X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.     

Genetic polymorphisms of glutathione S-transferases and cytochrome P450 enzymes as susceptibility factors to systemic lupus erythematosus in southern Brazilian patients

Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents several clinical manifestations, affecting multiple organs and systems. Immunological, environmental, hormonal and genetic factors may contribute to disease. Genes and proteins involved in metabolism and detoxification of xenobiotics are often used as susceptibility markers to diseases with environmental risk factors. Cytochrome P450 (CYP) enzymes activate the xenobiotic making it more reactive, while the Glutathione S-transferases (GST) enzymes conjugate the reduced glutathione with electrophilic compounds, facilitating the toxic products excretion. CYP and GST polymorphisms can alter the expression and catalytic activity of enzymes. This study aimed to investigate the role of genetic variants of CYP and GST in susceptibility and clinical expression of SLE, through the analysis of GSTM1 null, GSTT1 null, GSTP1*Ile105Val, CYP1A1*2C and CYP2E1*5B polymorphisms. 371 SLE patients from Hospital de Clínicas de Porto Alegre and 522 healthy blood donors from southern Brazil were evaluated. GSTP1 and CYP variants were genotyped using PCR–RFLP and GSTT1 and GSTM1 variants were analyzed by multiplex PCR. Among European-derived individuals, a lower frequency of GSTP1*Val heterozygous genotypes was found in SLE patients when compared to controls (p = 0.005). In African-derived SLE patients, the CYP2E1*5B allelic frequency was higher in relation to controls (p = 0.054). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*Ile/Val heterozygous genotype against the SLE in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived individuals.     

Trypan Blue Dye is an Effective and Inexpensive Way to Determine the Viability of Batrachochytrium dendrobatidis Zoospores

Batrachochytrium dendrobatidis (Bd) has been implicated in hundreds of amphibian declines and is the focus of a vast amount of research. Despite this, there is no reported efficient way to assess Bd viability. Discriminating between live and dead Bd would help determine the dose of live Bd zoospores and whether factors have lethal or sublethal effects on Bd. We tested whether trypan blue, a common stain to discriminate live and dead cells, could be used to assess Bd viability. We show that the proportion of live zoospores (zoospores that excluded the trypan blue dye) matched the proportion of known live zoospores added to cultures. In contrast, all of the zoosporangia stages of Bd stained blue. These results demonstrate that trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. We recommend using trypan blue to report the number of live zoospores to which hosts are exposed.     

Initiating Antiretroviral Therapy for HIV at a Patient’s First Clinic Visit: The RapIT Randomized Controlled Trial

BackgroundHigh rates of patient attrition from care between HIV testing and antiretroviral therapy (ART) initiation have been documented in sub-Saharan Africa, contributing to persistently low CD4 cell counts at treatment initiation. One reason for this is that starting ART in many countries is a lengthy and burdensome process, imposing long waits and multiple clinic visits on patients. We estimated the effect on uptake of ART and viral suppression of an accelerated initiation algorithm that allowed treatment-eligible patients to be dispensed their first supply of antiretroviral medications on the day of their first HIV-related clinic visit.ConclusionsOffering single-visit ART initiation to adult patients in South Africa increased uptake of ART by 36% and viral suppression by 26%. This intervention should be considered for adoption in the public sector in Africa.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01710397","term_id":"NCT01710397"}}NCT01710397, and South African National Clinical Trials Register DOH-27-0213-4177.     

Effects of nutrient supplementation on host‐pathogen dynamics of the amphibian chytrid fungus: a community approach

相似文献   

Elektronenmikroskopische Untersuchungen über den Sekretionsmodus des Parathormones

Zusammenfassung Durch EDTA-Injektion wird der Blutcalciumspiegel akut und befristet gesenkt. 150–200 g schwere Wistarratten erhalten je 2 ml EDTA (8%) i.p. und werden in verschiedenen Zeitpunkten nach der Injektion getötet. Die Veränderungen der Ultrastruktur der Epithelkörperchen werden untersucht.Zu Versuchsbeginn lassen sich lediglich Veränderungen an den osmiophilen Körpern nachweisen, die bevorzugt am Kapillarpol der Zelle liegen. Diese Einschlüsse zeigen eine Auflockerung und oft eine bläschenartige Umwandlung ihrer elektronendichten Innenstruktur. Sie treten später in enge räumliche Beziehungen zu den Fettkörpern. In der Spätphase läßt sich eine starke Entfaltung der vesikulär umgebildeten Golgizentren und des rauhen endoplasmatischen Retikulum beobachten. Somit kann eine Ausschüttungs- und eine Restitutionsphase unterschieden werden. Eine Beteiligung der Lysosomen an der Parathormonsekretion wird diskutiert.

---

Summary After EDTA-stimulation the blood calcium level is instantaneously lowered for a restricted time. EDTA- (8%) injected Wistar rats of 150–200 g body weight were killed in various intervals after the injections and the ultrastructural changes of the parathyroid glands were examined.In the beginning only changes in the so-called osmiophilic bodies are observed. The electron dense contents of these granules become flocculent and vesiculated. Later they gain close relation to lipid bodies. A confluence of the two bodies seems likely. In the final phase the vesicular Golgi field and the rough endoplasmic reticulum expand markedly, indicating an increased activity. Thus a phase of release and one of restitution can be distinguished. The participation of lysosomes in the secretion of the parathormone is discussed.

---

---

Ausgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

---

Wesentliche Teile der vorliegenden Arbeit werden von Brigitte Krässig der Medizinischen Fakultät der Universität Freiburg i. Br. als Inauguraldissertation vorgelegt.