Lysophosphatidylserine receptor P2Y10: A G protein-coupled receptor that mediates eosinophil degranulation.

BACKGROUND: P2Y10, along with GPR34 and GPR174, is a G protein-coupled receptor that is activated by an endogenous lipid mediator lysophosphatidylserine (LysoPS). Its expression pattern and its function are completely unknown. We have previously shown that P2Y10 is one of the highly up-regulated genes at the late differentiation stage during in vitro eosinophilopoiesis. OBJECTIVE: We explored the expression and functions of P2Y10 in human cord blood (CB)-derived and peripheral blood (PB) eosinophils. METHODS: Real-time PCR, FACS, Western blot, ELISA, and chemotaxis assays were performed to determine the expression and function of P2Y10. RESULTS: As CB cells differentiated towards eosinophils, P2Y10 mRNA and protein were abundantly expressed. P2Y10 was the most highly expressed in the granulocytes from PB, to a lesser extent in monocytes, and least in lymphocytes. Further fractionation of granulocytes revealed that eosinophils express P2Y10 much more strongly than do neutrophils. PB eosinophils solely expressed P2Y10 among the three LysoPS receptors, while PB neutrophils expressed the three at comparable levels. LysoPS activated both CB and PB eosinophils to induce a robust ERK phosphorylation. Importantly, LysoPS was capable of triggering degranulation of ECP in PB eosinophils. This response was significantly reduced by pharmacological inhibitors of TNF-alpha-converting enzyme (TACE), epidermal growth factor receptor (EGFR), and ERK1/2, which were known to be required in P2Y10-mediated signalling pathways. However, LysoPS had no effect on chemotaxis, differentiation, or eosinophil survival. CONCLUSIONS AND CLINICAL RELEVANCE: LysoPS provokes eosinophil degranulation through P2Y10. Therefore, P2Y10 is a potential therapeutic target to control eosinophil-associated diseases.

Emergence of Enterococcus species in the infectious microorganisms cultured from patients with endophthalmitis in South Korea.

PURPOSE: To investigate the microorganisms in culture-proven endophthalmitis and their susceptibilities to antimicrobial agents commonly used in South Korea. METHODS: Medical records of consecutive patients with culture-proven endophthalmitis at eight institutions between 1 January 2004 and 31 July 31 2010 were reviewed. Four categories of endophthalmitis were studied: postoperative, posttraumatic, endogenous, and unspecified. Outcome measures were culture-proven infectious organisms, antimicrobial susceptibilities, and final visual acuity in the patients. RESULTS: A total of 93 microorganisms were identified from 103 patients during the study period. The positive culture rate was 59.2 % (103/174). The most common organisms identified were Enterococcus faecalis (in 20.8 % of patients, 20/96), Staphylococcus epidermidis (18.8 %, 18/96), other coagulase-negative staphylococci (10.4 %, 10/96), Pseudomonas aeruginosa (6.3 %, 6/96), and Klebsiella pneumoniae (6.3 %, 6/96). Two cases of Enterococcus faecium (2.1 %) were recognized. Overall, 70 of 96 (73.0 %) isolates were Gram-positive bacteria, 22 (23.0 %) were Gram-negative bacteria, and 4 (4.2 %) were fungi. The most common organisms resulting in reduced light perception were E. faecalis and K. pneumoniae. CONCLUSIONS: The emergence of E. faecalis in endophthalmitis is mainly caused by the high incidence of E. faecalis in postoperative endophthalmitis. This increase also impacts the final visual acuity of the patients.

Genetic effect of single-nucleotide polymorphisms in the PPARGC1B gene on airway hyperreactivity in asthmatic patients.

BACKGROUND: Peroxisome proliferator-activated receptor gamma coactivator 1 beta (PPARGC1B) is a co-activator for intracellular receptors such as the estrogen receptor, PPAR, and glucocorticoid receptor, which are involved in asthma development. OBJECTIVES: Genetic association of single-nucleotide polymorphisms (SNPs) in the PPARGC1B gene with the risk of asthma and airway hyperreactivity (AHR) was investigated, as well as the functional effects of these SNPs on PPARGC1B gene and protein expression. METHODS: Direct sequencing of DNA from 24 Korean was performed to identify PPARGC1B SNPs. Genotyping was done in 264 controls and 949 asthmatics using single-base extension methods. PPARGC1B mRNA levels were measured using real-time PCR methodology. Luciferase and electrophoretic mobility shift assays (EMSA) were performed to functionally analyse PPARGC1B SNPs on promoter. RESULTS: Eighteen SNPs and one insertion/deletion polymorphism were identified, and seven SNPs were genotyped. No significant difference existed in the distribution of SNPs and haplotypes between the asthmatics and controls. However, the allele frequency of -427C>T and +102525G>A;R265Q showed a significant association with log-transformed PC(20) methacholine values in the asthmatics (P=0.005-0.0004). Real-time PCR demonstrated higher PPARGC1B mRNA levels in asthmatics having -427CC allele than in those having -427TT or CT alleles (P=0.048). The ratio of the mRNA expression for each PPARGC1B exon4-mRNA compared with the wild type was similar in peripheral blood mononuclear cells carrying the +102525G>A allele. Luciferase reporter assays revealed that -427C allele caused higher promoter activity than -427T allele. EMSA demonstrated that -427C allele exhibited stronger binding activity to a nuclear protein in 293T cells than did the -427T allele. CONCLUSIONS AND CLINICAL RELEVANCE: Polymorphisms of -427C>T on the promoter and those of +102525G>A on exon 5 of the PPARGC1B gene may affect the development of AHR through the modulation of PPARGC1B gene products. The PPARGC1B genotypes may serve as genetic markers for AHR.

An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer.

BACKGROUND: The cancer stem cell (CSC) hypothesis has important clinical implications for cancer therapeutics because of the proposed role of CSCs in chemoresistance. The aim of this study was to investigate changes in the CSC populations before and after primary systemic therapy (PST) and their prognostic role in human breast cancer. METHODS: Paired samples (before and after PST) of breast cancer tissue were obtained from clinical stage II or III patients (n=92) undergoing PST with the regimen of doxorubicin plus docetaxel (AD) (n=50) or doxorubicin plus cyclophosphamide (AC) (n=42) and subsequent breast resection. The proportions of putative CSCs with CD44+/CD24- or aldehyde dehydrogenase 1+ (ALDH1+) phenotypes were determined by immunohistochemistry. RESULTS: A higher proportion of CD44+/CD24- tumour cells and ALDH1 positivity in pre-chemotherapy tissue was correlated with higher histologic grade, oestrogen receptor (ER) negativity, high Ki-67 proliferation index and basal-like subtype of breast cancer. Aldehyde dehydrogenase 1 positivity in pre-chemotherapy biopsy was also associated with a higher rate of pathologic complete response following PST. In comparisons of putative CSC populations before and after PST, the proportions of CD44+/CD24- and ALDH1+ tumour cells were significantly increased after PST. The cases with increased CD44+/CD24- tumour cell populations after PST showed high Ki-67 proliferation index in post-chemotherapy specimens and those with increased ALDH1+ tumour cell population after PST were associated with ER negativity and p53 overexpression. Furthermore, cases showing such an increase had significantly shorter disease-free survival time than those with no change or a reduced number of CSCs, and the survival difference was most notable with regard to the changes of ALDH1+ tumour cell population in the patients who received AC regimen. CONCLUSION: The present study provides the clinical evidence that the putative CSCs in breast cancer are chemoresistant and are associated with tumour progression, emphasising the need for targeting of CSCs in the breast cancer therapeutics.

Association of angiotensin I-converting enzyme gene polymorphisms with aspirin intolerance in asthmatics.

BACKGROUND: Aspirin-intolerant asthma (AIA) refers to the development of bronchoconstriction in asthmatic individuals following the ingestion of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs). Angiotensin I-converting enzyme (ACE), a membrane-bound peptidase present in the lung, plays a pivotal role in the metabolism of the endogenous peptides involved in the pathogenesis of asthma. METHODS: We screened a Korean asthma cohort (581 asthmatics including 81 aspirin-intolerant asthmatics and 231 aspirin-tolerant asthmatics, and 181 normal controls) for four single nucleotide polymorphisms (SNPs; -262 A>T and -115 T>C in the 5'-flanking region and +5467 T>C [Pro450Pro] and+11860 A>G [Thr776Thr] in the coding region) and one ins/del (+21288 CT) in the ACE gene. RESULTS: None of the SNPs or haplotypes showed any association with the development of asthma, but they were significantly associated with the risk of AIA. Logistic regression indicated that the frequency of the rare alleles of -262 A>T and -115 T>C was higher in subjects with AIA than in subjects with aspirin-tolerant asthma (ATA) (P=0.003-0.01, P( corr)=0.015-0.05). Subjects homozygous for the rare alleles of -262 A>T and -115 T>C showed a greater decline in forced expiratory volume in 1 s (FEV(1)) after aspirin provocation than those homozygous for the common alleles (P<0.05). A luciferase reporter assay indicated that ACE promoters containing the rare -262 A>T allele possessed lower activity than did those containing the common allele (P=0.009). In addition, ACE promoters bearing the rare -115 T>C allele had no luciferase activity. DNA-protein binding assays revealed a band containing the ACE promoter region (including -262 A) and a protein complex. CONCLUSION: The -262 A>T polymorphism in the promoter of the ACE gene is associated with AIA, and the rare allele of -262 A>T may confer aspirin hypersensitivity via the down-regulation of ACE expression.

Triamcinolone acetonide protects the rat retina from STZ-induced acute inflammation and early vascular leakage.

Streptozotocin (STZ) has been commonly used to induce in vivo and in vitro hyperglycemic diabetes and its toxicity leads to inflammation and vascular injury. Triamcinolone acetonide (TA), as an anti-angiogenic/anti-inflammatory drug, is clinically used to improve the visual acuity in neovascular and edematous ocular diseases. The aim of this study was to investigate the effect of TA on early inflammation and vascular leakage in the retina of STZ-induced hyperglycemic rats. Hyperglycemia was induced in 8-week-old male Sprague-Dawley (SD) rats by a single intraperitoneal injection of STZ (65 mg/kg); only rats with blood glucose levels >13.9 mmol/l 1 day after STZ injection were included in STZ-hyperglycemic group. Sex- and age-matched SD rats injected with buffer were used as the control group. One day before STZ and buffer injection, 2 microl TA (4 mg/ml in saline) and 2 microl saline were intravitreal-injected into the right and the left eyes of rats, respectively. Retinal vascular leakage was measured using the Evans-blue method. Changes in pro-inflammatory target genes, such as tumor necrotic factor (TNF)-alpha, intracellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were assessed by immunoblottings, immunostaining, and ELISA analyses. Vascular hyperleakage and up-regulation of most pro-inflammatory genes peaked within a few days after STZ injection and had recovered. However, these changes were blocked by TA pretreatment. Our data suggest that TA controls STZ-induced early vascular leakage and temporary pro-inflammatory signals in the rat retina.

Triamcinolone suppresses retinal vascular pathology via a potent interruption of proinflammatory signal-regulated activation of VEGF during a relative hypoxia.

We examined the effect of triamcinolone acetonide (TA), a corticosteroid, on the relationship between vascular pathophysiology and vascular endothelial growth factor (VEGF) activation in the retina of a rat model of oxygen-induced retinopathy (OIR). OIR was induced by exposure of hyperoxia (80% oxygen) to Sprague-Dawley (SD) rats from P2 to P14 and then returned to normoxic conditions. TA was intravitreal-injected once into the right eye of OIR rats at P15. Effects of TA on vascular pathophysiology or changes of various genes in response to hypoxia and/or proinflammation under hypoxic retina were assessed by the Evans-blue method, fluorescein isothiocyanate-dextran (FITC-D) infusion, immunoblotting, and ELIZA. TA not only reduced retinal neovascularization and vascular leakage in the OIR-rat retina, but also blocked the induction of hypoxia-response proinflammatory genes before it negatively controlled VEGF activation. These findings suggest a potential that TA suppresses retinal neovascular pathophysiology via proinflammation-mediated activation of VEGF during hypoxia.

Effect and mechanism of lipopolysaccharide on allergen-induced interleukin-5 and eotaxins production by whole blood cultures of atopic asthmatics.

Interleukin (IL)-5 and eotaxin families regulate the development of eosinophilic inflammation of asthma in a co-operative manner. The exposure to airborne lipopolysaccharide (LPS) induces varying degrees of airflow obstruction and neutrophilic airway inflammation. Production of IL-5 and eotaxin subfamily chemokines was analysed in response to Dermatophagoides pteronyssinus allergen (D.p.) according to the presence of specific IgE to D.p., and investigated the mechanism underlying their LPS-mediated regulation of these cytokines in response to the specific allergen. Peripheral blood cells (PBCs) from asthmatics with (group 1) or without (group 2) specific IgE to D.p. and from non-asthmatics with (group 3) or without (group 4) were stimulated with D.p. or LPS. For LPS-mediated inhibition of IL-5 and eotaxin-2 production, LPS-induced cytokines were added to the D.p.-stimulated PBCs. IL-5 and eotaxin-2, but not eotaxin-1 and 3, were significantly increased by D.p.-stimulated-PBCs from group 1, while only eotaxin-2 was elevated in group 3. Eotaxin-2 production was found in monocytes and correlated with the level of specific IgE to D.p. LPS treatment resulted in the decrease in eotaxin-2 and IL-5 production by the D.p.-stimulated PBCs. LPS-induced IL-10 completely inhibited D.p.-stimulated production of eotaxin-2 and IL-5. The differential responses of the eotaxin family to specific antigens suggest that the predominant role of eotaxin-2 and LPS may attenuate eosinophilic inflammation by inhibiting IL-5 and eotaxin-2 synthesis through IL-10 production.

Effect of N-glycosylation on ligand binding affinity of rat V1a vasopressin receptor.

A rat Vla vasopressin (rVla) receptor has two putative N-glycosylation sites at 14th and 27th amino acid asparagine in the extracellular N-terminus. In the present study, we examined the possible roles of N-glycosylation of the N-terminus in the receptor function. Three point mutants for deglycosylated rVla receptor were generated in which the 14th and/or the 27th asparagine was replaced with glutamine, namely N14Q, N27Q, and N14:27Q, each tagged with an enhanced green fluorescent protein (EGFP) at their C-termini, and transfected to COS-7 or HEK292 cells. The two single mutants and a double mutant have progressively smaller molecular mass compared to the wild-type receptor as determined by immunoblot analysis, indicating that the two sites are effectively glycosylated in vivo. The maximal ligand binding capacities of three mutant receptors were comparable to that of wild-type (17.02 +/- 1.32 pmol/g protein) with modest changes in ligand binding affinities: N27Q and N14:27Q had decreased binding affinities compared to N14Q and wild-type receptors. The reduced binding affinities of the deglycosylated mutants are not likely due to the impaired intracellular transport since their traffickings were indistinguishable from one another. Taken together, these results suggest that the N-glycosylation at the two sites of the N-terminus of rV1a receptor minimally affects the surface expression and trafficking of the receptor.

The downregulation of Bcl-2 expression is necessary for theophylline-induced apoptosis of eosinophil.

Apoptosis of eosinophils is of increasingly important value in modulating allergic inflammatory airway diseases, such as asthma, and is suppressed by interleukin-5 (IL-5) in in vitro culture. In this study, we examined the effects of theophylline on survival/apoptosis, intracellular cAMP concentration, and Bcl-2 protein expression. Treatment with theophylline protected eosinophils against IL-5-mediated inhibition of apoptosis with a simultaneous suppression of survival in a dose-dependent manner. Theophylline caused an increase in the intracellular cAMP levels of IL-5-stimulated eosinophils. Enhancement of eosinophil apoptosis was consistent with an increase in DNA fragmentation in eosinophils treated with theophylline. On the other hand, the Bcl-2 protein appeared to be expressed constitutively in freshly isolated eosinophils. Bcl-2 expression was augmented by IL-5 stimulation, yet it was considerably inhibited by theophylline treatment. These data suggest that intracellular cAMP levels and Bcl-2 expression are involved in the suppression of eosinophil survival by theophylline.

The biochemical and molecular characterization of recombinant Bacillus subtilis tripeptidase (PepT) as a zinc-dependent metalloenzyme.

Aminopeptidases catalyze the release of N-terminal amino acid residue from polypeptides and peptides, and most of them are known to be metalloenzymes. A tripeptidase gene (pepT) of Bacillus subtilis was expressed in Escherichia coli, and the resulting recombinant PepT was purified in an active form through sequential chromatographies. The addition of Zn2+ or Co2+ increased the enzymatic activity by approximately two fold. The points at which Zn2+ and Co2+ stimulated a half-maximum activity of the PepT were 650 nM and 1,700 nM, respectively. The measurement of the metal content showed that this enzyme contained 0.26 atom of Zn2+ per molecule with essentially the absence of Co2+ and others, and 0.53 atom of Zn2+ with 1.5-fold increase of activity when reconstituted with Zn2+. Consistent with this result, this enzyme is much readily refolded in the presence of Zn2+ than Co2+. To further delineate the structure and function relations, we made serial deletion mutants and analyzed their enzymatic activities. Of eight deletion mutants, only a mutant lacking the N-terminal 66 amino acid residues retained enzymatic activity. The mutant enzyme, however, required a concentration of Zn2+ ion at least ten-fold higher to reach maximum activity without significantly affecting kinetic parameters such as Km and Vmax compared to the full length PepT. Taken together, these data suggest that the B. subtilis PepT is likely to be a Zn2+-dependent metalloenzyme and that the N-terminal region of the PepT stabilizes Zn2+-binding.

Role of cAMP-dependent pathway in eosinophil apoptosis and survival.

The survival and apoptosis of eosinophils is of pivotal importance for controlling allergic diseases such as asthma and rhinitis. In this study we have investigated the role for cAMP in regulating eosinophil survival and apoptosis in the absence of eosinophil-active cytokines. The treatment with dibutyryl cyclic AMP (dbcAMP) increased eosinophil survival with a concomitant decrease of apoptosis in a dose-dependent manner. The pretreatment with a protein kinase A (PKA) inhibitor blocked the effects of dbcAMP on survival and apoptosis of eosinophils. The catalytic subunit of PKA was translocated to nucleus in parallel with a robust increase of intracellular cAMP levels upon exposure to dbcAMP but not IL-5, suggesting the separation of PKA activation from the IL-5-induced suppression of eosinophil apoptosis. When eosinophils were treated with pharmacological inhibitors of protein kinases prior to exposure to dbcAMP or IL-5, only the mitogen-activating protein kinase (MAPK) inhibitor, PD098059, was partly able to block dbcAMP-induced augmentation of eosinophil viability, whereas both Janus kinase 2 and MAPK inhibitors effectively interrupted the IL-5-induced prolongation of eosinophil survival. The effects of dbcAMP and these protein kinase inhibitors on eosinophil apoptosis were confirmed by morphologic analysis. We propose that a cAMP-dependent pathway may constitute an important component for regulating eosinophil survival/apoptosisand that cAMP may inhibit eosinophil apoptosis through the activation of PKA and of subsequent MAPK in part.

Scale-up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies.

A cDNA encoding mature epidermal growth factor (EGF) was isolated and cloned into a pQE30 vector in which the His(6)-tagged EGF was expressed. pH-stat feeding of concentrated medium at the time of isopropyl beta-D-thiogalactoside induction and slug-feedings of the enriched medium during the induction resulted in a higher cell density and specific expression. Using a simple refolding protocol that consisted of 1 mM L-cysteine addition for a 1-h reduction followed by 5 mM L-cystine addition for oxidative refolding, we were able to convert nearly all EGF monomers into the oxidized form. Also, there folding aggregate was converted into the monomeric form. Approx. 50% overall yield was obtained from the dissolved inclusion bodies to a single peak under FPLC. We hope that the result of this study may provide information that is useful for the scale-up of the recombinant human EGF production process.

Interleukin (IL)-5 downregulates tumor necrosis factor (TNF)-induced eotaxin messenger RNA (mRNA) expression in eosinophils. Induction of eotaxin mRNA by TNF and IL-5 in eosinophils.

An eotaxin is a chemoattractant specific for eosinophils that are known to play a role in helminth infection and allergic responses. Although several cellular sources have been reported to produce eotaxin, it would be interesting to know whether eosinophils are able to produce their own eotaxin and participate in recruitment of themselves in response to inflammation. To this end, a cloned eotaxin complementary DNA was transcribed in vitro to use as a probe for detecting eotaxin messenger RNA (mRNA), and eotaxin protein levels were determined by enzyme-linked immunosorbent assay. Eotaxin mRNA was, as analyzed by in situ hybridization, rarely detectable in unstimulated eosinophils, but was strongly induced in eosinophils when stimulated with tumor necrosis factor (TNF). Interleukin (IL)-5, which is known to be a major factor of eosinophil survival in vivo and in vitro, was also able to induce a modest level of eotaxin mRNA but inhibited TNF-induced eotaxin mRNA expression in a dose-response manner. Dexamethasone inhibited TNF-induced eotaxin mRNA expression. This result was consistent with that from reverse transcription/polymerase chain reaction followed by Southern blot analysis. Unlike the little expression of eotaxin mRNA in the absence of stimuli, the measurement of eotaxin protein revealed that a considerable amount of eotaxin protein was constitutively produced in unstimulated eosinophils. Its expression was upregulated by TNF and IL-5 as well. However, the inhibitory effect of IL-5 on TNF-mediated eotaxin protein production was not as pronounced as that on eotaxin mRNA induction. Collectively, these data reflect the complex physiology of eosinophils in the expression of eotaxin gene upon the exposure to their survival and/or death factors.

A novel human ERK phosphatase regulates H-ras and v-raf signal transduction.

A cDNA encoding a novel human extracellularly-regulated kinase (ERK) phosphatase, designated B59, was isolated from a B5/589 human mammary epithelial cell cDNA library. The 1104 nucleotide open reading frame encodes 368 amino acids including the highly conserved catalytic site sequence of protein phosphotyrosine phosphatases (PTPs), VXVHCXXGXXR, at amino acid position 276-287. The predicted 70 amino acid stretch surrounding the HC motif shares significant sequence identity with other human dual specificity PTPs (dsPTPs), including the known ERK PTPs CL100, PAC1, B23, as well as the dsPTPs VH-1 and VHR. B59 protein synthesized in vitro in a rabbit reticulocyte lysate dephosphorylated rat ERK1 and ERK2 proteins whose phosphorylation had been stimulated by v-mos kinase added to the lysate. Ectopic expression of B59 in NIH3T3 fibroblasts inhibited the induction of an oncogene-responsive promoter by the dominant-activating raf mutant, raf-BXB. Moreover, cotransfection of NIH3T3 cells with B59 inhibited morphological transformation by H-ras and v-raf oncogenes. These results suggest that B59 suppresses the transforming activity of H-ras or v-raf oncogenes through ERK dephosphorylation and inactivation.

Identification of a domain within the carboxyl-terminal region of the beta platelet-derived growth factor (PDGF) receptor that mediates the high transforming activity of PDGF.

We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alpha PDGFR and the intracellular domain of the beta PDGFR (alpha 340 beta 342 R) was markedly more efficient than the wild type alpha PDGFR (alpha RWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of beta PDGFR that confers this higher transforming activity, we generated several additional alpha/beta PDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alpha PDGFR and the final 165 amino acids from the carboxyl-terminal of the beta PDGFR (alpha 933 beta 942 R) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alpha 340 beta 342 R. However, when chimeric PDGFRs in which either the kinase insert domain (alpha beta RKI) or the last 79 amino acids from the carboxyl-terminal end of the beta PDGFR (alpha 1024 beta 1028 R) were substituted into alpha PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alpha RWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of beta PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of beta PDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the beta PDGFR (alpha beta 942-1028R) were substituted for those in the alpha PDGFR. Cotransfection experiments indicated that alpha beta 942-1028R increased transforming activity of PDGF-A to similar extent as the alpha 933 beta 942R, or alpha 340 beta 342R. Therefore, our findings define a critical domain within the noncatalytic region of beta PDGFR intracellular domain that confers the higher focus forming activity mediated by the beta PDGFR.

Revascularization of the ischemic colon transplant using the internal mammary vessels.

We describe a successful reconstruction of the esophagus with the isoperistaltic right colon and terminal ileum, which had very poor continuity of the marginal artery. The stomach and the left colon were not available because of corrosive injury and intraabdominal adhesions. The blood supply of the ischemic transplant was augmented by anastomosis of the internal mammary vessels to the iliocolic vessels.

Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma in astrocytes.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein which can be induced on astrocytes, the major glial cell of the central nervous system (CNS). In this study, we examined the effect of three proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma), on the expression of ICAM-1 by primary rat astrocytes. Astrocytes constitutively express ICAM-1 mRNA and protein, which is enhanced by treatment with TNF-alpha, IL-1 beta and IFN-gamma. TNF-alpha is the most potent inducer of ICAM-1 expression, followed by IL-1 beta, then IFN-gamma. Kinetic analysis demonstrated optimum ICAM-1 mRNA expression after a 1-h exposure to TNF-alpha, 2 h exposure to IL-1 beta, and 4 h exposure to IFN-gamma. Peak ICAM-1 protein expression was detected 12-16 h after treatment with TNF-alpha or IL-1 beta, and after a 24-h exposure to IFN-gamma. Nuclear run-on analysis demonstrated that the ICAM-1 gene is transcribed under basal conditions in astrocytes, and that both TNF-alpha and IL-1 beta enhance transcriptional activation of the ICAM-1 gene. ICAM-1 mRNA stability studies determined that basal ICAM-1 mRNA has a half-life of about 1 h, and that TNF-alpha, IL-1 beta and IFN-gamma have a modest effect on stabilization of basal ICAM-1 mRNA expression. These results indicate that under inflammatory conditions in the CNS, such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), astrocytes can be induced to express the adhesion molecule ICAM-1, which can contribute to inflammatory events within the CNS.

Cloning and sequence analysis of the rat tumor necrosis factor-encoding genes.

We have isolated an approximately 22-kb TNF locus (encoding tumor necrosis factor) from a rat genomic library and sequenced the 7105-bp fragment that comprises the TNF-alpha and TNF-beta genes, including their flanking sequences. The two genes are tandemly arranged with TNF-beta 5' to TNF-alpha and separated by approximately 1.1 kb of intergenic space, and each gene consists of four exons and three introns, similar to those of the other species examined thus far. Comparison analysis showed that the rat TNF have high sequence homology with the mouse TNF (TNF-alpha, 86.5%; TNF-beta, 89.3%) and relatively low homology with the human, rabbit, and porcine TNF. The upstream sequence of rat TNF-alpha contains a number of sequence motifs implicated in the expression and regulation of eukaryotic genes, including binding sites for the transcription factors Sp-1, Ap-2, IFN.1 and NF-kappa B. The possible significance of potential regulatory sequence elements found in the rat TNF-alpha in the context of transcriptional regulatory mechanisms is discussed.