Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Δ⁵ to Δ⁷ or to Δ^5,7. Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24α-ethyl = trans-Δ²²-24α-ethyl > trans-Δ²²-24β-ethyl = 24α-E-ethylidene = 24α-methyl > 24β-methyl = trans-Δ²²-24β-methyl = 26-methyl = saturated C₇ side chain and C-20 R (17-αH, 20-αH, right-handed conformer) = cis-Δ²²-C₇ side chain and C-20 R > saturated C₇ side chain and C-20 S (17-αH, 20-βH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20α-hydroxycholesterol = 24ξ-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20α-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes.

We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.     

Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology

Currents through Ca channels were recorded in single canine atrial cells using whole-cell recording with patch pipettes. Two components of Ca channel current could be distinguished. One ("Ifast") was present only if cells were held at negative potentials, was most prominent for relatively small depolarizations, and inactivated within tens of milliseconds. The other ("Islow"), corresponding to the Ca current previously reported in single cardiac cells, persisted even at relatively positive holding potentials, required stronger depolarizations for maximal current, and inactivated much more slowly. Both currents were unaffected by tetrodotoxin and both were reduced by Co. Ifast had the same size and kinetics when Ca was exchanged for Ba, while Islow was bigger and slower with Ba as the charge carrier. In isotonic BaCl2, fluctuation analysis showed that Ifast had a smaller single channel current than Islow. Islow was much more sensitive to block by nitrendipine than was Ifast; also, Islow, but not Ifast, was increased by the dihydropyridine drug BAY K8644. Isoproterenol produced large increases in Islow but had no effect on Ifast.     

Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature.

In April 1983, an influenza virus of low virulence appeared in chickens in Pennsylvania. Subsequently, in October 1983, the virus became virulent and caused high mortality in poultry. The causative agent has been identified as an influenza virus of the H5N2 serotype. The hemagglutinin is antigenically closely related to tern/South Africa/61 (H5N3) and the neuraminidase is similar to that from human H2N2 strains (e.g., A/Japan/305/57) and from some avian influenza virus strains (e.g., A/turkey/Mass/66 [H6N2]). Comparison of the genome RNAs of chicken/Penn with other influenza virus isolates by RNA-RNA hybridization indicated that all of the genes of this virus were closely related to those of various other influenza virus isolates from wild birds. Chickens infected with the virulent strain shed high concentrations of virus in their feces (10(7) 50% egg infective dose per g), and the virus was isolated from the albumin and yolk of eggs layed just before death. Virus was also isolated from house flies in chicken houses. Serological and virological studies showed that humans are not susceptible to infection with the virus, but can serve as short-term mechanical carriers. Analysis of the RNA of the viruses isolated in April and October by gel migration and RNA-RNA hybridization suggested that these strains were very closely related. Oligonucleotide mapping of the individual genes of virulent and avirulent strains showed a limited number of changes in the genome RNAs, but no consistent differences between the virulent and avirulent strains that could be correlated with pathogenicity were found. Polyacrylamide gel analysis of the early (avirulent) isolates demonstrated the presence of low-molecular-weight RNA bands which is indicative of defective-interfering particles. These RNAs were not present in the virulent isolates. Experimental infection of chickens with mixtures of the avirulent and virulent strains demonstrated that the avirulent virus interferes with the pathogenicity of the virulent virus. The results suggest that the original avirulent virus was probably derived from influenza viruses from wild birds and that the virulent strain was derived from the avirulent strain by selective adaptation rather than by recombination or the introduction of a new virus into the population. This adaptation may have involved the loss of defective RNAs, as well as mutations, and thus provides a possible model for a role of defective-interfering particles in nature.     

Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi

Background

Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7–15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.

Methodology/Principal Findings

We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.

Conclusions/Significance

We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident that we can extend our efforts to the construction of strains with further filarial targets (in particular for those species that cannot be cultivated in the laboratory), and perform high-throughput drug screens to identify specific inhibitors of the parasite enzymes. By establishing synergistic collaborations with researchers working directly on different parasitic worms, we aim to aid antihelmintic drug development for both human and veterinary infections.     

急性心肌梗死患者冠脉搭桥术前中性粒细胞-淋巴细胞比率与围术期心肌损伤相关分析

目的：探讨急性心肌梗死患者冠脉搭桥（CABG）术前中性粒细胞．淋巴细胞比率（NLR）与围术期心肌损伤的关系,为,临床CABG围术期心肌保护提供参考依据。方法：选取2012年1月至2012年6月于首都医科大学附属北京安贞医院因急性心肌梗死接受冠脉搭桥手术（CABG）患者210例,收集术前血常规及术后肌钙蛋白I（cTnI）？Life酸激酶同工酶（CK-MB）,计算NLR;采用四分位法根据NLR水平将患者分为四组,比较各组cTnI及CK—MB峰值,多元逐步回归分析NLR与cTnI及CK-MB峰值的相关性。结果：随着NLR水平升高,高血压病史和射血分数〈50％患者比例逐渐增多;白细胞计数、术后CK-MB及cTnI峰值、术后血肌酐值均逐渐增加;多元逐步回归分析显示,NLR、WBC分别与cTnI峰值呈正相关（r=0．526,r=0．186,P〈0．05）。结论：术前NLR、WBC与cTnI峰值呈正相关,NLR可能是反应急性心肌梗死患者冠脉搭桥围术期心肌损伤的良好标志物。     

Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry.

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-alpha (des-Val-Val-TGF-alpha), a compact protein containing 48 residues and three disulfides.