scholarly journals Roles of the Two-MnSOD System of Stenotrophomonas maltophilia in the Alleviation of Superoxide Stress

2019 ◽  
Vol 20 (7) ◽  
pp. 1770 ◽  
Author(s):  
Herng-Woei Jair ◽  
Hsu-Feng Lu ◽  
Yi-Wei Huang ◽  
Sz-Yun Pan ◽  
I-Ling Lin ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Gene Replacement ◽  
Deletion Mutants ◽  
Aerobic Growth ◽  
Transcriptional Fusion ◽  
Bacterial Viability ◽  
Replacement Strategy ◽  
Qrt Pcr ◽  
The Individual ◽  
Logarithmic Growth

Manganese-dependent superoxide dismutase (MnSOD, SodA) and iron-dependent SOD (FeSOD, SodB) are critical cytosolic enzymes for alleviating superoxide stress. Distinct from the singular sodA gene in most bacteria, Stenotrophomonas maltophilia harbors two sodA genes, sodA1 and sodA2. The roles of SodA1, SodA2, and SodB of S. maltophilia in alleviating superoxide stress were investigated. The expression of sod genes was determined by promoter–xylE transcriptional fusion assay and qRT–PCR. SodA2 and sodB expressions were proportional to the bacterial logarithmic growth, but unaffected by menadione (MD), iron, or manganese challenges. SodA1 was intrinsically unexpressed and inducibly expressed by MD. Complementary expression of sodA1 was observed when sodA2 was inactivated. The individual or combined sod deletion mutants were constructed using the gene replacement strategy. The functions of SODs were assessed by evaluating cell viabilities of different sod mutants in MD, low iron-stressed, and/or low manganese-stressed conditions. Inactivation of SodA1 or SodA2 alone did not affect bacterial viability; however, simultaneously inactivating sodA1 and sodA2 significantly compromised bacterial viability in both aerobic growth and stressed conditions. SodA1 can either rescue or support SodA2 when SodA2 is defective or insufficiently potent. The presence of two MnSODs gives S. maltophilia an advantage against superoxide stress.

scholarly journals The testis-specific serine proteases PRSS44, PRSS46, and PRSS54 are dispensable for male mouse fertility†

2019 ◽  
Author(s):  
Richard J Holcomb ◽  
Seiya Oura ◽  
Kaori Nozawa ◽  
Katarzyna Kent ◽  
Zhifeng Yu ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Sperm Morphology ◽  
Homozygous Deletion ◽  
Developmental Expression ◽  
Sperm Function ◽  
Deletion Mutants ◽  
Es Cell ◽  
Functional Roles ◽  
Functional Genetics ◽  
The Individual

Abstract High-throughput transcriptomics and proteomics approaches have recently identified a large number of germ cell–specific genes with many that remain to be studied through functional genetics approaches. Serine proteases (PRSS) constitute nearly one-third of all proteases, and, in our bioinformatics screens, we identified many that are testis specific. In this study, we chose to focus on Prss44, Prss46, and Prss54, which we confirmed as testis specific in mouse and human. Based on the analysis of developmental expression in the mouse, expression of all four genes is restricted to the late stage of spermatogenesis concomitant with a potential functional role in spermiogenesis, spermiation, or sperm function. To best understand the male reproductive requirement and functional roles of these serine proteases, each gene was individually ablated by CRISPR/Cas9-mediated ES cell or zygote approach. Homozygous deletion mutants for each gene were obtained and analyzed for phenotypic changes. Analyses of testis weights, testis and epididymis histology, sperm morphology, and fertility revealed no significant differences in Prss44, Prss46, and Prss54 knockout mice in comparison to controls. Our results thereby demonstrate that these genes are not required for normal fertility in mice, although do not preclude the possibility that these genes may function in a redundant manner. Elucidating the individual functional requirement or lack thereof of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives.

scholarly journals The Yersinia enterocoliticaPhospholipase Gene yplA Is Part of the Flagellar Regulon

2000 ◽  
Vol 182 (8) ◽  
pp. 2314-2320 ◽  
Author(s):  
Deborah H. Schmiel ◽  
Glenn M. Young ◽  
Virginia L. Miller
Keyword(s):  
Virulence Genes ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Transcriptional Fusion ◽  
Logarithmic Growth Phase ◽  
Environmental Signals ◽  
Flagellar Regulon ◽  
Camp Receptor ◽  
Logarithmic Growth ◽  
Regulatory Sites

ABSTRACT Yersinia enterocolitica yplA encodes a phospholipase required for virulence. Virulence genes are often regulated in response to environmental signals; therefore, yplA expression was examined using a yplA::lacZYtranscriptional fusion. Maximal yplA expression occurred between pH 6.5 and pH 7.5 and was induced in the mid-logarithmic growth phase. Potential Fnr, cyclic AMP (cAMP)-cAMP receptor protein (Crp), and ςF regulatory sites were identified in the nucleotide sequence. Reduction of yplA expression by aeration, addition of glucose and sucrose, and application of high temperature and salt is consistent with Fnr-, cAMP-Crp-, and ςF-mediated regulation, respectively. Expression ofyplA was reduced in flhDC and fliAnull strains, indicating that yplA is part of the flagellar regulon.

Differences in vitro between fibroblast-like cells from cornea, heart, and skin of embryonic chicks

1977 ◽  
Vol 26 (1) ◽  
pp. 119-137
Author(s):  
G.W. Conrad ◽  
G.W. Hart ◽  
Y. Chen
Keyword(s):  
Corneal Stroma ◽  
Heart Cells ◽  
Heart Ventricle ◽  
Skin Cells ◽  
Single Organism ◽  
The Individual ◽  
Parallel Arrays ◽  
Logarithmic Growth ◽  
Different Tissues

Populations of fibroblast-like cells of corneal stroma, heart ventricle, and back skin of day-14 embryonic chicks were grown in vitro as primary and secondary cultures and were found to differ from one another by several criteria. Such cells were obtained from tissues either directly (cornea) by dissection or indirectly (heart and skin) by the rapid adhesion of the fibroblast-like cells to glass and plastic substrata. Individual fibroblast-like cells of cornea and heart were distinguishable from one another during their first 24–48 h in vitro. The morphologies of the individual cels of these 2 populations became indistinguishable during logarithmic growth, although each could be distinguished from individual fibroblast-like cells of skin. When the cultures reached saturation, corneal cells formed a monolayer of randomly oriented polygonal cells; skin cells formed a monolayer of long, narrow, ragged cells in parallel arrays with occasional double-layering; and heart cells formed multilayers of criss-crossed cells whose broad, smooth outlines were in parallel array in each layer. Saturation densities of the 3 fibroblast-like populations were different: heart greater than skin greater than cornea. By 3 methods of assay the cells were found to be differentially sensitive to treatment with trypsin and EDTA, and to EDTA alone, heart cells consistently being the least sensitive. Taken together, these data suggest that fibroblast-like cell populations isolated from different tissues of a single organism are different from one another and thus may behave differently from one another during in vitro studies.

scholarly journals A gene replacement strategy for engineering nisin

Microbiology ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 47-55 ◽  
Author(s):  
H. M. Dodd ◽  
N. Horn ◽  
C. J. Giffard ◽  
M. J. Gasson
Keyword(s):  
Gene Replacement ◽  
Replacement Strategy

scholarly journals Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Özgür Genç ◽  
Olexiy Kochubey ◽  
Ruud F Toonen ◽  
Matthijs Verhage ◽  
Ralf Schneggenburger
Keyword(s):  
Transmitter Release ◽  
Molecular Mechanisms ◽  
Gene Replacement ◽  
Target Protein ◽  
Phosphorylation Sites ◽  
Short Term ◽  
Calyx Of Held ◽  
Replacement Strategy ◽  
Kinase C ◽  
Post Tetanic Potentiation

Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a ‘conventional’ PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP.

scholarly journals Comprehensive Analysis of Peripheral Exosomal circRNAs in Large Artery Atherosclerotic Stroke

2021 ◽  
Vol 9 ◽  
Author(s):  
Qi Xiao ◽  
Ruihua Yin ◽  
Yuan Wang ◽  
Shaonan Yang ◽  
Aijun Ma ◽  
...  
Keyword(s):  
Area Under The Curve ◽  
Comprehensive Analysis ◽  
Circular Rnas ◽  
Large Artery ◽  
Rna Seq ◽  
Roc Curve Analysis ◽  
Qrt Pcr ◽  
Model Combining ◽  
Novel Biomarkers ◽  
The Individual

Exosomes are crucial vehicles in intercellular communication. Circular RNAs (circRNAs), novel endogenous noncoding RNAs, play diverse roles in ischemic stroke. Recently, the abundance and stability of circRNAs in exosomes have been identified. However, a comprehensive analysis of exosomal circRNAs in large artery atherosclerotic (LAA) stroke has not yet been reported. We performed RNA sequencing (RNA-Seq) to comprehensively identify differentially expressed (DE) exosomal circRNAs in five paired LAA and normal controls. Further, quantitative real-time PCR (qRT-PCR) was used to verify the RNA-Seq results in a cohort of stroke patients (32 versus 32). RNA-Seq identified a total of 462 circRNAs in peripheral exosomes; there were 25 DE circRNAs among them. Additionally, circRNA competing endogenous RNA (ceRNA) network and translatable analysis revealed the potential functions of the exosomal circRNAs in LAA progression. Two ceRNA pathways involving 5 circRNAs, 2 miRNAs, and 3 mRNAs were confirmed by qRT-PCR. In the validation cohort, receiver operating characteristic (ROC) curve analysis identified two circRNAs as possible novel biomarkers, and a logistic model combining two and four circRNAs increased the area under the curve compared with the individual circRNAs. Here, we show for the first time the comprehensive expression of exosomal circRNAs, which displayed the potential diagnostic and biological function in LAA stroke.

scholarly journals NUCLEOLAR AGING IN TETRAHYMENA DURING THE CULTURAL GROWTH CYCLE

1971 ◽  
Vol 48 (1) ◽  
pp. 143-154 ◽  
Author(s):  
Birgit Satir ◽  
Ellen Roter Dirksen
Keyword(s):  
Stationary Phase ◽  
Actinomycin D ◽  
Nuclear Periphery ◽  
Tetrahymena Pyriformis ◽  
Growth Cycle ◽  
Growth Phases ◽  
Stage 4 ◽  
The Individual ◽  
Stage 1 ◽  
Logarithmic Growth

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.

scholarly journals Interplay between OmpA and RpoN Regulates Flagellar Synthesis in Stenotrophomonas maltophilia

2021 ◽  
Vol 9 (6) ◽  
pp. 1216
Author(s):  
Chun-Hsing Liao ◽  
Chia-Lun Chang ◽  
Hsin-Hui Huang ◽  
Yi-Tsung Lin ◽  
Li-Hua Li ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Protein A ◽  
Underlying Mechanism ◽  
Hierarchical Organization ◽  
Qrt Pcr ◽  
Regulatory Cascade ◽  
Promising Target ◽  
And Function ◽  
The Relationship ◽  
Abundant Transcript

OmpA, which encodes outer membrane protein A (OmpA), is the most abundant transcript in Stenotrophomonas maltophilia based on transcriptome analyses. The functions of OmpA, including adhesion, biofilm formation, drug resistance, and immune response targets, have been reported in some microorganisms, but few functions are known in S. maltophilia. This study aimed to elucidate the relationship between OmpA and swimming motility in S. maltophilia. KJΔOmpA, an ompA mutant, displayed compromised swimming and failure of conjugation-mediated plasmid transportation. The hierarchical organization of flagella synthesis genes in S. maltophilia was established by referencing the Pseudomonas aeruginosa model and was confirmed using mutant construction, qRT-PCR, and functional assays. Distinct from the P. aeruginosa model, rpoN, rather than fleQ and fliA, was at the top of the flagellar regulatory cascade in S. maltophilia. To elucidate the underlying mechanism responsible for ΔompA-mediated swimming compromise, transcriptome analysis of KJ and KJΔOmpA was performed and revealed rpoN downregulation in KJΔOmpA as the key element. The involvement of rpoN in ΔompA-mediated swimming compromise was verified using rpoN complementation, qRT-PCR, and function assays. Collectively, OmpA, which contributes to bacterial conjugation and swimming, is a promising target for adjuvant design in S. maltophilia.

scholarly journals Identification of a Novel β-Lactamase Produced byXanthomonas campestris, a Phytopathogenic Bacterium

1999 ◽  
Vol 43 (7) ◽  
pp. 1792-1797 ◽  
Author(s):  
Shu-Fen Weng ◽  
Chun-Yi Chen ◽  
Yeong-Sheng Lee ◽  
Juey-Wen Lin ◽  
Yi-Hsiung Tseng
Keyword(s):  
Amino Acid ◽  
Stenotrophomonas Maltophilia ◽  
Xanthomonas Campestris ◽  
Southern Hybridization ◽  
Gene Replacement ◽  
Phytopathogenic Bacterium ◽  
Class A ◽  
Homologous Fragment ◽  
Group 2 ◽  
Lactamase Gene

ABSTRACT The Xanthomonas campestris pv. campestris 11 chromosome encodes a periplasmic β-lactamase of 30 kDa. Gene replacement and complementation confirmed the presence of this enzyme. Its deduced amino acid sequence shows identity and conserved domains between it andStenotrophomonas maltophilia L2 and other Ambler class A/Bush group 2 β-lactamases. Southern hybridization detected a single homologous fragment in each of 12 other Xanthomonasstrains, indicating that the presence of a β-lactamase gene is common among xanthomonads.

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