Different programs of activation-induced cell death are triggered in mature activated CTL by immunogenic and partially agonistic peptide ligands

Activation-induced cell death (AICD) of mature T cells plays an important role in the control of immune homeostasis and peripheral tolerance. TNFRs and Fas have been implicated in the induction of AICD. However, these molecules were shown to be dispensable, at least in some experimental systems, for downsizing of Ag-induced T cell expansions and development of tolerance in vivo. The conditions of T cell activation leading to T cell deletion in a death receptor-independent manner are not well characterized. Here we show that human CTLs die through a death receptor-independent apoptotic program upon triggering with a partially agonistic peptide ligand. This apoptotic process exhibits some features of T cell death due to lymphokine deprivation and is blocked by exogenous IL-2. Our data demonstrate that engagement of TCR by MHC-peptide complexes can trigger diverse apoptotic programs of AICD and that the choice between these programs is determined by the agonistic potency of MHC-peptide ligand.     

Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.     

Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.     

Nitroxyl anion regulation of the NMDA receptor

Nitric oxide (NO) is an important regulator of NMDA channel function in the CNS. Recent findings suggest that nitroxyl anion (NO(-)) may also be generated by nitric oxide synthase, which catalyzes production of NO. Using recombinant NMDA receptors (NMDA-r) transfected into human embryonic kidney cells, our data demonstrate that the nitroxyl anion donor, Angeli's salt (AS; Na(2)N(2)O(3)) dramatically blocked glycine-independent desensitization in NMDA-r containing NR1-NR2A subunits. AS did not affect glycine-dependent desensitization, calcium dependent inactivation or glutamate affinity for the NMDA-r. This effect could be mimicked by treatment with DPTA, a metal chelator and was not evident under hypoxic conditions. In contrast, receptors containing the NR1-NR2B subunits demonstrated an approximate 25% reduction in whole cell currents in the presence of AS with no apparent change in desensitization. Our data suggest that the regulation of NMDA-r function by nitroxyl anion is distinctly different from NO and may result in different cellular outcomes compared with NO.     

Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1

Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.     

Detection of infectious baboon cytomegalovirus after baboon-to-human liver xenotransplantation

Xenotransplantation is considered to be a solution for the human donor shortage. However, there is a potential risk of transmitting animal infections from the transplanted organ. The known transmissibility and clinical significance of human cytomegalovirus (HCMV) infection after allotransplantation led us to evaluate whether baboon cytomegalovirus (BCMV) transmission could occur after a baboon-to-human liver xenotransplant. We examined serial blood samples from a baboon liver recipient and isolated replication-competent CMV-like agents on days 29, 36, and 42 after xenotransplantation. BCMV and HCMV DNAs were detected in the day 29 isolate, while only HCMV DNA was detected in the other isolates. This is the first report of detecting a replication-competent virus from a source animal after xenotransplantation and is a concern with regard to potential zoonotic transmission to others.     

Parasite-host relationship of flies and small mammals in the Omsk region

A generalized analysis of data on a flea fauna, range of their hosts in various natural zones, and features of parasite-host relationships between fleas and small mammals obtained in the Omsk province during long term researches in 1963-1997 is given. 35 flea species are recorded. The most mass species both on animals and in their nest is Ctenophthalmus assimilis; the numerous species are Amalareus penicilliger, Megabothros rectangulatus, M. walkeri, Peromyscopsylla silvatica, Ctenophthalmus unciatus, Palaropsylla sorecis, Doratopsilla birulai, Neopsylla pleskei, Hystrichopsylla talpae; the usual species--Ceratopsyllus garei, M. calcarifer, M. turbidus, Frontopsylla elata, Amphipsylla sibirica, A. kuznetzowi, Peromyscopsylla dasycnema, Radinopsylla integella, Catalagia dacenkoi. Other species are less numerous or infrequent. The general infection rate of the flea populations on rodents and insectivores makes 30.4%. For certain species it reaches 65.1% (on red-backed vole Cletrionomys rutilus), for regular groups--86.9% (on shrews of the genus Sorex). The greatest variety of the flea populations is observed on Microtus arvalis, the least one--on Sorex caecutiens and S. daphaenodon. In the nests of small mammals the variety of fleas is significantly lower. Based on the index of flea species relative "loyalty" to small mammals and their nest we have recognized 6 groups of fleas.     

Differential substrate behaviour of phenol and aniline derivatives during conversion by horseradish peroxidase

For the first time saturating overall k(cat) values for horseradish peroxidase (HRP) catalysed conversion of phenols and anilines are described. These k(cat) values correlate quantitatively with calculated ionisation potentials of the substrates. The correlations for the phenols are shifted to higher k(cat) values at similar ionisation potentials as compared to those for anilines. (1)H-NMR T(1) relaxation studies, using 3-methylphenol and 3-methylaniline as the model substrates, revealed smaller average distances of the phenol than of the aniline protons to the paramagnetic Fe(3+) centre in HRP. This observation, together with a possibly higher extent of deprotonation of the phenols than of the anilines upon binding to the active site of HRP, may contribute to the relatively higher HRP catalysed conversion rates of phenols than of anilines.     

Holliday junctions are formed in concentrated solutions of purified products of DNA amplification

Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.     

Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.