A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.     

Carnosine and related dipeptides protect human ceruloplasmin against peroxyl radical-mediated modification

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. In a previous study, we showed that the aggregation of human ceruloplasmin was induced by peroxyl radicals. We investigated the effects of antioxidant dipeptides carnosine, homocarnosine and anserine on peroxyl radical-mediated ceruloplasmin modification. Carnosine, homocarnosine and anserine significantly inhibited the aggregation of CP induced by peroxyl radicals. When CP was incubated with peroxyl radicals in the presence of three compounds, ferroxidase activity, as measured by the activity staining method, was protected. All three compounds also inhibited the formation of dityrosine in peroxyl radicals-treated CP. The results suggest that carnosine and related compounds act as peroxyl radical scavenger to protect the protein modification. It is proposed that carnosine and related peptides might be explored as potential therapeutic agents for pathologies that involve CP modification mediated by peroxyl radicals generated in the lipid peroxidation.     

Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.     

UV-vulnerability of human papilloma virus type-16 E7-expressing astrocytes is associated with mitochondrial membrane depolarization and caspase-3 activation

The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.     

Reprogramming of the activity of the activator/dissociation transposon family during plant regeneration in rice

Many aspects of epigenetic phenomena have been elucidated via studies of transposable elements. An active transposable element frequently loses its ability to mobilize and goes into an inactive state during development. In this study, we describe the cyclic activity of a maize transposable element dissociation (Ds) in rice. In rice genome, Ds undergoes the spontaneous loss of mobility. However, an inactive state of Ds can be changed into an active state during tissue culture. The recovery of mobility accompanies not only changes in the methylation patterns of the terminal region of Ds, but also alteration in the steady state level of the activator (Ac) mRNA that is expressed by a constitutive CaMV 35S promoter. Furthermore, the Ds-reactivation process is not random, but stage-specific during plantlet regeneration. Our findings have expanded previous observations on Ac reactivation in the tissue culture of maize.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.     

Comparison of responses elicited by immunization with a Legionella species common lipoprotein delivered as naked DNA or recombinant protein

To evaluate the peptidoglycan-associated lipoprotein (PAL) antigen of Legionella pneumophila as a vaccine candidate, mice were immunized intramuscularly with pcDNA3-PAL and intraperitoneally with recombinant PAL (t-rPAL), which were compared for their ability to induce PAL-specific immune responses. The t-rPAL protein induced PAL-specific IgG antibody production significantly more than did pcDNA3-PAL. The IgG2a and IgG1 production was predominant after pcDNA3-PAL and t-rPAL administration, respectively. In particular, pcDNA3-PAL induced much higher PAL-specific cytotoxic T-lymphocyte responses than did t-rPAL. Furthermore, in vivo, CD19+ B-cell populations were dramatically increased by t-rPAL vaccination, suggesting a B-cell immunomodulatory activity of the lipoprotein. The PAL antigen was also conserved among Legionella species, as determined by PCR and immunoblot analyses. These results support a potential use of the t-rPAL protein and in particular DNA vaccines against Legionella infections.     

Proteome analysis of hepatocellular carcinoma

Development of hepatocellular carcinoma (HCC) is a complex process involving multiple changes in gene expression and usually occurs in the presence of liver cirrhosis. In this research, we observed proteome alterations of three tissue types isolated from livers of HCC patients: normal, cirrhotic, and tumorous tissue. Proteome alterations were observed using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparing the tissue types with each other, a significant change in expression level was found in 21 proteins. Of these proteins, sarcosine dehydrogenase, liver carboxylesterase, peptidyl-prolyl isomerase A, and lamin B1 are considered novel HCC marker candidates. In particular, lamin B1 may be considered as a marker for cirrhosis, because its expression level changes considerably in cirrhotic tissue compared with normal tissue. The proteins revealed in this experiment can be used in the future for studies pertaining to hepatocarcinogenesis, or as diagnostic markers and therapeutic targets for HCC.     

EGCG attenuates AMPA-induced intracellular calcium increase in hippocampal neurons

We have investigated the protective effect of (-)-epigallocatechin gallate (EGCG) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolo propionate (AMPA)-induced toxicity in cultured rat hippocampal neurons. Treatment of 24 h AMPA (10 microM) reduced the neuronal viability in both survival neuron counting and MTT reduction assay compared with control, with increase in cellular concentrations of hydrogen peroxide and malondialdehyde. These responses to AMPA were significantly blocked by co-treatments with EGCG (10 microM), which effect was very similar to the protective ability of a known antioxidant catalase (2000 U/ml). AMPA (50 microM) elicited the increase in intracellular calcium concentration ([Ca(2+)]i) on which EGCG significantly attenuated both peak amplitude and sustained nature of that [Ca(2+)]i increase in a dose-dependent manner. These data suggest that EGCG has a neuroprotective effect against AMPA through inhibition of AMPA-induced [Ca(2+)]i increase and consequent attenuation of reactive oxygen species production and lipid peroxidation as an antioxidant and a radical scavenger.