Knockout of the gene (<Emphasis Type="Italic">ste15</Emphasis>) encoding a glycosyltransferase and its function in biosynthesis of exopolysaccharide in <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15 ⁻) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis.     

Upregulation of PHLDA2 in Dicer knockdown HEK293 cells

It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer depletion rather than the consequence of growth arrest induced by Dicer knockdown. Considering the reports that there is consistent placental outgrowth in PHLDA2 knockout mice and that PHLDA2 overexpression in mice causes growth inhibition, we speculate that PHLDA2 may be a candidate for contributing to the reduced growth rate of Dicer-deficient cells and the very early embryonic lethality in Dicer knockout mice.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

Floral morphogenesis in Euptelea (Eupteleaceae, Ranunculales)

BACKGROUND AND AIMS: Based on molecular phylogenetic studies, the unigeneric family Eupteleaceae has a prominent phylogenetic position at or near the base of Ranunculales, which, in turn, appear at the base of eudicots. The aim of the present paper is to reveal developmental features of the flowers and to put the genus in a morphological context with other basal eudicots. METHODS: Flowers in all developmental stages of Euptelea pleiosperma were collected in the wild at intervals of 7-10 d in the critical stages and studied with a scanning electron microscope. KEY RESULTS: Remnants of a perianth are lacking throughout flower development. Floral symmetry changes from monosymmetric to asymmetric to disymmetric during development. Asymmetry is expressed in that the sequence of stamen initiation is from the centre to both lateral sides on the adaxial side of the flower but starting from one lateral side and proceeding to the other on the abaxial side. Despite the pronounced floral disymmetry, a dimerous pattern of floral organs was not found. The carpel primordia arise between the already large stamens and alternate with them. Stamens and carpels each form a somewhat irregular whorl. The carpels are ascidiate from the beginning. The stigma differentiates as two crests along the ventral slit of the ovary. The few lateral ovules alternate with each other. CONCLUSIONS: Although the flowers have some unusual autapomorphies (wind pollination, lack of a perianth, pronounced disymmetry of the floral base, long connective protrusion, long temporal gap between androecium and gynoecium initiation, small space for carpel initiation), they show some plesiomorphies at the level of basal eudicots (free carpels, basifixed anthers, whorled phyllotaxis), and thus fit well in Ranunculales.     

Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

The oligomeric state of c rings from cyanobacterial F-ATP synthases varies from 13 to 15

We isolated the c rings of F-ATP synthases from eight cyanobacterial strains belonging to four different taxonomic classes (Chroococcales, Nostocales, Oscillatoriales, and Gloeobacteria). These c rings showed different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), probably reflecting their molecular masses. This supposition was validated with the previously characterized c(11), c(14), and c(15) rings, which migrated on SDS-PAGE in proportion to their molecular masses. Hence, the masses of the cyanobacterial c rings can conveniently be deduced from their electrophoretic mobilities and, together with the masses of the c monomers, allow the calculation of the c ring stoichiometries. The method is a simple and fast way to determine stoichiometries of SDS-stable c rings and hence a convenient means to unambiguously determine the ion-to-ATP ratio, a parameter reflecting the bioenergetic efficacy of F-ATP synthases. AFM imaging was used to prove the accuracy of the method and confirmed that the c ring of Synechococcus elongatus SAG 89.79 is a tridecameric oligomer. Despite the high conservation of the c-subunit sequences from cyanobacterial strains from various environmental groups, the stoichiometries of their c rings varied between c(13) and c(15). This systematic study of the c-ring stoichiometries suggests that variability of c-ring sizes might represent an adaptation of the individual cyanobacterial species to their particular environmental and physiological conditions. Furthermore, the two new examples of c(15) rings underline once more that an F(1)/F(o) symmetry mismatch is not an obligatory feature of all F-ATP synthases.     

A DNA vaccine against chimeric AFP enhanced by HSP70 suppresses growth of hepatocellular carcinoma

Alpha-Fetoprotein (AFP) is produced principally in fetal liver, gastrointestinal tract and the yolk sac which is temporarily present during embryonic development. AFP is overexpressed in the majority of Hepatocellular Carcinoma (HCC) and thus offers an attractive target for immunotherapy against this neoplasm. Here, we report that anti-HCC effects were achieved in a therapeutic setting with a DNA vaccine encoding mouse AFP and co-expressing Heat Shock Protein 70 (HSP70) gene. We also demonstrated that this vaccine elicited a marked and highly effective AFP specific CTL response against AFP-positive target cells. This vaccine also induced the prolongation of life span in mice bearing the tumor and the eradication of HCC. It is anticipated that vaccine strategies such as this may contribute to the effective future treatment of Hepatocellular Carcinoma. Ying-hua Lan and Yong-guo Li contributed equally to this work.     

cGMP signals mainly through cAMP kinase in permeabilized murine aorta

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca(2+) sensitization, a process identified as Ca(2+) desensitization. Ca(2+) desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca(2+) desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca(2+)] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca(2+)] increased aortic tone in the absence and presence of 50 microM GTPgammaS with EC(50) values of 160 and 30 nM, respectively. In the absence of GTPgammaS, the EC(50) for [Ca(2+)] was shifted rightward from 0.16 microM to 0.43 and 0.82 microM by 1 and 300 microM 8-bromo-cGMP (8-Br-cGMP), and to 8 microM by 10 microM Y-27632. Contractions induced by 300 nM [Ca(2+)] were relaxed by 8-Br-cGMP with an EC(50) of 2.6 microM. Surprisingly, [Ca(2+)]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI(-/-) mice (EC(50) of 19 microM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI(-/-) mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI(-/-) mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca(2+)], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca(2+) sensitization in murine aortas.     

黄连属(毛茛科)花的形态发生

本文运用扫锚电子显微镜（SEM）观察了黄连属（Coptis）植物花的形态发生和发育过程,结果表明,该属植物所有的花部器官均为螺旋状发生,雄蕊为向心式发育,花瓣原基有微弱的延迟发育,心皮原基为对折型（即马蹄形）,子房为半封闭类型,子房柄是在发育过程中形成的。通过与其它具T．型染色体类群在花形态发生上的比较,认为黄连属表现出了某些原始的性状,这一结果与分子系统学研究认为黄连属为毛莨科的基部类群的结论一致。