Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice.

Two new chimeric flaviviruses were constructed from full-length cDNAs that contained tick-borne encephalitis virus (TBEV) CME or ME structural protein genes and the remaining genes derived from dengue type 4 virus (DEN4). Studies involving mice inoculated intracerebrally with the ME chimeric virus indicated that it retained the neurovirulence of its TBEV parent from which its pre-M and E genes were derived. However, unlike parental TBEV, the chimeric virus did not produce encephalitis when mice were inoculated peripherally, indicating a loss of neuroinvasiveness. In the present study, the ME chimeric virus (vME) was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence measured by direct inoculation of virus into the brain. We identified three distinct mutations that were each associated independently with a significant reduction of mouse neurovirulence of vME. These mutations ablated (i) the TBEV pre-M cleavage site, (ii) the TBEV E glycosylation site, or (iii) the first DEN4 NS1 glycosylation site. In contrast, ablation of the second DEN4 NS1 glycosylation site or the TBE pre-M glycosylation site or amino acid substitution at two positions in the TBEV E protein increased neurovirulence. The only conserved feature of the three attenuated mutants was restriction of virus yield in both simian and mosquito cells. Following parenteral inoculation, these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by the highly neurovirulent vME.     

Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.

The Saccharomyces cerevisiae CDC25 gene and closely homologous genes in other eukaryotes encode guanine nucleotide exchange factors for Ras proteins. We have determined the minimal region of the budding yeast CDC25 gene capable of activity in vivo. The region required for full biological activity is approximately 450 residues and contains two segments homologous to other proteins: one found in both Ras-specific exchange factors and the more distant Bud5 and Lte1 proteins, and a smaller segment of 48 amino acids found only in the Ras-specific exchange factors. When expressed in Escherichia coli as a fusion protein, this region of CDC25 was found to be a potent catalyst of GDP-GTP exchange on yeast Ras2 as well as human p21H-ras but inactive in promoting exchange on the Ras-related proteins Ypt1 and Rsr1. The CDC25 fusion protein catalyzed replacement of GDP-bound to Ras2 with GTP (activation) more efficiently than that of the reverse reaction of replacement of GTP for GDP (deactivation), consistent with prior genetic analysis of CDC25 which indicated a positive role in the activation of Ras. To more directly study the physical interaction of CDC25 and Ras proteins, we developed a protein-protein binding assay. We determined that CDC25 binds tightly to Ras2 protein only in the absence of guanine nucleotides. This higher affinity of CDC25 for the nucleotide-free form than for either the GDP- or GTP-bound form suggests that CDC25 catalyzes exchange of guanine nucleotides bound to Ras proteins by stabilization of the transitory nucleotide-free state.     

采用康宁克通—A治疗鼻息肉的临床疗效观察

鼻息肉与变态反应之间有密切联系。本文拟报道康克通—A用于鼻息肉摘除术前、后以延迟与防止息肉复发的临疗效观察。     

Rapid and reliable high-performance liquid chromatographic method for analysing human plasma serotonin, 5-hydroxyindoleacetic acid, homovanillic acid and 3,4-dihydroxyphenylacetic acid

The simultaneous measurement of homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in human plasma by an ultrafiltration and microbore high-performance liquid chromatography—electrochemical detection technique is established. Conventional preparation of blood is very tedious and time-consuming, but isocratic separation of the analytes in plasma ultrafiltrates using a microbore column could be achieved within 10 min. Hence, theoretically, over 140 analyses can be performed in a working day. The detection limit (signal-to-noise ratio = 3) of this method is about 0.1–0.5 pg per injection for all analytes. The required volume of plasma samples can be less than 100 μl. Hence, blood loss is minimal, especially in repeated blood sampling. This rapid, simple and sensitive method can, therefore, be used as a routine clinical tool in the simultaneous measurement of plasma homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid.     

Access to coronary catheterisation: fair shares for all?

OBJECTIVE--To determine the effects of patient''s sex and area''s material deprivation on utilisation rates of coronary catheterisation and angiography in the investigation of ischaemic heart disease. DESIGN--Retrospective analysis of routinely collected hospital statistics. SETTING--Acute hospitals throughout Northern Ireland. SUBJECTS--24,179 episodes of patients discharged from hospital with a primary diagnosis of ischaemic heart disease and 1270 episodes relating to patients with an underlying diagnosis of ischaemic heart disease who had either coronary catheterisation or angiography. MAIN OUTCOME MEASURES--Age standardised admission rates for heart disease and age standardised utilisation rates for catheterisation or angiography, or both, for 566 electoral wards ranked by Townsend "deprivation" scores. RESULTS--Catheterisation-angiography rates in men were over fivefold those of women, ranging from 85.5/100,000 v 16/100,000 in patients from "well off" areas to 123/100,000 v 22/100,000 for patients from deprived areas. After admission rates for heart disease were controlled for, the overall rate ratio for women was 0.48 (95% confidence interval 0.38 to 0.60). After differential admission rates for heart disease and other potential clinical confounders were controlled for, the investigation rates of patients from the least and most "deprived" areas were not significantly different (rate ratio 1.04 (0.87 to 1.25)). CONCLUSION--Although investigation rates were significantly lower in women than in men, further clinical data would be required before labelling this underutilisation as evidence of bias. There was no significant difference in invasive investigation rates for heart disease in areas of varying deprivation or affluence.     

Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Production enhancement of Pseudomonas amyloderamosa isoamylase

Isoamylase production in batchwise and fed-batch cultures of Pseudomonas amyloderomosa (strain WU7211-2) was investigated. By feeding maltose in a mode motivated by its structure gene (iam), the final isoamylase activity of 5100 U/ml was achieved, as compared to 4100 U/ml in batch cultivation and 3800 U/ml in shaken flasks. The enhancement may be due to the fact that the production of isoamylase can be induced effectively by the maltose only if the glucose concentration is maintained below the inhibitory level. Also, cultivations based on 500-ml shaken flasks, performed with different amount of proteimax HE90, showed that higher amounts of proteimax HE90 resulted in an increased value of pH that had an adverse effect on isoamylase yield.The authors wish to thank the Food Industry Research and Development Institute (FIRDI), Taiwan, R.O.C for the financial support (Cooperative Agreement 94M904-2B). Appreciation is also extended to Drs. C.C. Liao, W.S. Chu and L.L. Lin of FIRDI for their valuable discussions.