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991.
Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties 总被引:15,自引:0,他引:15
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Markus Wyss Roland Brugger Alexandra Kronenberger Roland Rmy Rachel Fimbel Gottfried Oesterhelt Martin Lehmann Adolphus P. G. M. van Loon 《Applied microbiology》1999,65(2):367-373
Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)−1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation. 相似文献
992.
Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, l-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the l-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus. 相似文献
993.
A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor
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Alexandra Trkola Jamie Matthews Cynthia Gordon Tom Ketas John P. Moore 《Journal of virology》1999,73(11):8966-8974
We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies. 相似文献
994.
The CC-Chemokine RANTES Increases the Attachment of Human Immunodeficiency Virus Type 1 to Target Cells via Glycosaminoglycans and Also Activates a Signal Transduction Pathway That Enhances Viral Infectivity
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Alexandra Trkola Cynthia Gordon Jamie Matthews Elizabeth Maxwell Tom Ketas Lloyd Czaplewski Amanda E. I. Proudfoot John P. Moore 《Journal of virology》1999,73(8):6370-6379
We have studied the mechanisms by which the CC-chemokine RANTES can enhance the infectivities of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses, when present at concentrations in excess of 500 ng/ml in vitro. Understanding the underlying mechanisms might throw light on fundamental processes of viral infection, in particular for HIV-1. Our principal findings are twofold: firstly, that oligomers of RANTES can cross-link enveloped viruses, including HIV-1, to cells via glycosaminoglycans (GAGs) present on the membranes of both virions and cells; secondly, that oligomers of RANTES interact with cell-surface GAGs to transduce a herbimycin A-sensitive signal which, over a period of several hours, renders the cells more permissive to infection by several viruses, including HIV-1. The enhancement mechanisms require that RANTES oligomerize either in solution or following binding to GAGs, since no viral infectivity enhancement is observed with a mutant form of the RANTES molecule that contains a single-amino-acid change (glutamic acid to serine at position 66) which abrogates oligomerization. 相似文献
995.
Elisabet Bjanes Reyna Garcia Sillas Rina Matsuda Benjamin Demarco Timothe Fettrelet Alexandra A. DeLaney Opher S. Kornfeld Bettina L. Lee Eric M. Rodríguez Lpez Daniel Grubaugh Meghan A. Wynosky-Dolfi Naomi H. Philip Elise Krespan Dorothy Tovar Leonel Joannas Daniel P. Beiting Jorge Henao-Mejia Brian C. Schaefer Kaiwen W. Chen Petr Broz Igor E. Brodsky 《PLoS pathogens》2021,17(10)
Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection. 相似文献
996.
Animal models are at the forefront of biomedical research for studies of viral transmission, vaccines, and pathogenesis, yet the need for an ideal large animal model for COVID-19 remains. We used a meta-analysis to evaluate published data relevant to this need. Our literature survey contained 22 studies with data relevant to the incidence of common COVID-19 symptoms in rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), African green monkeys (Chlorocebus aethiops), and ferrets (Mustela putorius furo). Rhesus macaques had leukocytosis on Day 1 after inoculation and pneumonia on Days 7 and 14 after inoculation in frequencies that were similar enough to humans to reject the null hypothesis of a Fisher exact test. However, the differences in overall presentation of disease were too different from that of humans to successfully identify any of these 4 species as an ideal large animal of COVID-19. The greatest limitation to the current study is a lack of standardization in experimentation and reporting. To expand our understanding of the pathology of COVID-19 and evaluate vaccine immunogenicity, we must extend the unprecedented collaboration that has arisen in the study of COVID-19 to include standardization of animal-based research in an effort to find the optimal animal model.Human research of disease presents a number of ethical dilemmas, prompting scientists to use animal models in their research with the primary goal of enhancing the understanding of a human disease or phenomenon. Animal models have been instrumental to our understanding of pathologies, the assessment of novel vaccines, and the testing of acute therapies. Of the past 222 Nobel prizes awarded in the physiology and medicine categories since 1901, all but 36 have been a direct result of animal-based research.31Insects, nematodes, fish, amphibians, and numerous mammals have enabled some of the most important advances in physiology and medicine since their introduction in disease research. Through genetic modification, surgical adaptation, xenografts, chemical induction, and infection models, these animals have been used to model human phenomena.31 However, although particular animal species are often chosen based on their ability to meet specific criteria in line with the research question, their size remains an important factor.26,31Small animals are often preferred in laboratory settings for their ease of use, shorter life cycle, easier handling and care, and short gestation.5 Rodents are the most commonly used animal for the study of human diseases for these very reasons, although they frequently fail to fully mimic the clinical signs and significant pathologic hallmarks of human diseases.11,18 For this reason, some researchers use large animal models. Nonhuman primates (NHPs), in particular, have been extremely useful in reproducing the clinical signs of human diseases due to their close phylogenetic relationship to humans and resulting genetic, behavioral, and biochemical similarities.14On March 11, 2020, the World Health Organization declared a SARS-CoV-2 pandemic. SARS-CoV-2 is a novel coronavirus causing symptoms similar to, but distinct from, those found in individuals infected with SARS-CoV, the coronavirus that caused the 2003 SARS pandemic. As of September 10, 2021, this coronavirus has infected 219 million individuals with the COVID-19 disease.10 Although vaccines have been developed and approved in record time, we still need to better understand the pathogenesis of the disease and the long-term implications of infections. To do this, and to increase our understanding of the immunogenicity of current vaccines, finding an animal that replicates the manifestation of COVID-19 in humans is imperative.Much of the research on COVID-19 thus far has been aided by previous SARS research. In both SARS-CoV and SARS-CoV-2 studies, mice33,45 and hamsters19,34 were small animal models of choice. Large animals such as ferrets, cats, pigs, chickens, dogs, and nonhuman primates have also been tested for their reproducibility of COVID-19, with varying degrees of success.27,41,49 While a perfect animal model of this viral infection is unlikely, the need remains to identify at least one large animal species as a frontrunner in reproducibility of the human clinical signs and significant pathologies of SARS-CoV-2 infection.The need for a large animal model to study COVID-19 does not imply a replacement for murine models, but rather an adjunct. The closer phylogenetic relationship of humans to NHPs makes them excellent candidates for the study of this disease. Vaccine trials have already shown that the responses of NHPs are closer to those of humans than are those of mice.23 This difference may be due to species differences in IgG antibody and T helper type 1 cell responses that influence virus-immune system interactions, which make small animal models problematic for studying SARS-CoV-2 infection and vaccine performance in humans.15 NHPs have potential high value as a model due to their homology to the human angiotensin‐converting enzyme‐2, which is the SARS-CoV-2 binding site.23,28 After the outbreak, the World Health Organization (WHO) formed the WHO COVID-19 modelling ad-hoc expert grouping. The working group identified various NHP models, including rhesus macaques, cynomolgus macaques and African green monkeys, in addition to ferrets as being susceptible to SARS Co-V-2 isolates that would result in reproducible infection, with mild to moderate disease.52 Therefore, the present article is focused on summarizing the results of multiple studies on rhesus macaque, cynomolgus macaque, African green monkey, and ferret infection with SARS-CoV-2. To highlight the species that best replicate the human clinical and laboratory findings of COVID-19, we synthesized the results of 22 animal studies to provide a comprehensive analysis of what is known about their infections to date. 相似文献
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The cytoarchitectural simplicity of the cerebral cortex makes it an attractive system to study central nervous system (CNS) histogenesis—the process whereby diverse cells are generated in the right numbers at the appropriate place and time. Recently, multipotent stem cells have been implicated in this process, as progenitor cells for diverse types of cortical neurons and glia. Continuous analysis of stem cell clone development reveals stereotyped division patterns within their lineage trees, highly reminiscent of neural lineage trees in arthropods and Caenorhabditis elegans. Given that these division patterns play a critical part in generating diverse neural types in invertebrates, we speculate that they play a similar role in the cortex. Because stereotyped lineage trees can be observed from cells growing at clonal density, cell-intrinsic factors are likely to have a key role in stem cell behavior. Cortical stem cells also respond to environmental signals to alter the types of cells they generate, providing the means for feedback regulation on the germinal zone. Evidence is accumulating that cortical stem cells, influenced by intrinsic programs and environmental signals, actually change with development—for example, by reducing the number and types of neurons they produce. Age-related changes in the stem cell population may have a critical role in orchestrating development; whether these cells truly self-renew is a point of discussion. In summary, we propose that cortical stem cells are the focus of regulatory mechanisms central to the development of the cortical cytoarchitecture. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 162–174, 1998 相似文献