High Levels In Lung Research Articles

Interactions between EGFR and EphA2 promote tumorigenesis through the action of Ephexin1

The cell signaling factors EGFR, EphA2, and Ephexin1 are associated with lung and colorectal cancer and play an important role in tumorigenesis. Although the respective functional roles of EGFR and EphA2 are well known, interactions between these proteins and a functional role for the complex is not understood. Here, we showed that Ephexin1, EphA2, and EGFR are each expressed at higher levels in lung and colorectal cancer patient tissues, and binding of EGFR to EphA2 was associated with both increased tumor grade and metastatic cases in both cancer types. Treatment with Epidermal Growth Factor (EGF) induced binding of the RR domain of EGFR to the kinase domain of EphA2, and this binding was promoted by Ephexin1. Additionally, the AKT-mediated phosphorylation of EphA2 (at Ser897) promoted interactions with EGFR, pointing to the importance of this pathway. Two mutations in EGFR, L858R and T790M, that are frequently observed in lung cancer patients, promoted binding to EphA2, and this binding was dependent on Ephexin1. Our results indicate that the formation of a complex between EGFR, EphA2, and Ephexin1 plays an important role in lung and colorectal cancers, and that inhibition of this complex may be an effective target for cancer therapy.

Resveratrol inhibits cell apoptosis by suppressing long noncoding RNA (lncRNA) XLOC_014869 during lipopolysaccharide-induced acute lung injury in rats.

BackgroundAcute lung injury (ALI) is a common clinical complication with a high mortality rate. Resveratrol (Res) has been shown to protect against ALI, but the role of long noncoding RNAs (lncRNAs) in this process is still unclear.MethodsMale rats (n=20) aged 7–8 weeks were randomly divided into four groups: control, lipopolysaccharide (LPS), LPS + Res, and LPS + dexamethasone (Dexa). Intragastric administration of Res (0.5 mg/kg) or Dexa (1.5 mg/kg) was performed 1 h before intraperitoneal injection of LPS (5 mg/kg). Lung tissue, serum, and bronchoalveolar lavage fluid were sampled 6 h after LPS treatment for inflammatory factor detection, pathological detection, lncRNA sequencing and bioinformatical analysis, and TdT-mediated dUTP Nick-End Labeling. Quantitative real time polymerase chain reaction and western blotting were used to verify the sequencing results. LPS, Res, and RNA interference were used in rat alveolar epithelial cells experiments to confirm the protective of Res/lncRNA against ALI.ResultsRes pretreatment inhibited lung injury and the increase of inflammatory cytokines induced by LPS. The differentially expressed lncRNAs and mRNAs (P<0.05 and |fold change| >2) were mainly involved in the signaling pathway of immunity, infection, signaling molecules and interactions. Among the lncRNAs and mRNAs, 26 mRNAs and 23 lncRNAs had high levels in lungs treated with LPS but decreased with Res, and 17 mRNAs and 27 lncRNAs were at lower levels in lungs treated with LPS but increased with Res. lncRNA and adjacent mRNA analysis showed that lncRNAs XLOC_014869 and the adjacent gene Fos, and the possible downstream genes Jun and Faslg were increased by LPS, but these changes were attenuated by Res. Pretreatment with Res reduced LPS-induced lung tissue apoptosis. Similarly, Res treatment and knockdown of lncRNA XLOC_014869 reduced LPS-induced apoptosis and the levels of Fos, c-Jun, and Fas-L.ConclusionsRes can inhibit the increase of lncRNAs XLOC_014869 caused by LPS stimulation and inhibit lung cell apoptosis. These effects may be due to lncRNA XLOC_014869 mediation of the pro-apoptotic factors (Fos, c-Jun, and Fas-L).

Key genes with prognostic values in suppression of osteosarcoma metastasis using comprehensive analysis

BackgroundOsteosarcoma is a primary malignant tumor originating from mesenchymal tissue, with a poor distant metastasis prognosis. The molecular mechanisms of osteosarcoma metastasis are extremely complicated.MethodsA public data series (GSE21257) was used to identify differentially expressed genes (DEGs) in osteosarcoma patients that did, or did not, develop metastases. Functional enrichment analysis, a protein-protein interaction network, and survival analysis of DEGs were performed. DEGs with a prognostic value were considered as candidate genes and their functional predictions, different expression in normal and malignant tissues, and immune infiltration were analyzed.ResultsThe DEGs were mainly enriched in the immune response. Three candidate genes (ALOX5AP, CD74, and FCGR2A) were found, all of which were expressed at higher levels in lungs and lymph nodes than in matched cancer tissues and were probably expressed in the microenvironment.ConclusionsCandidate genes can help us understand the molecular mechanisms underlying osteosarcoma metastasis and provide targets for future research.

Dynamic and unpredictable changes in mutant allele fractions of BRAF and NRAS during visceral progression of cutaneous malignant melanoma

BackgroundData indicate that primary cutaneous melanomas are characterized by clonal heterogeneity associated with oncogenic drivers. Less data are available on the clonal changes occurring during melanoma progression. We therefore wished to analyse these changes in skin melanomas in common sites of visceral metastases as compared to the primary tumor.MethodsAn autopsy cohort of 50 patients with BRAF- and NRAS-mutant cutaneous metastatic melanomas including 139 visceral metastases was analysed for mutant allele fractions (MAF), determined by pyrosequencing and corrected for tumor/normal ratio. MAF levels were also classified as high (> 40%), medium (15–40%) or low (< 15%).ResultsContrary to NRAS mutant cases, in BRAF-mutant melanomas MAFs were found to be significantly increased in visceral metastases compared to the primary due to the significantly higher levels in lung-, adrenal gland-, intestinal- and kidney metastases. The incidence of the three MAF variants in BRAF-mutant primaries was similar, whereas the high MAF cases were found to be increased in metastases. On the other hand, medium MAF levels were more common in case of NRAS-mutant tumors. Only 31.3% of BRAF mutant- and 50% of NRAS mutant cases maintained the MAF profile of the primary in metastasis. In the majority of multiple metastatic tumors, (BRAF:71.8%, NRAS:75%) metastases were relatively homogeneous regarding MAF. However, in 6/32(18.7%) of BRAF mutant cases low MAF primaries switched to high MAF in metastases. In heterogeneous BRAF mutant metastatic cases low to high or high to low MAF conversions occurred in a further 4/32(12.5%) cases in individual metastases as compared to the primary tumors. At lower frequency, in NRAS mutant tumor such changes also observed (2/12,16.7%).ConclusionWe provided evidence for the selection of BRAF-mutant melanoma cells during metastatic progression to the lung, intestine, adrenal gland and kidney. Our findings suggest that in visceral metastases of malignant melanoma BRAF- or NRAS-MAFs are rather heterogeneous and cannot be predicted from data of the primary tumor. These data may have clinical significance when using targeted therapies.

Quantification of unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples.

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation.QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types.Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits.In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings.

Screening of the different TNM stage-associated genes in lung adenocarcinoma by genomewide gene expression profile analysis in the Chinese population

Objective To screen for the differentially expressed genes associated with various TNM stages in lung adenocarcinoma of Chinese by comparing the expression profiles in tumor samples of different TNM stages. Methods This study was a case-case study.Lung adenocarcinoma specimens were collected from total of 240 patients receiving thoracic surgery in our hospital from May of 2008 to October of 2011.There were 90 samples (30 each for stageⅠ,ⅡandⅢA) for the gene array,and 150 samples (50 each for stageⅠ,ⅡandⅢA) for the further validation by qRT-PCR.A 35K Oligo gene array,covering about 25,100 genes,was used to screen the differentially expressed genes among 90 tumor samples of lung adenocarcinoma at various TNM stages.To verify the data from gene array,three genes of Zimp7,GINS2 and NAG-1 were confirmed by real-time RT-PCRin a different set of samples from the gene array.The expression levels of tested genes in different TNM stages were compared using ANOVA statistic method. Results Five hundreds and ninty-two differentially expressed genes in lung adenocarcinomas compared to the surrounding normal lung tissues were obtained,among which 264 genes were upregulated and 328 downregulated.From the 592 candidates,10 differentially expressed genes were identified among different TNM stages (StageⅠ,ⅡandⅢA),of which Zimp7,GINS2 and NAG-1 genes were first reported to be present at a high level in lung adenocarcinoma.The results of qRT-PCR for the three genes were consistent with those from the gene array. Conclusion Ten candidate genes associated with different TNM stages in lung adenocarcinoma were identified in the Chinese population,which would provides new insight into the molecular basis underlying the development of lung adenocarcinoma and may offer new targets for the diagnosis,therapy and prognosis prediction of lung adenocarcinoma.(Chin J Lab Med,2013,36:1104-1108) Key words: Lung neoplasms; Adenocarcinoma; Neoplasm staging; Gene expression profiling; Oligonucleotide array sequence analysis

MicroRNA-126 expression is decreased in cultured primary chicken hepatocytes and targets the sprouty-related EVH1 domain containing 1 mRNA

The microRNA-126 (miR-126) is a miRNA expressed in highly vascularized tissues, and it is believed to play a role in angiogenesis by repressing sprouty-related EVH1 domain containing 1 (Spred1). In the current study, we determined the expression pattern of chicken miR-126 (gga-miR-126) and predicted and validated its target genes. The quantitative reverse-transcription (qRT) PCR analysis showed that miR-126 was expressed in various chicken tissues with the highest level in lung. In liver, the expression level of miR-126 increased from 0 to 7 wk of age. The expression of miR-126 in primary chicken hepatocytes decreased with culturing. A miR-126 binding site was predicted in the 3' UTR (untranslated region) of chicken Spred1. Dual-luciferase reporter assays indicated that miR-126 could bind to the predicted site to repress the expression of Spred1. These data validate Spred1 as a target gene of chicken miR-126. These results will help further understand the function and regulation of miR-126 and Spred1 in chickens.

Study on the age-dependent tissue expression of FUT1 gene in porcine and its relationship to E. coli F18 receptor

Escherichia coli (E. coli) that produces adhesin F18 is the main pathogen responsible for porcine post-weaning diarrhea and edema disease. The receptor for E. coli F18 has not been described in pigs, however the alpha (1,2)-fucosyltransferase (FUT1) gene on chromosome 6 has been proposed as a candidate. The objective of this study, therefore, was to investigate the relationship between FUT1 gene expression and E. coli F18 receptor in Sutai pigs of different ages (8-, 18-, 30- and 35-day-old). FUT1 gene expression was detected in 11 pig tissues with the highest level in lung, and expressed consistently at the four time points. In most tissues, FUT1 gene expression levels decreased from days 8 to 18, then continually increased on days 30 and 35, with expression around weaning time higher than that on day 8. Gene ontology and pathway analysis showed that FUT1 was involved in 32 biological processes, mainly those integral to the membrane, or involved in glycosylation, as well as regulation of binding, interestingly participating in three pathways related to glycosphingolipid biosynthesis. From this analysis and the high linkage disequilibrium between the FUT1 gene and the E. coli F18 receptor locus, we can speculate that higher expression of the FUT1 gene in small intestine is beneficial to the formation of receptors to the E. coli F18 strain and is related to the sensitivity to the pathogen.

Dissecting the Roles of the Extracellular Regions in Apelin Peptide-Receptor Binding

The apelin receptor is a class A G-protein coupled receptor with homology to the angiotensin II receptor and the CXCR4 and CCR5 chemokine receptors. It is expressed at high levels in lung, heart, adipose tissue, kidney, spleen and in some areas of the brain. The apelin receptor gene is also upregulated and overexpressed in one-third of human tumours. The receptor is assumed to consist of a transmembrane region with 7 helical segments, an intracellular and an extracellular domain. The extracellular domain, made up of the N-terminal tail and three extracellular loops (EL1, 2 and 3), exhibits low sequence homology to other GPCRs. Several somatic mutations in loops have been linked to diseases, pointing out the importance of loops in the activation and/or binding of GPCRs to their cognate ligands. We carried out a physicochemical characterization of the N-terminus and extracellular loops of the apelin receptor in lipid mimetic environments. The N-terminus (attached to the first transmembrane domain) and EL2 were produced as recombinant proteins while the EL peptides and a fluorescently tagged apelin were synthesized by solid-phase peptide synthesis. All species were purified by high-performance liquid chromatography. Characterization both of the peptides in isolation and of the binding between apelin and extracellular loop peptides was carried out using circular dichroism spectroscopy, fluorescence resonance energy transfer and nuclear magnetic resonance spectroscopy. These results provide insight into understanding the role of the each of the extracellular regions of this receptor at the molecular level and constitute important information the apelin receptor for the development of therapeutics targeting this system.

Analysis of polymorphism in the porcine TLR4 gene and its expression related to Escherichia coliF18 infection

The genetic variation in exon 1 of the TLR4 gene was detected among a total of 893 animals, including Asian wild boars, 3 imported commercial and 10 Chinese indigenous pig breeds. The expression of TLR4 was assayed by RT-PCR and different expression between resistant and sensitive resource populations to ETEC F18 was analysed to discuss the role that the TLR4 gene plays in resistance. In this study, new alleles were detected in exon 1 of the TLR4 gene. These polymorphisms are significantly different between Chinese indigenous breeds and imported breeds. Based on the published TLR4 gene sequence (AB232527) in GenBank, a 93G &amp;gt; C mutation was found in 5&amp;rsquo;UTR and only a 194G &amp;gt; A synonymous mutation was found in the coding sequence of exon 1. In addition, TLR4 gene was broadly expressed in 11 tissues with the highest level in lung. The expression was relatively high in the lymph nodes, kidney and spleen. Generally, the expression of TLR4 gene in sensitive individuals was higher than that in resistant individuals. The results indicated that the downregulation of the mRNA expression of TLR4 gene had reduced the transmembrane signal transduction of LPS and then led to the responsive ability of the host to ETEC F18 in piglets.

Tissue distribution of 35S-labelled perfluorooctane sulfonate (PFOS) in C57Bl/6 mice following late gestational exposure

Exposure of rodents in utero to perfluorooctane sulfonate (PFOS) impairs perinatal development and survival. Following intravenous or gavage exposure of C57Bl/6 mouse dams on gestational day (GD) 16 to 35S-PFOS (12.5 mg/kg), we determined the distribution in dams, fetuses (GD18 and GD20) and pups (postnatal day 1, PND1) employing whole-body autoradiography and liquid scintillation counting. In dams, levels were highest in liver and lungs. After placental transfer, 35S-PFOS was present on GD18 at 2–3 times higher levels in lungs, liver and kidneys than in maternal blood. In PND1 pups, levels in lungs were significantly higher than in GD18 fetuses. A heterogeneous distribution of 35S-PFOS was observed in brains of fetuses and pups, with levels higher than in maternal brain. This first demonstration of substantial localization of PFOS to both perinatal and adult lungs is consistent with evidence describing the lung as a target for the toxicity of PFOS at these ages.

Improved oral bioavailability in rats of SR13668, a novel anti-cancer agent.

SR13668, a bis-indole with potent activity in vitro and in vivo against various cancers and promising cancer chemopreventive activity, was found to have very low oral bioavailability, <1%, in rats during pilot pharmacokinetic studies. The objective of these studies was to better understand the source of low oral exposure and to develop a formulation that could be used in preclinical development studies. An automated screening system for determining solubility in lipid-based vehicles, singly and in combination, was used to identify formulations that might enhance absorption by improving solubility of SR13668, and these results were confirmed in vivo using Sprague-Dawley rats. Pharmacokinetics of SR13668 was then determined in male and female Sprague-Dawley rats administered 1 mg/kg iv, 1, 10, and 30 mg/kg po formulated in PEG400:Labrasol (1:1 v/v). Blood was collected at time points through 24 h and the concentration of SR13668 determined using HPLC with UV and fluorescence detection. SR13668 was found to be resistant to plasma esterases in vitro and relatively stable to rat and human liver microsomal metabolism. SR13668 concentrates in tissues as indicated by significantly higher levels in lung compared to blood, blood concentrations ~2.5-fold higher than plasma levels, and apparent volume of distribution (V) of ~5 l/kg. A marked sex difference was observed in exposure to SR13668 with area under the curve (AUC) significantly higher and clearance (CL) lower for female compared to male rats, after both iv and oral administration. The oral bioavailability (F) of SR13668 was 25.4 ± 3.8 and 27.7 ± 3.9% (30 mg/kg), for males and females, respectively. A putative metabolite (M1), molecular weight of 445 in the negative ion mode (i.e., SR13668 + 16), was identified in blood samples from both the iv and po routes, as well as in vitro microsomal samples. In summary, while SR13668 does undergo metabolism, probably by the liver, the oral bioavailability of SR13668 in rats was dramatically improved by the use of formulation that contained permeation enhancers and promoted better solubilization of the drug.

DNA adduct kinetics in reproductive tissues of DNA repair proficient and deficient male mice after oral exposure to benzo(a)pyrene

Benzo(a)pyrene (B[a]P) can induce somatic mutations, whereas its potential to induce germ cell mutations is unclear. There is circumstantial evidence that paternal exposure to B[a]P can result in germ cell mutations. Since DNA adducts are thought to be a prerequisite for B[a]P induced mutations, we studied DNA adduct kinetics by (32)P-postlabeling in sperm, testes and lung tissues of male mice after a single exposure to B[a]P (13 mg/kg bw, by gavage). To investigate DNA adduct formation at different stages of spermatogenesis, mice were sacrificed at Day 1, 4, 7, 10, 14, 21, 32, and 42 after exposure. In addition, DNA repair deficient (Xpc(-/-)) mice were used to study the contribution of nucleotide excision repair in DNA damage removal. DNA adducts were detectable with highest levels in lung followed by sperm and testis. Maximum adduct levels in the lung and testis were observed at Day 1 after exposure, while adduct levels in sperm reached maximum levels at approximately 1 week after exposure. Lung tissue and testis of Xpc(-/-) mice contained significantly higher DNA adduct levels compared to wild type (Wt) mice over the entire 42 day observation period (P < 0.05). Differences in adduct half-life between Xpc(-/-) and Wt mice were only observed in testis. In sperm, DNA adduct levels were significantly higher in Xpc(-/-) mice than in Wt mice only at Day 42 after exposure (P = 0.01). These results indicate that spermatogonia and testes are susceptible for the induction of DNA damage and rely on nucleotide excision repair for maintaining their genetic integrity.

Extracellular Superoxide Dismutase Inhibits Inflammation by Preventing Oxidative Fragmentation of Hyaluronan

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.

Molecular characterization of a novel lysosomal enzyme degrading the anti-inflammatory lipid mediator N-acylethanolamine

N-Acylethanolamines (NAEs) represent a class of endogenous bioactive lipids generated from glycerophospholipids, and they include N-palmitoylethanolamine (an anti-inflammatory substance) and N-arachidonoylethanolamine (anandamide, an endogenous ligand of cannabinoid receptor). It is generally accepted that NAEs are hydrolyzed to free fatty acids and ethanolamine by the catalysis of fatty acid amide hydrolase (FAAH) acting principally at alkaline and neutral pH. Several years ago, our laboratory found another enzyme catalyzing the same reaction only at acidic pH. However, the molecular cloning and detailed analysis of this enzyme, termed “N-acylethanolamine-hydrolyzing acid amidase (NAAA)”, have not yet been performed. In the present study, we purified the enzyme from rat lung, and cloned its cDNA from human, mouse, and rat. The primary structures revealed that NAAA had no homology to FAAH, but revealed 33-35% amino acid identity to lysosomal acid ceramidase, which hydrolyzes ceramide to free fatty acids and sphingosine at acidic pH. Recombinant human NAAA hydrolyzed various NAEs, and exhibited the highest activity toward N-palmitoylethanolamine. In addition, a low ceramide-hydrolyzing activity was detected with NAAA. We also observed a lysosome-like distribution of NAAA-green fluorescence protein fusion protein, as expressed in HEK293 cells. The organ distribution of mRNA and enzyme activity in rats revealed its wide distribution with the highest level in lung. Interestingly, macrophages expressed NAAA abundantly, suggesting a role of this enzyme in inflammation. By using an NAAA-selective inhibitor which we developed, we showed that NAAA and FAAH cooperatively degraded NAEs in macrophages. Taken together, our results indicated that NAAA is a novel lysosomal enzyme having structural and functional similarity to acid ceramidase, and functions as a second NAE hydrolase in mammalian tissues.

Protein Phosphatase 6 Subunit with Conserved Sit4-associated Protein Domain Targets IκBϵ

Protein Phosphatase 6 Subunit with Conserved Sit4-associated Protein Domain Targets IκBϵ

Immunomodulation of experimental pulmonary fibrosis by intravenous immunoglobulin (IVIG)

Objectives: To assess the immunomodulatory effect of intravenous immunoglobulin (IVIG) using an experimental model of bleomycin-induced pulmonary fibrosis.Methods: Pulmonary fibrosis was induced in C57BL/6 mice by direct intratracheal injection of bleomycin. Mice were treated with IVIG 1 week prior to (prevention protocol), or 10 days following bleomycin injection, when the disease was in progress. The controls used in the study included mice given phosphate buffered saline (PBS) and mice subjected to a commercial individual-IgG. Collagen-I deposits in the affected lungs were detected by Sirius red staining of paraffin embedded lung sections. The collagen-I content was measured by employing the hydroxyproline assay.Results: Prevention of bleomycin-induced pulmonary fibrosis by IVIG has been demonstrated by reduced expression of collagen-I protein in the affected lungs. The hydroxyproline levels in the lungs of the IVIG-treated mice were 214.33 ± 13.56 μg/1 g tissue, compared to the higher levels in lungs of IgG treated mice (342.44 ± 35.60 μg/1 g tissue) or untreated controls 328.00 ± 45.55 μg/1 g tissue, (p < 0.0001). Effective treatment of bleomycin-induced pulmonary fibrosis by IVIG has been demonstrated by the reduced expression of collagen-I protein in the affected lungs, detected by sirius red histological staining. The hydroxyproline levels in the lungs of the IVIG-treated mice were 261.00 ± 18.81 μg/1 g tissue, in comparison to the higher levels in the lungs of the IgG treated mice (342.43 ± 32.89 μg/1 g tissue) and of untreated controls (344.33 ± 49.85 μg/1 g tissue), (p < 0.001).Conclusions: Based on these preliminary studies, we conclude that IVIG may have a beneficial effect in the down regulation of collagen-I levels in the lungs of mice with bleomycin-induced pulmonary fibrosis.

Differential allele expression of host defense genes, pulmonary surfactant protein-A and Osteopontin, in rat

Differential allele-specific expression has been observed in several genes involved in immunity. SP-A and OPN play a role in innate host defense. To determine whether SP-A and OPN are subject to differential allele-specific regulation, we investigated their gene or allele-specific expression in various tissues. The results showed: (1) Tissue-specific expression with high levels in lung ( SP-A) and kidney ( OPN). (2) Differences in allele-specific expression among individuals and tissues. SP-A showed an exclusively balanced biallelic expression (BB) in lung, but both BB and imbalanced biallelic (IB) expression in colon. Allele expression of OPN was more heterogeneous, e.g. in colon BB (22%), IB (64%), and monoallelic expression (MO) (14%). (3) Differential allele-specific expression was observed in all tissues studied ( OPN) or in all extrapulmonary tissues ( SP-A). (4) Family studies indicated that inheritable factor(s) may be involved in the regulation of allele-specific expression. (5) Analysis of co-expression of gene-specific alleles from double heterozygous rats revealed lack of coordinate allele expression among SP-A, SP-D, and OPN. We conclude that allele-specific expression occurs among genes of innate host defense. This may yet provide another level of regulatory complexity for molecules involved in the first line of defense.

Expression of the novel gene osteoactivin in rat and human liver tissue and isolated liver mononuclear phagocytes

The transmembrane glycoprotein osteoactivin is found in osteoblasts, dendritic cells and tumor cells and is supposed to plays a role in osteoblast maturation and cell adhesion. Methods: Total RNA was extracted from rat organs, and from cultured liver cell populations (hepatocytes, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells, myofibroblasts), and analyzed by Northern blot hybridization. Moreover, inflammatory mononuclear phagocytes (MNP) were isolated from carbon tetrachloride (CCl4)-treated rats. Cultured Kupffer cells and MNP were treated with lipopolysaccharide (LPS) or dexamethasone (DEX). Total RNA was extracted from human liver (acute liver failure, liver cirrhosis from different origin). Results: Organ expression of osteoactivin showed high levels in lungs and spleen, less in kidney and colon. Trace osteoactivin expression was found in normal rat liver. High levels of osteoactivin mRNA were detected in normal Kupffer cells. LPS and DEX treatment of Kupffer cells led to a decrease of osteoactivin mRNA. In CCl4-treated animals osteoactivin mRNA was abundantly detected in total liver RNA and in isolated inflammatory MNP of the liver. DEX treatment of animals and of cultured inflammatory MNP decreased osteoactivin mRNA expression. Human liver total RNA extracts contained elevated osteoactivin mRNA levels in fulminant hepatitis. Conclusions: This is the first description of abundant osteoactivin expression in Kupffer cells as well as in liver inflammatory MNP. The inhibitory effect of glucocorticoids suggests a role of osteoactivin in inflammatory processes.

Cloning and characterization of the full-length mouse Ptk7 cDNA encoding a defective receptor protein tyrosine kinase.

The 3.8-kb full-length mouse Ptk7 cDNA encoding a defective receptor protein tyrosine kinase was cloned by reverse transcription-PCR of mouse liver mRNA. The mouse PTK7 polypeptide showed 92.6% identity to human PTK7. The mouse Ptk7 gene consists of 20 exons and has exactly the same exon structure as the human PTK7 gene. Mouse PTK7 was shown to be phosphorylated neither by itself nor by other protein tyrosine kinases. In addition, its expression did not affect the phospho-tyrosine level of cellular proteins in COS-1 cells. The mouse Ptk7 mRNA was expressed at high levels in lung and un-pregnant uterus among adult tissues, and in the tail, limbs, somites, gut, and craniofacial regions among embryonic tissues. These data suggest that mouse PTK7, an orthologue of human PTK7, plays multiple roles in embryonic development.