Obesity and incidence of cancer: a large cohort study of over 145,000 adults in Austria.

We investigated the relation of overweight and obesity with cancer in a population-based cohort of more than 145 000 Austrian adults over an average of 9.9 years. Incident cancers (n=6241) were identified through the state cancer registry. Using Cox proportional-hazards models adjusted for smoking and occupation, increases in relative body weight in men were associated with colon cancer (hazard rate (HR) ratio 2.48; 95% confidence interval (CI): 1.15, 5.39 for body mass index (BMI) > or =35 kg m(-2)) and pancreatic cancer (HR 2.34, 95% CI: 1.17, 4.66 for BMI>30 kg m(-2)) compared to participants with normal weight (BMI 18.5-24.9 kg m(-2)). In women, there was a weak positive association between increasing BMI and all cancers combined, and strong associations with non-Hodgkin's lymphomas (HR 2.86, 95% CI: 1.49, 5.49 for BMI> or =30 kg m(-2)) and cancers of the uterine corpus (HR 3.93, 95% CI: 2.35, 6.56 for BMI> or =35 kg m(-2)). Incidence of breast cancer was positively associated with high BMI only after age 65 years. These findings provide further evidence that overweight is associated with the incidence of several types of cancer.

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities.

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.

Neuronal N-type calcium channel blockers: a series of 4-piperidinylaniline analogs with analgesic activity.

Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.

Structure-activity relationship at the leucine side chain in a series of N,N-dialkyldipeptidyl-amines as N-type calcium channel blockers.

Exploration of the SAR around the leucine side chain in a series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC is presented. A novel analog is disclosed which possesses improved aqueous solubility, in vivo activity in an audiogenic seizure model, and reversible blockade in electrophysiological assays.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Synthesis and biological activity of substituted bis-(4-hydroxyphenyl)methanes as N-type calcium channel blockers.

Voltage activated calcium channel (VACC) blockers have been demonstrated to have utility in the treatment of stroke and pain. A series of aminomethyl substituted phenol derivatives has been identified with good functional activity and selectivity for N-type VACC's over sodium and potassium channels. The methods of synthesis and preliminary pharmacology are discussed herein.

Structure-activity relationship at the proximal phenyl group in a series of non-peptidyl N-type calcium channel antagonists.

Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.

Structure-activity relationship of N-methyl-N-aralkyl-peptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.

N,N-dialkyl-dipeptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.

Accidental pleural puncture by a thoracic epidural catheter.

We report the occurrence of an accidental pleural puncture by an epidural catheter that happened during the attempted induction of thoracic epidural anaesthesia using a paramedian approach in an awake patient. The incorrect placement of the catheter was recognised while the patient was undergoing thoracoscopic surgery. The possibility of accidental pleural puncture during attempted thoracic epidural catheter placement by either the paramedian or the midline approach should be borne in mind. A misplaced catheter may injure lung tissue and result in a potentially dangerous intra-operative tension pneumothorax.

Accuracy and clinical performance of a continuous intra-arterial blood-gas monitoring system during thoracoscopic surgery.

Accuracy and performance of the only currently available intra-arterial blood-gas monitoring system (Paratrend 7, PT7) were assessed in 23 patients during thoracoscopic surgery using one-lung ventilation. Over a wide range of values for arterial PO2 (6.1-61.1 kPa), PCO2 (4.1-9.5 kPa) and pH (7.19-7.50), 138 arterial blood-gas values obtained by PT7 were compared with corresponding in vitro laboratory blood-gas measurements. We found good clinical performance with the PT7 and good agreement between PT7 values and in vitro measurements for arterial PO2 (bias (1.96 SD) = 0.38 (9.52) kPa), PCO2 (0.31 (0.76) kPa) and pH (-0.017 (0.065)). Also, the bias for sequential changes between two, consecutive times was not significantly different from the ideal value of 0. We conclude that the PT7 is helpful in monitoring patients during thoracoscopy.

Video-assisted thoracoscopic volume reduction surgery in patients with diffuse pulmonary emphysema: gas exchange and anesthesiological management.

Arterial blood gases were studied prospectively using continuous intraarterial blood gas monitoring during thoracoscopic volume reduction surgery (VRS) in 24 patients with advanced diffuse pulmonary emphysema. Additionally, the early postoperative course (48 h) of arterial blood gases was studied retrospectively. Twenty-six operations were performed using a combination of thoracic epidural and general anesthesia with left-sided double-lumen intubation for one-lung ventilation (OLV). Arterial blood gases were determined awake, during two-lung ventilation prior to surgery, during OLV (extreme values), and after tracheal extubation. Additionally, the extremes during the whole procedure were determined: avoiding excessive peak inspiratory pressures (26.4 +/- 7.0 cm H2O), minimum PaO2 was 77 +/- 39 mm Hg (mean +/- SD), maximum PaCO2 65 +/- 14 mm Hg (P < 0.0001 versus preoperative values), and minimum pHa 7.22 +/- 0.08 (P < 0.0001). One tension pneumothorax occurred during OLV. Immediate postoperative extubation was performed in 25 of 26 cases, reintubation was necessary in two cases. One patient with coronary artery disease died 36 h after surgery. Hypercapnia (maximum PaCO2 49 +/- 8 mm Hg, minimum pHa 7.37 +/- 0.04, P < 0.01) was still observed 48 h after surgery. These results demonstrate that adequate oxygenation can be preserved during OLV for VRS, but CO2 elimination is impaired. However, intraoperative hypercapnia and immediate postoperative tracheal extubation are well tolerated.

Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus.

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Characteristics of [125I]omega-conotoxin MVIIA binding to rat neocortical membranes.

[125I]omega-Conotoxin MVIIA (omega-CTM) binding to N-type voltage-sensitive calcium channels (VSCCs) was characterized using rat neocortical membranes. [125I]omega-CTM bound rapidly and with high affinity; these parameters were similar to binding using omega-conotoxin GVIA ([125I]omega-CTG). Unlike [125I]omega-CTG, however, [125I]omega-CTM readily dissociated from its binding site. Monovalent and divalent cations, polyamines, and aminoglycosides inhibited [125I]omega-CTM binding. Since [125I]omega-CTM appears to bind to the same site as [125I]omega-CTG in mammalian neurons, the reversibility of [125I]omega-CTM binding makes this ligand preferable for equilibrium binding analyses.

Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils.

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

Cyclic AMP inhibits secretion from electroporated human neutrophils.

It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.

Lipocortins are major substrates for protein kinase C in extracts of human neutrophils.

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.

Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins.

Investigations of protein kinase C (PKC) activity have focussed on protein phosphorylation using adenosine triphosphate (ATP), not guanosine triphosphate (GTP), as the phosphate donor. In a continuing study of the enzymology of the PKC of human neutrophils, we wanted to determine if there might be protein kinases that do use GTP as a phosphate donor. Soluble extracts or detergent-extracted fractions of human neutrophils were used as enzyme sources. Phosphorylation of histone using [gamma-32P]-GTP was 31% as effective as [gamma-32P]-ATP. Phosphorylation with GTP depended on Ca2+, Mg2+, and phospholipid, just as the ATP, and the Ca2+ requirements were similar. In all cases, H-7, an inhibitor of ATP-supported PKC activity, blocked GTP-utilizing activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that similar endogenous proteins were phosphorylated with ATP or GTP. The apparent Km and Vmax for the enzyme(s) for both phosphate donors were identical, although these were modified by treatment with Triton X-100. GTP competitively inhibited use of ATP by PKC; however, low concentrations of ATP enhanced GTP-utilizing kinase activity in some cases. Non-hydrolyzable forms of ATP and other nucleotide triphosphates were inhibitory. Detergent treatment also markedly altered the number of proteins phosphorylated by either nucleotide. The major protein phosphorylated in the soluble or detergent extract was a single polypeptide band in the 34 Kd range. These studies are the first to explicitly examine the possible phosphorylation by neutrophil PKC using GTP and point to a potential alternative mode of enzyme activity. Since high concentrations of GTP are available within neutrophils, the ability of PKC or a PKC-like enzyme to use this nucleotide may have important ramifications in signal transduction.