胰岛素受体亚型改变及相关通路活化在糖尿病小鼠肠上皮细胞过度增殖中的作用

 目的:探讨胰岛素受体亚型改变及其相关下游通路活化情况在糖尿病小鼠肠上皮细胞异常增殖中的作用。方法:用腹腔注射链脲霉素的方法制作糖尿病小鼠模型,采用增殖细胞核抗原标记法比较糖尿病小鼠及对照组小鼠肠上皮细胞的增殖情况。利用RT-PCR法测定胰岛素受体亚型表达比例在2组中的差异。采用real-time PCR及Western blot法分别从mRNA和蛋白质水平检测2组之间胰岛素受体相关通路各分子MEK1/2、ERK1/2、PI3K以及Akt的表达情况。结果:糖尿病组小鼠的小肠上皮细胞增殖指数显著升高(P<0.05),且细胞中胰岛素受体亚型IR-A/IR-B的比值也明显升高(P<0.05)。糖尿病小鼠肠上皮细胞中MEK1、MEK2和ERK1/2的mRNA水平及磷酸化蛋白水平均高于对照组(P<0.05)。结论:糖尿病小鼠肠上皮细胞过度增殖可能与其中胰岛素受体亚型IR-A/IR-B的比值增高及其相关MEK/ERK通路的激活有关。     

Fibroblast growth factor 8 induced downregulation of thrombospondin 1 is mediated by the MEK/ERK and PI3K pathways in breast cancer cells

Expression of fibroblast growth factor 8 (FGF-8) is increased in several forms of hormonal cancer. It was previously shown to regulate expression of thrombospondin 1 (TSP-1), an inhibitor of angiogenesis, in S115 breast cancer cells. Here, we studied the FGF-8-activated signalling pathways mediating TSP-1 repression in S115 cells and in non-tumorigenic MCF10A cells. Inhibition of FGF receptors or of MEK1/2 and PI3K with specific inhibitors (PD173074, U0126 or LY294002, respectively) restored TSP-1 mRNA expression in the presence of FGF-8 in S115 cells. Furthermore, U0126 and LY294002 increased TSP-1 mRNA expression in S115 cells over-expressing FGF-8. In MCF10A cells, FGF-8 treatment also decreased TSP-1 expression and the effect was dependent on active MEK1/2. In conclusion, FGF-8 suppresses TSP-1 expression through two independent pathways, MEK1/2 and PI3K. Repression of TSP-1 may be an important mechanism involved in induction of an angiogenic phenotype and growth of FGF-8-expressing breast cancer.     

Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease

Polycystic kidney (PCK) rats exhibit a multiorgan cyst pathology similar to human autosomal recessive polycystic kidney disease, and are proposed as an animal model of Caroli's disease with congenital hepatic fibrosis (CHF). This study investigated the expression and function of selected components of the mitogen activated protein kinase (MAPK) pathway in cultured intrahepatic biliary epithelial cells (BECs) of PCK rats. Compared to the proliferative activity of cultured BECs of control rats, those of the PCK rats were hyperresponsive to epidermal growth factor (EGF). The increase in BEC proliferation was accompanied by overexpression of MAPK/extracellular signal-regulated protein kinase (ERK) kinase 5 (MEK5), and subsequent phosphorylation of ERK5 in vitro. The increased proliferative activity was significantly inhibited by the transfection of short interfering RNA against MEK5 mRNA. An EGF receptor tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839), also significantly inhibited the abnormal growth of cultured BECs of PCK rats. By contrast, treatment with PD98059 and U0126, inhibitors for MEK1/2, was less effective. These results suggest that the activation of the MEK5-ERK5 cascade plays a pivotal role in the biliary dysgenesis of PCK rats, and also provide insights into the pathogenesis of Caroli's disease with CHF. As the MEK5-ERK5 interaction is highly specific, it may represent a potential target of therapy.     

Period is involved in the proliferation of human pancreatic MIA-PaCa2 cancer cells by TNF-alpha

Recent studies have found alterations to clock genes in several forms of cancer. Period (Per) gene plays an important role in the circadian system, the cell cycle, the induction of apoptosis, and DNA damage. However, the functions of Per in pancreatic cancer have not yet been elucidated. Here, we show that tumor necrosis factor-alpha (TNF-alpha) suppressed the expression of both Per1 and Per3 in MIA-PaCa2 cells, a human pancreatic cancer cell line. The levels of these proteins were 10% lower in the cells treated with 10 ng/mL TNF-alpha. Cell proliferation showed a 15%, 14%, and 16% decrease at 0.4, 2.0, and 10 ng/mL of TNF-alpha, respectively. In MTS-assays, MIA-PaCa2 cells transfected with siRNA against Per1 showed a 19% reduction in proliferation. However, the knockdown of Per3 did not significantly inhibit cell proliferation. The results suggest Per1 to be involved in the inhibition of the proliferation of MIA-PaCa2 cells by TNF-alpha.     

Specific phosphorylation and activation of ERK1c by MEK1b: a unique route in the ERK cascade

Extracellular signal-regulated kinases (ERKs) are key signaling molecules that regulate a large number of cellular processes, including mitosis. We showed previously that ERK1c, an alternatively spliced form of ERK1, facilitates mitotic Golgi fragmentation without the involvement of ERK1 and ERK2. Here we demonstrate that activation of ERK1c is mainly mediated by mitogen-activated protein kinase (MAPK)/ERK kinase 1b (MEK1b), which is an alternatively spliced form of MEK1 that was previously considered an inactive kinase. MEK1b phosphorylation and activity are preferentially stimulated by nocodazole, to induce its specific activity toward ERK1c. MEK1/2, on the other hand, preferentially target ERK1/2 in response to growth factors, such as EGF. As previously demonstrated for ERK1c, also MEK1b expression and activity are elevated during mitosis, and thereby enhance Golgi fragmentation and mitotic rate. MEK1 activity is also increased during mitosis, but this isoform facilitates mitotic progression without affecting the Golgi architecture. These results illustrate that the ERK cascade is divided into two routes: the classic MEK1/2–ERK1/2 and the splice-variant MEK1b–ERK1c, each of which regulates distinct cellular processes and thus extends the cascade specificity.     

Targeting the MEK1 cascade in lung epithelium inhibits proliferation and fibrogenesis by asbestos

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated after inhalation of asbestos. The effect of blocking this signaling pathway in lung epithelium is unclear. Asbestos-exposed transgenic mice expressing a dominant-negative mitogen-activated protein kinase kinase-1 (dnMEK1) (i.e., the upstream kinase necessary for phosphorylation of ERK1/2) targeted to lung epithelium exhibited morphologic and molecular changes in lung. Transgene-positive (Tg+) (i.e., dnMEK1) and transgene-negative (Tg-) littermates were exposed to crocidolite asbestos for 2, 4, 9, and 32 days or maintained in clean air (sham controls). Distal bronchiolar epithelium was isolated using laser capture microdissection and mRNA analyzed for molecular markers of proliferation and Clara cell secretory protein (CCSP). Lungs and bronchoalveolar lavage fluids were analyzed for inflammatory and proliferative changes and molecular markers of fibrogenesis. Distal bronchiolar epithelium of asbestos-exposed wild-type mice showed increased expression of c-fos at 2 days. Elevated mRNA levels of histone H3 and numbers of Ki-67-labeled proliferating bronchiolar epithelial cells were decreased at 4 days in asbestos-exposed Tg+ mice. At 32 days, distal bronchioles normally composed of Clara cells in asbestos-exposed Tg+ mouse lungs exhibited nonreplicating ciliated and mucin-secreting cells as well as decreased mRNA levels of CCSP. Gene expression (procollagen 3-a-1, procollagen 1-a-1, and IL-6) linked to fibrogenesis was also increased in lung homogenates of asbestos-exposed Tg- mice, but reduced in asbestos-exposed Tg+ mice. These results suggest a critical role of MEK1 signaling in epithelial cell proliferation and lung remodeling after toxic injury.     

MEK/ERK signaling is a critical mediator for integrin-induced cell scattering in highly metastatic hepatocellular carcinoma cells

The human hepatocellular carcinoma (HCC)-derived cell line KYN-2 is thought to provide a good model for studying the molecular basis of invasion and metastasis of human HCC, because it often shows cell scattering in vitro and intrahepatic metastasis in vivo. We previously found that integrin-mediated extracellular signals inactivated E-cadherin in KYN-2, and caused loss of cell-cell contact with gain of cell motility, which is considered to be a critical step in the process of cancer cell invasion and metastasis. To further understand molecular mechanisms involved in biological aggressiveness of HCC, we investigated intracellular signaling involved in integrin-mediated scattering of KYN-2 cells. Cultured KYN-2 cells formed trabecular aggregates in suspension, but when adhering to integrin-stimulating substrata, they scattered according to phosphorylation of extracellular signal-regulated kinase (ERK). Upon treatment with ERK kinase (MEK) inhibitor PD98059, adhered KYN-2 cell scattering was inhibited, tight cell-to-cell contact was recovered, and both E-cadherin and actin filaments accumulated in the area of intercellular contact zone. In contrast, constitutively active MEK1-transfected KYN-2 cells showed reduced E-cadherin and actin filaments in the intercellular contact zone, showing a flattened phenotype with broad lamellipodia. Enforced signaling of MEK-ERK pathway in KYN-2 cells suppressed cadherin-mediated homotypic adhesion and increased the potential of cell motility. An antibody-based protein microarray analysis revealed that the cytoplasmic protein c-Cbl was significantly downregulated in MEK1-transfected KYN-2 cells, suggesting that c-Cbl might be a candidate downstream mediator of integrin/MEK/ERK-mediated cell scattering. In conclusion, cell scattering of the highly metastatic cell line KYN-2 is regulated through the integrin-MEK-ERK signaling cascade, suggesting that this molecular pathway may be critical in intrahepatic metastasis of human HCC.     

Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer: MEK/ERK and p38 MAP kinase interaction in uPA synthesis

Increased expression of the hepatocyte growth factor (HGF) receptor (c-met) and urokinase type plasminogen (uPA) correlated with the development and metastasis of cancers. To investigate the role of HGF/c-met signaling on metastasis in cancer cells stimulated with HGF, we examined the effects of a specific MEK1 inhibitor (PD98059) and a p38 MAP kinase inhibitor (SB203580) on HGF-induced uPA expression in pancreatic cancer cell lines, L3.6PL and IMIM-PC2. Pretreatment of PD98059 decreased HGF-mediated phosphorylation of extracellular receptor kinase (ERK), uPA secretion and expression of matrix metalloproteinases (MMP-2 and MMP-9) in a dose-dependent manner. In contrast, SB203580 pretreatment increased HGF-stimulated ERK phosphorylation, uPA secretion and expression of MMPs. SB203580 also reversed the inhibition of HGF-mediated ERK activation and uPA secretion in the PD98059-pretreated cells. These results suggest that ERK activation by HGF might play important roles in the metastasis of pancreatic cancer and the p38 MAPK pathway also involved in the HGF-mediated uPA secretion and metastasis by regulation of ERK pathway. This revised version was published online in July 2006 with corrections to the Cover Date.     

LPS resistance in monocytic cells caused by reverse signaling through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway

The transmembrane form of tumor necrosis factor (mTNF), expressed on activated monocytes (MO) and macrophages (MPhi), is able to induce apoptosis in human endothelial cells (EC). Apoptosis is mediated by two distinct mechanisms: direct cell contact and a yet-unidentified soluble protein, death factor X. In addition, mTNF acts as a receptor that transduces a "reverse signal" into MO/MPhi when bound to the TNF receptor on EC. Reverse signaling by mTNF confers resistance to bacterial lipopolysaccharide (LPS). Stimulation of reverse signaling by mTNF blocks the ability of MO/MPhi to produce death factor X and proinflammatory cytokines. We have investigated which signaling pathways are used by mTNF acting as receptor. Reverse signaling triggers two independent pathways that can be distinguished by protein kinase C (PKC) inhibitors. The suppression of LPS-induced death factor X is dependent on PKC, whereas the suppression of LPS-mediated cytokine release is not. LPS and reverse signaling stimulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. It is interesting that the activation of reverse signaling by mTNF renders MO/MPhi refractory to a subsequent activation of the MAPK/ERK pathway by LPS. Thus, reverse signaling achieves LPS resistance in monocytic cells through interference with key signal-transduction pathways.     

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells' supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB-DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.     

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells’ supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB–DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.     

HGF mediates cell proliferation of human mesothelioma cells through a PI3K/MEK5/Fra-1 pathway

The ligand hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase, c-Met, are highly expressed in most human malignant mesotheliomas (MMs) and may contribute to their increased growth and viability. Based upon our observation that RNA silencing of fos-related antigen 1 (Fra-1) inhibited c-met expression in rat mesotheliomas (1), we hypothesized that Fra-1 was a key player in HGF-induced proliferation in human MMs. In three of seven human MM lines evaluated, HGF increased Fra-1 levels and phosphorylation of both extracellular signal-regulated kinase 5 (ERK5) and AKT that were inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042. HGF-dependent phosphorylation and Fra-1 expression were decreased after knockdown of Fra-1, whereas overexpression of Fra-1 blocked the expression of mitogen/extracellular signal-regulated kinase kinases (MEK)5 at the mRNA and protein levels. Stable MM cell lines using a dnMEK5 showed that basal Fra-1 levels were increased in comparison to empty vector control lines. HGF also caused increased MM cell viability and proliferating cell nuclear antigen (PCNA) expression that were abolished by knockdown of MEK5 or Fra-1. Data suggest that HGF-induced effects in some MM cells are mediated via activation of a novel PI3K/ERK5/Fra-1 feedback pathway that might explain tumor-specific effects of c-Met inhibitors on MM and other tumors.     

Pentoxifylline inhibits hepatic stellate cells proliferation via the Raf/ERK pathway

Pentoxifylline (PTX), which is a xanthine derivative, is a well-known suppressor of tumor necrosis factor-alpha (TNF-alpha) production in inflammatory cells and has also been shown to inhibit collagen synthesis in hepatic stellate cells (HSCs) in vitro. The present study aimed to evaluate the effects of PTX on proliferation in HSCs as mediated by the Raf/MEK/extracellular-signal-regulated kinase (ERK) signaling pathway. The rat hepatic stellate cell line T6 and activated primary rat HSCs were used in this study. The proliferation rate of the cells treated with 1 mM PTX significantly decreased compared with that of the control in T6 cells (78.3 ± 6.03% at 12 h, 61.0 ± 7.55% at 24 h, and 44.7 ± 2.08% at 48 h, p < 0.05). PTX (1 mM) also decreased the fraction of the HSC population in the S and G2/M-phases of the cell cycle in primary activated rat HSCs. The Raf-1 inhibitor GW5074 and the ERK inhibitor U0126 had inhibitory effects that were similar to those of PTX on HSC proliferation. In addition, PTX inhibited the phosphorylation of Raf-1 (p-Raf-1) and ERK (p-ERK) in a dose- and time-dependent manner in HSCs. These data provide evidence that PTX suppresses HSC proliferation via the Raf/MEK/ERK pathway.     

Tanshinone I inhibited growth of human chronic myeloid leukemia cells via JNK/ERK mediated apoptotic pathways

Tanshinone I (Tan I) is one of the main bioactive ingredients derived from Salvia miltiorrhiza Bunge, which has exhibited antitumor activities toward various human cancer cells. However, its effects and underlying mechanisms on human chronic myeloid leukemia (CML) cells still require further investigation. This study determined the effects and mechanisms of anti-proliferative and apoptosis induction activity induced by Tan I against K562 cells. The cytotoxic effect of Tan I at varying concentrations on K562 cells was evaluated via MTT assay. Cell apoptosis was further investigated through DAPI staining and flow cytometry analysis. The expression levels of apoptosis-related proteins and activities of JNK/ATF2 and ERK signaling pathways were analyzed by western blot. Quantitative PCR was performed to further determine mRNA expression levels of JNK1/2 and ERK1/2 after Tan I treatment. The results indicated that Tan I significantly inhibited K562 cell growth and induced apoptosis in a concentration- and time-dependent manner. It induced significant cellular morphological changes and increased apoptosis rates in CML cells. Tan I promoted the cleavages of caspase-related proteins, as well as increased the expression levels of PUMA. Furthermore, Tan I significantly activated JNK and inhibited ATF-2 and ERK signaling pathways. The mRNA expression levels of JNK1/2 and ERK1/2 were up-regulated by Tan I, further confirming its regulatory effects on JNK/ERK signaling pathways. Overall, our results indicated that Tan I suppressed cell viability via JNK- and ERK-mediated apoptotic pathways in K562 cells, suggesting that it might be a promising candidate as a novel anti-leukemia drug.     

ERK信号通路对人表皮干细胞增殖分化的影响

BACKGROUND:Epidermal stem cells (ESCs) cultured in vitro can easily become differentiated and aged, but have limited proliferation ability. These characters limit the application and development of ESCs in skin tissue engineering. Increasing understanding of the regulatory mechanism of ESCs proliferation and differentiation would lay the theoretical foundation for the clinical application of ESCs. OBJECTIVE:To identify the role of the ERK pathway in the proliferation and differentiation of ESCs. METHODS:ESCs were isolated and cultured in vitro. The activity of the ERK pathway was blocked by PD98059 and the activation of the ERK pathway was conducted by PMA or transfecting MEK1 recombinant plasmid to enable the overexpression of MEK1. The effect of activation or blockage of the ERK pathway on the proliferation and colony formation ability of ESCs was detected by MTT and colony formation assay, respectively. The expression of ESCs markers K19 and β1-integrin and differentiated cell marker K10 was detected by western blot. RESULTS AND CONCLUSION:When the activity of the ERK pathway was blocked by PD98059, the proliferation and colony formation abilities of ESCs were inhibited and the differentiation was enhanced. On the contrary, activating the ERK pathway by PMA or upregulation of MEK1 enhanced the proliferation and colony formation abilities of ESCs but inhibited the cell differentiation. These results indicate that the ERK pathway plays an important role in the proliferation and differentiation of ESCs.     

纳米羟基磷灰石可介导转染人肺癌A549细胞

目的 探讨纳米羟基磷灰石（nHAP）介导带有增强型绿色荧光蛋白（EGFP）基因的pGenesil-1质粒转染人肺癌A549细胞的可行性。方法 用均相沉淀法制备nHAP;透射电镜观察纳米粒结构;经超声分散及碳酸钠预处理后,在pH值7.4环境下应用多聚赖氨酸（PLL）修饰nHAP;实验分为nHAP-PLL组、脂质体组及对照组,分别配制转染复合物并转染A549细胞;荧光显微镜观察EGFP的表达;流式细胞仪检测pGenesil-1的转染率。结果 透射电镜下nHAP呈针状颗粒,粒径较均匀,约（15~20）nm×（60~80）nm,分散程度良好;nHAP-PLL组和脂质体组均见EGFP表达阳性的A549细胞,对照组未见EGFP表达阳性的A549细胞;nHAP-PLL组pGenesil-1转染A549细胞的转染率为(31.8±1.9)%,与脂质体组的转染率 (33.4±3.7)%无差异（P>0.01）,对照组未见转染阳性的A549细胞。结论 nHAP经PLL表面修饰后可介导人肺癌A549细胞基因转染。     

Thymosin alpha 1 enhancement of human lymphocyte proliferation is not mediated by prostaglandin E2

Prostaglandins of the E series have been implicated as possible mediators of thymosin alpha 1 (TA-1) effect on mitogen stimulated lymphoproliferation. In the present study human peripheral blood mononuclear cells were stimulated with concanavalin A in the presence of TA-1, prostaglandin E2 and indomethacin individually, or in combination. Both TA-1 and indomethacin significantly enhanced the proliferative response over that seen with mitogen alone (less than 0.001). The magnitude of enhancement observed with indomethacin (20 microM) was considerably less than that of TA-1 and it was abolished with prostaglandin E2. The combination of indomethacin and TA-1 did not alter TA-1 effect and prostaglandin E2 (up to 250 ng/ml) did not abolish thymosin effect. These results suggest that TA-1 and prostaglandin E2 have opposite effects and that thymosin enhancement of mitogen induced proliferation is not mediated by prostaglandin E2.     

Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5±2.8 pA/pF (n=29) in wild-type cells, and about half that value, i.e. 12.4±1.6 pA/pF (n=20) in heterozygotes (P=0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527±162 pA/pF (n=24) from wild-type, 316±35 pA/pF (n=21) from heterozygote and 419±83 pA/pF (n=7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.     

Efficient cell mediated lysis of human erythrocyte target cells is mediated by a human glycophorine A specific murine monoclonal antibody

A human glycophorine A specific murine monoclonal antibody mediates the antibody dependent killing of antibody sensitized human erythrocytes by human K cells (monocyte-free mononuclear cells). The overall level of killing is lower than that mediated by conventional, human anti-D antibodies but it is significant on each target cell.