Fatal duodenocaval fistula caused by peptic ulcer.

Gastrointestinal bleeding caused by a duodenocaval fistula is rare, diagnostically challenging, and associated with a high mortality rate. We describe the case of a patient with polymicrobial fungemia and fatal gastrointestinal bleeding related to a duodenocaval fistula caused by peptic ulcer. Polymicrobial fungemia, which has not previously been associated with this condition, raises the possibility of candidal endocarditis.

Azithromycin for empirical treatment of the nongonococcal urethritis syndrome in men. A randomized double-blind study.

OBJECTIVE: To evaluate the use of single-dose azithromycin for empirical treatment of nongonococcal urethritis. DESIGN: Randomized, double-blind, multicenter trial comparing azithromycin vs doxycycline therapy, with a 2:1 randomization ratio. Patients were evaluated clinically and microbiologically for Chlamydia trachomatis and Ureaplasma urealyticum infection before therapy and at 2 and 5 weeks after study entry. SETTING: Eleven sexually transmitted disease clinics throughout the United States. PATIENTS: A total of 452 men aged 18 years or older with symptomatic nongonococcal urethritis of less than 14 days' duration. INTERVENTION: Patients were treated with either 1.0 g of azithromycin as a single oral dose or 100 mg of doxycycline taken orally twice daily for 7 days. MAIN OUTCOME MEASURES: Clinical resolution of symptoms and signs of nongonococcal urethritis, microbiological cure of C trachomatis and U urealyticum, and occurrence of adverse experiences. RESULTS: Of the 452 patients enrolled, 248 in the azithromycin-treated group and 123 in the doxycycline-treated group were evaluable for clinical response. The two treatment groups were comparable in terms of age, weight, ethnic distribution, sexual preference, sexual activity, and history of prior nongonococcal urethritis or gonorrhea. Sixteen percent of the azithromycin group and 24% of the doxycycline group were culture positive for C trachomatis before therapy, while 38% and 28%, respectively, were culture positive for U urealyticum. The cumulative clinical cure rate was 81% (95% confidence interval [CI], 75% to 85%) in the azithromycin-treated group and 77% (95% CI, 69% to 84%) in the doxycycline-treated group. Clinical cure rates in the two groups were also comparable when patients were stratified by presence or absence of infection with C trachomatis or U urealyticum prior to therapy. Among those infected with C trachomatis, overall microbiological cure rates were 83% (95% CI, 65% to 94%) for azithromycin-treated patients (n = 30) and 90% (95% CI, 68% to 98%) for doxycycline-treated patients (n = 21). Among those infected with U urealyticum, overall microbiological cure rates were 45% (95% CI, 34% to 57%) for azithromycin-treated patients (n = 75) and 47% (95% CI, 30% to 65%) for doxycycline-treated patients (n = 32). Adverse reactions were generally mild to moderate and occurred in 23% of the azithromycin-treated group and 29% of the doxycycline-treated group. CONCLUSIONS: For empirical treatment of the acute nongonococcal urethritis syndrome in men, a single oral dose of azithromycin was as effective as a standard 7-day course of doxycycline in achieving clinical cure. Further, clinical cure rates were comparable with either regimen, regardless of the presence or absence of Chlamydia or Ureaplasma infection.

Regulation of hepatocyte plasma membrane alpha 1-adrenergic receptors by 4 beta-phorbol 12-myristate 13-acetate.

The effect of phorbol 12-myristate 13-acetate (PMA) on hepatocyte alpha 1-adrenergic receptors was determined by [3H]prazosin binding to plasma membranes from control and PMA-treated hepatocytes. Membranes from hepatocytes incubated with PMA (1 microgram/ml) for 1 h exhibited a 40% decrease in alpha 1-adrenergic receptors (481 +/- 10 fmol/mg of protein; mean +/- S.E.M. for three separate experiments) relative to vehicle-treated (dimethylformamide) hepatocytes (802 +/- 91 fmol/mg of protein; n = 3), with no significant effect on the KD. The PMA-induced decrease in alpha 1-adrenergic receptors was maximal by 30 min and half-maximal inhibition of [3H]prazosin binding occurred with a PMA concentration of approx. 15 ng/ml. Pretreatment of hepatocytes with staurosporine (5 microM) blocked the effect of PMA, and 4 beta-phorbol 13-monoacetate was ineffective, suggesting the involvement of protein kinase C (PKC). Treatment of hepatocytes with primaquine (300 microM) for 15 min decreased hepatocyte plasma membrane alpha 1-adrenergic receptors by 34.0 +/- 2.4% (mean +/- S.E.M. of three experiments). Removal of primaquine allowed essentially complete recovery (98 +/- 4%; mean +/- S.E.M. for five separate experiments) of plasma membrane [3H]prazosin binding within 20 min, suggesting that the alpha 1-adrenergic receptor undergoes endocytotic recycling. Addition of PMA (1 microgram/ml) to hepatocytes immediately after removal of primaquine, completely inhibited the increase in plasma membrane alpha 1-adrenergic receptors relative to control cells, but had no effect on hepatocytes whose cell surface alpha 1-receptors remaining after primaquine treatment had been inactivated by alkylation. These observations suggested that activation of PKC may facilitate the internalization of the alpha 1-adrenergic receptor in hepatocytes.

Intravenous streptomycin: a useful route of administration.

Streptomycin is an aminoglycoside antibiotic that is indicated for the treatment of tuberculous and nontuberculous infections. Intramuscular injection is the recommended route of administration. There are few reports on intravenous administration of streptomycin. We describe the use of intravenous streptomycin to treat endocarditis due to a strain of Enterococcus faecalis with high-level resistance to gentamicin. Physicians should consider the intravenous route as an alternate method of administering streptomycin.

Use of fluorescent latex microspheres to measure coronary blood flow distribution.

Radioactive microspheres have long been in use to measure blood flow distribution to various vascular beds. Their drawbacks are the short half-lives of the radioactive material, the need for appropriate care in handling and disposing of such material, and their relative expensiveness. We investigated the use of fluorescent microspheres as indicators of coronary blood flow distribution in a canine model. Radioactive (125I) microspheres were used as a comparison standard. Four colors of fluorescent microspheres were used: blue, yellow-green, orange, and red, having emission frequencies ranging from 385 to 605 nM. The experiments were carried out in dogs under pentobarbital anesthesia, in which the microspheres were given during pump perfusion of the left circumflex artery with the animal's own blood. The hearts were removed, fixed for 3 days in 10% formalin, and sectioned. Samples from the endocardial, myocardial, and epicardial layers were read on a gamma counter. The fluorescent microspheres were extracted from the same tissues into ethyl acetate, and read in a fluorescence spectrophotometer at the appropriate excitation/emission frequencies. Comparable results were obtained from the two methods, with good sensitivity and resolution of dye colors, using the fluorescent microspheres.

Rapid and reversible uncoupling of the hepatic alpha 1-adrenergic receptor and guanine-nucleotide-binding protein by phorbol 12,13-dibutyrate.

Phorbol 12,13-dibutyrate (PDBU), 1 microgram/ml for 2 min, abolished alpha 1-adrenergic receptor (AR)-mediated signal transduction in rat hepatocytes and converted 100% of alpha 1-ARs to a low-affinity state, similar to effects of a non-hydrolyzable GTP analog. Reversal of PDBU inhibition restored high-affinity alpha 1-ARs towards control values. The rapid uncoupling of the alpha 1-AR and guanine-nucleotide-binding protein by PDBU, and reversibility associated with reactivation of alpha 1-adrenergic signaling, identify this as an important inhibitory locus for PDBU.

Transforming growth factor-beta 1 rapidly activates phosphorylase in a calcium-dependent manner in rat hepatocytes.

Transforming growth factor-beta 1 (TGF-beta 1) rapidly activated phosphorylase in isolated rat hepatocytes (half-maximal rate of activation with approximately 0.1 ng/ml). Removal of Ca2+ from the external medium just before TGF-beta 1 addition markedly attenuated phosphorylase activation. TGF-beta 1 (1 ng/ml) produced a small increase in [Ca2+]i (approximately 10% increase after 30 s), which appears sufficient to account for phosphorylase activation. These observations indicate that activation of the TGF-beta 1 signal transduction system in hepatocytes is linked with a small increase in [Ca2+]i, and external Ca2+ may contribute in part to this increase.

Membrane-specific association of annexin I and annexin II in anterior pituitary cells.

Calpactins are members of the annexin family of structurally related Ca2(+)-dependent membrane binding proteins. Recent studies suggest a role for calpactins in the membrane fusion event of exocytosis. We show in this work that two members of the annexin family which are immunologically related to calpactin I (p36, annexin II) and calpactin II (p35, annexin I) are present in anterior pituitary cells. When sheep adenohypophyseal cells are disrupted in the absence of a Ca2+ chelator, immunoreactive calpactins associate with the crude vesicle fraction. Further purification of this subcellular fraction by sucrose density gradient centrifugation revealed a differential distribution: calpactin I was associated with secretory granule membranes and with plasma membranes, whereas calpactin II was found primarily with the plasma membrane fraction. Consistent with the Ca2+ and phospholipid binding properties of the calpactins, extraction of these proteins from the pituitary membranous fractions required sequential treatment with a detergent, octylglucoside, in the presence of 1 mM Ca2+ followed by solubilization with EGTA. Calpactins contain sites for phosphorylation by protein kinase C, and in this study we found phosphoprotein substrates for protein kinase C associated with secretory granule and plasma membranes which could be immunoprecipitated with calpactin antisera. In summary, the characteristics in anterior pituitary secretory cells of these two members of the annexin family lend support to the hypothesis that calpactins, potentially regulated by Ca2+ and by phosphorylation, may have a role in exocytosis.

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes.

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.

Protein kinase C phosphorylates leukemia RNA polymerase II.

Purified RNA polymerase II from chicken leukemia cells was found to be an effective substrate for protein kinase C but not cAMP-dependent protein kinase. Protein kinase C catalyzed the incorporation of 1-2 mol of phosphate per mol of polymerase II and the reaction was totally calcium and lipid dependent. Electrophoresis studies revealed a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 220 KDa, 180 KDa and 150 KDa, with a preferential phosphorylation of the 180 KDa polypeptide. The phosphorylated enzyme has a preference for using single-stranded DNA as the template for transcription, including transcription of the single-stranded myb oncogene sequence. Phosphoamino acid analysis indicated that both serine and threonine residues were phosphorylated at equal amounts. Phosphorylation by protein kinase C increased the affinity of substrate-polymerase binding and the initial rate of RNA synthesis, suggesting a mechanism by which gene expression can be activated by protein kinase C.

Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules.

Secretory granules isolated from anterior pituitary glands were examined for Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity as well as the occurrence of granule-associated substrate proteins. Sheep adenohypophyses were fractionated by differential and sucrose-density-gradient centrifugation to yield a granule fraction enriched for luteinizing-hormone (lutropin)-containing secretory granules. Marker-enzyme analysis showed no detectable cytosolic contamination, although there were small amounts of plasma membranes (2-4%) and lysosomes (4-6%) associated with the preparation. As determined by histone-H1 phosphorylation after DEAE-cellulose DE-52 chromatography, protein kinase C activity with a marked dependence on Ca2+ and lipid (4-fold increase in their presence) was evident in the secretory-granule fraction. Phosphorylation in vitro of the secretory-granule fraction by endogenous and exogenous protein kinase C revealed a protein of Mr 36,000, which by two-dimensional SDS/polyacrylamide-gel electrophoresis showed multiple sites of phosphorylation. The Mr-36,000 protein was not found in cytosolic or plasma-membrane fractions and was not phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Several secretory-granule proteins served as substrates for the catalytic subunit, the most prominent of which were of Mr 63,000, 23,000 and 21,000. From these data, we suggest that phosphorylation of secretory-granule-associated proteins by protein kinase C and by cyclic AMP-dependent protein kinase may be important in secretion regulation in the anterior pituitary gland.

Differential effects of phorbol ester on phenylephrine and vasopressin-induced Ca2+ mobilization in isolated hepatocytes.

Receptor-mediated breakdown of PtdIns(4,5)P2 produces two cellular signals, Ins(1,4,5)P3, which can release intracellular Ca2+, and diacylglycerol, which activates a Ca2+- and phospholipid-dependent protein kinase (protein kinase C). This study assesses the significance of protein kinase C in relation to phenylephrine- and vasopressin-induced Ca2+ mobilization in hepatocytes. Phorbol ester (4 beta-phorbol-12-myristate-13-acetate), which can directly activate protein kinase C, had no effect either on Ca2+ efflux from the cell (measured with arsenazo III) or on Ca2+ influx (measured with Quin-2), processes which are inhibited and stimulated, respectively, by both phenylephrine and vasopressin. No evidence of synergism between phorbol ester pretreatment of hepatocytes and the Ca2+ ionophore (ionomycin)-mediated effects on the increase of cytosolic free Ca2+ and phosphorylase activation could be obtained. These findings suggest that protein kinase C is not obligatorily involved in the regulation of hepatocyte Ca2+ fluxes. Pretreatment of hepatocytes with phorbol ester (PMA) or 1-oleoyl-2-acetylglycerol totally inhibited the effects of phenylephrine in elevating the cytosolic free Ca2+; half-maximal inhibitory effects occurred at PMA and 1-oleoyl-2-acetylglycerol concentrations of 1 ng/ml and 12 micrograms/ml, respectively. In contrast, pretreatment with PMA had a much smaller effect on Ca2+ mobilization induced by vasopressin. These observations suggest that protein kinase C may be involved in "down-regulation" of the alpha 1-receptor in hepatocytes and may thus exert a negative influence on the Ca2+-signalling pathway.

Inositol trisphosphate and diacylglycerol as intracellular second messengers in liver.

Receptor occupation by a variety of Ca2+-mobilizing hormones, such as alpha 1-adrenergic agents, vasopressin and angiotensin II, causes a rapid phosphodiesterase-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate in the plasma membrane with the production of the water soluble compound myo-inositol-1,4,5-trisphosphate (IP3) and the lipophilic molecule 1,2-diacylglycerol (DG). This review summarizes the recent evidence obtained in the liver that defines the roles of these products as intracellular messengers of hormone action. Intracellular Ca2+ mobilization is mediated by IP3, which releases Ca2+ from a subpopulation of the endoplasmic reticulum, resulting in a rapid increase of the cytosolic free Ca2+ concentration ( [Ca2+]i). Further effects of receptor occupancy are inhibition of the plasma membrane Ca2+-ATPase, despite net Ca2+ efflux, and an increased permeability of the plasma membrane to extracellular Ca2+. The activation of the phospholipid-dependent protein kinase C by DG does not alter Ca2+ fluxes across the plasma membrane. In contrast to some secretory cells, a synergism between protein kinase C activation and increased [Ca2+]i is not observed in liver. Activation of protein kinase C profoundly inhibits the response to alpha 1-adrenergic agonists, with only minimal effects on the vasopressin response. It is concluded that in liver the two inositol-lipid messenger systems, IP3 and DG, exert their effects by essentially separate pathways.

Risk of infection after penetrating abdominal trauma.

To identify the risk factors for the development of postoperative septic complications in patients with intestinal perforation after abdominal trauma, and to compare the efficacies of single-drug and dual-drug prophylactic antibiotic therapy, we studied 145 patients who presented with abdominal trauma and intestinal perforation at two hospitals between July 1979 and June 1982. Logistic-regression analysis showed that a higher risk of infection (P less than 0.05) was associated with increased age, injury to the left colon necessitating colostomy, a larger number of units of blood or blood products administered at surgery, and a larger number of injured organs. The presence of shock on arrival, which was found to increase the risk of infection when this factor was analyzed individually, did not add predictive power. Patients with postoperative sepsis were hospitalized significantly longer than were patients without infection (13.8 vs. 7.7 days, P less than 0.0001). Both treatment regimens--cefoxitin given alone and clindamycin and gentamicin given together--resulted in similar infection rates, drug toxicity, duration of hospitalization, and costs.

Phosphorylation of a 16-kDa protein by diacylglycerol-activated protein kinase C in vitro and by vasopressin in intact hepatocytes.

A 16-kDa protein present in a purified rat liver plasma membrane fraction and also in cytosol can be phosphorylated by endogenous diacylglycerol-activated protein kinase C. In intact hepatocytes prelabeled with 32P, vasopressin causes a rapid increase in the phosphorylation of a 16-kDa protein having a similar pI value to that observed in in vitro studies. These findings suggest that vasopressin-induced phosphorylation of the 16-kDa in the intact hepatocyte may reflect increased activity of protein kinase C, secondary to membrane polyphosphoinositide breakdown. Phosphorylation of the 16-kDa protein may thus be part of the coordinated mechanism associated with hormonal regulation of cellular Ca2+ fluxes.

Mechanisms underlying calcium homeostasis in isolated hepatocytes.

The steady state relationship between intra- and extramitochondrial free Ca2+ across the inner mitochondrial membrane has been investigated in isolated liver mitochondria. The extramitochondrial free Ca2+ concentration was essentially independent of the mitochondrial calcium content above 4 nmol/mg of protein. Below this value, a decrease in the mitochondrial calcium content was accompanied by a decrease in the extramitochondrial free Ca2+ concentration. The experimental data are compatible with a model in which the steady state distribution of calcium is described in terms of the kinetic parameters of the separate carriers catalyzing Ca2+ influx and efflux across the mitochondrial inner membrane. The corresponding relationship between cytosolic free Ca2+ concentration and the amounts of calcium in the mitochondria and endoplasmic reticulum was investigated in isolated river cells over a range of cellular Ca2+ contents by using a nondisruptive technique based on the selective release of calcium from mitochondrial and total cellular pools by addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and A23187, respectively. A net increase in cell calcium from 1 to 5 nmol/mg dry weight, increased the cytosolic free Ca2+ concentration from 0.1 to about 0.3 microM and increased the calcium contents of both mitochondria and endoplasmic reticulum. Above 5 nmol of calcium/mg cell dry weight, the endoplasmic reticulum calcium pool became filled, and further increases in calcium content were accounted for by increases of the mitochondrial pool but no further increase of the cytosolic free Ca2+ concentration. These studies and experiments with mixtures of isolated microsomes and mitochondria suggest that, in cells as normally isolated (containing 5 to 6 nmol of calcium/mg dry weight), the endoplasmic reticulum is saturated with calcium and is unlikely to play a major role as an intracellular calcium buffer. The in situ mitochondrial calcium content is sufficiently high (approximately 16 nmol/mg of protein) for these organelles to buffer effectively the cytosolic free Ca2+ concentration at a value of about 0.3 microM. In addition, it may be concluded that intramitochondrial Ca2+-dependent enzymes will be exposed to saturating concentrations of free Ca2+.