Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The stability of housekeeping genes is critical when performing gene expression studies. To date, there have been no studies that look at the stability of commonly used housekeeping genes in alveolar macrophages. Expression levels may be affected by culture, stimulation or disease severity. The present study investigated the expression level of 10 housekeeping genes and analysed the stability of their expression in alveolar macrophages from chronic obstructive pulmonary disease patients (n = 22) who were classified according to disease severity. Guanine nucleotide-binding protein, beta polypeptide 2-like 1 (GNB2L1), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and ribosomal protein L32 (RPL32) were the most stably expressed in alveolar macrophages, irrespective of disease severity. There was no difference in the expression levels of 10 housekeeping genes between mild and moderate/severe patients. GNB2L1, HPRT1 and RPL32 were also stably expressed in alveolar macrophages cultured with no stimulation, or with interleukin-1beta, lipopolysaccharide or tumour necrosis factor-alpha stimulation. In conclusion, as fluctuations in the expression of some housekeeping genes were observed, including glyceraldehyde-3-phosphate dehydrogenase, it is recommended that guanine nucleotide binding protein, beta polypeptide 2-like 1 be used as a reference gene for alveolar macrophages in similar study designs, or that the stability of housekeeping genes be validated in alveolar macrophages prior to expression studies.
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PMID:Stability of housekeeping genes in alveolar macrophages from COPD patients. 1645 84

Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.
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PMID:MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars. 1720 23

The prostaglandins are lipid mediators, discovered in the 1930s by von Euler in Sweden and Goldblatt in the United Kingdom. They are made by the bifunctional enzyme, cyclooxygenase, which has both cyclooxygenase and peroxidase activities in the same molecule. Prostaglandins are involved in physiological functions such as protection of the stomach mucosa, aggregation of platelets and regulation of kidney function. They also have pathological functions such as their involvement in inflammation, fever and pain. Vane in 1971 elegantly showed that the pharmacological actions of aspirin and similar drugs were due to the inhibition of cyclooxygenase. Thus, aspirin-like drugs exert their anti-inflammatory, antipyretic and analgesic effects by inhibition of cyclooxygenase. In 1991, Simmons and his colleagues identified a second cyclooxygenase enzyme, designated cyclooxygenase-2, derived from a separate gene from cyclooxygenase-1. Cyclooxygenase-2 is upregulated by inflammatory mediators and forms prostaglandins which intensify the inflammatory response. Cyclooxygenase-1 is, therefore, a 'housekeeping' enzyme making prostaglandins, which are important for maintaining physiological functions and cyclooxygenase-2 makes prostaglandins which are important in inflammation. The discovery of cyclooxygenase-2 and the establishment of its structure led to the development of selective inhibitors of this enzyme, such as celecoxib and rofecoxib, with potent anti-inflammatory actions but with reduced gastrotoxic effects. A putative cyclooxygenase-3, has also been characterised and cloned. This enzyme is a product of the cyclooxygenase-1 gene, but retains intron 1 after transcription and translates into a cyclooxygenase enzyme with 34 additional amino acids. It is more sensitive to inhibition by paracetamol, aspirin and some other non-steroid anti-inflammatory drugs than cyclooxygenase-1 or cyclooxygenase-2. A cyclooxygenase enzyme induced in cultured cells by some non-steroid anti-inflammatory drugs is also more sensitive to inhibition by paracetamol than cyclooxygenase-2 induced by bacterial lipopolysaccharide.
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PMID:Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. 1721 63

We previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor-alpha (TNF-alpha) gene expression. Here, we show that shikonin selectively inhibits the expression of TNF-alpha at the RNA splicing level. Treatment of lipopolysaccharide-stimulated human primary monocytes and THP-1 cells with shikonin resulted in normal transcriptional induction of TNF-alpha, but unspliced pre-mRNA accumulated at the expense of functional mRNA. This effect occurred with noncytotoxic doses of shikonin and was highly specific, because mRNA production of neither a housekeeping gene nor another inflammatory cytokine gene, interleukin-8 (IL-8), was affected. Moreover, cotreatment with lipopolysaccharide (LPS) and shikonin increased the endpoint protein production of IL-8, accompanied by suppressed activation of the double-stranded RNA-activated protein kinase (PKR) pathway. Because PKR inactivation has been shown to down-regulate the splicing process of TNF-alpha RNA and interfere with translation, our findings suggest that shikonin may achieve differential modulation of cytokine protein expression through inactivation of the PKR pathway and reveal that regulation of TNF-alpha pre-mRNA splicing may constitute a promising target for future anti-inflammatory application.
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PMID:Inhibition of tumor necrosis factor-alpha through selective blockade of Pre-mRNA splicing by shikonin. 1736 Aug 31

In the present study, we compared the levels of intravascular tissue factor (TF) present in cord versus adult whole blood (WB) prior and after lipopolysaccharide (LPS) stimulation. High levels of intravascular TF might help to explain the clinically observed efficient clotting of cord blood despite low levels of procoagulatory factors. Quantitative reverse transcription-polymerase chain reaction revealed same (basal) TF mRNA expression levels in both native cord and adult WB, and approximately same increase in TF mRNA expression owing to LPS incubation in both cord and adult WB (normalized to the housekeeping gene beta-actin). Flow-cytometric (fluorescence activated cell sorting) analysis revealed significantly higher surface TF antigen exposure on the neonatal monocyte membrane in native WB samples, and approximately same ability of neonatal and adult monocytes to express TF upon LPS-stimulation. Thrombelastography revealed significantly shorter clotting times of native cord versus adult WB (527+/-41 vs. 592+/-23 s, P<0.05). Moreover, shortening of clotting times owing to LPS-stimulation was significantly more pronounced in cord versus adult WB (29.65+/-3.35% vs. 12.03+/-6.23%, P<0.05). Because both quantitative reverse transcription-polymerase chain reaction and fluorescence activated cell sorting analysis revealed same capability of both neonatal and adult monocytes to express TF upon LPS-stimulation, this efficient shortening effect in cord WB might be explained by the constitutively high number of monocytes present in neonates. We suggest that the high levels of intravascular TF present in neonates (prior and after LPS-stimulation) might help to explain the clinically observed efficient clotting of cord blood despite low levels of procoagulatory factors.
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PMID:High availability of intravascular tissue factor in neonates. 1748

To establish an easy, fast and reliable RT-PCR for the analysis of human cytokine expression, we made use of the recently developed technique of TaqMan PCR. This technique is based on the cleavage of fluorochrome-labelled internal oligodeoxynucleotide probes by the 5'-->3' nuclease activity of Taq DNA polymerase. Measurement of fluorescence intensity during each cycle of the PCR reaction with a Sequence Detection System allows the determination of a threshold cycle at which an increase in fluorescence intensity is first detectable. From these values, a starting amount of template DNA can be calculated. Here, we established specific primers and corresponding internal, fluorogenic probes for the human cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma), and for the constant region of the T-cell receptor beta chain (TCRbeta) and the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalization of mRNA expression levels. Titrations of the cDNA input showed a strict inverse correlation between the threshold cycles obtained and the starting amount of template. This in turn allowed the generation of a standard curve, and thus quantification of mRNA abundance in cDNA samples. Evaluation of the method using cDNAs from peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or phytohae-magglutinin (PHA) showed basal expression of TNF-alpha and IL-1beta in untreated PBMC while IFN-y was not detectable or only weakly expressed. After stimulation with LPS, a strong induction of IL-1beta and TNF-alpha was measured, while IFN-gamma was induced to a lesser extent. PHA treatment, in contrast, led to an induction of all three cytokines with IFN-gamma being the most prominent. The method has a large dynamic range, requires no post-PCR processing and gives reliable results.
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PMID:Semiquantitative determination of human cytokine mRna expression using TaqMan RT-PCR. 1765 27

Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.
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PMID:Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens. 1827 35

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.
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PMID:S-layer nanoglycobiology of bacteria. 1833 1

The omptin family of Gram-negative bacterial transmembrane aspartic proteases comprises surface proteins with a highly conserved beta-barrel fold but differing biological functions. The omptins OmpT of Escherichia coli, PgtE of Salmonella enterica, and Pla of Yersinia pestis differ in their substrate specificity as well as in control of their expression. Their functional differences are in accordance with the differing pathogenesis of the infections caused by E. coli, Salmonella, and Y. pestis, which suggests that the omptins have adapted to the life-styles of their host species. The omptins Pla and PgtE attack on innate immunity by affecting the plasminogen/plasmin, complement, coagulation, fibrinolysis, and matrix metalloproteinase systems, by inactivating antimicrobial peptides, and by enhancing bacterial adhesiveness and invasiveness. Although the mechanistic details of the functions of Pla and PgtE differ, the outcome is the same: enhanced spread and multiplication of Y. pestis and S. enterica in the host. The omptin OmpT is basically a housekeeping protease but it also degrades cationic antimicrobial peptides and may enhance colonization of E. coli at uroepithelia. The catalytic residues in the omptin molecules are spatially conserved, and the differing polypeptide substrate specificities are dictated by minor sequence variations at regions surrounding the catalytic cleft. For enzymatic activity, omptins require association with lipopolysaccharide on the outer membrane. Modification of lipopolysaccharide by in vivo conditions or by bacterial gene loss has an impact on omptin function. Creation of bacterial surface proteolysis is thus a coordinated function involving several surface structures.
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PMID:Invited review: Breaking barriers--attack on innate immune defences by omptin surface proteases of enterobacterial pathogens. 1931 17

beta-Actin has been frequently used as an internal control (gene) or as a housekeeping gene to normalize the expression of the target gene(s) or mRNA levels between different samples. However, the beta-actin expression has been shown to be influenced by the sample type and experimental conditions. If beta-actin could be used as a reference gene for the half-smooth tongue sole Cynoglossus semilaevis remains ill-defined. Here we evaluate the tissue-specific beta-actin gene expression pattern in C. semilaevis when challenged with antigenic agents namely, lipopolysaccharide (LPS) or Vibrio anguillarum employing absolute quantitative real-time PCR. The real-time PCR was performed based on the standard curve generated from recombinant plasmids. No significant differences in beta-actin expression were found between treated and untreated tissue samples. We thus conclude that beta-actin could be used as a reliable internal reference gene for real-time PCR based quantitation of gene expression studies in various tissue samples of C. semilaevis challenged with LPS or pathogenic bacteria.
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PMID:beta-Actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum. 2022 7


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