Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.     

Comparative pulmonary toxicity of cadmium and nickel: histopathological and bronchoalveolar lavage analysis

Pulmonary toxicity of cadmium and nickel was evaluated in rat lungs following intratracheal instillation of their chlorides. Concentration of both the metals varied from 0.2-5 mM. Both the metals increased total number of cells, number of polymorphonuclear neutrophils, total protein, sialic acid and the activity of lactate dehydrogenase and beta-glucuronidase in bronchoalveolar lavage 3 days after exposure. Increase in the levels of the selected parameters was more following Cd exposure than in Ni exposed rats. Histologically there was an inflammatory response and interstitial fibroblastic proliferation in the lungs of Cd exposed animals. These changes were mild in Ni-exposed animals and higher concentrations of Ni were needed to produce changes similar to those produced by smaller concentrations of Cd.     

EBV structural antigens, gp350 and gp85, as targets for ex vivo virus-specific CTL during acute infectious mononucleosis: potential use of gp350/gp85 CTL epitopes for vaccine design

For many years, EBV vaccine development efforts have concentrated on the use of structural Ag, gp350, and have been directed toward Ab-mediated blocking virus attachment to the target cell. There is increasing evidence to suggest that the development of neutralizing Abs in vaccinated animals does not always correlate with protection; nevertheless, it has been postulated that gp350-specific T cell-mediated immune responses may have an effector role in protection. This hypothesis has largely remained untested. In the present study, we demonstrate that CTL from acute infectious mononucleosis patients display strong ex vivo reactivity against the EBV structural Ags, gp85 and gp350. Moreover, long-term follow up studies on infectious mononucleosis-recovered individuals showed that these individuals maintain gp350- and gp85-specific memory CTL, albeit at low levels, in the peripheral blood. These results strongly suggest that CTL specific for EBV structural proteins may play an important role in the control of EBV infection during acute infection. More importantly, we also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results have important implications for future EBV vaccine design and provides evidence, for the first time, that CTL epitopes from EBV structural proteins may be used for establishing strong antiviral immunity against EBV infection.     

Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.     

Isolation and Characterization of <Emphasis Type="Italic">Citrobacter</Emphasis> Strain HPC255 for Broad-Range Substrate Specificity for Chlorophenols

This study aims at development of an approach for selection of strain, which has capability for oxidation of broad-range of chloro-substitute phenols. A multiplex PCR was optimized targeting loci involved in phenol and chlorophenol degradation, which was used to select activated sludge samples and also to assess the degradative genotype of isolates. The isolated strains were screened on the basis of RAPD analysis. In parallel, physiological experiments were carried out with activated sludge samples and isolated bacteria by respirometric analysis. Based on cluster analysis of RAPD pattern and respirometric data, the isolate G20 was selected and identified by using 16S rDNA sequence analysis as Citrobacter freundii strain HPC255. The strain could oxidize different substituted chlorophenol molecules. Such strains could provide the pool of intermediates, which can further be degraded by the associated population, thus helping in maintaining the synergistic association of catabolic activity in activated sludge.     

Pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates. Synthesis, DNA binding and cytotoxicity

New pyrrolobenzodiazepine-anthraquinone hybrids have been designed and synthesized, found to effectively bind to DNA and also exhibit cytotoxicity against many cancer cell lines     

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.     

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.     

14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule-associated tau phosphorylation complex

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an approximately 400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 zeta co-elutes with the tau phosphorylation complex components tau and GSK3 beta. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3 zeta, GSK3 beta, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3 beta, 14-3-3 zeta co-precipitates with GSK3 beta, indicating that GSK3 beta binds to 14-3-3 zeta. From HEK-293 cells transfected with tau, GSK3 beta, and 14-3-3 zeta in different combinations, tau co-immunoprecipitates with GSK3 beta only in the presence of 14-3-3 zeta. In vitro, approximately 10-fold more tau binds to GSK3 beta in the presence of than in the absence of 14-3-3 zeta. In transfected HEK-293 cells, 14-3-3 zeta stimulates GSK3 beta-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 zeta dimer simultaneously binds and bridges tau and GSK3 beta and stimulates GSK3 beta-catalyzed tau phosphorylation.