Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Current concepts on Escherichia coli K1 translocation of the blood-brain barrier

The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively. With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E. coli requires a high-degree of bacteremia, E. coli binding to and invasion of HBMEC, and E. coli traversal of the BBB as live bacteria. Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E. coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo. We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E. coli translocation of the BBB is the result of specific pathogen-host cell interactions. Recent studies using functional genomics techniques have identified additional E. coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis. In this review, we summarize the current knowledge on the mechanisms underlying the successful E. coli translocation of the BBB.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Comparing the performance of one-column process and four-zone simulated moving bed by computer simulation

A new one-column chromatography process, analogous to a four-zone simulated moving bed (SMB), was presented. The basic principle of the process was identical to that of a four-zone SMB. The process consisted of one chromatographic column and four tanks, instead of the four columns in the four-zone SMB (1-1-1-1), and has been used for the separation of two amino acids, phenylalanine and tryptophan, using an ion exchange resin. The operating parameters for the one-column process and four-zone SMB were obtained from equilibrium theory. Computer simulations were used to compare the performances of the new one column process to that of the general four-zone SMB, using Aspen Chromatography^™ v 11.1. The differences between the one-column and SMB processes in terms of the purities and yields of phenylalanine and tryptophan were less than 4 and about 6%, respectively. The lower purities of the one-column process were due to the loss of the developed concentration profiles in the column when the liquid was stored in tanks. The one-column process gave great flexibility, and would be useful for reconstructing an existing conventional chromatography process to one of a SMB.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site

gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site.     

Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking

Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.