Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.


Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase. DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.


A post-translational modification, unrelated to hydroxylation, in the collagenous domain of nonhelical pro-alpha 2(I) procollagen chains secreted by chemically transformed hamster fibroblasts. Transformed Syrian hamster embryo (NQT-SHE) fibroblasts do not synthesize the pro-alpha 1 subunit of type I procollagen, but secrete two modified forms of the pro-alpha 2(I) subunit that migrate more slowly than the normal chain during gel electrophoresis (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). By electrophoretic analysis of cyanogen bromide and V8 protease-derived peptides from the collagenous domains of intra- and extracellular pro-alpha 2(I) chains, we find that the modification occurs almost exclusively in secreted molecules, is located in the region spanned by the cyanogen bromide peptide CB3,5, and persists when hydroxylation is inhibited. Thus, modification is due to a post-translational reaction other than hydroxylation. The modified chains appear to be secreted in the denatured state since: 1) helical structures formed at 4 degrees C under acidic conditions were unstable under neutral conditions at 37 degrees C; 2) conditions that destabilize the type I procollagen helix and thus inhibit its secretion, i.e. inhibition of proline hydroxylation or incorporation of the proline analog cis-hydroxyproline, did not affect secretion of the modified chains. The time courses for secretion of nonhelical modified chains from NQT-SHE and of hydroxylated helical procollagen I from control cells, as a proportion of total collagen synthesized, were similar. Although cis-hydroxyproline did not inhibit the secretion of the modified chains, it induced their rapid intracellular degradation.


Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae. When growing in a sodium-rich environment, wild-type Enterococcus hirae extrudes sodium by two mechanisms, ATP-driven sodium extrusion, and NaH-antiport. Mutant 7683 is unable to grow on sodium-rich media. This is due to two mutations, one inactivating ATP-driven sodium transport and a second rendering NaH-antiport inoperative. 7683 was transformed by electroporation with a gene bank, derived from E. hirae, in an Escherichia coli-E. hirae shuttle vector. Transformants which had regained the ability to grow on sodium-rich media were selected for and the transforming plasmids analyzed. A gene able to restore NaH-antiport activity in 7683 was identified. This gene was named napA. It codes for an extremely hydrophobic protein of 383 amino acids. Hydropathy analysis of this protein indicates that it probably forms 12 transmembraneous helices. In a mutant, possessing only the NaH-antiporter, the napA gene was disrupted by homologous recombination. The resultant strain failed to grow in sodium-rich media, and vesicles isolated from these cells exhibited a defect in sodium proton antiport activity. We conclude that the napA gene codes for a NaH-antiporter. The NapA protein does not exhibit significant homology to any protein in the EMBL genetic data bank.


Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11. A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.


Mevalonate kinase is localized in rat liver peroxisomes. Recent data suggest that rat liver peroxisomes play a critical role in cholesterol synthesis. Specifically, peroxisomes contain a number of enzymes required for cholesterol synthesis as well as sterol carrier protein-2. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol from mevalonate and contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. In this study we have investigated the subcellular localization of mevalonate kinase (EC 2.7.1.36; ATP:mevalonate-5-phosphotransferase). Mevalonate kinase is believed to be a cytosolic enzyme and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. Mevalonate kinase has been purified from rat liver cytosol and a cDNA clone coding for rat mevalonate kinase has also been isolated and characterized. In this study, utilizing monoclonal antibodies made against the purified rat mevalonate kinase, we demonstrate the presence of mevalonate kinase in rat liver peroxisomes and in the cytosol. Each of these compartments contained a different form of the protein. The pI and the Mr of the peroxisomal protein is 6.2 and 42,000, respectively. The pI and Mr of the cytosolic protein is 6.9 and 40,000, respectively. The peroxisomal protein was also significantly induced by a number of different hypolipidemic drugs. In addition, we present evidence for the unexpected finding that the purified mevalonate kinase (isolated from the cytosol and assumed to be a cytosolic protein) is actually a peroxisomal protein.


Alternative multimeric structures affect myogenin DNA binding activity. The native molecular weight of the basic helix-loop-helix (bHLH) proteins myogenin, MyoD, and E12 was calculated from their mobilities on sucrose gradients and molecular sieve chromatography. The muscle bHLH proteins associate to form a variety of higher order complexes, most of which are larger than dimers. Homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer whereas homotetramers and larger forms do not recognize this DNA sequence. The ubiquitous bHLH protein E12 forms monomers or homodimers with little evidence for higher order complexes. Mixtures of myogenin and E12 show some heterodimeric structures, but most of the myogenin remains in large complexes. This result using purified proteins is also obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin is present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein containing only the myogenin HLH region forms large homomeric complexes. A model is presented in which each helix associates with a different subunit to form chains or ring structures to explain these observations. The partition of myogenin in nuclear extracts into dimers that recognize known DNA sequences and higher order complexes that do not raises important new issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.


Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells. The location of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary (CHO) cells made deficient in phosphatidylcholine was determined by immunofluorescence techniques. A rabbit polyclonal antibody was raised against a synthetic peptide corresponding to the amino-terminal 17 amino acid residues of rat liver cytidylyltransferase. The antibody recognized both native and denatured cytidylyltransferase from both rat liver and CHO cells. CHO cells were treated with phospholipase C to alter the lipid composition of the plasma membrane and to elicit translocation of cytidylyltransferase from the less active soluble pool to an activated membrane fraction. Visualization of cytidylyltransferase by indirect immunofluorescence revealed staining of the nuclear envelope in phospholipase C-treated cells but not in untreated cells. CHO cells were also starved for choline and supplemented with a choline analogue to provide an alternative technique of rendering the cells phosphatidylcholine-deficient. Although this treatment should affect different cellular membranes than those affected by phospholipase C treatment, cytidylyltransferase still translocated to the nuclear envelope, as shown by indirect immunofluorescence. These results indicate that activated, membrane-bound cytidylyltransferase is associated with the nuclear membrane and suggest that the nuclear membrane may be a site of de novo phosphatidylcholine synthesis.


Culture of human osteoblasts on demineralised human bone. Possible means of graft enhancement. We cultured human osteoblasts from trabecular bone explants and confirmed their phenotype by alkaline phosphatase assay, increased cyclic adenosine monophosphate production in response to prostaglandin E2 and radiographic micro-analysis of nodules of calcification. The osteoblasts were seeded on to demineralised human bone fragments and examined at ten-day intervals over a 50-day period by scanning electron microscopy. During this time the bank bone became progressively repopulated by the cultured osteoblasts. This system may offer a means of graft enhancement in elective orthopaedic and maxillofacial surgery by delivery of cultured autologous human osteoblasts to bone defects.


Effects of regional systolic asynchrony on left ventricular global diastolic function in patients with coronary artery disease. Patients with coronary artery disease often have impaired left ventricular diastolic filling despite normal global systolic function. The influence of regional systolic asynchrony on diastolic function was assessed by radionuclide angiography in 60 patients with coronary artery disease and normal ejection fraction at rest: group 1 (n = 30) with normal wall motion at rest and group 2 (n = 30) with abnormal wall motion. Data were compared with those obtained from 19 normal volunteers. Age, heart rate, ejection fraction and echocardiographic end-diastolic dimension did not differ among the three groups. Peak filling rate in group 1 and group 2 was similar (2.5 +/- 0.5 and 2.3 +/- 0.6 end-diastolic counts/s, respectively) and significantly lower than that in the normal subjects (2.8 +/- 0.7 end-diastolic counts/s; p less than 0.01 vs. group 2, p less than 0.05 vs group 1). Time to peak filling rate was prolonged in group 2 (184 +/- 27 ms) compared with that in normal subjects (162 +/- 19 ms; p less than 0.01) and group 1 (172 +/- 15 ms; p less than 0.05). Left ventricular end-diastolic pressure was significantly higher in group 2 than in group 1 (14 +/- 7 vs. 10 +/- 5 mm Hg, respectively; p less than 0.05). Asynchrony was assessed by sector analysis of the radionuclide left ventricular region of interest. Diastolic asynchrony was similar in the two patient groups (30 +/- 23 ms in group 2, 26 +/- 16 ms in group 1) and was higher in both groups than in the normal subjects (16 +/- 8 ms; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


MRI of small hepatocellular carcinoma: comparison with US, CT, DSA, and Lipiodol-CT. Thirteen cirrhotic patients with 27 nodules of hepatocellular carcinoma less than 3 cm (small HCC) were examined with ultrasonography (US), MR, pre- and postcontrast CT, digital subtraction angiography (DSA), and CT after injection of Lipiodol (Lipiodol-CT). The accuracy of MR was compared with other diagnostic modalities and MR morphologic and the signal intensity features of HCC were investigated. The detection rate by MR was 63%, by US 67%, by CT 50%, by DSA 74%, and by Lipiodol-CT 93%. The Mc Nemar test showed no difference between the detection rates of MR and CT, MR and DSA, MR and US, and Lipiodol-CT and DSA; however, the differences between the detection rates of MR and Lipiodol-CT and CT and Lipiodol-CT were statistically significant (p less than or equal to 0.05). The difference in sensitivity between the detection rates of Lipiodol-CT and US was just above the level considered significant (P less than or equal to 0.065). On T1- and T2-weighted spin echo images 83% of small HCC were hyperintense relative to the surrounding liver parenchyma. Pseudocapsule was observed in 58% of lesions on T1-weighted images in particular. We believe that US is still the best diagnostic technique for the screening of HCC. We prefer MR to CT as a second level examination to support US in noninvasive diagnosis of small HCC, since MR gives the same or slightly better results than CT without the need of ionizing radiation and large amounts of iodized contrast medium. In our opinion, more invasive examinations, such as DSA and Lipiodol-CT, cannot be avoided in cases where an exact knowledge of the number of lesions is essential for the choice of therapy.


An experimental model of acute and subacute viral myocarditis in the pig. Twenty-six young pigs were infected with encephalomyocarditis virus, observed clinically, studied at intervals by noninvasive and invasive methods to assess cardiac function and eventually examined pathologically. All infected animals appeared ill, usually manifesting diminished appetite, lethargy and fever. Spontaneous mortality occurred either 1 to 4 or 20 to 21 days after infection. Electrocardiographic abnormalities, seen in the majority of animals, comprised ST-T wave changes, conduction disturbances or ventricular ectopic rhythm. The majority of animals manifested echocardiographic evidence of left ventricular dilation and decreased systolic function, which improved with time in some animals. Hemodynamic studies revealed elevation of biventricular filling pressures in 3 of 10 animals; as a group, infected animals manifested significantly elevated right ventricular filling pressures. In selected animals, the feasibility of gallium scans as well as left ventriculography and coronary angiography was demonstrated. At autopsy, heart weight/body weight ratio was significantly elevated in infected animals. The heart of all but two animals showed active myocarditis associated with fibrosis and focal calcification in the later stages. In general, the cardiovascular manifestations were parallel with those seen in acute and subacute myocarditis in humans. It is concluded that encephalomyocarditis infection in the pig is a large animal model of viral myocarditis suitable for assessing alterations in the structure and function of the cardiovascular system and the effects of interventions.


The effects of eosinophil-granule major basic protein on lung-macrophage superoxide anion generation. The effects of major basic protein (MBP) on superoxide anion release in vitro from normal guinea pig (GP) alveolar macrophages (AM) was determined. Native MBP at 55 micrograms/ml had an immediate effect (reduction) on phorbol myristate acetate (PMA)-induced (p less than 0.01) and spontaneous (p = 0.055) superoxide (O2-) release by GP AMs. A similar effect was not observed from AMs incubated for 24 hours with MBP before assessment of O2- release. However, after AMs were incubated with MBP for 48 hours, again there was a significant reduction observed in both PMA-induced (p = 0.01) and spontaneous (p = 0.002) O2- release compared to that of control cultures. The immediate effect of MBP on AM O2- release was not due to cytotoxicity of MBP for AM. In contrast, the effect observed after 48 hours of culture was due, in part, to a direct toxic effect of the MBP on the AM because the viability of AM cultured for 48 hours with MBP (55 micrograms/ml) was 63.6% +/- 13% compared to the viability of control cultures of AM that was 83.9% +/- 7%; p = 0.03. The effect of MBP on AM O2- release at 48 hours was progressive over concentrations ranging from 2 to 55 micrograms/ml. These data suggest that native MBP can affect adversely PMA-induced and spontaneous release of O2- by GP AMs and that this effect depends only in part on the cytotoxic properties of MBP.


Differentiation between hepatoma and hemangioma with inversion-recovery snapshot FLASH MRI and Gd-DTPA. Thirty-eight patients with focal liver tumors (20 hepatomas, 18 hemangiomas) were studied by dynamic sequential inversion recovery (IR) snapshot fast low angle shot (FLASH) MR imaging with Gd-DTPA. Immediately after 0.05 mmol/kg Gd-DTPA was administered intravenously for 2-3 s followed by flushing with normal saline for 4-5 s, 10 images were obtained in the first 20 s (time zero is the end of flush, early phase). Then, one image every 30 s from 1 to 3 min (late phase) and images at 5 min and 7 min (delayed phase) were obtained serially. Hepatomas showed total enhancement in 18 of 20 patients in the early phase, and isointense or low intensity enhancement with respect to the surrounding liver parenchyma in 18 patients in the late to delayed phases. Hemangiomas showed peripheral enhancement in 14 patients in the early phase, but did not show total enhancement (except for two hemangiomas less than 3 cm in size) in the early phase, and showed high intensity enhancement in 15 patients in the late phase. Ninety percent of hepatomas and 82% of hemangiomas showed their characteristic enhancement patterns in the early to delayed phases. We conclude that dynamic sequential IR snapshot FLASH MR images enhanced with Gd-DTPA can facilitate differentiation between hepatomas and hemangiomas.


Delayed enhancement of fibrotic areas in hepatic masses: CT-pathologic correlation. We report a histological analysis of the areas of high density in the postequilibrium and delayed phase CT in 43 focal hepatic lesions. The cases consisted of 16 cholangiocellular carcinomas, 9 hepatocellular carcinomas (including a sclerosing type of hepatocellular carcinoma and a combined hepatocellular-cholangiocellular carcinoma), 13 metastases, 2 granulomas, an inflammatory pseudotumor, a malignant lymphoma, and an epithelioid hemangioendothelioma. Computed tomography was performed after hepatic angiography using 40-50 g iodine and arteriographic CT using 35 g iodine. The areas of delayed enhancement corresponded histologically to fibrotic tissues, from inflammatory change to extensive fibrosis.


Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation. Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth. The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP. CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag. Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event. Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1. Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane. rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression. Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1. CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant. These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.


Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells. When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.


Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors. Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.


Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes. Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.


Galactose receptors and presentation of HIV envelope glycoprotein to specific human T cells. Recognition of viral Ag and of the envelope glycoprotein of HIV (gp120) in particular by human Th cells is critical in the immune response to the viral Ag which includes antibody production and generation of cytotoxic cells. Procedures to increase antigenicity of gp120 are highly desirable in a vaccine perspective. Therefore, to induce activation of gp120-specific T cells by a liminal dose of Ag we enhanced uptake of gp120 by exploiting the galactose receptors on APC. Terminal sialic acid residues were removed by neuraminidase treatment from the carbohydrate side chains of the heavily glycosylated gp120. Galactose residues were exposed and hence recognized by galactose receptors on APC. The experiments demonstrated that 1) human monocytes and dendritic cells, but not cells of the B lineage, bear galactose receptor; 2) galactose receptors are indeed involved because enhanced presentation is inhibited by galactose and acetylgalactosamine and competed for by other asialoglycoproteins; 3) galactose receptors mediate internalization of Ag in intracellular compartments that intersect the processing and presenting pathways, resulting in activation of specific T cells; 4) antigenicity of gp120 for specific T cells can be enhanced by the exposure of galactose residues.


Epidermal cell-polymorphonuclear leukocyte cooperation in the formation of leukotriene B4 by transcellular biosynthesis. The cellular origin of Leukotriene B4, a potent pro-inflammatory agent that is present in psoriatic lesions, has not been completely ascertained. The present study was performed in order to assess the possible contribution of epidermal cells to leukotriene B4 synthesis through 5-lipoxygenase or by means of transcellular metabolism of the epoxide intermediate leukotriene A4 from activated polymorphonuclear leukocytes. The metabolism of exogenous arachidonic acid in fresh human epidermal cell, polymorphonuclear leukocyte or mixed suspensions was determined by means of high-performance liquid chromatography. Epidermal cells transformed arachidonic acid mainly into 12-hydroxy-eicosatetraenoic acid and prostaglandin E2. Formation of prostaglandins F2 alpha and D2, 12-hydroxy-eptadecatrienoic acid, and 15- and 11-hydroxy-eicosatetraenoic acids was also detected. We did not detect any eicosanoid derived from 5-lipoxygenase pathway. Mixed suspensions of polymorphonuclear leukocytes and epidermal cells (ratio 1:4) produced 1.72 times more leukotriene B4 than leukocytes alone under the same experimental conditions. Epidermal cells incubated with 5 microM authentic leukotriene A4 for 3 min yielded 2.954 +/- 0.27 pmoles/10(6) cells of leukotriene B4, which was characterized by co-elution with authentic standard and its ultraviolet absorption spectrum. These data demonstrate the existence of a leukotriene A4 epoxide hydrolase activity in human epidermal cells. Our results suggest that epidermal cells could cooperate in leukotriene B4 biosynthesis by transcellular metabolism of leukotriene A4 in lesions of psoriasis, and possibly other inflammatory dermatoses characterized by increased leukotriene B4 levels and prominent polymorphonuclear leukocyte infiltrates.


All-trans-retinoic acid treatment and retinoic acid receptor alpha gene rearrangement in acute promyelocytic leukemia: a model for differentiation therapy. All-trans-retinoic acid (ATRA), a vitamin A derivative, is a safe and effective drug in the obtention of complete remission in acute promyelocytic leukemia (APL). ATRA is able to activate the maturation of malignant cells from patients with APL either in vitro or in vivo. Complete remission was obtained without any feature of aplastic phase and the severe bleeding diathesis rapidly disappeared. The major adverse effect is the occurrence of hyperleukocytosis which is prevented by the addition of chemotherapy. A progressive acquired resistance appears during ATRA treatment and prolonged event free survival time is obtained after consolidation with cytotoxic drugs. In APL the retinoic acid receptor alpha gene is rearranged and fused with a novel gene called PML. The hybrid PML-RAR product could have a role in the leukemogenesis blocking the effect of the normal RAR on target genes. Retinoic acid exerts a differentiating effect either by the induction of a normal activity of the aberrant product in the presence of pharmacological concentration, or by an over-expression of the normal allele. The results obtained by cellular and molecular biology gave opportunities to confirm the diagnosis, to follow the assessment of the minimal residual disease and to understand the acquired resistance.


[Antiproliferative actions of medroxyprogesterone acetate (MPA) to human ovarian cancer cell lines in vitro]. The cytostatic effect of medroxyprogesterone acetate (MPA; an oral luteohormone preparation) on two cell lines of ovarian carcinoma cultured in vitro (SHIN-3 and MN-1) was studied. At the same time, changes in the morphology and colony formation of these cancer cells after exposure to the drug were examined. 1) The doubling time of SHIN-3 cells in the logarithmic growth phase was prolonged 2.5 times at a MPA dose of 10(-8)M and 3.2 times at a MPA dose of 10(-5)M. The drug, however, exerted no significant cytostatic effect on MN-1 cells. 2) When the IC50 of MPA was assessed by counting live SHIN-3 cells in an FCS-added medium, it was 2.7 x 10(-5)M. When the same assessment was done with a medium without serum, IC50 was 6.4 x 10(-6)M. 3) After 120 hours of incubation in a medium containing 10(-8)M of MPA, CA125 (a tumor marker) production by SHIN-3 cells was suppressed by 35%. The suppression rate was 79% for SHIN-3 cells incubated in a medium containing 10(-5) of MPA. 4) The cytoplasm of SHIN-3 cells, incubated in a MPA-added medium, showed the formation of small mucous vacuoles and expansive degeneration, accompanied by a marked increase in size and thinning of the nucleus. 5) In an experiment on colony formation with collagen gel, the colony size decreased MPA concentration dependently, and was accompanied by the appearance of lobulated colonies.(ABSTRACT TRUNCATED AT 250 WORDS)


[Usefulness of magnetic resonance imaging (MRI) in the detection of the lesions of gestational trophoblastic disease--comparison with computed tomography and digital subtraction angiography]. Twenty patients with gestational trophoblastic disease (GTN) were examined by Magnetic Resonance Imaging (MRI), Computed Tomography (CT) and Digital Subtraction Angiography (DSA), to evaluate their usefulness in the diagnosis of the disease. The lesions of hydatidiform mole were mainly composed of molar vesicles, dilated vessels and hemorrhage which were depicted as small round high intensity lesions on the T2-weighted images and as tree-like low intensity lesions and high or low intensity lesions of various shapes in the T1-, T2-weighted images. These MRI findings closely corresponded to the histopathological findings. On the other hand, CT findings obtained with hydatidiform mole were characterized by filling defects or a small round low density area on contrast enhanced images. The detection ratio for intramural lesions of invasive mole and choriocarcinoma by MRI was 83% (5/6), while that by CT was 50% (3/6). The obliteration of the junctional zone and interruption of the myometrium observed in MRI were significant signs suggesting intramural invasion of the disease. In fact, these signs in MRI were observed in all of the six cases with invasive mole or choriocarcinoma examined. In conclusion, MRI is a powerful means for the determining the intramural invasive mole and choriocarcinoma. Thus more accurate diagnosis of GTN will be obtained with the combined use of MRI and DSA.


An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA. The product of the ie1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS-PAGE, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as beta-galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions.


Characterization of the varicella-zoster virus gene 61 protein. The protein predicted to be encoded by varicella-zoster virus (VZV) gene 61 exhibits limited amino acid sequence similarity to the herpes simplex virus type 1 nuclear phosphoprotein Vmw110, which functions as a transcriptional activator. The gene 61 protein was expressed in its entirety, or as an amino- or carboxy-terminal fragment in Escherichia coli and vaccinia virus recombinants, and monospecific rabbit antisera were raised against an E. coli fusion between beta-galactosidase and the majority of the gene 61 protein. Use of the antisera showed that the gene 61 protein is present in VZV-infected cell nuclei as a heterogeneous phospho-protein of Mr62K to 65K. Phosphorylation occurs in the amino- and, to a lesser extent, carboxy-terminal portions of the protein. The carboxy-terminal region directs transport of the protein to the nucleus, whereas the amino-terminal region, which contains a potential zinc-binding domain, is responsible for a punctate distribution. Preliminary mapping data indicated that gene 61 is transcribed as a 1.8 kb mRNA which initiates about 65 bp upstream from the translation initiation codon, at a position located appropriately with respect to potential regulatory elements.


On the cellular localization of the components of the herpes simplex virus type 1 helicase-primase complex and the viral origin-binding protein. We constructed recombinant viruses based on the herpes simplex virus type 1 mutant tsK which individually were able to express the products of four viral DNA replication genes (UL5, UL8, UL9 and UL52) in the absence of any of the other proteins required for viral DNA synthesis. These viruses were used in immunofluorescence experiments to investigate the cellular localization of the four replication proteins expressed. The results demonstrated that all three components of the viral helicase-primase complex (UL5, UL8 and UL52 proteins) must be co-expressed to allow their efficient localization to the nucleus. Since the UL5 and UL52 proteins together form a complex which is enzymatically indistinguishable from a complex formed from all three proteins, a possible role of the UL8 protein may be in facilitating nuclear uptake. The UL9 protein (origin-binding protein) efficiently entered the cell nucleus when expressed alone. Both UL9 protein and the tripartite helicase-primase complex exhibited patterns of fluorescence which resembled the 'pre-replicative sites' described previously.


The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle. Herpes simplex virus type 1 (HSV-1) gene UL11 encodes a myristylated virion protein. In this paper we have characterized the UL11 product further and investigated its role in the virus life cycle. Wild-type HSV-1 strain 17syn+ expresses three electrophoretically distinguishable UL11 polypeptide species. Analysis of single plaque isolates demonstrated that two virus populations exist within the 17syn+ stock: a major population encoding only the two higher Mr species, and a minor population encoding the lowest Mr species alone. DNA sequence analysis suggests that the latter polypeptide differs from the former ones at a single amino acid residue only. The UL11 polypeptides are synthesized as delayed early gene products and are phosphorylated in vitro. Following subcellular fractionation of infected cells, they are found predominantly associated with membranes. Within the virus particle, they appear to reside within the tegument. An insertion mutant containing the lacZ gene from Escherichia coli within the UL11 open reading frame is viable in tissue culture, although it gives smaller plaques and is impaired for growth compared to the wild-type parent or revertant viruses; it does not have a temperature-sensitive or host-range phenotype. Thus, although required for efficient replication, the myristylated HSV-1 virion protein, in contrast to those of many other viruses, is not essential for virus growth in tissue culture.


Distribution and substrate specificity of intracellular proteolytic processing enzyme(s) for paramyxovirus fusion glycoproteins. Intracellular proteolytic processing of fusion glycoprotein precursors (F0) of paramyxoviruses, i.e. a virulent strain of Newcastle disease virus (NDV), parainfluenza virus type 3 (PIV3) and simian virus 5 (SV5), was examined in NALM6 and BSC40 cells and compared with that in LLCMK2 cells to investigate the distribution of the virus-activating protease(s) among the cells and its substrate specificity. BSC40 cells lack a processing endoprotease of the neuropeptide precursor, pro-opiomelanocortin (POMC), which possesses multiple cleavage sites at pairs of basic residues, Lys-Arg and Arg-Arg, a motif similar to that found in the cleavage site of the F0 proteins. In NALM6 cells, only small amounts of the F0 protein of virulent NDV was cleaved whereas those of PIV3 and SV5 were efficiently cleaved. In BSC40 cells the F0 proteins of these three viruses were cleaved normally as well as in LLCMK2 cells. The processing inhibitors monensin, chloroquine and A23187 suppressed the F0 cleavage in the three cell types. These results indicate that both NALM6 and BSC40 cells possess virus-activating proteases similar to that of LLCMK2 cells, but suggest that the enzyme of NALM6 may be slightly different in its substrate specificity from those of BSC40 and LLCMK2. The results also suggest that the virus-activating proteases are different in their distribution and substrate specificity from the processing enzyme of POMC.


Internalization of intact poliovirus by HeLa cells as shown by subcellular fractionation in isoosmotic Nycodenz gradients. HeLa cells were infected with radiolabelled poliovirus at different temperatures, and the intracellular distribution of input radioactivity was studied. To this end, homogenates were fractionated by rate zonal centrifugation in linear isoosmotic (2 to 30%) Nycodenz gradients. Further purification of subcellular fractions was achieved by recentrifugation to equilibrium in 10 to 30% Nycodenz. Temperatures were kept below 30 degrees C to prevent virus capsid modification. Under these conditions, the cell-associated virions remained fully infectious. Below 18 degrees C, most of the viral label was recovered from a bottom region (BR) of the rate zonal gradients. Marker enzyme analysis and antibody accessibility showed that the BR consisted of virions bound to the plasma membrane. Between 18 degrees C and 26 degrees C, viral label also accumulated in a top region (TR) of the rate zonal gradients. According to the criterion of antibody accessibility, the virions associated with the TR were present within intracellular structures, probably lipid membranes. Electron microscopy confirmed the presence of vesicles and tubules in this region of the gradient. No correlation was found between the TR and endosomal, lysosomal or plasma membrane markers. The TR equilibrated at low density (1.10 g/ml) in Nycodenz (free virus, 1.31 g/ml). The results confirm that intact poliovirions can enter the cell and do so via lipid-bound vesicles.


Identification and characterization of foot-and-mouth disease virus O1 Burgwedel/1987 as an intertypic recombinant. The foot-and-mouth disease virus field isolate Burgwedel/1987 subtype O1 was found to differ genetically from the antigenically related strain O1 Kaufbeuren within the region encoding the non-structural proteins. This genetic difference was indicated by the RNase mismatch cleavage method and confirmed by nucleotide sequencing. An alignment of sequences encoding proteinase 3C of the Burgwedel isolate and several other virus strains identified this isolate as an intertypic recombinant; the parent strains were O1 Kaufbeuren and a subtype C1 strain. Recombination occurred between nucleotide positions 5493 and 5521, within the region encoding peptide 3B1. Thus, the 5' three-quarters of the O1 genome were fused to the 3'-terminal quarter of the C1 genome. Other contemporary isolates from the same district are not recombinants. Sequence alignment distinguished four patterns of proteinase 3C-coding sequences among the virus strains analysed: subtypes A12, C1 and O1 exhibit one pattern each, and another pattern is common to subtypes A5, A10 and O2.


The nucleotide sequences of wild-type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant. We have shown previously that, compared to other enteroviruses, the coxsackievirus A9 (CAV-9) prototype strain, Griggs, contains a C-terminal extension to the capsid protein VP1 and that within this extension there is an RGD (arginine-glycine-aspartic acid) motif. To determine whether these features are found in other CAV-9 strains and therefore analyse whether they are likely to be functionally important, we have determined the nucleotide sequence of the appropriate region from five strains, isolated over a 25 year period. The results indicate that there is considerable diversity between the strains and there is little correlation between nucleotide sequence identity and date of isolation. All isolates exhibit the VP1 extension and although its amino acid sequence is otherwise variable, the RGD motif is common to all. This conservation of sequence, within a region which can otherwise vary, implies that the RGD sequence must be functionally significant. The VP1 extension shows similarity to sequences found in foot-and-mouth-disease virus strains and to part of the precursor of the cellular protein, human transforming growth factor beta, and the possible significance of these observations is discussed.


Shedding of a rhinovirus minor group binding protein: evidence for a Ca(2+)-dependent process. Soluble rhinovirus minor group binding activity was found to be shed into the medium upon incubation of HeLa cells at 37 degrees C. Although substantial amounts of this protein were released, no decrease of virus binding to the cell surface was seen. When the membrane-associated receptor was stripped from the cells with trypsin, virus binding was rapidly restored from an intracellular pool even in the absence of de novo protein synthesis. The release of this 85K virus-binding activity was inhibited by metal chelators such as EDTA, EGTA or 1,10-phenanthroline. The potential involvement of a Ca(2+)-dependent protease and/or a phospholipase in this process is discussed.


Nucleotide sequences of normal and rearranged RNA segments 10 of human rotaviruses. Normal and rearranged RNA segments 10 of group A rotaviruses isolated from a chronically infected immunodeficient child were amplified by the polymerase chain reaction as full-length cDNA copies, and were subsequently cloned and sequenced. Compared with the nucleotide sequence of the normal RNA segment 10, the rearranged form contains a partial non-coding duplication at its 3' end and several point mutations. The normal RNA segment 10 was similar to that of bovine rotavirus.


Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts. An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma. The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74). An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer. The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli. Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80. There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA. These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.


Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. Based on variation in nucleotide sequence within restricted regions in the putative C (core) gene of hepatitis C virus (HCV), four groups of HCV have been postulated in a panel of 44 HCV isolates. They were provisionally designated types I, II, III and IV. A method for typing HCV was developed, depending on the amplification of a C gene sequence by polymerase chain reaction using a universal primer (sense) and a mixture of four type-specific primers (antisense). HCV types were determined by the size of the products specific to each of them. Type II was found in HCV samples from 131 (82%) of 159 blood donors, more often than in those from 48 (60%) of 80 patients with non-A, non-B (NANB) liver disease in Japan (P less than 0.01). In 11 haemophiliacs who had received imported coagulation factor concentrates, type I was found in five, as against type II in four. Double infection with two different HCV types was found in two patients with chronic NANB liver disease (types I and II; II and III) and two haemophiliacs (types I and II; I and III). HCV types were identical in mother and baby in each of two examples of perinatal transmission, and were also identical in donor and recipient in a case of accidental needle exposure.


Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction. The genomic RNA of hepatitis C virus (HCV) in the plasma of volunteer blood donors was detected by using the polymerase chain reaction in a fraction of density 1.08 g/ml from sucrose density gradient equilibrium centrifugation. When the fraction was treated with the detergent NP40 and recentrifuged in sucrose, the HCV RNA banded at 1.25 g/ml. Assuming that NP40 removed a lipid-rich surface coat from HCV, the 1.08 g/ml and 1.25 g/ml HCV RNA may correspond to intact HCV virions and nucleocapsids, respectively. The extraordinarily low density of the virion is unusual in comparison to the density of classified viruses.


Baculovirus-expressed glycoprotein H of herpes simplex virus type 1 (HSV-1) induces neutralizing antibody and delayed type hypersensitivity responses, but does not protect immunized mice against lethal HSV-1 challenge. We have shown previously that herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) expressed by a baculovirus recombinant is transported to the cell surface in the absence of other HSV-1 gene products, and that the expressed gH has an apparent Mr similar to that of authentic HSV-1 gH. We report here that antibodies raised in mice to this baculovirus-expressed gH neutralize the infectivity of HSV-1 in vitro; this neutralizing activity was not complement-dependent. Mice vaccinated with gH also developed delayed type hypersensitivity (DTH) to HSV-1. This is the first report of expressed HSV-1 gH inducing neutralizing antibody or DTH responses in vaccinated animals. In contrast to the gH expressed in mammalian systems, the ability of this baculovirus-expressed gH to induce a neutralizing antibody response may be due to the inability of the mammalian expression system to transport gH to the cell surface. Despite inducing anti-HSV-1 neutralizing antibody and DTH responses, vaccination of mice with gH did not protect the mice against lethal intraperitoneal challenge with HSV-1.


The herpes simplex virus type 1 tegument protein VP22 is encoded by gene UL49. VP22 is a major tegument protein of herpes simplex virus type 1 and is highly phosphorylated in the infected cell. Indirect evidence exists to suggest that it is encoded by gene UL49, present in the BamHI F fragment of the genome. Using the polymerase chain reaction we have cloned the UL49 open reading frame into a mammalian expression vector under the control of the human cytomegalovirus immediate early gene promoter. After transfection into COS-7 cells expression of the gene product was detected by means of Western blotting and immunofluorescence. The results clearly indicate that the protein encoded by UL49 is VP22, and that in transfected cells it appears to have characteristics similar to those of the protein synthesized in infected cells.


Comparison between in vitro neutralization titres and in vivo protection against homologous and heterologous challenge induced by vaccines prepared from two serologically distinct variants of foot-and-mouth disease virus, serotype A22. Guinea-pigs were challenged with homologous or heterologous strains of foot-and-mouth disease virus (FMDV) following vaccination with baby hamster kidney (BHK) monolayer cell-adapted or BHK suspension cell-adapted strains of FMDV serotype A22 Iraq 24/64. The protection afforded by these vaccines was analysed as a function of antigen dose and the in vitro serum virus neutralization titres achieved. The results show that the level of neutralizing antibody induced that afforded 50% protection was similar for both vaccines in homologous or heterologous challenge situations. However, although the dose of antigen required to achieve this titre against homologous virus was similar for the two vaccines, approximately 20-fold more of the suspension cell-adapted virus was required to elicit a protective titre against heterologous challenge compared to the dose of monolayer cell-adapted virus required. A synthetic peptide representing the amino acid sequence 135 to 167 of VP1, which is identical in the A22 Iraq 24/64 variant viruses, was shown to induce protection against both homologous and heterologous virus challenge.


Sequence analysis of M2 mRNA of bovine respiratory syncytial virus obtained from an F-M2 dicistronic mRNA suggests structural homology with that of human respiratory syncytial virus. The nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94% identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features of BRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43% amino acid identity. As described for HRSV, the 3'-terminal end of the M2 mRNA overlaps with the 5' end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75%. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.


The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. The expression patterns of 13 GABAA receptor subunit encoding genes (alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta) were determined in adult rat brain by in situ hybridization. Each mRNA displayed a unique distribution, ranging from ubiquitous (alpha 1 mRNA) to narrowly confined (alpha 6 mRNA was present only in cerebellar granule cells). Some neuronal populations coexpressed large numbers of subunit mRNAs, whereas in others only a few GABAA receptor-specific mRNAs were found. Neocortex, hippocampus, and caudate-putamen displayed complex expression patterns, and these areas probably contain a large diversity of GABAA receptors. In many areas, a consistent coexpression was observed for alpha 1 and beta 2 mRNAs, which often colocalized with gamma 2 mRNA. The alpha 1 beta 2 combination was abundant in olfactory bulb, globus pallidus, inferior colliculus, substantia nigra pars reticulata, globus pallidus, zona incerta, subthalamic nucleus, medial septum, and cerebellum. Colocalization was also apparent for the alpha 2 and beta 3 mRNAs, and these predominated in areas such as amygdala and hypothalamus. The alpha 3 mRNA occurred in layers V and VI of neocortex and in the reticular thalamic nucleus. In much of the forebrain, with the exception of hippocampal pyramidal cells, the alpha 4 and delta transcripts appeared to codistribute. In thalamic nuclei, the only abundant GABAA receptor mRNAs were those of alpha 1, alpha 4, beta 2, and delta. In the medial geniculate thalamic nucleus, alpha 1, alpha 4, beta 2, delta, and gamma 3 mRNAs were the principal GABAA receptor transcripts. The alpha 5 and beta 1 mRNAs generally colocalized and may encode predominantly hippocampal forms of the GABAA receptor. These anatomical observations support the hypothesis that alpha 1 beta 2 gamma 2 receptors are responsible for benzodiazepine I (BZ I) binding, whereas receptors containing alpha 2, alpha 3, and alpha 5 contribute to subtypes of the BZ II site. Based on significant mismatches between alpha 4/delta and gamma mRNAs, we suggest that in vivo, the alpha 4 subunit contributes to GABAA receptors that lack BZ modulation.


The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. In an effort to determine subunit compositions of in vivo GABAA receptors, the cellular localization of 13 subunit encoding mRNAs (alpha 1-alpha 6, beta 1-beta 3, gamma 2-gamma 3, delta) was determined in the rat olfactory bulb and cerebellum. Cerebellar granule cells expressed significant quantities of alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta mRNAs. They contained very much lower levels of alpha 4, beta 1, and gamma 3 mRNAs, and the alpha 2, alpha 3, alpha 5, and gamma 1 genes appeared to be silent. Purkinje cells contained only the alpha 1, beta 2, beta 3, and gamma 2 mRNAs. Putative Bergmann glial cells were found to contain the gamma 1 mRNA and possibly the alpha 2 mRNA. In the molecular layer, only the alpha 1, beta 2 and gamma 2 mRNAs were expressed in stellate/basket cells. The alpha 3 probe hybridized weakly to targets in the molecular layer. The inferior olivary nucleus contained significant quantities of alpha 2, alpha 4, and gamma 1 transcripts, with the alpha 1, alpha 3, beta 2, beta 3, and gamma 2 mRNAs also present. In the olfactory bulb, mitral cells were found to express the alpha 1, beta 1, beta 2, beta 3, and gamma 2 mRNAs strongly and the alpha 3 mRNA weakly. Tufted cells contained alpha 1, alpha 3, beta 2, beta 3, and gamma 2 mRNAs and, occasionally, the alpha 2 mRNA. In the internal granule cells the alpha 2, alpha 4, alpha 5, beta 3, and delta mRNAs were all present. Low levels of alpha 3, gamma 1, gamma 2, and gamma 3 mRNAs were also noted in these cells. Periglomerular cells expressed low levels of alpha 2, alpha 3, alpha 4, beta 2, beta 3, gamma 1, gamma 2, and gamma 3 mRNAs. No alpha 6 mRNA was present in the olfactory bulb. Correlations that are general ones from other brain regions are the colocalizations of alpha 1 beta 2, alpha 2 beta 3, and alpha 4 delta mRNAs. In both the olfactory bulb and cerebellum, alpha 1 beta 2 gamma 2 receptor cores are probably employed. The delta-subunit mRNA appears to codistribute with alpha-subunit mRNAs (alpha 4 and alpha 6) associated with GABAA subunits that fail to bind benzodiazepine agonists.


Pharmacological characterization of endothelin-stimulated phosphoinositide breakdown and cytosolic free Ca2+ rise in rat C6 glioma cells. Because increasing evidence indicates that glial cells are a target of endothelin, we have characterized endothelin-induced phosphoinositide (PI) turnover and Ca2+ homeostasis in C6 glioma cells. Endothelin-1 (ET) increased formation of 3H-inositol phosphate (IP) from PI and elicited an increase in cytosolic free Ca2+ ([Ca2+]i) in rat C6 glioma. In the presence of Li+, the increase in 3H-inositol trisphosphate formation was rapid, reaching its peak at 5 min after stimulation. ET also elicited a rapid and sustained increase in [Ca2+]i in a dose-dependent manner (1-100 nM). The rank orders of efficacy for ET-related peptides in increasing [Ca2+]i were ET = ET-2 greater than sarafotoxin greater than ET-3. Both ET-mediated stimulation of IP formation and [Ca2+]i increase were largely inhibited in the absence of external Ca2+ but unaffected by the depletion of external Na+ and the presence of dihydropyridine derivatives or verapamil. Inorganic Ca2+ channel blockers Cd2+, La3+, and Mn2+ at 1 mM inhibited both responses induced by ET. Cross-desensitization and nonadditivity were observed for both events among ET-related peptides tested, but not between ET and ATP. Pretreatment of cells with pertussis toxin (PTX) attenuated the PI response to ET, but had no effect on ET-elicited [Ca2+]i increase. ET-induced Ca2+ mobilization (measured in Ca(2+)-free medium) was only transient and was inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate. Moreover, the intracellular Ca2+ pools mobilized by ET and ATP appeared to overlap, as indicated by their partial heterologous desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)


Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor. Phosphorylation of the rat brain ryanodine receptor was studied using a monoclonal antibody, Ry-1, against the cardiac ryanodine receptor. A large polypeptide with the same SDS-PAGE mobility as that of the canine cardiac receptor was detected in rat brain membranes by immunoblotting. The brain ryanodine receptor was solubilized from the microsomal membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and more than 85% of the solubilized receptor was immunoprecipitated by Ry-1. Immunoprecipitated receptors were phosphorylated by cAMP-dependent protein kinase. The ryanodine receptor was also expressed in cultured fetal rat brain neurons and was phosphorylated by treating the cells with dibutyryl cAMP. The number of cells showing a caffeine-induced Ca2+ transient was increased significantly in the phosphorylating condition. These results suggest that the Ca channel activity of the brain ryanodine receptor is regulated by cAMP-dependent phosphorylation.


Acetylcholine synthesis and release is enhanced by dibutyryl cyclic AMP in a neuronal cell line derived from mouse septum. Cholinergic properties of the SN56.B5.G4 cell line derived from the fusion of neurons of the mouse postnatal day 21 septum and the murine neuroblastoma cell line N18TG2 were investigated and correlated with morphological differentiation. In basal serum-containing growth medium, few cells developed neurites. Neurite extension occurred in cells grown for 2 d with forskolin or dibutyryl cAMP (dbcAMP) but not with butyrate. In cells treated with these compounds, the activity of ChAT and ACh content were two- to threefold higher relative to controls. The cells synthesized ACh from choline taken up by the sodium-dependent high-affinity transport. Forskolin-, dbcAMP-, and butyrate-treated cells (but not the controls) were capable of spontaneous and depolarization-evoked ACh release. The results indicate that the morphological and the neurochemical aspects of SN56.B5.G4 cell differentiation are independently regulated.


Energy metabolism of rabbit retina as related to function: high cost of Na+ transport. Experiments designed to examine the energy requirements of neurophysiological function were performed on isolated rabbit retina. Function was altered by photic stimulation or by function-specific drugs, and the response of energy metabolism was assessed by simultaneous measurements of O2 consumption and lactate production. In other experiments, the supply of O2 or glucose was reduced and the effect on energy metabolism and electrophysiological function was observed. Energy requirements under control conditions in darkness were high, with O2 consumption (per gm dry wt) at 11.3 mumol min-1, with lactate production at 14.8 mumol min-1, and with the derived value for glucose consumption at 9.3 mumol min-1 and for high-energy phosphate (approximately P) generation at 82.6 mumol min-1. Energy reserves were small. Removing glucose abolished the b-wave of the electroretinogram (ERG) with a t1/2 of 1 min, but did not immediately affect O2 consumption or the PIII of the ERG. Removing O2 caused increases of up to 2.7-fold in glycolysis (Pasteur effect) and caused both PIII and b-wave to fail, with a t1/2 of about 5 min. Neurotransmission through the inner retina was supported almost entirely by glycolysis, as evidenced by large increases in lactate production in response to flashing light and decreases in response to transmitter blockers (2.3-fold overall change), with no change in O2 consumption. Phototransduction, on the other hand, was normally supported by oxidative metabolism. The dark current accounted for 41% of the retina's O2 consumption. With O2 reduced, the dark current was partially supported by glycolysis, which accounts (at least in part) for the large Pasteur effect. Na+ transport by NaK ATPase accounted for about half of all energy used, as evidenced by the response to strophanthidin, that is, for 49% of the oxidative energy and 58% of the glycolytic energy. The t1/2 for the turnover of intracellular Na+ was calculated from these data to be less than 1 min. Changes in temperature caused changes in the amplitude of light-evoked electrical responses of 6.5% per degree and caused changes in both O2 consumption and glycolysis of 6.8% per degree (Q10 = 1.9). A surprisingly large fraction of oxidative energy, corresponding to about 40% of the total energy generated, could not be assigned to phototransduction, to neurotransmission, to Na+ transport for other purposes, or to vegetative metabolism. We cannot account for its usage, but it may be related to the (previously reported) rapid turnover of the gamma-phosphate of retinal GTP, the function of which also remains unknown.(ABSTRACT TRUNCATED AT 400 WORDS)


Demonstration of coexisting catecholamine (dopamine), amino acid (GABA), and peptide (NPY) involved in inhibition of melanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze-substitution immunocytochemical study. This quantitative ultrastructural immunocytochemical study demonstrates the coexistence of a catecholamine [dopamine (DA)], an amino acid (GABA), and a neuropeptide [neuropeptide Y (NPY)] in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin color during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculostellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Monolabeling studies on routinely fixed and freeze-substituted tissues showed that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger, electron-dense ones. Double and triple labeling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA, and NPY coexist within almost all varicosity profiles and (2) DA and NPY are costored within electron-dense vesicles. Varicosity profiles that about melanotrope cells show a much higher ratio between the numbers of electron-lucent and electron-dense vesicles than varicosities contacting folliculostellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculostellate cells.


Calcium/calmodulin-activated phosphodiesterase expressed in olfactory receptor neurons. We show that calmodulin-dependent phosphodiesterase (CAM-PDE) is selectively expressed in mature olfactory receptor neurons within the olfactory mucosa. Immunocytochemical staining reveals neuronal immunoreactivity that is most pronounced within cilia, dendritic knobs, and axon bundles. Neither sustentacular cells nor basal cells display immunoreactivity. The extent of loss of neuronal immunoreactivity following bulbectomy resembles loss of the neuronal population. High-affinity CAM-PDE activity in olfactory cilia is fivefold greater than in brain, when assayed at low micromolar cAMP. This activity is depleted in turbinates following bulbectomy. Olfactory mucosal PDE activity is composed of a minimum of two major forms. In the absence of Ca(2+), rolipram-sensitive PDE comprises 65% of total activity. Following stimulation by Ca2+, CAM-PDE activity is elevated sixfold to become the predominant form, thereby increasing total activity 300%, with half-maximal effect at 1 microM Ca2+. We propose that Ca2+ stimulation of CAM-PDE may be necessary for termination of olfactory signals.


Spatiotemporal patterns of expression of NGF and the low-affinity NGF receptor in rat embryos suggest functional roles in tissue morphogenesis and myogenesis. We show here that NGF and its low-affinity receptor (p75NGFR) are expressed during rat embryogenesis at sites that are known to have important roles in tissue morphogenesis and myogenesis. The developing skin of the maxilla, the mandible, and the limb showed very similar patterns of NGF and p75NGFR expression. However, NGF and p75NGFR expression in the developing limb initiated at the limb bud stage and was concentrated at proximal and distal developmental sites that have been reported to be involved in limb morphogenesis. Expression at the proximal/distal ends of the limb persisted throughout limb development, with some of the highest levels of expression occurring at the limb axillary sites, which were not highly innervated. We have also found p75NGFR expression at sites of mesenchymal/epithelial interactions in several developing organs that do not appear to have an adjacent source of NGF and may therefore be sites that bind and respond to the other members of the NGF family (brain-derived neurotrophic factor and neurotrophin-3). These organs include the lung, testes, and kidney, where expression of p75NGFR occurred during the morphogenesis of specific epithelial structures and was coexpressed with the cell adhesion molecule NCAM. In addition, we found that NGF and p75NGFR were expressed during myogenesis. p75NGFR was observed in myoblast cells expressing MyoD1, a myoblast differentiation marker, and NGF transcripts in cells just adjacent to the developing myoblasts. When the myoblasts differentiate into myotubes, p75NGFR and MyoD1 cease to be expressed and the adjacent cells concomitantly cease to be make NGF. However, NGF and p75NGFR were not present in the early muscle precursor cells of the myotome of the somites but were observed in the dermatome and sclerotome, respectively. These results suggest that NGF and p75NGFR have functional roles in developmental processes that affect morphogenesis and cell differentiation.


Aberrant projection induced by otocyst removal maintains normal tonotopic organization in the chick cochlear nucleus. Nucleus magnocellularis (NM), a second-order nucleus in the chick auditory system, is topographically and tonotopically organized. The basilar papilla (cochlea) projects onto the ipsilateral NM via the auditory nerve. The anteromedial region of NM is innervated by the proximal end of the basilar papilla and responds to high-frequency sounds; more posterolateral regions receive input from more distal locations along the papilla and respond to progressively lower frequencies. NM projects exclusively to the third-order neurons of nucleus laminaris (NL). Otocyst removal prevents the formation of the ipsilateral cochlea and cochlear nerve and results in the development of an aberrant functional projection from the contralateral NM to the "deafferented" NM on the operated side of the brain (Jackson and Parks, 1988). In the present experiment, the otocyst was removed unilaterally and the tonotopic organization of the deafferented NM was physiologically mapped at 17-18 d of embryonic age (E17-E18). Quantitative analyses revealed that the frequency organization of the deafferented NM is almost identical to that in normal embryos. Progressively higher characteristic frequencies were recorded at successively more anterior and more medial locations in the nucleus, and the orientation of the tonotopic axis was indistinguishable from normal. Furthermore, the correlation between characteristic frequency and anatomical location is comparable in the deafferented (r = 0.91) and normal (r = 0.87) NM. The only noticeable discrepancy is that characteristic frequencies in NM on both sides of the brain of operated embryos are higher than the frequencies observed previously at comparable regions of the nucleus in unoperated controls.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of tumor necrosis factor-alpha on human malignant glial cells. The influence of human recombinant tumor necrosis factor-alpha has been assessed on a cell line (U-251) derived from a human malignant glial tumor. The results of this study demonstrate that tumor necrosis factor-alpha at doses of 50 and 100 ng/ml: 1) did not have cytotoxic or cytostatic effects on the U-251 cell line; 2) significantly increased the intracellular activity of manganese superoxide dismutase but had no effect on copper and zinc superoxide dismutase, catalase, or glutathione peroxidase activity; and 3) did not significantly alter the intracellular or extracellular general protease and collagenase type IV activity of these cells. The resistance of the U-251 cell line to tumor necrosis factor-alpha cytotoxicity may be related in part to the high intrinsic manganese superoxide dismutase activity present in this cell line combined with the ability of this cell line to induce substantial amounts of protective manganese superoxide dismutase activity in response to tumor necrosis factor-alpha.


Developing national guidelines for nurse practitioner education: an overview of the product and the process. In 1988, the National Organization of Nurse Practitioner Faculties appointed an ad hoc education committee (AHEC) to review existing nurse practitioner (NP) education curricula and to formulate national guidelines. An overview of the new NP guidelines are presented along with the philosophy, content, objectives, and competencies that are viewed as the foundation for NP education today. The guidelines are presented within the structure outlined in Brykczynski's (1985) research about NP practice. Thus, as a "product" of the Advanced Nursing Practice: Nurse Practitioner Curriculum Guidelines (NPCG), a new NP graduate for the 1990s could be described as one who demonstrates beginning competencies in primary health care theory and research within the five domains of practice identified by Brykczynski.


Linking theory and practice in teaching basic nursing skills. In an attempt to integrate theory and practice in baccalaureate nursing education, students were taught nursing skills with two cognitive strategies (Vee heuristics and concept maps) that consciously identify and reinforce connections between scientific theory and practice. The research showed that students using Vee heuristics and concept maps, rather than traditional modes, were significantly better able to identify scientific principles to describe why specific steps of a nursing skill were done.


Use of clinical logs by beginning nursing students and faculty to identify learning needs. Students entering a nursing program bring with them an increasing variety of life experiences. This study compares the learning needs of nontraditional and traditional nursing students during the first clinical experience. Twenty sophomore students wrote weekly reactions to their clinical experiences in the form of a log. The logs analyzed each week's clinical experience, assessed attainment of the week's goals, and identified goals for the following week. Descriptive analysis identified the following major themes and patterns of learning needs: learning from negative experiences, awareness of contextual influences, recognition of improved communication skills, identification of locus of motivation, and ability to conduct client teaching. Building on students' prior experiences can provide opportunities for self-reflection and promote the development of self-directed learning.


Graduate home health education: a survey of home health educators and agency personnel. To determine if there is a body of essential content that should be common to master's-level home care programs, a purposive sample of 200 nurses involved in home health or community health nursing was selected. The respondents (N = 118), including faculty and educational administrators (N = 61) and supervisors and administrators of home health agencies (N = 57), were asked their perceptions of the knowledge needed and their priorities for curriculum content for master's-prepared home care specialists in both clinical and administrative roles. There was agreement in most content areas, but significant differences were found between faculty and agency personnel in their priorities for community health concepts, and in their evaluation of nursing theory and epidemiology as essential content in both roles. We believe these results have important implications for nurse educators. As schools prepare students for leadership roles in today's home care environment, it is critical that pertinent content be well integrated into the curriculum.


The relationship among multiple assessments of nursing education outcomes. Three internal and three external outcome measures generated data for this longitudinal study. The internal outcome measures were nursing GPA, nonnursing GPA, and clinical evaluations of students during eight clinical rotations. The external evaluations were NCLEX scores, the graduates' self-rating of their competency in meeting program objectives, and their immediate supervisors' ratings on the identical competency rating scale. Data analyzed using Pearson correlation coefficients revealed significant correlations among all internal measures of academic outcomes with one external measure, the NCLEX score. There was no correlation between any nursing measure of outcome and the supervisor's ratings. There was a modest correlation between the supervisors' rating and the nonnursing GPA. The faculty clinical evaluation was the only significant correlation found between the alumni rankings and any other measure of outcome. There was no correlation between the supervisors' and the graduates' ratings.


Sexual harassment in academia: legal and administrative challenges. Guidelines and institutional policies regarding sexual harassment in academia have a relatively short and controversial background. Deference to Equal Employment Opportunity Commission (EEOC) guidelines in employment sexual harassment incidents guides much of the thinking in contemporary courts. Title IX of the Educational Amendments and the Civil Rights Restoration Act of 1987 are but two of the legal redresses available to students with harassment grievance complaints. Lack of definition of the term as well as research studies in nursing complicate the issue of sexual harassment. The potential impact of harassment on nursing students both in the classroom and in the practice area is significant. Nursing administrators and educators must be proactive in writing and implementing policies regarding sexual harassment.


Mortality among dust-exposed Chinese mine and pottery workers. A cohort study of approximately 68,000 persons employed during 1972 to 1974 at metal mines and pottery factories in south central China was conducted to evaluate mortality from cancer and other diseases among workers exposed to different levels of silica and other dusts. A follow-up of subjects through December 31, 1989 revealed 6,192 deaths, a number close to that expected based on Chinese national mortality rates. There was, however, a nearly 6-fold increase in deaths from pulmonary heart disease (standard mortality ratio, 581; 95% confidence interval 538 to 626), and a 48% excess of mortality from nonmalignant respiratory diseases (standard mortality ratio, 148; 95% confidence interval, 139 to 158), primarily because of a more than 30-fold excess of pneumoconiosis. Pulmonary heart disease and noncancerous respiratory disease rates rose in proportion to dust exposure. Cancer mortality overall was not increased among the miners or pottery workers. There was no increased risk of lung cancer, except among tin miners, and trends in risk of this cancer with increasing level of dust exposure were not significant. Risks of lung cancer were 22% higher among workers with than without silicosis. The findings indicate that respiratory disease continues to be an occupational hazard among Chinese miners and pottery workers, but that cancer risks are not as yet strongly associated with work in these dusty trades.


MMPI profiles of women in codependency treatment. The Minnesota Multiphasic Personality Inventory (MMPI) profiles of 73 women presenting themselves for treatment of codependency in a 10-day residential setting were examined. Results suggest a significant amount of psychopathology to be present in this population. More than 50% of these individuals produced clinically significant elevations of five or more scales, and 38% produced elevations on six or more scales.


MCMI differences between alcoholics and cocaine abusers: effect of age, sex, and race. Samples of alcohol-dependent-only subjects (n = 164), cocaine-dependent-only subjects (n = 63), and subjects dependent on multiple substances, including alcohol and cocaine (n = 103), were administered the Millon Clinical Multiaxial Inventory (MCMI) as part of a routine clinical evaluation. The alcohol-only group was older (M = 38.7 years) and had a higher percentage of White subjects than other groups. The multiple-use (60%) group also had a higher percentage of White subjects than the cocaine only group (38%) and a higher percentage of male subjects (81%) than the alcohol-only group (67%). Although the MCMI characteristics of each diagnostic group paralleled previous findings, few MCMI differences were found after controlling for age, sex, and race effects. The implications of these results for treatment development are discussed.


Gastrointestinal motor alterations induced by precipitated benzodiazepine withdrawal in rats. The effects of benzodiazepine withdrawal on intestinal motor activity and propulsion were investigated in two groups of diazepam-dependent rats (15 mg/kg/day for 8 days). Withdrawal was precipitated by injection of two benzodiazepine antagonists (Ro 15.1788 and PK 11.95) acting on central and peripheral-type receptors, respectively. Intestinal motor activity was assessed by implanting electrodes for long-term electromyographic recordings. Gastrointestinal transit was evaluated after gavage by a marker (51CrO4Na2) and radioactivity counting. Both RO 15.1788 (15 mg/kg) and PK 11.195 (5 mg/kg) triggered an abstinence syndrome with behavioral and autonomic signs. At the intestinal level, Ro 15.1788 induced a phase of strong irregular spiking activity (173 +/- 63 min) which remained located in the duodenum. In contrast, PK 11.195 induced a period of propagated myoelectric complexes characterized by phases II and III of high amplitude. The cecal frequency was doubled during the 1st hr after withdrawal induced by the two antagonists. Both Ro 15.1788 and PK 11.195 at this dosage had no effect per se on intestinal motility in vehicle-treated rats. In the second group of rats, gastric emptying was enhanced by 49.4 and 45.6% by Ro 15.1788 and PK 11.195, respectively. In contrast, PK 11.195 was able to accelerate the intestinal transit more than did Ro 15.1788 (geometric center, 5.9 +/- 0.43 and 5.3 +/- 0.49, respectively, vs. 4.1 +/- 0.31 in control rats). Our study shows that precipitated benzodiazepine withdrawal in diazepam-dependent rats induces alterations of the intestinal myoelectrical activity leading to an increase of the gastrointestinal transit. Central and peripheral-type receptors are involved in these effects.


The effect of sensory nerve depletion on cholinergic neurotransmission in guinea pig airways. C-fiber primary afferent sensory nerves, containing neuropeptides such as substance P and neurokinin A, mediate excitatory nonadrenergic, noncholinergic (e-NANC) bronchoconstrictor responses in guinea pigs. We have studied the effects of sensory nerve depletion in vivo and at four different airway levels in vitro. Capsaicin pretreatment significantly reduced the cholinergic responses to electrical field stimulation at all airway levels studied when compared to similar responses obtained in tissues from both untreated animals and animals treated with the vehicle for capsaicin (at 8 Hz, 27.2 +/- 4.9% of maximal contraction to 10(-2) M acetylcholine in lower trachea from vehicle-pretreated animals compared with 11.6 +/- 2.9% in lower trachea from capsaicin-pretreated animals, P less than .01). Stimulation of lower tracheal segments at 30-min intervals revealed a cholinergic response which was followed by a small, delayed e-NANC component. In vivo cholinergic bronchoconstrictor responses to vagal stimulation were also significantly diminished (at 5 Hz from 11.6 +/- 2.2 cm H2O in vehicle-pretreated animals to 3.3 +/- 1.4 cm H2O in capsaicin-pretreated animals). Bronchoconstrictor responses to acetylcholine or substance P in normal, vehicle-pretreated or capsaicin-pretreated animals were not significantly different either in vitro or in vivo. Sensory neuropeptides released from C-fiber afferent nerves may facilitate cholinergic neurotransmission in airways. This effect appears more marked at airway levels in which e-NANC responses are demonstrable.


Attention deficit hyperactivity disorder in children: diagnosis, treatment, and follow-up. Hyperactive, and fidgeting behavior in children, is not a new syndrome, but has been present in children for centuries. Only in recent years have health providers begun to observe these features and behaviors in children and recommend some therapies. Children labeled with Attention Deficit Hyperactivity Disorder (ADHD) display the symptoms of inattention, impulsivity, and hyperactivity; however, these behaviors are seen in a variety of conditions. This article describes some of the symptoms associated with ADHD, as well as the evaluation, therapeutic modalities (behavioral, psychodynamic and pharmacological), and the follow-up of children with ADHD.


Characterization of the contractile serotonergic receptor in guinea pig trachea with agonists and antagonists. 5-Hydroxytryptamine (5-HT)2 receptors can be partially characterized by their sensitivity to ketanserin blockade and increase in phosphoinositide turnover upon stimulation. Previously, the contraction of guinea pig trachea to 5-HT was shown to be antagonized by the 5-HT2 receptor antagonists ketanserin and LY53857. However, 5-HT did not dramatically increase phosphoinositide turnover in guinea pig trachea, suggesting that the contractile receptor may be different from the classically defined 5-HT2 receptor. The present in vitro studies better characterize this receptor, using diverse serotonergic agonists and antagonists to profile in more detail the contractile serotonergic receptor in guinea pig trachea. With regard to agonists, the 5-HT2 receptor agonists DOI and alpha-methyl-5-HT contracted guinea pig trachea with greater potency than quipazine, 5-methoxytryptamine, 5-carboxamidotryptamine, 8-hydroxy-2-(di-N-propylamino)tetralin and 2-methyl-5-HT. Sumatriptan and 1-(3-chlorophenyl)-piperazine (10 nM-100 microM) were inactive as agonists. A strong correlation between agonist potency (EC50) and reported 5-HT receptor binding affinities was found for both the 5-HT1C (r = 0.890) and 5-HT2 (r = 0.831) receptor. Ketanserin, spiperone, ritanserin, LY53857, 1-napthylpiperazine, 1-(3-chlorophenyl)-piperazine, rauwolscine, ICS 205-930, cyanopindolol and sumatriptan all blocked 5-HT-induced contractions in guinea pig trachea. As occurred with agonist potencies, strong correlations were found between reported 5-HT1C (r = 0.814) and 5-HT2 (r = 0.912) receptor binding affinities in brain membranes and apparent dissociation constants (KB) for the 10 antagonists of 5-HT induced contraction in guinea pig trachea.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurally mediated relaxation of opossum internal anal sphincter: influence of superoxide anion generator and the scavenger. The present investigation was undertaken to examine the influence of super oxide anion generator hydroquinone and the superoxide anion scavenger superoxide dismutase (SOD) on the neurally mediated relaxation of the internal and sphincter (IAS). The studies were performed in vitro on IAS smooth muscle strips of opossums. Neural stimulation with appropriate parameters of electrical field stimulation is known to cause IAS relaxation by the activation of nonadrenergic noncholinergic inhibitory neurons. Hydroquinone caused a concentration-dependent suppression of IAS relaxation in response to neural stimulation. The suppression of the IAS relaxation was reversed by SOD in a concentration-dependent manner. The fall in the IAS tension by nitric oxide (NO) was also suppressed by hydroquinone and reversed by SOD. SOD alone caused an augmentation of the fall in the IAS tension by NO. The fall in the IAS tension in response to the candidate inhibitory neurotransmitter vasoactive intestinal polypeptide was not modified by SOD in the basal state. Hydroquinone, however, caused a significant suppression of vasoactive intestinal polypeptide-induced fall in the IAS tension which was reversed by SOD. The fall in the resting IAS tension by the directly acting smooth muscle relaxants, beta adrenoceptor agonist isoproterenol and atrial natriuretic factor were not significantly modified by hydroquinone or SOD. From the present data we conclude: NO or an NO-like factor plays a significant role in the nonadrenergic noncholinergic nerve-mediated IAS relaxation.


Prostaglandin E1-induced vasorelaxation in porcine coronary arteries. The mechanism of prostaglandin E1 (PGE1)-induced vasorelaxation was studied in the porcine left anterior descending artery (diameter = 2.0-3.0 mm) and its branches (diameter = 0.6-1.0 mm). In the large coronary arteries, PGE1 increased the basal tone at concentrations from 10(-8) to 3 x 10(-7) M and decreased the tone at concentrations above 3 x 10(-7) M. However, in the small coronary arteries, PGE1 did not affect the basal tone at concentrations below 3 x 10(-6) M and evoked only relaxation at above 10(-5) M. PGE1 caused dose-dependent relaxation of vessels previously contracted by prostaglandin F2 alpha or endothelin-1 at concentrations above 10(-7) M in large coronary arteries and above 10(-8) M in small coronary arteries. Removal of the endothelium did not influence the relaxant response to PGE1 of either type of artery. In addition, 10(-4) M aspirin did not influence the PGE1-induced vasorelaxation of large and small coronary arteries. However, treatment with 5 x 10(-6) M ouabain or partial replacement of Na+ with Li+ (Na+ concentration, 25 mEq/l) significantly attenuated the relaxant response to PGE1 in large and small coronary arteries. These results indicate that the responsiveness of large and small coronary arteries to PGE1 differs in the pig and that the electrogenic Na+ pump has a primary role in the relaxant effect of PGE1 on both small and large arteries.


Pharmacological evaluation of JO 1870: relation to the potential treatment of urinary bladder incontinence. The effect of p.o. administered JO 1870 [(-)-1-(p-chlorophenyl)-N,N- dimethyl-1-ethyl(4-phenyl)-but-3-en-1-ylamine, hydrochloride] on the urinary bladder cystometrogram has been investigated in the unanesthetized rat. The effects of JO 1870 have been compared with those of morphine and reference drugs currently used for the treatment of urinary incontinence in humans. JO 1870 (2.5-25 mg/kg) dose-dependently increased the bladder capacity and the threshold pressure responsible for urination. These effects were antagonized by the i.v. administration of naloxone (0.2 mg/kg). In comparison, morphine (2.5-25 mg/kg) moderately increased urinary volume and threshold pressure. Different anticholinergic drugs (propantheline bromide, 10 mg/kg; terodiline, 25 mg/kg; dicyclomine, 25 mg/kg) had no significant effects on either parameter and the antidepressant clomipramine (25 mg/kg) decreased both. In vitro, JO 1870 specifically displaced [3H]-[D-Ala2,MePhe4,Gly5-(ol)]enkephalin from binding sites in guinea pig whole-brain membranes and rat thalamus; the sodium shift ratio obtained from [3H]naloxone binding was 16. These results suggest that JO 1870 has some opioid agonist activity. JO 1870 (2.5-300 mg/kg) had few effects on cardiorespiratory, gastrointestinal or nociceptive systems in rats. Together, these results indicate that JO 1870 is a nonanticholinergic agent that potently increases bladder capacity, likely through an opioid mechanism, and may have potential use in the treatment of urinary incontinence.


Zolpidem behavioral pharmacology in baboons: self-injection, discrimination, tolerance and withdrawal. This study examined in baboons various behavioral effects of zolpidem, a short-acting imidazopyridine hypnotic which has selectivity for subtypes of the benzodiazepine receptor. Intravenous drug self-injection was studied under a fixed-ratio 80- or 160-response schedule with a 3-hr timeout after each injection. Maximal rates of self-injection maintained by zolpidem (0.01-1 mg/kg) were consistently higher than those maintained by vehicle and the benzodiazepine hypnotic triazolam. Substitution of vehicle after about 2 weeks of zolpidem self-injection (7-8 mg/kg/day) resulted in a time-limited suppression of food pellet intake, indicating a drug withdrawal effect. In a drug discrimination study, baboons were trained to discriminate either lorazepam (1.8 mg/kg p.o.) or pentobarbital (10 mg/kg p.o.) from the no-drug condition. Zolpidem (0.1-18 mg/kg p.o.) occasioned both lorazepam- and pentobarbital-appropriate responding (greater than 80%) in a dose-dependent manner. In a final experiment, zolpidem (3.2 or 5.6 mg/kg i.m.) produced ataxia and sedation that progressively decreased over 7 consecutive days of administration. The withdrawal, discriminative stimulus effects and tolerance shown with zolpidem were similar to those shown previously with benzodiazepines under similar conditions. The rates of self-injection of zolpidem were similar to those maintained by intermediate duration barbiturates (e.g., pentobarbital) and higher than those maintained by 11 benzodiazepines studied previously under similar conditions. Further research on the reinforcing effects of zolpidem may provide useful insights into mechanisms underlying the maintenance of behavior by compounds acting through the benzodiazepine receptor.


Effects of ketamine on sensory perception: evidence for a role of N-methyl-D-aspartate receptors. The chiral forms of ketamine were applied as probes for N-methyl-D-aspartate receptor-mediated neurotransmission in humans. Both enantiomers, in clinically relevant concentrations, displaced [3H]dizocilpine (MK 801) from specific binding sites (phencyclidine sites) in membrane fractions of brain homogenates. (S)-Ketamine was at least 4 times as potent as (R)-ketamine in this respect. In healthy volunteers, the most obvious effect of subanesthetic doses of both enantiomers was altered sensory perception. (S)-Ketamine was 4 times as potent as (R)-ketamine in reducing pain perception and in causing auditory and visual disturbances. Both enantiomers caused proprioceptive disturbances (feelings of detachment from the body) and slightly reduced the ability to recall objects seen after administration of the drugs. The ability to recall objects seen immediately before drug exposure was unaffected. The results are in accordance with the hypothesis that inhibition of sensory perception by ketamine in subanesthetic concentrations is due to N-methyl-D-aspartate receptor blockade. It is suggested that N-methyl-D-aspartate receptor-mediated transmission is involved in the processing of sensory information in the human brain.


Extremely long-lasting antagonistic actions of nor-binaltorphimine (nor-BNI) in the mouse tail-flick test. The duration of antagonistic action of nor-binaltorphimine (nor-BNI), a kappa antagonist, of antinociception resulting from selective opioid agonists, was examined using the mouse tail-flick assay as the endpoint. Nor-BNI (1 nmol, i.c.v. at -20 min) antagonized equiantinociceptive doses of the opioid kappa agonists (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl) benzeneacetamide (U69,593) (70 nmol i.c.v.) or bremazocine (25 nmol i.c.v.), but did not antagonize antinociception produced by the mu opioid-selective [D-Ala2, NMePhe4, Gly-ol]enkephalin or the delta opioid-selective [D-Pen2, D-Pen5]enkephalin. Pretreatment with nor-BNI (1 nmol i.c.v.) antagonized the antinociceptive effects of U69,593 and bremazocine for up to 28 days. At all pretreatment times, the antinociceptive dose-response lines for these kappa agonists were displaced to the right to various degrees in a parallel fashion; an increasing rightward displacement of the U69,593 and bremazocine antinociceptive dose-response lines was observed at 1 and 3 days after a single nor-BNI pretreatment, with a gradual return toward the control level at later times after pretreatment. Increasing the dose of nor-BNI to 10 nmol produced only a transient blockade of equiantinociceptive doses of the mu selective agonist [D-Ala2, NMePhe4, Gly-ol]enkephalin and the delta selective agonist [D-Pen2, D-Pen5]enkephalin (at 20-30 min post-nor-BNI pretreatment).(ABSTRACT TRUNCATED AT 250 WORDS)