Reversible chemical modification of the scrapie agent

The scrapie agent causes a degenerative nervous system disease in sheep and goats. Studies with extensively purified preparations demonstrated that the agent contains a protein that is required for infectivity. Chemical modification of the scrapie agent by diethyl pyrocarbonate reduced the titer 1000-fold. Exposure of the inactivated agent to hydroxylamine, a strong nucleophile, resulted in complete restoration of infectivity. Presumably, nucleophilic residues within a scrapie agent protein undergo carbethoxylation on reaction with diethyl pyrocarbonate, and subsequent addition of hydroxylamine displaces these carbethoxy groups.     

朊病毒增殖机制的研究现状

朊病毒为一不含核酸的蛋白感染因子，能引起哺乳动物中枢神经组织病变，它是由正常形式的蛋白（PrPC)错误折叠成致病蛋白（PrPSc)而组成的，两种结构异型蛋白来源于同一基因，朊病毒抚养殖是通过PrPC构象转变成为PrPSc而实现的。     

Infection-specific particle from the unconventional slow virus diseases

Scrapie-associated fibrils, first observed in brains of scrapie-infected mice, were also observed in scrapie-infected hamsters and monkeys, in humans with Creutzfeldt-Jakob disease, and in kuru-infected monkeys. These fibrils were not found in a comprehensive series of control brains from humans and animals affected with central nervous system disorders resulting in histopathologies, ultrastructural features, or disease symptoms similar to those of scrapie, kuru, and Creutzfeldt-Jakob disease. These fibrils are also found in preclinical scrapie and in the spleens of scrapie-infected mice; they are a specific marker for the "unconventional" slow virus diseases, and may be the etiological agent.     

A general model of prion strains and their pathogenicity

Prions are lethal mammalian pathogens composed of aggregated conformational isomers of a host-encoded glycoprotein and which appear to lack nucleic acids. Their unique biology, allied with the public-health risks posed by prion zoonoses such as bovine spongiform encephalopathy, has focused much attention on the molecular basis of prion propagation and the "species barrier" that controls cross-species transmission. Both are intimately linked to understanding how multiple prion "strains" are encoded by a protein-only agent. The underlying mechanisms are clearly of much wider importance, and analogous protein-based inheritance mechanisms are recognized in yeast and fungi. Recent advances suggest that prions themselves are not directly neurotoxic, but rather their propagation involves production of toxic species, which may be uncoupled from infectivity.     

Infectious nucleic acids,a new dimension in virology

Viral nucleic acids have been found to be infectious for tissues and animals, yet are nonantigenic and resistant to antibodies against whole virus. Other unique properties, such as their host range and their susceptibility to nuclease action, render them a wholly new dimension to be reckoned with in virology. These properties may explain a number of conditions which at present are considered anomalous. The release from infected tissues of even a small proportion of total virus as free nucleic acid could, in an otherwise immune individual, lead to a low level of infection which would, perhaps, explain permanent immunity. If the proportion of nucleic acid is higher, or if the nucleic acid is resistant to nucleases because of an inert envelope, such conditions as "carrier" states-that is, viremias with or without antibodies-are possible. Note added in proof: The recent article by J. D. Ebert and F. H. Wilt (54) came to my attention after the present article was written. Ebert and Wilt's excellent article indicates the impact of the newer knowledge of viral nucleic acids on the ideas developing in the field of embryology.     

Photodynamic inactivation of infectious nucleic acid

Tobacco mosaic virus-infectious nucleic acid causes a color shift when combined with acridine orange, methylene blue, and safranine. A high concentration of acridine orange inactivates infectious nucleic acid even in darkness, while a mixture of nucleic acid with a low concentration of the dye must be exposed to visible light prior to inoculation for inactivation to occur.     

Reciprocal interference between specific CJD and scrapie agents in neural cell cultures

Infection of mice with an attenuated Creutzfeldt-Jakob disease agent (SY-CJD) interferes with superinfection by a more virulent human-derived CJD agent (FU-CJD) and does not require pathological prion protein (PrPres). Using a rapid coculture system, we found that a neural cell line free of immune system cells similarly supported substantial CJD agent interference without PrPres. In addition, SY-CJD prevented superinfection by sheep-derived Chandler (Ch) and 22L scrapie agents. However, only 22L and not Ch prevented FU-CJD infection, even though both scrapie strains provoked abundant PrPres. This relationship between particular strains of sheep- and human-derived agents is likely to affect their prevalence and epidemic spread.     

Nucleic acid guanine: reaction with the carcinogen N-acetoxy-2-acetylaminofluorene

Reaction of N-acetoxy-2-acetylaminofluorene with DNA or RNA at pH 7 causes marked increase in absorption at 280 to 320 millimicrons and marked decrease in guanine content. Reaction with guanosine-8-(14)C yields a radioactive fluorescent derivative. The data suggest that metabolic esters of N-hydroxy-2-acetylaminofluorene may be intermediates in the binding of this proximate carcinogen to nucleic acids in vivo.     

武汉单极虫巢式聚合酶链反应检测方法的建立与优化

应用巢式聚合酶链反应(nested PCR)建立了一种检测异育银鲫武汉单极虫(Thelohanellus wuhanensis)基因组DNA的方法,提取的DNA作为模板进行两次扩增,第一次扩增用单极虫18S rRNA序列通用引物:5'-CTGCGGACGGCTCAG TAAATCAGT-3'和5'-CCAGGACATCTTAGGGCATCACAGA-3',扩增长度为1 584 bp;第二次依据第一次扩增产物中武汉单极虫特有的高度保守区设计特异性引物:5'-ACCCACTTCTGTGGC CTTTC-3'和5'-AATCCGACCTACAACGCTGG-3',扩增长度为853 bp。结果表明,通过PCR反应体系中退火温度、dNTP、Mg2+、延伸时间、扩增循环数和模板浓度的优化,优化后的巢式PCR最低DNA检测量达10 fg,优化后比常规PCR检测的灵敏度高104倍。因此,本文所建立的巢式PCR检测方法适合于水环境或水生动物中武汉单极虫的微量检测,也为异育银鲫武汉单极虫病的诊断和流行病调查提供了一种高灵敏的检测技术。     

Aggregations of 35-nanometer particles associated with neuronal cytopathic changes in natural scrapie

Neuronal vacuolation and intravacuolar budding of vesicles and cytoplasmic processes appear to be the most characteristic cellular lesion in natural scrapie, a chronic degenerative disease of the central nervous system of sheep which is transmissible by injection. Membrane-bound accumulations of the 35-nm particles are fotnd in the cytoplasmic processes that project inside the vacuoles of the neuronal perikaryon. Such particles are present only in a small number of vacuolated neurons.     

A bacterium that can grow by using arsenic instead of phosphorus

Life is mostly composed of the elements carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus. Although these six elements make up nucleic acids, proteins, and lipids and thus the bulk of living matter, it is theoretically possible that some other elements in the periodic table could serve the same functions. Here, we describe a bacterium, strain GFAJ-1 of the Halomonadaceae, isolated from Mono Lake, California, that is able to substitute arsenic for phosphorus to sustain its growth. Our data show evidence for arsenate in macromolecules that normally contain phosphate, most notably nucleic acids and proteins. Exchange of one of the major bio-elements may have profound evolutionary and geochemical importance.     

Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2

A protein designated p14 was purified from a simian immunodeficiency virus (SIVMne) and was shown by amino acid sequence analysis to be nearly identical to the predicted translational product of a unique open reading frame (X-ORF) in the nucleotide sequences of SIVmac and human immunodeficiency virus type 2 (HIV-2). Thus the X-ORF is proven to be a new retroviral gene. The p14 is present in SIVMne in molar amounts equivalent to those of the gag proteins. This is the first example of a retrovirus that contains a substantial quantity of a viral protein that is not a product of the gag, pro, pol, or env genes. SIV p14 and its homolog in HIV-2 may function as nucleic acid binding proteins since purified p14 binds to single-stranded nucleic acids in vitro. Antisera to the purified protein detected p14 in SIVMne, SIVmac, and a homologous protein (16 kilodaltons) in HIV-2 but did not react with HIV-1. Diagnostic procedures based on this novel protein will distinguish between HIV-1 and HIV-2.     

Cloning of a gene whose expression is increased in scrapie and in senile plaques in human brain

A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.     

A replication cycle for viroids and other small infectious RNA's

Experimental data concerning viroid-specific nucleic acids accumulating in tomato plants establish, together with earlier studies, the major features of a replication cycle for viroid RNA in plant cells. Many features of this pathway, which involves multimeric strands of both polarities, may be shared by other small infectious RNA's including certain satellite RNA's and "virusoid" RNA's which replicate in conjunction with conventional plant viruses. The presence, in host plans, of an elaborate machinery for replicating these disease agents suggests a role for endogenous small RNA's in cellular development.     

CTAB法用于染病烟草植株中烟草丛顶病毒RNA的提取

 CTAB法是一种广泛用于从染病植物中提取总核酸,并用于后续相关病原物PCR检测的核酸提取法,已普遍运用于DNA病毒、植原体、细菌、真菌侵染植物的总DNA提取和病原检测。本文尝试将CTAB法用于一种由RNA病毒（烟草丛顶病毒）引起的烟草丛顶病染病组织总核酸的提取,以总核酸进行烟草丛顶病毒的RT-PCR检测,并与常规RNA病毒核酸提取法——Trizol法作比较。结果表明CTAB法和Trizol法提取的核酸用于烟草丛顶病毒RT-PCR检测时,均能扩增到目的条带,检测结果一致。在对复合侵染材料进行检测时发现,CTAB法提取的染病植物组织总核酸既包括DNA也包含RNA,有利于对DNA和RNA病原物同时进行检测;CTAB法所用试剂较Trizol法便宜,毒性小,且提取的核酸量较多、保存时间较长。因此CTAB法是一种高效、简便、经济的从烟草丛顶病染病组织中提取烟草丛顶病毒RNA的方法。     

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.     

磁性纳米颗粒在DNA分离纯化中的应用

为了解磁性纳米颗粒在DNA分离纯化中的应用,采用文献综述和归纳总结的方法,阐述了1997—2021年以铁氧化物为核心的磁性纳米材料的研究进展,同时比较了材料结构和表面相关活性官能团的修饰对DNA提取效率的影响。结果表明：1）应用磁性纳米颗粒的固相萃取有着安全无毒、操作简单、可重复使用、能够实现自动化与高通量操作等多种优势,提取DNA效率相比于商业化试剂盒也有优势。2）目前磁性纳米材料已有比较全面的研究。磁性纳米颗粒的尺寸、孔径大小、磁化强度及引入的活性官能团不同等性质均对DNA的提取效率有影响,改变这些性质研发新型且高效的功能化磁性纳米颗粒或改变磁性纳米材料的制备方法、所提取DNA的形态性质、溶液的pH或盐浓度等试验条件,以提高DNA与材料的解吸率,不断优化磁性分离过程,提高DNA分离效率和质量。3）发展高通量商业化的核酸提取程序,是磁性固相萃取可观的发展前景。4）磁性纳米颗粒除了在核酸提取中的应用,在其他生物医学应用中也有着广泛的研究。以上结果均表明磁性纳米材料有着极强的研究发展前景。     

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.     

Cell biology. Sowing the protein seeds of prion propagation

Ever since Prusiner first proposed his radical "protein-only" hypothesis to explain how certain infectious proteins (prions) are transmitted from one mammal to another in the absence of DNA or RNA, scientists have been trying to prove him right (or wrong). The study of mammalian prions, such as those causing Creutzfeldt-Jakob disease in humans, scrapie in sheep and mad cow disease in cattle, has been slow to yield answers. However, as Tuite discusses in his Perspective, the Sup35p and Ure2p proteins of yeast that exist in both normal and infectious forms are providing evidence that the "protein-only" hypothesis may be right (Sparrer et al.).