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J L Farber

Publications and source records attributed to J L Farber.

At least 19 recordsLinked to original sources
PubMed2006

The presence of B-cell nodules does not necessarily portend a less favorable outcome to therapy in patients with acute cellular rejection of a renal allograft.

The presence of B-cell nodules in kidney biopsies of patients undergoing acute renal allograft rejection has been reported to be associated with glucocorticoid resistance and a high risk of graft failure. In an attempt to corroborate this observation, biopsies of renal transplants that evidenced Banff grade I A acute rejection were examined for the presence of B- or T-cell nodules, the detection of which was correlated with the therapeutic response. Biopsies from 14 consecutive renal transplant recipients with a diagnosis of acute cellular rejection were examined for the presence of T (CD3-positive) or B (CD20-positive) cells by immunohistochemistry. All patients were biopsied because of a rise in serum creatinine. No biopsy showed evidence of acute humoral rejection. Immunofluorescence microscopy was negative for C4d deposition in peritubular capillaries. There were no neutrophils in the peritubular or glomerular capillaries. Five patients had T-cell nodules; four had B-cell nodules; three had both T- and B-cell nodules; two had no nodules. All biopsies contained CD3-positive cells in the tubules and in the interstitium. In all but one of the patients, episodes of acute rejection were treated with steroids (one received thymoglobulin). Furthermore two patients received mycophenolate mofetil and one, sirolimus. There were no significant differences among the groups in either the initial creatinine or the creatinine after therapy. The presence of B-cell nodules in renal allograft biopsies of patients experiencing acute cellular rejection did not portend a less favorable outcome.

Acute Disease↗
PubMed2006

c-Kit-positive mast cells in portal tracts cannot be used to distinguish acute cellular rejection from recurrent hepatitis C infection in liver allografts.

Cirrhosis secondary to chronic hepatitis C virus (HCV) is the most common indication for liver transplantation. Recurrence of HCV infection in the liver allograft occurs at a high rate. The differentiation of recurrent HCV infection from acute cellular rejection (ACR) represents a difficult challenge in transplantation pathology. The c-Kit receptor is a tyrosine kinase membrane protein encoded by the c-Kit proto-oncogene, which is expressed on mast cells and on hematopoietic stem and progenitor cells. Mast cells are important effector cells of a broad range of immune responses. Recently, c-Kit+ mast cells were shown to form part of the inflammatory infiltrate in acute liver allograft rejection. A strong relationship was found between c-Kit+ cell densities and increasingly severe rejection. The present study sought to determine whether the presence of c-Kit+ cells could be used to distinguish between ACR and recurrent HCV in liver allografts. Immunohistochemical staining for c-Kit was performed on 20 transplant biopsy specimens from 10 patients with mild to moderate ACR and 10 other patients with recurrent hepatitis C. The number of c-Kit+ cells per portal tract varied with the density of the overall inflammatory infiltrate. There was no significant difference between the number of c-Kit+ cells in the biopsy specimens that carried a diagnosis of ACR and those from patients who had been diagnosed as having recurrent HCV. It was concluded that immunohistochemical staining for the presence of c-Kit+ mast cells cannot be used to differentiate between ACR and recurrent HCV infection in liver allograft biopsy specimens.

Biopsy↗
PubMed2006

Acute respiratory distress syndrome due to pulmonary involvement by neoplastic plasma cells in multiple myeloma.

Pulmonary involvement with multiple myeloma occurs infrequently and may be difficult to distinguish from more common primary lung tumours, metastatic disease, or other pleural and parenchymal abnormalities. A patient who developed acute respiratory distress syndrome (ARDS) was subsequently found to have multiple myeloma with involvement of lung parenchyma by neoplastic plasma cells. Only one other report of ARDS in association with multiple myeloma was found, and there are no previous reports where the appearance of ARDS antedated a diagnosis of multiple myeloma. In patients with ARDS, parenchymal involvement from multiple myeloma should be included in the differential diagnosis.

Aged↗
PubMed2001

Esophageal cancer prevention in zinc-deficient rats: rapid induction of apoptosis by replenishing zinc.

BACKGROUND: Nutritional zinc deficiency in rats increases esophageal cell proliferation and the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors. Replenishing zinc with a zinc-sufficient diet reduces these effects in zinc-deficient (ZD) rats. We investigated whether apoptosis was involved in the reduction of NMBA-induced esophageal tumors when ZD rats consumed a zinc-sufficient diet. METHODS: Weanling rats were fed a ZD diet (zinc at 3-4 ppm) for 5 weeks to establish esophageal cell proliferation, then treated once with NMBA (2 mg/kg body weight), and divided into the following five groups (47-100 per group). One ZD group was fed the ZD diet, and four zinc-replenished (ZR) groups, ZR(1), ZR(24), ZR(72), and ZR(432), were fed a zinc-sufficient diet (zinc at 74-75 ppm) beginning 1, 24, 72, and 432 hours, respectively, after NMBA treatment. From 24 hours to 2 weeks after beginning a zinc-sufficient diet, esophagi from all ZR groups were analyzed for apoptosis and cell proliferation; ZD esophagi were the controls. Tumor incidence was determined 15 weeks after zinc replenishment. All statistical tests were two-sided. RESULTS: Zinc replenishment initiated shortly after NMBA treatment effectively reduced esophageal tumorigenesis; 8% (three of 37) of ZR(1), 14% (five of 37) of ZR(24), 19% (five of 26) of ZR(72), and 48% (19 of 40) of ZR(432) rats developed esophageal tumors compared with 93% (14 of 15) of ZD animals (all P<.001). Importantly, 24 and 30 hours after zinc replenishment, esophagi had numerous apoptotic cells (% apoptotic cells: 0 hour = 2.9%, 95% confidence interval [CI] = 2.5% to 3.3%; 24 hours = 9.4%, 95% CI = 8.2% to 10.6%), and the expression of the proapoptotic Bax protein doubled. Within 48 hours, the ZR(1) epithelium was three to five cell layers thick compared with 10-20 layers before zinc replenishment. CONCLUSIONS: Zinc replenishment of NMBA-treated ZD rats rapidly induces apoptosis in esophageal epithelial cells and thereby substantially reduces the development of esophageal cancer.

Animals↗
PubMed2001

Induction of the mitochondrial permeability transition mediates the killing of HeLa cells by staurosporine.

The role of the mitochondrial permeability transition (MPT) in the killing of HeLa cells by staurosporine (STR) was assessed with the use of bongkrekic acid (BK), an inhibitor of the MPT. BK prevented cell killing as well as biochemical manifestations of the MPT: (a) the loss of the mitochondrial membrane potential (deltapsim); (b) the release of cytochrome c from the intramembranous space to the cytosol; and (c) the release of malate dehydrogenase from the mitochondrial matrix. Stable transfectants that overexpressed Akt were also resistant to cell killing and did not develop an MPT. STR inhibited the phosphorylation of Bad, whereas Bad phosphorylation was preserved in cells that overexpress Akt. In wild-type HeLa cells treated with STR, the content of Bax in the cytosol decreased as that in the mitochondria increased, a result that was again prevented by overexpression of Akt. Bid accumulation in the mitochondria with STR was not affected by overexpression of Akt. The pan-caspase inhibitor Z-Val-Ala-Val-Asp(OMe) fluoromethylketone prevented cell killing bu not induction of the MPT. The data document the central role of the MPT in the killing of HeLa cells by STR. The data are consistent with the hypothesis that induction of the MPT is a consequence of the movement of Bax to the mitochondria. Phosphorylation of Bad prevents Bax translocation. Caspases participate in the events related to cell killing that occur subsequent to induction of the MPT.

Apoptosis↗
PubMed2001

Bronchial atresia with relapsing pulmonary infection in a middle-aged man.

Congenital bronchial atresia (CBA) is a rare disorder, first reported in 1953. Less than 100 cases are reported in the literature, mostly in young, asymptomatic male patients with involvement of the apical-posterior segment of the left upper lobe. Patients may complain of fever, cough, or shortness of breath, symptoms that result from post-obstructive, sometimes recurrent, infections. Chest radiography and computed tomography reveal a tubular branching density representing mucus impaction or mucocele with surrounding focal hyperinflation. Surgical excision is reserved for symptomatic cases. We report an unusual case of CBA in a middle-aged man with a history of relapsing infections, who was found to have an atretic superior segment of the left lower lobe, with surrounding areas of organizing pneumonia.

Activities of Daily Living↗
PubMed2000

A possible role for centrosome overduplication in radiation-induced cell death.

Radiotherapy plays a key role in the treatment of many tumors; however, the precise mechanisms responsible for radiation-induced cell death remain uncertain. We have reported previously that ionizing radiation induces centrosome overduplication in human tumor cells. The present study was designed to elucidate a possible link between centrosome dysregulation and radiation-induced cell death. Exposure to 10 Gy gamma-radiation resulted in a substantial increase in cells containing an abnormally high number of centrosomes in a variety of cell lines derived from different types of human solid tumors. These aberrant centrosomes contribute to the assembly of multipolar spindles, thereby causing an unbalanced division of chromosomes and mitotic cell death characterized by the appearance of multi- or micronucleated cells. An extensive analysis of a panel of 10 tumor cell lines revealed a positive correlation between the fraction of cells with multiple centrosomes and the fraction with these nuclear abnormalities after irradiation. When the centrosome overduplication was blocked by enforced expression of p21Waf1/Cip1, the radiation-induced lethality was drastically rescued. Taken together, these results indicate that centrosome overduplication may be a critical event leading to mitotic failure and subsequent cell death following exposure to ionizing radiation.

Adenoviridae↗
PubMed2000

Early deregulation of the the p16ink4a-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesis in zinc-deficient rats.

The p16ink4a-cyclin D1/cyclin-dependent kinase 4 (Cdk4)-retinoblastoma (Rb) pathway has emerged as a critical target in oncogenesis. The zinc-deficient (ZD), N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer model provides a tool to study cell proliferation and cell cycle control in cancer initiation. Weanling rats were fed a ZD or zinc-sufficient (ZS) diet for 5 weeks, and then given a dose of NMBA. After 14 weeks, esophageal tumor incidence was 88% in ZD rats with highly proliferative esophagi versus 0% in ZS rats. Expression of p16ink4a, cyclin D1, Cdk4, and Rb in relation to that of proliferating cell nuclear antigen was characterized in esophagi by immunohistochemistry at 0, 24, and 48 h, and 1, 3, 7, 10, and 14 weeks after NMBA treatment. As early as 24 h, proliferating cell nuclear antigen-positive focal hyperplastic lesions were detected in the suprabasal layers of ZD esophagi. At the same time, overexpression of cyclin D1, Cdk4, and Rb was found in the corresponding lesion in adjacent esophageal sections. By contrast, p16ink4a expression was reduced or absent. At all time points, p16ink4a showed reduced nuclear staining in ZD esophagi compared with that in ZS esophagi. In addition, increased expression of the hyperphosphorylated forms of Rb was detected in ZD esophagi by immunoblotting. Importantly, tumors were consistently observed in ZD esophagi at very early time points. These data, obtained using a unique in vivo model for esophageal cancer with rapid tumor induction, provide strong evidence for a link between deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and the initiation of esophageal tumors.

Animals↗
PubMed2000

Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition.

The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-alpha) in the presence of 0.5 microg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A(2) inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-alpha alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-alpha causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-alpha can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8.

Animals↗
PubMed1999

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.

The overexpression of Bax kills cells by a mechanism that depends on induction of the mitochondrial permeability transition (MPT) (Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol. Chem. 273, 7770-7775). In the present study, purified, recombinant Bax opened the mitochondrial permeability transition pore (PTP). Depending on its concentration, Bax had two distinct effects. At a concentration of 125 nM, Bax caused the release of the intermembranous proteins cytochrome c and adenylate kinase and the release from the matrix of sequestered calcein, effects prevented by the inhibitor of the PTP cyclosporin A (CSA). At this concentration of Bax, there was no detectable mitochondrial swelling or depolarization. These effects of low Bax concentrations are interpreted as the consequence of transient, non-synchronous activation of the PTP followed by a prompt recovery of mitochondrial integrity. By contrast, Bax concentrations between 250 nM and 1 microM caused a sustained opening of the PTP with consequent persistent mitochondrial swelling and deenergization (the MPT). CSA prevented the MPT induced by Bax. Increasing concentrations of calcium caused a greater proportion of the mitochondria to undergo the MPT in the presence of Bax. Importantly, two known mediators of apoptosis, ceramide and GD3 ganglioside, potentiated the induction by Bax of the MPT. The data imply that Bax mediates the opening of the mitochondrial PTP with the resultant release of cytochrome c from the intermembranous space.

Adenylate Kinase↗
PubMed1999

Tumor necrosis factor induces phosphorylation and translocation of BAD through a phosphatidylinositide-3-OH kinase-dependent pathway.

Tumor necrosis factor (TNF) induced the phosphorylation of BAD at serine 136 in HeLa cells under conditions that are not cytotoxic. BAD phosphorylation by TNF was dependent on phosphatidylinositide-3-OH kinase (PI3K) and was accompanied by the translocation of BAD from the mitochondria to the cytosol. Blocking the phosphorylation of BAD and its translocation to the cytosol with the PI3K inhibitor wortmannin activated caspase-3 and markedly potentiated the cytotoxicity of TNF. Transient transfection with a PI3K dominant negative mutant or a dominant negative mutant of the serine-threonine kinase Akt, the downstream target of PI3K and the enzyme that phosphorylates BAD, similarly potentiated the cytotoxicity of TNF. By contrast, transfection with a constitutively active Akt mutant protected against the cytotoxicity of TNF in the presence of wortmannin. Phosphorylation of BAD prevents its interaction with the antiapoptotic protein Bcl-XL. Transfection with a Bcl-XL expression vector protected against the cytotoxicity of TNF in the presence of wortmannin. The mechanism by which the inhibition of the phosphorylation of BAD is likely linked to the induction of lethal mitochondrial damage in TNF-intoxicated cells is discussed.

Androstadienes↗
PubMed1998

The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition.

Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpression of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA-preventable loss of the mitochondrial membrane potential (DeltaPsim). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of DeltaPsim and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation.

Apoptosis↗
PubMed1998

Zinc replenishment reduces esophageal cell proliferation and N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence in zinc-deficient rats.

Previous work has shown that sustained increased and decreased cell proliferation, induced by dietary zinc deficiency and caloric restriction respectively, influence the course of N-nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis in rats. The present study considered whether the increased cell proliferation and esophageal tumor incidence induced by zinc deficiency are reversed upon zinc replenishment. Weanling rats were maintained initially on a deficient diet containing 4 p.p.m. zinc. After 5 weeks, carcinogen-treated animals were given six intragastric doses of NMBA (2 mg/kg twice weekly). Controls were untreated. After the second NMBA dose, the rats were divided into three dietary groups. One group was continued on the deficient diet, while the other two groups were switched to diets containing either 75 or 200 p.p.m. zinc, with half of the members in each group fed ad libitum and half pair-fed with deficient rats. NMBA-untreated controls were similarly replenished. At various time points, esophageal cell proliferation was assessed in five animals from each group by immunohistochemical detection of cells in S phase, with in vivo 5-bromo-2'deoxyuridine labeling. At 11 weeks after the first dose, esophageal tumor incidence was greatly reduced, from 100% in the deficient group to 26 and 14% respectively in the replenished groups fed ad libitum 75 and 200 p.p.m. zinc and to 14 and 11% respectively in the replenished groups pair-fed 75 and 200 p.p.m. zinc. In addition, the number of tumors per esophagus was reduced from 9.93 +/- 4.25 in deficient rats, to a range of 0.11 +/- 0.31-0.30 +/- 0.54 in replenished animals. Following zinc replenishment, esophageal cell proliferation, as measured by labeling index (LI), the number of labeled cells and the total number of cells, was markedly decreased in NMBA-untreated and -treated esophagi as compared with those in corresponding deficient esophagi. Thus, the esophageal cell proliferation induced by zinc deficiency is reversed by zinc replenishment and replenished animals have a markedly lower incidence of esophageal tumors.

Animals↗
PubMed1998

Analysis of cell type and radiographic presentation as predictors of the clinical course of patients with bronchioalveolar cell carcinoma.

STUDY OBJECTIVES: Bronchioloalveolar carcinoma is a primary lung neoplasm of variable histopathologic, radiologic, and clinical expression. There are three cell types described in bronchioloalveolar carcinoma: Clara cells, mucin-producing cells, and alveolar type II epithelial cells. It is unclear whether these three tumor cell types are associated with a specific radiologic presentation and clinical course. In this study, we investigated whether tumor cell type, identified by transmission electron microscopy, correlated with a specific radiologic pattern, and whether tumor cell type or radiologic presentation correlated with the patient's clinical course and outcome. DESIGN: Transmission electron microscopy was used to restudy tissue blocks from the original surgical histopathologic specimens in 54 patients with primary bronchioloalveolar carcinoma diagnosed over a 10-year period (1980 to 1990). The pretreatment radiographs were reviewed in each case, and the first chest radiograph obtained at the time of the discovery of the tumor in each patient was compared with the results of the ultrastructural study. The medical records of each patient were examined to obtain pertinent radiologic, clinical, and patient outcome information. MEASUREMENT AND RESULTS: There were 32 Clara cell tumors, 10 mucin-producing cell tumors, and 1 alveolar type II epithelial cell tumor in this series. Eleven additional tumors had mixtures of two or more cell types. No statistically significant relationship was detected between tumor cell type and radiologic presentation or patient mortality pattern. There was increased mortality among patients who presented radiologically with segmental, lobar, multifocal, or diffuse disease compared with those patients exhibiting a solitary pulmonary nodule at presentation. CONCLUSION: Radiologic presentation, rather than tumor cell type, provides prognostic information that aids in predicting patient outcome.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1997

Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis.

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.

Animals↗
PubMed1997

Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition.

The present and a previous study [J. W. Snyder, J. G. Pastorino, A. M. Attie, and J. L. Farber, Am. J. Physiol. 264 (Cell Physiol. 33): C709-C714, 1993] define two mechanisms whereby ATP depletion promotes liver cell death. ATP depletion and cell death are linked by the mitochondrial permeability transition (MPT). Mitochondrial deenergization promotes the MPT, and ATP maintains a membrane potential by reversal of ATP synthase. With an increased influx of Ca2+ induced by the ionophore A-23187, oligomycin depleted the cells of ATP without loss of the mitochondrial membrane potential and further elevated the intracellular Ca2+ concentration. Cyclosporin A (CyA) prevented the accompanying cell killing. Fructose also preserved the viability of the cells. With the increased cytosolic Ca2+ imposed by A-23187, viability is maintained by ATP-dependent processes. Upon depletion of ATP, Ca2+ homeostasis cannot be maintained, and the MPT is induced. Rotenone also depleted the cells of ATP, and A-23187 accelerated the loss of the mitochondrial membrane potential occurring with rotenone alone. CyA and fructose prevented the cell killing with rotenone and A-23187. Oligomycin did not prevent this action of fructose. We conclude that ATP is needed to maintain Ca2+ homeostasis to prevent the MPT and the resultant liver cell death. ATP is also needed to maintain mitochondrial energization when electron transport is inhibited.

Adenosine Triphosphate↗
PubMed1997

Human- and mouse-inducible nitric oxide synthase promoters require activation of phosphatidylcholine-specific phospholipase C and NF-kappa B.

BACKGROUND: The production of nitric oxide by type II inducible nitric oxide synthase (type II NOS) gene is controlled at least in part by transcriptional activation. Although the murine and human type II NOS genes share significant sequence homology, they differ in the induction stimuli required for activation. MATERIALS AND METHODS: The A549 human and murine RAW 264.7 cell lines were cultured in the presence of inducers of the type II NOS gene and exposed to specific inhibitors of phosphatidyl choline-specific phospholipase C, NF-kappa B, and endocytosis, as well as to reagents that deplete stores of ATP or prevent the acidification of endosomes. The effect of these reagents on the induction of the type II NOS gene transcription, translation, and NO expression was studied using electromobility shift assays, Western blotting, and the detection of NO as nitrates, as appropriate. Additionally, the ability of the native human type II NOS NF-kappa B recognition sequence to bind NF-kappa B was compared with a concensus sequence and with a mutated oligomer. RESULTS: Type II NOS production by both human and mouse cells could be prevented by the addition of the specific inhibitor of phosphatidylcholine-specific phospholipase C, D609, and of agents that interfere with the activation of NF-kappa B. Both mouse and human cells also required acidic endosome formation and the production of 1,2-diacylglycerol for type II NOS expression. Additionally, the native human type II NOS NF-kappa B recognition sequence bound NF-kappa B with significantly less affinity than did the recognition sequence derived from the human immunoglobulin light-chain gene promoter. CONCLUSIONS: These experiments show that whereas mouse cells can be activated by lipopolysaccharide to produce nitric oxide, and human cells require activation by a mixture of cytokines to produce nitric oxide, the intracellular activation pathway following receptor binding of these heterologous stimuli is shared. Additionally, NF-kappa B activation is necessary but not sufficient for inducible nitric oxide synthase production in human cells, in contrast to murine cells in which it serves as a complete inducer.

Animals↗