TheCampylobacter fetusS layer is not essential for initial interaction with HEp-2 cells

1998 ◽  
Vol 44 (3) ◽  
pp. 244-250 ◽  
Author(s):  
Lori L Graham ◽  
K L MacDonald
Keyword(s):  
Wild Type ◽  
Cell Numbers ◽  
Campylobacter Fetus ◽  
Time Period ◽  
Significant Difference ◽  
Mutant Strains ◽  
Fetus Subsp ◽  
Initial Interaction ◽  
Wild Type Parent

In vitro adherence assays were used to determine whether the S layer mediated interactions between Campylobacter fetus subsp. venerealis strains and HEp-2 cells. At multiplicity of infection ratios ranging from 0.1:1 through 100:1, quantitation of bacterial adherence by light microscopy revealed that S layer deficient isogenic C. fetus 809K and C. fetus 810K were not less efficient in their attachment to HEp-2 cells; either S layer deficient C. fetus strains interacted with HEp-2 cells in greater numbers than the corresponding wild-type parent strains 809 and 810 or there was no significant difference in adherence levels between wild-type and mutant strains. Adherence of C. fetus strains to HEp-2 cells increased most during the first 2 h of a 22-h incubation period with only a slight increase in C. fetus cell numbers occuring subsequent to 2 h. At each assay point throughout this 22-h time period, equivalent numbers of wild-type and S layer deficient C. fetus strains were observed associated with HEp-2 cells. Prior to 2 h, adherence levels of all C. fetus strains exceeded those of Escherichia coli AB264 and Salmonella typhimurium SL1344. And, unlike S. typhimurium, C. fetus did not undergo significant replication following initial adherence to HEp-2 cells. Campylobacter fetus did not adhere to HEp-2 cells in a localized or aggregative pattern but were randomly distributed over individual HEp-2 cells and at no time during the assay with C. fetus were changes in HEp-2 cell morphology apparent. These data suggest that the S layer is not essential for mediating initial interactions between C. fetus and HEp-2 cells.Key words: Campylobacter fetus, S layer, HEp-2.

The correlation between serum Ca-125 level and clinical pregnancy in in vitro fertilization (IVF): A meta-analysis

2019 ◽  
Author(s):  
Xin-Lei Wang ◽  
Zhuo Li ◽  
Han Zhang ◽  
Ce Shi ◽  
Tong Tong ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Transfer ◽  
Meta Analysis ◽  
Clinical Pregnancy ◽  
Ca 125 ◽  
Vitro Fertilization ◽  
Time Period ◽  
Central Register ◽  
Significant Difference

Abstract Background Several studies had investigated the role of serum Ca-125 in clinical pregnancy of patients undergoing in vitro fertilization (IVF); however, their conclusions had been inconsistent. This study aimed to evaluate the correlation between serum Ca-125 level and clinical pregnancy in IVF.Methods We systematically review the studies in the databases of Mediline OvidSP, EMBASE OvidSP and Cochrane (CENTRAL Central Register of Controlled Trials). Studies on the correlation between serum Ca-125 level and clinical pregnancy in patients underging IVF with or without Intracytoplasmic sperm injection (ICSI) were considered. The pooled standardized mean difference (SMD) with 95% confidence intervals (CIs) was used in the analysis.Results Seven studies involving 558 patients were included. The meta-analysis showed that there was no significant difference in the serum Ca-125 level before embryo transfer (ET) between clinical pregnant group and nonpregnant group (SMD 0.72; 95% CI [0.01, 1.43], P = 0.05, I 2 = 88%), and the same conclusion was also reached in patients without endometriosis (SMD 0.31; 95% CI [-0.53, 1.16], P = 0.47, I 2 = 89%); However, after embryo transfer, the result showed that the Ca-125 level has a small but significantly increase in the clinical pregnant group than in the nonpregnant group (SMD 0.39; 95% CI [0.09, 0.69], P = 0.01, I 2 = 0%).Conclusions Berore ET, there was no significant correlation between serum Ca-125 level and clinical pregnancy in IVF; After ET, the Ca-125 level has a small but significantly increase in the clinical pregnant group than in the nonpregnant group, and it might reflect a successful interaction between the embryo and the endometrium in that time period.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

In vitro Fertilization of Bovine Ova in the Presence of Campylobacter fetus subsp. venerealis

1994 ◽  
Vol 29 (6) ◽  
pp. 488-493 ◽  
Author(s):  
A. Bielanski ◽  
M. Sampath ◽  
C. Gradil ◽  
M. D. Eaglesome ◽  
M. Garcia
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization ◽  
Campylobacter Fetus ◽  
Fetus Subsp

158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Cell Types ◽  
Cell Number ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
In Vitro Embryo Production

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

scholarly journals Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

2020 ◽  
Vol 8 (1) ◽  
pp. 70 ◽  
Author(s):  
Bhumika Shokeen ◽  
Jane Park ◽  
Emily Duong ◽  
Sonam Rambhia ◽  
Manash Paul ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Fusobacterium Nucleatum ◽  
Interspecies Interaction ◽  
Wild Type ◽  
Oral Epithelial Cells ◽  
Small Proteins ◽  
Mutant Strains ◽  
Oral Epithelial ◽  
Wild Type Parent

RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

scholarly journals Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen

2003 ◽  
Vol 197 (6) ◽  
pp. 735-742 ◽  
Author(s):  
Loïc Coutte ◽  
Sylvie Alonso ◽  
Nathalie Reveneau ◽  
Eve Willery ◽  
Brigitte Quatannens ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Wild Type ◽  
Early Step ◽  
Bacterial Surface ◽  
Extracellular Milieu ◽  
Wild Type Parent

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.

scholarly journals Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

2000 ◽  
Vol 191 (5) ◽  
pp. 859-870 ◽  
Author(s):  
Melanie Allen ◽  
Linne Svensson ◽  
Marsha Roach ◽  
John Hambor ◽  
John McNeish ◽  
...  
Keyword(s):  
Map Kinase ◽  
Es Cells ◽  
Interleukin 1 ◽  
Embryonic Stem ◽  
Wild Type ◽  
Antiinflammatory Drugs ◽  
Gene Encoding ◽  
Significant Difference ◽  
Induced Apoptosis

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.

CEP701 and PKC412 Predictably and Reliably Inhibit FLT3 Phosphorylation in Primary AML Blasts but Their Induction of a Cytotoxic Response Appears to Be Much More Variable.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Steven Knapper ◽  
Alan K. Burnett ◽  
Amanda F. Gilkes ◽  
Kenneth I. Mills ◽  
Val Walsh
Keyword(s):  
Statistical Significance ◽  
Annexin V ◽  
Expression Level ◽  
Wild Type ◽  
Response Curves ◽  
Allele Ratio ◽  
Cytotoxic Response ◽  
Significant Difference ◽  
Flt3 Expression

Abstract Activating mutations of the receptor tyrosine kinase FLT3 are present in approximately one-third of AML cases and are associated with an adverse prognosis. FLT3 is expressed in over 90% of cases of AML and many non-mutants show evidence of FLT3 activation, which may play a significant signalling role in leukaemogenesis, making FLT3 an attractive therapeutic target. CEP701 (Cephalon) and PKC412 (Novartis) are orally-bioavailable indolocarbazole derivatives that potently inhibit FLT3 phosphorylation. We studied the relationship between in vitro inhibition of FLT3 phosphorylation and induction of cytotoxicity in primary AML blasts from 12 patients. 7 of the cases were FLT3 mutants (6 ITDs and 1 D835 point mutant), the amount of mutant RNA varying between 7% and 84% of total FLT3 RNA expressed. The blasts were exposed for 1 hour to a range of concentrations of CEP701 and PKC412, lysed and immunoprecipitated with an anti-FLT3 antibody. After sequential immunoblotting with anti-phosphotyrosine and anti-FLT3 antibodies, inhibition of FLT3 phosphorylation was measured by densitometry. Both drugs inhibited FLT3 phosphorylation in all samples with lower concentrations required in FLT3 mutants. CEP701 inhibited FLT3 phosphorylation with median IC50s of 3.7nM and 11.9nM in mutant and wild type (WT) cases respectively (p=0.0006). IC50s for PKC412 were 7.7nM and 59.8nM in mutant and WT cases (p=0.0268). Induction of cytotoxicity was assessed by MTS assay following 72-hour exposure of blasts to a range of concentrations of CEP701 and PKC412. Cytotoxic responses to both drugs were greater in FLT3 mutants than WT cases at each dose studied and in terms of IC50 dose (median IC50s in mutant and WT cases: 95nM and 231nM with CEP701, 1.24 μM and 1.61μM with PKC412) although these differences did not reach statistical significance. Annexin V binding apoptosis assay produced similar dose response curves. Both agents showed greater inter-case variability in cytotoxic response than in sensitivity to inhibition of FLT3 phosphorylation. A lack of cytotoxic response to FLT3 inhibition with CEP701 was seen in the ITD mutant with the lowest ratio of mutant to WT FLT3 RNA (0.08) and several WT samples displayed resistance to in vitro induction of cytotoxicity despite almost complete inhibition of FLT3. Induction of cytotoxicity with PKC412 in both mutant and WT cases generally required doses well in excess of those required to fully inhibit FLT3 phosphorylation. Cases were further stratified by flow cytometric measurement of surface FLT3 expression, and by immunoblotting to measure STAT5 dephosphorylation in response to both drugs. No significant difference in overall FLT3 expression was seen between mutant and WT cases. Interestingly the highest FLT3 expression level was seen in a wild type case that was highly sensitive to CEP701. Inhibition of STAT5 phosphorylation appeared closely linked to FLT3 inhibition, although in some cases a good cytotoxic response was achieved despite failure to inhibit STAT5, suggesting involvment of other signalling pathways. In summary, although both CEP701 and PKC412 predictably and reliably inhibit FLT3 phosphorylation in primary AML blasts, their induction of cytotoxicity appears to be much more variable. A number of factors may influence this including variations in level of dependency on FLT3 signalling for blast survival, mutant to WT allele ratio and overall FLT3 expression level. Effects on targets other than FLT3 also need to be considered.

scholarly journals Salmonella enterica Serovar Enteritidis Pathogenicity Island 1 Is Not Essential for but Facilitates Rapid Systemic Spread in Chickens

2009 ◽  
Vol 77 (7) ◽  
pp. 2866-2875 ◽  
Author(s):  
Taseen S. Desin ◽  
Po-King S. Lam ◽  
Birgit Koch ◽  
Claudia Mickael ◽  
Emil Berberov ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Systemic Infection ◽  
Poultry Meat ◽  
Wild Type ◽  
Secretion Systems ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Systemic Spread ◽  
Mutant Strains ◽  
Vitro Analysis

ABSTRACT Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both ΔSPI-1 and ΔinvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and ΔSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or ΔSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the ΔSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.

scholarly journals Construction and Characterization of Transposon Insertion Mutations in Corynebacterium diphtheriae That Affect Expression of the Diphtheria Toxin Repressor (DtxR)

2002 ◽  
Vol 184 (20) ◽  
pp. 5723-5732 ◽  
Author(s):  
Diana Marra Oram ◽  
Ana Avdalovic ◽  
Randall K. Holmes
Keyword(s):  
Oxidative Stress ◽  
Diphtheria Toxin ◽  
Corynebacterium Diphtheriae ◽  
Insertion Mutant ◽  
Toxin Gene ◽  
Wild Type ◽  
Transposon Insertion ◽  
Diphtheria Toxin Repressor ◽  
Mutant Strains

ABSTRACT Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe2+ concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C. diphtheriae genes cloned in Escherichia coli or analyzed in vitro. We modified a Tn5-based mutagenesis technique for use with C. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion within dtxR does not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion within dtxR abolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.

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