Cytotoxic activity of clinical Stenotrophomonas maltophilia

AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.     

Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

Fusarium Nail and Skin Infection: A Report of Eight Cases from Natal, Brazil

Fusarium spp. are non-dermatophytic hyaline moulds distributed worldwide and recovered from the nature as soil saprophytes and plant pathogens. Human infections are usually precipitated by local or systemic predisposing factors and disseminated infection is associated with impaired immune responses. We report eight cases of cutaneous lesions caused by Fusarium spp. All patients were immunocompetent. Seven cases with presented onychomycosis and one patient with interdigital intertrigo. It is important to alert the medical community about the relevance of the opportunistic fungi, such as Fusarium spp., which have emerged as human infectious agents, emphasizing the importance of correct etiological identification, allowing for appropriate treatment.     

Prevalence and Antifungal Susceptibility of Yeasts Obtained from the Oral Cavity of Elderly Individuals

Infections caused by Candida yeasts are common in elderly individuals. Seventy-five isolates of Candida spp. were obtained from saliva samples of 136 institutionalized elderly individuals resident in six retirement homes of Belo Horizonte, Brazil. Forty-seven isolates (62.66%) were identified as Candida albicans, 15 (20%) as C. tropicalis, 7 (9.33%) as C. glabrata, 4 (5.33) as C. parapsilosis, and 2 (2.67%) as C. guilliermondii. Among the 136 elderly individuals studied, 49 (36%) were male and 87 (64%) were female. Ages ranged from 60 to 90 years old. Sixty-three (46.3%) of the institutionalized individuals were denture wearers and, among them, 53 (84.1%) carried Candida yeasts in the oral cavity. Forty-four subjects presented lesions in the oral mucosa and among these, 36 (82%), had positive culture for Candida spp. The samples were tested for the in vitro susceptibility to amphotericin B, itraconazole, fluconazole and 5-flucytosin, and great variations were observed in the minimum inhibitory concentrations (MIC) of these drugs according to the species.     

Keep the traffic moving: mechanism of the Hsp70 motor

Hsp70s are ubiquitous chaperones that use ATP hydrolysis to drive a variety of protein processing reactions, including a number of steps in protein trafficking. Recent studies have shed light on how ATP might generate conformational changes in an Hsp70 molecule and how such changes might be harnessed to drive processes as diverse as protein import into subcellular organelles and uncoating of clathrin-coated vesicles.     

Ecological resistance, seed density and their interactions determine patterns of invasion in a California coastal grassland

Relatively little experimental evidence is available regarding how ecological resistance and propagule density interact in their effects on the establishment of invasive exotic species. We examined the independent and interactive effects of neighbour cover (biotic resistance), winter vs. spring water addition (abiotic resistance) and seed density on the invasion of the European perennial grass Holcus lanatus into a California coastal grassland dominated by exotic annual grasses. We found that decreased competition from resident exotic grasses had no effect. In contrast, increased late-season water availability eroded the abiotic resistance offered by naturally dry conditions, facilitating invasion. Finally, watering treatment and seed density interacted strongly in determining seedling survival: while seedling mortality was close to 100% in ambient and winter water addition plots, survivor numbers increased with seed density in spring-watered plots. Thus, decreased abiotic resistance can amplify the effect of increased propagule density on seedling establishment, thereby increasing the likelihood of invasion.     

Generation of stella-GFP transgenic mice: a novel tool to study germ cell development

The relationship between germ cells and pluripotent embryonic stem (ES) cells is of particular interest, together with approaches to generate primordial germ cell (PGCs) from ES cells. A critical requirement in these experiments is the ability to unambiguously detect PGCs with the use of, for example, reporter genes. The currently available transgenic reporters do not show exclusive expression in PGCs at their earliest developmental stages. Here we describe the use of germline-restricted expression of stella, which is currently the best marker gene for PGCs. We generated two stella-GFP reporters and show that both transgenes surpass other reporters in terms of timing and specificity of expression in PGCs. Additionally, we demonstrate the usefulness of stella-GFP during the derivation of PGCs from ES cells.     

Microplate assay for inhibitors of the transpeptidase activity of PBP1b of Escherichia coli

The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the beta-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un-cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un-cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, followed by removal of unbound ampicillin. The substrate for the TP, un-cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin scintillation proximity assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1bcatalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme assay, it has the potential to detect novel, non-beta-lactam TP inhibitors and could lead to the discovery of new antibacterial agents.     

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).     

Physiological Indicators of Plant Water Status of Irrigated and Non-irrigated Grapevines Grown in a Low Rainfall Area of Portugal

Water is a key resource in commercial wine production and both large excesses and deficits have undesirable effects upon the amount and quality of the wine produced. A balance between the water requirements of a fully developed canopy and the induced stress necessary for the commercial quality of the wine must be reached. Thus we need a physiological indicator that integrates both soil and climatic conditions to use as a management tool. An experimental field was established in the eastern part of the Demarcated Region of Douro – Portugal, to study the effect of water supply on the quality of the musts produced and we need a physiological indicator that relates to the water use and stress of the grapevines (Vitis vinifera L.) and to the later evaluation of the effect of irrigation practices upon the quality of the musts. We chose as indicators sap flow, leaf water potential at pre-dawn (0600 h), mid-morning (1000 h), solar noon (1400 h) and sunset (1900 h), stomatal conductance and leaf transpiration both measured at mid-morning and at solar noon, and related them to our experimental treatments that induce differences in soil water content, evaluated with time-domain reflectometry probes, with the objective of selecting the indicator that best describes the plant water status under different amounts of available water. Sap flow, leaf water potential and leaf transpiration rate measured at solar noon had highly significant correlations with soil water content and their regression on soil water content was also highly significant. Each of these parameters has shortcomings and none has a clear advantage over the other two as an integrator of the environmental conditions under these experimental conditions. Further studies of the parameters and their relationship with the quality characteristics of the produced musts are needed to achieve the ultimate objective of manipulating the soil water content.