Pinpointing disulfide connectivities in cysteine-rich proteins.

A simple MD-based protocol is presented to accurately predict both the sequence and order of disulfide bond formation in proteins containing multiple cysteine residues. It provides a detailed description of their dynamical and structural features, which can be used to perform ensemble-averaged ECD calculations. Plant cyclotides are used as model compounds.

Can a simple test of functional capacity add to the clinical assessment of diabetes?

AIM: To identify impairment in functional capacity associated with complicated and non-complicated diabetes using the 6-min walk distance test. METHODS: We enrolled 111 adults, aged ≥40 years, with Type 2 diabetes from a hospital facility and 150 healthy control subjects of similar age and sex from a community site in Lima, Peru. All participants completed a 6-min walk test. RESULTS: The mean age of the 261 participants was 58.3 years, and 43.3% were male. Among those with diabetes, 67 (60%) had non-complicated diabetes and 44 (40%) had complications such as peripheral neuropathy, retinopathy or nephropathy. The mean unadjusted 6-min walk distances were 376 m and 394 m in adults with and without diabetes complications, respectively, vs 469 m in control subjects (P<0.001). In multivariable regression, the subjects with diabetes complications walked 84 m less far (95% CI -104 to -63 m) and those without complications walked 60 m less far (-77 to -42 m) than did control subjects. When using HbA1c level as a covariate in multivariable regression, participants walked 13 m less far (-16.9 to -9.9 m) for each % increase in HbA1c . CONCLUSIONS: The subjects with diabetes had lower functional capacity compared with healthy control subjects with similar characteristics. Differences in 6-min walk distance were even apparent in the subjects without diabetes complications. Potential mechanisms that could explain this finding are early cardiovascular disease or deconditioning.

Type 2 diabetes and cardiac autonomic neuropathy screening using dynamic pupillometry.

AIM: To determine if changes in pupillary response are useful as a screening tool for diabetes and to assess whether pupillometry is associated with cardiac autonomic neuropathy. METHODS: We conducted a cross-sectional study with participants drawn from two settings: a hospital and a community site. At the community site, individuals with newly diagnosed diabetes as well as a random sample of control individuals without diabetes, confirmed by oral glucose tolerance test, were selected. Participants underwent an LED light stimulus test and eight pupillometry variables were measured. Outcomes were diabetes, defined by oral glucose tolerance test, and cardiac autonomic dysfunction, determined by a positive readout on two of four diagnostic tests: heart rate response to the Valsalva manoeuvre; orthostatic hypotension; 30:15 ratio; and expiration-to-inspiration ratio. The area under the curve, best threshold, sensitivity and specificity of each pupillometry variable was calculated. RESULTS: Data from 384 people, 213 with diabetes, were analysed. The mean (±sd) age of the people with diabetes was 58.6 (±8.2) years and in the control subjects it was 56.1 (±8.6) years. When comparing individuals with and without diabetes, the amplitude of the pupil reaction had the highest area under the curve [0.69 (sensitivity: 78%; specificity: 55%)]. Cardiac autonomic neuropathy was present in 51 of the 138 people evaluated (37.0%; 95% CI 28.8-45.1). To diagnose cardiac autonomic neuropathy, two pupillometry variables had the highest area under the curve: baseline pupil radius [area under the curve: 0.71 (sensitivity: 51%; specificity: 84%)], and amplitude of the pupil reaction [area under the curve: 0.70 (sensitivity: 82%; specificity: 55%)]. CONCLUSIONS: Pupillometry is an inexpensive technique to screen for diabetes and cardiac autonomic neuropathy, but it does not have sufficient accuracy for clinical use as a screening tool.

Avaliação das atividades antioxidante, anti e pró-hemolítica do extrato etanólico das folhas de Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae) / Evaluation of antioxidant, anti- and pro-hemolytic activities of ethanol extract from the leaves of Pterogynenitens Tul. (Fabaceae-Caesalpinioideae)

A pesquisa de produtos naturais permite a descoberta de novos princípios ativos, ou ainda, a descoberta de novas atividades para extratos de plantas (amplamente utilizados pela população brasileira) e princípios ativos naturais já conhecidos. Pterogyne nitens é uma planta cuja descrição das atividades é relativamente recente e, portanto, tem no extrato bruto boa fonte para pesquisas na área de produtos naturais. Desta forma, o objetivo deste trabalho foi estudar o perfil antioxidante do extrato bruto etanólico das folhas de P. nitens e possível interferência sobre a hemólise provocada pelo radical AAPH•. No estudo da ação antioxidante das espécies estudadas, ABTS•+, DPPH•, H2O2 e HOCl, encontrou-se os valores de IC50 de 5,0 µg mL-1, 17 µg mL-1, sem ação e 3,9 µg mL-1, respectivamente, valores relativamente baixos e que indicam bom potencial antioxidante. Foram encontradas atividades pró-hemolítica e anti-hemolítica para o extrato de forma concentração-dependente. O extrato estudado mostro boa fonte de moléculas naturais com potencial de ação biológica.

The search for natural products as a widespread practice enables the discovery of new active principles, or the discovery of new activities for plant extracts (extensively used by the population) and natural active principles already known. Pterogynenitensis is a plant whose descriptions of activities are relatively recent and therefore has in its crude extract a good source for research in the field of natural products. Thus, the aim of this study was to evaluate the antioxidant profile of crude ethanol extract from P. nitens leaves and a possible influence on the hemolysis caused by AAPH• radical. For the studied oxidant species, ABTS•+, DPPH•, HOCl and H2O2, the IC50 values were found of 5.0 µg mL-1, 17 µg mL-1, no action at all, and 3.9 µg mL-1, respectively, relatively low values, indicating a good antioxidant potential. Pro- and anti-hemolytic activities were found for the extract in a concentration-dependent way. The studied extract showed to be a good source of natural molecules with potential biological action.

Heteroantagonismo entre estirpes de Enterobacter agglomerans isoladas de urubu (Coragyps atratus) como produtoras e Pseudomonas aeruginosa como reveladoras / Evaluation of the heteroantagonism between Enterobacter agglomerans strains isolated from vulture (Coragyps atratus) for production and Pseudomonas aeruginosa as developed

Avaliou-se o heteroantagonismo entre Enterobacter agglomerans isolada do trato gastrintestinal de urubu (Coragyps atratus) e Pseudomonas aeruginosa isoladas do ambiente hospitalar. Foram utilizados o método de sobrecamada ou lento e a técnica direta ou de poços. Pelo método da sobrecamada, de 196 testes realizados para pesquisa da atividade antagonista, foi detectada a presença de halos de inibição relacionados ao fenômeno de heteroantagonismo em 118 deles (60,2 por cento). Pelo método de poços, obtiveram-se resultados semelhantes. As sete amostras de E. agglomerans foram capazes de realizar heteroantagonismo nas condições testadas, que foram detectados pela formação de halos claros de inibição. O extrato de levedura adicionado a 1 por cento no meio de cultura foi um suplemento adequado para a demonstração do antagonismo.

The heteroantagonism between Enterobacter agglomerans, isolated from the gastrointestinal tract of American vulture Coragyps atratus, and Pseudomonas aeruginosa isolated from a hospital environment was evaluated. The slow (layer) and the wells (direct) techniques were tested, using agar and soy tryptone broth pH 7.3 at 37ºC. Through the slow method from 196 tests, inhibition growth halos, related heteroantagonism phenomenon observed in 118, corresponding to 60.2 percent positive results. Equivalent positive results were detected using wells (direct) methodology. The seven samples of E. agglomerans tested were capable of revealing heteroantagonism in the experimental conditions; antagonism reveled by the presence of a clear growth inhibition halo. The added 1 percent yeast extract to media was adequate for revealing antagonisms best.

Morphofunctional alterations in endocrine pancreas of short- and long-term dexamethasone-treated rats.

Long-term dexamethasone therapy may induce peripheral insulin resistance (IR), which in turn elicits increased beta-cell function and proliferation. However, whether such adaptive compensations occur during short-term treatment with dexamethasone is unclear. Here, we compared morphofunctional parameters in endocrine pancreas after short- and long-term dexamethasone administration. Groups of rats received daily i. p. injection of 1 mg/kg b. w. dexamethasone for 1 (DEX-1), 3 (DEX-3), or 5 consecutive days (DEX-5), whilst control rats were saline-treated (CTL). Despite the absence of apparent IR in DEX-1 rats, this group exhibited increased circulating insulin levels and glucose-stimulated insulin secretion (GSIS), compared to the CTL group (p<0.05). Evident IR as well as marked hyperinsulinemia and GSIS, as judged by the static and dynamic insulin secretion values, were observed in DEX-3 and DEX-5 rats (p<0.05). GSIS in islets cultured with 1 µM dexamethasone was lower compared to the control (p<0.05). Marked increases in beta-cell proliferation were observed in DEX-3 and DEX-5 rats, compared to CTL and DEX-1 rats (p<0.05). The alterations observed in DEX-3 rats were more pronounced in DEX-5 rats, which also exhibited a higher content of islet Cdk4 and Cd2 proteins, compared to the CTL group (p<0.05). We conclude that short-term dexamethasone treatment (DEX-1) induces an increase in beta-cell function that does not require the presence of discernible IR. As the treatment continues, the IR develops rapidly, and increased insulin secretion as well as beta-cell hyperplasia is demanded for the appropriate maintenance of glucose homeostasis.

A molecular study on the evolution of a subtype B variant frequently found in Brazil.

In spite of the remarkable diversity of HIV-1 env genes, several amino acids are extremely conserved, probably due to functional constraints. One example is the proline found at the second position of the GPGR motif. Several viruses, however, bear substitutions at this site, for instance, GWGR subtype B variant. GWGR viruses are described in Brazil since the beginning of the epidemics, but the extent of their dispersion or the geographical origin of the variant remains unknown. In the present study, phylogenetic trees were constructed in order to study the origin and spread of this variant. All GWGR sequences as well as a subset of subtype B sequences available were included in the analyses. Analyses of differential selection were also performed on GWGR and non-GWGR sequences in order to unveil evolutionary novelties due to the action of positive selection. Although the GWGR variant was found at least in 23 countries, its expansion probably has a single origin, and Brazil is the epicenter.

Exposure of young rats to high estrogen doses leads to degeneration of elongated spermatids.

Single high doses of estrogen (35 mg/kg body weight) were administered to young rats aiming to exacerbate its effects on germ cell populations. The short-term (1 week) and medium-term (7 weeks) consequences of this estrogenic treatment (ET) on the testis were evaluated using light and electron microscopies, quantitative methods and TUNEL reaction. Short-term ET led to 50% atrophy of the testis, however, in the medium term the gonado-somatic index was recovered. No histopathological alterations were found at seminiferous epithelium except for short-term severe degeneration of elongated spermatids (EL) and low frequency of these cells in both time intervals. Two morphologically distinct patterns of degeneration were observed: (1) clusters of EL which were TUNEL-negative and exhibited bizarre appearance and nuclear fragmentation, (2) isolated apoptotic EL within the cytoplasm of Sertoli cells (SC). Both degenerative phenomena were more frequent in stages III-VIII of seminiferous cycle, whereas at stages I and II only coiling of flagellum was observed. One week after ET, small amounts of EL were detected in stages IX-XII, suggesting spermiation failure. Signs of functional SC damage such as an accumulation of myelin-like inclusions in their cytoplasm were observed in the short but not medium-term. However, the apoptotic rates still remained five times higher and the number of elongated spermatids was three-fold lower. Our data indicate that exposure to a high dose of estrogen around puberty has stage-specific effects on the testis and causes massive degeneration of elongated spermatids.

Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids.

The application of high-performance liquid chromatography (HPLC) using a C30 reverse-phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.

[Detection of pyrogenic exotoxin SpeA, SpeB and SpeC genes in Chilean streptococci isolates and their association with clinical manifestations]. / Detección de los genes de las exotoxinas pirogénicas SpeA, SpeB y SpeC en cepas chilenas de Streptococcus pyogenes y su asociación con la clínica.

BACKGROUND: The virulence of Streptococcus pyogenes is determined by a variety of structural molecules, toxins and complex enzymes. Pyrogenic exotoxins cause fever, erythematous reactions, cytotoxic and immunological effects. AIM: To assess the frequency of speA, SpeB and SpeC genes in Chilean Streptococcus pyogenes strains and their association with the invasiveness of infections. MATERIAL AND METHODS: The genes for pyrogenic exotoxins SpeA, SpeB and SpeC were determined by polymerase chain reactions in 114 strains of group A Streptococcus pyogenes isolated from Chilean patients with invasive or non invasive infections. RESULTS: The gene for SpeA was present in 30.7% of isolates, the gene for SpeB was present in 69.3% and the gen for SpeC in 44.7% of isolates. The gene for SpeA was present in 20 of 33 invasive infections and in 15 of 81 non invasive infections (p < 0.0001). On the contrary, the gene for SpeC was present in 11 of 33 invasive infections and in 41 of 81 non invasive infections (p < 0.05). The frequency of speB was similar in invasive and non invasive infections. CONCLUSIONS: There is a clear relationship between the presence of SpeA genes and the severity of infections caused by Streptococcus pyogenes.

Indirect modulation of dopamine D2 receptors as potential pharmacotherapy for schizophrenia: II. Glutamate (Ant)agonists.

OBJECTIVE: To summarize the published preclinical and clinical data that suggest the possible use of glutamate receptor agonists or antagonists as novel antipsychotic agents. DATA SOURCES: Primary and review articles were identified by MEDLINE search (from 1966 to December 1999) and through secondary sources. STUDY SELECTION AND DATA EXTRACTION: All of the articles identified from the data sources were evaluated and all information deemed relevant was included. DATA SYNTHESIS: The standard antipsychotic drugs, whose clinical activity correlates with affinity for dopamine D2 receptors, alleviate some of the positive symptoms of schizophrenia, but have limited impact on negative symptoms. Several lines of evidence implicate glutamate-receptor system dysfunction(s) in schizophrenia, either as causative or contributory factors. In addition, several standard antipsychotic drugs modulate glutamate or glutamate receptor activity, suggesting an alternative view of their mechanism of antipsychotic action. Preliminary studies have shown that drugs which modulate glutamate brain concentrations have positive effects in animal models of schizophrenia. CONCLUSIONS: A role for glutamate in the pathogenesis or pharmacotherapy of schizophrenia is suggested from anatomic (interactions between glutamatergic and dopaminergic systems in relevant brain regions), physiologic (implication of glutamate-receptor dysfunction), and pharmacologic (modulation of glutamate or glutamate receptors) evidence. Therefore, compounds that function at glutamate receptors might represent a novel approach to the treatment of the disease or to the amelioration of symptoms, either as monotherapy or as an adjunct to dopamine D2 receptor antagonists.

Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria.

We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.

Could a common biochemical mechanism underlie addictions?

The subject of 'drug addiction' is multifaceted and many aspects of it (even some of the definitions) are controversial. Collateral medical problems include the spread of HIV and hepatitis C virus secondary to i.v. drug abuse and effects on prenatal brain development (1). Progress in the understanding of the causes of addictions and its treatment has been impeded by the lack of a unifying biochemical theory. However, recent evidence suggests that some common mechanism might underlie addictions to otherwise apparently unrelated drugs. A major hypothesis has emerged suggesting that the neurotransmitter dopamine (DA) might play a central role in the molecular mechanisms of at least some addictions. If so, it would represent an important target for discovery of effective pharmacotherapy and revolutionize the pharmacist's role in treating addictions. This short overview outlines the status of the theory of a common biochemical mechanism of drug addiction.

Seroprevalence of Chlamydia pneumoniae in Chile.

We used microimmunofluorescence to survey the prevalence of antibodies to Chlamydia pneumoniae in 403 serum samples from asymptomatic subjects aged 6 months to 89 y in Santiago, Chile. The results suggest that Chlamydia pneumoniae infection is endemic in Chile, with a seroprevalence of 60% which does not differ by gender.

Effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme in bean (Phaseolus vulgaris L.) grown under different nitrogen conditions.

The effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme have been examined in different cultivars of Phaseolous vulgaris L. grown under 1 and 12 mM nitrogen. Low nitrogen nutrition reduces chlorophyll and soluble protein contents in the leaves and thus the photosynthesis rate and dry-matter accumulation. Chlorophyll, soluble protein and Rubisco contents and photosynthesis rate are not significantly altered by ambient levels of UV-B radiation (17 microW m-2, 290-320 nm, 4 h/day for one week). Comparative studies show that under high nitrogen, UV-B radiation slightly enhances leaf expansion and dry-matter accumulation in cultivar Pinto, but inhibits these parameters in Vilmorin. These results suggest that the UV-B effect on growth is mediated through leaf expansion, which is particularly sensitive to UV-B, and that Pinto is more tolerant than Vilmorin. The effect of UV-B radiation on UV-B-absorbing compounds and on NADP-malic enzyme (NADP-ME) activity is also examined. Both UV-B radiation and low-nitrogen nutrition enhance the content of UV-B-absorbing compounds, and among the three cultivars used, Pinto exhibits the highest increases and Arroz the lowest. The same trend is observed for the specific activity and content of NADP-ME. On a leaf-area basis, the amount of UV-B-absorbing compounds is highly correlated with the enzyme activity (r2 = 0.83), suggesting that NADP-ME plays a key role in biosynthesis of these compounds. Furthermore, the higher sensitivity of Vilmorin than Pinto to UV-B radiation appears to be related to the activity of NADP-ME and the capacity of the plants to accumulate UV-B-absorbing compounds.

[Surveillance of gram positive cocci susceptibility to betalactams, glycopeptides, and other antimicrobials]. / Vigilancia de susceptibilidad de cocáceas grampositivas a betalactámicos, glicopéptidos y otros antimicrobianos.

BACKGROUND: During the last decade, there has been a progressive increase in the resistance of gram (+) cocci to betalactamics and other antimicrobials. Therefore, vancomycin and teicoplanin have incorporated as alternative antimicrobial drugs. AIM: To assess the susceptibility of gram (+) cocci to different antimicrobials including vancomycin and teicoplanin. MATERIAL AND METHODS: We studied 447 strains of gram (+) cocci coming from ambulatory and hospitalized patients. These included 308 Enterococcus sp strains, 99 Staphylococcus aureus strains and 40 coagulase negative Staphylococci strains. Enterococci susceptibility was measured using minimal inhibitory concentrations in agar and that of Staphylococci, through diffusion. Susceptibility to vancomycin and teicoplanin was measured using minimal inhibitory concentrations in all strains. RESULTS: Enterococcus faecalis was 100% susceptible to ampicillin, penicillin, vancomycin and teicoplanin, 23% susceptible to tetracyclin and 47% to chloramphenicol. Susceptibility of E faecium was 61% to penicillin, 49% to chloramphenicol, 41% to tetracyclin, 100% to vancomycin and teicoplanin. Of 19 Enterococcus spp strains, 90% were susceptible to ampicillin, 80% to penicillin, 55% to chloramphenicol and 45% to tetracyclin. Only one E casseiflavus strain had a low level resistance to vancomycin and was susceptible to teicoplanin. No Staphylococcus aureus strain was resistant to vancomycin or teicoplanin. CONCLUSIONS: A permanent surveillance of gram (+) cocci antimicrobial susceptibility is required to update therapeutic schemes.

Epidemiology of invasive pneumococcal infections in infants and young children in Metropolitan Santiago, Chile, a newly industrializing country.

AIM: To study the epidemiology of invasive pneumococcal infections in infants and young children in Santiago, Chile, as a representative pediatric population in a newly industrializing country where pneumococcal conjugate vaccines may be used in the future. METHODS: A 5-year retrospective laboratory-based review (1989 to 1993) was followed by a 3-year prospective laboratory and hospital surveillance study in two of the six health administrative areas of Santiago to detect all hospitalized cases of invasive pneumococcal disease (defined as Streptococcus pneumoniae isolated from blood, cerebrospinal fluid or another normally sterile site) among infants and children (0 to 23 months of age in the retrospective and 0 to 59 months of age in the prospective study). RESULTS: During the 5-year retrospective survey the incidence of invasive pneumococcal disease was 90.6 cases per 10(5) infants 0 to 11 months old and 18.5 cases per 10(5) toddlers 12 to 23 months old. Similar rates (60.2 per 10(5) infants and 18.1 per 10(5) toddlers) were recorded during the 3 years of prospective surveillance. Among the 110 cases in children 0 to 59 months of age detected during the 3-year prospective surveillance, 2 clinical forms, pneumonia and meningitis, accounted for 87.2% of all cases; 13 of the 49 pneumonia patients (26%) had empyema as a complication. Notably 40 of the 110 cases (36.4%) occurred before 6 months of age (63.4% of the 63 infant cases). Serotypes 1, 14, 5 and 6B were the most prevalent. Overall 76 and 69%, respectively, of S. pneumoniae isolates were antigenic types that would be covered by the 11- or 9-valent conjugate vaccines under development. CONCLUSIONS: Invasive pneumococcal infections in Santiago, Chile, exhibit an epidemiologic pattern intermediate between that of developing and industrialized countries. The high burden of disease in early infancy dictates that an accelerated immunization schedule (beginning in the perinatal period) or maternal immunization with pneumococcal vaccines should be explored.

Adherence of Acinetobacter baumannii to rat bladder tissue.

Acinetobacter baumannii, an important nosocomial pathogen, causes severe infections in patients of intensive care units, but its pathogenic attributes are unknown. Previously, the adherence of A. baumannii to cell lines has been negative in the authors' laboratory. In this work, the adherence of strains of A. baumannii of various biotypes to small pieces of rat bladder tissue was investigated. Tissue pieces were submerged into cultures of A. baumannii and sessile cells were counted after removing planktonic bacteria. Fimbriae and sessile cells were examined by transmission and scanning electron microscopy, respectively. In contrast to a uropathogenic strain of Escherichia coli, all cultures exhibited a mannose- and galactose-resistant agglutination of human group O red blood cells as well as mannose- and galactose-resistant adherence to the bladder tissue. Inhibition of exopolysaccharide synthesis did not modify adherence. Indeed, adherence, apparently unrelated to these fimbriae or to the exopolysaccharide, may be a factor contributing to the pathogenicity of A. baumannii in the urinary tract or in other tissues.