scholarly journals Transcription of Candidate Virulence Genes ofHaemophilus ducreyi during Infection of Human Volunteers

2001 ◽  
Vol 69 (3) ◽  
pp. 1483-1487 ◽  
Author(s):  
Robert E. Throm ◽  
Stanley M. Spinola
Keyword(s):  
Experimental Infection ◽  
Virulence Genes ◽  
Human Model ◽  
Specific Gene ◽  
Gene Products ◽  
Virulence Determinants ◽  
Reverse Transcription Pcr ◽  
Human Volunteers

ABSTRACT Haemophilus ducreyi expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (momp, ftpA, losB, lst, cdtC, and hhdB) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with H. ducreyiwere subjected to reverse transcription-PCR. Transcripts corresponding to momp, ftpA, losB, lst, cdtB, and hhdA were expressed in vivo. In addition, transcripts for other putative virulence determinants such as ompA2, tdhA, lspA1, andlspA2 were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.

scholarly journals The Late Osteoblast/Preosteocyte Cell Line MLO-A5 Displays Mesenchymal Lineage PlasticityIn VitroandIn Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Dongqing Yang ◽  
Stan Gronthos ◽  
Sandra Isenmann ◽  
Howard A. Morris ◽  
Gerald J. Atkins
Keyword(s):  
Cell Line ◽  
Alcian Blue ◽  
Culture Conditions ◽  
Specific Gene ◽  
Stem Cell Markers ◽  
Osteogenic Cell ◽  
Reverse Transcription Pcr ◽  
Safranin O

The process of osteoblast switching to alternative mesenchymal phenotypes is incompletely understood. In this study, we tested the ability of the osteoblast/preosteocyte osteogenic cell line, MLO-A5, to also differentiate into either adipocytes or chondrocytes. MLO-A5 cells expressed a subset of skeletal stem cell markers, including Sca-1, CD44, CD73, CD146, and CD166. Confluent cultures of cells underwent differentiation within 3 days upon the addition of osteogenic medium. The same cultures were capable of undergoing adipogenic and chondrogenic differentiation under lineage-appropriate culture conditions, evidenced by lineage-specific gene expression analysis by real-time reverse-transcription-PCR, and by Oil Red O and alcian blue (pH 2.5) staining, respectively. Subcutaneous implantation of MLO-A5 cells in a gel foam into NOD SCID mice resulted in a woven bone-like structure containing embedded osteocytes and regions of cartilage-like tissue, which stained positive with both alcian blue (pH 2.5) and safranin O. Together, our findings show that MLO-A5 cells, despite being a strongly osteogenic cell line, exhibit characteristics of skeletal stem cells and display mesenchymal lineage plasticityin vitroandin vivo. These unique characteristics suggest that this cell line is a useful model with which to study aging and disease-related changes to the mesenchymal lineage composition of bone.

scholarly journals Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

1991 ◽  
Vol 11 (7) ◽  
pp. 3633-3641 ◽  
Author(s):  
T Chakraborty ◽  
T J Brennan ◽  
L Li ◽  
D Edmondson ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Gene Activation ◽  
Binding Activity ◽  
Specific Gene ◽  
Gene Products ◽  
Basic Domain ◽  
High Affinity ◽  
Dna Binding Activity

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

Rapid changes in gene expression in response to microbial elicitation

1986 ◽  
Vol 314 (1166) ◽  
pp. 411-426 ◽  
Keyword(s):  
Phenylalanine Ammonia Lyase ◽  
Chalcone Synthase ◽  
Blot Hybridization ◽  
Enzyme Synthesis ◽  
Chalcone Isomerase ◽  
Colletotrichum Lindemuthianum ◽  
Specific Gene ◽  
Gene Products

Treatment of cell suspension cultures of French bean ( Phaseolus vulgaris ) with polysaccharide elicitor molecules from cell walls of the anthracnose fungus, Colletotrichum lindemuthianum , results in the rapid accumulation of isoflavonoid phytoalexins, deposition of wall-bound phenolic compounds and synthesis of hydroxyproline-rich glycoproteins. These changes are dependent upon a highly selective induction of gene products, including the enzymes L-phenylalanine ammonia-lyase, cytochrome P450-dependent cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, prolyl hydroxylase and protein: arabinosyl transferase. Use of in vivo labelling, in vitro translation and RNA blot hybridization techniques has shown that these elicitormediated changes arise from rapid but transient induction of enzyme synthesis, resulting from the accumulation of specific mRNAs. Similar phenomena are observed in bean hypocotyls at the onset of phytoalexin synthesis in response to infection by incompatible and compatible races of C. lindemuthianum . In bean, both L-phenylalanine ammonia-lyase and chalcone synthase are encoded by multigene families and, at the protein level, both exhibit subunit and intact enzyme polymorphism. A number of less than full-length phenylalanine ammonialyase copy DNAs containing identical open reading frames have been produced from mRNA from elicitor-induced bean cells. Analysis of phenylalanine ammonia-lyase genomic clones predicts the presence of enzyme forms of differing amino acid sequence. In cultured bean cells, elicitor differentially induces the two apparent phenylalanine ammonia-lyase iso-forms with the lowest K m values. In addition to transcriptional control of the appearance of specific gene products, post-translational processes may result in increased subunit polymorphism for phenylalanine ammonia-lyase, and in the activation of chalcone isomerase. Changes in endogenous phenylpropanoid intermediate pools may signal the rapid removal of phenylalanine ammonia-lyase activity, in addition to exerting less specific inhibitory effects on the formation and/or activity of the mRNAs encoding phenylalanine ammonia-lyase and other phytoalexin biosynthetic enzymes.

scholarly journals First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

2009 ◽  
Vol 77 (5) ◽  
pp. 1854-1865 ◽  
Author(s):  
Anne E. Kizy ◽  
Melody N. Neely
Keyword(s):  
Streptococcus Pyogenes ◽  
Large Scale ◽  
Virulence Genes ◽  
Insertion Site ◽  
Disease Model ◽  
Invasive Infection ◽  
Virulence Determinants ◽  
Infectious Disease Model

ABSTRACT The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.

scholarly journals Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding

1991 ◽  
Vol 11 (7) ◽  
pp. 3633-3641
Author(s):  
T Chakraborty ◽  
T J Brennan ◽  
L Li ◽  
D Edmondson ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Gene Activation ◽  
Binding Activity ◽  
Specific Gene ◽  
Gene Products ◽  
Basic Domain ◽  
High Affinity ◽  
Dna Binding Activity

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals A novel BRD4 inhibitor suppresses osteoclastogenesis and ovariectomized osteoporosis by blocking RANKL-mediated MAPK and NF-κB pathways

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Ying Liu ◽  
Wenjie Liu ◽  
Ziqiang Yu ◽  
Yan Zhang ◽  
Yinghua Li ◽  
...  
Keyword(s):  
Bone Loss ◽  
Osteoporosis Treatment ◽  
Inhibitory Effect ◽  
Specific Gene ◽  
Actin Ring ◽  
Treatment Target ◽  
Significant Inhibitory Effect ◽  
Promising Treatment

AbstractBromodomain-containing protein 4 (BRD4) has emerged as a promising treatment target for bone-related disorders. (+)-JQ1, a thienotriazolodiazepine compound, has been shown to inhibit pro-osteoclastic activity in a BRD4-dependent approach and impede bone loss caused by ovariectomy (OVX) in vivo. However, clinical trials of (+)-JQ1 are limited because of its poor druggability. In this study, we synthesized a new (+)-JQ1 derivative differing in structure and chirality. One such derivative, (+)-ND, exhibited higher solubility and excellent inhibitory activity against BRD4 compared with its analogue (+)-JQ1. Interestingly, (-)-JQ1 and (-)-ND exhibited low anti-proliferative activity and had no significant inhibitory effect on RANKL-induced osteoclastogenesis as compared with (+)-JQ1 and (+)-ND, suggesting the importance of chirality in the biological activity of compounds. Among these compounds, (+)-ND displayed the most prominent inhibitory effect on RANKL-induced osteoclastogenesis. Moreover, (+)-ND could inhibit osteoclast-specific gene expression, F‐actin ring generation, and bone resorption in vitro and prevent bone loss in OVX mice. Collectively, these findings indicated that (+)-ND represses RANKL‐stimulated osteoclastogenesis and averts OVX-triggered osteoporosis by suppressing MAPK and NF-κB signalling cascades, suggesting that it may be a prospective candidate for osteoporosis treatment.

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