Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Scrapie resistance in ARQ sheep

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.     

代谢、血液碱化和纯氧影响呼吸调控的人体实验研究II： 血液碱化后运动试验*

目的: 在完成吸入室内空气状态下症状限制性最大极限心肺运动试验(CPET)和动脉血气指标动态变化规律的基础上,进一步探讨体液酸碱度和CO₂含量对呼吸调控的影响。方法: 选正常志愿者5名,给予5%NaHCO₃(总量0.3 g/kg)分次口服,每5 min口服75 ml(3.75g )。总量服完1 h后,重复CPET。于静息、热身、运动及恢复期,连续测定肺通气指标及每分钟动脉取样的血气指标变化,并与本人在非碱化血液条件下对照数据进行配对t检验比较。结果: 碱化血液之后,CPET期间随着运动功率逐步递增,气体交换和血气指标的反应模式与非碱化血液对照相似（P>0.05）;即与静息状态比较,每分通气量、潮气量、呼吸频率、VO₂、VCO₂均呈现近于线性渐进性递增(P<0.05~0.001)。与碱化血液前吸入室内空气的对照比较：在碱化血液条件下,所有时间点血红蛋白浓度,PaCO₂与pH均显著提高(P<0.05);除无氧阈PaCO₂减低外,只有热身状态呈增高态势,统计学有显著差异(P<0.05);而PaO₂无差异(P>0.05),各状态均较对照状态减低,除恢复期外均有统计学差异(P<0.05)。与非碱化血液对照比较,除静息每分通气量低于对照(P<0.05）外,所有通气指标均无统计学差异(P>0.05)。结论: 碱化血液条件下, 尽管有更高的CaCO₂, PaCO₂ 和 pHa平均水平及更低的Hba和[H⁺]a平均水平,机体对CPET的呼吸反应模式基本相似。     

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.      

Prion gene sequence variation within diverse groups of U.S. sheep,beef cattle,and deer

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene (PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer (Odocoileus virginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

代谢、血液碱化和纯氧影响呼吸调控的人体实验研究III： 血液碱化后纯氧运动试验*

目的: 在急性血液碱化前、后空气吸入下完成症状限制性最大极限心肺运动试验(CPET)的基础上,本文探讨在血液碱化后吸入纯氧对呼吸调控的影响。方法: 正常志愿者5名在碱化血液后呼吸纯氧CPET,在静息、热身、运动及恢复期,连续测定肺通换气指标及每分钟动脉取样的血气指标,对CPET期间的呼吸气体交换和血气指标的动态变化进行分析,同时与急性碱化血液前、后空气CPET数据比较。结果: 碱化血液后吸入纯氧运动呼吸反应与急性碱化血液前、后空气CPET呼吸反应基本一致。CPET期间,各运动状态下的每分通气量均与对照组相似(P>0.05);仅静息每分通气量较血液碱化空气CPET略高(P<0.05),而其它状态和恢复2min时均相近(P>0.05)。潮气量仅峰值运动时较对照和血液碱化空气CPET略低(P<0.05);而运动过程和恢复2min时的潮气量均相近(P>0.05)。呼吸频率在各个时间与血液碱化前后CPET均无差异(P>0.05)。在碱化血液后吸入纯氧运动各个时期的PaO₂和SaO₂较碱化血液前后空气CPET时明显提高(P<0.001,P<0.05)。血红蛋白浓度虽然较急性血液碱化前后均低,但仅较血液碱化前显著降低(P<0.05),比血液碱化后差异不显著(P>0.05) ; 开始时的PaCO₂较碱化血液前后空气CPET时降低(P<0.05),无氧阈时相近(P>0.05),但到峰值及恢复2 min时明显增高(P<0.05);pH仅较对照增高(P<0.05),但与碱化血液空气试验时无差异;乳酸水平较对照略高,但仅在热身和恢复期有差异(P<0.05)。纯氧提高了两人无氧阈和三人峰值运动的功率和时间。结论: 虽然血液碱化给予纯氧, CPET呼吸反应与碱化血液前、后空气CPET呼吸反应模式相似,表明运动中呼吸反应主要取决于代谢变化,而非动脉血气平均值高低。     

Detection of femtomolar quantities of the ganglioside GM1 on thin-layer chromatography plates by native choleratoxin and labeled antisera

After separation of gangliosides by thin-layer chromatography, femtomolar quantities of G_M1 were detected by incubating the plate with native choleratoxin, followed by anticholeratoxin and species-specific labeled antiserum. Only stable reagents were involved when antiserum labeled with horseradish peroxidase was used. Native choleratoxin rather than iodinelabeled toxin ensured good reproducibility of the method.