Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin. Presence of specific cation-binding sites

Single and multiple mutants of extracellular Glu side chains of bacteriorhodopsin were analyzed by acid and calcium titration, differential scanning calorimetry, and thermal difference spectrophotometry. Acid titration spectra show that the second group protonating with Asp(85) is revealed in E204Q in the absence of Cl(-) but is not observed in the triple mutant E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu(9) as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(a) of Asp(85) protonation in water and in the deionized forms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type bacteriorhodopsin, differential scanning calorimetry reveals a lack of the pretransition in the multiple mutants, whereas in E9Q it appears at lower temperature and with lower cooperativity. Additionally, at neutral pH the band at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsin having as ligands Glu(9), Glu(194), Glu(204), and water molecules.     

Fuzzy modeling in symptomatic HIV virus infected population

This paper introduces a model for the evolution of positive HIV population and manifestation of AIDS (acquired immunideficiency syndrome). The focus is on the nature of the transference rate of HIV to AIDS. Expert knowledge indicates that the transference rate is uncertain and depends strongly on the viral load and the CD4+ level of the infected individuals. Here, we suggest to view the transference rate as a fuzzy set of the viral load and CD4+ level values. In this case the dynamic model results in a fuzzy model that preserves the biological meaning and nature of the transference rate λ. Its behavior fits the natural history of HIV infection reported in the medical science domain. The paper also includes a comparison between the fuzzy model and a classic Anderson’s model using data reported in the literature.     

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus

Bacteria communicate at high cell density through quorum sensing, however, there are no reports about this mechanism in archaea. The archaeon Natronococcus occultus produces an extracellular protease at the end of growth. Early production of protease activity was observed when a low density culture was incubated with late exponential conditioned medium suggesting the presence of factor(s) inducing this activity. Conditioned medium and ethyl acetate extracts corresponding to the transition from exponential to stationary phase showed a positive signal in Agrobacterium biosensor. We report the detection of potential autoinducer molecules of the acylated homoserine lactone type in the archaeon N. occultus. These molecules may be responsible for the production/activation of extracellular protease.     

Chromosomal polymorphism,syntenic relationships,and ploidy in the pathogenic fungus Paracoccidioides brasiliensis

Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas. Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. This represents the first comparative mapping study among different isolates. The results are indicative of the existence of genetic differences among natural isolates. DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy. Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus.     

DNA cleavage studies of mononuclear and dinuclear copper(II) complexes with benzothiazolesulfonamide ligands

Copper-based transition metal complexes performing single- and double-strand scission of DNA have been studied. The dinuclear complexes [Cu(2)(L)(2)(OCH(3))(2)(NH(3))(2)] and [Cu(2)(L)(2)(OCH(3))(2)(DMSO)(2)] are more active than the corresponding mononuclear [Cu(L)(2)(py)(2)] (where HL= N-(4-methylbenzothiazol-2-yl)benzenesulfonamide), suggesting that the dinuclearity is an important factor in the oxidative cleavage of DNA. The cleavage efficiency of the complexes depends on the reducing agent used in the process, the tandem ascorbate/H(2)O(2) being the most efficient. PAGE analyses have shown that these complexes cleave DNA without sequence selectivity. The DNA degradation process takes place mainly by C1' oxidation, but C4' and C5' oxidations cannot be ruled out as minor pathways. These copper complexes preferably oxidize guanine under mild conditions, but under more drastic conditions the oxidation reactivity appears to be T>G>C>A, suggesting the intervention of hydroxyl radicals as active species.     

Induction of astaxanthin accumulation by nitrogen and magnesium deficiencies in Haematococcus pluvialis

Haematococcus pluvialis was cultured under N– and Mg+2-deficient conditions with two light intensities: 40 and 230 mol m² s–1. Highest astaxanthin concentration, 49.5 g·ml–1, was obtained when high light was applied under N-deficient conditions. N-deficiency has a greater effect than high light intensity on astaxanthin synthesis by exerting a stronger blocking effect on cell division. The effect of high light was synergetic with the other stress conditions in stimulating the synthesis of astaxanthin. Mg+2 deficiency also stimulated the synthesis of astaxanthin but produced lower concentrations: 7 and 26 g·ml–1 for low and high light intensities respectively. When both N and Mg+2 were absent from the culture media the concentration of astaxanthin was lower than with N-deficiency alone but higher than with Mg+2-deficiency. © Rapid Science Ltd. 1998     

Passive Ca binding in ventricular myocardium of neonatal and adult rats

In this study, passive Ca²⁺ binding was determined in ventricular homogenates (VH) from neonatal (4–6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca²⁺ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca²⁺ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca²⁺] ([Ca²⁺]_F was measured with Indo-1 and bound Ca²⁺ ([Ca²⁺]_B) was the difference between [Ca²⁺]_F and total Ca²⁺. Apparent Ca²⁺ dissociation constants (K_d) for BAPTA and Indo-1 were increased by 10–20 mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl₂ additions (2.5–10 nmoles) yielded δ[Ca²⁺]_B/δ[Ca²⁺]_F for the sum of [Ca²⁺]_B at all three classes of binding sites. From this function, the apparent number of endogenous sites (B_en) and their K_d (K_en) were determined. Similar K_en values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, B_en was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ∼300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that B_en determined in VH underestimates cellular B_en by 29%. Although B_en values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing B_en per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca²⁺ binding capacity at high-affinity sites is ∼300 and 176 mmol/I cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca²⁺ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.