scholarly journals Differential Cell Lysis Among Periodontal Strains of JP2 and Non-JP2 Genotype of Aggregatibacter actinomycetemcomitans Serotype B Is Not Reflected in Dissimilar Expression and Production of Leukotoxin

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 211 ◽  
Author(s):  
Anne Birkeholm Jensen ◽  
Marianne Lund ◽  
Niels Nørskov-Lauritsen ◽  
Anders Johansson ◽  
Rolf Claesson ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Western Blotting ◽  
Quantitative Methods ◽  
Cell Lysis ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Comprehensive Examination ◽  
Differential Cell ◽  
A Cell ◽  
Serotype B ◽  
Comparison Of The Results

Leukotoxic potential of Aggregatibacter actinomycetemcomitans strains has been studied by the use of several methods, and results differ depending on the methods used. The aim of the present study was to perform a comprehensive examination of the leukotoxic potential of a collection of A. actinomycetemcomitans strains by use of three quantitative methods, Western blotting, ELISA, and mRNA expression assay and compare these results with previous data obtained by a cell lysis assay. A higher leukotoxic potential among JP2 genotype strains compared to non-JP2 genotype strains of A. actinomycetemcomitans was found by Western blotting, ELISA and mRNA expression assay. Leukotoxicity as determined by cell lysis assay showed a variation among strains examined, not only depending on being part of JP2 genotype vs. non-JP2 genotype group of A. actinomycetemcomitans. The leukotoxicity of A. actinomycetemcomitans strains as determined by cell lysis assay did not correspond to the leukotoxic potential of A. actinomycetemcomitans strains as determined by three quantitative methods. A comparison of the results obtained by ELISA and mRNA expression assay showed a reasonable correlation between these two methods. It seems important to use more than one method to assess the LtxA-related virulence capacity of A. actinomycetemcomitans in order to obtain comprehensive understanding of the leukotoxic potential of A. actinomycetemcomitans strains.

scholarly journals Determination of Total Interleukin-8 in Whole Blood after Cell Lysis

2000 ◽  
Vol 46 (9) ◽  
pp. 1387-1394 ◽  
Author(s):  
Jochen Reinsberg ◽  
Jörg Dembinski ◽  
Christoph Dorn ◽  
Daniela Behrendt ◽  
Peter Bartmann ◽  
...  
Keyword(s):  
Whole Blood ◽  
Healthy Subjects ◽  
Room Temperature ◽  
Sample Pretreatment ◽  
Cell Lysis ◽  
Interleukin 8 ◽  
Simple Method ◽  
Sample Handling ◽  
A Cell

Abstract Background: It has been shown that a high percentage of interleukin-8 (IL-8) in blood is cell associated. Recently, a simple method for determination of cell-associated IL-8 in whole blood after cell lysis has been described. The purpose of this study was to evaluate this method, to examine the influence of preanalytic sample handling, and to establish the concentration range of total IL-8 and its relation to age and sex in healthy subjects. Methods: Total IL-8 content of whole blood was determined after lysing blood cells with Milenia® cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE® IL-8 immunoassay. Results: When freshly drawn blood was stored up to 48 h on ice, no significant changes in total IL-8 were measured in the subsequently prepared lysate, whereas with storage at room temperature, total IL-8 increased after 3 h from 94 ± 13 ng/L to 114 ± 16 ng/L (n = 10). In lysate stored for 48 h at 4 °C, marginal changes of the IL-8 concentration were noted, with storage at room temperature, only 76% ± 5% (n = 12) of initial concentration was recovered. From lysate frozen at −20 and −80 °C, respectively, 84% ± 4% and 93% ± 2% of initial IL-8 was recovered after 70 days (n = 10). IL-8 was measured with comparable precision in plasma (CV, 3.2–4.2%) and blood lysate (CV, 3.7–4.1%). When plasma was diluted with cell lysis solution, a slightly overestimated recovery (125% ± 3%) was observed; for lysate specimens with a cell lysis solution content ≥75%, the recovery after dilution was 98% ± 2%. In lysate prepared from 12 blood samples with exogenous IL-8 added, IL-8 recovery was 104% ± 2% (recovery from plasma <35%). The median total IL-8 in blood lysates from 103 healthy subjects (22–61 years) was 83 ng/L of blood (2.5–97.5 percentile range, 49–202 ng/L of blood). In females but not in males, total IL-8 increased significantly with advancing age (P <0.002). We found grossly increased total IL-8 in six pregnant women with amniotic infection syndrome. Conclusions: The evaluated method allows the assessment of total IL-8 in blood with good performance when appropriate conditions of sample pretreatment are considered. The values in healthy volunteers all were above the detection limit of the IL-8 assay; therefore, slight changes of total IL-8 could be noted. Thus, the present method is a suitable tool to study the diagnostic relevance of total IL-8 in blood.

scholarly journals Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution

2003 ◽  
Vol 177 (2) ◽  
pp. 305-317 ◽  
Author(s):  
D Schams ◽  
S Kohlenberg ◽  
W Amselgruber ◽  
B Berisha ◽  
MW Pfaffl ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Mrna Expression ◽  
Western Blotting ◽  
Mammary Gland Development ◽  
Mammary Tissue ◽  
Positive Staining ◽  
Bovine Mammary Gland ◽  
Total Rna ◽  
Gland Development

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.

scholarly journals Fetal Tracheal Occlusion Induces a Cell-Specific Increase in IGF-1 and TGF-β2 mRNA Expression

1999 ◽  
Vol 45 (4, Part 2 of 2) ◽  
pp. 315A-315A ◽  
Author(s):  
Bruno Piedboeuf ◽  
Marie Gamache ◽  
Stuart B Hooper
Keyword(s):  
Mrna Expression ◽  
Tracheal Occlusion ◽  
A Cell ◽  
Specific Increase

scholarly journals Galactose Residues on the Lipooligosaccharide of Moraxella catarrhalis 26404 Form the Epitope Recognized by the Bactericidal Antiserum from Conjugate Vaccination

2008 ◽  
Vol 76 (9) ◽  
pp. 4251-4258 ◽  
Author(s):  
Shengqing Yu ◽  
Hang Xie ◽  
Anup Datta ◽  
Natasha Naidu ◽  
Xin-Xing Gu
Keyword(s):  
Mutant Strain ◽  
Western Blotting ◽  
Moraxella Catarrhalis ◽  
Critical Role ◽  
Kanamycin Resistance ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Oligosaccharide Chain ◽  
Serotype B ◽  
Conjugate Vaccination

ABSTRACT Lipooligosaccharide (LOS) from Moraxella catarrhalis has the potential to elicit bactericidal antibodies against the pathogen. We generated LOS-based conjugate vaccines that elicited bactericidal antibodies in animal models. However, epitopes on the LOS recognized by the functional anti-LOS antibodies remain unidentified. In this study, a mutant strain, D4, which lost the recognition by a bactericidal anti-LOS rabbit serum in Western blotting was generated from a serotype C strain 26404 by random transposon mutagenesis. Sequence analysis revealed there was an insertion of a kanamycin resistance gene in the lgt2 gene of D4, which encodes β(1-4)-galactosyltransferase. An isogenic lgt2 mutant, 26404lgt2, was constructed. Structural analysis indicated that the mutant strain produced a truncated LOS lacking terminal galactoses from 4- and 6-linked oligosaccharide chains of strain 26404. Further studies showed that the antiserum lost the recognition of both mutant cells and LOSs in Western blotting, an enzyme-linked immunosorbent assay (ELISA), or a flow cytometry assay. The antiserum also lost the ability to kill the mutant strain in a bactericidal assay, whereas it showed a bactericidal titer of 1:80 to strain 26404. In an inhibition ELISA, d-(+)-galactose or 26404lgt2 LOS showed no inhibition. However, the 26404 LOS and a serotype A O35E LOS with terminal galactoses on its 6-linked oligosaccharide chain showed >90% inhibition, while a serotype B 26397 LOS showed >60% inhibition. These studies suggest that the terminal α-Gal-(1→4)-β-Gal on the 6-linked oligosaccharide chain of 26404 LOS plays a critical role in forming the epitope recognized by the bactericidal antiserum induced by immunization with our conjugate vaccine.

Osmotic regulation of betaine homocysteine-S-methyltransferase expression in H4IIE rat hepatoma cells

2007 ◽  
Vol 292 (4) ◽  
pp. G1089-G1098 ◽  
Author(s):  
Christine Schäfer ◽  
Lars Hoffmann ◽  
Katrin Heldt ◽  
Mohammad Reza Lornejad-Schäfer ◽  
Gernot Brauers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Tyrosine Kinases ◽  
Hepatoma Cells ◽  
Pcr Analysis ◽  
Osmotic Regulation ◽  
A Cell ◽  
Gene Expression Studies ◽  
Rat Hepatoma ◽  
Rat Hepatoma Cells

Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase ( Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups.

scholarly journals AGR3 Regulates Airway Epithelial Junctions in Patients with Frequent Exacerbations of COPD

2021 ◽  
Vol 12 ◽  
Author(s):  
Rui Ye ◽  
Cuihong Wang ◽  
Pengbo Sun ◽  
Shuang Bai ◽  
Li Zhao
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Mrna Expression ◽  
Western Blotting ◽  
Human Lung ◽  
Airway Epithelial Cells ◽  
Copd Exacerbation ◽  
Airway Epithelial ◽  
Copd Exacerbations ◽  
E Cadherin

Background: The mechanisms underlying differences in the susceptibility to chronic obstructive pulmonary disease (COPD) exacerbations between patients are not well understood. Recent studies have shown that the patients with frequent COPD exacerbations is related to specific protein expression in lung tissue. Anterior gradient 3 (AGR3) is expressed in airway epithelial cells in the lung and proteomic analysis revealed that its expression is decreased in patients with frequent COPD exacerbations. Moreover, the loss of epithelial integrity might facilitate trans-epithelial permeability of pathogens in such patients. This study was performed to determine that AGR3 protein play a role in COPD frequency exacerbators.Methods: Human lung tissues were collected from current-smoking patients (Control; n = 15) as well as patients with infrequent COPD exacerbations (IFCOPD; n = 18) and frequent COPD exacerbations (FCOPD; n = 8). While AGR3 protein expression was measured by immunohistochemistry and western blotting, AGR mRNA expression was determined by real time quantitative polymerase chain reaction (RT-qPCR). Furthermore, adherent junctions (AJs) and tight junctions (TJs) protein expression in human lung tissues were measured by immunohistochemistry. The effects of cigarette smoke extract (CSE) on AJ and TJ protein and mRNA expression in BEAS-2B cells were assessed by western blotting and RT-qPCR. In addition, the effect of AGR3 overexpression and knockdown on AJ and TJ protein expression was determined.Results: AGR3 was mainly expressed in the airway epithelium and AGR3-positive products were localized in the cytoplasm. Western blotting and RT-qPCR results showed that AGR3 protein (p = 0.009) and mRNA (p = 0.04) expression in the FCOPD group was significantly lower than that in the IFCOPD group. Moreover, E-cadherin, occludin, and zonula occludens-1 (ZO-1) expression was lower in the FCOPD group than in the IFCOPD group. The protein and mRNA expression of E-cadherin, occludin, and ZO-1 was decreased within 24 h post-CSE exposure. AGR3 overexpression rescued CSE-induced downregulation of E-cadherin, occludin, and ZO-1.Conclusion: Difference in AGR3 expression in the lung tissue might be correlated with increased susceptibility to COPD exacerbation. AGR3 can prevent CSE-induced downregulation of E-cadherin, occludin, and ZO-1 in airway epithelial cells. Loss of AGR3 might promote viral and bacterial infection and induce immune inflammation to increase COPD exacerbation.

scholarly journals Aquaporin Expression in the Fetal Porcine Urinary Tract Changes During Gestation

2018 ◽  
pp. 283-292 ◽  
Author(s):  
L. K. JAKOBSEN ◽  
K. F. TRELBORG ◽  
P. S. KINGO ◽  
S. HØYER ◽  
K.-E. ANDERSSON ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Mrna Expression ◽  
Western Blotting ◽  
Gestational Age ◽  
Renal Pelvis ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Protein Levels ◽  
Water Handling ◽  
Aqp1 Expression

The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive on RT-PCR), and immunohistochemistry. Bladder samples were additionally examined by Western blotting. RNA was extracted from 76 tissue samples obtained from 19 fetuses. Gestational age was 60 (n=11) or 100 days (n=8). PCR showed that AQP1, 3, 9 and 11 mRNA was expressed in all locations. The expression of AQP3 increased significantly at all four locations with gestational age, whereas AQP11 significantly decreased. AQP1 expression increased in the ureter, bladder and urethra. AQP9 mRNA expression increased in the urethra and bladder, but decreased in the ureter. AQP5 was expressed only in the urethra. Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide insights into normal and pathophysiological water handling mechanisms in the fetus.

scholarly journals Mathematical models of Influenza A. virus infections in vitro: Invesigating defective interfering particles and virus release

2021 ◽  
Author(s):  
Laura Liao
Keyword(s):  
Mathematical Models ◽  
Influenza A Virus ◽  
Release Rate ◽  
Influenza A ◽  
Quantitative Methods ◽  
Virus Release ◽  
Virus Infections ◽  
Defective Interfering Particles ◽  
A Cell

In this work, two studies were performed where mathematical models (MM) were used to re-examine and refine quantitative methods based on in vitro assays of influenza A virus infections. In the first study, we investigated the standard experimental method for counting defective interfering particles (DIPs) based on the reduction in standard virus (STV) yield (Bellett & Cooper, 1959). We found the method is valid for counting DIPs provided that: (1) a STV-infected cell’s co-infection window is approximately half its eclipse phase (it blocks infection by other virions before it begins producing progeny virions); (2) a cell co-infected by STV and DIP produces less than 1 STV per 1,000 DIPs; and (3) a high MOI of STV stock (>4 plaque-forming units/cell) is added to perform the assay. Prior work makes no mention of these criteria such that the counting method has been applied incorrectly in several publications discussed herein. We determined influenza A virus meets these criteria, making the method suitable for counting influenza A DIPs. In the second study, we compared a MM with an explicit representation of viral release to a simple MM without explicit release, and investigated whether parameter estimation and the estimation of neuraminidase inhibitor (NAI) efficacy were affected by the use of a simple MM. Since the release rate of influenza A virus is not well-known, a broad range of release rates were considered. If the virus release rate is greater than ∼0.1 h−1, the simple MM provides accurate estimates of infection parameters, but underestimates NAI efficacy, which could lead to underdosing and the emergence of NAI resistance. In contrast, when release is slower than ∼0.1 h−1, the simple MM accurately estimates NAI efficacy, but it can significantly overestimate the infectious lifespan (i.e., the time a cell remains infectious and producing free virus), and it will significantly underestimate the total virus yield and thus the likelihood of resistance emergence. We discuss the properties of, and a possible lower bound for, the influenza A virus release rate. Overall, MMs are a valuable tool in the exploration of the known unknowns (i.e., DIPs, virus release) of influenza A virus infection.

scholarly journals Mathematical models of Influenza A. virus infections in vitro: Invesigating defective interfering particles and virus release

2021 ◽  
Author(s):  
Laura Liao
Keyword(s):  
Mathematical Models ◽  
Influenza A Virus ◽  
Release Rate ◽  
Influenza A ◽  
Quantitative Methods ◽  
Virus Release ◽  
Virus Infections ◽  
Defective Interfering Particles ◽  
A Cell

In this work, two studies were performed where mathematical models (MM) were used to re-examine and refine quantitative methods based on in vitro assays of influenza A virus infections. In the first study, we investigated the standard experimental method for counting defective interfering particles (DIPs) based on the reduction in standard virus (STV) yield (Bellett & Cooper, 1959). We found the method is valid for counting DIPs provided that: (1) a STV-infected cell’s co-infection window is approximately half its eclipse phase (it blocks infection by other virions before it begins producing progeny virions); (2) a cell co-infected by STV and DIP produces less than 1 STV per 1,000 DIPs; and (3) a high MOI of STV stock (>4 plaque-forming units/cell) is added to perform the assay. Prior work makes no mention of these criteria such that the counting method has been applied incorrectly in several publications discussed herein. We determined influenza A virus meets these criteria, making the method suitable for counting influenza A DIPs. In the second study, we compared a MM with an explicit representation of viral release to a simple MM without explicit release, and investigated whether parameter estimation and the estimation of neuraminidase inhibitor (NAI) efficacy were affected by the use of a simple MM. Since the release rate of influenza A virus is not well-known, a broad range of release rates were considered. If the virus release rate is greater than ∼0.1 h−1, the simple MM provides accurate estimates of infection parameters, but underestimates NAI efficacy, which could lead to underdosing and the emergence of NAI resistance. In contrast, when release is slower than ∼0.1 h−1, the simple MM accurately estimates NAI efficacy, but it can significantly overestimate the infectious lifespan (i.e., the time a cell remains infectious and producing free virus), and it will significantly underestimate the total virus yield and thus the likelihood of resistance emergence. We discuss the properties of, and a possible lower bound for, the influenza A virus release rate. Overall, MMs are a valuable tool in the exploration of the known unknowns (i.e., DIPs, virus release) of influenza A virus infection.

scholarly journals A Simple and Efficient Mechanical Cell Disruption Method Using Glass Beads to Extract β-Glucans from Spent Brewer’s Yeast

2022 ◽  
Vol 12 (2) ◽  
pp. 648
Author(s):  
Ionut Avramia ◽  
Sonia Amariei
Keyword(s):  
Cell Suspension ◽  
Cell Lysis ◽  
Glass Beads ◽  
Small Scale ◽  
Optimal Parameters ◽  
Brewer’S Yeast ◽  
Absorption Bands ◽  
Suspension Concentration ◽  
Brewer's Yeast ◽  
A Cell

β-glucan extraction from spent brewer’s yeast is a long process that starts with the lysis of yeast cells, this step lasting up to 36 h and can be disadvantageous when working on a small scale. In this study, a rapid cell rupture method was selected for the lysis of spent brewer’s yeast to obtain β-glucans. Optimal parameters were determined for the lysis of a cellular suspension of spent brewer’s yeast by vortexing with glass beads. Thus, parameters such as the number of 10 min vortex cycles from 1 to 3, the concentration of cell suspension (5, 10, and 15%), and the ratio of yeast/glass beads (1:1, 1:2, and 1:3) were varied in a Box-Behnken design. A cell lysis mechanism using glass beads allows the cell to rupture and permits the removal of intracellular content. An increase in yeast suspension concentration decreased the disruption efficiency, while a proportional increase was observed with the yeast/glass beads ratio and the increasing number of vortexing cycles. The optimal parameters for cell lysis were found to be a cell suspension concentration of 5%, a ratio of yeast/glass beads of 1:2, and a vortexing cycle of 3, with a disruption efficiency of 99.8%. The β-glucan fraction extracted from the optimal sample showed characteristic absorption bands at 1370.77 and 1153.92 cm−1, the content of β-glucan being 78.53%.

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