中国西北部毛莨科新资料

本文发表了中国西北部毛莨科新资料, 即巴里坤毛莨Ranunculus balikunensis J.G.Liou, sp.nov.;髯毛蒙古白头翁Pulsatilla ambigua Turcz.ex pritz.var.barbata J.G.Liou, var.nov.。     

几株杆状病毒包涵体蛋白双向高压电泳指纹图谱比较

用蔗糖密度梯度离心提纯的6种昆虫病毒包涵体(BsNPV1,BsNPV2,BtNPV,EpNPV,PrGV,PxGV)经DAS碱解,粗提纯的包涵体蛋白经Sepharose-6B柱层析和Caaalco-prep-disc法进一步纯化后,在SDS-PAGE中显示为一条分子量约28,000d的带。用常规双向免疫扩散法检查各种包涵体蛋白之间的差异,结果表明血清学BsNPV与BtNPV,PrGV与PxGV之间都是不可分辩的。6种包涵体蛋白的双向高压指纹图谱表明,它们的指纹图谱都不同程度地存在着一些相同或十分相似的肽点。就每种病毒的指纹图谱又都是独特的,NPV间相似性低于GV间相似性。我们认为NPV和GV的包涵体蛋白在结构上存在着一些相同的或相似的保守区域,但不同种之间在整个一级结构上是有差异的。利用包涵体蛋白的指纹图谱鉴定杆状病毒是个灵敏可行的方法。     

Salicylic acid-independent plant defence pathways

Salicylic acid is an important signalling molecule involved in both locally and systemically induced disease resistance responses. Recent advances in our understanding of plant defence signalling have revealed that plants employ a network of signal transduction pathways, some of which are independent of salicylic acid. Evidence is emerging that jasmonic acid and ethylene play key roles in these salicylic acid-independent pathways. Cross-talk between the salicylic acid-dependent and the salicylic acid-independent pathways provides great regulatory potential for activating multiple resistance mechanisms in varying combinations.     

DEAD box RhlB RNA helicase physically associates with exoribonuclease PNPase to degrade double-stranded RNA independent of the degradosome-assembling region of RNase E

The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.     

Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.     

人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化

 本文报道了培养的人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化方法。Bowes株人黑色素瘤细胞的分泌产物,经CM-Sephadex C--50层析,赖氨酸-Sepharose 4B,苯甲眯-sepharose 4B亲和层析后,即可得到纯化470倍的蛋白纯品。样品经聚丙烯酰胺凝胶电泳鉴定为均一单带,测得其分子量约为72kD。纯化的t-PA与尿激酶相比较,发现前者有更高亲和纤维蛋白的能力。     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer

Change of DNA cytosine methylation (5mC) is an early event in the development of cancer, and the recent discovery of a 5-hydroxymethylated form (5hmC) of cytosine suggests a regulatory epigenetic role that might be different from 5-methylcytosine. Here, we aimed at elucidating the role of 5hmC in breast cancer. To interrogate the 5hmC levels of the leucine zipper, putative tumor suppressor 1 (LZTS1) gene in detail, we analyzed 75 primary breast cancer tissue samples from initial diagnosis and 12 normal breast tissue samples derived from healthy persons. Samples were subjected to 5hmC glucosyltransferase treatment followed by restriction digestion and segment-specific amplification of 11 polymerase chain reaction products. Nine of the 11 5′LZTS1 fragments showed significantly lower (fold change of 1.61–6.01, P < .05) 5hmC content in primary breast cancer tissue compared to normal breast tissue samples. No significant differences were observed for 5mC DNA methylation. Furthermore, both LZTS1 and TET1 mRNA expressions were significantly reduced in tumor samples (n = 75, P < .001, Student''s t test), which correlated significantly with 5hmC levels in samples. 5hmC levels in breast cancer tissues were associated with unfavorable histopathologic parameters such as lymph node involvement (P < .05, Student''s t test). A decrease of 5hmC levels of LZTS1, a classic tumor suppressor gene known to influence metastasis in breast cancer progression, is correlated to down-regulation of LZTS1 mRNA expression in breast cancer and might epigenetically enhance carcinogenesis. The study provides support for the novel hypothesis that suggests a strong influence of 5hmC on mRNA expression. Finally, one may also consider 5hmC as a new biomarker.