The proteomic alterations of Thermoanaerobacter tengcongensis cultured at different temperatures.

Thermoanaerobacter tengcongensis, one of many thermophilic organisms, survives harsh living conditions in temperatures ranging from 50 to 80 degrees C. In this comprehensive analysis, we present a robust approach, 2-DE and MALDI-TOF MS, to compare and identify the bacterial proteins responding to the temperature stress. In total, 164 spots of 2-DE were found with the significant changes in spot volume at three culture temperatures, 55, 75, and 80 degrees C, respectively; furthermore, 87 unique proteins were characterized by MS. Our results reveal that the electrophoretic images of the bacterial proteins, extracted from two culture temperatures (55 and 75 degrees C), had similar patterns; however, the bacteria cultured at 80 degrees C had dramatically decreased their spot volumes. Additionally, the temperature-sensitive proteins are broadly divided into two groups: specific expression at certain temperatures and consistent changes of expression responsive to temperature. For instance, three proteins closely related with redox regulation, dihydrolipoamide acyltransferase, NADH:ubiquinone oxidoreductase, and ferredoxin, were only detected in the bacteria cultured at 55 degrees C. Whereas, two chaperonins, GroES and GroEL, were found to show a consistent increase during the elevated temperatures with the determinations, either by MS or Western blot. The proteomic information, thus expedites our understanding of the molecular mechanisms regarding how thermophilic bacteria adapt to the alterations in living environment.

A proteomic investigation into a human gastric cancer cell line BGC823 treated with diallyl trisulfide.

Garlic is generally used as a therapeutic reagent against various diseases worldwide. Although a great effort is made to understand the pharmaceutical mechanisms of garlic and its derivatives, there are many mysteries to be uncovered. Using proteomic means, herein we have systematically studied the responses of protein expression in BGC823 cells, a gastric cancer cell line, induced by diallyl trisulfide (DATS), a major component of garlic derivatives. A total of 41 unique proteins in BGC823 were detected with significant changes in their expression levels corresponding with DATS administration. Of these proteins, five typical ones, glutathione S-transferase-pi (GST-pi), voltage-dependent anion channel-1 (VDAC-1), Annexin I, Galectin and S100A11, were further examined by Western blotting, resulting in coincident data with the proteomic evidence. Moreover quantitative real-time RT-PCR experiments offered dynamic data of mRNA expression, indicating the responses of Annexin I and GST-pi expression within a short period after DATS treatment. Interestingly, approximately 50% of DATS-sensitive proteins (19/41) in BGC823 are tightly associated with apoptotic pathways. These proteomic results presented, therefore, provide additional support to the hypothesis that garlic is a strong inducer of apoptosis in tumor cells.

Major vault protein, in concert with constitutively photomorphogenic 1, negatively regulates c-Jun-mediated activator protein 1 transcription in mammalian cells.

Constitutively photomorphogenic 1 (COP1), a RING finger ubiquitin ligase with substrates including c-Jun and p53, was recently found to be overexpressed in a number of breast and ovarian tumor samples. In addition to its E3 activity, COP1 was also shown to be able to inhibit activator protein 1 (AP-1) transcription. Through an affinity purification method, we have identified major vault protein (MVP) as a novel interacting partner for COP1 in mammalian cells. MVP, also known as lung resistance protein, is the main component of a ribonucleoprotein organelle called vault, and has been implicated in multiple drug resistance in many cancer cell lines and primary tumor samples. The interaction between COP1 and MVP is detectable at the endogenous level and occurs mostly in the cytoplasm. Similar to COP1, MVP inhibits c-Jun accumulation and AP-1 transcription activity. MVP knockout or knockdown cells contain elevated amount of c-Jun and increased AP-1 transcription activity. UV irradiation enhances MVP tyrosine phosphorylation, causes dissociation of COP1 from MVP, and alleviates the inhibitory activity of MVP on AP-1 transcription. Taken together, we propose that MVP, most likely through its interaction with COP1, suppresses c-Jun-mediated AP-1 transcription under unstressed conditions, thereby preventing cells from undergoing stress response.

Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys.

Snake venom is a complex mixture of proteins and peptides, and a number of studies have described the biological properties of several venomous proteins. Nevertheless, a complete proteomic profile of venom from any of the many species of snake is not available. Proteomics now makes it possible to globally identify proteins from a complex mixture. To assess the venom proteomic profiles from Naja naja atra and Agkistrodon halys, snakes common to southern China, we used a combination strategy, which included the following four different approaches: (i) shotgun digestion plus HPLC with ion-trap tandem MS, (ii) one-dimensional SDS/PAGE plus HPLC with tandem MS, (iii) gel filtration plus HPLC with tandem MS and (iv) gel filtration and 2DE (two-dimensional gel electrophoresis) plus MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS. In the present paper, we report the novel identification of 124 and 74 proteins and peptides in cobra and viper venom respectively. Functional analysis based upon toxin categories reveals that, as expected, cobra venom has a high abundance of cardio- and neurotoxins, whereas viper venom contains a significant amount of haemotoxins and metalloproteinases. Although approx. 80% of gel spots from 2DE displayed high-quality MALDI-TOF-MS spectra, only 50% of these spots were confirmed to be venom proteins, which is more than likely to be a result of incomplete protein databases. Interestingly, these data suggest that post-translational modification may be a significant characteristic of venomous proteins.

Inactivated SARS-CoV vaccine prepared from whole virus induces a high level of neutralizing antibodies in BALB/c mice.

We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.

Preparation, characterization and preliminary in vivo studies of inactivated SARS-CoV vaccine.

A large quantity of SARS-CoV virus was proliferated in Vero cells, inactivated with ß-propiolactone, then purified by Sepharose 4FF column chromatography to prepare inactivated vaccine. The vaccine was identified by Western blot, mass spectrographic analysis, ELISA and electron microscopy. The vaccine with or without aluminum hydroxide adjuvant was inoculated into female BALB/c mice at different dosages. The result showed that the antibodies to SARS-CoV were induced in the mice. The antibody levels induced by the vaccine with aluminum hydroxide were higher than those without aluminum hydroxide.