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PubMed2006

EGFR intragenic loss and gene amplification in astrocytic gliomas.

We have studied EGFR gene amplification and allelic status of chromosome 7 in 68 tumors consisting of 34 WHO grade IV glioblastomas (26 primary and 8 secondary), 14 WHO grade III anaplastic astrocytomas, and 20 WHO grade II astrocytomas, by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), quantitative PCR, and microsatellite analysis. EGFR gene amplification was present in 27 of these tumors (40%), and we identified allelic losses at 7p11 approximately p14 in 38 of the 68 cases (56%), including 17 tumors displaying loss for EGFR intragenic markers. The positive correlation (P < 0.05, chi(2)) between tumors with EGFR intragenic loss and EGFR gene amplification, frequently displaying the EGFR vIII form, suggests that EGFR gene rearrangement leading to intragenic loss is a molecular event that participates in the amplification process of this gene.

Astrocytoma↗

PubMed2005

Characterization of novel splice variants of the PAC1 receptor in human neuroblastoma cells: consequences for signaling by VIP and PACAP.

Expression of VPAC and PAC1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC1 receptor splice variants and the VPAC2 receptor, a number of novel PAC1 receptor splice variants were identified. The comparatively low level expression of the VPAC2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.

Adult↗

PubMed2004

An exploratory study on pharmacogenetics of inosine-monophosphate dehydrogenase II in peripheral mononuclear cells from liver-transplant recipients.

Mycophenolate mofetil (MMF) is an immunosuppressant used for the prophylaxis of rejection in renal, pancreas, and liver transplantation. It inhibits the inducible isoform of the enzyme inosine-monophosphate dehydrogenase (IMPDH II) via its active metabolite mycophenolic acid (MPA). IMPDH II is necessary for de novo purine synthesis in activated lymphocytes. The aims of the present study were to evaluate the feasibility of a real-time polymerase chain reaction (PCR) quantitative assessment of IMPDH II gene expression in liver transplant recipients as well as to provide a preliminary evaluation of possible correlations with drug tolerability. RNA was extracted from peripheral blood mononuclear cells of liver recipients after at least 6 months of MMF administration. IMPDH II gene expression was assessed using quantitative, real-time PCR and normalized using glyceraldheyde-3-phosphate dehydrogenase (GAPDH). Finally, adverse events associated with MMF administration were recorded. Real-time PCR quantitation of IMPDH II gene expression was reliable, sensitive, and specific. The intrapatient variability for both IMPDH II and GAPDH assays was lower than 0.6% in all patients. The results demonstrated a wide interpatient variability, with the mean value +/- standard deviation of 0.949 +/- 0.525 (95% confidence interval, 0.669-1.229) and a median value of 0.797. Patients with treatment-related toxicities displayed a trend to a higher level of IMPDH II expression than those without toxicity (mean, 1.126 vs 0.771). In conclusion, pharmacogenetic analysis of IMPDH II may represent a novel approach to MMF therapeutic monitoring.

Adult↗

PubMed1998

Increased levels of IFN-gamma in the trigeminal ganglion correlate with protection against HSV-1-induced encephalitis following subcutaneous administration with androstenediol.

Androstenediol (AED) is a metabolic product of dehydroepiandrosterone (DHEA), an adrenal steroid known to possess immunomodulatory characteristics. The present study was undertaken to assess the efficacy of AED treatment in mice ocularly infected with herpes simplex virus type 1 (HSV-1). The subcutaneous administration of 320 mg/kg AED 4 h prior to viral inoculation was found to enhance the survival of HSV-1-infected mice while lower doses (32.0-100.0 mg/kg) were without effect. However, there were no apparent differences in the viral load in the eye or trigeminal ganglion (TG) 3 or 6 days post infection (p.i.) in vehicle- or AED (320 mg/kg)-treated mice. Likewise, there were no differences in the expression of cytokine or chemokine mRNAs in the eyes or TG early (i.e., 3 days p.i.) following infection. However, by 6 days p.i., there was a significant increase in the expression of the chemokines IP-10, MCP-1, and RANTES and the cytokines interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) in the AED (320 mg/kg)-treated mice compared to vehicle-treated controls as determined by reverse transcription (RT)-polymerase chain reaction (PCR) and quantitative PCR (for IFN-gamma). Likewise, there was a corresponding increase in IFN-gamma and IL-2 but not IL-12 protein in the TG of AED-treated mice 6 days p.i. AED-treatment also induced a rise in splenic natural killer activity in a dose- and time-dependent fashion. Collectively, these results suggest that the protective effect following subcutaneous administration of AED is associated in a rise in selective type 1 cytokines (IL-2 and IFN-gamma) as well as natural killer activity.

Anabolic Agents↗

PubMed1999

The role of polymerase chain reaction and its newer developments in feline medicine.

We give a brief overview on the principles of the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative competitive PCR and real-time PCR (TaqMan technology). The literature dealing with PCR and its role in the diagnosis, pathogenesis and research of infectious diseases of the domestic cat is reviewed. Cross-contaminations which occasionally occur during handling of amplified DNA may be an important problem in the PCR laboratory. In many infectious diseases, PCR results are difficult to interpret as their predictive positive and negative values are not always known. Newer assays, such as TaqMan procedures, are becoming increasingly reliable and cost-effective. It can be expected that additional knowledge on how to interpret PCR results will soon be available.

Animals↗

PubMed2002

The coexpression of the apoptosis-related genes bcl-2 and wt1 in predicting survival in adult acute myeloid leukemia.

The Wilms tumor gene wt1 and the protooncogene bcl-2 are upregulated in acute myeloid leukemia (AML) and are known to regulate or to inhibit the onset of apoptosis. Since wt1 has been shown to regulate the expression of bcl-2, we investigated the association of the expression of these genes and their prognostic relevance in AML. Leukemic blasts from the bone marrow of 152 patients with newly diagnosed AML were analyzed for bcl-2 and wt1 mRNA expression using RT-PCR and quantitative PCR. Therapy outcome was correlated with the level of bcl-2 and wt1 transcripts. Bcl-2-specific mRNA was detectable in 127/152 (84%) patients and wt1 mRNA in 113/152 (74%) patients with AML. In monocytic subtypes the frequency of bcl-2 and wt1 transcripts was significantly lower. The expression of bcl-2 mRNA was correlated significantly with that of wt1 mRNA (P < 0.0001). In AML patients <60 years, high expression of bcl-2 and wt1 was associated with a reduced rate of continuing complete remission (CCR, P = 0.002 and P = 0.005, respectively) and increased death rate (P = 0.0002 and P = 0.04, respectively) in contrast to patients >60 years, where the expression of bcl-2 or wt1 had no prognostic impact. Based on the coexpression of bcl-2 and wt1, we established a prognostic model defining three risk groups with significant differences in CCR rate (P = 0.01), overall survival (P < 0.04) and disease-free survival (P < 0.03). Thus, bcl-2 and wt1 mRNA expression are associated with response and long-term outcome in AMLs. The coexpression of these genes allows determination of prognostic groups with high predictive value for overall and disease-free survival.

Acute Disease↗

PubMed1998

Interferon-gamma plays a role in pancreatic islet-cell destruction of reovirus type 2-induced diabetes-like syndrome in DBA/1 suckling mice.

Reovirus type 2 (Reo-2) infection in DBA/1 suckling mice causes insulitis, which leads to pancreatic islet-cell destruction, resulting in a diabetes-like syndrome. T-helper (Th) 1 cytokines are thought to play a key role in islet inflammation in insulin-dependent diabetes mellitus. We examined this hypothesis in the Reo-2-induced diabetes-like syndrome. We used reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR techniques to examine mRNA expression of interferon (IFN)-gamma (Th1 type cytokine), and interleukin (IL)-4 (Th2 type cytokine) in splenic cells. We observed that in Reo-2 infected mice the level of IFN-gamma expression increases with the development of insulitis, whereas expression of message for IL-4 is minimal to detectable with the immuno-inflammatory process 10 days after infection. The treatment of monoclonal antibody (mAb) against mouse IFN-gamma during the expansion phase of insulitis (5-9 days after infection) inhibited the development of insulitis and the elevation of blood glucose concentrations in a dose dependent manner. Furthermore altered CD4+/CD8+ cell ratio compared with uninfected mice in the splenic cells by the infection was recovered to the ratio of uninfected mice by the treatment of mAb against mouse IFN-gamma, suggesting normalization of T cell balance in immune system. These results suggest that Reo-2-triggered autoimmune insulitis may be mediated by Th1 lymphocytes and IFN-gamma may play a role in islet inflammation leading to islet cell destruction.

Animals↗

PubMed2005

Enhancing effects of Scutellaria baicalensis and some of its constituents on TGF-beta1 gene expression in RAW 264.7 murine macrophage cell line.

Transforming growth factor beta1 (TGF-beta1) has been implicated as an inhibitor of cell proliferation and a potent inducer of apoptosis. Scutellaria baicalensis Georgi (SbG) has been widely used in Asia and recent investigations have shown that SbG has anticancer, antiviral, and anti-inflammatory effects. The aim of this study is to investigate the modulatory effect of SbG on TGF-beta1 gene expression. By using RAW 264.7 cell line as an in vitro model, the effects of SbG on TGF-beta1 gene expression were evaluated by ELISA, reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR. Many inhibitors such as mitogen-activated protein kinase (MAPK) inhibitor (PD98059), p38-MAPK inhibitor (SB203580), NF-kappaB inhibitor (aspirin) and protein kinase C inhibitor (H7) were used to determine the possible signal transduction pathways. The results showed that crude extracts of SbG as well as its pure compounds, baicalin, baicalein and chrysin up-regulated TGF-beta1 gene expression on RAW 264.7 cells in a concentration-dependent manner. However, the flavonoid of SbG, wogonin, did not up-regulate TGF-beta1 expression on gene and protein levels on RAW264.7 cells. The facts that aspirin and H7 but not PD98059 and SB203580 blocked the enhancing effect suggested that NF-kappaB and PKC might be involved in baicalin-enhanced TGF-beta1 gene expression. We conclude that SbG up-regulates TGF-beta1 gene expression on RAW264.7 cells through NF-kappaB and PKC pathways and this might provide evidence to explain the therapeutic effect and potential adverse effects on the clinical use of Scutellaria baicalensis Georgi.

Animals↗

PubMed2000

Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs.

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.

Adolescent↗

PubMed1990

Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer.

A method is described for preparing site-specific mutants using a polymerase chain reaction (PCR) based protocol. The protocol requires a single mutant primer, and has been used to introduce mutations into DNA fragments ranging in size from 200 bp to 1569 bp in length in the GM-CSF, beta-actin, human growth hormone and erythropoietin genes. Sequence analysis of PCR derived mutant fragments shows an error rate of less than one bp change per 1500 bp incorporated. Single base pair mutations have been introduced into these genes which create unique restriction sites. We demonstrate that these mutant templates may be used for competitive PCR to quantitate mRNA and DNA. This method thus offers a rapid means for producing competitive templates for use in quantitative PCR.

Actins↗

PubMed2005

Correlation of HER2 gene amplification with immunohistochemistry in breast cancer as determined by a novel monoplex polymerase chain reaction assay.

Amplification of the HER2 oncogene in breast cancer identifies patients who are likely to respond to anti-HER2 mAb therapy. Current clinical practice dictates that all breast cancers first undergo HER2 screening by IHC. Strongly positive (3+ on a 0-to-3+ scale) IHC cases are considered as HER2-amplified tumors and are not evaluated further because of the strong correlation between HER2 gene amplification as measured by FISH and 3+ IHC. This strong correlation has recently been questioned, and some data suggest that over 50% of 3+ IHC HER2 immunostains may not be due to HER2 gene amplification. To help resolve this discrepancy, the authors developed a quantitative PCR assay for HER2. Quantitative PCR was used to determine the amount of HER2 DNA relative to a control gene, IF2 (eukaryotic translation initiation factor, 2p11.1-q11.1). The PCR assay is performed on genomic DNA isolated from paraffin-embedded breast cancer tissue. The PCR assay developed is a monoplex assay in which the HER2 and IF2 PCRs are performed in separate cuvettes. Cases of HER2 FISH amplified breast cancer and HER2 FISH nonamplified breast cancer were chosen for study by monoplex HER2 PCR. HER2 overexpression was evaluated by IHC. Twenty-two cases of HER2-positive and 22 cases of HER2-negative breast cancer, as determined by FISH, were assayed for HER2 by PCR and IHC. Sixteen of the 44 cases were interpreted as 3+ IHC. All 16 showed HER2 amplification by PCR and 15 showed HER2 amplification by FISH. One FISH negative case was found to be HER2 amplified by PCR and showed 3+ IHC stain, suggesting the FISH result in this case was underinterpreted. Two FISH positive cases were found to be negative by PCR and negative in IHC as well, suggesting the FISH result in these cases was overinterpreted. The authors conclude that 3+ IHC membrane staining correctly identifies neoplasms showing HER2 gene amplification. Monoplex HER2 PCR may offer significant advantages over both IHC and FISH for HER2 testing in breast cancer.

Breast Neoplasms↗

PubMed2006

Transcytosis of Human immunodeficiency virus 1 across the placenta is enhanced by treatment with tumour necrosis factor alpha.

The human placenta is relatively resistant to Human immunodeficiency virus 1 (HIV-1), but obstetric complications associated with inflammatory processes, including chorioamnionitis and spontaneous preterm delivery, are associated with increased rates of vertical transmission. It was hypothesized that the pro-inflammatory mediator tumour necrosis factor alpha (TNF-alpha), which promotes HIV-1 transmission across endothelial membranes, increases HIV-1 transmission across the placenta. Flow cytometry and immunostaining studies were performed, which demonstrated that the HIV-1 receptors CD4, CCR5 and CXCR4 were not expressed by villous trophoblast cells. Consequently, primary villous trophoblast cells were not infected with cell-free HIV-1 isolates, as measured by in situ PCR and quantitative PCR, but villous trophoblast cells were infected by HIV-1-infected peripheral blood mononuclear cells (PBMC). HIV-1 from infected PBMC was rapidly transported across confluent transformed trophoblast cell monolayers by transcytosis, and TNF-alpha significantly upregulated transcytosis of HIV-1 across the trophoblast layer without disrupting cell viability or confluency. Inhibitors of TNF-alpha (antibodies against TNF-alpha and TNF-alpha receptors) and an anti-inflammatory drug (tenidap) significantly reduced transcytosis rates. It was concluded that the villous trophoblast is resistant to infection by cell-free HIV-1 but susceptible to transcytosis of HIV-1 from infected PBMC, and inflammatory mediators such as TNF-alpha may play a critical role in promoting maternal-fetal transmission of HIV-1.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Nicotinic acetylcholine receptors on basophils and mast cells.

Anaphylaxis in response to drugs administered during anaesthesia is a rare but potentially catastrophic event. The anaesthetic drugs most commonly associated with anaphylaxis are neuromuscular blocking agents. As these drugs act on the nicotinic acetylcholine receptor of the neuromuscular junction, potentiation of anaphylaxis by a nicotinic receptor on basophils and mast cells is plausible. The aim of this study was to investigate whether nicotinic acetylcholine receptors are present on a human basophil and mast cell lines as their presence may suggest a mechanism of associated anaphylaxis. Nicotinic receptors were demonstrated on a basophil and a mast cell line using an alpha-bungarotoxin-fluorescein conjugate by flow cytometry and by both conventional and confocal microscopic techniques. The identity of this receptor was confirmed by reverse transcriptase PCR and quantitative PCR.

Basophils↗

PubMed2006

Cladophora (Chlorophyta) spp. harbor human bacterial pathogens in nearshore water of Lake Michigan.

Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 x 10(3) cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 x 10(2) Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.

Campylobacter↗

PubMed2004

Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater.

The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.

Animals↗

PubMed1995

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants.

Amino Acid Sequence↗

PubMed2007

IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro.

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.

Animals↗

PubMed2004

Molecular characterization of porcine hyaluronidase genes 1, 2, and 3 clustered on SSC13q21.

Hyaluronidase genes (HYAL) encode hyaluronidase enzymes required for hyaluronan degradation. Both in humans and in mouse, clustered hyaluronidase genes have been identified. Here, the porcine hyaluronidase cluster consisting of genes HYAL1, HYAL2 and HYAL3 was characterized. The porcine cDNA sequences and proteins share homologies to human orthologs of 85 and 81% for HYAL1, 87 and 89% for HYAL2 and 86 and 83% for HYAL3, respectively. The porcine hyaluronidase proteins approximately share a 40% homology with each other. Furthermore, genes FUS1 and FUS2 were found within this cluster, which was assigned to SSC13q21. A total of seven SNPs were detected in the genes (four in HYAL1, two in HYAL2 and one in HYAL3). Three of the four SNPs in HYAL1 led to amino acid exchanges (C622G --> Asp24 to Glu; C633T --> Pro28 to Leu, and G1298T --> Ala250 to Ser). The amino acid replacements induce putative changes in the extended strand at Asp24, in the extended strand and the random coil at Pro28, and finally in the random coil and the alpha helix at Ala250. Frequency estimations for four SNPs located in genes HYAL1 and HYAL3 using animals (n = 295) of nine European and six Chinese pig breeds indicated several significant deviations. For example, there were no significant differences in allele frequencies between pigs representing breeds Hampshire and Jiangquhai at SNP C633T (HYAL1), but between Hampshire respectively Jiangquhai animals and Rongchang pigs. Analysis of the same breeds at SNP C588T (HYAL3) indicates significant differences between Hampshire and Jiangquhai respectively Rongchang, but not between Jiangquhai and Rongchang. The breed Göttingen Minipig displayed significant differences concerning two SNPs with respect to the other European pig breeds tested. For all three hyaluronidase genes, N-glycosylation sites are typical. For HYAL2 the lysosomal character was proven. The catalytic site responsible for HAase activity is conserved in the three enzymes. Expression of hyaluronidases was determined by RT-PCR and quantitative PCR. Broad gene expression was observed in different tissues for the three genes, respectively.

Amino Acid Sequence↗