutathione reductase activity increased significantly in response to water deficit, suggesting that this enzyme plays a protective role in roots during drought stress by catalyzing the catabolism of reactive oxygen species. Clone CN5 has stress avoidance mechanisms that account for its lower sensitivity to drought compared with Clone ST51.

Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug.

The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli. One variant, dsFv3 (VL L46C VH D101C0, has similar affinity for antigen (Kd = 0.7 nM) as the wild-type Fv and was used to construct a fusion protein in which beta-lactamase, RTEM-1, is joined to the carboxy terminus of VH. The dsFv3-beta-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, kcat/km = 1.5 x 10(5) s-1 M-1). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cell lines SK-BR-3 and MCF7, which express p185HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-beta-lactamase fusion protein specifically enhances the toxicity level of PRODOX to that of doxorubicin against SK-BR-3 but not MCF7 cells. The fusion protein retains both antigen-binding plus kinetic activity in murine serum and is cleared rapidly as judged by pharmacokinetic analysis in nude mice (initial and terminal half-lives of 0.23 and 1.27 h, respectively). Development and characterization of the dsFv3-beta-lactamase fusion protein is an important step toward targeted prodrug therapy of p185HER2-overexpressing tumors.

Synthesis and beta-lactamase-mediated activation of a cephalosporin-taxol prodrug.

BACKGROUND: Enzyme-activatable prodrugs in conjunction with antibody-enzyme fusion proteins may enhance the anti-tumor efficacy of antibodies and reduce the toxic side effects of conventional chemotherapeutics. Cephalosporins have proven to be highly versatile triggers for the enzymatic activation of such prodrugs. RESULTS: A cephem prodrug of taxol (PROTAX) was synthesized by substituting the C-3' position of cephalothin with 2'-(gamma-aminobutyryl) taxol. Hydrolysis of PROTAX by beta-lactamase rapidly released 2'-(gamma-aminobutyryl) taxol (kcat/K(M) = (1.4 +/- 0.1) x 10(5) s-1 M-1), which yielded taxol following intramolecular displacement. PROTAX is inactive in a microtubule assembly assay in vitro but has similar activity to taxol following prolonged activation with beta-lactamase. PROTAX is approximately 10-fold less toxic than taxol against SK-BR-3 breast tumor cells in vitro but has activity approaching that of taxol following prolonged activation with a fusion protein comprising beta-lactamase fused to a tumor-targeting antibody fragment. CONCLUSIONS: Tubulin polymerization activity is abolished and cytotoxicity is reduced in the PROTAX prodrug compared to taxol. Activation of PROTAX by beta-lactamase followed by self-immolation restores the activity of PROTAX to that of free taxol.

Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex.

Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson's disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 A resolution on a synchrotron-radiation source and belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 A, beta = 91.14 degrees.

Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms.

Cryptococcus neoformans is the causative agent of cryptococcosis and cryptococcal meningitis, which are serious pathological conditions affecting up to 10% of patients with AIDS. Mechanisms of pathogenicity of C. neoformans and the host defenses against this fungus are reviewed, incorporating recent data and perspectives.

Correlation between adrenal central vein parasitism and heart fibrosis in chronic chagasic myocarditis.

The correlation between Trypanosoma cruzi parasitism of the adrenal central vein (ACV) wall and fibrous connective tissue neoformation in the left ventricular myocardium (LVM) of patients with chronic Chagas' disease who were autopsied was evaluated using the following procedures: 1) a comparison of the incidence of fibrosis in the LVM among 18 chagasic patients with ACV parasitism and 18 individuals without phleboparasitism; 2) a determination of fibrosis intensity in the LVM in 12 cases with ACV parasites and in 12 cases without phleboparasitism, matched with respect to age, sex, race, and anatomoclinical form of the disease (indeterminant, cardiac, and digestive forms); and 3) in the cases with ACV parasitism, a calculation of Pearson's correlation coefficient between T. cruzi nests in the vessel and the intensity of fibrous connective tissue neoformation in the LVM. Among chagasic individuals with adrenal phleboparasitism, there was an increased incidence and intensity of fibrous connective tissue neoformation in the LVM, both highly significant, compared with patients without adrenal phleboparasitism. Furthermore, there was a positive, though nonsignificant, correlation (r = +0.19) between the density of nests in the ACV and the intensity of myocardial fibrosis. These results are consistent with previous data showing a higher intensity of the leukocyte exudate in the LVM and increased heart weight in individuals with T. cruzi nests in the ACV, suggesting a role of parasitism at that site in terms of the development of chronic chagasic cardiopathy.

Possible role of an adrenal parasite reservoir in the pathogenesis of chronic Trypanosoma cruzi myocarditis.

The possible contribution of parasitism of the central vein of the adrenal gland (CVAG) to chronic Trypanosoma cruzi myocarditis was assayed by comparing the occurrence of nests of amastigotes in the left ventricular myocardium (LVM) and determining the number and extent of areas of focal leucocyte exudate (FLE) in Chagas disease patients with and without CVAG parasitism. The frequency of occurrence of T. cruzi nests in the LVM, as well as the FLE number and area, were greater among patients with CVAG parasitism. We therefore suggest that CVAG parasitism plays a role in the onset and intensity of chronic T. cruzi myocarditis.

Serum vitamin A levels in patients with ocular lesions attributable to non-complicated malaria in the Brazilian Amazon region.

In order to evaluate the association between serum vitamin A levels and ocular lesions attributable to non-complicated malaria, 200 patients seen consecutively at the Malaria Outpatient Clinic of FUNASA, Manaus, Amazonas, Brazil were included in this study. Ophthalmologic examination consisted of indirect binocular ophthalmoscopy under medicamentous mydriasis, biomicroscopy with a portable slit lamp and measurement of central visual acuity. Vitamin A serum concentration was determined by HPLC, and deficiency was defined as serum values equal to or lower than 0.35 micromol/l. Serum vitamin A values between 0.36 and 0.70 micromol/l were considered as marginal levels. Hypovitaminosis A (

Transferrin production by the ciliary body of rabbits: a biochemical and immunocytochemical study.

PURPOSE: We have previously reported that transferrin is one of several glycoproteins synthesized within the eye and secreted into the vitreous. The present investigation was designed to determine the role of the ciliary body in the production of this vitreous transferrin. METHODS: Isolated ciliary body-iris were incubated with 3H-fucose, 3H-tyrosine or 35S-methionine and afterwards the culture media were processed for affinity chromatography using columns of Sepharose conjugated with antibody to rabbit plasma transferrin. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from fresh ciliary body-iris and primers constructed on the basis of the known sequence of transferrin mRNA from rabbit liver. The fragment obtained was employed as a probe in northern-blots of total RNA of ciliary body-iris. Furthermore, paraffin sections of eyes were treated for immunocytochemical visualization of transferrin. RESULTS: A labeled polypeptide, specifically eluted from the antitransferrin columns, was detected in the incubation medium, transferrin mRNA was found in extracts of whole ciliary body-iris, and transferrin antigenicity was identified in the ciliary and iridial epithelial cells by immunocytochemistry. CONCLUSIONS: These results demonstrate the ciliary epithelium as one of the sources of the vitreous transferrin.