Septins, a conserved family of cytoskeletal GTPbinding proteins, were presented in diverse eukaryotes. Here, a comprehensive phylogenetic and evolutionary analysis for septin proteins in metazoan was carried out. First, we demonstrated that all septin proteins in metazoan could be clustered into four subgroups, and the representative homologue of every subgroup was presented in the non-vertebrate chordate Ciona intestinalis, indicating that the emergence of the four septin subgroups should have occurred prior to divergence of vertebrates and invertebrates, and the expansion of the septin gene number in vertebrates was mainly by the duplication of pre-existing genes rather than by the appearance of new septin subgroup. Second, the direct orthologues of most human septins existed in zebrafish, which suggested that human septin gene repertoire was mainly formed by as far as before the split between fishes and land vertebrates. Third, we found that the evolutionary rate within septin family in mammalian lineage varies significantly, human SEPT1, SEPT 10, SEPT 12, and SEPT 14 displayed a relative elevated evolutionary rate compared with other septin members. Our data will provide new insights for the further function study of this protein family.

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Activation of b-catenin, the central effector of the canonical wingless-type (Wnt) pathway, has been implicated in hepatocellular carcinoma (HCC). However, the transcription regulation mechanism of the b-catenin gene in HCC remains unknown. Here we report that human zinc finger protein 191 (ZNF191) is a potential regulator of b-catenin transcription. ZNF191, a Krüppel-like protein, specifically interacts with the TCAT motif, which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene ex vivo. We demonstrate that ZNF191 is significantly overexpressed in human HCC specimens and is associated with growth of human HCC cells. Global profiling of gene expression in ZNF191 knockdown human hepatic L02 cells revealed that the important Wnt signal pathway genes b-catenin and cyclin D1 messenger RNAs (mRNAs) are significantly downregulated. In agreement with transcription level, b-catenin and cyclin D1 proteins are also down-regulated in transient and stable ZNF191 knockdown L02 and hepatoma Hep3B cell lines. Moreover, significant correlation between ZNF191 and b-catenin mRNA expression was detected in human HCCs. Promoter luciferase assay indicated that ZNF191 can increase transcription activity of the full-length b-catenin (CTNNB1) promoter, and nucleotide (nt)-1407/-907 of the CTNNB1 promoter exhibited the maximum transcriptional activity. Electrophoretic mobility shift assay showed that purified ZNF191 protein can directly bind to the CTNNB1 promoter, and the binding region is located at nt-1254/-1224. Finally, we demonstrate that the key binding sequence of ZNF191 in vivo is ATTAATT. Conclusion: ZNF191 can directly bind to the CTNNB1 promoter and activate the expression of b-catenin and its downstream target genes such as cyclin D1 in hepatoma cell lines. This study uncovers a new molecular mechanism of transcription regulation of the b-catenin gene in HCC.

The use of an ultrasonic nebulizer to assist electrospray ionization mass spectrometry (ESI-MS) has been described and demonstrated with the analysis of a transfer RNA digest by microcolumn LC. The restricted range of mobile-phase compositions amenable to the electrospray process has traditionally placed a severe limitation on the types of LC applications that can be used with ESI-MS. For this reason, an ultrasonic nebulizer configured for LC has been developed that can generate the fine dispersion of liquid required for ESI-MS from any type of mobile phase. In the case presented here, a transfer RNA was enzymatically digested into its substituent nucleosides, which were then analyzed by microcolumn LC. The required mobile-phase gradient (beginning at 5% methanol) falls outside the solvent range that can be used with conventional electrospray. The ultrasonic nebulizer, however, resolves this problem. The fundamental behavior of the four most common nucleosides (cytidine, adenosine, guanosine, uridine) was studied, and conclusions concerning the effects of solution chemistry were drawn. Specifically, signal from the H+ adducts of these species seems to be strongly dependent on the pKa value. Also, effects from several source operating variables were examined. These included capillary exit voltage, drying and focusing gases, and nebulizer frequency. Performance was found to be consistent over a wide range (0-100% methanol) of mobile-phase compositions. The limit of detection for adenosine injected onto a microcolumn was found to be 100 amol. Finally, nucleosides from as little as 150 fmol of RNA (amount prior to digestion) could be detected.

Genetic interference by double-stranded RNA (dsRNA) or RNA interference (RNAi) triggers the sequence-specific degradation of cellular transcripts. The mediators of mRNA degradation are small interfering RNAs (siRNAs). Here, we report that in Trypanosoma brucei, 10%-20% of siRNAs cosediment with polyribosomes. Preventing the assembly of ribosomes on mRNAs results in a concomitant decrease of siRNAs sedimenting as high-molecular-weight complexes. We further provide evidence that siRNAs are associated with translating ribosomes and that this association is mediated by a 70-kD ribonucleoprotein complex.

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.