scholarly journals The URA5 Gene Is Necessary forHistoplasma capsulatum Growth during Infection of Mouse and Human Cells

1999 ◽  
Vol 67 (2) ◽  
pp. 624-629 ◽  
Author(s):  
Diane M. Retallack ◽  
Elizabeth L. Heinecke ◽  
Reta Gibbons ◽  
George S. Deepe ◽  
Jon P. Woods
Keyword(s):  
Biosynthetic Pathway ◽  
Histoplasma Capsulatum ◽  
Fragment Length Polymorphism ◽  
Human Cells ◽  
Uv Mutagenesis ◽  
Allelic Replacement ◽  
In Trans ◽  
Ura5 Gene ◽  
Uracil Auxotrophs

ABSTRACT The Histoplasma capsulatum URA5 gene, which has recently been cloned and disrupted by allelic replacement, encodes orotidine-5′-monophosphate pyrophosphorylase. Inactivation ofURA5 by either targeted or UV mutagenesis results in disruption of the pyrimidine biosynthetic pathway and uracil auxotrophy. We examined the effect of uracil auxotrophy due to aura5 mutation on H. capsulatum virulence in both cell culture and whole-animal models. Uracil auxotrophs of two H. capsulatum restriction fragment length polymorphism classes were found to be avirulent in cultured murine and human cells, as well as in mice. Moreover, virulence could be restored either by supplying a functional URA5 gene in trans or by supplying exogenous uracil during infection in vitro. These experiments demonstrate that the pyrimidine biosynthetic pathway is essential for H. capsulatum growth and virulence.

The URA5 Gene Is Necessary for Histoplasma capsulatum Growth during Infection of Mouse and Human Cells

2000 ◽  
Vol 68 (1) ◽  
pp. 426-426 ◽  
Author(s):  
Diane M. Retallack ◽  
Elizabeth L. Heinecke ◽  
Reta Gibbons ◽  
George S. Deepe ◽  
Jon P. Woods
Keyword(s):  
Histoplasma Capsulatum ◽  
Human Cells ◽  
Ura5 Gene

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 503-512 ◽  
Author(s):  
Hongbo Liu ◽  
Stephen R Hewitt ◽  
John B Hays
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Uv Mutagenesis ◽  
Repair Protein ◽  
Mismatch Repair System

Abstract Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

scholarly journals RTEL1 influences the abundance and localization of TERRA RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fiorella Ghisays ◽  
Aitor Garzia ◽  
Hexiao Wang ◽  
Claudia Canasto-Chibuque ◽  
Marcel Hohl ◽  
...  
Keyword(s):  
Cell Viability ◽  
Telomere Length ◽  
Human Cells ◽  
Helicase Domain ◽  
Telomere Repeat ◽  
Disease Phenotypes ◽  
G Quadruplex ◽  
Non Coding Rnas ◽  
Subtelomeric Regions

AbstractTelomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.

Late-onset X-linked sideroblastic anemia following hemodialysis

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4623-4624 ◽  
Author(s):  
Kazumichi Furuyama ◽  
Hideo Harigae ◽  
Chiharu Kinoshita ◽  
Toshihiko Shimada ◽  
Kazuko Miyaoka ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Late Onset ◽  
The Elderly ◽  
Nutritional Deficiencies ◽  
Maintenance Hemodialysis ◽  
Sideroblastic Anemia ◽  
Ringed Sideroblasts ◽  
Aminolevulinate Synthase ◽  
Deficient Activity

Abstract X-linked sideroblastic anemia (XLSA) is due to deficient activity of erythroid-specific 5-aminolevulinate synthase (ALAS2). We report here a patient who developed sideroblastic anemia at the age of 81 years while undergoing hemodialysis. The diagnosis of sideroblastic anemia was established by the presence of ringed sideroblasts in the bone marrow, and treatment with oral pyridoxine completely eliminated the ringed sideroblasts. We identified a novel point mutation in the fifth exon of this patient's ALAS2 gene, which resulted in an amino acid change at residue 159 from aspartic acid to asparagine (Asp159Asn). In vitro analyses of recombinant Asp159Asn ALAS2 revealed that this mutation accounted for the pyridoxine-responsiveness of this disease. The very late onset in this case of XLSA emphasizes that nutritional deficiencies caused either by dietary irregularities in the elderly or, as in this case, by maintenance hemodialysis therapy, may uncover occult inherited enzymatic deficiencies in the heme biosynthetic pathway.

scholarly journals Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine

2004 ◽  
Vol 78 (5) ◽  
pp. 2502-2509 ◽  
Author(s):  
Linda Scobie ◽  
Samantha Taylor ◽  
James C. Wood ◽  
Kristen M. Suling ◽  
Gary Quinn ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Germ Line ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
Miniature Swine ◽  
Dna Libraries ◽  
Porcine Endogenous Retroviruses ◽  
Perv Transmission

ABSTRACT The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.

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