Isolation of calicivirus from the joint of a kitten with arthritis. Calicivirus was isolated from the joint of a kitten that had pyrexia, upper respiratory tract disease, and severe shifting-limb lameness. Marked mononuclear inflammation was found in the synovial fluid. Calicivirus infection or live-virus vaccination previously had been associated with arthropathy, but virus had not been recovered from affected joints. Calicivirus infection should be considered as a diagnosis in kittens with fever and arthralgia, especially if there is a history of recent vaccination or upper respiratory tract disease. Lameness associated with calicivirus infection may be severe, but appears to be self-limiting and is associated with an excellent prognosis for recovery.


Pituitary tumor size, neurologic signs, and relation to endocrine test results in dogs with pituitary-dependent hyperadrenocorticism: 43 cases (1980-1990). Pituitary neoplasm was identified in 43 dogs with pituitary-dependent hyperadrenocorticism via necropsy (n = 33), diagnostic imaging with computerized tomography or magnetic resonance imaging (n = 5), or diagnostic imaging and necropsy (n = 5). All dogs had clinical signs and clinicopathologic test results typical of hyperadrenocorticism. Thirty-seven dogs had grossly visible pituitary tumors, and 6 dogs had microscopic pituitary tumors. Fifteen dogs had developed neurologic signs typical of those resulting from an enlarging pituitary mass. Twenty-three dogs had pituitary tumors greater than or equal to 1 cm in diameter. Provocative testing of the pituitary-adrenocortical axis was performed on all dogs. Dogs with grossly visible pituitary tumors and dogs with neurologic signs had significantly (P less than 0.05) higher mean plasma endogenous ACTH concentrations, compared with values from dogs with microscopic tumors and dogs without neurologic signs, respectively. Dogs with grossly visible pituitary tumors and dogs with tumors greater than or equal to 1 cm in diameter had significantly (P less than 0.05) lower adrenocortical responsiveness to exogenous ACTH, compared with dogs with microscopic pituitary tumors and dogs with tumors less than 1 cm in diameter, respectively. Despite these differences, there was overlap between test results among dogs. On the basis of endocrine test results, it would appear difficult to distinguish dogs with pituitary-dependent hyperadrenocorticism and large pituitary tumors from those with pituitary-dependent hyperadrenocorticism and microscopic pituitary tumors prior to onset of neurologic signs.


Preparation and characterization of a murine monoclonal antibody (MDR3M) reactive with mdr3 gene product. A murine monoclonal antibody (MDR3M) (isotype: IgM) reactive with mdr3 gene product was generated by immunizing mice with mdr3-specific peptide (H2N-12WRPTSAEGDFELGISSKQKRKKTKTVKMI41G-COOH) and hybridizing the primed mouse splenic B cells with X63-Ag8,6.5.3 mouse plasmacytoma cells. MDR3M did not cross-react with mdr1 gene product. This monoclonal antibody may be useful for analyzing the role of mdr3 gene product in cells and tissues.


New hepatocellular carcinoma cell line SUHC-1 established from a patient with hepatitis C virus RNA in serum. A new human hepatocellular carcinoma cell (HCC) line, designated SUHC-1, was derived from a Japanese patient with hepatocellular carcinoma having antibody to hepatitis C virus (HCV) and HCV-RNA in his serum, and established in tissue culture. This cell line exhibited typical epithelial cell morphology in culture as observed by phase-contrast and electron microscopy. The SUHC-1 cells produced albumin and alpha 2-macroglobulin. Chromosomal analysis showed several rearrangements at short and long arms of chromosome 1, 17 and 20 (1p-, 1q-, i(1q), i(17q) and 20q+) with a modal number of 91. HCV-RNA was not detected in the supernatant of SUHC-1 cells by nested polymerase chain reaction assay or in the SUHC-1 cells by the in situ hybridization method. We concluded that complete HCV does not exist in the SUHC-1 cell line. The SUHC-1 cell line is the first line of HCC to have been derived from a patient with persistent HCV infection, and may provide a suitable model for studies of hepatocarcinogenesis related to HCV.


The performance of gilts in a new group housing system: endocrinological and immunological functions. The effect of a new group housing system on performance (132 gilts and litters) and endocrinological (35 gilts) and immunological functions (28 gilts) was studied. Animals were randomly assigned to a conventional system (control), involving greater than 2 mo in individual stalls, or to the Hurnik-Morris (H-M) housing system, involving continuous housing in small groups, for breeding-gestating swine. The gilts were reared throughout gestation in their respective housing systems and moved 3 to 5 d prefarrowing to a common farrowing facility. Various production data were collected, including sow weight and backfat measurements, number of pigs born, number born alive, number weaned, litter birth weight, and litter weaning weight. An adrenal function test using dexamethasone pretreatment and ACTH1-24 challenge was imposed on gilts 5 d prebreeding and once between d 81 to 87 of gestation. Plasma progesterone was measured at the same time. Immune function was measured by serum antibody response to hen egg white lysozyme (HEWL) and delayed-type hypersensitivity (DTH) to tuberculin. Gilts reared in the H-M housing system exhibited a number of pigs weaned per litter and litter weaning weights comparable to the number and weights in the control system (7.3 +/- .33 vs 6.9 +/- .38, P = .421 and 56.9 +/- 2.42 kg vs 51.3 +/- 2.76 kg, P = .132, respectively). Prefarrowing and weaning backfat measurements were significantly reduced in group-housed gilts (15.8 +/- .45 mm vs 17.8 +/- .55 mm, P = .005 and 14.6 +/- .4 mm vs 16.2 +/- .42 mm, P = .008, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship of fetal position within the uterus to fetal weight, placental weight, testosterone, estrogens, and thymosin beta 4 concentrations at 70 and 104 days of gestation in swine. Fetal intrauterine position relative to the sex of adjacent fetuses has an effect on reproductive performance in rodents. An experiment was conducted to determine whether sex of adjacent fetuses in utero has an influence on fetal and placental weights and whether the hormonal mechanisms documented in rodents are similar in fetal pigs. Sows were slaughtered at 70.1 +/- 1.7 d (n = 123) and 104.5 +/- .05 d (n = 135) of gestation. The fetuses and placentas were removed from the uterus and the position and sex of each fetus was recorded to indicate whether the fetus was between two males, two females, or a male and a female. Fetal blood was sampled for later hormonal analysis. At 70 d of gestation, male fetal and placental weights were heavier than those of females (P less than .05), but no differences were detected relative to the sex of adjacent fetuses. At 104 d of gestation, a fetus surrounded on each side in utero by fetuses of the opposite sex (two males or two females) was lighter in weight than a fetus surrounded by fetuses of the same sex (P less than .01). Differences in fetal weight due to the sex of adjacent fetuses were not related to placental function because placental weights were generally not different at 104 d of gestation. By 104 d of gestation, most placentas were not separated by necrotic regions and were in close apposition with surrounding placentas. No differences in growth or development could be related to hormonal effects (testosterone, estrone, or estrone sulfate) from surrounding fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of hydrated sodium calcium aluminosilicate on fescue toxicosis and mineral absorption. The possibility of supplementing livestock diets with an aluminosilicate to protect them from fescue toxicosis was investigated. An in vitro study showed that hydrated sodium calcium aluminosilicate (HSCAS) removed greater than 90% of the ergotamine from aqueous solutions at pH 7.8 or lower, indicating a high affinity of ergotamine for HSCAS in vitro. Rats fed diets containing tall fescue seed infested (E+) with the endophytic fungus Acremonium coenophialum had lower (P less than .05) feed intakes and weight gains than did rats fed diets containing uninfested (E-) tall fescue seed. When feed intake by rats fed the E- seed diet was limited to that of rats fed the E+ seed diet, weight gains did not differ, but testes weights and serum prolactin (PRL) concentrations were lower (P less than .05 and .10, respectively) in rats receiving E+ seed. Supplementing E+ seed diets with HSCAS did not eliminate effects of E+ seed on intake, PRL, or testes weights. Sheep fed E+ tall fescue hay had higher (P less than .05) rectal temperatures than did sheep fed an equal amount of E- tall fescue hay, but OM and N digestion coefficients did not differ between the two hays. Supplementing E+ hay diets with HSCAS did not eliminate the effect of E+ hay on rectal temperatures. Addition of 2% HSCAS to tall fescue hay diets did not affect apparent absorption by sheep of OM, N, Ca, P, Na, K, or Cu, but it reduced (P less than .05) the apparent absorption of Mg, Mn, and Zn.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of alkaline hydrogen peroxide treatment on cell wall composition and digestion kinetics of sugarcane residues and wheat straw. Our objective was to characterize changes in cell wall composition and digestibility of sugarcane bagasse, pith from bagasse, and wheat straw after treatment with alkaline hydrogen peroxide (AHP). The AHP treatment solution contained 1% H2O2 (wt/vol) maintained at pH 11.5 with NaOH. The H2O2 in solution amounted to 25% of the quantity of substrate treated. After treatment, residues were washed and dried. Detergent fiber composition, total fiber components (neutral sugars, uronic acids, Klason lignin, and noncore lignin phenolic acids), IVDMD, in vitro digestion kinetics of NDF, and monosaccharide digestibilities (24 and 120 h) were determined. Total fiber (TF) and NDF concentrations of all treatment residues were increased (P less than .05) over control substrates by AHP because of greater losses of cell solubles than of cell wall constituents. Hemicellulose:cellulose ratio in NDF of treatment residues was decreased (P less than .05) by AHP for all substrates, but the neutral sugar composition of TF did not agree with this preferential loss of hemicellulose components. Klason lignin, ADL, and esterified noncore lignin, especially ferulic acid, were reduced (P less than .05) by AHP, whereas etherified noncore lignin composition was unchanged. Treatment increased (P less than .05) IVDMD, extent of NDF digestion, and monosaccharide digestibilities of all crop residues. The rate of NDF digestion was increased (P less than .05) for the sugarcane residues but not for wheat straw. Alkaline hydrogen peroxide improved crop residue digestibility, probably as a result of the removal of core and noncore lignin fractions.


Intergeneric conjugation in Thiobacillus versutus. In plate matings with Escherichia coli HB101/pUW965::Tn5 (KmR) Thiobacillus versutus reacted as an efficient recipient, producing 10(-2) to 10(-3) kanamycin resistant (KmR) T. versutus exconjugants per donor cell. Analysis of agarose gels of plasmid DNA extracted from the exconjugants confirmed that the suicide vector pUW964 did not persist in the recipient, implying that the kanamycin resistance of the exconjugants is based on effective transposition of Tn5 in T. versutus as well as function of the E. coli kanamycin gene. Transfer was equally efficient when a nalidixate-resistant T. versutus mutant was used as recipient. Hybridization evidence for the presence of Tn5 was consistently negative. The significance of this anomalous result is discussed.


Isolation of inclusion bodies from vegetative Clostridium perfringens: partial purification of a 47 kDa inclusion protein. A refractile inclusion body produced by vegetative cells of Clostridium perfringens at temperatures above 40 degrees C was isolated and partially characterized. The inclusion was composed of protein and could be solubilized by sodium dodecyl sulphate plus either dithiothreitol or beta-mercaptoethanol. The solubilized inclusion showed no antigenic relationship with Cl. perfringens enterotoxin. One major band with an apparent MW of 47 kDa was demonstrated after polyacrylamide gel electrophoresis of the solubilized inclusion. Both enterotoxin-positive and enterotoxin-negative strains produced the inclusion body. No effect on the morphology of several eucaryotic cell lines was observed when solubilized or intact inclusion was added to the cell cultures.


Development changes to gut microflora metabolism in mice. Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and beta-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4-24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal beta-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or beta-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice. We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.


Effects of itraconazole on phagocytosis and killing of Candida glabrata by polymorphonuclear leucocytes from guinea pigs. Itraconazole, a systemically active antifungal, was tested for its effects on microscopically assessed phagocytosis and killing of Candida glabrata 233 in vitro. Yeast cells were exposed to itraconazole in culture and guinea-pig peritoneal polymorphonuclear leucocytes were exposed to the drug injected intraperitoneally in vivo. At a concentration of 10(-7) M and with exposure times of 1 h, itraconazole pre-treatment of the leucocytes had no effect on the ability of PMNL to ingest or kill C. glabrata. However, pre-treatment of the growing C. glabrata cells under the same conditions significantly increased their vulnerability to both phagocytosis and intracellular killing. Longer exposures of the yeasts to itraconazole further increased their susceptibility to leucocyte phagocytosis, and it also rendered the cells vulnerable to killing merely by immersion in sodium deoxycholate solution. These findings indicate that short exposures of C. glabrata to low itraconazole concentrations damages the cells sublethally and renders them highly susceptible to leucocyte killing. Itraconazole had no direct effects on leucocyte function itself.


Pharmacokinetics and biliary elimination of temafloxacin in pigs. The purpose of the present work was to investigate the 48 h pharmacokinetic and biliary elimination of temafloxacin, administered intraduodenally at a single dose of 10 mg/kg to six conscious pigs provided with a double terminal choledocal fistula allowing the collection and the reinstillation of bile. The concentrations of temafloxacin in biological fluids were measured by both HPLC and bioassay. The mean serum half-life of temafloxacin was 11.45 +/- 4.4 h and the apparent oral clearance (assuming a 95% bioavailability) was 171 +/- 66 mL/min. The penetration of temafloxacin into bile is significant as indicated by a bile/serum AUC ratio of 9.1 +/- 2.2. The 0-45 h biliary excretion amounted to 2.002 +/- 0.8 mg, representing 0.9 +/- 0.3% of the administered dose. Biliary peak concentrations ranged from 8.0 to 37.5 mg/L and the 24 h mean biliary concentration was 3.7 +/- 2.4 mg/L. No statistically significant differences between HPLC and bioassay concentrations or AUCs could be observed in bile, indicating that temafloxacin is not extensively biotransformed into active metabolites in this model. These data, together with data in man, suggest that temafloxacin could be a useful agent for the treatment of biliary tract infections.


The in-vitro activity of new streptogramins, RP 59500, RP 57669 and RP 54476, alone and in combination. RP 59500 is a 30:70 mixture of RP 57669 and RP 54476. The activity of RP 59500 and its two components against Gram-positive and Gram-negative organisms was compared with that of clarithromycin, roxithromycin, azithromycin and rokitamycin. RP 59500 inhibited 90% of erythromycin-susceptible and resistant Staphylococcus aureus and coagulase-negative staphylococci at less than or equal to 1 mg/L (range 0.06-2 mg/L). Both inducibly and constitutively-resistant strains of S. aureus, as well as strains resistant to rifampicin, gentamicin and ciprofloxacin, were inhibited. Streptococcus pyogenes, including erythromycin-resistant isolates, and group C and G streptococci were inhibited by 0.5 mg/L. Streptococcus pneumoniae and viridans group streptococci were inhibited by 1 mg/L. The MIC90 was 4 mg/L for Haemophilus influenzae and 1 mg/L for Moraxella catarrhalis. RP 59500 did not inhibit Enterobacteriaceae or Pseudomonas aeruginosa. The activity of RP 59500 against streptococci was less than that of the four other macrolides. Clostridium perfringens strains were highly susceptible, as were Bacteroides spp. RP 59500, when combined with ciprofloxacin, cefotaxime or gentamicin, did not have altered activity against susceptible species or alter the activity of the other component of the combination against susceptible species. MBCs in serum were increased two- to four-fold for S. pyogenes, S. pneumoniae and S. aureus, compared with MBCs in broth, but RP 59500 was as active at pH 6 as at pH 7, and there was not an appreciable inoculum effect. RP 59500 has potential use as an agent against inducibly and constitutively erythromycin-resistant isolates of Gram-positive species and selected anaerobic organisms.


Differential effects of general anesthesia on cGMP-mediated pulmonary vasodilation. We investigated the effects of an intravenous (pentobarbital sodium) and an inhalational (halothane) general anesthetic on guanosine 3',5'-cyclic monophosphate- (cGMP) mediated pulmonary vasodilation compared with responses measured in the conscious state. Multipoint pulmonary vascular pressure-flow plots were generated in the same nine dogs in the fully conscious state, during pentobarbital sodium anesthesia (30 mg/kg iv), and during halothane anesthesia (approximately 1.2% end tidal). Continuous intravenous infusions of bradykinin (2 micrograms.kg-1.min-1) and sodium nitroprusside (5 micrograms.kg-1.min-1) were utilized to stimulate endothelium-dependent and -independent cGMP-mediated pulmonary vasodilation, respectively. In the conscious state, both bradykinin and nitroprusside decreased (P less than 0.01) the pulmonary vascular pressure gradient (pulmonary arterial pressure-pulmonary arterial wedge pressure) over the entire range of flows studied; i.e., bradykinin and nitroprusside caused active flow-independent pulmonary vasodilation. Pulmonary vasodilator responses to bradykinin (P less than 0.01) and nitroprusside (P less than 0.05) were also observed during pentobarbital anesthesia. In contrast, during halothane anesthesia, the pulmonary vasodilator responses to both bradykinin and nitroprusside were abolished. These results indicate that, compared with the conscious state, cGMP-mediated pulmonary vasodilation is preserved during pentobarbital anesthesia but is abolished during halothane anesthesia.


Urinary leukotriene E4 excretion in exercise-induced asthma. Recent evidence suggests that the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) may be important in the pathogenesis of exercise-induced asthma. To evaluate the role of these mediators further, nine asthmatic subjects with exercise-induced bronchoconstriction were studied on two occasions. On visit 1, subjects performed 6 min of treadmill exercise; the mean maximal percent fall in FEV1 was 38.0 +/- 5.3%. On visit 2, maximal bronchoconstriction observed after exercise was matched with aerosolized methacholine. Urine was collected in two 90-min fractions (0-90 and 90-180 min) after challenges and analyzed by high-performance liquid chromatography-radioimmunoassay for LTE4. There were no significant differences in urinary LTE4 excretion between exercise and methacholine challenges for the periods 0-90 min (16.9 +/- 5.4 vs. 20.4 +/- 4.2 ng/mmol urinary creatinine), 90-180 min (24.9 +/- 8.2 vs. 20.1 +/- 5.5), or 0-180 min (21.5 +/- 6.5 vs. 18.8 +/- 4.1). Thus in contrast to allergen-induced bronchoconstriction, there is little evidence for enhanced cysteinyl-leukotriene generation in exercise-induced bronchoconstriction as assessed by urinary LTE4. If local release and subsequent participation of functionally active cysteinyl-leukotrienes in the pathways that ultimately lead to bronchoconstriction after exercise challenge do occur, these are of insufficient magnitude to perturb urinary LTE4 excretion.


Alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do monocytes. Alveolar macrophages release greater amounts of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) after A23187 stimulation than do blood monocytes. The mechanisms for this enhanced 5-lipoxygenase activity in alveolar macrophages are unknown. In these studies, we determined whether alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do blood monocytes. We confirmed that alveolar macrophages released greater amounts of LTB4 after A23187 stimulation than did equivalent numbers of blood monocytes. In both the presence and absence of A23187, alveolar macrophages had greater amounts of immunoreactive 5-lipoxygenase, determined by Western analysis, on a per cell and a per protein basis than did blood monocytes. The amounts of 5-lipoxygenase enzyme in the cells roughly correlated with the amounts of LTB4 released by both types of cells. These observations suggest that A23187 stimulates alveolar macrophages to release greater amounts of LTB4 and 5-HETE than blood monocytes, in part, due to the greater amounts of 5-lipoxygenase.


Clinical spectrum of viral infections in hemophilic patients. Although both hemophilia A and B do not impair immunity, the use of human blood products to treat bleeding has resulted in a potential exposure to numerous viral agents. The course of infections with HIV, hepatitis B, and hepatitis C are described in this article, with comments on the interaction of these viruses in the hemophilic host. The impact of these infections on the family and community is also described.


Treatment of venous thromboembolism with low molecular weight heparins. The accumulating evidence indicates that certain LMW-heparins administered subcutaneously may replace classical intravenous heparin therapy. Certain of these subcutaneously administered LMW-heparins do not require monitoring. The simplified care offered by LMW-heparin therapy offers the possibility of transferring care from in the hospital to out of the hospital in uncomplicated patients with deep vein thrombosis. The advantages to the patient of avoiding in-hospital care and its associated hazards are obvious. Outpatient LMW-heparin therapy will likely prove to be highly cost-effective. It is uncertain at the present time whether the findings associated with an individual LMW-heparin preparation can be extrapolated to a different LMW-heparin. For this reason the findings of clinical trials apply only to the particular LMW-heparin evaluated and cannot be generalized to the LMW-heparins at large.


Deep negative T waves associated with reversible left ventricular dysfunction in acute adrenal crisis. We report two cases of reversible left ventricular dysfunction associated with deep negative T waves during acute adrenal crisis due to isolated deficiency of adrenocorticotrophic hormone. There were no symptoms suggestive of heart disease in either case and left ventricular wall motion abnormalities, present mainly around the left ventricular apex, returned to normal in 1-2 weeks. Deep negative T waves normalized 4 weeks after corticosteroid administration. Acute adrenal crisis should be considered when deep negative T waves are associated with left ventricular dysfunction without cardiac symptoms.


Cardiovascular actions of OPC-18790: a novel positive inotropic agent with little chronotropic action. OPC-18790 [(+/-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]- 2(1H)-quinolinone], a novel positive inotropic agent, was investigated in several in vitro and in vivo experiments to elucidate its cardiovascular effects and its mechanism of action. In isolated blood-perfused dog heart preparations, OPC-18790 increased contractile force at 10 to 1,000 nmol i.a.; increased coronary arterial blood flow at 30 to 1,000 nmol; and decreased sinus rate slightly at 1,000 nmol. Atrio-ventricular nodal conduction was slightly facilitated with OPC-18790 (10 to 1,000 nmol), whereas ventricular automaticity tended to decrease. OPC-18790 (10(-6) to 10(-4) M) increased contractile force in isolated ventricular muscles of dogs, cats, rabbits and guinea pigs but not rats. OPC-18790 increased left ventricular contractile force dose-dependently in anesthetized open-chest dogs and in conscious dogs with slight or no changes in heart rate and blood pressure. The positive inotropic effect of OPC-18790 was not affected by beta-blockade. OPC-18790 (10(-5) to 10(-4) M) prolonged the duration of action potential in guinea pig papillary muscles. Na+, K(+)-ATPase was not inhibited, but peak-III phosphodiesterase (low Km cyclic AMP specific fraction, inhibited by cyclic GMP) was inhibited by OPC-18790 (IC50 = 0.41 x 10(-6) M) in dog myocardium. However, such an inhibitory action of phosphodiesterase can hardly be reconciled with the lack of a positive chronotropic effect shown by OPC-18790. Thus, these results suggest that OPC-18790 may have an additional mechanism. The cardiovascular effects revealed by this study suggest that OPC-18790 may exert a beneficial effect in the treatment of congestive heart failure.


Intracardiac thrombus in murine Coxsackievirus B3 myocarditis. We studied the appearance of intracardiac mural thrombi with time and the relationship between thrombosis and congestive heart failure (CHF) in murine coxsackievirus B3 (CB3) myocarditis. Four- to six-week-old C3H/He mice were inoculated intraperitoneally with CB3 and were observed for 90 days. Mice were sacrificed periodically on days 4, 8, 14, 30, and 90. Among 129 mice with myocarditis, 35 (27.1%) developed CHF and 40 (31.0%) demonstrated thrombi after day 8. The total incidence of thrombosis was significantly higher in mice with CHF (71.4%; 25/35) than in those without CHF (16.0%; 15/94) (P less than 0.001). The present study suggests that CB6 myocarditis carries a significant risk of thromboembolism, and that CHF is a risk factor for the appearance of thrombi.


Neurochemistry and child and adolescent psychiatry. This article reviews some of the neurochemistry and neurophysiology of three neurotransmitters: dopamine, norepinephrine, and serotonin. These neurotransmitters are selected because they appear to be involved in the regulation of several important behavioral systems that help regulate the interaction of the organism with its external environment, because many of the psychotropic drugs' modes of action may be result from their effects on these neurotransmitter systems, and because the majority of neurochemical studies in child psychiatry have focused on these three neurotransmitters. After the review of the neurotransmitter systems, neurochemical studies in several child psychiatric disorders are reviewed to illustrate possible biochemical/behavioral relationships in child psychiatry.


Diagnostic value of CT in the detection of brain metastasis in small cell lung cancer patients. Fifty-seven patients with histologically proven small cell lung carcinoma were prospectively evaluated for signs of brain metastasis by neurological examination and computerized tomographic (CT) brain scanning. The reliability of demonstrating brain metastases by means of neurological examination was compared with that of CT. Three (5%) patients, two with cerebrovascular infarcts and one with leptomeningeal metastases, were excluded from the study. Four (7%) patients, all with extensive disease, showed neurological symptoms and signs of brain metastases, which were confirmed in three cases by brain CT. The fourth patient had neurological symptoms and signs pointing to cerebral metastasis, but no sign of this was detected on CT at the time of diagnosis. However, six months later, after completion of chemotherapy, CT revealed signs of brain metastasis. The other fifty neurologically asymptomatic patients had no brain metastases on CT. This clinical study suggests that routine CT of the brain is not useful in neurologically asymptomatic patients with small cell lung carcinoma.


Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism. Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.


Evidence that a plasmid from a hyperthermophilic archaebacterium is relaxed at physiological temperatures. A plasmid of 3.45 kb (pGT5) was recently discovered in a strain of hyperthermophilic archaebacterium which was isolated from samples collected in a deep-sea hydrothermal vent. This strain (GE5) grows within a temperature range of 68 to 101.5 degrees C, and we show here that it contains a strong ATP-dependent reverse gyrase activity (positive DNA supercoiling). By comparison with eubacterial plasmids of known superhelical densities, we estimated the superhelical density of the archaebacterial plasmid pGT5 to be -0.026 at 25 degrees C. The equation which relates the change of the rotation angle of the DNA double helix with temperature was validated at 95 degrees C, the optimal growth temperature of the GE5 strain. Considering these new data, the superhelical density of plasmid pGT5 was calculated to be -0.006 at the physiological temperature of 95 degrees C, which is close to the relaxed state. This finding shows that the DNA topology of a plasmid isolated from a hyperthermophilic archaebacterium containing reverse gyrase activity is strikingly different from that of typical eubacterial plasmids.


Gene structure of Enterococcus hirae (Streptococcus faecalis) F1F0-ATPase, which functions as a regulator of cytoplasmic pH. Enterococcus hirae (formerly Streptococcus faecalis) ATCC 9790 has an F1F0-ATPase which functions as a regulator of the cytoplasmic pH but does not synthesize ATP. We isolated four clones which contained genes for c, b, delta, and alpha subunits of this enzyme but not for other subunit genes. It was revealed that two specific regions (upstream of the c-subunit gene and downstream of the gamma-subunit gene) were lost at a specific site in the clones we isolated, suggesting that these regions were unstable in Escherichia coli. The deleted regions were amplified by polymerase chain reaction, and the nucleotide sequences of these regions were determined. The results showed that eight genes for a, c, b, delta, alpha, gamma, beta, and epsilon subunits were present in this order. Northern (RNA) blot analysis showed that these eight genes were transcribed to one mRNA. The i gene was not found in the upper region of the a-subunit gene. Instead of the i gene, this operon contained a long untranslated region (240 bp) whose G + C content was only 30%. There was no typical promoter sequence such as was proposed for E. coli, suggesting that the promoter structure of this species is different from that of E. coli. Deduced amino acid sequences suggested that E. hirae H(+)-ATPase is a typical F1F0-type ATPase but that its gene structure is not identical to that of other bacterial F1F0-ATPases.


Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster. DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.


Decreased function of the class B tetracycline efflux protein Tet with mutations at aspartate 15, a putative intramembrane residue. The aspartate 15 residue within the first predicted intramembrane helix of the tetracycline efflux protein Tet has been conserved in four tetracycline resistance determinants from gram-negative bacteria. Its replacement in class B Tet by tyrosine, histidine, or asparagine resulted in a 60 to 85% loss of tetracycline resistance and a similar loss of tetracycline-proton antiport. The tyrosine and histidine substitutions lowered the Vmax of the efflux system by some 90% but did not alter the Km. The asparagine substitution raised the Km over 13-fold, while the Vmax was equal to or greater than that of the wild type. Therefore, although the nature of its role is unclear, aspartate 15 is important for normal Tet function.


Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding. The mercury resistance (mer) operon of the gram-negative transposon Tn21 encodes not only a mercuric reductase and regulatory genes but also two inner membrane proteins (MerT and MerC) and a periplasmic protein (MerP). Although the merT, merP, and merC genes have been implicated in Hg(II) transport, the individual roles of these genes have not been established. We created in vitro precise deletion and frameshift mutations that eliminated each of the genes singly and in combination. Our results show that both merT and merP are required for Hg(II) binding but that merC is not. Both merT and merP are required for full expression of Hg(II) resistance, but loss of merP is less deleterious than loss of merT. Furthermore, mutations eliminating both merT and merP decrease resistance more than the single mutations do. In contrast, mutating merC had no effect on Hg(II) resistance. Both the merT and merP mutations increase the threshold Hg(II) concentration for induction of merA-lacZ transcriptional fusions and cause an increase in the maximal expression level. In contrast, the merC mutation had little effect on the threshold inducing concentration of Hg(II) but decreased the level of expression. Our results show that merT and merP alone are sufficient to specify a mercury transport system. The role of merC remains obscure.


Cloning, sequencing, and expression of the pantothenate kinase (coaA) gene of Escherichia coli. Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. The structural gene (coaA) located at 90 min of the Escherichia coli chromosome was cloned and sequenced. The coaA gene was transcribed in the opposite direction to the flanking genes birA and thrU and produced a single 1.1-kb transcript. Translation of the coaA gene produced two protein products (36.4 and 35.4 kDa) that differed by eight amino acids at the amino terminus. The poor homology of the coaA promoter region to consensus E. coli promoter sequences and the low frequency of optimal codon usage (0.565) were consistent with the low abundance of pantothenate kinase. Strains containing multiple copies of the coaA gene possessed 76-fold-higher specific activity of pantothenate kinase; however, there was only a 2.7-fold increase in the steady-state level of CoA. These data corroborate the conclusion that regulation of pantothenate kinase activity by feedback inhibition is the critical factor controlling the intracellular CoA concentration.


arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon. In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype). Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d). The Ts- growth phenotype was also shown to be associated with previously described cyd alleles. In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide. Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature. Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background. Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3). Two extragenic suppressors were shown to be null alleles in either arcA or arcB. One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function. Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.


Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium. The genes required for use of ethanolamine as a carbon and nitrogen source are encoded by a single operon (eut) whose expression is induced by the simultaneous presence of both ethanolamine and cobalamin (vitamin B12). The action of B12 as an inducer of this operon reflects the fact that this cofactor is required by the degradative enzyme ethanolamine lyase (eutBC). The eutR gene encodes a protein that activates transcription of the eut operon in response to the simultaneous presence of B12 and ethanolamine. The eutR gene is expressed by a weak constitutive promoter activity (PII) and by the main regulated promoter (PI). Because it is encoded within the operon that it activates, the EutR protein controls its own production. Initial induction of the eut operon by ethanolamine plus B12 causes an increase in expression of the eutR gene; this increase acts as part of a positive feedback loop that is required for maximal operon expression. Because of this mode of regulation, constitutive regulatory mutations, described here, include mutations that generate new internal promoters and thereby increase the basal level of eutR gene expression. In mutants with an increased level of activator protein, each inducer (B12 or ethanolamine), presented singly, is sufficient for partial operon induction.


Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.


Two transcriptionally active OmpR mutants that do not require phosphorylation by EnvZ in an Escherichia coli cell-free system. D55Q-T83A and D55Q-G94S, two pseudorevertants of the D55Q mutant OmpR, an Escherichia coli transcriptional activator, were isolated previously by R. Brissette, K. Tsung, and M. Inouye (J. Bacteriol. 173:3749-3755, 1991). These pseudorevertant OmpR proteins were purified and examined for their function as transcriptional activators in a cell-free system with an ompF DNA fragment. These proteins were transcriptionally active, even after acid treatment, whereas the wild-type OmpR was completely inactive after the same treatment. Phosphorylation of acid-treated wild-type OmpR with an EnvZ11 membrane fraction and ATP restored transcriptional activity, whereas the activities of the mutant OmpR proteins did not change after phosphorylation.


Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus. Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe.


Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403. A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.


Identification of tms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine 1-phosphate uridyltransferase in Bacillus subtilis. The temperature-sensitive Bacillus subtilis tms-26 mutant strain was characterized biochemically and shown to be defective in N-acetylglucosamine 1-phosphate uridyltransferase activity. At the permissive temperature (34 degrees C), the mutant strain contained about 15% of the wild-type activity of this enzyme, whereas at the nonpermissive temperature (48 degrees C), the mutant enzyme was barely detectable. Furthermore, the N-acetylglucosamine 1-phosphate uridyltransferase activity of the tms-26 mutant strain was much more heat labile in vitro than that of the wild-type strain. The level of N-acetylglucosamine 1-phosphate, the substrate of the uridyltransferase activity, was elevated more than 40-fold in the mutant strain at the permissive temperature compared with the level in the wild-type strain. During a temperature shift, the level of UDP-N-acetylglucosamine, the product of the uridyltransferase activity, decreased much more in the mutant strain than in the wild-type strain. An Escherichia coli strain harboring the wild-type version of the tms-26 allele on a plasmid contained increased N-acetylglucosamine 1-phosphate uridyltransferase activity compared with that in the haploid strain. It is suggested that the gene for N-acetylglucosamine 1-phosphate uridyltransferase in B. subtilis be designated gcaD.


Further evidence that transposition of Tn5 in Escherichia coli is strongly enhanced by constitutively activated RecA proteins. We have shown that excision and transposition of Tn5 in Escherichia coli are greatly increased by recA(Prtc) genes, which encode constitutively activated RecA proteins (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). Contrary results, showing a significant decrease in Tn5 transposition under SOS conditions, were subsequently reported (M. D. Weinreich, J. C. Makris, and W. S. Reznikoff, J. Bacteriol. 173:6910-6918, 1991). We have extended our studies to examine the following: (i) transposition of Tn5 from sites in the phoA, phoB, proC, trpD, and ilvD genes; (ii) the effect of gene transcription; (iii) the comparative effect of dinD+ and dinD(Def) alleles; (iv) the use of a mating-out assay of transposition; (v) the effect of a recA(Prtc) allele located at the normal chromosomal site; and (vi) the effect at 41.5 degrees C of the recA441(Prtc) allele. The new results fully confirm our previous conclusions, including the fact that the frequency of Tn5 transposition under constitutive SOS conditions is site dependent.


Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli. Anaerobic expression of the tdcABC operon of Escherichia coli requires cyclic AMP and the catabolite gene activator protein (CAP). Purified CAP binds to a 30-bp sequence in the tdc promoter between positions -55 and -26, and a mutant CAP site with base substitutions at positions -48, -47, and -45 failed to bind CAP and also drastically reduced the beta-galactosidase expression from a tdcB'-'lacZ fusion plasmid. Recently, we showed that efficient expression of the tdc operon also requires a functional integration host factor (IHF) and an IHF-binding site in the tdc promoter between positions -118 and -88. The levels of beta-galactosidase activity from the tdcB'-'lacZ fusion plasmids were also reduced in an IHF-deficient strain with the wild-type or mutant plasmid CAP sequence. In vitro footprinting experiments revealed that CAP and IHF occupy their specific binding sites on tdc DNA when they are present separately or together. These regulatory proteins also induced significant bending of the tdc promoter DNA. Our results suggest that CAP and IHF act in concert as positive transcription factors for tdc operon expression in vivo.


Conditional growth of Escherichia coli caused by expression of vaccinia virus DNA topoisomerase I. Active vaccinia virus topoisomerase I is expressed in Escherichia coli containing plasmid p1940 (S. Shuman, M. Golder, and B. Moss, J. Biol. Chem. 263:16401-16407, 1988). Growth curves showed a decline of 2 to 3 logs in the number of viable cells at 42 degrees C after shift from 30 degrees C because of increased vaccinia virus topoisomerase I level. Mutations in the gyrA and gyrB genes allowed cells to grow equally well at 42 and 30 degrees C. The presence of gyrase inhibitor also improved growth at 42 degrees C.


Permeation of neutral molecules through calcium channel in sarcoplasmic reticulum vesicles. Permeation of neutral molecules as well as Ca2+ through the Ca2+ channel in sarcoplasmic reticulum vesicles has been studied by the tracer and/or by the light scattering methods. In the absence of KCl, the Ca2+ channel was found not to be able to pass neutral molecules such as glucose, xylose, and glycine under the condition that the channel was open, although the channel could pass Ca2+. On the other hand, submolar concentrations of KCl made the channel become permeable to neutral molecules as well as Ca2+. Since the effect of KCl could be replaced by NaCl and KNO3, but not by sucrose and glucose, this effect of KCl is considered to be due to ionic strength and not to osmotic pressure. These results suggest that low ionic strength transforms the Ca2+ channel protein in such a manner as to block the permeation of neutral molecules without modifying the gating mechanism of the channel.


Structural characterization of the dihydropyridine receptor-linked calcium channel from porcine heart. Ca(2+)-channel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691-1707 of the rabbit cardiac alpha 1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle alpha 2 delta subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca(2+)-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 55, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250-270 kDa (approximately 270 kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (alpha 1) is responsible for photoaffinity labeling with [3H]diazepine, since minor photolabeled approximately 270 kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (alpha 2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with alpha 1 through disulfide linkage and the other was not covalently associated with any other component.(ABSTRACT TRUNCATED AT 250 WORDS)


Mitochondrial porin can be translocated across both endoplasmic reticulum and mitochondrial membranes. Mitochondrial porin is a major integral membrane protein of the outer membrane. To assess the stop-transfer sequence in the yeast porin molecule (P), we constructed the following chimeric proteins. (i) The signal sequence of interleukin 2, a secretory protein, was fused to the amino-terminus of porin (SP). (ii) The matrix targeting presequence of cytochrome c oxidase subunit IV was fused to the amino-terminus of porin (CP). (iii) The amino-terminal segment consisting of 42 amino acid residues of "70 kDa protein" of yeast mitochondria, a major membrane protein of the outer membrane, was introduced into the middle portion of interleukin 2 (IL70). These chimeric proteins were expressed with an in vitro transcription-translation system and their integration into microsomal membrane or mitochondrial membranes was examined. When the proteins were synthesized in vitro with wheat germ cell-free system in the presence of rough microsomal membrane (RM), SP was completely translocated across the membrane, processed by the signal peptidase, and glycosylated. The translocation of IL70 molecule across RM was stopped at the introduced amino-terminal segment of 70 kDa protein. The authentic porin did not interact with the microsomal membrane. To assess the interaction with mitochondria, porin and CP were synthesized with the reticulocyte lysate system and subjected to posttranslational import reaction with isolated rat liver mitochondria. The authentic porin was integrated into the outer membrane in an alkali-resistant fashion. CP was imported into the mitochondria and its presequence was cleaved by the processing protease in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)


A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state. A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.


Inhibition of CDP-DG: inositol transferase by inostamycin. Inostamycin, a novel microbial secondary metabolite, inhibited [3H]inositol and 32P1 incorporation into phosphatidylinositol (PtdIns) induced by epidermal growth factor (EGF) in cultured A431 cells, the IC50 being 0.5 micrograms/ml, without inhibiting macromolecular synthesis. The drug inhibited cellular inositol phosphate formation only when it was added at the same time as labeled inositol. It was found to inhibit in vitro CDP-DG:inositol transferase activity of the A431 cell membrane, the IC50 being about 0.02 micrograms/ml. It did not inhibit tyrosine kinase, PtdIns phospholipase C, or PtdIns kinase. Therefore, inhibition of PtdIns turnover by inostamycin must be due to the inhibition of CDP-DG:inositol transferase. Thus, inostamycin is a novel inhibitor of CDP-DG:inositol transferase.


The Na,K-ATPase.

The Na,K-ATPase. The energy dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na+ into and for K+ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an alpha-subunit of 112-kD and a beta-subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the alpha-subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na+ and K+.


Na,K-ATPase: isoform structure, function, and expression. An interesting feature of the Na,K-ATPase is the multiplicity of alpha and beta isoforms. Three isoforms exist for the alpha subunit, alpha 1, alpha 2, and alpha 3, as well for the beta subunit, beta 1, beta 2, and beta 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the alpha subunit are present in the brain, while only alpha 1 is present in kidney and lung, and alpha 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na+ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell. Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the alpha subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural features of cation transport ATPases. Several cation transport ATPases, sharing the common feature of a phosphorylated intermediate in the process of ATP utilization, are compared with respect to their subunit composition and amino acid sequence. The main component of these enzymes is a polypeptide chain of MW slightly exceeding 100,000, comprising an extramembranous globular head which is connected through a stalk to a membrane-bound region. With reference to the Ca2+ ATPase of sarcoplasmic reticulum, it is proposed that the catalytic (ATP binding and phosphorylation) domain resides in the extramembranous globular head, while cation binding occurs in the membrane region. Therefore, these two functional domains are separated by a distance of approximately 50 A. Alignment of amino acid sequences reveals extensive homology in the isoforms of the same ATPases, but relatively little homology in different cation ATPases. On the other hand, all cation ATPases considered in this analysis retain a consensus sequence of high homology, spanning the distance between the phosphorylation site and the preceding transmembrane helix. It is proposed that this sequence provides long-range functional linkage between catalytic and cation-binding domains. Thereby, translocation of bound cation occurs through a channel formed by transmembrane helices linked to the phosphorylation site. Additional sequences at the carboxyl terminal provide regulatory domains in certain ATPases.


An alignment of 17 deduced protein sequences from plant, fungi, and ciliate H(+)-ATPase genes. Seventeen protein sequences of H(+)-ATPases from plants (Arabidopsis thaliana, Nicotiana plumbaginifolia, Lycopersicum esculentum), fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Zygosaccharomyces rouxii, Neuropora crassa, Candida albicans), and a parasitic ciliate (Leishmania donovani) have been aligned. Twenty sequence fragments were identified which were conserved in H(+)-, Na+/K(+)-, and Ca++ plasma membrane-ATPases. In addition, a total of 118 residues not located in these fragments were found to be conserved in all H(+)-ATPases. Among those, 38 amino acid residues were screened out as being priority targets for site-directed mutagenesis experiments aimed at the identification of the amino acid residues specifically involved in cation specificity.


The Ca(2+)-transport ATPases from the plasma membrane. The initial studies on the plasma membrane (PM) Ca(2+)-transport ATPases were made in the erythrocyte, a structure that can not be taken as representing a typical eukaryotic cell. In other cell types however, the study of the PM Ca(2+)-transport ATPase is complicated by the simultaneous expression of related Ca(2+)-pumps in intracellular stores. Whereas there are as yet no known specific inhibitors for the PM Ca(2+)-transport ATPase, a number of selective inhibitors for the endo(sarco)plasmic reticulum Ca2+ pumps have been described: thapsigargin, cyclopiazonic acid and 2,5-di-(tert-butyl)-1,4-benzohydroquinone. With the recent introduction of the molecular biological approach, it became quickly obvious that a family of at least 5 different PM Ca(2+)-transport ATPase genes govern the tissue-dependent expression of PM Ca2+ pumps. Moreover alternative splicing of the primary gene transcripts was found to further enhance the number of pump variants. The PM Ca(2+)-transport ATPase are subject to modulatory control by calmodulin, by acidic phospholipids, and by the known families of protein kinases. Each of the ensuing effects are mutually related and interdependent. The wide variety PM Ca2+ pump isoforms and their regulation by such an intricate modulatory network allows the distinct tissues to adapt most adequately to the prevailing tissue and stimulus specific requirements.


MgtA and MgtB: prokaryotic P-type ATPases that mediate Mg2+ influx. The gram-negative bacterium Salmonella typhimurium possesses three distinct Mg2+ transport systems, encoded by the corA, mgtA, and mgtB loci. The CorA transport system is the constitutive Mg2+ influx system. It can also mediate Mg2+ efflux at very high extracellular Mg2+ concentrations. In contrast, the MgtA and MgtB Mg2+ transport systems are normally expressed only at low extracellular Mg2+ concentrations. A strain of S. typhimurium was constructed by mutagenesis which lacks Mg2+ transport and requires 100 mM Mg2+ for growth. Using this strain, both the MgtA and MgtB transport systems were cloned by complementation of the strains inability to grow without Mg2+ supplementation. After sequencing and further genetic analysis, the MgtB system appears to be an operon composed of the mgtC and mgtB genes (5' to 3'). The downstream mgtB gene encodes the 102 kDa MgtB protein which by sequence analysis is clearly a P-type ATPase. Interestingly, while MgtB has relatively poor homology to other known prokaryotic P-type ATPases, it is highly homologous to mammalian reticular Ca(2+)-ATPases. MgtC is a 22.5 kDa hydrophobic membrane protein that lacks homology to any known protein. Transposon insertions in this gene abolish uptake by the MgtB transport system. We hypothesize that MgtC is a subunit of the MgtB ATPase involved either in proper insertion of MgtB into the membrane or possibly in binding of extracellular Mg2+ for delivery to the ATPase subunit. The sequence of the MgtA gene has recently been completed, and it too is a P-type ATPase more similar to eukaryotic than prokaryotic P-type ATPases. Expression of both MgtA and MgtB are highly regulated by the concentration of extracellular Mg2+. Transcription of mgtB can be increased about 1000 fold by lowering Mg2+ from 1 mM to 1 microM. Likewise, when mgtB is expressed from a multicopy plasmid, a similar decrease in extracellular Mg2+ greatly increases transport. Under growth conditions of limiting Mg2+, MgtB becomes the dominant Mg2+ influx system in S. typhimurium. Even so, since MgtB (and MgtA) mediate only influx of Mg2+, it is unclear why the cell requires energy from ATP to mediate Mg2+ entry into the cell down a large electrochemical gradient. Further studies of the structure-function and energetics of these novel Mg2+ influx P-type ATPases should yield insights into the function of P-type ATPases in general as well as information about the regulation of cellular Mg2+ fluxes.


Isolation of a mouse "5HT1E-like" serotonin receptor expressed predominantly in hippocampus. Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors that interact with G proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor, the 5HT1E beta receptor. Amino acid sequence comparisons revealed that its closest relatives were the recently characterized 5HT1E receptor (S31) and the 5HT1B and 5HT1D receptors. When expressed transiently in Cos-7 cells, the 5HT1E beta receptor displayed a high affinity for the nonspecific serotonergic radioligand 2-[125I]iodolysergic acid diethylamide (Kd = 980 pM). The pharmacological profile of the 5HT1E beta receptor resembled that of previously reported 5HT1E sites that have a low affinity for 5-carboxamidotryptamine and that have been found in human and rat brain. When stably expressed in NIH-3T3 cells, the 5HT1E beta receptor was negatively coupled to adenylate cyclase. In situ hybridization experiments revealed that the 5HT1E beta transcripts were detected only in the CA1, CA2, and CA3 layers of the hippocampus. Our results therefore demonstrate that the 5HT1E receptors constitute a heterogeneous family of receptors.


Studies on p40, the leucine zipper motif-containing protein encoded by the first open reading frame of an active human LINE-1 transposable element. Full-length human LINE-1 retrotransposons encode p40 proteins with varying electrophoretic mobilities under denaturing conditions. The p40 expressed from the first open reading frame in the LINE-1 copy designated L1.2A co-electrophoreses with the endogenous p40 in human teratocarcinoma cells. This finding is consistent with previous data indicating that L1.2A is an active element. The amino acid sequence in the central region of the L1.2A p40 accounts, at least in part, for its characteristic mobility. This region includes sequences which can, in principle, form a leucine zipper.


Ty4, a new retrotransposon from Saccharomyces cerevisiae, flanked by tau-elements. We have isolated and sequenced a genomic clone from Saccharomyces cerevisiae that shows structural features of a novel retrotransposon, designated Ty4. The element is 6.2 kilobases in length, and its genetic organization of the deduced functional domains is similar to Ty1 and Ty2 and thus different from Ty3. In contrast to hitherto known Ty elements from yeast, Ty4 is flanked by long terminal tau-element repeats instead of delta or sigma sequences. Ty4 contains two overlapping open reading frames. The first open reading frame, TYA4, is 1230 base pairs long and encodes a protein with a motif found in the nucleic acid-binding gag-protein of retroviruses. The second 4395-base pair open reading frame, TYB4, encodes a polyprotein that has domains with significant homology to retroviral protease, integrase, reverse transcriptase, and RNase H, structurally arranged in that order. The deduced amino acid sequence shows the greatest similarity with Ty2 and Ty1. The overall identity of the deduced functional protein domains is 28% with Ty2, 25% with Ty1, 19% with copia from Drosophila, and 18% with Ty3. Examination of genomic DNA from several laboratory strains indicates that Ty4 is present in two to four copies. Ty4 mRNA is of low abundance as compared to other Ty retrotransposons. At the 3' end of Ty4, two "solo" delta-elements, a full length and an overlapping, truncated one, are associated.


1,3-Butadiene oxidation by human myeloperoxidase. Role of chloride ion in catalysis of divergent pathways. 1,3-Butadiene was oxidized by human myeloperoxidase in the absence of KCl to yield butadiene monoxide (BM) and crotonaldehyde (CA), but at KCl concentrations higher than 50 mM, 1-chloro-2-hydroxy-3-butene (CHB) was the major metabolite detected; metabolite formation was dependent on incubation time, pH, KCl, 1,3-butadiene, and H2O2 concentrations. The data are best explained by 1,3-butadiene being oxidized by myeloperoxidase by two different mechanisms. First, oxygen transfer from the hemoprotein would occur to either C-1 or C-4 of 1,3-butadiene to form an intermediate which may cyclize to form BM or undergo a hydrogen shift to form 3-butenal, an unstable precursor of CA. Further evidence for this mechanism was provided by the inability to detect methyl vinyl ketone, a possible product of an oxygen transfer reaction to C-2 or C-3 of 1,3-butadiene, and by the finding that CA was not simply a decomposition product of BM under assay conditions. In the second mechanism, however, chloride ion is oxidized by myeloperoxidase to HOCl which reacts with 1,3-butadiene to yield CHB. Further evidence for this mechanism was provided by the finding that CHB was readily formed when 1,3-butadiene was added to the filtrate of a myeloperoxidase/H2O2/KCl incubation and when 1,3-butadiene was allowed to react with authentic HOCl. In addition, CHB was not detected when BM or CA was incubated with myeloperoxidase, H2O2, and KCl for up to 60 min, or when 1,3-butadiene and KCl were incubated with chloroperoxidase and H2O2 or with mouse liver microsomes and NADPH, enzyme systems which catalyze 1,3-butadiene oxidation to BM and CA, but unlike myeloperoxidase, do not catalyze chloride ion oxidation to HOCl. These results provide clear evidence for novel olefinic oxidation reactions by myeloperoxidase.


Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor. In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and human epidermal carcinoma (A431) cells with thapsigargin. Intracellular free calcium was monitored by single cell image analysis using fura-2 and correlated with MAP kinase stimulation as assessed by immunoprecipitation, kinase renaturation assays and immunoblotting. Thapsigargin stimulated the 44- and 42-kDa MAP kinase isozymes in both cell types with kinetics that were slightly delayed relative to enzyme stimulated by epidermal growth factor. Removal of external calcium did not significantly affect the activation of the MAP kinases by thapsigargin, indicating that intracellular calcium mobilization is sufficient to stimulate the enzymes. However, treatment of cells with EGTA under conditions which deplete both intra- and extracellular calcium inhibited stimulation by thapsigargin but not epidermal growth factor. Stimulation of the MAP kinases by the calcium ionophore ionomycin paralleled the activation observed with thapsigargin in both calcium-containing and calcium-free conditions. These results indicate that there are at least two independent pathways for stimulation of MAP kinase: one that is dependent on intracellular calcium mobilization, and one that is mediated by the tyrosine kinase epidermal growth factor receptor and is calcium-independent.


The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol. The respiratory burst oxidase is a multimeric enzyme responsible for O2- production by stimulated neutrophils and a few other cell types. In the resting neutrophil, the oxidase is dormant, and its subunits are distributed between the cytosol, in which they appear to exist in the form of a multisubunit complex, and the plasma membrane; but, when the neutrophil is activated, the cytosolic complex translocates to the membrane to assemble the active enzyme. Using a cell-free system in which oxidase activity was elicited with SDS, we examined the effects of GTP gamma S and dioctanoylglycerol (DiC8) on both the activation of O2- production and the transfer of the cytosolic oxidase components p47phox and p67phox to the plasma membrane. GTP (added as undialyzed cytosol) and GTP gamma S augmented the transfer of the oxidase components to the plasma membrane and was essential for the acquisition of O2- producing activity by the oxidase. DiC8 also supported the SDS-mediated transfer of oxidase components to the membrane, but O2- production did not take place unless GTP or GTP gamma S was present. In the presence of these nucleotides, however, DiC8 augmented both translocation and O2- production. We interpreted these results in terms of a mechanism in which 2 membrane-binding sites are created during the activation of the cytosolic complex, one for diacylglycerol and the other for a second site on the membrane. Development of the second membrane-binding site depends upon the action of a G protein and is essential for the expression of oxidase activity. The results further suggested that the priming of the respiratory burst oxidase in intact neutrophils might be due to an increase in membrane diacylglycerol concentration that occurs in response to the priming stimulus. Because of the increased diacylglycerol content, a larger than usual amount of active respiratory burst oxidase could be assembled on the primed plasma membrane when the neutrophil is fully activated.


Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate. Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.


Identification of a phosphodiesterase that converts inositol cyclic 1:2-phosphate to inositol 2-phosphate. Inositol 2-phosphate (Ins(2)P) has been identified in several cell types. The cellular levels of Ins(2)P appear to be directly correlated with the levels of inositol 1:2-cyclic phosphate (cIns(1:2)P) (Ross, T. S., Wang, F. P., and Majerus, P. W. (1992) J. Biol. Chem. 267, 19919-19923). In this study we have detected an enzyme in extracts from CV-1 cells and rat cerebellum that converts cIns(1:2)P to Ins(2)P and inositol 1-phosphate. This enzyme (designated cyclic hydrolase II) is not the same protein previously designated cIns(1:2)P 2-phosphohydrolase (cyclic hydrolase I). The products, heat inactivation curves, pH optima, and metal dependence of these two activities are different, and the two activities were separated by DEAE and gel filtration chromatography. Mixing of cyclic hydrolase I with cyclic hydrolase II does not effect the activity of either. The Km of the CV-1 cyclic hydrolase II for D-cIns(1:2)P is 10 microM. The enzyme is approximately 55 kDa as estimated by gel filtration analysis in the presence of sodium chloride and 120 kDa in its absence.


Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study. Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.


Role of the A protein-binding sites in the in vitro transposition of mu DNA. A complex circuit of interactions involving the mu ends and the transpositional enhancer. To investigate the role of the A protein-binding sites at the Mu ends in the DNA strand transfer reaction, we constructed mutant mini-Mu molecules in which these sites were deleted (L3 or R3) or substituted (L2 or R2) to conserve the spacing arrangements at the adjacent sites. The single site mutants are poor substrates for phosphodiester bond hydrolysis at the Mu ends in Type 1 reactions in the absence of Escherichia coli integration host factor (IHF). Addition of IHF to the reaction stimulates Type 1 cleavage more than 10 times for the delta-R3, delta-L3, S-L2 mutants and more than five times in the case of the S-R2 mutant under alternate conditions. The site of IHF stimulation resides within the transpositional enhancer which implicates the end-binding sites L2, L3, R2, and R3 in interactions with the enhancer. At least two of the L2, L3, and R3 sites are required for proficient reaction in the presence of IHF. By combining the single site mutants with O1 or O2 partially deleted enhancer elements, we have tentatively localized some of the interactions to each side of the functional enhancer revealing a complex circuit of end-enhancer interactions. The R3 site is suggested to be involved in interactions only with O2 and the L3 site only with O1. The data also suggest the possibility that L2 and R2 may be involved in interactions with both O1 and O2. Finally, our working model predicts that the L3-O1 and R3-O2 interactions may be required contacts for discriminating between the Mu left and right ends in transpososome formation.


Nitrogenase-catalyzed ethane production and CO-sensitive hydrogen evolution from MoFe proteins having amino acid substitutions in an alpha-subunit FeMo cofactor-binding domain. Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the wild-type and altered MoFe proteins), which were monitored by EPR spectroscopy and resulting catalytic activity.


Characterization of a rapidly dissociating inositol 1,4,5-trisphosphate-binding site in liver membranes. The binding of inositol 1,4,5-trisphosphate (InsP3) to a specific receptor induces the release of Ca2+ from an intracellular store. In the liver, the KD of a low affinity state of the receptor (RL) found at low Ca2+ concentration ([Ca2+]) is in close agreement with the EC50 of the InsP3-induced Ca2+ release. We have developed an experimental procedure for measuring the rate of dissociation of this low affinity [32P]InsP3-receptor complex in less than 1 s. When the receptor was in the RL state, two kinetic components, RL1 and RL2, were identified with respective rate constants (k(off)) of 1-2 s-1 and 0.03-0.06 s-1. Increasing the [Ca2+] up to 1 microM transformed the receptor into the high affinity state (RH) and decreased the dissociation rate constant to 2 x 10(-2) min-1. We also investigated the time course of the transformation of the receptor from the high affinity (RH) to the low affinity state (RL) after decreasing the [Ca2+] to less than 10 nM. This reversion was dramatically dependent on temperature: at 4 degrees C, the receptor was locked in the RH state, whereas at 37 degrees C the receptor reverted to the RL state with a half-time of less than 1 s. The reversion from the RH state to the RL one is associated to a recovery of InsP3-induced 45Ca2+ release on permeabilized hepatocytes. The rapid and reversible transformation of the InsP3 receptor from an active to an inactive state may be a key event in the Ca2+ release process in intact cells.


Up-regulation of the vitamin D receptor in response to 1,25-dihydroxyvitamin D3 results from ligand-induced stabilization. Several studies have shown that the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor protein levels increase in response to 1,25-(OH)2D3. We have studied the mechanism of this regulation in both mouse fibroblasts and rat intestinal epithelial cells. Cell extracts and total RNA were prepared at varying times after addition of 1,25-(OH)2D3. The 1,25-(OH)2D3 receptor protein levels, measured using an immunoradiometric assay, rose significantly 2-3 h posttreatment and had risen 3-fold at 8 h. Concurrently, the 1,25-(OH)2D3 receptor mRNA content, measured using a ribonuclease protection assay, was not altered by 1,25-(OH)2D3 during this time. In cycloheximide-blocked cells, the administration of 1,25-(OH)2D3 markedly reduced the degradation rate of previously formed receptor. The 1,25-(OH)2D3 receptor protein half-life was determined as 4 h in the absence of 1,25-(OH)2D3 and increased to at least 8 h in the presence of 1,25-(OH)2D3. We also measured the 1,25-(OH)2D3 receptor mRNA levels in the duodena and kidney of vitamin D-deficient rats after a single 150-pmol injection of 1,25-(OH)2D3. Again, we found that 1,25-(OH)2D3 receptor mRNA levels were not changed in these tissues after 1,25-(OH)2D3 treatment. Therefore, the elevation of the 1,25-(OH)2D3 receptor protein following 1,25-(OH)2D3 administration is apparently the result of increased receptor protein lifetime and not increased transcription.


Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. Hepatocyte growth factor (HGF) is a heterodimeric protein consisting of a heavy chain and a light chain held by a disulfide bond. These chains are produced by endoproteolytic processing from a single chain precursor. In this study, we examined whether the processing is a prerequisite for the mitogenic activity of HGF on hepatocytes in primary culture. Single chain HGF was proteolytically converted to the heterodimeric form during incubation with hepatocytes and was as mitogenic as the heterodimeric form. When the conversion was inhibited by serine-protease inhibitors, the mitogenic activity of single chain HGF was markedly reduced. Furthermore, a mutant resistant to the proteolytic processing, which was prepared by in vitro mutagenesis, completely lost the mitogenic activity. From these results, we concluded that the single chain form of HGF is endoproteolytically processed by a serine-protease and that this processing is a prerequisite for the mitogenic activity of HGF.


Stimulation of the thrombin receptor of human glomerular mesangial cells by Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe peptide. We have studied the effects of thrombin (alpha-thrombin) and Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe (SFLL), a peptide agonist of the platelet thrombin receptor in cultured human mesangial cells, and find that SFLL can reproduce the biochemical and morphological effects of thrombin. Treatment of mesangial cells with cAMP-elevating agents causes fragmentation of stress fibers, loss of the vitronectin receptor from sites of focal adhesion, and produces a change in shape from a flat to a more arborized configuration. These effects are prevented by both thrombin and SFLL. Thrombin and SFLL also initiate biochemical signaling events in mesangial cells by stimulating the metabolism of phospholipids. Both thrombin and SFLL stimulate release of inositol phosphates from [3H]inositol-labeled cells, elevation of cytosolic calcium, the formation of [3H]myristic acid-labeled diacylglycerol, an increase in the mass of diacylglycerol, 32P incorporation into phospholipids, and release of unesterified [3H]arachidonic acid from cells prelabeled with [3H]arachidonic acid. When present together, the effects of SFLL and thrombin on diacylglycerol formation, arachidonic acid production, and inositol phosphate production were not additive. This suggested that SFLL and thrombin were acting on the same receptor. This was further supported by our observations that cells pretreated with SFLL and subsequently exposed to thrombin (or vice versa) did not show elevated cytosolic calcium. We also show that phospholipase D is activated by demonstrating production of radiolabeled phosphatidylethanol when cells are treated with SFLL in the presence of ethanol. These findings indicate that SFLL can be used to study the receptor-mediated effects of thrombin in mesangial cells, thereby avoiding thrombin's proteolytic actions.


Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1. Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.


Identification of a novel retinoid-responsive element in the promoter region of the medium chain acyl-coenzyme A dehydrogenase gene. To study the mechanisms involved in regulation of nuclear genes encoding mitochondrial enzymes in oxidative energy pathways, the promoter region of the medium-chain acyl-CoA dehydrogenase (MCAD) gene was analyzed. A series of hexamer sequences known to bind and confer responsiveness to a subset of members of the nuclear receptor superfamily of transcription factors was identified. Cotransfection of an MCAD promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid with retinoic acid receptor (RAR)alpha, beta, or retinoid X receptor alpha (RXR alpha) resulted in 10-15-fold transcriptional activation in response to retinoic acid. The retinoic acid-induced activation was 3-4-fold higher with RXR alpha than with either RAR alpha or RAR beta. Deletional analysis confirmed that a region between -341 and -308 base pairs upstream of the MCAD gene cap site conferred the RA-responsive transcriptional activation to homologous and heterologous promoters. Gel mobility shift assays demonstrated that the MCAD RARE interacted directly with overexpressed receptors. Mutational analysis of the RARE delineated three hexamer binding sequences with unique orientation and spacing compared to other reported retinoid responsive elements. These results indicate that the MCAD gene promoter region contains a novel regulatory element that interacts with members of the retinoid receptor family, with preferential activation by RXR alpha. This element likely plays a role in the transcriptional regulation of this gene and perhaps others involved in oxidative energy metabolism.


Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system. Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.


Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.


Cloning of a novel somatostatin receptor, SSTR3, coupled to adenylylcyclase. The gene encoding a novel mouse somatostatin receptor termed mSSTR3 was isolated and characterized. The sequence of mSSTR3 shows 46 and 47% identity with mSSTR1 and mSSTR2, respectively. mSSTR3 binds somatostatin-14 and somatostatin-28 with high affinity, but shows very low affinity for the somatostatin analogs MK-678 and SMS-201-995. In addition, mSSTR3 is coupled to pertussis toxin-sensitive G proteins and mediates somatostatin inhibition of forskolin-stimulated and dopamine D1 receptor-stimulated cAMP formation, indicating that it is coupled to adenylylcyclase. The pharmacological properties of mSSTR3 and its ability to couple with adenylylcyclase distinguish SSTR3 from the other cloned somatostatin receptors and indicates that it mediates biological functions different from SSTR1 or SSTR2. In situ hybridization indicates that SSTR3 mRNA is widely distributed in the mouse brain, and its expression in the nucleus of the lateral olfactory tract and in the piriform cortex, the primary olfactory cortex in the rodent brain, suggests that SSTR3 may participate in the processing and modulation of primary sensory information.