Hormonal regulation of calcium current in freshly isolated airway smooth muscle cells.

Single freshly isolated smooth muscle cells of adult bovine trachea were voltage clamped, and the calcium inward current was separated from K+ currents by blocking the large outward currents with intra- and extracellular Cs+ and extracellular tetraethylammonium chloride. Isoproterenol stimulated peak calcium current (ICa) in a dose-dependent manner through the beta-adrenergic receptor. The isoproterenol effect was not mediated or caused by the stimulation of a K+ or Na+ current, a decrease in the intracellular concentrations of Ca2+ or H+, the stimulation of the Na(+)-H+ or the Na(+)-Ca2+ exchanger. Neither basal nor isoproterenol-stimulated ICa was affected by internal dialysis of the cell with adenosine 3',5'-cyclic monophosphate (cAMP), cAMP analogues, or the catalytic subunit of cAMP-kinase. Internal dialysis of the cells with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) blocked the stimulation of isoproterenol whereas dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) induced an isoproterenol-like maximal increase of ICa. These results show that the beta-adrenergic receptor stimulates the L-type calcium current of isolated tracheal smooth muscle cells independent of cAMP and cAMP-kinase through a GTP/GDP regulated protein.


The use of oral mexiletine for the treatment of pain after peripheral nerve injury.

Neuropathic pain is often a difficult condition to treat. Clinical and laboratory studies using intravenously administered local anesthetics or antiarrhythmic agents support the use of these drugs for the treatment of neuropathic pain. The availability of the oral antiarrhythmic medication, mexiletine, has made it possible to study the effects of an orally administered medication on chronic neuropathic pain. The study used a double-blind placebo-controlled design to examine 11 subjects in whom treatment with conventional pain medications had been unsuccessful. Subjects had a history of peripheral nerve injury or dysfunction, and all complained of symptoms consistent with neuropathic pain. After baseline pain measurements, mexiletine or placebo was given in gradually increasing doses to a maximum daily dose of 750 mg mexiletine. After 1 month at steady state, the subject received the alternative medication. Mexiletine was found to produce a statistically significant reduction in reported pain when compared to baseline or placebo. Pain scores were rated on a scale from 0 (no pain) to 10 (unbearable pain). Median pain scores prior to mexiletine were 7, after placebo treatment 7, and while receiving mexiletine (750 mg/day) 4. Side effects were mild and well-tolerated. Mexiletine may be effective in reducing neuropathic pain for patients in whom alternative pain medications have been unsatisfactory.


Halothane effects on human malignant hyperthermia skeletal muscle single calcium-release channels in planar lipid bilayers.

Malignant hyperthermia (MH) may be life-threatening when genetically predisposed individuals are administered triggering anesthetic agents that are believed to produce intracellular calcium release. To test this theory, the effects of halothane on normal and MH human skeletal muscle calcium-release channels were studied. Single calcium-release channels were incorporated from isolated sarcoplasmic reticulum membrane vesicles into a planar lipid bilayer, and halothane effects on the conductance and gating properties were measured by electrophysiologic techniques. Among the subjects studied, seven were MH-susceptible, and 13 channels were recorded from this group. Five subjects were negative for MH, and 10 channels were recorded from this group. Among the 13 channels recorded from the MH group, 7 were affected by halothane, which increased the probability of the channel to change from the inactive, closed state to an open state. This effect of halothane to increase open-state probability was associated with an overall increase in channel conductance. Thus, halothane affected the activation/inactivation process of the halothane-sensitive calcium-release channel from MH muscle as well as the gating properties of the MH calcium-release channel, as evidenced by the increased conductance. In 6 of the 13 channels recorded from MH muscle, halothane (2.2-17.6 microM) was without effect on these properties of the channel. Halothane (2.2-17.6 microM or 0.0057-0.0456 vol%) also had no measurable effect on the 10 channels from the negatively diagnosed subjects. Results of this study support a defect in the ryanodine-sensitive calcium-release channel from MH human muscle.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of overproduction of superoxide dismutases in Escherichia coli on inhibition of growth and on induction of glucose-6-phosphate dehydrogenase by paraquat.

Stationary phase inocula were more susceptible to the growth inhibitory effect of paraquat than were log phase inocula and this difference was exacerbated in strains overproducing superoxide dismutases (SOD). Glucose-6-phosphate dehydrogenase (G-6-PD), a member of the soxR regulon, was induced by paraquat promptly in the case of log phase cells; but only after a lag in stationary phase cells and this difference was also exaggerated in strains overproducing SOD. The negative consequences of overproduction of SOD on the adaptation of stationary phase cells to paraquat may be attributed to competition for cellular resources with an attendant delay in biosynthesis of other components of soxR. Since overproduction of SOD did not prevent log phase cells from inducing G-6-PD in response to paraquat, it appears likely that soxR can respond to aspects of redox status other than O2-. This conclusion is in accord with data which is already in the literature.


Effects of adenosine administration on the function and membrane composition of liver mitochondria in carbon tetrachloride-induced cirrhosis.

The effect of chronic carbon tetrachloride (CCl4) administration on liver mitochondria function and the protective action of adenosine on CCl4-induced damage were assessed in rats made cirrhotic by long-term exposure to the hepatotoxin (8 weeks). The CCl4 treatment decreased the ADP-stimulated oxygen consumption, respiratory control, and ADP/O values, mainly for substrates oxidation of site I, in isolated mitochondria. This impaired mitochondrial capacity for substrate oxidation and ATP synthesis was accompanied by an important diminution (approximately 30 mV) of membrane electrical potential. Disturbances of the mitochondrial membrane, induced by CCl4 treatment, were also evidenced as increased mitochondria swelling and altered oscillatory states of mitochondrial volume, both energy-linked processes. The deleterious effects of CCl4 on mitochondrial function were also reflected as a deficient activity of the malate-aspartate shuttle that correlated with abnormal distribution of cholesterol and phospholipids in membranes obtained from submitochondrial particles. Adenosine treatment of CCl4-poisoned rats partially prevented the alterations in mitochondria membrane composition and prevented, almost completely, the impairment of mitochondria function induced by CCl4. Although the nature of the protective action of adenosine on CCl4-induced mitochondria injury remains to be elucidated, such action at this level might play an important role in the partial prevention of liver damage induced by the CCl4.


Effect of lipophilic chelators on oxyradical-induced DNA strand breaks in human granulocytes: paradoxical effect of 1,10-phenanthroline.

Strand breaks can be produced in the DNA of intact granulocytes by a flux of oxyradicals (O2- and H2O2) generated by tetradecanoylphorbol acetate (TPA) or by a flux of H2O2 generated by glucose oxidase. The mechanism by which such breaks are induced is still uncertain. Lipophilic chelators such as dipyridyl and 1,10-phenanthroline (OP) strongly inhibit strand breaks induced by H2O2, presumably because of their ability to chelate intracellular iron. We now report that dipyridyl also partially inhibits strand breaks in TPA-stimulated granulocytes while a "copper-specific" lipophilic chelator, neocuproine, has no effect. As opposed to these effects, OP increases the number of strand breaks in TPA-stimulated granulocytes. Superoxide dismutase (SOD) (but not catalase) partially blocks this increase. Both the cell-impermeable chelator, EDTA, and neocuproine strongly block the increase also. In fact, in the presence of EDTA, OP behaves like dipyridyl and inhibits strand breaks. Preformed OP2-copper(II) complex causes DNA breaks in TPA-stimulated granulocytes. The paradoxical effect of OP may be explained by assuming that OP may form two different metal complexes, a DNA-damaging complex with copper or an inhibitory complex with iron. If copper(II) and O2- are present, the first complex may form and the net effect may be an increase in strand breaks. If the formation of this complex is prevented by SOD, EDTA, or neocuproine, then OP may complex iron and the net effect may be (like dipyridyl) an inhibition of strand breaks. The source of the copper responsible for the formation of OP2-copper complex is unknown.


Electrostatic modulation of the kinetics of electron transfer from cytochrome c to cobalt phenanthroline by binding to lipid bilayers: effects of ionic strength and extent of incorporation of various negatively charged lipids.

The effect of electrostatically binding ferrous cytochrome c to anionic liposomes, composed of dimyristoyl phosphatidylglycerol (DMPG-), dioleoyl phosphatidyl-glycerol (DOPG-), or cardiolipin (CL2-) mixed with varying amounts of egg phosphatidylcholine (PC), on the kinetics of cytochrome oxidation by the positively charged cobalt phenanthroline ion has been measured using stopped-flow spectrophotometry. The rate of electron transfer is enhanced as much as 3000-fold by increasing the number of negatively charged binding sites on the liposome surface, and by as much as 1000-fold by decreasing the ionic strength of the buffer. The sigmoidal shape of the curve of rate constant vs mole percent anionic lipid is consistent with a positively cooperative effect of the negative surface charge. The rate stimulation is greater for DOPG(-)- and CL2(-)-containing liposomes than for DMPG- vesicles; this is most likely due to structural differences in the respective liposomes. The results do not provide any support for a role of structural changes in the bound cytochrome in influencing oxidation kinetics, a possibility suggested by recent spectroscopic measurements, although relatively small conformational effects cannot be completely ruled out.


Purification of Chlamydomonas 28-kDa ubiquitinated protein and its identification as ubiquitinated histone H2B.

One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 28-kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlamydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those Chlamydomonas ubiquitin [corrected] and wheat histone H2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease V8-digested polypeptide fragments of the 28-kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonas 28-kDa ubiquitinated protein is monoubiquitinated histone H2B.


Detergents affect insulin binding, tyrosine kinase activity and oligomeric structure of partially purified insulin receptors.

Insulin receptor activities, i.e., insulin binding and tyrosine kinase activation depend on the lipid environment of the receptor. As detergent may disrupt or interfere with this environment, we investigated the effect of various common detergents on insulin receptor properties. Experiments were carried out (i) on solubilized and partially purified insulin receptor and (ii) on the receptor reconstituted into phosphatidylcholine vesicles. The detergents tested, Triton X-100, octyl-beta-D-glucopyranoside, octyl-beta-D-thioglucopyranoside, 3[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps), and Na deoxycholate affected the insulin receptor properties differently when compared with the control receptor in the absence of detergent. On the partially purified insulin receptor, Na deoxycholate inhibited both insulin receptor activities; octyl-beta-D-glucopyranoside and octyl-beta-D-thioglucopyranoside decreased insulin binding and kinase activation as their concentration increased, particularly above their respective critical micellar concentration (CMC). Triton X-100 was the only detergent which allowed an increase of insulin binding and kinase activation throughout the whole range of concentrations assayed. Reconstitution of the receptor into phosphatidylcholine vesicles protected the receptor from the direct effects of the detergents, for both the stimulation observed with Triton X-100 and the inhibition produced by the other detergents. In order to determine the effect of detergents on the oligomeric forms of the soluble insulin receptor, we investigated a new rapid sucrose gradient centrifugation technique. Insulin receptors were detected on the gradient by 125I insulin binding. For low concentrations of detergent, i.e., near the CMC, octylglucoside, Chaps, and Triton X-100 favored the (alpha 2 beta 2)2 oligomeric form of the receptor. Higher concentrations of Triton X-100 did not modify the polymeric state of the receptor. In contrast, octylglucoside and Chaps induced an increase in the sedimentation coefficient of the receptor which appeared as (alpha 2 beta 2)3 and (alpha 2 beta 2)4 forms. These alterations in the oligomerization status of the insulin receptor may explain the deleterious effects observed with both Chaps and octylglucoside at higher concentrations.


A two-step mechanism of fluoride inhibition of rat liver inorganic pyrophosphatase.

Product formation curves for inorganic pyrophosphatase-catalyzed hydrolysis of pyrophosphate in the presence of fluoride were analyzed in order to get insight into the mechanism of its inhibitory action on this enzyme. The enzymatic reaction was monitored with a phosphate analyzer operating on the time scale of seconds. Inhibition patterns were virtually identical for cytosolic and mitochondrial pyrophosphatases. The effect of fluoride was biphasic: it caused a rapid (t 1/2 less than 1 s) decrease in the initial velocity of the reaction followed by slow (t 1/2 greater than or equal to 4 s) inactivation of the enzyme during catalysis. The slow phase resulted in trapping intact substrate at the active site, and the resulting complex could be isolated by gel filtration. Pyrophosphatase remained active when incubated with fluoride in the absence of pyrophosphate or in the presence of its bisphosphonate analogs, which are bound to but not hydrolyzed by this enzyme. These features of the inhibition are consistent with the mechanism in which rapid binding of the inhibitor to the enzyme.substrate complex is followed by its slow isomerization. Kinetic parameters obtained in this work indicate that appreciable inactivation of pyrophosphatase can occur at fluoride concentrations found in human plasma. This effect may therefore be one of the major factors contributing to fluoride toxicity.


Expression and turnover of acylphosphatase (muscular isoenzyme) in L6 myoblasts during myogenesis.

Acylphosphatase (muscular isoenzyme) levels have been measured in L6J1 myoblasts either proliferating or differentiating into myotubes. Results indicated that the increase in enzyme levels during differentiation is very similar to that of creatine kinase, a specific muscular enzyme. The half-lives of acylphosphatase in myoblasts and myotubes were also determined; t1/2 values of 3 h 30 min (myoblasts), and 2 h 18 min (myotubes) were found. These results indicate that acylphosphatase could be considered a short-lived muscle-specific protein and that its increase in myotubes must be accompanied by an activation of its breakdown.


Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.


Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors.

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.


Hydroxyl radical generation by red tide algae.

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-t-butyl-alpha-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O2-) (DMPO-OOH) and that of hydroxyl radical (.OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.


Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides.

To clarify the nature of cytocidal molecular species among the radicals generated in the iron-catalyzed reactions of peroxides (ROOH), we examined the cytocidal effects of these radicals against gram-positive and gram-negative bacteria in the presence or absence of various radical scavengers. Three organic peroxides, t-butyl hydroperoxide (t-BuOOH), methyl ethyl ketone peroxide (MEKOOH), and cumene hydroperoxide, were used. Each radical generated from these peroxides was identified and quantitated by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The major cytotoxic radical species generated in the mixtures of various peroxides and heme iron, especially methemoglobin, metmyoglobin, or hemin, was the alkyl peroxyl radical (ROO.). Strong bactericidal action against gram-positive bacteria was observed in the peroxide-heme iron system, especially in the case of t-BuOOH and MEKOOH. Killing curves for gram-positive bacteria showed an initial lag period, which may indicate the multihit/multitarget kinetics of cell killing. When the diethylenetriamine pentaacetic acid (DTPA)-Fe2+ complex was used as a catalyst for decomposition of various peroxides, alkyl, alkoxyl, and alkyl peroxyl radicals were identified by spin-trapping analysis. However, study of the time course of alkyl peroxyl radical production in the DTPA-Fe2+ complex system revealed that radical species generated in this system were very short lived: a maximal level was achieved within 1 min and then declined sharply, and no bactericidal activity was observed after 10 min. In contrast, the alkyl peroxyl radical level generated by the organic peroxide-heme iron system remained high for 30 min or longer. The generation of alkyl peroxyl radicals quantified by ESR correlated quite well with the bactericidal effect of the system of peroxide plus iron. In addition, bactericidal activity was completely inhibited by the addition of the spin trap DMPO, as well as of other various radical scavengers (alpha-tocopherol and L-ascorbic acid), into the peroxide-heme iron system, but this effect was not observed with superoxide dismutase, beta-carotene, dimethyl sulfoxide, diphenylamine, or butylated hydroxyltoluene. In view of these results, it is assumed that alkyl peroxyl radicals are the potent molecular species that are cytotoxic against bacteria, whereas alkoxyl radicals (RO.) generated in this system do not affect bacterial viability.


On the nature of leukotriene C4 synthase in human platelets.

Leukotriene C4 is considered to play a major role in several important pathophysiological conditions, e.g., allergy, asthma, and shock. The present investigation demonstrates the presence in human platelets of a membrane-associated enzyme catalyzing the final step in the biosynthesis of leukotriene C4. This leukotriene C4 synthase was shown to be distinct from previously characterized "microsomal" and soluble glutathione transferases. The latter enzymes did not contribute significantly to the leukotriene A4 conjugating activity in platelets. As determined with leukotriene C4 synthase of a crude membrane fraction from human platelets, the Km value was 7 microM and the V value was 0.56 nmol x min-1 x mg-1 with leukotriene A4 as substrate. The enzyme was 20-fold more efficient with leukotriene A4 than with leukotriene A5 and 30-fold more efficient than with the unphysiological derivative leukotriene A4 methyl ester, as measured by the corresponding V/Km values; 14,15-leukotriene A4 was not a substrate. Platelets should be a useful source for the purification and further characterization of human leukotriene C4 synthase.


The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage.

Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.


Serum concentration and urinary excretion of "classical" estrogens, catecholestrogens and 2-methoxyestrogens in normal human pregnancy.

Catecholestrogens, 2-methoxyestrogens and "classical" estrogens (estrone, estradiol, estriol) were measured simultaneously in serum and urine samples of 220 pregnant women from the 8th week of pregnancy until to delivery. From these data we established the central 0.80 centile intervals as time specified reference intervals for each substance analyzed. Serum and urinary estradiol rise steadily during the progress of pregnancy, whereas estrone, catecholestrogens and 2-methoxyestrogens reach a plateau during the last trimester. These observations support the hypothesis, that the amount of the latter compounds may be regulated by separate mechanisms. The values of concentration and excretion of 2- and 4-substituted estrogens varied widely throughout pregnancy. Even very high or very low concentrations of these substances had no recognizable relation to the outcome of pregnancy. This supports the assumption that catecholestrogens and their methylethers are metabolites without any regulatory function in pregnancy.


White blood cell activation in Raynaud's phenomenon of systemic sclerosis and vibration induced white finger syndrome.

Systemic sclerosis (SSc) and vibration induced white finger syndrome (VWF) are common causes of secondary Raynaud's phenomenon. Previous studies have suggested an increase in inflammation in patients with SSc. Vibration induced white finger syndrome occurs in workers exposed to vibration and is now a prescribed disease. In VWF, although it is recognised that vibration can cause direct damage to blood vessels, it does not explain why only some workers are affected. It is possible that an inflammatory process develops in these patients in the same way as is seen in SSc. Leukotriene B4, produced mainly by polymorphonuclear cells, is a potent mediator of inflammation. The plasma thiol concentration shows the degree of oxidation of plasma and a decreased concentration indicates the increased production of free radicals which are capable of oxidative damage. In this work, the white blood cell (WBC) production of leukotriene B4 and plasma thiol concentrations were measured in patients with SSc and VWF. Fifty nine patients were studied: 34 had SSc and 25 had VWF. The results were compared with 25 matched normal controls and are expressed as the median and range. After stimulation of the polymorphonuclear cells with calcium ionophore A23187 (1 microgram/ml), patients with SSc and VWF were found to have significantly increased leukotriene B4 production (23.5 (14.4-44.0) and 26 (14.4-39.4) ng/10(6) cells, respectively) compared with normal controls (17.2 (6.0-38.0) ng/10(6) cells). The plasma thiol concentration was shown to be significantly lower in patients with SSc and VWF (445 (375-475) and 450 (417-510) mumol/l, respectively) compared with normal controls (480 (418-555) mumol/l). Our results show increased leukotriene B4 production and increased free radical activity in patients with SSc and VWF. Although previous indirect evidence has suggested increased WBC activity in patients with SSc, this is reported directly here for the first time. In addition, the possibility of an inflammatory process occurring in patients with VWF, as shown here, has not previously been studied. This may be a further mechanism to explain the poor circulation in the fingers of these patients.


Structure of the sugar-phosphate moiety of lipid A from lipooligosaccharide of Neisseria meningitidis group B, strain BC5S No. 125. Hydrolytic stability of phosphate and pyrophosphate substituents.

On the basis of chemical and NMR data the partial structure of lipid A from lipooligosaccharide (LOS) of Neisseria meningitidis group B, strain BC5S No 125 was established. Lipid A consisted of disaccharide 2-deoxy-6-O-[2-deoxy-2-(3-hydroxytetradecanoylamino)- beta-gluco-pyranosyl]-2-(3-hydroxytetradecanoylamino)- alpha-glucopyranose carrying the beta-(2-aminoethyl)pyrophosphate residue at 0-4' and the pyrophosphate or phosphate residue at 0-1. On hydrolysis of the acidic form of LOS with 1% acetic acid the substituent at 0-1 was practically completely removed whereas that at 0-4' was stable. The analogous hydrolysis of the Mg-salt of LOS was accompanied by splitting off the pyrophosphate linkage in the substituent at 0-4'. Hydrolysis of LOS at pH 4.5 in the presence of SDS led mainly to a lipid A preparation retaining both pyrophosphate residues.


Total vascular exclusion for hepatic resection in cirrhotic patients. Application of venovenous bypass.

Hepatic vascular exclusion with double venovenous bypass using a centrifugal force pump was used in major hepatic resections in eight patients with hepatocellular carcinoma combined with cirrhosis, and results were compared with those in four patients with hepatocellular carcinoma without cirrhosis and eight with metastatic tumors without cirrhosis among 521 patients undergoing liver resection. Concomitant resection of the retrohepatic inferior vena cava was performed in three of eight patients with cirrhosis and five of 12 patients without cirrhosis. All patients, except one with cirrhosis, tolerated major resection without any hemodynamic impairment, which is often observed in hepatic vascular exclusion without venovenous bypass. One patient, whose complete inflow occlusion period was 70 minutes, died of liver failure. In this patient, the recovery of the arterial ketone body ratio above 1.0 was delayed until 3 days after recirculation, whereas the ratio in the others recovered promptly. Postoperative complications such as increased bilirubin level, pleural effusion, and gastrointestinal tract bleeding were observed in seven of eight patients with cirrhosis compared with six of 12 without cirrhosis. Hepatic vascular exclusion is feasible even in cirrhotics as long as it is applied with venovenous bypass and is kept within the time limit of 60 minutes.


Localization of herpes simplex virus type 1 in sebaceous glands of mice.

The distribution of HSV-1 during the development of zosteriform skin lesions in SCID mice was analyzed by immunofluorescence and electron microscopy. The virus initially appeared within certain keratinocytes, sometimes surrounded by keratinocytes whose surfaces were also positive for the antigens, in the lower epidermal layers including the hair follicles, and then extended upward to the entire epidermis and downward to the sebaceous glands 1-2 days later, when no macroscopic skin lesion was seen. The affected epidermal cells subsequently degenerated and lost their viral antigens within a day, when the zosteriform lesion then became evident. This was followed by a degeneration of the dermis. The sebaceous glands eventually degenerated in 10 days, but some glands in the necrotic skin areas preferentially retained HSV-1. The horizontal spread of the virus in the epidermis beyond the first invaded dermatome occurred much later. In mice passively immunized with specific immune serum, viral antigens were observed even 20 days after the infection in sebaceous glands in necrotized areas. Therefore, HSV-1 appears to spread first via the extracellular fluid among the keratinocytes after being shed from nerve endings, and then produces a successive degeneration of the affected keratinocytes which may prevent any further extension of horizontal viral spread. The pilosebaceous apparatus is possibly acting as a site not only for the replication of HSV-1 with a delayed cytopathic effect, but also as an area that is temporarily sheltered from host defense mechanisms.


Correlation between gI, gII, gIII, and gp 50 antibodies and virus excretion in vaccinated pigs infected with pseudorabies virus.

Eleven groups of 8 pigs were vaccinated with different vaccinal strains of pseudorabies virus deleted or not for non-essential glycoproteins (gI, gX, gp 63), then were challenged 8-9 weeks later with a virulent strain. Antibodies against the major viral glycoproteins (gII, gIII, gp 50) were titrated at the day of challenge. Excretion of the challenge strain, growth performance of the pigs, and gI antibody responses from pigs vaccinated with a gI minus strain, were monitored after the challenge. The results demonstrated a strong association between gII and gIII antibody titres before the challenge, with the amount of the challenge strain excreted and clinical protection after the challenge. Furthermore, gI antibody titres after the challenge were negatively correlated with the level of gII and gIII antibodies at the day of challenge, the virus excretion and the clinical protection after the challenge. Implications of these results for vaccination of pigs and gI antibody screening for the infection are discussed.


Infectious laryngotracheitis virus growth, DNA replication, and protein synthesis.

The polypeptides associated with infection of primary chicken kidney (CK) cells with infectious laryngotracheitis virus (ILTV) were examined by metabolic labelling with [35S]methionine and SDS-PAGE. Polypeptide synthesis was followed over the first 24 h post-infection (p.i.) as this was shown to be the period of viable virus production. A total of 16 ILTV encoded or induced polypeptides were identified using metabolic labelling. The use of inhibitors of protein and DNA synthesis in conjunction with metabolic labelling and viral DNA replication studies enabled a cascade pattern of gene expression, similar to that of herpes simplex virus type 1 (HSV-1), to be established for ILTV. Representatives of alpha, beta, gamma 1 and gamma 2 classes of genes were identified. In contrast to infection with HSV types 1 and 2, which leads to a rapid inhibition of total host cell polypeptide synthesis, ILTV infection of CK cells did not result in a complete inhibition of cellular protein synthesis, with only a small number of host cell polypeptides absent from infected cells.


Herpes simplex virus type 1 (HSV-1) HSZP interferes also after antibody neutralization with early shutoff of host protein synthesis induced by HSV-1 KOS.

The HSZP strain of herpes simplex virus type 1 (HSV-1) is defective with respect to the early shutoff of host protein synthesis. However, in superinfection experiments using Vero cells, the HSZP strain was effective, even after neutralization by antibody, at interfering with the early shutoff function of the HSV-1 KOS strain. Evidence was given that the observed interference was not due to exclusion of the KOS by HSZP at the level of adsorption or penetration. The neutralized KOS strain failed to induce early shutoff of host protein synthesis.


Restriction endonuclease and genetic mapping studies indicate that the vegetative genome of the temperate, Salmonella-specific bacteriophage, epsilon 15, is circularly-permuted.

Data from physical and genetic mapping studies show that the vegetative genome of the temperate, Group E 1 Salmonella bacteriophage, epsilon 15, is circularly-permuted. Preliminary evidence suggests that the circular permutation of the epsilon 15 genome is non-random.


In vitro recognition of an orf virus early promoter in a vaccinia virus extract.

DNA fragments containing varying lengths of the 5' end of an orf virus early gene (ORF3) and its associated promoter were introduced into sodium deoxycholate-solubilized vaccinia virus extracts capable of initiating transcription in vitro from vaccinia virus early promoters. After separation of the radiolabelled products of the reactions on a 5% polyacrylamide/7 M urea gel, discrete transcripts were detected the sizes of which were consistent with initiation of transcription from the orf virus early promoter. This is the first demonstration in a functional assay of the conservation of early transcriptional promoters between an orthopoxvirus and a parapoxvirus.


The effects of beta-2-microglobulin on the infectivity of murine cytomegalovirus.

The role of beta-2-microglobulin (beta 2m) in murine cytomegalovirus (MCMV) infection of susceptible (H-2d) and resistant (H-2k) murine embryo fibroblasts (MEF) and peritoneal macrophages was evaluated using serum-free virus (SF-MCMV). The infectivity of SF-MCMV was significantly lower than virus propagated in serum, although the concentrations of virions were similar. Infection of cells with SF-MCMV was assessed by measuring the proportion of cells expressing viral antigens, the sizes of plaques formed in fibroblast monolayers and TCID50 titers. Infection of susceptible fibroblasts was significantly increased 1.6-4.7 fold by the addition of whole FCS, a less than 20 kDa FCS fraction, or purified human beta 2m. These supplements also significantly enhanced infection of susceptible macrophages and increased TCID50 titers by 3.5-10 fold in susceptible MEF. In relatively resistant H-2k cells, the TCID50 titer and the proportion of cells expressing viral antigens after infection with SF-MCMV were not affected by beta 2m or FCS, but plaque sizes were increased 2.5-3 fold in resistant BALB.K MEF. When human or murine beta 2m was added to infected cultures, immunogold electron microscopy revealed these proteins to be always associated extracellularly with the tegument material of disrupted multicapsid virions, but rarely with the envelope of intact virions. However, no murine beta 2m was found in association with the envelope or tegument of SF-MCMV. These relatively modest effects of beta 2m which were restricted to genetically susceptible cells, may be due to tegument-bound beta 2m facilitating infection by capsids, or the stabilisation of the conformation of Class 1 molecules by exogenous beta 2m, promoting MCMV binding to the target cell.


Molecular characterization and heterogeneity of feline immunodeficiency virus isolates.

We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, in gag, pol, and env genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96,95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly, tat- and rev-like short open reading frames contained inframe stop codons in the FIV Petaluma but not in the FIV TM2.


Mesenteric fibromatosis preventing restorative proctectomy.

A case is reported illustrating the dilemma of encountering mesenteric fibromatosis during restorative proctectomy for familial adenomatous polyposis.


Rat lung possesses the mineralocorticoid receptor.

Lung cytosol from male, adrenalectomized rats was screened for the mineralocorticoid receptor (MCR) by a polyclonal antiserum raised in the rabbit against rat renal antigen. Western blot analysis revealed a single 98 kDa band, like the MCR purified biochemically. The MCR could also be photolabelled for the first time by 3H-R 5020 in this very 98 kDa region that was displaced by RU 26752 specific to MCR. Immune IgG was able to precipitate the MCR-3H-RU 26752 complex, and to displace the same to high molecular weight regions during gel permeation chromatography on Sephacryl columns. Thus, MCR mediated actions need to be redefined. Furthermore, the technique of photochemical labelling forms a novel tool to assess MCR specificity, and to dissect its structure and function.


Expression and transforming activity of a variant of the heparin-binding fibroblast growth factor receptor (flg) gene resulting from splicing of the alpha exon at an alternate 3'-acceptor site.

Splicing at an alternate 3'-acceptor site results in deletion of a CCCAG in the 5'-sequence of the exon coding for the NH2-terminal immunoglobulin-like disulfide loop of the heparin-binding fibroblast growth factor receptor (flg) alpha isoform. The result is an in-frame stop codon 138 base pairs after the first flg consensus translational initiation site. The next more favorable site predicts the same two loop intracellular receptor isoform, gamma, which was predicted from two different human cDNAs that arise by alternate use of two exons at the same site. Although expressed in normal tissue, the gamma mRNA is increased in rat prostate tumors and confers ability of anchorage-dependent cells expressing non-secreted heparin-binding fibroblast growth factors to grow in soft agar.


Protamine kinase phosphorylates eukaryotic protein synthesis initiation factor 4E.

Up to 1 mol of phosphoryl groups was incorporated per mol of eukaryotic protein synthesis initiation factor (eIF) 4E following incubation of purified preparations of this factor with purified preparations of a protamine kinase from bovine kidney cytosol. By contrast, purified preparations of two forms of mitogen-activated protein kinase, casein kinase II and two forms of a distinct autophosphorylation-activated protein kinase exhibited little activity, if any, with eIF-4E. Together with previous observations, the results indicate that the protamine kinase could contribute to the insulin-stimulated phosphorylation of eIF-4E.


The reducing activity of S-aminoethylcysteine ketimine and similar sulfur-containing ketimines.

S-aminoethylcysteine ketimine and other sulfur-containing similar ketimines reduce molecular oxygen and phospho-18-tungstate (Folin Marenzi reagent), although the sulfur atom is formally present in the non reducing thioether form. We have now found that 2,6-diclorophenolindophenol, some ferrihemoproteins and other reagents are also reduced by this group of ketimines. Ferricytochrome c is reduced faster than methemoglobin, metmyoglobin and free hematin, whereas horse radish peroxidase compound I is reduced at once. These results indicate a wider reducing activity of this type of ketimine. The oxidation of ketimines by ferric cytochrome c appears a relevant finding pointing to a new possible way of enzymatic modification of sulfur-ketimines in tissues.


Plasma membrane calcium pump expression in human placenta and small intestine.

To identify the forms of the plasma membrane calcium pump present in tissues that transport calcium, cDNA from human placenta and proximal small intestine was amplified by the polymerase chain reaction using a pair of mixed primers based on all the known human and rat plasma membrane calcium pump sequences. Clones were identified from the two human forms HPMCA1 and HPMCA4, but no new sequences were found in either tissue. RNA blots probed with HPMCA1 showed two bands in both tissues; probing with HPMCA4 gave a single, larger species. In placenta, HPMCA4 was the more abundant form and similar expression was found in full-term and second-trimester placentas. In contrast, in the small intestine, HPMCA1 was more abundant, suggesting that calcium absorption is not associated with any one specific isoform in calcium transporting cells.


Endothelin and structurally related analogs distinguish between endothelin receptor subtypes.

The endothelin (ET) analog ET-1[1,3,11,15-Ala] was compared with ET-1, ET-2, ET-3 and sarafotoxins (SRTX) S6b and S6c for receptor binding and function. All the peptides exhibited high affinity binding and contracted rabbit pulmonary artery with near equal potency. In rat aorta both ET-3 and ET-1 [1,3,11,15-Ala] bound with much lower affinity than ET-1 while ET-3 displayed weak contractile potency and ET-1 [1,3,11,15-Ala] and SRTX-c were inactive. In rat left atria, ET-1 [1,3,11,15-Ala] and SRTX-c were weak inhibitors of binding and were also functionally inactive, whereas ET-1, ET-2, ET-3, and SRTX-b were equipotent in producing contractile responses. The data support the idea of there being a predominance of ETA receptors in rat aorta and ETB receptors in rabbit pulmonary artery. In rat left atria, the ET receptor could not be readily classified into ETA or ETB and suggests the existence of a new receptor subtype.


Identification of guanidino succinate as a putative endogenous source of the endothelium derived relaxing factor.

Using a specific HPLC analysis for guanidines, we find that rat aorta contains guanidino succinate (GS), guanidino acetate (GA), guanidino propionate (GP), guanidino butyrate (GB), methyl guanidine (MG) and guanidine. The concentration of L-arginine (0.05 nmol/mg tissue) is significantly lower than the other guanidines. GS is found to be the most potent vasodilator-guanidine in the rat aorta preparation and this vasodilation depends predominantly on the presence of the endothelium. This effect of GS is antagonized by NG-monomethyl L-arginine (L-NMMA), NW-nitro L-arginine benzyl ester (L-NABA), hemoglobin and by methylene blue, all of which are known to block or attenuate endothelium dependent relaxation. Further, the relaxation mediated by GS is accompanied by the formation of cGMP in the rat aorta. From these results we suggest that GS may be a major endogenous source of EDRF.


Protease inhibitors but not proteases inhibit the glucose-6-phosphatase of native rat liver microsomes.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.


Is the osteoclast calcium "receptor" a receptor-operated calcium channel?

Elevated extracellular calcium levels ([Ca2+]e) inhibit osteoclast function by elevating cytosolic free calcium levels ([Ca2+]i), presumably via the activation of a surface Ca2+ "receptor". It is unclear whether or not Ca(2+)-induced [Ca2+]i elevation involves the direct gating, by the putative "receptor", of a divalent cation channel. The results show that [Ca2+]i elevation in response to elevated [Ca2+]e comprises a distinct component of Ca2+ influx, the magnitude of which can be decreased and increased, respectively, by depolarising (100 mM-[K+]) and hyperpolarising (1 microM-[valinomycin]) the osteoclast membrane. In addition, activation of the putative Ca2+ "receptor" by elevated [Ca2+]e causes influx of the related divalent cation, magnesium (Mg2+). We suggest that Ca2+ influx induced by Ca2+ "receptor" activation is a major component of the observed [Ca2+]i response.


A Ca2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor.

Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1 microM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+ 98 +/- 9%) when binding was performed at 0.5 degrees C instead of 21 degrees C as already observed in non PKC-desensitized cells. PMA (100 nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1 microM increased Bmax (53 +/- 10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC50 = 1 +/- 0.35 10(-6) M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34 +/- 6% and 36.5 +/- 7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC50 = 1 +/- 0.5 10(-5) M and IC50 = 0.8 +/- 0.2 10(-6) M). We conclude that a Ca2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.


Molecular cloning and characterization of a novel rat activin receptor.

We report the isolation of a full-length rat cDNA for a new activin receptor. The deduced amino acid sequence of this receptor shows 67 percent overall identity with that of a previously identified mouse activin receptor. As predicted for the mouse activin receptor, the amino acid sequence of the rat receptor is consistent with a polypeptide containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and a serine/threonine kinase intracellular domain. In an expression assay, this new receptor was found to bind I125 radiolabeled activin.


Characterization of the promoter region of the murine fibroblast growth factor receptor 1 gene.

To obtain some clue for the regulatory mechanism by which fibroblast growth factor (FGF) receptor 1 (FGFR 1) gene is expressed, we have cloned the promoter region of this gene from genomic library of mouse FGF-responsive cell lines. The genomic clone isolated here includes the FGFR 1 gene from position -868 to +697 relative to transcription initiation site. Sequence analysis reveals the presence of various consensus sequences for the binding sites of transcriptional factors such as SP 1, GCF, Oct-I, AP 1 and AP 2, but the absence of TATA and CAAT sequence motif. The transfection of this promoter-CAT constructs into NIH 3T3 cells demonstrates its promoter activity which is at least located between base -106 and +104.


Calmodulin-binding proteins of calcium-independent type in rat brain synaptosomal membranes: their localization and properties.

Some properties of calmodulin(CaM)-binding proteins (CaMBPs) of the Ca(2+)-independent type were investigated in the synaptosomal membrane (SM) from rat brain using the [125I]CaM gel overlay method. When SM was prepared in the presence of Ca2+, Ca(2+)-independent CaM binding was decreased, whereas the Ca(2+)-dependent type was not altered. All Ca(2+)-independent-type CaMBPs were membrane-bound and scarcely present in the soluble fractions. When SM was heat-denatured, the 24/22.5-kDa CaMBPs could no longer be detected by [125]CaM binding and a new component with higher molecular mass (greater than 200 kDa) was shown to bind CaM in a Ca(2+)-independent manner. A possible effect of cAMP- and Ca2+/CaM-dependent phosphorylation on CaM binding was also examined.


Reactivation of denatured fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase with E. coli ribosome.

Fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase were denatured either by physical or by chemical means. In vitro reconstitution of these denatured enzymes was assisted by 70S E. coli ribosome, as shown by the recovery of their catalytic competence. Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively.


Whole rat electron paramagnetic resonance imaging of a nitroxide free radical by a radio frequency (280 MHz) spectrometer.

Low frequency (280 MHz) electron paramagnetic resonance spectroscopy has been used to follow uptake, distribution and reduction of the nitroxyl spin label PCA in the rat. No difference of half life was found in seven rats submitted to three administrations of PCA (11.3 +/- 0.4; 11.0 +/- 0.6 and 11.5 +/- 0.7 min). Transversal two-dimensional images of PCA distribution in the rat body were obtained over 6 min by means of field gradients. PCA was observed in three regions by projections along the longitudinal axis of the rat. PCA accumulation was found in the lower abdomen 12 min after the start of the PCA injection.


Protein kinase C activity in neonatal cultured rat cardiomyocytes supplemented with docosahexaenoic acid.

In vitro studies have indicated that the 1-stearoyl, 2-arachidonyl diacylglycerol (DAG) is the most effective one for the activation of protein kinase C, although many other DAGs having a different fatty acid composition are active, but to a different extent. Using cultures of neonatal rat ventricular cells, grown in a medium enriched in docosahexaenoic acid (DHA), we previously obtained a cell population that, after alpha 1-adrenoceptor stimulation, produced a DHA enriched DAG. In this study, we have tested the "in vivo" ability of this modified DAG as protein kinase C activator, demonstrating a lower but more persistent translocation of the enzyme from cytosol to particulate fraction in the DHA treated cells. The differences in the PKC activation pattern could be explained by a different metabolism of the DHA enriched DAG by DAG kinase.


Interaction of interferon-alpha with interleukin-1 beta or tumor necrosis factor-alpha on hepatitis B virus enhancer activity.

The interaction of IFN-alpha with IL-1 beta or TNF-alpha on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-alpha, IL-1 beta or TNF-alpha, and synergistically depressed when IFN-alpha was used in combination with IL-1 beta or TNF-alpha. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-alpha in combination with IL-1 beta or TNF-alpha. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-alpha in combination with IL-1 beta or TNF-alpha caused a much greater suppression of HBV enhancer activity than IFN-alpha, IL-1 beta or TNF-alpha alone in both hepatoma cells. These findings suggest that the interaction of IFN-alpha with IL-1 beta or TNF-alpha synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.


Cumulative trauma disorders--an overview.

1. CTDs are a major problem in some industries. Evidence exists for an association between symptoms of CTDs and occupational risk factors such as forceful exertion, repetitiveness, vibration, cold, and extreme postures. 2. The efficacy of ergonomic interventions or programs in reducing CTDs must be evaluated using the same methods as those used to identify problems initially; user feedback regarding job or tool redesigns is essential. Implementing ergonomic interventions is an ongoing, iterative process. 3. The ability of the occupational health nurse to carry out basic nursing functions of health promotion and prevention of injury and illness will be enhanced through the application of the principles of ergonomics.


Single vs. repeated microwave exposure: effects on benzodiazepine receptors in the brain of the rat.

We studied the effects of single (45 min) and repeated (ten daily 45-min sessions) microwave exposures (2450-MHz, 1 mW/cm2, average whole-body SAR of 0.6 W/kg, pulsed at 500 pps with pulse width of 2 microseconds) on the concentration and affinity of benzodiazepine receptors in the cerebral cortex, hippocampus, and cerebellum of the rat. We used a receptor-binding assay with 3H-flunitrazepam as ligand. Immediately after a single exposure, an increase in the concentration of receptor was observed in the cerebral cortex, but no significant effect was observed in the hippocampus or cerebellum. No significant change in binding affinity of the receptors was observed in any of the brain-regions studied. In rats subjected to repeated exposures, no significant change in receptor concentration was found in the cerebral cortex immediately after the last exposure, which may indicate an adaptation to repeated exposures. Our data also show that handling and exposure procedures in our experiments did not significantly affect benzodiazepine receptors in the brain. Because benzodiazepine receptors in the brain are responsive to anxiety and stress, our data support the hypothesis that low-intensity microwave irradiation can be a source of stress.


Ethanol inhibition of the chemiluminescent response of stimulated macrophages.

The effect of ethanol on the luminol-dependent chemiluminescence and cAMP level in mice peritoneal macrophages was investigated. Ethanol was shown to inhibit the chemiluminescence of macrophages by acting both as a "trap" for active radicals and as a suppressor of the cellular functional activity. A short preincubation of macrophages with ethanol results in a dose-dependent decrease of the chemiluminescent response to the stimulatory agent (opsonized zymosan). Ethanol was also shown to induce a peakwise rise of the intracellular cAMP after a 2-min incubation. The observed effects are correlated both in time and concentration, which allows the presumption that inhibition of the functional activity of macrophages is mediated by the increase of the intracellular cAMP level.


Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.


Exogenous synapsin I promotes functional maturation of developing neuromuscular synapses.

We have investigated the possible role of synapsin I, a nerve terminal-specific protein, in the maturation of neuromuscular synapses in Xenopus cell cultures. Purified synapsin I was loaded into embryonic spinal neurons by injection of the protein into one of the early blastomeres of a Xenopus embryo. At synapses made by synapsin I-loaded neurons, spontaneous synaptic currents occurred with higher frequency and amplitude, and the amplitude exhibited an earlier appearance of a bell-shaped distribution. These characteristics are indicative of more mature quantal secretion. Impulse-evoked synaptic currents also showed a significant increase in amplitude. Using cell manipulation techniques, enhanced transmitter release from synapsin I-loaded neurons was shown to occur at the onset of synaptogenesis, suggesting a presynaptic developmental action of synapsin I prior to synaptic contact. Taken together, these results suggest that endogenous synapsin I may participate in the functional maturation of synapses.


Mechanisms of p185HER2 expression in human non-small cell lung cancer cell lines.

To identify mechanisms that allow p185HER2 expression in lung cancer, we performed Western, Southern, and Northern blot analyses of 14 cell lines derived from human non-small cell lung carcinomas and one cell line derived from a human mesothelioma. Human bronchiole epithelial cells and rat type II pneumocytes were found to express p185HER2 at low to undetectable levels by Western blot technique. In contrast, 13 lung cancer cell lines expressed p185HER2, and eight of these 13 expressed p185HER2 at levels at least 2-fold higher than that found in normal bronchiole epithelial cells or type II pneumocytes. Genomic Southern analysis showed that amplification of the HER2 gene was present in only one of the eight cell lines that expressed p185HER2 at these higher levels. Increased levels of steady-state HER2 mRNA occurred in the remaining seven cell lines. We conclude that in human non-small cell lung carcinoma cell lines the most common mechanism resulting in increased p185HER2 expression is due to mechanisms that increase HER2 mRNA levels, with HER2 gene amplification occurring less commonly.


Enhanced generation of free radicals from phagocytes induced by mineral dusts.

Several studies have suggested that pulmonary toxicity to asbestos and silica may be mediated through oxidant-induced cell injury. We have reported recently that surface radicals associated with freshly fractured silica may be an important factor in cell injury and induction of pulmonary disease. Although the generation of oxygenated radicals in dust-cell interactions has been demonstrated, there are no data correlating the toxicity of a dust with the level of oxygen radical generation by the dust during its interaction with phagocytic cells. In the present study, we have investigated the in vitro generation of oxygen free radicals from human neutrophils and rat alveolar macrophages stimulated with freshly fractured silica, aged silica, amosite, crocidolite, chrysotile, and nontoxic dust, barite. Electron spin resonance (ESR) with the aid of a spin trap phenyl-N-tert-butyl nitrone (PBN) was used to measure the oxygen radicals generated during phagocytosis of the dusts. The relative toxicity index and ESR peak heights, on an equal surface area basis and normalized to barite as one, showed a direct relationship. The normalized toxicity indices and peak heights were: silica, 3.5 versus 2; chrysotile, 4 versus 2; crocidolite, 11 versus 8; and amosite, 26 versus 13. Addition of hydroxyl radical scavengers such as catalase, dimethyl sulfoxide, 1,3 dimethyl-2-thiourea (DMTU), sodium benzoate, and mannitol prevented the radical generation. Carmustine, a glutathione reductase-glutathione peroxidase inhibitor, caused a 5-fold increase in the radical generation. These results indicate that a nontoxic dust such as barite generates toxic oxygen radicals at a minimal level that can be quenched by the normal cellular defense system. For toxic dusts such as silica, amosite, chrysotile, and crocidolite, the potential for oxygen radical generation is enhanced by their surface properties, physical dimensions, and the surface-based radical-generating redox sites. The enhanced radical generation may impair the cellular defense system, resulting in cell injury. Use of scavengers, chelators, and potentiating agents suggests the membrane-based oxidase system as the probable primary source of the radical-generating system. The data presented herein suggest the generation of oxygen free radicals as an important primary event in silica- as well as asbestos-induced cell injury.


Alterations in eicosanoid production by rat alveolar type II cells isolated after silica-induced lung injury.

Although alveolar type II cells in primary culture have been shown to produce eicosanoids and exposure of type II cells to silica in vitro alters eicosanoid production, the production of eicosanoids by alveolar type II cells isolated after acute lung injury in vivo has not been evaluated. Therefore, we investigated the production of arachidonic acid (AA) metabolites by alveolar type II cells isolated after silica-induced lung injury. Alveolar type II cells were isolated from rats 14 days after intratracheal silica instillation and from untreated animals. Type II cells were separated into normotrophic and hypertrophic populations by centrifugal elutriation, and secreted eicosanoids were determined under basal and stimulated conditions by enzyme immunoassay on the day of isolation and after 1 day in culture. Under basal conditions, freshly isolated type II cells from silica-treated animals produced more prostaglandin (PG) E2 than 6-keto-PGF1 alpha or thromboxane B2 (TxB2). Production of all three prostanoids increased with increasing cell size. The calcium ionophore A23187 stimulated a less than 2-fold increase in PGE2 and 6-keto-PGF1 alpha production in all groups of cells. In contrast, this calcium ionophore greatly enhanced TxB2 and leukotriene C4 (LTC4) production by normotrophic type II cells from both untreated and silica-treated animals. Incubation with exogenous AA suggested that the increased capability of the hypertrophic cells to synthesize PGE2 and TxB2 was due primarily to an increase in arachidonate availability. The hypertrophic type II cells also appear to have increased prostacyclin synthase activity. There were no differences in the catabolism of PGE2 between the normotrophic and the hypertrophic type II cells.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of saturation of ACE binding sites on the pharmacokinetics of enalaprilat in man.

1. Eight healthy male volunteers received oral enalapril, 10 mg, in the presence and absence of pretreatment with captopril, 50 mg, twice daily for 5 days. 2. Enalaprilat pharmacokinetics were characterised after both doses of enalapril to investigate the effect of saturating ACE binding sites by pretreatment with captopril. 3. The pharmacokinetics of enalaprilat were best described by a one compartment model with zero order input incorporating saturable binding to plasma and tissue ACE. 4. Values of AUC (0.72 h) for enalaprilat were 419 +/- 97 and 450 +/- 87 ng ml-1 h in the presence and absence of captopril, respectively. The difference was not statistically significant nor were there any other differences in model parameters. 5. Induction of ACE by captopril resulting in an increase in the number of ACE binding sites, may have obscured any effect of captopril on the occupancy of ACE binding sites by enalapril.


Lisinopril and nifedipine administration inhibits the ex vivo uptake of [45Ca2+] by platelets from hypertensive diabetic patients.

1. The effect of administration of the angiotensin converting enzyme inhibitor (ACEI), lisinopril (Carace; 10-40 mg twice daily) and the calcium channel blocker, nifedipine (Adalat Retard; 20-40 mg twice daily) on ex vivo [45Ca2+] uptake by platelets from hypertensive diabetic (type 1 and 2) patients was investigated. 2. At the end of at least 3 months treatment, blood was collected prior to the patient taking the morning dose of medication and washed platelets prepared. [45Ca2+] uptake was monitored following the addition of adrenaline, isoprenaline and dibutyryl cAMP (dbcAMP), as well as in unstimulated (zero) platelets. 3. Both nifedipine and lisinopril significantly inhibited the ex vivo uptake of [45Ca2+] by platelets when this process was stimulated by adrenaline, isoprenaline and dibutyryl cAMP. Basal uptake was also inhibited in both groups. 4. These data consolidate the hypothesis that ACE inhibitors may possess calcium channel/calcium mobilisation blocking properties. Apart from its hypertensive action, lisinopril may also reduce platelet activity via modulation of calcium dynamics, thereby reducing the incidence of vascular complications associated with diabetes mellitus.


Genital human papillomavirus infections in adolescents: implications for evaluation and management.

Genital infections caused by human papillomaviruses (HPV) have become the most prevalent sexually transmitted disease in the United States. Rates of infection with these agents are particularly high among adolescents. The most common clinical expression of genital HPV infection is condyloma acuminata, a condition that dermatologists are frequently called on to treat. The identification of external genital warts in a teenager raises questions that impact directly on the patient's evaluation and management. Issues of importance include the likelihood that occult infection exists at other sites in the genital tract, the risks associated with HPV infection, and the need, if any, for further diagnostic assessment, including that necessary to detect the presence of coexisting sexually transmitted infections. This article examines these and associated issues and provides information that will be of value to the dermatologist caring for adolescent patients with genital warts.


Prognostic value of flow cytometrically determined DNA-ploidy, intracellular pH and esterase activity of non-small cell lung carcinomas.

30 surgical specimens of patients with non-small cell lung carcinomas (NSCLC) were investigated. Significant increases of intracellular pH values in epithelial and inflammatory cells, in the percentage of dead epithelial and inflammatory cells and in the cell volume of vital inflammatory cells in cancerous lung tissue were encountered. Furthermore, decreases of the esterase activity of vital epithelial cells and of the percentage of free cell nuclei were observed. The DNA aneuploidy in 36.6% of the tumours was frequently associated with non-squamous cell carcinomas and stage II, III, IV tumours. Patients with DNA aneuploid tumours had a significantly shorter survival rate than those with DNA euploid tumours. Within the different tumour stages a similar tendency was observed which was, however, only significant in stage III tumour patients. Stage III tumours constitute therefore a heterogeneous entity with a worse prognosis for DNA aneuploid tumour patients. The intracellular pH values and esterase activity as well as the cell volume, the percentage of free cell nuclei and dead inflammatory or epithelial cells contained no significant prognostic information.


Characterization of the multiple EPR line shapes of iron-semiquinones in photosystem 2.

We have compared the temperature-dependence characteristics of the EPR signals of Qa and Qb iron-semiquinones from both purple bacterial and plant photosystems. The data obtained were analyzed and estimates of the splitting parameters of the non-heme Fe2+ spin sublevels obtained. The study confirms the similarities of the g = 1.8 Qa iron-semiquinone signal (D/k = 15.6 K, E/k = 3.3 K) formed in formate-treated plant photosystem 2 to the signal found in purple bacteria. However, the g = 1.9 Qa iron-semiquinone signal (D/k = 7.1 K, E/k = much less than 1 K), formed in photosystem 2 when bicarbonate remains bound, has a unique temperature behavior. A series of spectral features associated with the iron-semiquinone in bicarbonate-bound photosystem 2 appear as the temperature is lowered, and the analysis of these data requires that some of these features be assigned to the higher spin states. The results are discussed in terms of the requirement for bicarbonate to be a ligand of the non-heme iron.


Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)


Retroviral nucleocapsid protein specifically recognizes the base and the ribose of mononucleotides and mononucleotide components.

The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS [Secnik, J., Wang, Q., Chang, C.-M., & Jentoft, J.E. (1990) Biochemistry 29, 7991-7997]. The estimated binding affinities were unexpectedly similar for nucleotides, nucleosides, and bases (Ka greater than 10(6) M-1). AMP, UMP, GMP, and CMP bound to NC with essentially equal affinity, indicating that NC does not discriminate between bases. This is consistent with its role in coating, condensing, and packaging the RNA within virions. Nucleosides, bases, riboses, and ribose phosphate bind to NC with 1000-fold higher affinity than inorganic phosphate, indicating that the NC binding site includes elements that recognize nucleotide base and ribose components in addition to phosphate ions. However, the binding affinities of components are not additive, i.e., the Kapp values for adenine and deoxyribose are very similar to that for deoxyadenosine, indicating that the interaction between the NC subsite and the base and the sugar components is complex. The stoichiometry of the complex between bis-ANS and NC was established to be NC.(bis-ANS)3.(ABSTRACT TRUNCATED AT 250 WORDS)


EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.


Voltage-dependent stimulation of Na+/K(+)-pump current by external cations: selectivity of different K+ congeners.

Currents generated by the endogenous Na+/K+ pump in the oocytes of Xenopus laevis were determined under voltage-clamp as currents activated by different K+ congeners. The voltage dependence of the pump current reflects voltage-dependent steps in the reaction cycle. The decrease of K(+)-activated pump current at positive potentials has been attributed to voltage-dependent stimulation by the external K+ (Rakowski, Vasilets, LaTona and Schwarz (1991) J. Membr. Biol. 121, 177-187). In Na(+)-free solution, activation of the pump by external cations seems to be the dominating voltage-dependent and rate-determining step in the reaction cycle. Under these conditions, the voltage dependence of apparent Km values for pump activation can be analyzed. The dependence suggests voltage-dependent binding of extracellular cations assuming that an effective charge of about 0.4 of an elementary charge is moved in the electrical field during a step associated with the cation binding. The apparent Km values at 0 mV differ for various cations that stimulate pump activity. The values are in mM: 0.10 for Tl+, 0.63 for K+, 0.71 for Rb+, 9.3 for NH4+, and 12.9 for Cs+. The corresponding apparent affinities follow the same sequence as the cation permeability of the K(+)-selective delayed rectifier channel of nerve cells. The results are compatible with the interpretation that the cations have to pass an ion-selective access channel to reach their binding sites in the pump molecule.


Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.


The role of Mg2+ and K+ in the phosphorylation of Na+,K(+)-ATPase by ATP in the presence of dimethylsulfoxide but in the absence of Na+.

We have previously demonstrated that Na+,K(+)-ATPase can be phosphorylated by 100 microM ATP and 5 mM Mg2+ and in the absence of Na+, provided that 40% dimethylsulfoxide (Me2SO) is present. Phosphorylation was stimulated by K+ up to a steady-state level of about 50% of Etot (Barrabin et al. (1990) Biochim. Biophys. Acta 1023, 266-273). Here we describe the time-course of phosphointermediate (EP) formation and of dephosphorylation of EP at concentrations of Mg2+ from 0.1 to 5000 microM and of K+ from 0.01 to 100 mM. The results were simulated by a simplified version of the commonly accepted Albers-Post model, i.e. a 3-step reaction scheme with a phosphorylation, a dephosphorylation and an isomerization/deocclusion step. Furthermore it was necessary to include an a priori, Mg(2+)- and K(+)-independent, equilibration between two enzyme forms, only one of which (constituting 14% of Etot) reacted directly with ATP. The role of Mg(2+) was two-fold: At low Mg2+, phosphorylation was stimulated by Mg2+ due to formation of the substrate MgATP, whereas at higher concentrations it acted as an inhibitor at all three steps. The affinity for the inhibitory Mg(2+)-binding was increased several-fold, relative to that in aqueous media, by dimethylsulfoxide. K+ stimulated dephosphorylation at all Mg(2+)-concentrations, but at high, inhibitory [Mg2+], K+ also stimulated the phosphorylation reaction, increasing both the rate coefficient and the steady-state level of EP. Generally, the presence of Me2SO seems to inhibit the dephosphorylation step, the isomerization/deocclusion step, and to a lesser extent (if at all) the phosphorylation reaction, and we discuss whether this reflects that Me2SO stabilizes occluded conformations of the enzyme even in the absence of monovalent cations. The results confirm and elucidate the stimulating effect of K+ on EP formation from ATP in the absence of Na+, but they leave open the question of the molecular mechanism by which Me2SO, inhibitory Mg2+ and stimulating K+ interact with the Na+,K(+)-ATPase.


Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody.

Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phospholipid. A sulfhydryl group in the hinge region of a monoclonal antibody fragment, was covalently linked to derivatives of phosphatidylethanolamines and phosphatidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which we have named 'Fab-lipids' in liposomal and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface and techniques used for their manipulation are outlined. We describe two possibilities for building a monomolecular receptor layer, consisting of two-dimensional pattern of oriented Fab-fragments with their artificial hydrophobic anchor embedded in a lipid matrix. In the first method a monomolecular film at the air/water interface was allowed to form from a vesicular suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were qualified using micro-fluorimetry.


Transforming growth factors-beta as regulators of cellular growth and phenotype.

An increasing number of cDNAs and polypeptides have been ascribed to the transforming growth factor-beta (TGF-beta) superfamily. It has become evident that this evolutionally well-conserved gene family has representatives in many organisms, from invertebrates to mammalians. As new members are being identified, the knowledge of their biological significance is derived not only from studies of their effects in various in vitro models, but also of their organismal expression during different stages of embryogenesis and morphogenesis. The major described functions of the TGF-beta and like proteins seem to be the regulation of cell proliferation, phenotype, and differentiation. Part of these effects may be attributed to changes in the adhesive properties of the cells and integrity of their extracellular matrices modulated by TGF-beta. Identification of the new members of the TGF-beta family, their biological effects, cell-surface receptors, and cell or tissue-specific expression will be invaluable in trying to understand the purpose of many seemingly alike multiregulators.


Antisense inhibition of oncogene expression.

To understand the role of individual genes in regulating biological processes, one must be able to interfere specifically with either their expression or function. While monoclonal antibodies have proven very useful in studying cell surface proteins, the specific inhibition of intracellular proteins in viable cells is a much more difficult problem. The goal of antisense technology is to develop small oligonucleotides, plasmids, or retroviral vectors which can be readily introduced into living cells in order to inhibit specific gene expression. In this review, we briefly describe the principles of antisense usage, including problems of cellular uptake and intracellular distribution, mechanism of antisense action, and the properties of various oligonucleotide derivatives. In addition we present several examples of the biological effects of antisense administration used to study the role of specific genes in the regulation of cell growth and differentiation.


Hepatocyte growth factor: molecular structure, roles in liver regeneration, and other biological functions.

Hepatocyte growth factor (HGF) is the most potent mitogen for mature hepatocytes and seems to act as a hepatotropic factor that has not been purified over the past 30 years. HGF was first purified from rat platelets in 1986. HGF is a hetrodimer molecule composed of 69-kDa alpha-subunit and 34-beta-subunit. In 1989, cDNAs of both human and rat HGF were cloned and primary structure of HGF was determined. HGF is derived from preproprecursor of of 728 amino acids, which is proteolytically processed to form mature HGF. The alpha-chain contains four kringle domains and it has 38% homology with plasmin. HGF mRNA and HGF activity increase markedly in the liver of rats after various liver injuries such as hepatitis, ischemia, physical crush, and partial hepatectomy. Production of HGF in the liver occurs in Kupffer cells and sinusoidal endothelial cells, but not in parenchymal hepatocytes. HGF mRNA is also markedly increased even in the intact lung, kidney, and spleen after injuries of the liver. Therefore, HGF may act as a trigger for liver regeneration through two mechanisms: a paracrine mechanism and an endocrine mechanism. Moreover, HGF mRNA increases markedly in the kidney after various renal injuries, thus it suggests that HGF may act not only as a hepatotropic factor but also as a renotropic factor. HGF receptor with a Kd of 20 to 30 pM is widely distributed in various epithelial cells including hepatocytes. HGF receptor was recently identified as the product of c-met protooncogene, which encodes a 190-kDa transmembrane protein possessing tyrosine kinase domain. HGF has recently been shown to be a pleiotropic factor. HGF stimulates growth of various epithelial cells, including renal tubular cells (Mitogen). It is worth noting that HGF strongly enhances motility of epithelial cells (Motogen) and induces epithelial tubule formation (Morphogen), while it strongly inhibits growth of several tumor cells. All these findings indicate that HGF may have important roles in organogenesis, morphogenesis, carcinogenesis, as well as in organ regeneration.


The parameter identification problem for the somatic shunt model.

The somatic shunt model, a generalized version of the Rall equivalent cylinder model, is used commonly to describe the passive electrotonic properties of neurons. Procedures for determining the parameters of the somatic shunt model that best describe a given neuron typically rely on the response of the cell to a small step of hyperpolarizing current injected by an intrasomatic recording electrode. In this study it is shown that the problem of estimating model parameters for the somatic shunt model using physiological data is ill-posed, in that very small errors in measured data can lead to large and unpredictable errors in parameter estimates. If the somatic shunt is assumed to be a real property of the intact neuron, the effects of these errors are not severe when predicting EPSP waveshapes resulting from synaptic input at a given location. However, if the somatic shunt is assumed to be a consequence of a leakage pathway around the recording electrode, and a correction for the shunt is applied, then the instability of the inverse problem can introduce large errors in estimates of EPSP waveshape as a function of synaptic location in the intact cell. Morphological constraints can be used to improve the accuracy of the inversion procedure in terms of both parameter estimates and predicted EPSP responses.


Mediastinal germ cell tumors.

Mediastinal germ cell tumors are among the most common tumors in the anterior mediastinum. This tumor should be considered strongly in the differential diagnosis of anterior mediastinal masses. Benign teratomas are common in this site, and complete surgical resection results in cure of nearly all patients. Malignant mediastinal seminomas are highly curable and either radiotherapy (mediastinal disease) or cisplatin-based chemotherapy (metastatic disease) will result in long-term survival in 80% or more of patients. Mediastinal nonseminomatous germ cell malignancies represent a clinically and biologically distinct subset of mediastinal germ cell neoplasms. Relative to their testicular counterparts, these tumors carry a poor prognosis, but, with newer refinements in cisplatin-based chemotherapy, approximately half of these patients will survive their illness. The history of success with chemotherapy should prompt thorough pathologic and serologic evaluation of all patients with mediastinal malignancies in hopes of defining a curable process. In particular, poorly differentiated carcinomas at this site should be treated as germ cell tumors, and so long-term survival will be attainable. Mediastinal nonseminomatous germ cell tumors are associated with the development of nongerm cell malignancies such as embryonal rhabdomyosarcomas and hematologic malignancies such as acute megakaryocytic leukemia and malignant histiocytosis. The development of these associated malignancies is not related to therapy and represents a true biological link between these malignancies. It is likely that the development of these malignancies is an expression of the multipotential nature of primitive germ cells. Careful clinical and biologic investigations of these rare transformations may lead to greater understanding of the regulation of events in the malignant process.


[The diagnosis of lymphatic vascular dysplasia and lymphatic fistula formation].

The importance of lymphangiography for diagnosing the formation of lymphatic fistulas and vascular dysplasias of the lymphatic vessels is on the increase within the scope of therapeutic invasiveness. Its importance and ranking in diagnosing lymphatic circular disorders is demonstrated and exemplified by case reports on three patients suffering from chylothorax of various origin, on one patient with chyluria, and one patient with lymphoedema in the soft parts of the neck. In two chylothorax patients, lymphangiography resulted in successful surgery. It is the decisive method in diagnosis and therapy planning in fistulas and dysplasias of the lymphatic vessel system.


Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.


cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2.

To determine how the human cdc25 gene product acts to regulate p34cdc2 at the G2 to M transition, we have overproduced the full-length protein (cdc25Hs) as well as several deletion mutants in bacteria as glutathione-S-transferase fusion proteins. The wild-type cdc25Hs gene product was synthesized as an 80-kDa fusion protein (p80GST-cdc25) and was judged to be functional by several criteria: recombinant p80GST-cdc25 induced meiotic maturation of Xenopus oocytes in the presence of cycloheximide; p80GST-cdc25 activated histone H1 kinase activity upon addition to extracts prepared from Xenopus oocytes; p80GST-cdc25 activated p34cdc2/cyclin B complexes (prematuration promoting factor) in immune complex kinase assays performed in vitro; p80GST-cdc25 stimulated the tyrosine dephosphorylation of p34cdc2/cyclin complexes isolated from Xenopus oocyte extracts as well as from overproducing insect cells; and p80GST-cdc25 hydrolyzed p-nitrophenylphosphate. In addition, deletion analysis defined a functional domain residing within the carboxy-terminus of the cdc25Hs protein. Taken together, these results suggest that the cdc25Hs protein is itself a phosphatase and that it may function directly in the tyrosine dephosphorylation and activation of p34cdc2 at the G2 to M transition.