The use of synthetic oligodeoxynucleotide primers in cloning and sequencing segment of 8 influenza virus (A/PR/8/34)

Complete double-stranded DNA copies of the RNA genes of the human influenza virus A/PR/8/34 have been synthesized by using two synthetic oligodeoxynucleotide primers. The gene encoding the non-structural proteins NS1 and NS2, prepared with these primers, has been cloned into the bacteriophage M13mp7 and sequenced. The sequence is compared with that from another human strain and from an avian strain.     

Spontaneous thrombosis of an unruptured anterior communicating artery aneurysm. An unusual cause of ischemic stroke

BACKGROUND: Stroke caused by spontaneous thrombosis of an unruptured intracranial aneurysm is a rare event. CASE DESCRIPTION: A 66-year-old woman experienced a transient ischemic attack and cerebral infarctions due to spontaneous thrombosis of an unruptured anterior communicating artery aneurysm. Extension of thrombus into both anterior cerebral arteries and the left middle cerebral artery, resulting in ischemic infarction in all three vascular territories, was diagnosed by CT scanning, MRI, and cerebral angiography and confirmed at autopsy. CONCLUSIONS: This case illustrates a rare complication of an unruptured saccular aneurysm with neuroimaging and pathological correlation. Morphological and hemodynamic factors that may have precipitated aneurysm thrombosis are discussed with reference to experimental models.     

Water governance,ecosystems and sustainability: a review of progress in South Africa

It is approaching two decades since South Africa's democratic transformation introduced a visionary approach to water resources management. The earlier challenges of water law reform have given way to the greater undertaking of implementation. This paper reviews progress in relation to 7 of the 10 key challenges of the Africa Water Vision 2025. Progress in many areas has been slow, including establishment of catchment management institutions and implementation of environmental flows. Concerns remain regarding poor water quality and aquatic ecosystem health. A significant threat is the persistent shortage of skilled water professionals.     

Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.     

Protein engineering of the hydrophobic domain of human factor IX

Vitamin K-dependent plasma proteins contain a highly conservedhydrophobic domain located between the -carboxyglutamic acid(Gla) domain and the first epidermal growth factor (EGF)-likedomain. Here we have used protein engineering of the hydrophobicdomain in human factor IX to investigate its function in intactfactor IX. Mutant proteins were generated by site-directed mutagenesisand in vitro expression in Madin-Darby canine kidney (MDCK)cells. All of our mutants, including one with a deletion ofthe entire hydrophobic domain, were activated by factor XIa,showing that this domain is not required for factor IX activation.The results with the mutant Phe41 Val suggest that the hydrophobicdomain interacts with the adjacent EGF-like domain. Our datafor the Phe41 Asp mutant is consistent with, but cannot prove,a role for this residue in the maintenance of a phospholipid-bindingstructure required for factor IX function.     

Ligand requirements for Ca2+ binding to EGF-like domains

Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant K_d=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. ¹H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (K_d=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (K_d=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the K_d for calcium (K_d=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IX_Alabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (K_d=37 mM,I=0.15), suggesting that the defect in factor IX_Alabama is dueto impaired calcium binding to its first EGF-like domain.     

Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo

A persistent obstacle that has hampered gene transfer experiments is the short-term nature of transgene expression in vivo. In this article we present evidence for sustained expression from primary human keratinocytes, using the retroviral vector MFG. Primary keratinocytes were transduced in culture with the MFG retroviral vector containing the coding region from factor IX cDNA. Transduced keratinocytes, which secreted on average 830 ng of factor IX/10(6) cells/24 hr in tissue culture, were used to form a bilayered skin equivalent and grafted onto nude mice under a silicone transplantation chamber. Between 0.1 and 2.75 ng of human factor IX per milliliter was found in mouse plasma for more than 1 year, suggesting that keratinocyte stem cells were both transduced and grafted. The results show, for the first time, that long-term expression is obtainable in retrovirally transduced keratinocytes after transplantation.     

可提供短路保护的浪涌限幅器

对于包含有大电容量的装置而言，控制浪涌电流成为一大难题。最简单的方法就是将浪涌限幅电阻器与电容器组串联，但电阻器会浪费功率并增加压降。图1所示电路解决了这一难题并能提供其它优势。在启动时，双极型PNP晶体管Q2使N沟道功率MOSFET晶体管Q1保持关断，直到电容器C1两端的电压高到足以关断Q2的电平为止。在此时间间隔内，电阻器R1为C1及电路其它元件提供启动电流。当Q2关断时，Q1导通并在R1两端提供一条低阻通道。当关闭外部电源时，电路随C1放电而复位。     

Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the effects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H2O2, there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 micromol/l H2O2 (+AG 0 vs. 10 micromol/l, NS), and reduced the percentage of apoptotic cells in response to 50 micromol/l H2O2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dose-dependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H2O2-induced benzoate hydroxylation in a dose-dependent manner. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.     

Understanding Internet traffic streams: dragonflies and tortoises

We present the concept of network traffic streams and the ways they aggregate into flows through Internet links. We describe a method of measuring the size and lifetime of Internet streams, and use this method to characterize traffic distributions at two different sites. We find that although most streams (about 45 percent of them) are dragonflies, lasting less than 2 seconds, a significant number of streams have lifetimes of hours to days, and can carry a high proportion (50-60 percent) of the total bytes on a given link. We define tortoises as streams that last longer than 15 minutes. We point out that streams can be classified not only by lifetime (dragonflies and tortoises) but also by size (mice and elephants), and note that stream size and lifetime are independent dimensions. We submit that ISPs need to be aware of the distribution of Internet stream sizes, and the impact of the difference in behavior between short and long streams. In particular, any forwarding cache mechanisms in Internet routers must be able to cope with a high volume of short streams. In addition ISPs should realize that long-running streams can contribute a significant fraction of their packet and byte volumes-something they may not have allowed for when using traditional "flat rate user bandwidth consumption" approaches to provisioning and engineering.