Oxidative stress and expression of chaperones in aging mollusks

The mechanisms of aging are not well understood in animals with continuous growth such as fish, reptiles, amphibians and numerous invertebrates, including mollusks. We studied the effects of age on oxidative stress, cellular defense mechanisms (including two major antioxidant enzymes, superoxide dismutase (SOD) and catalase), and molecular chaperones in two mollusks--eastern oysters Crassostrea virginica and hard clams Mercenaria mercenaria. In order to detect the age-related changes in these parameters, correction for the effects of size was performed where appropriate to account for growth-related dilution. Fluorescent age pigments accumulated with age in both species. Protein carbonyls did not change with age or size indicating that they are not a good marker of aging in mollusks possibly due to the fast turnover and degradation of oxidized proteins in growing tissues. SOD did not show a compensatory increase with aging in either species, while catalase significantly decreased with age. Mitochondrial heat shock protein (HSP60) decreased with age in mollusks suggesting an age-related decline in mitochondrial chaperone protection. In contrast, changes in cytosolic chaperones were species-specific. HSP70 increased and HSP90 declined with age in clams, whereas in oysters HSP70 expression did not change, and HSP90 increased with aging.     

Hemoptysis--a rare complication of pacemaker implantation

We describe a case of hemoptysis as a rare complication of pacemaker lead insertion via the axillary approach in a patient with difficult chest anatomy.     

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Oxidative stress causes membrane phospholipid rearrangement and shedding from RBC membranes--an NMR study

Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the relationship between oxidative stress experienced by RBCs and their phospholipid content and shedding. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio, an indicator of oxidative stress, in normal RBCs treated with oxidants (t-butylhydroxyperoxide and H2O2) as well as in beta-thalassemic RBCs. Using 31P-NMR, we found 30% more phosphatidylcholine (PC), and unexpectedly, 35% less phosphatidylserine (PS) in the thalassemic RBCs. PS was decreased by treatment with oxidants and increased by anti-oxidants (vitamin C and N-acetyl cysteine); PC showed the opposite behavior. Thalassemic RBCs incubated in phosphate buffered saline produced more PS in the supernatant than normal RBCs. Anti-oxidants reduced the PS in the supernatant while oxidants increased it. Plasma of thalassemic patients contained 2.6-fold and 1.8-fold more PS and PC, respectively, than normal plasma. These results indicate that the decreased PS in RBCs resulted from increased shedding. The nature of the shed PS was studied by purifying and analyzing membranous microparticles from the plasma and RBC supernatants. More PS was found in microparticles purified from thalassemic plasma and RBC supernatants (5.6- and 4.8-fold, respectively) than in their normal counterparts. However, the bulk (80-90%) of the shed PS was not associated with microparticles. The significance of PS shedding for RBC survival needs further clarification.     

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.     

Mitochondrial cytochrome b sequence variations and population structure of Siberian chipmunk (Tamias sibiricus) in Northeastern Asia and population substructure in South Korea

Twenty-five chipmunk species occur in the world, of which only the Siberian chipmunk, Tamias sibiricus, inhabits Asia. To investigate mitochondrial cytochrome b sequence variations and population structure of the Siberian chipmunk in northeastern Asia, we examined mitochondrial cytochrome b sequences (1140 bp) from 3 countries. Analyses of 41 individuals from South Korea and 33 individuals from Russia and northeast China resulted in 37 haplotypes and 27 haplotypes, respectively. There were no shared haplotypes between South Korea and Russia--northeast China. Phylogenetic trees and network analysis showed 2 major maternal lineages for haplotypes, referred to as the S and R lineages. Haplotype grouping in each cluster was nearly coincident with its geographic affinity. In particular, 3 distinct groups were found that mostly clustered in the northern, central and southern parts of South Korea. Nucleotide diversity of the S lineage was twice that of lineage R. The divergence between S and R lineages was estimated to be 2.98-0.98 Myr. During the ice age, there may have been at least 2 refuges in South Korea and Russia--northeast China. The sequence variation between the S and R lineages was 11.3% (K2P), which is indicative of specific recognition in rodents. These results suggest that T. sibiricus from South Korea could be considered a separate species. However, additional information, such as details of distribution, nuclear genes data or morphology, is required to strengthen this hypothesis.