Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B

Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.     

Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B

Mannose-binding lectin (MBL) is a serum protein that has been demonstrated to activate the classical complement pathway and to function directly as an opsonin. Although MBL deficiency is associated with a common opsonic defect and a predisposition to infection, the role of the protein in bacterial infection remains unclear. We have investigated MBL binding to Neisseria meningitidis serogroup B1940 and three isogenic mutants, and the subsequent activation of the two major isoforms of C4 (C4A and C4B) by an associated serine protease, MASP. The mutants lacked expression of the capsular polysaccharide (siaD-), the lipo-oligosaccharide (LOS) outer core that prevented LOS sialylation (cpsD-), or both capsule and LOS outer core (cps-). Using flow cytometry, it was possible to detect strong MBL binding to the cps- and cpsD- mutants over a wide range of concentrations. In contrast, minimal or no MBL binding was detected on the parent organism, with binding to siaD- only at higher MBL concentrations. C4 was activated and bound by mutants that had previously bound MBL/MASP, but there was no significant difference in the amounts of C4A and C4B bound. When sialic acid residues were removed from the parent organism by neuraminidase treatment, the binding of both MBL and C4 increased significantly. Our results suggest that MBL may bind to and activate complement on these encapsulated organisms, and the major determinants of these effects are the LOS structure and sialylation.     

A Mechanism for Carbon Depletion at Bondline of High-Frequency Electric-Resistance-Welded X70 Pipeline Steel

Metallurgical and Materials Transactions A - The bondline of electric-resistance-welded (ERW) linepipe steel, often etched white (i.e., ferrite) in optical microscopy, is generally believed to be...     

Three rules for bone adaptation to mechanical stimuli

The primary mechanical function of bones is to provide rigid levers for muscles to pull against, and to remain as light as possible to allow efficient locomotion. To accomplish this bones must adapt their shape and architecture to make efficient use of material. Bone adaptation during skeletal growth and development continuously adjusts skeletal mass and architecture to changing mechanical environments. There are three fundamental rules that govern bone adaptation: (1) It is driven by dynamic, rather than static, loading. (2) Only a short duration of mechanical loading is necessary to initiate an adaptive response. (3) Bone cells accommodate to a customary mechanical loading environment, making them less responsive to routine loading signals. From these rules, several mathematical equations can be derived that provide simple parametric models for bone adaptation.     

Cerebral organization for language in deaf and hearing subjects: biological constraints and effects of experience

Highly enriched methanotrophic communities (> 25 serial transfers) were obtained from acidic ombrotrophic peat bogs from four boreal forest sites. The enrichment strategy involved using media conditions that were associated with the highest rates of methane uptake by the original peat samples, namely, the use of diluted mineral medium of low buffering capacity, moderate incubation temperature (20 degrees C), and pH values of 3 to 6. Enriched communities contained a mixture of rod-shaped bacteria arranged in aggregates with a minor contribution of Hyphomicrobium-like cells. The growth stoichiometry of isolates was characteristic of methanotrophic bacteria (CH4/O2/CO2 = 1:1.1:0.59), with an average apparent yield of 0.41 +/- 0.03 g of biomass C/g of CH4-C. DNA from each enrichment yielded a PCR product of the expected size with primers for both mmoX and mmoY genes of soluble methane monooxygenase. Two types of sequences were obtained for PCR-amplified fragments of mmoX. One of them exhibited high identity to the mmoX protein of the Methylocystis-Methylosinus group, whereas the other showed an equal level of divergence from both the Methylosinus-Methylocystis group and Methylococcus capsulatus (Bath) and formed a distinct branch. The pH optimum for growth and for CH4 uptake was 4.5 to 5.5, which is very similar to that for the optimum CH4 uptake observed in the original peat samples. These methanotrophs are moderate acidophiles rather than acidotolerant organisms, since their growth rate and methane uptake were much lower at neutral pH. The growth of the methanotrophic community was enhanced by using media with a very low salt content (20 to 200 mg/liter), more typical of their natural environment. All four enriched communities grew on N-free medium.     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

BACKGROUND: Carotid angioplasty (CA) has been suggested to be a safer and more cost-effective alternative to carotid endarterectomy (CEA) in the management of symptomatic severe internal carotid artery (ICA) disease. METHODS: The study was conducted as a prospective consecutive randomized trial of CEA versus CA for symptomatic severe ICA disease in a university teaching hospital. All patients were assessed before and after surgery by a neurologist. The study consisted of 23 patients with focal carotid territory symptoms and severe ICA stenosis (> 70%) who were randomized to either CEA or CA. However, only 17 had received their allocated treatment before trial suspension. CEA with patching or CA with stenting were used as interventions. The main outcome measures were death or disabling or nondisabling stroke within 30 days. RESULTS: All 10 CEA operations proceeded without complication, but 5 of the 7 patients who underwent CA had a stroke (P=.0034), 3 of which were disabling at 30 days. CONCLUSIONS: After referral, the Data Monitoring Committee invoked the stopping rule and the trial was suspended. The investigators and the Ethics Committee subsequently concluded that the trial could not be restarted--even in an amended format-primarily because of problems with informed consent. We review many of the ethical dilemmas encountered in the performance of this study. If future trials do suggest a selected role for CA, it is essential that both the inclusion and the exclusion criteria are fully documented.     

CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav

Germ cells are distinct from somatic cells in their immortality, totipotency, and ability to undergo meiosis. Candidates for components that guide the unique germline program are the distinctive granules observed in germ cells of many species. We show that a component of germ granules is essential for fertility in C. elegans and that its primary function is in germline proliferation. This role has been revealed by molecular and genetic analyses of pgl-1. PGL-1 is a predicted RNA-binding protein that is present on germ granules at all stages of development. Elimination of PGL-1 results in defective germ granules and sterility. Interestingly, PGL-1 function is required for fertility only at elevated temperatures, suggesting that germline development is inherently sensitive to temperature.     

Clinic characteristics associated with reduced hospitalization of drug users with AIDS

The purpose of this study was to develop a substantive grounded theory of courage among middle-aged adults with long-term health concerns. Twenty-five persons from rural and non-metropolitan areas of Central Illinois were selected to participate in this study based on theoretical sampling procedures. Interviews of 1 to 2 hours using openended questions were audiotaped and transcribed verbatim. The data were analysed using grounded theory methods. Courage among middle-aged adults with long-term health concerns was determined to consist of an ongoing progressive-regressive process of becoming and being courageous. Being courageous involves being fully aware of and accepting the threat of a long-term health concern, solving problems using discernment, and developing enhanced sensitivities to personal needs and the world in general. Courageous behaviour consists of taking responsibility and being productive. Courage is not limitless, and the process of becoming and being courageous is dependent on intrapersonal and interpersonal factors. Health-care providers facilitate this process by demonstrating competence and communicating effectively. Outcomes of being courageous include personal integrity and thriving in the midst of normality.