Biweekly administration of docetaxel and carboplatin for advanced or recurrent endometrial and ovarian carcinomas.

OBJECTIVE: To examine efficacy and safety of biweekly administration of docetaxel and carboplatin for advanced or recurrent en- dometrial and ovarian carcinomas. MATERIAL AND METHODS: The recommended doses were determined in the phase I study. In the phase II feasibility study, the primary end-point was safety, and the secondary end-point was response rate and progression-free survival (PFS). RESULTS: The recommended doses of docetaxel and carboplatin were determined to be 45 mg/n(2) and AUC 3.0, respectively, in phase I study. In phase II feasibility study, no treatment-related death was observed. Most non-hematotoxicity cases were mild or moderate. Grade 4 neutropenia was confirmed in 13 patients (31.0%), whereas all cases showed tolerability with 2.6 days delay of anticancer drugs administration in both groups. Response rate was 55.0% in the ovarian carcinoma group, and average PFS was 8.7 months. In the endometrial carcinoma group, response rate was 50.0% and average PFS was 32.0 months. CONCLUSION: The present results showed that biweekly administration of docetaxel and carboplatin for advanced and recurrent endometrial and ovarian carcinomas results in acceptable side effects, response rate, and PFS.

Crystal structure and enzymatic activity of an ADAMTS-13 mutant with the East Asian-specific P475S polymorphism.

BACKGROUND: An East Asian-specific P475S polymorphism in the gene encoding ADAMTS-13 causes an approximately 16% reduction in plasma ADAMTS-13 activity. OBJECTIVES: To demonstrate the impact of this dysfunctional polymorphism by characterizing the structure and activity of the P475S mutant protein. METHODS: We determined the crystal structure of the P475S mutant of ADAMTS-13-DTCS (DTCS-P475S, residues 287-685) and compared it with the wild-type structure. We determined the enzymatic parameters of ADAMTS-13-MDTCS (residues 75-685) and MDTCS-P475S, and further examined the effects of denaturants and reaction temperature on their activity. We also examined the cleavage of shear-treated von Willebrand factor (VWF) by MDTCS-P475S. RESULTS: MDTCS-P475S showed a reaction rate similar to that of wild-type MDTCS, but showed two-fold lower affinity for the peptidyl substrate, indicating that the Pro475-containing V-loop (residues 474-481) in the CA domain is a substrate-binding exosite. Structural analysis showed that the conformation of the V-loop was significantly different in DTCS-P475S and the wild type, where no obvious interactions of Ser475 with other residues were observed. This explains the higher susceptibility of the enzymatic activity of MDTCS-P475S to reaction environments such as denaturants and high temperature. MDTCS-P475S can moderately cleave shear-treated VWF. CONCLUSIONS: We have provided structural evidence that the P475S polymorphism in ADAMTS-13 leads to increased local structural instability, resulting in lowered affinity for the substrate without changing the reaction rate. The moderate activity of ADAMTS-13-P475S for shear-treated VWF is sufficient to prevent thrombotic thrombocytopenic purpura (TTP) onset.

Carvedilol accelerate elevation of serum potassium in chronic heart failure patients administered spironolactone plus furosemide and either enalapril maleate or candesartan cilexetil.

OBJECTIVE: To retrospectively investigate the effect of carvedilol and spironolactone plus furosemide, administered concomitantly with an angiotensin II converting enzyme inhibitor (ACE-I) or an angiotensin II receptor blocker (ARB) to patients with chronic heart failure (CHF). METHODS: Patients with CHF, who visited Departments of Cardiovascular Internal Medicine at the National Hospital Organization Osaka Medical Center, were enrolled for this study. Serum potassium, blood urea nitrogen (BUN), serum creatinine (Scr) and serum sodium were measured in every patient at the time of start of treatment and after 3 and 12 months of treatment. Data from patients in groups A (20 mg/day carvedilol + 25 mg/day spironolactone + 40 mg/day furosemide + an ACE-I) and B (20 mg/day carvedilol + 25 mg/day spironolactone + 40 mg/day furosemide + ARB) were compared. RESULTS: When 20 mg/day carvedilol plus 25 mg/day spironolactone plus 5 mg/day enalapril maleate (enalapril, group A) or 8 mg/day candesartan cilexetil (candesartan, group B) plus 40 mg/day furosemide were used concomitantly, the mean serum potassium increased significantly in both groups of patients. Seven of 59 (11.9%) patients had hyperkalemia (>5.5 mEq/L) during 12 months of treatment whereas 8.5% of patients (five of 59) had hypokalemia (< or =3.5 mEq/L). CONCLUSION: When carvedilol is used concomitantly with spironolactone, furosemide and enalapril or candesartan, it is necessary to monitor serum potassium concentration, even if spironolactone is administered at a low dose of 25 mg/day.

Nitric oxide and cyclic guanosine monophosphate stimulate apoptosis via activation of the Fas-FasL pathway.

BACKGROUND: Inappropriately exaggerated response of pulmonary vascular cells to inflammatory mediators may be one mechanism that leads to acute (or adult) respiratory distress syndrome. Nitric oxide (NO) is induced following such exaggerated responses and may have a variety of biological effects, including induction of apoptosis. The mechanism by which NO causes apoptosis is unknown; however, Fas (CD95) and Fas ligand (FasL) (CD95L) have been implicated. We hypothesized that NO-induced apoptosis in pulmonary vascular smooth muscle cells is mediated through a Fas-FasL pathway. MATERIALS AND METHODS: Cultured human and rat pulmonary artery smooth muscle cells (PASMCs) were exposed to soluble FasL (0-5 ng/ml), the NO donor S(G)-nitroso-N-acetyl pencillamine (SNAP) (0-50 microg/ml), and/or anti-FasL (0-100 microg/ml) for 12 h. Apoptosis was measured using in situ DNA nick end labeling and flow cytometry. Changes in Fas and FasL protein levels were assessed via Western blot analysis. Messenger RNA (mRNA) abundance of apoptosis-related genes was determined using a ribonuclease protection assay. RESULTS: Rat PASMCs exposed to FasL show a dose-dependent increase in apoptosis. Human PASMCs are less responsive to FasL. Addition of anti-FasL to rat PASMCs treated with 10(-5) M SNAP decreases apoptosis levels compared to SNAP treated alone. FasL and Fas receptor proteins are increased in response to 10(-3) to 10(-4) M SNAP or 10(-6) M 8-bromo-cyclic guanosine monophosphate (cGMP). The mRNA abundance of Fas, FasL, and other apoptosis-related genes is increased in response to 10(-6) M 8-bromo-cGMP but not 8-bromo-cyclic adenosine monophosphate. CONCLUSIONS: Nitric oxide-induced apoptosis in rat and human PASMCs is mediated, at least in part, through the Fas-FasL pathway, with cGMP increasing the expression of Fas and FasL.

Proliferation and functional changes of pancreatic gastrin cells in neonatal rat.

INTRODUCTION: Although gastrin cells are not found in the adult pancreas, they are found transiently in the neonatal pancreas. It has been suggested that gastrin may play a role in pancreatic development. However, cell kinetics as well as the fate and the role of gastrin cells are not clear. METHODOLOGY: Proliferation and functional changes of pancreatic gastrin cells in neonatal Wister rats were studied by immunohistochemistry and [(3)H]thymidine autoradiography. RESULTS: Numbers of pancreatic gastrin cells in neonatal rats showed a peak immediately after birth and then decreased rapidly. Gastrin cells were observed within approximately 2 weeks after birth in islets and within approximately 4 weeks after birth among exocrine cells. In contrast with the decrease of gastrin cell numbers, numbers of duodenal cholecystokinin cells increased remarkably after 7 days of age. Proliferative activity of acinar cells showed two peaks at age 2 days and 9 days. Despite a decrease in gastrin cell numbers, gastrin cells maintained a certain degree of proliferative activity. The "re-staining method" for gastrin and insulin revealed that immunoreactive cells for both gastrin and insulin were rarely found a few days after birth. CONCLUSION: These results suggest that pancreatic gastrin cells do not die off or change to another type of endocrine cell and that some gastrin cells change to insulin cells.

Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro.

An in vitro assay system to detect tyrosylprotein sulfotransferase (TPST) activity of higher plant cells was established, using synthetic oligopeptides based on the deduced amino acid sequence of a phytosulfokine-alpha (PSK-alpha) precursor. TPST activity was found in microsomal membrane fractions of rice, asparagus and carrot cells and it was confirmed that acidic amino acid residues adjacent to the tyrosine residues of acceptor peptides were essential to the sulfation reaction. The asparagus TPST exhibited a broad pH optimum of 7.0-8.5, required manganese ions for maximal activity and appeared to be a membrane-bound protein localized in the Golgi apparatus. These enzymes should be defined as a new class of plant sulfotransferases that catalyze tyrosine O-sulfation of a PSK-alpha precursor and other unknown proteins.

Cyclic nucleotides and inducible nitric oxide synthesis in pulmonary artery smooth muscle.

BACKGROUND: Nitric oxide (NO), cGMP, and cAMP affect the synthesis, metabolism, and cellular effects each other. We wanted to study how cGMP and cAMP interact to affect the induced synthesis of NO in response to interleukin-1 beta (IL-1 beta) in rat pulmonary artery smooth muscle cells. To further dissect the relative contributions of each cyclic nucleotide, and to detect any possible "crossover" effect of one cyclic nucleotide activating the other protein kinase, we tested how pharmacological inhibition of cGMP-dependent and cAMP-dependent protein kinases (PKG and PKA, respectively) affected responses. MATERIALS AND METHODS: We tested the effects of IL-1 beta, dibutyryl (db)-cAMP (1-100 micro) and 8-bromo (Br)-cGMP (1 microM-1 mM) on NO synthesis in cultured rat pulmonary artery smooth muscle cells. Positive effects were then tested in the presence of KT5720 (10(-9)-10(-5) M), the pharmacological inhibitor of PKA, and KT5823 (10(-9)-10(-5) M), the pharmacological inhibitor of PKG. NO production was measured using the Greiss reaction, and mRNA abundance of the inducible NO synthase (iNOS), using semiquantitative RT-PCR. RESULTS: IL-1 beta caused nitrite levels to increase nearly 10-fold over basal levels at 24 h (P < 0.05). Nitrite levels increased with the addition of either db-cAMP (100 microM, an 8-fold increase) or 8-Br-cGMP (100 microM, a 3-fold increase) to IL-1 beta (P < 0.05). PKA inhibition with KT5720 (10(-5) M) completely inhibited NO synthesis in response to the combination of IL-1 beta and cAMP, while KT5823 had less effect at all doses tested. NO synthesis in response to IL-1 beta plus cGMP also decreased to PKA inhibition, but not PKG inhibition, indicating that cGMP responses are a crossover effect. Both cAMP and cGMP in combination with IL-1 beta increased iNOS mRNA abundance above basal levels on reverse transcription polymerase chain reaction. KT5720, but not KT5823, decreased iNOS mRNA to basal levels. CONCLUSION: Both cAMP and cGMP augment cytokine induction of NO synthesis through activation of PKA: cAMP does so directly; cGMP, through a crossover stimulation of PKA.

Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells.

Nitric oxide (NO), a product of certain cytokine-activated cells, affects rates of apoptosis, a mechanism of programmed cell death. We asked whether NO affected rates of apoptosis in pulmonary vascular cells. Using rat pulmonary artery smooth muscle cells, we studied direct effects of the NO donor SG-nitroso-acetyl-D,L-penicillamine (SNAP) and the effects of NO endogenously synthesized in response to bacterial lipopolysaccharide (LPS) and inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha (a combination called cytomix for convenience). We determined apoptosis on the basis of light microscopy and the bromodeoxyuridine terminal deoxynucleotidyl transferase reaction (BrdUTdT). Both SNAP- and cytomix-induced synthesis of NO resulted in histologic evidence of apoptosis based upon fluorescence microscopy using propidium iodide. SNAP (10(-5) M) increased BrdUTdT-positive cells from 17.5 to 78.4% compared with basal medium alone, with the maximal response occurring at 15 h or exposure. Exposing cells to LPS and cytokines induced NO production (from 0.1 +/- 0.1 to 24.6 +/- 0.5 microM, P < 0.05) caused cytological changes consistent with apoptosis and led to an increase of increased BrdUTdT-positive cells from 11 to 41% at 12 h compared with basal medium alone. The competitive NO synthase inhibitor NG-monomethyl-L-arginine inhibited both NO synthesis and NO apoptosis, returning the proportion of BrdUTdT-positive cells (6%) to levels below control. L-Arginine (0.5 mM) restored percentages to those increase in response to endogenously synthesized NO, and NO is a potential mechanism of acute lung injury in response to inflammatory cytokines.

Sustained nitric oxide exposure decreases soluble guanylate cyclase mRNA and enzyme activity in pulmonary artery smooth muscle.

BACKGROUND: The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanoside 3':5'-cyclic monophosphate (cGMP). Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung. Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilatation, and septic shock. In the pulmonary bed the opposite response, pulmonary hypertension, may occur. We hypothesized that sGC activity decreases in the face of sustained levels of NO. MATERIALS AND METHODS: We used the NO-donor S-nitroso-acetyl-D-L-penicillamine to study the effects of NO on sGC mRNA abundance and enzyme activity in cultured rat pulmonary artery smooth muscle cells. RESULTS: NO caused a prompt rise in extracellular cGMP production. Pretreating cells with NO for >/=45 min inhibited subsequent cGMP synthesis. NO-pretreated cells recovered the capacity for cGMP synthesis after removal of NO for 120 min. When actinomycin or cycloheximide was added to NO pretreatment, cells retained cGMP synthetic capacity. NO pretreatment decreased sGC mRNA abundance, but did not totally eliminate it. CONCLUSION: NO has important regulatory effects on cGMP synthesis at the level of enzyme activity and mRNA abundance. NO causes an immediate synthesis of large amounts of cGMP. With prolongation of exposure (>/=60 min) sGC enzyme activity decreases and cGMP production drops significantly. Soluble GC mRNA abundance also decreases and may result in decreased responsiveness of cells to NO with regard to cGMP production.

Monozygotic twins discordant for single umbilical artery and congenital heart disease.

We report a case of monozygotic twins. One twin had a single umbilical artery and the other co-twin had congenital heart disease. This case and a review of the literature suggest that local environmental factors are important in the pathogenesis of these malformations although genetic factors cannot be excluded.

Nitric oxide inhibits peroxide-mediated endothelial toxicity.

BACKGROUND: Oxidant molecules and nitric oxide (NO) have each been implicated as mediators of endothelial cell damage, but the biologic effect of these molecules acting in concert is incompletely understood. MATERIALS AND METHODS: We studied the effects of an NO donor, S-nitroso-acetyl-D,L-penicillamine (SNAP), in combination with the peroxidants tert-butyl hydroperoxide (TBH) and hydrogen peroxide (H2O2) on rabbit aortic endothelial cells in culture. Cell viability was assessed using Alamar blue, a nontoxic dye indicator of cell metabolism. Lipid peroxidation was assessed using a chemiluminescent single-photon counting technique. RESULTS: After 90 min exposure to test reagents, there was concentration-dependent cytotoxicity for both TBH and H2O2. Peroxidant-induced cytotoxicity was significantly ameliorated by SNAP (10(-4)-10(-3)M). N-Acetylpenicillamine and NO-depleted SNAP failed to demonstrate a cytoprotective effect against peroxidant cellular injury, thus implicating NO as the agent responsible for the protective effect. SNAP reduced lipid peroxidation caused by 10(-3) M TBH in a dose-dependent manner. Preincubation of cells with SNAP before exposure to peroxidants alone had no effect on toxicity. CONCLUSIONS: NO is cytoprotective to the endothelium in the presence of peroxidants through a reduction of lipid peroxidation.

Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle.

The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanosine 3':5'-cyclic monophosphate (cGMP). Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung. Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilation, and septic shock. In the pulmonary bed the opposite response may occur, pulmonary hypertension. We hypothesized that sGC activity becomes downregulated in the face of Escherichia coli lipopolysaccharide (LPS). We tested the effects of LPS on alpha1-subunit sGC mRNA abundance, Western analysis, and enzyme activity in cultured rat pulmonary artery smooth muscle cells. LPS increased extracellular cGMP production by pulmonary artery smooth muscle cells, with increased levels being first detectable at 3-6 h (10 microg/ml LPS) and exceeding 140 pmol/ml by 24 h (P < 0.05). The response was inhibited by 0.05 mM l-NG-monomethyl-l-arginine (l-NMA) and, in turn, restored by 1 mM l-arginine, indicating a NO synthase-dependent response. Pretreating cells with LPS for >/= 3 h inhibited subsequent cGMP synthesis in response to 10(-4) M SNAP for 60 min. Coincubating cells with 0.05 mM l-NMA also reversed this effect. Soluble GC enzyme activity in cells exposed to basal medium alone measured 0.74 pmol cGMP/ml per minute; activity in cells exposed to 10 microg/ml LPS for 24 h decreased to 0.04 pmol cGMP/ml per minute (P < 0.05). LPS pretreatment decreased sGC mRNA abundance and protein mass, but did not totally eliminate them. It is concluded that LPS affects cGMP synthesis at the level of enzyme activity, enzyme mass, and mRNA abundance. Over the short term (<24 h) LPS causes the synthesis of large amounts of cGMP. As the duration of exposure progresses (>/=3 h), mechanisms come into play that decrease cGMP production significantly and include decreases in mRNA abundance, enzyme mass, and enzyme activity.

Lymphoid hyperplasia causing recurrent rectal prolapse.

An 8-year-old girl had a 5-month history of recurrent rectal prolapse. On colonoscopy, two submucosal masses were noted in the distal rectum and diagnosed by biopsy as benign lymphoid hyperplasia. These were excised by limited dissection superficial to the submucosa, and the histologic diagnosis was confirmed. The child has done well after removal of the nodules, with no subsequent prolapse for more than 2 years.

Efficacy of inhaled nitric oxide in oleic acid-induced acute lung injury.

OBJECTIVE: To assess the efficacy of inhaled nitric oxide in improving pulmonary hypertension and gas exchange following oleic acid-induced acute lung injury. DESIGN: Prospective, pharmacologic study. SETTING: Surgical research laboratory at the University of Pittsburgh, Pittsburgh, PA. SUBJECTS: Instrumented, intubated pigs weighing 16 to 27 kg. INTERVENTIONS: Intravenous oleic acid and inhaled nitric oxide. MEASUREMENTS AND MAIN RESULTS: All pigs treated with intravenous oleic acid (0.11 mL/kg) developed a severe lung injury with pulmonary hypertension, accompanied by impaired oxygenation, intrapulmonary shunting, and increased extravascular lung water (p < .05 compared with baseline). Following nitric oxide inhalation, although pulmonary hypertension decreased in a dose-dependent fashion, no amelioration in pulmonary gas exchange was observed, as reflected by PaO2 and intrapulmonary shunt. Plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, did not increase following nitric oxide exposure in this model of severe lung injury. CONCLUSIONS: The effect of inhaled nitric oxide, restricted to relieving pulmonary vasoconstriction in this model of lung injury, may have limited benefit in improving pulmonary gas exchange when diffusion is impaired by severe lung injury and inflammatory thickening of the alveolar-capillary barrier. Nitric oxide inhalation may have better results when used at an earlier, less severe stage of acute lung injury.

Nitric oxide and the pulmonary artery smooth muscle cell.

Nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS), which exists in different isoforms in various tissues. The inducible NOS (iNOS) isoform of the enzyme is expressed in vascular smooth muscle in response to lipopolysaccharide and inflammatory mediators. When this expression of iNOS occurs in the lung, the NO produced may play a role in the inflammatory process of acute lung injury. This article reviews the research that characterizes iNOS in rat pulmonary artery smooth muscle and discusses current investigation into the role of NO in sepsis and injury.