Long-lasting enhancement of metabotropic excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the amygdala/pyriform cortex of deep prepiriform cortical kindled rats. We have previously demonstrated that ibotenate (IBO)-stimulated polyphosphoinositide (PPI) hydrolysis is increased for a long period in the amygdala/pyriform cortex (AM/PC) of amygdala (AM)- and hippocampal (HIPP)-kindled rats. This finding indicates that enhanced function of the PPI-coupled excitatory amino acid (EAA) receptor may be associated with the long-lasting seizure susceptibility of kindling. The present study further examined PPI hydrolysis induced by trans-ACPD, a selective agonist of the metabotropic EAA receptor, as well as by IBO in brain slices of rats kindled from the deep prepiriform cortex (DPC). IBO-stimulated accumulation of [3H]inositol monophosphate ([3H]InsP) was significantly increased in the AM/PC by 162 (P less than 0.0001), 130 (P less than 0.005) and 81% (P less than 0.03) at 24 h, 7 days and 28 days, respectively, after the last kindled seizure, whereas it was increased significantly only at 24 h after the last seizure in the HIPP and did not change at any time in the limbic forebrain (LFB). The IBO-stimulated accumulation of [3H]InsP was significantly increased by 55% (P less than 0.01) in the AM/PC of partially kindled rats reaching an average stage of 3.7, but not in the AM/PC of those remaining at stage 1, 7 days after the last kindled seizure. Trans-ACPD-stimulated PPI hydrolysis was significantly increased in the AM/PC of DPC-kindled rats by 65 (P less than 0.05) and 45% (P less than 0.005) at 7 and 28 days, respectively, after the last kindled seizure. Cis-ACPD-stimulated PPI hydrolysis was also significantly increased in the AM/PC of DPC-kindled rats by 45 (P less than 0.03) and 30% (P less than 0.04) at 7 and 28 days, respectively, after the last seizure. There was no increase in trans-ACPD- or cis-ACPD-stimulated PPI hydrolysis in the HIPP or LFB. These results further confirm our previous studies showing that the metabotropic EAA receptor-stimulated PPI hydrolysis exhibited a long-lasting increase in the AM/PC irrespective of the primary stimulation site for kindling.


Glutamatergic postsynaptic block by Pamphobeteus spider venoms in crayfish. The effects of toxins from venom glands of two south american spiders (Pamphobeteus platyomma and P. soracabae) on glutamatergic excitatory synaptic transmission were studied in the neuromuscular junction of the opener muscle of crayfish. The toxins selectively and reversibly blocked both excitatory postsynaptic currents and potentials in a dose-dependent manner. They also reversibly abolished glutamate-induced postsynaptic membrane depolarization. They had no effect on resting postsynaptic membrane conductance nor on postsynaptic voltage-gated currents. The synaptic facilitation and the frequency of miniature postsynaptic potentials were unaffected by the toxins, indicating that presynaptic events were not modified. Picrotoxin, a selective antagonist of the gamma-aminobutyric acid (GABA)A receptor, did not modify toxin effects. We conclude that both toxins specifically block the postsynaptic glutamate receptor-channel complex.


Longevity of synaptic depression in the hippocampal dentate gyrus. This study used urethane-anesthetized rats to investigate the longevity of heterosynaptically evoked depression of the monosynaptic response generated by synapses between entorhinal cortical (EC) afferents and the cells of the dentate gyrus (DG). Brief, high-frequency activation of the converging ipsilateral EC-DG input depressed the synaptic response of the contralateral EC-DG synapses without prior experimentally induced potentiation. This depression lasted for hours. Such observations are consistent with a role for heterosynaptically induced long-term depression in the encoding functions of synapses.


Activation of beta-adrenergic receptor induces Na(+)-dependent inward currents in acutely dissociated motoneurons of bullfrog spinal cord. Effects of adrenergic drugs on single motoneurons acutely dissociated from the lumbar enlargement of adult bullfrogs were examined. The dissociated large cells were identified as motoneurons by retrograde labeling with a fluorescent dye. Adrenaline caused membrane depolarization with a decrease in input resistance. Under whole-cell voltage clamp conditions at a holding potential of -70 mV, adrenergic drugs induced inward currents in a dose-dependent manner. Adrenaline was more potent than noradrenaline. Under K(+)-free conditions, adrenaline (10(-6)-10(-5) M) induced inward currents which were blocked by propranolol (10(-6) M) but not by phentolamine (10(-5) M). CoCl2 (1 mM) did not affect the currents. Substitution of choline+ in the recording solution for Na+ abolished the currents, but tetrodotoxin (TTX, 10(-6) M) had no effect on them. The adrenaline-induced currents exhibited a characteristic voltage-dependency: the conductance became large at hyperpolarized membrane potential (-150 to -30 mV) and approached zero at the depolarized membrane potential (greater than -30 mV), but was never reversed up to 30 mV, suggesting that the currents are different from non-specific cation currents. Substitution of isethionate- for Cl- in the recording solution had no effect on the voltage-dependency of the adrenaline-induced currents, whereas substitution of choline+ for Na+ apparently attenuated the voltage-dependency of the currents. These results indicate that adrenaline induces Na(+)-dependent inward currents through activation of beta-adrenergic receptors in bullfrog motoneurons.


Purification of squid synaptic vesicles and characterization of the vesicle-associated proteins synaptobrevin and Rab3A. Two proteins associated with mammalian synaptic vesicles, the integral membrane protein synaptobrevin and the GTP-binding protein rab3A, are identified in squid nervous tissue using Western blotting and subcellular fractionation of synaptosomes. They both copurify with synaptic vesicles prepared from squid optic lobe. Synaptobrevin (18.1 kDa) is present at high levels in synaptic terminals but at very low levels in axon. Rab3A (27.5 kDa) is a member of the rab family of low-molecular weight GTP-binding proteins which regulates vesicle traffic in secretory and endocytic processes. As resolved with 2-dimensional gels, squid neurons contain at least 16 GTP-binding species (19-29 kDa), and most of these are present in both soluble and particulate fractions. The 24 kDa class of GTP-binding proteins is highly enriched in axonal transport organelles. The characterization of synaptobrevin and rab3A in squid synaptic vesicles extends their known distributions to invertebrates and points to a fundamental importance of these proteins in neurotransmitter release.


Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by mu-opioid agonists, but not by delta- and kappa-opioid agonists. To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered mu-opioid agonists, such as morphine (10 micrograms) and [D-Ala2,N-Me Phe4,Gly-ol5]enkephalin (DAMGO, 0.5 micrograms). However, i.c.v. administration of [D-Pen2,5]enkephalin (DPDPE, 5 micrograms), a delta-opioid agonist, and U-50,488H (50 micrograms), a kappa-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 microgram), DAMGO (10 micrograms), DPDPE (0.5 micrograms) or U-50,488H (50 micrograms) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal mu-opioid receptor-mediated antinociception, but they are normally responsive to activation of delta- and kappa-opioid receptors.


Differential effects of intermittent or continuous exposure to cocaine on the hypothalamic-pituitary-adrenal axis and c-fos expression. Accumulating evidence indicates that acute administration of cocaine alters neuroendocrine functions. In order to ascertain the long-term effects of cocaine on the male rat's hypothalamic-pituitary-adrenal (HPA) axis, a series of experiments were performed utilizing two different paradigms of cocaine administration for 6 days. In the first paradigm, rats received daily intravenous injections of cocaine (5 mg/kg), while in the second, they were continuously exposed to the drug (5 or 100 mg/kg/day) via osmotic pumps. We measured plasma adrenocorticotropin hormone (ACTH) and corticosterone levels, as well as the brain pattern of the proto-oncogene c-fos expression in response to either mode of drug administration. Repeated, intermittent injections of cocaine caused consistent increases in ACTH and corticosterone secretion over a 30-min sampling period on days 2, 4 and 6. This paradigm of drug administration also induced considerable, short-lasting and reversible c-fos expression in the caudate putamen but not in hypothalamic regions associated with endocrine function. In contrast, we consistently failed to observe any measurable increases in ACTH or corticosterone secretion at any time during continuous exposure to the drug. Administration of cocaine by osmotic pumps also had no effect on c-fos expression in the caudate putamen, indicating that c-fos expression as well as activation of the HPA axis are dependent upon the mode and frequency in which cocaine is administered. We conclude that continuous exposure to cocaine does not appear to activate the HPA axis, while intermittent injections of the drug induce repeated increases in plasma ACTH and corticosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuronal firing patterns of organotypic rat spinal cord cultures in normal and in ACTH/alpha-MSH(4-10) analog (BIM 22015)-supplemented medium. The spontaneous and evoked electrical patterns of spinal cord explants from 13- to 14-day old rat fetuses grown from 2 to 8 weeks in vitro were compared when fed either with a standard or with an adrenocorticotropic hormone/alpha-melanocyte stimulating hormone (4-10) analog (BIM 22015)-supplemented medium. Standard and BIM 22015-treated cultures developed similar patterns of extracellularly recorded activity which consisted of mostly phasic but also tonic discharges. The standard cultures when treated by BIM 22015 in acute experiments (100 micrograms/ml) showed a decrease in their frequency of discharges which fired in a regular tonic pattern. These effects were neither age- nor dose-dependent but were increased in Ca2+ free medium. The ventral cord neurons chronically fed with BIM 22015 showed a strongly bursting pattern resembling strychnine-induced synchronized bursts. Both these effects, inhibitory (acute) and excitatory (chronic), of the BIM upon spinal cord cultured ventral horn neurons are discussed as possible calcium-dependent phenomena.


In vivo occupation of mouse brain opioid receptors by endogenous enkephalins: blockade of enkephalin degrading enzymes by RB 101 inhibits [3H]diprenorphine binding. With the aim of possibly studying the local activity of brain enkephalinergic pathways by autoradiography and positron emission tomography, preliminary competition experiments of [3H]diprenorphine binding in mouse brain were carried out after i.v. administration of the first systemically-active mixed inhibitor of enkephalin degrading enzymes RB 101 (N(R,S)-2-benzyl-3[(S)-(2-amino-4-methylthiobutyldithio]-1-oxoprop yl]- L-phenylalanine benzyl ester). Although devoid of affinity for the opioid binding sites, RB 101 inhibited the [3H]diprenorphine binding to the opioid receptors in a dose-dependent manner. This effect, very likely due to an RB 101-induced increase in extracellular levels of enkephalins, reached a plateau at a dose of 10 mg/kg, where almost 30% displacement was observed. Intravenous administration of either 5 or 20 mg/kg of RB 101 in mice submitted to warm-swim stress led to an additional [3H]diprenorphine displacement, which reached 45% compared to unstressed controls. This ceiling effect could account for the reported minimal morphine-like side effects induced by mixed inhibitors. A large increase in endogenous enkephalin levels induced by RB 101, associated or not with stress, was also indirectly demonstrated by the analgesic responses elicited by i.v. injection of the mixed inhibitor. This effect was blocked by naloxone but not by the delta antagonist naltrindole (NTI), supporting a preferential implication of mu receptors in supraspinal analgesia. Taken together, these results suggest that RB 101 could be used to determine the precise in vivo localization of enkephalinergic pathways recruited by various stimuli.


Voltage- and Ca(2+)-activated ionic currents in acutely dissociated cells of the chick pineal gland. Previous research on cultured chick pineal cells suggests that melatonin production is modulated by Ca2+ influx through voltage-dependent Ca2+ channels. The possible existence of other ionic currents was investigated by means of whole-cell recordings from acutely isolated cells. Several different inward and outward currents were identified. Inward currents included L-type Ca2+ currents and voltage-activated tetrodotoxin (TTX)-sensitive Na+ currents. Sodium currents have not been reported previously in pineal cells of any species. These two inward currents were present in the majority of cells. Chick pineal cells also expressed several types of voltage-dependent and Ca(2+)-dependent K+ currents that differed in voltage dependence, kinetics, and pharmacology. These included two Ca(2+)-dependent outward currents which differed in sensitivity to tetraethylammonium chloride (TEA), and at least two distinct voltage-activated K+ currents. Considerable cell-to-cell variation in the amplitude and nature of the evoked outward currents was observed. These ionic currents may be important for the regulation of melatonin synthesis and the modulation of circadian rhythmicity.


Stress-induced analgesia prevents the development of the tonic, late phase of pain produced by subcutaneous formalin. Subcutaneous injection of formalin produces a biphasic pain response: a transient early phase followed by a tonic late phase. It has recently been suggested that development of the late phase depends upon the presence of the early one. In support of this suggestion, we now demonstrate that blocking the early phase by stress-induced analgesia prevents development of the late phase, whereas the same stressor given after the first phase does not. Both phases are manifested when stress-induced analgesia is blocked by the N-methyl-D-aspartate (NMDA) or opiate antagonists, MK-801 and naloxone.


CNS cell groups projecting to the submandibular parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study. The retrograde transneuronal viral tracing method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons controlling the submandibular gland in the rat. A genetically engineered beta-galactosidase expressing Bartha strain of pseudorabies virus (PRV) was injected into the submandibular gland of rats. After 4 days, PRV infected tissues were reacted with the Bluo-Gal substrate (halogenated indolyl-beta-D-galactoside) and labeled cell bodies were identified throughout the brain. In the medulla oblongata, cell body labeling was seen in the superior salivatory nucleus, and throughout the medullary reticular formation as well as in the nucleus of the solitary tract, spinal trigeminal nucleus, and deep cerebellar nuclei. In the pons, PRV labeled neurons were found bilaterally in the locus ceruleus, subceruleus region, and parabrachial complex. In the mesencephalon, labeled cells were found in the Edinger-Westphal nucleus, deep mesencephalic nucleus, and central grey matter. Several hypothalamic regions were labeled including the lateral, perifornical and paraventricular hypothalamic nuclei. In the telencephalon, PRV-positive cell bodies were observed in the substantia innominata, bed nucleus of the stria terminalis and central nucleus of the amygdala. The results suggest that widespread areas of the CNS are involved in control of salivation.


Induction of feeding by 7-chlorokynurenic acid, a strychnine-insensitive glycine binding site antagonist. Two experiments examined the involvement of the strychnine-insensitive glycine binding site (SIGBS) of the N-methyl-D-aspartate (NMDA) receptor/channel complex in feeding behavior. The first experiment demonstrated that the SIGBS antagonist, 7-chlorokynurenic acid (7CK), dose-dependently increased food intake in rats without producing significant locomotor deficits or stereotypies. The second experiment showed that D-serine, a SIGBS agonist, dose-dependently antagonized 7CK induced feeding, such that at the highest dose of D-serine used 7CK induced feeding was completely abolished, demonstrating that the effects of 7CK are specific to an action at the SIGBS.


Glial fibrillary acidic protein (GFAP) expression and nucleolar organizer regions (NORs) in human gliomas. The frequency of nucleolar organizer regions (NORs) in each glioma tissue and the relation between the expression of glial fibrillary acidic protein (GFAP) and the frequency of NORs was investigated. The number of Ag-NORs per cell for glioblastoma multiforme was significantly higher than that for anaplastic astrocytoma (P less than 0.05) and that for astrocytoma (P less than 0.01). The number of Ag-NORs per cell for GFAP-positive cells was significantly lower than that for GFAP-negative cells in each histopathological grade (P less than 0.01). Moreover, the linear relationship was demonstrated between the Ag-NORs numbers of GFAP-negative cells and bromodeoxyuridine (BUdR) labeling indices. From these results, it is concluded that many GFAP-positive glioma cells may have low growth potential in glioma tissue and GFAP-negative cells may have a close relation to cell proliferation. The combination of immunohistochemical and silver colloid staining is a useful method for investigating the biological characteristics of brain tumors.


Selective distribution pattern of gamma-hydroxybutyrate receptors in the rat forebrain and midbrain as revealed by quantitative autoradiography. Using quantitative autoradiography to study the precise distribution of gamma-hydroxybutyrate high-affinity binding sites, the present results showed the heterogeneous localization of these sites in cortical and hippocampal layers and also in some diencephalic and mesencephalic nuclei. In frontal, parietal and temporal cortex, GHB binding sites are generally distributed in three distinct layers. The olfactory system, the amygdala, septum, basal ganglia and substantia nigra also exhibited significant amounts of GHB receptors. In thalamus, the radioactivity was heterogeneously distributed, the highest amounts being in the lateral posterior nucleus. Hypothalamus, cerebellum, colliculi and pons-medulla were apparently devoid of binding sites. This more accurate mapping of GHB high-affinity receptors in rat brain is due to some technical improvements and the use of [3H]GHB of higher specific activity.


Postnatal change in a Ca(2+)-mediated decrease in (Na+ + K+)-ATPase activity in rat brain slices. The treatment of brain slices from immature rats with veratrine and monensin did not cause any change in (Na+ + K+)-adenosine triphosphatase (ATPase) activity or [3H]ouabain binding in membranes prepared from the slices, though these reagents remarkably stimulated Ca2+ uptake in the slices. Exposure of the slices from adult rats to a glucose-free, hypoxic or both glucose-free and hypoxic medium resulted in a decrease in the enzyme activity, but the enzyme from immature rats was resistant to the conditions.


Testosterone prevents castration-induced reduction in peripheral benzodiazepine receptors in Cowper's gland and adrenal. The effect of surgical castration of adult male Sprague-Dawley rats on peripheral and central benzodiazepine (BZ) receptors was studied. Following removal of the testes, a significant decrease in the density of peripheral BZ receptors (PBR) was observed in Cowper's glands (71%; P less than 0.005) and the adrenal (31%; P less than 0.01), but not in the heart. Administration of testosterone acetate (TA) prevented castration-induced PBR depletion. Orchiectomy per se, as well as TA administration to castrated rats, had no effect on central or peripheral BZ receptors in whole brain without the cerebellum. These results indicate the regulatory role of testosterone in the regulation of PBR in Cowper's glands and adrenal.


Memory-enhancing effects of post-training dipivefrin and epinephrine: involvement of peripheral and central adrenergic receptors. These experiments examined the effects, in mice, of post-training i.p. injections of dipivefrin (DPE), a lipophilic prodrug of epinephrine, and epinephrine (EPI) on 48-h retention assessed in inhibitory avoidance and Y-maze discrimination tasks. DPE, in doses of 0.3-10 micrograms/kg significantly facilitated retention: the effects were approximately 10-fold more potent than those of EPI obtained with similar experimental conditions. The alpha-adrenergic antagonists prazosin (alpha 1; 3.0 mg/kg; i.p.), yohimbine (alpha 2; 3.0 mg/kg; i.p.) and phentolamine (alpha 1 and alpha 2; 3.0 mg/kg; i.p.) did not block the enhancement of retention induced by either DPE (10.0 micrograms/kg; i.p.) or EPI (0.1 mg/kg; i.p.). However, the beta-adrenergic antagonist propranolol (2.0 mg/kg; i.p.) attenuated the effects of both DPE and EPI. Sotalol (2.0 mg/kg; i.p.), a peripherally-acting beta-adrenergic antagonist, attenuated the effects of EPI but not those of DPE. These findings suggest the DPE-induced enhancement of memory involves central beta- but not alpha-adrenergic mechanisms while EPI's effects are initiated by activation of peripheral beta-adrenergic systems.


Correlation between calmodulin-like protein, phospholipids, and growth in glucose-grown Mycobacterium phlei. In Mycobacterium phlei TMC 1548 supplementation of growth medium containing 2% v/v glycerol with glucose (up to 5% w/v) resulted in an increase in growth (yield of cells), in amount of total phospholipids, and in each of the individual phospholipids (cardiolipin, phosphatidylethanolamine, phosphatidylinositol and its mannosides, and phosphatidylglycerol). However, when the medium was supplemented with a higher concentration (7.5% w/v) of glucose, both growth and phospholipid levels decreased to near control values (2% v/v glycerol alone). Cyclic AMP levels, which decreased at all concentrations of glucose, had no relation to phospholipid content or growth. The presence of a protein that possesses the property of stimulating c-AMP phosphodiesterase activity was recently demonstrated in Mycobacterium smegmatis (Falah et al. 1988. FEMS Microbiol. Lett. 56: 89-93). In M. phlei the level of this calmodulin-like protein (assayed by radioimmunoassay) changed with different concentrations of glucose in the growth medium in a manner identical with that of phospholipids. We suggest that in mycobacteria (i) intracellular calmodulin-like protein levels are affected by glucose concentration in the growth medium and (ii) there is a positive correlation between the levels of calmodulin-like protein, total and individual phospholipids, and growth (yield of cells) in glucose-grown M. phlei.


Biochemical defects of outermost layer deficient mutants during sporulation of Bacillus megaterium. To determine the regulation of morphogenesis of the outermost layer, the thick layer outside the inner coat, of the Bacillus megaterium spore, we isolated 15 outermost layer deficient mutants of B. megaterium using transposon Tn917. Three mutant strains lacked both synthesis of the 48-kDa outermost layer protein and induction of two initial enzymes for galactosamine-6-phosphate polymer synthesis, evidence that these biochemical events are regulated in the cascade system during morphogenesis of the outermost layer.


Generation of cytotoxic monoclonals against AK-5 histiocytoma: conjugation with daunomycin and use in chemotherapy. We have successfully generated cytotoxic monoclonal antibodies against a rat histiocytoma, AK-5. Monoclonal antibodies obtained after fusing immunized rat splenocytes with SP2/0 myeloma, were cytotoxic to AK-5 cells in the presence of complement. These monoclonals were highly specific and did not show any cross reactivity with normal cells and ascitic tumors such as Zajdela ascites hepatoma or Meth A. One of the antibodies was conjugated to daunomycin and used in the chemotherapeutic treatment. Total regression of AK-5 histiocytoma was obtained after injection of daunomycin-MAb conjugate into tumor bearing animals suggesting the specific targeting of the antineoplastic drug to the tumor. The histology of the tumor sections showed extensive necrosis of the tumors after treatment of the animals with drug-MAb conjugate.


Value of the facial nerve latency test in the prognosis of childhood Bell's palsy. In the present study we evaluated the facial nerve latency test (FNLT) as a prognostic tool in cases of childhood Bell's palsy. Twenty-five children aged 4-14 years were studied. We divided our subjects into three groups according to duration of latency time (LT). Group A patients had an LT within the normal range, with average of 3.27 ms, group B a slightly prolonged LT averaging 5.7 ms, and group C a markedly prolonged LT averaging 10.5 ms. Analysis of the recovery index by group showed that group A patients experienced complete and quick recovery, while in group B 50% had complete but delayed recovery and 50% slightly impaired facial nerve function, and in group C 50% had slightly impaired function and 50% incomplete recovery. The more prolonged the LT, the worse the clinical results. The FNLT is thus a valuable prognostic tool in cases of Bell's palsy in childhood.


Seroreactivity to HPV-16 proteins in women with early cervical neoplasia. Although serological reactivity to human papillomavirus type 16 (HPV-16) proteins has been demonstrated in patients with invasive cervical carcinoma, the degree of seroreactivity to these proteins in women with preinvasive disease and its relationship to the HPV type associated with the disease are unclear. We obtained sera from 27 women undergoing cone biopsy for cervical precursor lesions and 22 controls and analyzed seroreactivity by Western blot to fusion proteins containing portions of the HPV-16 E4, L1 and L2 open-reading frames (ORFs). Positives were analyzed by scanning densitometry and intensity values for each case plotted relative to controls. Cervical biopsy specimens from patients were analyzed for HPV-16 nucleic acids by DNA.DNA in situ hybridization. Mean intensity values for seroreactivity to the pATH-E4 protein approached significance (P = 0.058) and a significantly higher proportion of cases vs controls registered values over 4.0 for pATH-E4 (26% vs 4.5%; P = 0.04) and pATH-L2 (48% vs 18%; P = 0.03) proteins. A significantly higher mean intensity value for E4 was observed for cases containing HPV-16 DNA vs HPV-16 negative cases or controls. Thus, seroreactivity to HPV-16-derived proteins may be more common in women with preinvasive cervical disease, and for some protein targets (E4) may indicate a relatively type-specific response.


Loss of endoplasmic reticulum membrane integrity: an image analysis of the glucose-6-phosphatase system in human hepatocyte. Histochemical and cytochemical methods induce a loss of endoplasmic reticulum (ER) membrane integrity in hepatocytes. In order to evaluate the degree of ER membrane integrity, glucose-6-phosphatase (G6P-A) was localized in light and electron microscopy using glucose-6-phosphate (G6P) and mannose-6-phosphate (M6P) as substrates. In case of ER membrane alteration, M6P diffuses inside the ER and is hydrolysed by a non-specific phosphohydrolase. G6P and M6P hydrolysis was quantified with image analysis methods. In light microscopy, the ratio of reaction of M6P hydrolysis/G6P hydrolysis gave 75% of non specific reaction. In electron microscopic study this ratio was about 30%. These results showed that enzyme localization methods in electron microscopy produced less ER membrane alteration than light microscopic methods.


Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum. The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium. All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.


The role of ions and second messengers in circadian clock function. The fact that single cells can exhibit circadian rhythmicity simultaneously in quite different processes, such as those of photosynthesis, bioluminescence, and cell division, suggests that membrane-bound compartmentalization is important for temporal organization. Since these rhythms, as well as others, are known to be affected by changes in the ionic environment and are probably membrane-bound systems, it is not surprising that transmembrane ion transport or flux has been proposed to be a key feature of the underlying circadian oscillator(s). Likewise, signal transduction along the entrainment pathway leading to the clock, among the elements, or "gears," of the timing loop itself, and within the output pathway between the oscillator and its "hands" likely is mediated by ions and second messengers. In this overview, we examine the theoretical and experimental evidence supporting the possible roles of intracellular free calcium and cyclic AMP in these capacities, particularly in view of the fact that oscillations in the concentrations of both species have been proposed to form the basis of pacemaker activity and other biological rhythms.


Synthetic studies of vitamin D analogues. X. Synthesis and biological activities of 1 alpha,25-dihydroxy-21-norvitamin D3. 1 alpha,25-Dihydroxy-21-norvitamin D3 (3) was synthesized from 1 alpha-hydroxydehydroepiandrosterone (4). Certain biological properties of 3 were examined in comparison with those of 1 alpha,25-dihydroxyvitamin D3 (1) and 1 alpha,25-dihydroxy-21-nor-20-oxavitamin D3 (2) to evaluate the effect of the 21-methyl substituent on biological activities. The differentiation-inducing activity of 3 towards human myeloid leukemia cells was approximately one-fifth of that of 1, while in the binding affinity with chick intestinal cytosolic receptor, 3 was about one-tenth of that of 1. The rather weak effect of 3 on serum calcium levels in normal mice at a dosage of 500 micrograms/kg (intravenous administration) indicates that the essential importance of the 21-methyl moiety may lie in its effect on the regulation of calcium metabolism.


Synthesis and structure-activity relationships of substituted 2-[(2-imidazolylsulfinyl)methyl]anilines as a new class of gastric H+/K(+)-ATPase inhibitors. II. A series of 2-[(2-imidazolylsulfinyl)methyl]anilines (2) having various substituents on their imidazole and aniline rings was synthesized and examined for their H+/K(+)-ATPase (adenosine triphosphatase) inhibitory effects and antisecretory activity against histamine-stimulated gastric acid secretions in Heidenhain pouch dogs. Although substitutions on the imidazole ring did not enhance biological activity, substitutions on the aniline ring by electron-donating substituents potently enhanced the enzyme inhibitory activity and also showed an inhibitory effect on histamine-stimulated gastric acid secretion after oral administration. In particular, the in vitro activity of the dimethyl (2u--w) and trimethyl (2ac) derivatives was about 10 times that of omeprazole. Also, 4-methyl (2k), 4-methoxy-5-methyl (2y) and 3,5-dimethyl-4-methoxy (2ab) derivatives showed a potent antisecretory effect of more than 80% after oral administration at 6 mg/kg. Although these aniline derivatives have relatively low stabilities in aqueous solution, replacement of the isobutyl group at the aniline nitrogen atom with N-(2-methoxyethyl) group enhanced the stability.


Ultrasonically-guided fine-needle biopsy of gastrointestinal organs: indications, results and complications. Percutaneous ultrasonically (US) guided fine-needle biopsy can be considered the first invasive diagnostic step for hepatic malignancies. This procedure may also be useful for benign hepatic lesions. In pancreatic tumors the success rate of this technique is lower than that obtained in hepatic masses. US-guided puncture shows low risk even though fatal and major complications, although rare, may occur.


The effect of stainless steel, cobalt-chromium, titanium alloy, and titanium on the respiratory burst activity of human polymorphonuclear leukocytes. Although infection is a serious complication associated with the use of orthopedic prosthetic implants, the impact of the metal used in these devices on host defense is poorly understood. The authors investigated the effect of stainless steel, titanium, titanium alloy, and cobalt-chromium alloy on the respiratory burst of polymorphonuclear leukocytes (PMN), a vital component of bactericidal activity. In the presence of stainless steel powder or supernatants obtained from the incubation of stainless steel in buffer, superoxide production by PMN was significantly impaired. Titanium, titanium alloy, and cobalt-chrome alloy had no significant effect on superoxide production. Nickel and chromium, the only metal ions detectable in the stainless steel supernatant, did not impair superoxide production when tested at concentrations similar to those found in the supernatant. Inhibition of PMN superoxide production may play a role in the establishment and persistence of stainless steel device-related infections.


[Acute posterior interosseous nerve paralysis with constrictions possibly due to twists in the nerve trunk]. This report is concerning a case of acute spontaneous paralysis of the posterior interosseous nerve (P.I.O.N.), possibly caused by twists in the nerve trunk, in a 23-year-old woman. The subject felt a tingling pain over the lateral epicondyle of the right forearm when grasping and lifting a basin, and noticed that the fingers of the right hand could not be extended three days later. The fingers of the left hand also experienced paralysis 3 months after the first injury when she pronated and extended left forearm. Neurological examination revealed bilateral P.I.O.N. paralysis. When, after a period of time, the bilateral P.I.O.N. paralysis had not improved, surgical exploration of both P.I.O.N. was performed. It revealed that the right P.I.O.N. underwent a severe constriction at 2 cm proximal to the superficial portion of the supinator muscle, and that the proximal portion of the right nerve was swollen. The constricted portion of the right nerve was resected 5 mm in length, and nerve suturing was performed. The left P.I.O.N. was also constricted at the same location, and was found to be a sausage-like neurinoma with two constrictions. In the histological examination of the right resected P.I.O.N., edema of the interstitial tissue and a great number of regenerating cluster formations, including swollen axons, were observed proximal to the constriction. Distal to this, severe Wallerian degeneration was found. These histological findings were the same as those of chronic compression neuropathy. The authors reviewed and analysed reports on 20 other cases of P.I.O.N. paralysis that had compressions at 2 cm proximal to the superficial portion of the supinator muscle.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunohistochemical and ultrastructural classification of peripheral neuropathies with onion-bulbs. By immunochemically and ultrastructurally identifying cellular components of onion-bulbs (Obs), this study attempts to delineate and classify types of hypertrophic neuropathies which are otherwise obscured by proliferation of the morphologically similar cells forming Obs. Fourteen cases with different forms of chronic localized or generalized hypertrophic neuropathy with Obs were retrospectively studied to include: one Dejerine-Sottas (D-S); two Charcot-Marie-Tooth (CMT); three chronic inflammatory demyelinating polyneuropathy (CIDP); one inflammatory localized hypertrophic mononeuritis (LHM); two perineuriomas (PNM); one traumatic amputation neuroma (TAN); one chronic diabetic neuropathy (CDN); two neurofibromas (NF); and one chronic arsenic polyneuropathy (AsPN). By immunostaining with 7 selected antibodies, we can distinguish at least 4 nosologically distinct types of neuropathy by identifying cellular components of given Obs: 1) primarily Schwann cells with no perineurial cells or macrophages in CMT or D-S, 2) predominantly Schwann cells, activated macrophages and a few fibroblasts in CIDP and LHM, 3) primarily perineurial and a few central Schwann cells with no macrophages in PNM and TAN, and 4) exclusively perineurial cells in the hamartomatous PNM associated with NF.


Diagnostic reliability of radionuclide ventriculography in detecting left ventricular hypertrophy. Echocardiographic and ECG correlations. Left ventricular hypertrophy (LVH) is frequently present in patients referred for radionuclide ventriculography (RVG) for evaluation of left ventricular function. During interpretation of these studies, the nuclear medicine physician may have the subjective impression that increased septal thickening is present because of the abnormally prominent separation of the right and left ventricular blood pools. To examine the diagnostic reliability of this finding, we retrospectively reviewed the RVG studies of 43 consecutive patients and correlated the finding of subjectively increased septal thickness with established echocardiographic (ECHO) criteria and commonly used electrocardiographic (ECG) indices of LVH. Using standard ECHO measurements of septal thickness as a gold standard, RVG interpretation of septal thickening demonstrated a sensitivity of 0.69, specificity of 0.70, and accuracy of 0.70. When compared with standard ECG criteria for LVH, RVG performed quite favorably in the diagnosis of LVH confirmed by ECHO left ventricular mass index. We conclude that scintigraphic evidence of LVH should be reported when RVG studies are interpreted.


Sural nerve bioptic findings in essential cryoglobulinemic patients with and without peripheral neuropathy. Peripheral neuropathy often occurs in cryoglobulinemia but the pathogenesis of the peripheral nerve involvement is not completely understood, so that the relation between the reported endoneural changes and neuropathy is not clear. In this study we compared the sural nerve biopsies of 6 cryoglobulinemic patients with or without signs of peripheral neuropathy and all affected by the essential mixed type II form (ECII) and, moreover, of 8 age-matched controls. We found that in all the patients with neuropathy, axonopathy occurred and it was invariably associated with endoneural vessel damage. Moreover, the fiber losses were patchily distributed within the nerve fascicles. On the contrary both nerve fibers and vessels were normal in the patients without clinical and neurophysiological evidence of neuropathy and in controls. Our results support the hypothesis that the endoneural damage observed during ECII is not simply coincidental, but is relevant in the pathogenesis of cryoglobulinemic neuropathy. They also favor the assumption that ischemic damage of the nerve fibers occurs during ECII.


An autopsied case of juvenile parkinsonism and dementia, with a widespread occurrence of Lewy bodies and spheroids. An autopsied case of juvenile parkinsonism and dementia is described. The patient is a 48-year-old man who had a ten-year history of parkinsonian syndrome and progressive dementia. Neuropathological examination revealed a widespread occurrence of Lewy bodies and spheroids in the central nervous system. Lewy bodies were found not only in the brain stem and diencephalon, but also in the cerebral cortex. Massive numbers of small spheroids were observed in the globus pallidus, substantia nigra, mamillary bodies and hippocampus. Electron microscopical examination showed that most spheroids were composed of degenerative organelles with only a few neurofilaments, and were different from those of Hallervorden-Spatz disease. There was also marked neuronal loss with gliosis in the CA3-4 of the hippocampus. Some neurofibrillary tangles occurred in the hippocampus, subcortical and brain stem nuclei, but senile plaques were absent. This case may represent an atypical form of pure diffuse Lewy body disease.


Assessment of gelatin-supplemented BACTEC blood culture medium in a pediatric hospital. Sodium polyanetheolesulfonate (SPS), an anticoagulant used in blood culture media, adversely affects the isolation of Neisseria meningitidis. The addition of gelatin appears to counteract this effect. Studies using the radiometric BACTEC system, however, have noted a lower isolation rate of other bacteria from gelatin-supplemented media. We wished to evaluate the effect of the addition of gelatin (1.2%) to a nonradiometric BACTEC aerobic medium (NR6A) on the recovery of N. meningitidis and other pathogens. The NR6A medium with gelatin (NR6A analogue) also contained a lower concentration of SPS (0.025% vs 0.035%). We did 6045 paired comparisons of blood cultured in routine NR6A medium and the NR6A analogue. Eight isolates of N. meningitidis were recovered, five only from the gelatin-supplemented medium and three from both bottles. There was no statistically significant difference in total recovery of aerobic and facultative bacteria or Candida species from both bottles. Haemophilus influenzae was detected earlier in the nonsupplemented NR6A medium. We conclude that the use of the NR6A analogue medium appeared to increase the yield of N. meningitidis without adversely affecting the recovery of other common pathogens, although the recovery of H. influenzae was slightly delayed.


The utility of restriction endonuclease analysis and phage typing in the epidemiologic investigation of a Staphylococcus aureus outbreak in a neonatal nursery. Outbreaks of Staphylococcus aureus infections in neonatal units require prompt investigation and implementation of control measures. From January to March 1990, a marked increase in the number of S. aureus infections was observed in a neonatal nursery. Twenty-seven S. aureus isolates from 23 patients were analyzed by phage typing and restriction endonuclease analysis (REA). Only nine strains were differentiated by phage type. However, REA with HindIII, CfoI, and ClaI differentiated 20 strains. The REA results indicated that the outbreak was due to several different S. aureus strains and did not represent transmission of a single epidemic strain. REA may enable more accurate determination of the presence or absence of an epidemic strain during an outbreak than would traditional methods such as phage typing.


Evaluation of variables in immunofluorescence procedures for the detection of antibodies against human herpesvirus 6 (HHV-6). Ten human sera were used to study different parameters, namely, methods of smear preparation and fixation, and age of infected HSB-2 cells in order to optimize indirect immunofluorescence assay (IFA) and anticomplement immunofluorescence (ACIF) procedures to measure antibody levels against HHV-6. Results showed a greater sensitivity of rapid smear drying and methanol fixation over conventional acetone smear preparation. Cells harvested 6 days after infection and fixed with methanol exhibited a sharper and more intense fluorescence. IFA titers were higher than those obtained with ACIF, although the latter procedure enabled the distinction between three fluorescent sites. Reactivity pattern of individual sera against infected cells was variable and indicated that the human immune response to HHV-6 is directed against different antigens. An easier interpretation and a better definition of the fluorescence of HSB-2 cell line infected with HHV-6 strain Dv is obtained with the following conditions: cells should be harvested at 5-8 days after infection (at the giant cell stage of infection), cell smears have to be dried quickly before fixation with methanol at -20 degrees C, and finally, they should be stained by IFA.


The efficacy of case management services for severely mentally disabled clients. A comprehensive review of published and unpublished research studies of case management for severely mentally disabled adults was undertaken. This yielded 20 studies of four models of case management, Full Support, Personal Strengths, Rehabilitation and Expanded Broker Models. This article reviews these models, the research studies, the outcomes of these studies and conclusions that can be drawn regarding the efficacy of case management services for severely mentally disabled adults. Future directions for research in this area are recommended.


Immunopurification of gastric parietal cell tubulovesicles. 1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.


The effect of exercise and in vivo treatment with ACTH and norepinephrine on the lipolytic responsiveness of guinea pig (Cavia porcellus) adipose tissue. 1. In adipose slices of the guinea pig (Cavia porcellus), adrenocorticotrophic hormone (ACTH) produced a significantly higher lipolytic response (increased Vmax and decreased Km) as compared to norepinephrine (NE), thus indicating a marked difference with human adipose tissue. 2. Exercise-training caused a significant increase in the affinity of the lipolytic response in adipose tissue towards both ACTH and NE, but had no significant effect on Vmax. 3. Prolonged in vivo treatment with both ACTH and NE significantly decreased the responsiveness (affinity and capacity) of adipose slices towards ACTH. Responsiveness towards NE was much less affected.


In vitro activity of DNA gyrase inhibitors, singly and in combination, against Mycobacterium avium complex. The in vitro activities of the DNA gyrase inhibitors ciprofloxacin, coumermycin, and novobiocin against 31 clinical isolates of Mycobacterium avium complex were studied using a microdilution technique. Minimal inhibitory concentrations (MICs) were determined in 4 days using Middlebrook 7H9 broth, and minimal bactericidal concentrations (MBCs) were determined by subculturing to Middlebrook 7H10 agar. MICs were: ciprofloxacin, 0.5-greater than 16 (mean, 4.1) micrograms/ml; novobiocin, 4-greater than 128 (mean, 54.7) micrograms/ml; and coumermycin, 2-greater than 16 (mean, 17.5) micrograms/ml. MBCs were usually more than two dilution steps higher than MICs. Checkerboard studies failed to reveal synergistic or antagonistic inhibitory activity of DNA gyrase-A and DNA gyrase-B inhibitors in vitro.


The effect of prolonged exercise and in vivo treatment with ACTH and norepinephrine on plasma cortisol and on the levels of energy metabolites in liver, muscle and blood of the guinea pig (Cavia porcellus). 1. Exercise and in vivo treatment with adrenocorticotrophic hormone (ACTH) showed a marked tendency to increase in vivo plasma cortisol levels in the guinea pig (Cavia porcellus). 2. However, in vivo norepinephrine (NE) treatment did not have any notable effect on plasma cortisol levels. 3. Metabolite levels (glycogen, glycerol and lactate) in liver and plantaris and soleus muscle, and the levels of glucose, glycerol and lactate in blood, were determined in response to the same treatments. 4. A number of statistically significant changes, as well as certain trends, in metabolite levels were observed in response to the treatments and are discussed.


A numerical reconstruction of the effects of late stimulation on a cardiac ventricular action potential. Numerical simulations of a propagating cardiac action potential utilizing Beeler-Reuter and Drouhard-Roberge physiological routines for the membrane current have been performed. These action potentials show increases in action potential duration when subjected to strong late stimuli of either positive or negative polarity. The mechanism is the same as that reported in an earlier paper which utilized a different physiological approach: repolarizing stimuli can reset the fast sodium gates locally so that they can be retriggered by diffusive return of charge from surrounding tissue. This results in a large depolarizing transient that lengthens action potential duration.


In vitro activity of lomefloxacin and other antimicrobials against bacterial enteritis pathogens. Lomefloxacin is a new, difluoroquinolone. In this study, the in vitro activity of lomefloxacin against clinical isolates of a variety of bacterial species associated with acute diarrheal disease was determined and compared with that of ciprofloxacin, ofloxacin, amoxicillin, sulphamethoxazole, trimethoprim, tetracycline, and chloramphenicol. Bacterial isolates were obtained from different geographical areas, including Western Europe and the United Kingdom, Southern Europe, Africa, the Middle East, South and Southeast Asia, and South America, and were included to reflect the range of susceptibility seen throughout the world. Minimum inhibitory concentrations (MICs) were determined using an agar incorporation technique in Mueller-Hinton medium supplemented when necessary with saponin-lysed horse blood at a final concentration of 10% vol/vol. Lomefloxacin was highly active against all the species examined which included Salmonella spp., Shigella spp., Escherichia coli enterotoxigenic [(ETEC), enteroinvasive (EIEC), and enteropathogenic (EPEC) strains], Yersinia enterocolitica, Campylobacter jejuni, Vibrio spp., and Aeromonas hydrophila, with all isolates inhibited by 1 mg/L or less. This activity was similar to ofloxacin and slightly less than that of ciprofloxacin. By contrast, many of the isolates were resistant to one or more of the other, unrelated animicrobials. No cross-resistance between lomefloxacin and any of the nonfluoroquinolone antimicrobials examined in the study was observed.


A recurrent marker chromosome involving chromosome 1 in two mammary tumors of the dog. An apparently identical marker chromosome resulting from a chromosome 1. translocation was found in the mammary carcinomas of two bitches. Although these karyotypic aberrations were the sole clonal aberrations detected, it was not possible to unambiguously identify the material translocated to the chromosome 1 in either animal. Our observations, however, represent the first report of a recurring marker chromosome in mammary tumors of the dog and suggest that these tumors may become an interesting model for human breast cancer.


Interpretive criteria for CI-960 (AM-1091, PD127391) disk diffusion tests using 5-microgram disks. CI-960, a new potent fluoroquinolone, was evaluated to develop disk diffusion susceptibility criteria for a 5-micrograms content disk. Preliminary interpretive zone diameter criteria were proposed for two possible susceptible breakpoint MICs (less than or equal to 1 and less than or equal to 2 micrograms/ml). Both sets of interpretive criteria performed well having 96.8%-98.4% absolute agreement and no false-susceptible errors. The more conservative less than or equal to 1-microgram/ml breakpoint criteria had correlate zones of greater than or equal to 23 mm for susceptible and less than or equal to 19 mm for resistant. These criteria produced no false resistance, whereas 0.7% of strains exhibited major errors at the less than or equal to 2-micrograms/ml breakpoint.


US experience with itraconazole in Aspergillus, Cryptococcus and Histoplasma infections in the immunocompromised host. Itraconazole has emerged as an important new oral agent in the treatment of systemic fungal infections. This paper summarizes the data available on its use in aspergillosis, cryptococcosis and histoplasmosis, compiled in the United States with particular attention to the immunocompromised host. Data have been accrued in open-label studies including 57 patients with cryptococcal disease where the overall response rate among patients with meningitis was 86%, and in 28 patients (8 with acquired immune deficiency syndrome (AIDS) or human immunodeficiency virus (HIV) infection) with invasive aspergillosis where the overall response rates were 80% in patients without AIDS and 86% in patients with AIDS. Data are summarized on 6 patients with allergic bronchopulmonary aspergillosis, 5 of whom demonstrated marked improvement on therapy, and 12 patients with histoplasmosis including 8 with AIDS, 11 of whom responded and 1 recrudesced on therapy. In summary, itraconazole showed activity in human studies of aspergillosis, cryptococcosis and histoplasmosis with minimal toxicity. Itraconazole offers a new oral alternative to conventional amphotericin B therapy in these infections. Comparative studies are needed to clarify its role.


Itraconazole in neutropenic patients.

Itraconazole in neutropenic patients. Treatment of fungal infections in neutropenic patients continues to be a major problem for the clinician. Treatment of such infections with amphotericin B is difficult, because of its many side-effects. In a neutropenic mouse model, itraconazole appeared to be as effective as amphotericin B against Candida albicans, and was more effective than amphotericin B in treating an Aspergillus infection in a patient with chronic granulomatous disease. In a randomized, comparative trial of itraconazole and amphotericin B as treatment for Candida and Aspergillus infections, 32 neutropenic patients were evaluated. Patients received either oral itraconazole, 200 mg twice daily, intravenous amphotericin B, 0.6 mg/kg/day, or in some cases of Candida infection intravenous amphotericin B, 0.3 mg/kg/day, plus flucytosine, 150 mg/kg/day. The causative organism of fungal infection was Candida spp. in 16 patients and Aspergillus spp. in 13 patients; 27 patients had pneumonia. The median duration of treatment was 13 days with amphotericin B and 20 days with itraconazole. Nine of 16 patients responded to amphotericin B, and 10 of 16 patients responded to itraconazole. Of the patients with Aspergillus infection, 6/8 treated with itraconazole and 2/5 treated with amphotericin B responded. Three patients with Aspergillus infection died in the amphotericin B arm and none in the itraconazole arm; 2 patients treated with itraconazole died from candidal infections. Absorption of itraconazole was unreliable in these seriously ill patients with disturbed gastrointestinal function. These results suggest that itraconazole could be an effective drug against systemic fungal infections in neutropenic patients. One retrospective study also suggest that itraconazole is superior to ketoconazole in prophylaxis for Aspergillus infections. Further studies are needed.


Tropical mycoses.

Tropical mycoses. The most common tropical subcutaneous and deep mycoses include chromomycosis, sporotrichosis and mycetoma. All are commonly found in Natal and in other subtropical countries. Although blastomycosis is endemic in North America, only 4 cases have been identified in Natal during the last 25 years, and all presented with atypical clinical features. African histoplasmosis, caused by Histoplasma capsulatum var. duboisii and limited mainly to central and western Africa, has been found in only 1 patient in Natal. Paracoccidioidomycosis, though the most common deep mycosis in Latin America, is limited to that area and there is no experience of this disease in South Africa. Over the past 8 years, itraconazole has been used in clinical trials for all 3 mycoses. The results in sporotrichosis, non-meningeal blastomycosis and paracoccidioidomycosis suggest that for these diseases itraconazole may be the drug of choice. The results in histoplasmosis are encouraging, as are the results in chromomycosis, particularly those cases associated with Cladosporium carrionii. Where Fonsecaea pedrosoi is the causal agent and in mycetomas, however, successful management still remains a therapeutic problem. Enhanced efficacy by combining flucytosine and itraconazole was seen in 3 patients. Even over prolonged periods, itraconazole has an impressive safety profile. In the present series of 41 patients, no side-effects were observed, no adverse reactions occurred, and serum chemistry values remained within normal limits. It appears, therefore, that itraconazole, though not the final answer to management of deep mycoses, is certainly a major improvement on previous drugs.


In vitro antifungal spectrum of itraconazole and treatment of systemic mycoses with old and new antimycotic agents. Itraconazole is a lipophilic triazole with potent in vitro activity. It is also effective after topical, oral and parenteral administration. The antifungal activity of itraconazole has been evaluated against more than 6,500 different strains, belonging to more than 260 fungal species, using the serial decimal dilution test in fluid broth medium (brain-heart infusion broth). Candida spp., Torulopsis spp., Cryptococcus neoformans, Pityrosporum spp. (Dixon broth), various other yeasts, dermatophytes, Aspergillus spp., Penicillium spp., Sporothrix schenckii, dimorphic fungi (mycelium phase and yeast phase), Phaeohyphomycetes, Entomophthorales and various Hyalohyphomycetes are sensitive. Most strains of Fusarium and Zygomycetes are poorly sensitive. Itraconazole was administered orally and parenterally in normal and immunocompromised guinea-pigs infected with C. albicans, Cr. neoformans, Histoplasma duboisii, S. schenckii, P. marneffei and A. fumigatus. It was effective in terms of both survival of the animals and elimination of the fungi from the various tissues. Itraconazole was superior to fluconazole in candidosis, cryptococcosis, sporotrichosis and aspergillosis, and to amphotericin B and to flucytosine in candidosis, cryptococcosis and aspergillosis. No comparative studies have yet been undertaken for other deep mycoses. The results of combination therapy with itraconazole and fluconazole in cryptococcosis were indifferent; with flucytosine or amphotericin B, additive or synergistic effects were seen in systemic candidosis, cryptococcosis and aspergillosis. No drug-related side-effects were observed after oral or parenteral administration of itraconazole.


Opportunistic fungal infections in patients with acquired immune deficiency syndrome. The recently developed antifungal agents itraconazole and fluconazole have been evaluated for primary and maintenance therapy for mycoses in patients with acquired immune deficiency syndrome (AIDS) in comparative and non-comparative trials. In oropharyngeal candidosis, ketoconazole may have to be given at a dose of 400 mg/day for comparable efficacy with fluconazole, 50 mg/day, because many patients with AIDS lack the gastric acid secretion necessary for drug absorption. Relapse rates were high. Itraconazole, 200 mg/day for 4 weeks, was as effective as ketoconazole, 400 mg/day, in achieving clinical remission and was better tolerated, but relapse rates were also high. An oral formulation of itraconazole in cyclodextrin has given clinical and mycological remissions in 39/39 patients after 7 days of treatment. In primary treatment of cryptococcosis, fluconazole is well tolerated, but is effective in only 50% of patients. Pilot studies of itraconazole, 200-400 mg/day, gave responses in 11/12 patients, of whom 8 had meningitis. Itraconazole combined with flucytosine gave a response in 12/13 patients, of whom 9 had meningitis. This combination shortened the time to cure. Both itraconazole and fluconazole are effective as maintenance therapy given after successful primary therapy of cryptococcosis. Itraconazole, 200 mg/day, was successful in preventing relapse in 34/39 patients over a mean 12-month period. The serum antigen titre fell progressively in 14, and fell to zero in 18 patients. Limited non-comparative trials suggest that triazoles are effective in coccidioidomycosis, and in particular that itraconazole is active against histoplasmosis in patients with AIDS; 8/9 patients with histoplasmosis responded to itraconazole, 400 mg/day, with a mean follow-up of 1 year.(ABSTRACT TRUNCATED AT 250 WORDS)


Alpha-melanocyte-stimulating hormone antagonizes the neuroendocrine effects of corticotropin-releasing factor and interleukin-1 alpha in the primate. alpha-Melanocyte stimulating hormone (alpha-MSH), a peptide derived from POMC has previously been shown to antagonize the action of exogenously administered beta-endorphin (beta-EP) on pituitary PRL and LH release in the primate. In this study, we have tested the ability of alpha-MSH to block some of the acute pituitary effects of CRF and interleukin-1 alpha (IL-1 alpha), effects which are thought in part to result from the release of endogenous beta-EP. Experiments were performed in ovariectomized rhesus monkeys bearing a chronically implanted lateral ventricular cannula for peptide infusion. Peripheral blood samples for LH, cortisol, and PRL RIA were obtained at 15-min intervals during a 3-h control period when saline was infused into the ventricle, followed by a 5-h experimental period. CRF (15 micrograms/h) infused alone for 5 h caused a significant suppression of pulsatile LH release; by the fifth hour, LH secretion was reduced to 32.5 +/- 2.4% of the control saline infusion. The CRF-induced suppression of LH was prevented by coinfusion of alpha-MSH (60 micrograms/h); by the fifth hour LH was 89.0 +/- 3.6% of the control (P less than 0.05 vs. CRF alone). alpha-MSH also prevented the CRF-induced decrease in LH pulse frequency (P less than 0.05). IL-1 alpha (4.2 micrograms) was infused alone for 30 min or in combination with alpha-MSH (120 micrograms/h for 2 h). After IL-1 alpha alone, LH decreased to 30.1 +/- 2.4% of baseline at 5 h. This decrease was prevented by alpha-MSH; by 5 h LH was 101 +/- 5.1% of baseline (P less than 0.005 vs. IL-1 alpha alone). IL-1 alpha did not affect LH pulse frequency but pulse amplitude was reduced; this reduction was prevented by alpha-MSH (P less than 0.05). IL-1 alpha also stimulated PRL release. PRL rose from a mean baseline of 3.5 +/- 0.3 ng/ml to a peak of 13.8 +/- 2.7 ng/ml; after coinfusion of alpha-MSH the mean peak PRL response was only 4.4 +/- 1.5 ng/ml (P less than 0.001 vs. IL-1 alpha alone). After CRF infusion, cortisol increased to 136 +/- 7.9% of the mean morning baseline concentration. This increase was not prevented by alpha-MSH coinfusion; after CRF plus alpha-MSH, cortisol increased to 121 +/- 6.0% of baseline. In contrast, alpha-MSH prevented the IL-1 alpha-induced increase in cortisol: 167 +/- 15.5% vs. 91.7 +/- 8.3% (P less than 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)


The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin. We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.


Identification of somatostatin and gastrin receptors on enterochromaffin-like cells from Mastomys gastric tumors. Histamine-secreting enterochromaffin-like (ECL) cells of the gastric fundus of the Mastomys can develop into solid ECL cell tumors, either spontaneously or after induction by acid inhibition. We used this tumor tissue to perform in vitro receptor autoradiography for somatostatin (SS), gastrin, and substance-P, using, respectively, [125I]Tyr3-octreotide, [125I]gastrin-17, and [125I]Bolton-Hunter-labeled substance-P as radioligands. A high density of SS receptors was found in the nontumor fundic mucosa, where gastrin receptors were only barely detectable. However, in the group of spontaneously developing ECL cell tumors, a high density of SS and gastrin receptors was observed, homogeneously distributed in the tumor tissue. In addition, the loxtidine-induced ECL cell tumors expressed a high density of SS and gastrin receptors. The receptors were specific for the respective peptide and of high affinity, with a dissociation constant (Kd) of 0.90 nM for SS receptor and 0.87 nM for gastrin receptors. No substance-P receptors were detected on the ECL cell tumors, although they were present in the muscle layers of the Mastomys gastric fundus. These results demonstrate that ECL-derived tumors express receptors for both SS and gastrin. This observation is consistent with the proposal that there is substantial regulation of the histamine-producing ECL cell by SS and gastrin. The presence of gastrin receptors is compatible with a role for gastrin as a trophic factor in ECL cell hyperplasia and neoplasia. The expression of SS receptors may be of diagnostic and therapeutic relevance in the regulation of ECL function and neoplastic transformation.


Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cells: in vitro correlate of hyperreninemic hypoaldosteronism. In some critically ill patients, aldosterone secretion is diminished despite hyperreninemia. These same patients demonstrate appropriately elevated plasma ACTH and cortisol levels. In addition, infusion of ACTH or angiotensin-II (AII) fails to elicit the normal aldosterone response, implying that the defect is at the level of the zona glomerulosa (ZG) cell. To test the hypothesis that elevated ACTH levels induce this defect, Percoll-purified bovine ZG cells were plated in serum-free defined medium and cultured for 5 days. On days 1-4, cells were exposed to various concentrations of ACTH for 1 h. On the fifth day of culture, half of the wells pretreated with ACTH were treated for 1 h with AII (10(-7) M); the other half of the wells received another dose of ACTH for 1 h. Additionally, cells were exposed to daily 1-h pulses of AII (10(-7) M) alone or in combination with ACTH (10(-8) M) for 5 days. Acutely dispersed bovine ZG cells showed dose-dependent increases in aldosterone when incubated with ACTH, potassium, or AII, with minimal cortisol production. Acutely dispersed bovine fasciculata cells produced no aldosterone, but demonstrated a dose-dependent cortisol response to ACTH and AII, but not potassium. On day 1 of culture, the ZG cells demonstrated a significant (P less than 0.001 in all cases) dose-related increase in aldosterone secretion in response to ACTH. However, continued daily pulsation with ACTH resulted in a dose-dependent decrease in aldosterone secretion, with a concomitant dose- and time-related rise in cortisol production. Indeed, ACTH-induced cortisol production in ZG became similar to ACTH-induced cortisol production in zona fasciculata cells. The addition of AII to the daily ACTH pulse did not significantly alter the aldosterone or cortisol response patterns to ACTH alone. In contrast, ZG cells treated with AII alone for 5 days showed a minimal change in cortisol production and no reduction in aldosterone production until day 4. Northern blot analysis of total RNA isolated from ZG cells pulsed with ACTH for 5 days demonstrated a parallel dose-dependent increase in 17 alpha-hydroxylase mRNA, which did not occur in cells pulsed with AII alone. These in vitro results suggest that elevated ACTH levels over time induce 17 alpha-hydroxylase activity in ZG cells, thereby shifting steroid biosynthesis from an aldosterone-producing to a cortisol-producing pathway. It is likely that the chronically elevated ACTH levels in critically ill patients induce a similar change in ZG cell biosynthesis, resulting in their hyperreninemic hypoaldosterone state.


Intermediate filaments and steroidogenesis in adrenal Y-1 cells: acrylamide stimulation of steroid production. The possible role of intermediate filaments in steroidogenesis was investigated in Y-1 mouse adrenal tumor cells by treatment with acrylamide, which is thought to disrupt intermediate filaments without directly affecting microtubules or microfilaments. Treatment of cells with 5 mM acrylamide increases steroidogenesis after a lag period of 4-6 h and induces rounding of the cells at approximately the same time. The effect of acrylamide on steroidogenesis is not cAMP mediated and occurs before pregnenolone formation. DNA synthesis is inhibited, while protein synthesis is not. Acrylamide does not affect polymerization/depolymerization of microtubules in vitro. Acrylamide stimulation of steroidogenesis is additive with that produced by either colchicine or ACTH, implying that acrylamide, ACTH, and colchicine act at different rate-limiting steps in steroidogenesis. In addition, acrylamide stimulation is additive with that of forskolin. Pretreatment of cells with taxol, an agent that specifically promotes microtubule polymerization, decreases acrylamide-stimulated (as well as colchicine or ACTH-stimulated) steroidogenesis, implying that there must also be some shared elements in the stimulating pathways. We hypothesize that regulation of steroidogenesis in the Y-1 cell depends on 1) disruption of a vimentin or tubulin coat surrounding lipid droplets and 2) possible functional shortening of the distance between cholesterol droplets and the mitochondrion. However, because of interactions between cytoplasmic fibers, it is currently impossible to say whether interruption of any one of them is a direct or indirect stimulus of steroidogenesis.


Changes in retinoic acid receptor messenger ribonucleic acid levels in the vitamin A-deficient rat testis after administration of retinoids. Recently, we have reported that retinoic acid (RA), similarly to retinol acetate, is able to reinitiate spermatogenesis in vitamin A-deficient rats. Here, we investigated the expression of RA receptors RAR alpha, RAR beta, RAR gamma, and retinoid X receptor RXR alpha by Northern blot analysis of poly(A)+ RNA of testes of vitamin A-deficient rats before and after reinitiation of spermatogenesis induced by injection of retinol acetate or RA and testes of 21-day-old and 10-week-old normal rats. In the testis of vitamin A-deficient rats 1.9-, 2.8-, and 3.8-kilobase (kb) transcripts of RAR alpha; 2.8- and 3.3-kb transcripts of RAR beta; 1.8-, 2.8-, and 3.4-kb transcripts of RAR gamma; and two transcripts of RXR alpha of 2.5 and 4.8 kb are expressed. When vitamin A-deficient rats receive RA or retinol acetate, a 3-fold increase in the amount of poly(A)+ RNA per testis can be observed after 8 h, while the amounts of glyceraldehyde-3-phosphate dehydrogenase and sulfated glycoprotein-1 mRNA hardly change. Also, the expression of several transcripts of each RAR type is significantly increased from 1.8- up to 3.6-fold. Moreover, additional transcripts of RAR beta and RXR alpha (1.8 and 1.0 kb, respectively) can be detected. In the testes of 21-day-old rats, three transcripts of each RAR type and two RXR alpha transcripts are expressed. In contrast, in the normal adult rat testis the expression of all RARs, if present, is lower than that in the 21-day-old rat testis or the adult vitamin A-deficient rat testis. The expression of all transcripts of each RAR in the testis of 21-day-old rats shows great similarity with the expression in the testis of the vitamin A-deficient rat after replacement of retinol acetate or RA. These changes in expression indicate that RARs and RXR alpha may play a role in the process of proliferation and differentiation of A spermatogonia, which is induced in vitamin A-deficient rats shortly after replacement of RA or retinol acetate.


Stimulatory role of cyclic adenosine monophosphate as a mediator of meiotic resumption in rabbit oocytes. The present study was undertaken to assess the role of alterations of intraoocyte cAMP concentrations in the meiotic maturation of isolated and follicle-enclosed oocytes. In isolated oocyte culture, the intracellular cAMP content of denuded oocytes declined within 15 min of incubation, whereas the cAMP content of cumulus-enclosed oocytes did not change substantially for 1.5 h of incubation, and then declined abruptly. Commitment to meiotic maturation was preceded by reduced concentrations of intraoocyte cAMP. Forskolin inhibited the spontaneous maturation of cumulus-enclosed oocytes in a dose-dependent manner. However, this inhibition was attenuated as the duration of incubation increased. Forskolin significantly stimulated the intracellular cAMP content of denuded and cumulus-enclosed oocytes, but intraoocyte cAMP returned to pretreatment values within 4 h. The decline in intraoocyte cAMP was followed by the meiotic maturation of isolated oocytes. In in vitro perfused rabbit ovaries, exposure to forskolin at 10(-4) M, as well as to 50 IU human CG, accelerated the meiotic maturation of follicle-enclosed oocytes. The intraoocyte cAMP content increased significantly within 30 min and reached its maximum 2 h following exposure to forskolin. Thereafter, cAMP decreased abruptly and returned to pretreatment levels by 6 h. These alterations of intraoocyte cAMP contents following exposure to forskolin paralleled those observed in human CG-treated ovaries. The decline in cAMP content of follicle-enclosed oocytes was followed by their meiotic maturation. In conclusion, the sustained elevation of intraoocyte cAMP levels inhibits the initiation of meiotic maturation in isolated and follicle-enclosed oocytes. Within the follicle, resumption of meiosis is triggered via a transient increase in intraoocyte cAMP, but commitment to meiosis must await the decline of intraoocyte cAMP.


Effects of interleukin-1 on the stress-responsive and -nonresponsive subtypes of corticotropin-releasing hormone neurosecretory axons. Administration of interleukin-1 (IL-1) induces increases in plasma ACTH and glucocorticoids. Numerous experiments have implicated the hypothalamic CRH neurosecretory system in these responses, but have failed to provide evidence for involvement of the ACTH secretagogue vasopressin (VP). The rat CRH neurosecretory system contains two types of cells: VP expressing and VP deficient. Hence, the above findings suggested that IL-1 may selectively activate the VP-deficient subtype of CRH neurosecretory cells. In this study we employed postembedding electron microscopic immunocytochemistry to directly assay IL-1-induced depletion of secretory vesicles from identified VP-expressing and VP-deficient CRH neurosecretory axons. IL-1-induced depletion of secretory vesicles from these axons was correlated with increases in plasma ACTH and decreases in plasma PRL. No dose of IL-1 was found that could selectively activate one subtype of CRH neurosecretory axons; at doses of 0.67 microgram/100 g and above for both IL-1 alpha and IL-1 beta, equal depletion of vesicles from the two subtypes was observed. Similar results were previously found after the injection of bacterial lipopolysaccharide, which induces the release of IL-1 from macrophages. The findings unequivocally establish for the first time that IL-1 activates hypothalamic CRH neurosecretory cells in the absence of surgical stress, anesthesia, disruption of the infundibular area, or administration of toxic drugs. In addition, these data clearly demonstrate that IL-1 induces the release of VP from neurosecretory axons in the portal capillary zone of the external zone of the median eminence. Previous studies have shown that the VP-deficient subtype of CRH neurosecretory axons is not strongly activated by several types of stress; therefore, activation of the system by inflammatory mediators involves mechanisms different from those mediating the stress response.


Thyrotropin and triiodothyronine regulate iodothyronine 5'-deiodinase messenger ribonucleic acid levels in FRTL-5 rat thyroid cells. We investigated the regulation of type I iodothyronine 5'-deiodinase (5'-D) gene expression by TSH and T3 in FRTL-5 rat thyroid cells. Northern blot analysis revealed that these cells express a 5'-D messenger RNA (mRNA) species of 2.1 kilobases. Readdition of TSH to FRTL-5 cells, precultured in both thyroid hormones and TSH-depleted medium for 4 days, increased 5'-D mRNA levels, reaching a maximum (2.8-fold compared to control) after 12 h of TSH (10 microU/ml) stimulation. Dibutyryl cAMP (DBC) and forskolin mimicked this stimulatory effect of TSH on 5'-D mRNA levels. T3 also increased the 5'-D mRNA levels, reaching a maximum (2-fold compared to control) after 8 h of T3 (10(-9) M) stimulation. Addition of TSH (10 microU/ml) or DBC (1 mM) together with T3 (10(-9) M) further increased 5'-D mRNA levels, reaching a maximum (5-fold compared to control) after 12 h of stimulation. Examination of the rate of disappearance of 5'-D mRNA levels after inhibition of mRNA transcription by actinomycin-D revealed that neither TSH nor T3 significantly affected the rate of disappearance. Cycloheximide, a protein synthesis inhibitor, almost completely blocked the induction of 5'-D mRNA by TSH and DBC, but did not block the induction by T3. These results suggest that both TSH and T3 increase 5'-D mRNA levels probably by increasing transcription rate, and that TSH regulates it, in part via the second messenger cAMP, for which cycloheximide-sensitive de novo protein synthesis is required, whereas T3 does without requiring it.


Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads. When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.


Ligand-mediated internalization of glucagon receptors in intact rat liver. The ligand-induced internalization of the hepatic glucagon receptor has been studied in rats in vivo using cell fractionation. Injection of glucagon (11 nmol/100 g BW) led to a 2- to 3-fold increase in glucagon-binding activity in Golgi-endosomal (GE) fractions along with a 10-20% decrease in binding activity in plasma membrane (PM) fractions. These changes were time and dose dependent, reaching a maximum by 12-24 min and undergoing reversal in 2 h. Glucagon injection also caused a 20% decrease in glucagon binding to the total particulate fraction, which did not occur when binding was measured in the presence of the detergent octylglucoside. The change in glucagon-binding activity in PM and GE fractions resulted mainly from a change in receptor number; affinity remained unaffected (apparent Kd, 0.5 and 5 nM, respectively). A 5- to 10-fold increase in the glucagon content of GE fractions was observed in glucagon-treated rats. Neither the distribution of PM and Golgi marker enzymes nor that of the asialoglycoprotein receptor was affected by glucagon treatment. Regardless of glucagon treatment, glucagon receptors in GE fractions were less sensitive to GTP than receptors in PM fractions with respect to both inhibition of steady state binding and dissociation of prebound ligand. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, glucagon-receptor complexes formed in PM and GE fractions and subsequently cross-linked showed the same apparent mol wt (57 kilodaltons). In addition, they were identically sensitive to N-glycanase treatment, with two major species of lower mol wt generated. However, only cross-linked complexes associated with PM fractions showed detectable GTP sensitivity. GE fractions displayed a GTP-sensitive adenylate cyclase activity that was about 12 times lower than that of PM fractions. In both fractions, activity was stimulated by the addition of forskolin (8-fold) and, to a lesser extent, glucagon (3-fold). In vivo glucagon treatment led to an increase in activity in GE, but not PM, fractions. These results are consistent with the view that upon acute occupancy, hepatic glucagon receptors are rapidly and specifically internalized along with their ligand. During this process, receptor retained structural integrity and uncouple, albeit partially, from other components of the adenylate cyclase system.


Changes in the effect of gamma-aminobutyric acid on prolactin secretion during sexual maturation in female rats. To evaluate the effects of the gamma-aminobutyric acid (GABA)ergic system on PRL secretion during sexual development in female rats, aminooxyacetic acid (an inhibitor of GABA-transaminase which increases the hypothalamic GABA concentration) was administered to 12- to 16- and 30-day-old female rats. The increased GABA level in the hypothalamus was accompanied by a significant increase in the serum PRL concentration in rats 16 days of age and a decrease in the serum concentration of the hormone in 30-day-old rats. No changes in PRL levels were observed at 12 days of age. Muscimol, a GABA-A receptor agonist, increased serum PRL concentrations in 16-day-old rats. By contrast, in rats 30 days of age, muscimol induced a significant decrease in PRL levels. On the other hand, the administration of baclofen, a GABA-B receptor agonist, induced a pattern of PRL modification similar to that observed with muscimol, i.e. stimulation in rats 16 days of age and inhibition in 30-day-old rats. These findings indicate that both the stimulatory and inhibitory effects of the GABAergic system on PRL secretion during sexual maturation are mediated by GABA-A and GABA-B receptors. The present results show that during sexual maturation there is a qualitative modification of the effect of the GABAergic system on PRL secretion. These findings could be related to the complex neuroendocrine mechanisms involved in the onset of puberty in the female rat.


Inhibition by human interleukin-1 alpha of parathyroid hormone-related peptide effects on renal calcium and phosphorus metabolism in the rat. Humoral hypercalcemia of malignancy (HHM) is at least partly caused by tumor secretion of PTH-related peptide (PTHrP), but there is growing evidence for cosecretion with PTHrP of other bone-resorbing peptides, such as the cytokine interleukin-1 alpha (IL-1 alpha). Administration of PTHrP in vivo and in vitro generally mimics the actions of PTH itself, with increases in both resorption and formation of bone. However, bone in HHM is characterized by uncoupling of bone turnover, with increased resorption and decreased formation. We performed experiments to determine whether IL-1 alpha might alter the effects of PTHrP and produce uncoupling. Thus, we administered to 100-g male rats by sc osmotic minipumps synthetic PTHrP-(1-34) alone (2 micrograms/100 g/day), recombinant IL-1 alpha alone (1.5 micrograms/100 g/day), both peptides together at the previous doses, or vehicle only. We infused 5 groups of 12 rats each (PTHrP, IL-1 alpha, PTHrP plus IL-1 alpha, ad libitum fed control, and controls pair-fed to the PTHrP plus IL-1 alpha group) for 14 days. At the end of the study, blood and urine were taken for chemical measurements, and tibias and femurs were harvested for histomorphometry and extraction of RNA from periosteal cells. As expected, PTHrP induced hypercalcemia, relative hypophosphatemia, phosphaturia, and reduced bone mass. Osteoblast number was increased, but osteoclast number was not. Indices of bone formation were unchanged or reduced. The dose of IL-1 alpha chosen had no statistically significant effect, except for reduced longitudinal bone growth, but when combined with PTHrP, IL-1 alpha reduced hypercalcemia, hypophosphatemia, and phosphaturia. In contrast to the blood and urine effects, IL-1 alpha did not interact significantly with PTHrP's effect on bone measurements. Northern analysis of periosteal cell mRNA showed that PTHrP reduced expression of osteocalcin, but not glyceraldehyde-3-phosphate dehydrogenase; IL-1 alpha had no additional effect. These data suggest that 1) continuously administered PTHrP alone may induce uncoupled bone turnover with decreased cortical bone formation; 2) IL-1 alpha appears to inhibit strongly the renal effects of PTHrP and weakly (if at all) its actions on bone and, thus, to decrease its hypercalcemic, phosphaturic, and hypophosphatemic actions; and 3) cosecretion of IL-1 alpha, and possibly other peptide cytokines, with PTHrP may modify the clinical expression of HHM.


Adenosine 3',5'-monophosphate suppresses metastatic spread in nude mice of steroidogenic rat granulosa cells transformed by simian virus-40 and Ha-ras oncogene. 8-Bromo-cAMP and substances elevating cAMP levels within cells, such as forskolin, cholera toxin, and Bordetella pertussis-invasive adenylate cyclase (BPAC), suppress the growth of cultured granulosa cells cotransfected by simian virus-40 (SV40) DNA and Ha-ras oncogene concomitantly with the induction of steroidogenesis and without affecting oncogene expression. We, therefore, tested the hypothesis that cAMP can modulate tumorigenesis and metastatic spread of these cells in vivo. The cotransfected cells induced rapid development of tumors when injected sc in nude mice. Tumor development was faster in less differentiated cotransfected cells originating from preantral ovarian follicles than in those obtained from highly differentiated transformed cells originating from preovulatory follicles. Cells transfected by SV40 DNA alone produced only slow-growing small tumors. Metastatic lesions of cotransfected cells were most abundant in lung and less frequent in ovaries, kidney, and spleen. No metastatic lesions were found in the liver. However, metastatic spread was dramatically suppressed when cotransfected cells injected into nude mice were pretreated with the invasive BPAC. In contrast, no suppression of metastases was observed when the cells were pretreated with 8-bromo-cAMP, forskolin, or cholera toxin. Removal of forskolin in cultured cotransfected cells yielded a rapid decrease in cAMP levels. In contrast, high levels of cAMP persist in cell cultures even several hours after 1-h pretreatment and subsequent removal of BPAC from the medium of culture cotransfected cells. It is suggested that the inhibitory effect of BPAC on the metastatic spread of these cells is due to prolonged elevation of cAMP in vivo. The newly established granulosa cell lines transformed by SV40 and the Ha-ras oncogene can serve as a model for further studies of cAMP modulation of carcinogenesis in ovarian malignancies.


Feedback and facilitation in the adrenocortical system: unmasking facilitation by partial inhibition of the glucocorticoid response to prior stress. Previously stressed animals remain responsive to subsequent stressors, despite secreting an adequate corticosteroid signal during the first stress which should act to damp the response to a second stress. We have previously postulated that stress acts to facilitate subsequent responses in the adrenocortical system, and that this facilitation is balanced by the corticosteroid feedback signal. To test this hypothesis directly, we treated young male rats with cyanoketone (CK) to partially block the adrenal capacity to synthesize corticosterone (B). Subsequently, groups of CK- or vehicle (VEH)-treated rats were exposed to the FIRST stress of 30-min restraint with small blood samples collected at 0, 15, and 30 min. The FIRST stress was given to subgroups of rats 12, 9, 6, or 3 h before lights off (12 h) or lights on (24 h). At 12 or 24 h, rats were again restrained with blood samples at 0 ("basal") and 30 min (SECOND stress). Control groups were stressed for the first time when the experimental groups received their SECOND stress. Plasma ACTH and B concentrations were measured. Although in the absence of stress, basal B concentrations were normal in CK-treated compared to VEH-treated rats throughout the day, the B response to the FIRST stress was reduced by 60% in the CK- compared to the VEH-treated group. When the FIRST stress was performed during the time of lights on, "basal" plasma ACTH was elevated in CK groups at 12 h (lights off) compared to levels in both previously stressed VEH groups and unstressed CK controls. There was no difference at this time of day in the magnitude of the ACTH response to the SECOND stress in CK rats compared to that in CK rats receiving their only stress (controls) or that in VEH-treated rats receiving the SECOND stress. When first stress was performed during the time of lights off, "basal" plasma ACTH at 24 h (lights on) in CK and VEH rats were not different compared to levels in their respective unstressed controls. The ACTH response to the SECOND stress at 24 h was elevated in all previously stressed CK groups compared to that in either CK control or VEH groups. At neither time of day were SECOND stress ACTH concentrations in VEH rats different from those in control VEH rats. At 12 h (lights off), but not at 24 h (lights on), "basal" ACTH was significantly elevated in VEH rats above the unstressed VEH control values.(ABSTRACT TRUNCATED AT 400 WORDS)


Direct secretagogue effect of corticotropin-releasing factor on the rat adrenal cortex: the involvement of the zona medullaris. CRF dose-dependently enhanced corticosterone (B) secretion by rat adrenal slices including both cortex and medulla. Conversely, CRF did not exert any B response by fragments of adrenocortical autotransplants, which are completely deprived of chromaffin tissue. However, autotransplant quarters exhibited a dose-dependent response to ACTH qualitatively similar to that of adrenal slices, although markedly less intense. The maximal B response of adrenal slices to CRF (10(-8) M) was completely annulled by corticotropin-inhibiting peptide (10(-6) M), a competitive inhibitor of ACTH, which totally blocked the secretory response to ACTH (10(-8) M) of both kinds of preparations. ACTH immunoreactivity was present in the adrenal gland of control rats, but was undetectable in autotransplanted adrenocortical nodules. Moreover, adrenal fragments mainly composed of chromaffin tissue released detectable amounts of ACTH in response to high concentrations of CRF (10(-8)/10(-6) M). These findings suggest that chromaffin medullary cells play a pivotal role in the direct adrenocortical secretagogue effect of CRF, probably by releasing ACTH, which, in turn, may evoke, in a paracrine manner, the glucocorticoid response.


Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons. Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 +/- 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 +/- 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 microM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 microM was 28.9 +/- 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 +/- 0.38 microM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 microM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 microM omega-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)