Clinicopathologic significance of tumor microenvironment CD11c, and FOXP3 expression in diffuse large B-cell lymphoma patients receiving rituximab, cyclophosphamide, anthracycline, vincristine, and prednisone (R-CHOP) combination chemotherapy

BACKGROUND/AIMS: CD11c is a dendritic cell marker in humans, which potentially induces a cytotoxic effect on lymphoma cells. Forkhead boxP3 (FOXP3) is a regulator of T lymphocyte in the microenvironment of the lymphoma. The principal objective of this study was to determine whether the tumors' microenvironment expressions of CD11c and FOXP3 are predictive of clinical outcomes in diffuse large B-cell lymphoma (DLBCL) patients receiving treatment with rituximab, cyclophosphamide, anthracycline, vincristine, and prednisone (R-CHOP) combination chemotherapy. METHODS: The study population consisted of 100 patients with DLBCL. The CD11c and FOXP3 expression in primary tumors' microenvironment were evaluated using an immunohistochemistry (IHC). RESULTS: CD11c and FOXP3 expression positivity in microenvironment were 25% and 35%, respectively. Each one counted for 1 point. In CD11c and FOXP3 stain, positive was counted as 0 and negative was 1. The points were separated into low risk (0 to 1) and high risk (2) groups. Only the extranodal DLBCL patient group analysis conveyed significant differences of progression-free survival (p = 0.019) and overall survival (p = 0.039) between the two groups. CONCLUSIONS: We can achieve possible clinical significance of lymphoma tumor microenvironments through CD11c and FOXP3 IHC stains in extranodal DLBCL patients receiving R-CHOP therapy.

Association Between MicroRNA196a2 rs11614913 Genotypes and the Risk of Non-Small Cell Lung Cancer in Korean Population / 예방의학회지

OBJECTIVES: The microRNA (miRNA) miR-196a2 may play an important role in lung cancer development and survival by altering binding activity of target mRNA. In this study, we evaluated their associations with the susceptibility of non-small cell lung cancers (NSCLC) by case-control study in a Korean population. METHODS: We performed genotyping analyses for miR-196a2 rs11614913 T/C at miRNA regions in a case-control study using blood samples of 406 NSCLC patient and 428 cancer-free control groups. RESULTS: The total C allele frequencies for miR-196a2 were 48.8% for the patients and 45.6% for the controls; and the genotype frequencies of TT, TC, and CC were 23.7%, 55.2%, and 21.1% for the patients and 31.1%, 46.35%, and 22.4% for the controls (p<0.05). Participants who possesses TC/CC genotypes showed high risk for NSCLC compared to those possessed TT genotypes (OR, 1.42; 95% CI, 1.03 to 1.96). The association was persisted in 60 and older age group, male, smokers, those without family history for cancer. However, no significant association of CC genotypes in recessive genetic model was observed. CONCLUSIONS: In conclusion, this case-control study provides evidence that miR-196a2 rs11614913 C/T polymorphisms are associated with a significantly increased risk of NSCLC in a dominant model, indicating that common genetic polymorphisms in miR-196a2 rs11614913 are associated with NSCLC. The association of miR196a2 rs11614913 polymorphisms and NSCLC risk require confirmation through additional larger studies.

Mercury Concentrations of Maternal and Umbilical Cord Blood in Korean Pregnant Women: Preliminary Study / 대한산업의학회지

OBJECTIVES: To evaluate the level of maternal and prenatal mercury exposure and to analyze the related factors. METHODS: Fifty-nine pregnant women were recruited into this study after obtaining informed consent. Samples were collected at delivery from normal pregnant women who were living in the city of Busan, Korea. Mercury concentrations in maternal and umbilical cord blood samples were measured using a gold-amalgam collection method. The total and methyl mercury levels of 36 of the 59 pregnant women were analyzed after randomization, and the results were compared. RESULTS: The mean total mercury concentration was 3.16+/-1.21 ppb and 5.43+/-2.22 ppb in maternal and cord blood, respectively. The average, maternal blood mercury level was lower than the prescribed toxic limit for human (WHO, 5 ppb), whereas the cord blood mercury was higher. The mercury exposure level exceeded the WHO recommendation in 5 (8.47%) cases of maternal blood and 29 of (49.15%) cord blood. There was a significant correlation between maternal and cord blood mercury concentrations. Total mercury and methyl mercury concentrations of the 36 random pregnant women were 3.06+/-1.17 ppb, and 2.60+/-1.11 ppb in maternal blood, and 5.20+/-2.36 ppb, and 4.70+/-1.97 ppb in cord blood, respectively. Methyl mercury accounted for 85.0% of the total mercury in maternal blood and 90.4% in cord blood. There was a significant correlation between total and methyl mercury concentrations. CONCLUSIONS: The study results suggest that mercury concentrations of cord blood may be regarded as indicative of high prenatal mercury exposure. Therefore, further studies are necessary to explain the cause of high mercury concentrations in cord blood, and to examine its relationship with various health indices.

Expression of dendritic cell markers on cultured neutrophils and its modulation by anti-apoptotic and pro-apoptotic compounds

Neutrophils are also known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. In this study, neutrophils were cultivated in vitro in the presence or absence of compounds modulating their survival in an attempt to characterize the expression profile of the DC markers. Higher MHC-II, CD80, CD86, CD83, and CD40 expression levels were detected on the surface of the cultured neutrophils for 24 h than on the freshly isolated cells. The annexin V-positive cells showed a higher expression level of the DC markers than the annexin V-negative cells. The population of neutrophils double stained with annexin V and the DC markers increased after being incubated with agonistic anti-Fas Ab. LPS, the anti-apoptotic compound, decreased the CD86 and MHC-II expression levels but 50-60% of the DC marker-positive cells were detected in the annexin V-positive cells. In contrast, CD80, CD86, CD83, and HLA-DR mRNA levels increased in the GM-CSF-treated neutrophils but not in the anti-Fas Ab-treated neutrophils. T cell proliferation was inhibited by co-culturing them with anti-Fas Ab- or LPS-treated neutrophils at a high neutrophil:T cell ratio. However, the superantigen-mediated T cell proliferation was increased by the LPS-treated neutrophils but decreased by the anti-Fas Ab-treated neutrophils. There was a lower level of interferon-gamma production in the T cells co-cultured with anti-Fas Ab-treated neutrophils than with the LPS-treated neutrophils. This suggests that apoptotic neutrophils express DC markers on their surface and the differential expression of DC markers might have a detrimental effect on the immune reaction.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1alpha were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.

Hypermethylation of p16(INK4a) in Korean Non-small Cell Lung Cancer Patients

Promoter hypermethylation of the p16(INK4a) gene was investigated in 81 sets of samples of tumor tissue and adjacent normal tissue from Korean patients with primary lung cancer, using the modified real-time polymerase chain reaction (PCR)/ SYBR Green detection method. The results showed hypermethylation of p16(INK4a) in 27.2% of tumor tissues, and in 11.1% of adjacent normal tissue. No significant association was found between the overall aberrant methylation in tumor and corresponding normal specimens (r=0.137, p=0.219). In 22 cases with p16(INK4a) hypermethylation in tumor tissues, only 4 (18.1%) cases were found to have a hypermethylated normal tissue specimen. The findings of this study show that smoking can influence the methylation level of the promoter region of p16(INK4a), and that this occurs in tumor tissues more frequently than in normal tissues. Other clinicopathological characteristics, including age, sex, tumor stage, and histologic type were not found to be correlated with p16(INK4a) methylation.

The effects of peritoneal fluid from patients with advanced endometriosis on HLA-DR expression of monocytes / 대한산부인과학회지

OBJECTIVE: Endometriosis is a common gynecologic disorder characterized by the presence of endometrial tissue outside the uterine cavity. Many theories have been suggested to explain the mechanism for the development of this disease. Although no single theory can explain all cases of endometriosis, retrograde menstruation theory has gained the widest acceptance. But the discrepancy between the incidence of retrograde menstruation and prevalence of endometriosis suggests that additional factor(s) may determine susceptibility to endometriosis in the peritoneal cavity and the survival and implantation of endometrial cells seem to be related with immunologic factors. For evaluation of the immunologic factors, we investigated the effects of peritoneal fluid from patients with advanced endometriosis (EPF) on the HLA-DR expression of monocytes. METHODS: EPF (n=20) and peritoneal fluid from the control group (CPF) (n=10) were collected prior to main laparoscopic operation. CPF were obtained from fertile women with various gynecologic problem other than endometriosis, such as dermoid cyst and carcinoma in situ of the uterine cervix. Peripheral blood mononuclear cells were collected by density-gradient centrifugation of whole blood over a peripheral blood mononuclear cell separation medium (Histopaque-1077), and THP-1 cells derived from a monocyte/macrophage cell line (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI-1640 medium. The expression of HLA-DR was evaluated by confocal microscopy, and the levels of IL-10, TGF-beta and VEGF were measured by standard enzyme-linked immunosorbent assay kits (R and D system). RESULTS: Compared to CPF, the addition of 10% EPF (n=10) to culture medium significantly reduced the percentage of HLA-DR positive cells in cultures of a THP-1, monocytic cell line at 48 hours. The effect of EPF was dose-dependent, and similar effect was observed in the peripheral blood monocytes. An inverse correlation was found between the HLA-DR expression level and IL-10 concentration in EPF (r=-0.518; P=0.019) and in the supernatant of peripheral blood monocyte cultured in EPF (n=20) (r=-0.459; P=0.042). IL-neutralizing antibody significantly abrogated the effect of EPF on HLA-DR expression level. CONCLUSION: These results suggest that HLA-DR expression level on monocytes is down-regulated by EPF, and that IL-10 may be one of the factors in EPF to modulate HLA-DR expression level.

Detection of single nucleotide insertion of BCR/ABL region in imatinib-resistant human myelogenous leukemia SR-1 cells

Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.

Delay of Spontaneous Neutrophil Apoptosis by Vascular Endothelial Growth Factor

PURPOSE: Vascular endothelial growth factor (VEGF) stimulates angiogenesis and vascular permeability. Tissue damage is related to angiogenesis, and induced by a delay in neutrophil apoptosis. This study was performed to investigate the effect of VEGF on the spontaneous neutrophil apoptosis via the activation of VEGFR-1 and phosphorylation of the p38-MAPK pathway. METHODS: Neutrophils were prepared from 10 healthy young donors, cultured for 20 h, and the apoptosis measured by the morphological changes and flow cytometry. The VEGF receptor expression and phosphorylation of mitogen activated protein kinase (MAPK) were measured using a Western blotting method. RESULTS: VEGF dose-dependently delayed the spontaneous neutrophil apoptosis, but this effect was blocked by pre-treatment of the cells with a VEGF receptor antagonist. VEGF increased the phosphorylated forms of the extracellular stress related kinase (Erk) and p38-MAPK. However, the VEGF-induced delay in apoptosis was not affected by the Erk inhibitor, PD98059 but was affected by the p38- MAPK inhibitor, SB203580. The VEGF receptor-1, but not the VEGF receptor-2, was detected in neutrophils, but its level was reduced in cultured neutrophils. CONCLUSION: VEGF delays neutrophil apoptosis through p38- MAPK activation.

Trp-Lys-Tyr-Met-Val-Met stimulates phagocytosis via phospho-lipase D-dependent signaling in mouse dendritic cells

Dendritic cells (DCs) play a key role in activating the immune response against invading pathogens as well as dying cells or tumors. Although the immune response can be initiated by the phagocytic activity by DCs, the molecular mechanism involved in this process has not been fully investigated. Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) stimulates the activation of phospholipase D (PLD) via Ca2+ increase and protein kinase C activation in mouse DC cell line, DC2.4. WKYMVM stimulates the phagocytic activity, which is inhibited in the presence of N-butanol but not t-butanol in DC2.4 cells. Furthermore, the addition of phosphatidic acid, an enzymatic product of PLD activity, enhanced the phagocytic activity in DC2.4 cells. Since at least two of formyl peptide receptor (FPR) family (FPR1 and FPR2) are expressed in DC2.4 as well as in mouse bone marrow-derived dendritic cells, this study suggests that the activation of FPR family by WKYMVM stimulates the PLD activity resulting in phagocytic activity in DC2.4 cells.

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.

Hepatopulmonary Syndrome Induced by Common Bile Duct Ligation in a Rabbit Model: Correlation between Pulmonary Vascular Dilatation on Thin-Section CT and Angiography and Serum Nitrite Concentration or Endothelial Nitric Oxide Synthase (eNOS) 1 Expression

OBJECTIVE: To investigate the correlation between radiologic vascular dilatation and serum nitrite concentration and eNOS expression in the endothelial cell and pneumocyte in a rabbit model of hepatopulmonary syndrome induced by common bile duct ligation (CBDL). MATERIALS AND METHODS: Thin-section CT scans of the lung and pulmonary angiography were obtained 3 weeks after CBDL (n=6), or a sham operation (n=4), and intrapulmonary vasodilatation was assessed. The diameter and tortuosity of peripheral vessels in the right lower lobe by thin-section CT and angiography at the same level of the right lower lobe in all subjects were correlated to serum nitrite concentration and eNOS (endothelial nitric oxide synthase) expression as determined by immunostaining. RESULTS: The diameters of pulmonary vessels on thin-section CT were well correlated with nitrite concentrations in serum (r = 0.92, p < 0.001). Dilated pulmonary vessels were significantly correlated with an increased eNOS expression (r = 0.94, p < 0.0001), and the severity of pulmonary vessel tortuosity was found to be well correlated with serum nitrite concentration (r = 0.90, p < 0.001). CONCLUSION: The peripheral pulmonary vasculature in hepatopulmonary syndrome induced by CBLD was dilated on thin-section CT and on angiographs. Our findings suggest that peripheral pulmonary vascular dilatations are correlated with serum nitrite concentrations and pulmonary eNOS expression.

Protein Kinase C-delta Mediates Nitric Oxide- induced Proliferation of Gastric Cancer Cells, MKN-2

PURPOSE: The purpose of this study was to investigate the effect of exogenous nitric oxide (NO) on the proliferation of gastric carcinoma cells and the signaling pathways that regulate these responses. METHODS: MKN-28 cells were obtained from the Korean Cell Line Bank (KCLB) and maintained in DMEM culture media. The effect of sodium nitroprusside (SNP), a NO donor, on the proliferation of a serum-starved gastric carcinoma cell line, MKN-28, was examined by [3H]thymidine incorporation. Western blot was performed to analyze the translocation of protein kinase C (PKC)-deltafrom the cytosol to the plasma membrane of the MKN-28 cells. RESULTS: The proliferation of MKN-28 cells was significantly increased by SNP. It was also found that the proliferation was significantly inhibited by the protein kinase A (PKA) inhibitor, KT5720, and the protein kinase G inhibitor (PKG), KT5823, in SNP-treated cells. The SNP-induced proliferation was also inhibited by the PKC-deltaspecific inhibitor, rottlerin (1mu), but was increased by the PKC-beta inhibitor, Go6976 (1muM). The amount of translocated PKC-deltaprotein in the plasma membrane from the cytosol increased time-dependently after treating the cells with SNP, suggesting that NO activates PKC-delta Anti-inflammatory drugs, including dexamethasone, aspirin, indomethacin, mephenamic acid, and acetaminophen inhibited the SNP-induced proliferation of the cells and blocked of PKC-deltaactivation. CONCLUSION: NO stimulates the proliferation of serum- starved gastric cancer cells. The NO-induced proliferation may be mediated by PKC-delta The inhibitory effect of anti-inflammatory drugs on cell proliferation may be related to the inhibition of PKC-deltaactivity.

Correlation of Pulmonary Vascular Dilatation on HRCT to Expression of eNOS in a Rabbit Model of Hepatopulmonary Syndrome

PURPOSE: To investigate the correlation between pulmonary vascular dilatation on high-resolution computed tomography (HRCT) and expression of endothelial nitric oxide synthase (eNOS) after common bile duct ligation (CBDL) in the rabbit as a model of hepatopulmonary syndrome. MATERIALS AND METHODS: CBDL was done in 11 rabbits (2 weeks after CBDL, n = 5; 3 weeks after CBDL, n = 6). Four rabbits were done by abdominal incision with peritoneal suture only as a control group. HRCT scans were performed in the both groups. We evaluated peripheral pulmonary vascular dilatation in the upper and lower lobe. Tissue samples were immediately obtained from both upper and lower lobes of the lung and the liver after sacrifice. Dilatation of peripheral pulmonary vessel was correlated with the expression of endothelial nitric oxide synthase (eNOS) determined by Western blot. We also compared the degree of pulmonary vascular dilatation between the groups with administration of L-arginine (n = 5) and without administration of L-arginine (n = 6) after CBDL. RESULTS: Two weeks after CBDL, pulmonary vascular dilatation on HRCT was seen in three rabbits (60%) and the increase of eNOS expression was shown in two rabbits (40%) in the lower lobe. Three weeks after CBDL, pulmonary vascular dilatation on HRCT was seen in four rabbits (66.7%) and five rabbits (83.3%) each upper and lower lobe, respectively. Expression of eNOS was coincidently increased. The pulmonary vascular dilatation was noted more frequently in the lower lobe than in the upper lobe. Pulmonary vascular dilatation on HRCT was highly correlated with increase of expression of eNOS in the upper (r = 1.00, p = .0001) and lower lobe (r = .83, p = .0015). In contrast, control group of four rabbits developed neither pulmonary vascular dilatation on HRCT nor increase of eNOS expression. The grade of pulmonary vascular dilatation in the group with L-arginine administration was higher than that without administration of L-arginine (p < .05). CONCLUSION: Pulmonary vascular dilatation on HRCT is significantly correlated with increase of eNOS expression in a rabbit lung after CBDL. These results suggest that NO, derived from pulmonary eNOS, contributes to pulmonary vascular dilatation in a rabbit model of hepatopulmonary syndrome. Index words : Lung, CT Lung, effect of drugs on Lung, vascular disease

Study in the Relationship between Angiogenic Factor and Expression of Cyclooxygenase and Nitric Oxide Synthase in Gastric Cancer

PURPOSE: Secretion of angiogenic factors from tumor cells is know to play an important role in neo-vascularization and metastasis. However, which angiogenic factor is related with the formation of neo-vasculature in gastric carcinomas is not well known. This study was performed to observe changes in the expression of vascular endothelial growth factor (VEGF), cyclooxygenase (COX), and nitric- oxide synthase (NOS). METHODS: Expressions of VEGF, COX, and NOS in thirty specimens resected from patients with a gastric carcinoma were investigated using the western blot method. Cultured MKN28 gastric cancer cells were treated with 100 ng/ml VEGF, and changes in the expression of COX and NOS were examined. Changes in VEGF expression were also investigated after treatment of the cells with inhibitors of COX and NOS. RESULTS: Expressions of VEGF, COX, and eNOS were increased up to 10, 60, and 30%, respectively, in tumors compared to surrounding normal tissues. VEGF-positive tumors showed a higher expression of COX-2. Human recombinant VEGF induced the expression of COX-2, but not eNOS, in the cultured MKN28 cells. The increase in expression was blocked with actinomycin D, the VEGF antibody, and anti-VEGF peptide. VEGF-induced expression of COX-2 was also blocked by pretreatment of cells with aspirin and indomethacin, suggesting that these anti-inflammatory drugs inhibit VEGF. The expression of eNOS was decreased by indomethacin in VEGF-treated cells, but COX-2 expression was not affected by inhibitors of NO production, N-arginine methylester (NAME). However, the protein level of VEGF was increased by indomethacin and NAME. CONCLUSION: This study showed that COX-2 and eNOS in gastric carcinomas seem to play an important role in the production of VEGF and that their expressions may also be affected by VEGF.

Modulation of Apoptosis of Polymorphonuclear leukocyte by Plasma and Peritoneal fluid from Patients with Endometriosis / 대한산부인과학회잡지

OBJECTIVE: The effects of plasma and peritoneal fluid from endometriosis patients on apoptosis of neutrophils were investigated. METHODS: Neutrophils and plasma were obtained from women with endometriosis (n=20) and healthy control donors (n=20). Peritoneal fluid from endometriosis patients (n=10) and the controls (n=5) were collected prior to laparoscopic operation. Apoptotic changes of the cells were evaluated by morphological changes using Giemsa staining. DNA electrophoretic analysis were used to confirm apoptosis. RESULTS: Compared to the plasma of healthy controls, the addition of 10% plasma from patients with endometriosis to culture medium reduced the percentage of apoptotic cells from 65.3+/-6.6 % to 27.2+/-4.6% in cultures of neutrophils maintained in vitro at 24 h. The addition of 10% peritoneal fluid obtained from patients with endometriosis to culture medium also inhibited the apoptosis of neutrophils compared with control group (10.5+/-4.3% vs. 45.3+/-4.8%). 10 ng of interleukin-8 (IL-8) in the presence of plasma from controls and endometriosis patients further delayed the spontaneous apoptosis of neutrophils. Neutralizing IL-8 antibody abrogated the delay of apoptosis of neutrophils induced by peritoneal fluid but not in the plasma of endometriosis patients. Neutrophils obtained from patients with endometriosis showed delayed apoptosis in the absence and in the presence of plasma or peritoneal fluid from either source and this delay was not abrogated by neutralizing IL-8 antibody. CONCLUSION: These findings show that IL-8 is one of the neutrophil survival factors in the peritoneal fluid of endometriosis patients, which contains both pro-apoptotic and anti-apoptotic cytokines. However, our results indicate that an unidentified survival factor is present in the plasma of patients with endometriosis, and also suggest that the impairment of innate immunity, such as the delayed apoptosis of neutrophils may be related with pathogenesis of endometriosis.

Dexamethasone enhances phospholipase D activity in M-1 cells

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.

Preliminary Results for Changes in the Expression of Angiogenic Factors with Stage in Gastric Cancer

BACKGROUND: It has been suggested that the expression of angiogenic factors by tumor cells contributes to the increased neovascularization and vessel permeability that are associated with tumor vasculature. However, the significance of protein expression involving tumor angiogenesis in gastric cancer has yet not to be classified. METHODS: In this study, the expression of vascular endothelial growth factor (VEGF), cyclooxygenase (Cox), and nitric-oxide synthase (NOS) were investigated in 14 surgically resected human gastric carci nomas by using western blotting. RESULTS: In 6 of 14 paired cases, VEGF expression in the tumor tissue was slightly increased compared with the nonneoplastic counterpart in the same specimen. However, the expression of inducible-type Cox-2 was significantly increased in tumor tissue while the expression of constitutive-type Cox-1 was decreased. The expression of endothelial-type eNOS in cancer tissue was shown to be higher than in normal gastric resected tissues, but the expression of nNOS in cancer was lower than it was in a normal gastric mucosa. CONCLUSION: Although a direct positive correlation between VEGF expression and cyclooxygenase or nitric oxide synthase was not found in differnt stages of gastric tumor development, the cyclooxygenase and nitric-oxide synthase may play an important role in gastric cancer development.

Activation of Neutrophil Menbrane Phospholipase D by Soluble Proteins: Comparison of Cytosolic Neutrophil 50 kDa Factor , ADP-ribosylation Factor and a Novel Brain Factor / 대한면역학회지

GTPrS-dependent phospholipase D activity in human neutrophils was investigated using exogenous phospholipid as a substrate. Both cytosolic and membrane- associated phospholipase D activities were identified. The previously described 50 kDa cytosolic activating factor was resolved chromatographically from the cytosolic phospholipase D. Using exogenous phospholipid as substrate along with chromatographically resolved 50 kDa factor and recombinant ADP-ribosylation factor 1, plasma membrane was required for activity, indicating that the activity which was previously seen using endogenous phospholipid substrate was due to a phospholipase D located in the plasma membrane. In addition, ADP-ribosylation factor and the 50 kDa factor activated synergistically. Using neutrophil plasma membranes, a third regulator of neutrophil membrane phospholipase D was identified from bovine brain cytosol. This factor was resolved from ADP-ribosylation factor and Rho A by successive column chromatographies. The brain factor showed a synergistic effect with the 50 kDa neutrophil activator but an additive effect with recombinant ADP- ribosylation factor. Whether or not ADP-ribosylation factor or the brain factor were present, high activities were seen only when the 50 kDa factor was present, indicating that the 50 kDa cytosolic factor is a major activating factor for the neutrophil plasma membrane phospholipase D.

The Action Mechanism of Growth Hormone on Regeneration of Nitiric Oxide Synthase(NOS)-containing Nerves after Cavernous Neurotomy in the Rat / 대한비뇨기과학회지

PURPOSE: As growth hormone was reported to improve cavernosal nerve regeneration, we studied the action mechanism of growth hormone(GH) on the regeneration of nitric oxide synthase(NOS)-containing penile nerves after unilateral cavernous nerve neurotomy in rats. MATERIALS AND METHODS: Male rats were divided into three groups: sham operation(n=10); unilateral neurotomy of a 5mm segment of the cavernous nerve(n=10) and unilateral neurotomy with GH injection(n=10). Electrostimulation of the intact cavernous nerve was performed at 1 and 3 months after operation. Nicotinamide adenine dinucleotide phosphate(NADPH) diaphorase staining was used to identify nNOS in penile nerve fibers of the mid-shaft segment. The gene expression for nNOS, insulin like growth factor(IGF)-I and nerve growth factor(NGF) were investigated in corporal tissue by reverse transcriptase-polymerase chain reaction(RT-PCR) using specific oligonucleotide primers. RESULTS: Electrostimulation in the GH-treated group revealed a greater maximal intracavernosal pressure and a shorter latency period than in those given neurotomy alone at 3 months after operation. One month after unilateral neurotomy, both neurotomy alone and the GH-treated groups showed a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves on the side of neurotomy, however mRNA expression of nNOS and IGF-I showed a significant increase in GH-treated group. At 3 months, the number of NOS-containing nerve fibers in the neurotomy alone group did not increase while the GH-treated group showed a significant increase. CONCLUSIONS: These results show that GH significantly enhances the regeneration of NOS-containing fibers in the dorsal and intracavernosal nerves after unilateral cavernous nerve injury via IGF-I.