QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Noradrenaline suppression of the spinal efferent component of the pressor reflexes

Noradrenaline applied to the dorsal surface of spinal cord segments C6-T1 suppressed the pressor components of the blood pressure reflexes evoked by stimulation of radial nerve afferents in anesthetized cats. Noradrenaline applied to spinal cord segments L4-S1 suppressed pressor reflexes elicited by stimulation of tibial nerve afferents. The increase in noradrenaline concentration from 0.05 to 0.2% enhanced the duration and intensity of this effect.     

Production of glycerinated models of muscle fibers with phosphorylated myosin light chains

A simple method for obtaining glycerinated muscle fibres of m. psoas of rabbit containing regulatory myosin light chains (LC2) of different levels of phosphorylation. The glycerination conditions stimulated endogenic kinase LC2 or phosphatase LC2. Glycerinated muscle fibres contained phosphorylated and dephosphorylated (levels of phosphorylation are 95 +/- 5%, and 5 +/- 5%, respectively) LC2 of myosin. To determine the level of phosphorylation the method of polyacrylamide gel electrophoresis in 8 M urea was modified.     

RAPID SCREENING FOR SALMONELLA IN FISHMEAL BY ENRICHMENT-IMMUNOASSAY

We previously described an enrichment-immunoassay utilizing a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Here we evaluated it for the rapid screening for Salmonella in fishmeal obtained from the national Animal and Plant Quarantine service in the People's Republic of China. In this method, the number of Salmonella present is first expanded by appropriate enrichment cultures, and the pathogens are then directly detected by the T6 immunoassay. In a total of 94 enrichment cultures of fishmeal, we obtained an overall concordance of 98% between the results obtained in parallel by this method and by conventional test method. The positive prediction by this method was 92% and the negative prediction was 100%. The turn around time for the new test was 27 h which is a significant improvement from the turn around time exceeding 96 h required for the conventional test method. This test proved to be compatible with the routine work flow in the practical setting of a quarantine laboratory.     

eIF3j facilitates loading of release factors into the ribosome

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.     

Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells

Background  

Mannose binding lectin (MBL) is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro.     

A conserved surface loop in type I dehydroquinate dehydratases positions an active site arginine and functions in substrate binding

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.     

Optimization of in vivo crosslinking technique for the study of AlpB-protein interactions in Lysobacter sp. XL1 cells

The bacterium Lysobacter species strain XL1 is known as a producer of extracellular lytic enzymes, which are capable of degrading cell wall components of other bacteria and simple eukaryotes. This ability determines the ecological, medical and agricultural relevance of Lysobacter sp. XL1. However, the molecular mechanism of secretion of lytic exoenzymes from Lysobacter cells is yet unknown, which in turn necessitates the search of protein–protein interactions that occur during exoenzyme secretion. The current paper is concerned with investigation of protein complexes that are likely formed during the secretion of AlpB lytic protease from the cells of Lysobacter sp. XL1. In this study, we have optimized the method of stabilization of protein complexes formed in the intact cells of Lysobacter sp. XL1 by using crosslinking reagent dithiobis(succinimidylpropionate) (DSP) and detected DSP-linked protein complexes by the monoclonal antibodies against AlpB propeptide.