New Olimycins from a Cold-Seep-Derived Streptomyces olivaceus.

Cold-seeps are areas of the ocean floor in which hydrogen sulfide and methane are released into the open water. The cold-seep microbes are an emerging source of novel bioactive natural products. Four new ansa-ring opened linear ansamycin analogues, named olimycins E-H (1-4) were isolated from the cold-seep-derived Streptomyces olivaceus OUCLQ19-3. The planar and stereochemical structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses together with ECD calculations.

Antibiotic Dixiamycins from a Cold-Seep-Derived Streptomyces olivaceus.

Two new (1 and 2) along with six known (3-8) dixiamycins were isolated from the culture broth of a cold-seep-derived actinomycete, Streptomyces olivaceus OUCLQ19-3. Structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses together with ECD calculations. In the antibacterial test, compounds 1-8 exhibited notable growth inhibitions against a panel of multi-drug-resistant (MDR) strains with MIC values of 0.78-6.25 µg/mL, among which 1, 2, and 5-7 were more potent than the positive control tetracycline.

Expanding the Structural Diversity of Drimentines by Exploring the Promiscuity of Two N-methyltransferases.

Methylation is envisioned as a promising way to rationally improve key pharmacokinetic characteristics of lead compounds. Although diverse tailoring enzymes are found to be clustered with cyclodipeptide synthases (CDPSs) to perform further modification reactions on the diketopiperazine (DKP) rings generating complex DKP-containing compounds, so far, a limited number of methyltransferases (MTs) co-occurring with CDPS have been experimentally characterized. Herein, we deciphered the methylation steps during drimentines (DMTs) biosynthesis with identification and characterization of DmtMT2-1 (from Streptomyces sp. NRRL F-5123) and DmtMT1 (from Streptomyces youssoufiensis OUC6819). DmtMT2-1 catalyzes N4-methylation of both pre-DMTs and DMTs; conversely, DmtMT1 recognizes the DKP rings, functioning before the assembly of the terpene moiety. Notably, both MTs display broad substrate promiscuity. Their combinatorial expression with the dmt1 genes in different Streptomyces strains successfully generated eight unnatural DMT analogs. Our results enriched the MT tool-box, setting the stage for exploring the structural diversity of DKP derivatives for drug development.

Differential roles for ETS, CREB, and EGR binding sites in mediating VEGF receptor 1 expression in vivo.

Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.

Bronchoalveolar lavage fluid analysis provides diagnostic information on pulmonary Langerhans cell histiocytosis.

Histiocytes of Langerhans cell type are recovered from the bronchoalveolar lavage fluid (BALF) of patients with interstitial lung diseases in a nonspecific manner. Langerhans cells (LCs) can be identified through immunostaining for S-100, CD1a, and, more specifically, langerin. To evaluate the diagnostic value of BALF in pulmonary Langerhans cell histiocytosis (PLCH), we performed a retrospective clinicopathological study of 5 patients with biopsy-confirmed PLCH or Hand-Schüller-Christian disease involving the lung. As a control study, we examined BALF cells from 23 patients with various diseases, including sarcoidosis, hypersensitivity pneumonitis, collagen vascular disease, idiopathic pulmonary fibrosis, and adenocarcinoma of the lung. Cytospins obtained from BALF were stained with Giemsa or Papanicoloau and others were immunostained. In general, cytospins showed a monomorphous and dispersed cell population containing mononucleated or binucleated and occasionally multinucleated histiocytes. LCs recovered from BALF were characterized by clear and velvety cytoplasm; oval or kidney-shaped, vesicular nuclei with irregular shapes; nucleoli; and frequent grooves and indentations. Radiography and high-resolution computed tomography showed multiple bilateral nodular or cystic lesions in the middle and upper lung zones. The mean percentage of LCs in 9 lavages from the 5 patients was 8.0%, whereas that from the control group was only 0.3% (maximum, 1.6%). The percentage of cells positive for S-100 or CD1a was comparable to the percentage of Langerhans-like histiocytes stained with Giemsa stain. The present results indicate that the survey of LCs in BALF with the aid of immunocytochemical evaluation and corresponding clinical data could play a critical role in establishing the diagnosis of PLCH, thus providing a less invasive approach than lung biopsy, which carries a risk of complications.

Evaluation of fractional analysis of bronchoalveolar lavage combined with cellular morphological features.

BACKGROUND: The value of bronchoalveolar lavage (BAL) still remains controversial, prompting a need for further improvement. The purpose of this study was to develop and evaluate a sequential analysis of cell content in fractional BAL (FBAL) from the airways and alveolar sacs with incorporation of the cellular morphologic features. METHODS: Initially, 30 ml saline was infused into a subsegmental lobe of the lung and the recovered fluid was assigned as FBAL-I being mainly originated from whole airways. The second and third lavages (FBAL-II and FBAL-III) each were performed using 50 ml saline being from more distal portions of airways and alveolar sacs respectively in the same lobe. Total cell number/ml and percentages of macrophages, lymphocytes, neutrophils, and eosinophils in each fraction together with their morphological alterations and mast cells, basophils and Masson bodies were assessed. RESULTS: In the 12 controls, percentage of neutrophils was high and lymphocytes and macrophages were low in FBAL-I while in FBAL-III, neutrophils decreased to nearly nil and lymphocytes and macrophages were increased. Analysis of FBAL from 76 patients with sarcoidosis and 14 with hypersensitivity pneumonitis (HP) revealed that a predominance of small, round and well-differentiated lymphocytes with relative absence of neutrophils, basophils and Masson bodies correlated best with sarcoidosis. In contrast, neutrophil predominance and presence of lymphocytes having deep nuclear indentations and abundant cytoplasm with a process resembling a "hand-mirror" correlated well with HP. CONCLUSIONS: Evaluation of FBAL together with cellular morphological features especially characteristics of lymphocytes provides valuable information for establishing the diagnosis in interstitial lung diseases.

Induction of pulmonary thromboembolism by neutrophil elastase in collagen-induced arthritis mice and effect of recombinant human soluble thrombomodulin.

We previously reported that during total knee arthroplasty in rheumatoid arthritis (RA) patients, the use of tourniquet might promote local release of neutrophil elastase (NE) from neutrophils, which may contribute to the development of pulmonary thromboembolism (PTE) and tissue injury. The aim of this study was to develop PTE by the use of NE in a mouse model of collagen-induced arthritis (CIA) and investigate the relationship between thrombus and endothelial cells as well as the effect of recombinant human soluble thrombomodulin (rhs-TM) in reducing the risk of PTE. Male DBA/1J mice were injected intracutaneously at several sites with an emulsion containing bovine collagen and later a booster shot to produce CIA mice. Subsequently, NE was injected intravenously 2 times a day for 3 days and after a further 4 days, mice were sacrificed. A group of mice received rhs-TM injections prior to NE injections. We divided the mice into four groups of normal, CIA control, CIA + NE, and CIA + rhs-TM + NE mice and evaluated thrombus formation status. All CIA + NE mice developed PTE. In contrast, no thrombosis was found in normal control, CIA control and CIA + rhs-TM + NE mice. Plasma thrombin level, fibrinogen expression and neutrophil count were increased in CIA + NE mice. Double staining for anticoagulant TM and procoagulant von Willebrand factor (vWF) in pulmonary endothelial cells in normal mice showed a TM-dominant expression while in both CIA control and CIA + NE mice a vWF-dominant expression compatible with coagulant status was observed. Injection of rhs-TM into CIA + NE mice resulted in a phenotypic conversion of endothelial cells from vWF-dominant to TM-dominant expression and a reduction in fibrinogen deposition. These findings demonstrate that by repeated use of NE in CIA mice, it is feasible to produce PTE and to study its pathogenesis and that rhs-TM reduces the risk of PTE. We suggest that in surgical operations of upper and lower extremities in RA patients, the use of a tourniquet should be avoided as it may trigger NE release.

A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo.

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.

Spatial and phenotypic characterization of vascular remodeling in a mouse model of asthma.

Asthma is a chronic inflammatory disease characterized by airway wall remodeling in which vascular remodeling is thought to be a main contributor. Vascular endothelial growth factor (VEGF) is known as a major regulator of angiogenesis and enhancer of vascular permeability. Here, we define the spatial nature of vascular remodeling and the role of VEGF and its receptors (Flt-1 and Flk-1) in the allergic response in mice (A/J) susceptible to the development of allergen-induced airway hyperresponsiveness using morphometric and quantitative approaches. Increased vascularity, vasodilatation, and endothelial cell proliferation were found in the tracheal and bronchial walls in the early and late phases of asthma. Vascular changes were observed not only in small vessels but also in larger vessels. In contrast to normal control, lung tissue from the asthma model showed dual expression for CD31 and von Willebrand factor in the endothelial cells and alpha-smooth muscle actin and desmin in the mural cells of the vessels, suggesting a phenotypic and functional transformation. The mRNA levels of VEGF isoforms, VEGF(164) and VEGF(188), were significantly increased in the tracheal and lung tissue, respectively. In addition, the mRNA level of VEGF receptor Flk-1 was significantly increased in the trachea. These results establish the existence of vascular remodeling in the airways in a mouse model of allergic asthma and support a key role for the expression of unique VEGF isoform genes as mediators of structural changes.

Anti-apoptotic PTD-FNK protein suppresses lipopolysaccharide-induced acute lung injury in rats.

The present study was aimed at clarifying the effects of an anti-apoptotic protein for modulating symptoms in acute lung injury (ALI). From Bcl-x(L), a Bcl-2 family member, we constructed an artificial protein (FNK) and fused it with the protein transduction domain (PTD) of the HIV/Tat protein (PTD-FNK) to facilitate its permeation into cells. ALI was induced by intratracheal infusion of lipopolysaccharide (LPS) into Sprague-Dawley male rats. PTD-FNK was injected into the peritoneal cavity of the animals either 2 h before, or 3 h or 6 h after LPS challenge. All rats were sacrificed 24 h after the last treatment. Cell differential ratios and albumin concentration were estimated in bronchoalveolar lavage fluid. We examined histological change, myeloperoxidase activity, TUNEL assay, caspase-3/caspase-3-like activity and immunohistochemical reaction for caspase 3 (active form). In animals with PTD-FNK treatment, the albumin leakage was significantly attenuated with protection of tissue damage. Also, the apoptosis of alveolar wall cells was reduced by PTD-FNK treatment, while a total cell number and the neutrophil ratio were not changed. Human umbilical vein endothelial cells (HUVEC) and cells of an alveolar epithelial cell line (A549) were exposed to LPS or TNF-alpha with or without PTD-FNK treatment in vitro. Cell survival rates examined by trypan-blue exclusion assay were increased by PTD-FNK treatment in a concentration-dependent manner. Thus, PTD-FNK could play a protective role in ALI by suppressing apoptosis of alveolar epithelial cells and capillary endothelial cells despite of some effect on neutrophil activity.

Angiogenic switching in the alveolar capillaries in primary lung adenocarcinoma and squamous cell carcinoma.

The status of angiogenic switching was examined in alveolar capillaries of primary lung adenocarcinoma (ADC) from 10 patients and primary squamous cell carcinoma (SCC) from 11 patients, using immunostaining for CD31, thrombomodulin, von Willebrand factor (vWF), collagen types IV and VII, and alpha-smooth muscle actin (alpha-SMA). We applied the TdT-mediated dUTP nick-end labeling assay and the reverse transcription-polymerase chain reaction for vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). In bronchioloalveolar and papillary subtypes of ADC, the neoplastic cells, replacing the normal alveolar epithelial cells, had spread over alveolar walls and adhered firmly to alveolar interstitium as shown by the development of type IV collagen. Neoplastic cells of SCC were characterized by local proliferation in alveolar sacs without firm attachment to alveolar walls. Tumor lesions of SCC had often developed necrotic foci of various size. In ADC and SCC, alveolar capillary endothelial cells newly obtained reactivity to vWF. Such segments of endothelial cells lost surface thrombomodulin expression. CD31 was consistently expressed in normal and ADC tissues, but each endothelial cell marker was often attenuated or even lost in SCC, suggesting degeneration or necrosis of the alveolar capillaries. The capillary pericytes and interstitial fibroblasts were often hypertrophic and developed alpha-SMA in the cytoplasm in ADC, but they became atrophic in SCC. In ADC, apoptosis occurred in cells of alveolar capillaries more frequently in the peripheral zone than in the deeper zone of the tumor, whereas the frequency was not consistent in SCC. In microdissected alveolar wall tissues, mRNA expression patterns of VEGF isoforms and VEGFRs were similar in both ADC and SCC. In ADC, de novo angiogenic switching took place in cytoplasm as a unit of cells segments in alveolar capillary endothelium. Suppression of angiogenic switching in SCC implies that factors other than VEGF-VEGFR interaction, such as physical contact and compression of tumor cells, might play a critical role in alveolar capillaries.

A three-kilobase fragment of the human Robo4 promoter directs cell type-specific expression in endothelium.

Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5'-flanking region directs endothelial cell-specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at -2528 and -2941, 2 SP1 consensus motifs at -42 and -153, and an ETS consensus motif at -119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA-protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (approximately 50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination. Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell-specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3-kb upstream promoter of human Robo4 contains information for cell type-specific expression in the intact endothelium.

Critical roles of capillary endothelial cells for alveolar remodeling in nonspecific and usual interstitial pneumonias.

To characterize the relationship between angiogenesis factors and alveolar remodeling in interstitial lung diseases, we examined alveolar capillary endothelial cells in the normal lung (n=5) and in lungs with nonspecific interstitial pneumonia (NSIP) (n=4) or usual interstitial pneumonia (UIP) (n=6) using immunofluorescence staining for thrombmodulin and von Willebrand factor (vWF). With three-dimensional images of alveolar capillaries, the diameter of capillary tubes and their branching frequency per unit length were determined to define rearrangement of the capillary meshwork. Alveolar capillary endothelial cells in normal lungs expressed surface thrombomodulin, and those in lungs with cellular NSIP often showed coexpression of surface thrombmodulin and cytoplasmic vWF. In the alveolar septa of fibrotic NSIP and UIP, capillary endothelial cells demonstrated vWF in only the cytoplasm. Capillary branching frequencies in NSIP and UIP were decreased to 45% and 22%, respectively, of the normal level (p<0.002). Compared with normal lungs, in NSIP and UIP lungs alveolar capillaries containing TUNEL-positive endothelial cells (p<0.05) showed increases of 3.6-fold and 4.3-fold, respectively, indicating a close correlation between endothelial cell apoptosis and remodeling of alveolar capillary frameworks. The analysis of mRNA expression of vascular endothelial growth factors (VEGF) and their receptors (VEGFR1 and VEGFR2) showed a significant decrease in each VEGF isoform and in VEGFR2 mRNA in representative alveolar wall tissues microdissected from the normal, NSIP, and UIP lungs. These results suggest that decreased expression of VEGF mRNA is associated with a reduction in the number of capillary tubes via endothelial cell apoptosis that possibly results in alveolar remodeling in NSIP and UIP. However, whether VEGF is related to fibroblastic activation in the interstitial matrix remains unclear.

Activation of PAR4 induces a distinct actin fiber formation via p38 MAPK in human lung endothelial cells.

Protease-activated receptors (PARs) are multifunctional G protein-coupled receptors. Among the four existing PARs, PAR4 is preferentially expressed in the human lung tissue. However, the function of PAR4 has not been defined in the lung endothelial cells. Because PAR1-mediated cellular effects are deeply related to the morphological changes, we focused on the actin fiber and p38 mitogen-activated protein kinase (MAPK) signaling involved in actin polymerization to elucidate the role of PAR4. RT-PCR and Western blot analyses identified PAR4 expression in human pulmonary artery endothelial cells and in human microvascular endothelial cells from lung. We then examined the changes in actin fibers in endothelial cells treated with PAR4-activating peptide. PAR1-activating peptide was used for comparison. Activation of PAR4 and PAR1 by their corresponding peptides induced actin fiber formation; however, the actin filaments were broadly bundled in PAR4 as compared with the ringlike actin filaments in PAR1 activation. Correspondingly, the magnitude of p38 MAPK phosphorylation was different between cells treated with PAR4 and PAR1, with PAR4-activating peptide showing a significantly higher sensitivity to p38 MAPK inhibitor, SB203580. Taken together, these results demonstrate that activation of PAR4 results in the formation of actin fiber distinct from that by PAR1 activation, suggesting PAR4 may play specific roles in the lung endothelial cells.

Up-regulation of growth factor receptor-bound protein 10 in cervical squamous cell carcinoma.

The Grb10 gene on chromosome 7p11.2-p12 belongs to a family of adapter proteins known to interact with a number of receptor tyrosine kinases, such as EGF, ErbB2/Her2, platelet-derived growth factor (PDGF), IGF-I receptors and vascular endothelial growth factor (VEGF) receptor, KDR (kinase insert domain containing receptor). In addition to receptor tyrosine kinases, Grb10 has also been found to interact with non-receptor tyrosine kinases such as Tec and Bcr-Abl, other cellular signaling molecules such as Raf-1, and the mitogen-activated protein (MAP) kinase, MEK. We demonstrated increased expression of Grb10 mRNA in more than one half of primary cervical squamous cell cancers (12 of 15 cases) when compared to corresponding non-cancerous uterine squamous cell tissues. In addition, immunohistochemical staining demonstrated that the Grb10 protein was prominent in the cytoplasm of cancer cells, whereas it was unreactive in the surrounding normal cervical squamous cells. In addition, its interruption by siRNA exhibited marked cell growth inhibition. These data indicate that amplification and increased expression of the Grb10 gene may play a role in the development of a portion of human cervical squamous cell cancer.

Role of cdk4, p16INK4, and Rb expression in the prognosis of bronchioloalveolar carcinomas.

BACKGROUND: The p16(INK4) protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. OBJECTIVES: Loss of p16(INK4) has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. METHODS: We examined immunohistochemical expression of p16(INK4), cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. RESULTS: All BACs expressed cdk4, while 89 and 82% expressed p16(INK4) and Rb proteins, respectively. None of the clinicopathological factors correlated with p16(INK4), cdk4, or Rb expression separately. A low p16(INK4)/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16(INK4)/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16(INK4)/cdk4 ratio (p = 0.01). CONCLUSIONS: The results suggest a role of the cdk4/p16(INK4) pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16(INK4)/cdk4 ratio may identify tumors that are destined to behave unfavorably.

Demonstration of microvessel networks and endothelial cell phenotypes in the normal murine lung.

The vascular endothelial cells (ECs) express various antigens related to coagulation factors, including factor VIII-related antigen or von Willebrand factor (vWF) in the cytoplasm and thrombomodulin (TM; a thrombin receptor)along the plasma membrane. CD34 (a hematogenic stem cell marker) is also expressed along the surface membrane of the ECs. Using these EC markers and fluorescein-isothiocyanate-labeled dextran (FITC-dextran)(Sigma Co., St. Louis, MO), we attempted to demonstrate the complex network of microvessels and their EC phenotypes in tracheo-bronchial trees and lung parenchyma of the normal adult ICR male mice. Under anesthesia, saline with heparin was infused slowly through left ventricle to drain off the blood. Following brief fixation with 4% buffered paraformaldehyde solution (PFA) through the same route, one group of animals received, 1) FITC-dextran injection via left ventricle, and the large airways and lungs were further fixed in PFA, or 2) The airways and lungs of the other group were rapidly frozen, and the thin sections were stained with two antibodies of vWF and Alexa Fluor 594-labeled CD34. The vWF antibody was later labeled by FITC. The microvessels of airways and lungs were observed by a laser scanning confocal microscope (TC-SP, Leica, Heidelberg, Germany). The phenotypic characteristics of microvessel ECs appeared mostly identical with those described previously in the human lung, although CD34 was applied instead of TM in the present study. The topographical heterogeneity of immunohistochemical properties of ECs would suggest functional differences at different sites of the lung, that would provide a novel insight for understanding the pathogenesis of human lung diseases.

Amplification, up-regulation and over-expression of C3G (CRK SH3 domain-binding guanine nucleotide-releasing factor) in non-small cell lung cancers.

The Ras-CRK-Rap1 cellular signal-transduction system is regulated by guanine nucleotide exchange factors (GEFs). Transcription of C3G on chromosome 9q34 and a key member of the GEF gene family is activated by the CRK-adaptor protein; the C3G product is a CRK SH3 domain-binding guanine nucleotide-releasing factor. We document here the amplification of C3G in five of 18 primary non-small cell lung cancers examined and its increased expression in 18 of 28 tumors in comparison to corresponding non-cancerous lung tissues. Immunohistochemical staining revealed prominent C3G protein in the cytoplasm of cancer cells, associated with faint staining at the nucleolar membrane, but C3G was not detectable in normal bronchial mucoepithelial cells or in broncholoalveolar cells of the bronchial/bronchiolar ducts or alveoli. These data indicate that amplification and increased expression of the C3G gene may play some role in human lung carcinogenesis through derangement of the CRK-Rap1 signaling pathway.

Hypermethylation-associated inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human pancreatic cancers.

BACKGROUND: SOCS-1, a JAK-binding protein (SSI-1/SOCS-1/JAB), regulates the JAK/STAT signal transduction pathway that relays signals from various cytokines in the extracellular matrix into the cell. Inactivation of the SOCS-1 gene by methylation has been previously described in hepatocellular carcinomas and multiple myeloma. The purpose of the present work was to analyze the expression of the SOCS-1 gene and identify inactivation of this gene by methylation in pancreatic cancers. METHODS: 20 samples were analyzed. We identified the expression of SOCS-1 gene using RT-PCR and the mechanism of inactivation in this gene by methylation assay. RESULTS: We documented marked suppression of SOCS-1 mRNA and reduction of SOCS-1 protein in 7 of 14 primary pancreatic cancers examined; moreover, CpG-rich regions upstream of the SOCS-1 gene were hypermethylated in 8 of the 14 tumors. CONCLUSIONS: The results suggested that this gene is silenced in a substantial portion of pancreatic cancers through mechanisms that cause methylation in the promoter region.

Human airway trypsin-like protease induces PAR-2-mediated IL-8 release in psoriasis vulgaris.

Human airway trypsin-like protease (HAT), a novel serine protease in the airways, enhances cell growth and IL-8 production. The expression and role of HAT in the skin however, is unknown. Immunofluorescence staining and reverse transcription (RT)-PCR were done to know HAT production in normal and psoriatic tissues and keratinocyte cell lines. Cell growth and/or IL-8 release analyses were made by bromo-deoxyuridine (BrdU) uptake and ELISA. Psoriatic epidermis showed more extensive immunofluorescence expression of HAT, and less extensive expression of protease-activated receptor (PAR)-2. RT-PCR demonstrated a higher HAT and a lesser PAR-2 mRNA expressions in psoriatic epidermis. Normal keratinocyte and epidermoid carcinoma cell lines expressed HAT and PAR-2 mRNA, and immortalized keratinocytes (HaCaT) expressed PAR-2, but not HAT mRNA. PAR-2 was detected along the keratinocyte surface in culture and became invisible upon HAT stimulation, suggesting a process of its internalization. HAT or PAR-2 activating peptide did not enhance BrdU uptake, but induced an IL-8 release. Treatment with HAT and IL-1beta synergistically increased the effect of IL-8 release. Inhibition of PAR-2 resulted in a decreased HAT-induced IL-8 release. Thus, HAT might promote PAR-2-mediated IL-8 production to accumulate inflammatory cells in the epidermal layer of psoriasis.