Actions of somatostatin on GABA-ergic synaptic transmission in the CA1 area of the hippocampus.

Somatostatin and gamma-aminobutyric acid (GABA) are co-localized in some neurons in the CA1 area of the hippocampus. Since it is possible that the peptide and the amino acid are co-released, the interactions between the actions of somatostatin and GABA-ergic inhibitory post-synaptic potentials (IPSPs) in the CA1 pyramidal neurons of guinea pig hippocampal slices have been investigated. Somatostatin (2 microM) induced a hyperpolarization of the CA1 neurons associated with a reduction in the input resistance of the cells. These effects were not blocked by picrotoxinin (20 microM) or phaclofen (1 mM). Chelation of intracellular Ca2+ (Ca2+i) with BAPTA or the inhibition of protein kinase C (PKC) with sphingosine (30 microM) had no significant effects on the hyperpolarizing actions of somatostatin. The peptide suppressed the GABAA receptor-mediated fast IPSPs and the GABAB receptor-mediated slow IPSPs, but had no significant effect on the excitatory post-synaptic potentials (EPSPs). Somatostatin-induced depression of the IPSPs was not due to the hyperpolarization of the neurons. Baclofen (20 microM) suppressed the EPSP, as well as the fast and the slow IPSPs. The hyperpolarization of the CA1 neurons caused by somatostatin was greatly reduced in the presence of baclofen, an effect that was not due to the hyperpolarization of the cell by baclofen. The presence of QX-314 in the CA1 neurons, which suppressed the Na+ spikes and the slow IPSPs, prevented the hyperpolarization of the neurons by somatostatin and baclofen.(ABSTRACT TRUNCATED AT 250 WORDS)


Phosphorylation and activation of tyrosine hydroxylase in PC18 cells: a cell line derived from rat pheochromocytoma PC12 cells.

Treatment of rat pheochromocytoma PC18 cells (a variant subclone of PC12 cells) with forskolin produced increased activity and phosphorylation of tyrosine hydroxylase. In contrast, treatment of the PC18 cells with 56 mM K+, A23187, phorbol-12-myristate-13-acetate (PMA) or phorbol-12,13-dibutyrate (PDB) did not affect the activity and only slightly increased the phosphorylation of tyrosine hydroxylase. None of the treatments except forskolin increased cyclic AMP levels in PC18 cells. Furthermore, 45Ca2+ uptake into PC18 cells was not affected by 56 mM K+, PDB or forskolin; however, A23187 increased 45Ca2+ uptake 4-fold over basal uptake. Nevertheless, no activation and little increase in phosphorylation of tyrosine hydroxylase was observed in PC18 cells treated with A23187. When tyrosine hydroxylase levels in PC18 cells were elevated by treatment with dexamethasone, activation of tyrosine hydroxylase by 56 mM K+, PDB or A23187 was still not observed. Both purified Ca2+/calmodulin-dependent protein kinase and cyclic AMP-dependent protein kinase catalyzed the phosphorylation of tyrosine hydroxylase purified from PC18 cells in vitro. Furthermore, crude cell extracts from PC12 cells and PC18 cells possessed Ca2+/calmodulin-dependent protein kinase activity that catalyzed the phosphorylation of purified tyrosine hydroxylase. These results suggest that tyrosine hydroxylase activity in PC18 cells is regulated by a cyclic AMP-dependent mechanism. However, due to a number of abnormalities the Ca(2+)-dependent mechanisms do not result in the activation of tyrosine hydroxylase and only slightly increase the phosphorylation of the enzyme in PC18 cells.


Role of catecholamines in the modafinil and amphetamine induced wakefulness, a comparative pharmacological study in the cat.

Seventeen adult cats were chronically implanted with electrodes for polygraphic recordings in order to assess the role of catecholamines in the arousal effects of oral administrations of modafinil, a presumed noradrenergic agonist, and amphetamine, a well-known catecholamine-releasing agent. Whereas both modafinil (1, 2.5 and 5 mg/kg) and amphetamine (0.25, 0.5 and 1 mg/kg) caused a significant and dose-dependent increase in wakefulness and brain temperature, amphetamine, but not modafinil, elicited marked signs of behavioral excitation. Pretreatments with alpha-methyl-DL-p-tyrosine methyl ester (50 mg/kg, i.p.), an inhibitor of catecholamine synthesis, almost completely prevented the effects of amphetamine (0.25 and 1 mg/kg), but only slightly reduced the duration of the waking effect of modafinil (2.5 and 5 mg/kg). Pretreatments with phentolamine (10 mg/kg, i.p.), prazosin (1.5 mg/kg, per os) and propranolol (5 mg/kg, i.p.), an alpha-, alpha 1- and beta-receptor antagonist, respectively, attenuated significantly the arousal effect of modafinil (1 mg/kg, the same as below) but not of amphetamine (0.25 mg/kg, the same as below). Intraperitoneal injections of haloperidol (0.5 mg/kg), a dopamine-receptor antagonist, blocked significantly the arousal of amphetamine but not of modafinil. The effects of both modafinil and amphetamine were enhanced by a pretreatment with yohimbine (1 mg/kg, i.p.), an alpha 2-receptor antagonist. These results suggest that the arousal effect of modafinil does not depend on the availability of the endogenous catecholamines but results from an enhancement of alpha 1- and beta-receptor activity and that the waking and behavioral effects of amphetamine may be mainly due to an increase in dopamine release.


Theta burst stimulation is optimal for induction of LTP at both apical and basal dendritic synapses on hippocampal CA1 neurons.

The efficacy of stimulation patterns consisting of brief high frequency bursts repeated at various intervals to induce long-term potentiation (LTP) at synapses on apical and basal dendrites of CA1 hippocampal neurons was tested in vitro. Both apical and basal dendritic synapses exhibited maximal LTP after bursts repeated at 5-10 Hz, i.e. close to the frequency of the endogenous hippocampal theta rhythm. As at apical dendritic synapses, LTP at basal dendritic synapses was blocked by an antagonist of NMDA receptors. Basal dendritic LTP was significantly greater in magnitude than apical dendritic LTP, although the reason for this is unknown.


Muscarinic nature of cholinergic receptors in the cerebellar flocculus involved in the enhancement of the rabbit's optokinetic response.

Intrafloccular micro-injection of the aselective cholinergic agonist carbachol enhances the optokinetic reflex (OKR)17. Histochemical and physiological studies have identified cholinergic receptors of the muscarinic as well as nicotinic type in the cerebellar cortex, and both have been implicated in cholinergic transmission. The present study was undertaken to elucidate the receptor type involved in the control of OKR. For that purpose, effects of injections of the nicotinic N1 agonist DMPP on the OKR and vestibulo-ocular reflex (VOR) were compared with injections of the muscarinic agonist betanechol and the aselective cholinergic agonist carbachol. Injection of betanechol mimicked the enhancement of the OKR by carbachol, while DMPP had no effect. We conclude that muscarinic receptors are involved in the positive modulatory action of the cholinergic system in the cerebellar flocculus.


Cardioprotective effects of carvedilol, a novel beta adrenoceptor antagonist with vasodilating properties, in anaesthetised minipigs: comparison with propranolol.

The aim was to evaluate in a minipig model of acute myocardial infarction the cardioprotection provided by the beta adrenoceptor blocking and vasodilating activities present in carvedilol; comparison was made to the pure beta adrenoceptor antagonist, propranolol. Experiments were performed in 25 Yucatan minipigs (9-12 kg), randomly assigned to receive vehicle (n = 7), carvedilol 0.3 mg.kg-1 (n = 6), carvedilol 1 mg.kg-1 (n = 6), or propranolol 1 mg.kg-1 (n = 6). Myocardial infarction was produced by occlusion of the left anterior descending coronary artery for 45 min followed by 4 h of reperfusion. Vehicle, carvedilol (0.3 and 1 mg.kg-1) or propranolol (1 mg.kg-1) were given intravenously 15 min before the coronary artery occlusion. At the end of the reperfusion period, infarct size was determined using Evans blue dye and triphenyltetrazolium chloride staining. Infarct volumes were visualised using computer assisted three dimensional image analysis of the stained myocardial tissue sections. Myeloperoxidase activity was measured in tissue samples removed from normal, infarcted, and at risk areas. Carvedilol (1 mg.kg-1) reduced infarct size by over 90% without producing pronounced changes in systemic haemodynamic variables. The ability of carvedilol to reduce infarct size was clearly dose dependent. Thus infarct size, which represented 27.5(SEM 2.3)% of the area at risk in the vehicle treated group, was only 13.1(4.0)% (p < 0.05) and 2.4(1.5)% (p < 0.01) in pigs treated with carvedilol at 0.3 and 1 mg.kg-1, respectively. In animals treated with propranolol (1 mg.kg-1), infarct size represented 10.9(2.4)% of the area at risk (p < 0.05). The 60% and 91% reductions in infarct size produced by propranolol (1 mg.kg-1) and carvedilol (1 mg.kg-1), respectively, were clearly evident upon three dimensional image analysis. The reduction in infarct size was significantly greater for carvedilol (1 mg.kg-1) compared to propranolol (1 mg.kg-1) at equivalent beta adrenoceptor blocking doses. Pretreatment with propranolol did not reduce the increases in myeloperoxidase activity observed in the area at risk or in the infarcted area. In contrast, carvedilol produced a dose dependent reduction in myeloperoxidase activity in these areas. Carvedilol limits myocardial necrosis resulting from coronary artery occlusion and reperfusion in a more pronounced manner than the pure beta adrenoceptor antagonist, propranolol. The cardioprotective effect of carvedilol, which reduced infarct size by 91%, may result from the combined effects of beta adrenoceptor blockade and vasodilatation, and possibly also from inhibition of intracellular calcium overload in cardiac cells resulting from antagonism of myocardial alpha 1 adrenoceptors and/or calcium channel blockade. The cardioprotection provided by carvedilol may ultimately be of benefit in hypertensive patients who are at risk for acute myocardial infarction.


Studies on antiulcer drugs. VI. 4-Furyl-2-guanidinothiazoles and related compounds as potent histamine H2-receptor antagonists.

A series of 4-furyl-2-guanidinothiazole derivatives and related compounds were synthesized and evaluated for histamine H2-receptor antagonist and gastric acid antisecretory activities. Among them, compounds I-17, I-48 and I-49 showed high activities in these tests. In addition, compound I-17 possessed potent inhibitory activities on each of the gastric ulcers induced by stress, ethanol and HCl-aspirin. On the other hand, compound I-48 demonstrated antimicrobial activity against Helicobacter Pylori and the potency was far stronger than that of clinically used H2-antagonists. Some structure-activity relationships are discussed.


Keratinized epithelial transport of beta-blocking agents. I. Relationship between physicochemical properties of drugs and the flux across rat skin and hamster cheek pouch.

The maximum fluxes (Jmax) of beta-blockers through keratinized membranes were determined in vitro and compared with their physiochemical parameters such as lipophilicity (log k'0) and melting point (mp). Rat abdominal skin and hamster cheek pouch mucosa were used as the model membranes. Propranolol, metoprolol, timolol, pindolol, nadolol and agenolol were used as beta-blockers with a variety of physicochemical characters. Linear relations of Jmax with either log k'0 or mp were observed both in intact rat skin and in intact hamster cheek pouch, suggesting that the lipophilicity and thermodynamic activity of a drug in the crystal state primarily affect the drug's permeation through these membranes. However, the slope, dJmax/d(log k'0), for cheek pouch mucosa was greater than that for rat skin, corresponding to the lack of appendigeal shunt pathways in cheek pouch. Penetration studies using the delipidized membranes and the isolated stratum corneum sheet of hamster cheek pouch mucosa clarified that the primary rate-limiting barrier function might exist in the lipid layer of the stratum corneum. Jmax values for the tape-stripped and delipidized skins correlated with both the solubilities of drugs in the vehicle and with the mp, suggesting the polar porous characteristics of both model membranes. However, a theoretical approach confirmed that the contribution of an intracellular or aqueous pore route in the intact membrane to the permeation of drugs with positive lipophilic indexes is negligible.


Renin gene restriction fragment length polymorphisms in a Japanese family with a high incidence of essential hypertension.

1. Human renin gene restriction fragment length polymorphisms (RFLP) were compared in a Japanese family which has a high incidence of essential hypertension. 2. RFLP of human renin gene were observed in three restriction enzymes, Mbo I, Bgl I and Hind III; 1.4 kb and 1.0 kb [Mbo I], 5.0 kb and 9.0 kb [Bgl I] and 9.0 and 6.2 kb [Hind III]. 3. Plasma renin activity was not associated with blood pressure. 4. Renin gene RFLP were not cosegregated with essential hypertension in this Japanese family.


The roles of intracellular buffers and bone mineral in the regulation of acid-base balance in mammals.

1. Regulation of intracellular and extracellular pH may conflict in their requirements for movement of acid or base. 2. Cells make a positive or a negative contribution to 'tissue buffering' of extracellular fluid (ECF), depending on their internal buffer value, on the tightness of their internal pH control by membrane mechanisms, and on the nature of the acid-base disturbance. 3. A role is suggested for electrogenic Na-HCO3 co-transport in some of the ion shifts that occur in acid-base disturbances. 4. The time course of 'tissue buffering' in nephrectomized mammals in hypercapnia is variable, and it is far from clear in intact, unanaesthetized mammals. 5. Buffering of ECF by Ca salts of bone mineral in acidosis can only be substantial if accompanied by Ca excretion; the release of HCO3 with Na and K is more significant. 6. The relative importance of cells and of bone mineral in the buffering of ECF is unclear.


A new source of biologically active compounds--earthworm tissue (Eisenia foetida, Lumbricus rubelus).

1. Earthworms possess immunological recognition and memory as well as high regenerative abilities. Coelomic fluid was used as a source of biologically active compounds. 2. A biologically active glycolipoprotein extract from a whole earthworm tissue homogenate was isolated and named G-90. 3. G-90 forms precipitation arcs in gel with different animal and human sera. 4. It alters murine cell growth rate in vitro in serum in a dose-dependent manner and slows murine tumor growth in vivo. 5. G-90 does not contain mutagens or carcinogens.


Basic biochemical data on blood from antarctic Weddell seals (Leptonychotes weddelli): ions, lipids, enzymes, serum proteins and thyroid hormones.

1. Standard laboratory values of blood samples taken from Weddell seals in Antarctica were determined. 2. Numerous blood parameters are similar to those observed in man. 3. Comparatively high cholesterol levels but normal triglyceride levels were observed when compared with humans. 4. In comparison to laboratory findings in humans, T4 levels were decreased although T3 levels were normal. 5. The levels of alkaline phosphatase are considerably higher than those in humans. 6. The data obtained indicate different lipid and thyroid hormone metabolism in Weddell seals when compared with humans.


Correlations between serum corticosterone concentration and reproductive conditions in the white-footed mouse (Peromyscus leucopus noveboracensis).

1. Adrenal size and activity in reproductively inhibited young born into laboratory populations of the white-footed mouse were examined. Measurements were made of body weight, reproductive organ weight and adrenal weight. Serum corticosterone was measured by radioimmunoassay. 2. Data from reproductively inhibited animals were compared with corresponding values from reproductively capable animals of the same sex. The mean paired testis and seminal vesicle, or paired ovary and uterus, weights, were significantly reduced in reproductively inhibited animals of both sexes. The paired adrenal weights were not different in any comparison. 3. Reproductively inhibited males selected from laboratory populations had a mean corticosterone concentration that was significantly higher than the corresponding value for reproductively capable males. Females showed no significant difference in mean serum corticosterone concentrations. 4. The results are discussed in relation to earlier studies in two species of Peromyscus and the apparent paradox of elevated serum corticosterone in the absence of adrenal hypertrophy.


A possible relationship between KCl symport and basolateral K(+)-conductance in Necturus gallbladder epithelial cells.

1. Apical membrane potential (Va), transepithelial potential (VT), fractional apical voltage ratio (FVa = delta Va/delta VT), tissue resistance (RT), and intracellular Cl- (aiCl) and K+ (aiK) activities were measured in isolated gallbladders maintained between oxygenated bicarbonate-free physiological media (23 degrees C, pH 7.2 or 8.2) in a divided chamber. The basolateral membrane potential (Vb) was calculated from the measured values of Va and VT. 2. Cl- removal from the serosal medium (which should accelerate coupled basolateral KCl exit) significantly depolarized Vb, decreased aiCl, decreased FVa, increased RT, and attenuated the depolarization of Vb (delta Vb) induced by high K+ added to the serosal side. These changes are consistent with a decrease in the K(+)-conductance of the basolateral membrane (gbK). 3. Addition of furosemide (an inhibitor of KCl cotransport) to the serosal medium induced significant increases in Vb, FVa, and high K(+)-induced delta Vb, indicating an increase in gbK. 4. In the presence of serosal furosemide, Cl- removal from the serosal medium did not significantly alter Vb, aiCl or delta Vb from their corresponding values when serosal Cl- was present. 5. Serosal furosemide had no significant effect on aiK and aiCl measured with double-barreled ion-selective microelectrodes. 6. These results suggest the possibility of a reciprocal relationship between gbK and the rate of basolateral KCl cotransport. This may contribute to the maintenance of aiK in gallbladder epithelial cells.


Comparative hematology: studies on guinea-pigs (Cavia porcellus).

1. Guinea-pig blood clots rapidly and the clots retract in glass tubes. The prothrombin time is long and the activated partial thromboplastin time short compared to human. The Russel viper venom time is similar to human. 2. Factors VII and X assay at levels far below and factors V, VIII and XII assay far above human levels. Other coagulation factors (fibrinogen, II, IX, XI, Fletcher and Fitzgerald) assay within or close to the human range. 3. The thromboplastin generation test results for guinea-pigs and humans are similar. 4. Platelets are numerous and small. They aggregate with ADP, arachidonic acid and pig plasma, variably with ristocetin and poorly with bovine collagen or thrombin. On electron microscopy, platelets appear small with many dark granules (dense bodies). There is an open canicular system. Glycogen particles are sparse. Microtubules are occasionally seen, mitochondria are rare and alpha-granules are not readily distinguished from dark granules. 5. Ristocetin cofactor is very low, assaying at < 16% of human (< 0.16 U/ml). 6. Leukocyte counts are variable (6300-17,000 per microliters) and differential counts show neutrophils slightly lower and lymphocytes slightly higher than average human counts. 7. Guinea-pig erythrocyte parameters fall within human ranges. 8. Protein electrophoresis shows total protein and albumin to be slightly lower than human. 9. Antithrombin III, Protein C and alpha 2-antiplasmin assay within the human range and plasminogen at very low levels. 10. Bleeding times are consistently about 4 min.


Auditory-evoked brainstem responses in the hibernating woodchuck Marmota monax.

1. This study measured the changes in the auditory-evoked brainstem responses in the woodchuck (Marmota monax) during hibernation and arousal. 2. The auditory brainstem response of the euthermic woodchuck consisted of four waves occurring in a 10 msec time window after stimulation. 3. In the hibernating woodchuck, waves I and II could be traced down to the lowest body temperatures. 4. As temperatures increased all the components of the ABR emerged. The latencies of all the waves showed systematic decrease with temperature increments, the effect being cumulative across the time window. 5. These findings reflect activity in the VIIIth cranial nerve and the cochlear nuclei during hibernation and restoration of the functional integrity of the brainstem auditory pathway during arousal.


The effects of established diabetes on the growth of the fetal liver and skin in the rat.

1. The effects of uncontrolled maternal diabetes on the growth of the whole rat fetus and its liver and skin were studied over the last 4 days of gestation. 2. Smaller fetuses, with growth-retarded livers and skins, were consistently found between 18 and 21 days in the diabetic pregnancies. 3. The smaller diabetic livers and skins (i.e. combined epidermis and dermis) possessed lower protein, RNA and DNA contents, compared with the same fetal tissues from normal pregnancies. 4. The growth retardation of the livers in diabetic fetuses was attributed to fewer normally sized hepatic cells. 5. Whilst hepatic rates of protein synthesis (measured in vivo) remained largely unchanged, these were actually increased in the skin, compared with normal control tissues. 6. These findings point to an elevated rate of protein degradation in both diabetic tissues as the most likely cause of their retarded growth.


Effects of recombinant human growth hormone on anorexic Nile crocodiles (Crocodylus niloticus).

1. Eleven-month-old Nile crocodiles with poor appetite and retarded growth were injected with 0.325 micrograms/g recombinant human growth hormone (hGH) twice a week for 4 weeks. 2. The treated animals had a mean intake per meal of 29.8 g/kg, while the controls ate only 2.8 g/kg. 3. The treated group gained 8.1% of their initial body weight, while the controls lost 6.3%. 4. During 4 weeks of treatment the body and head length increased by 3.93 and 1.29%, respectively, while no linear growth took place in the controls. 5. The treated group had higher contents of skeletal muscle protein and liver glycogen than the control group. 6. In conclusion, recombinant hGH induces appetite and growth in anorexic crocodiles.


Fibre digestion and digesta retention from different physical forms of the feed in the rabbit.

1. Digestibility of fibre was higher in guinea-pigs than in rabbits, however, the digestibility of hemicellulose fraction containing agar was similar in both animals. 2. The digestibility of fibre of a fine particle diet was higher than that of a large particle diet in the rabbit permitted coprophagy, whereas it was lower in the rabbit prevented from coprophagy. 3. The fine particle diet tended to cause shorter retention time of digesta in the rabbit prevented from coprophagy. 4. These suggest that the digestibility of fine components relates to their physical properties which affect the retention time of digesta in the rabbit.


Effects of pantethine on lipogenesis and CO2 production in the isolated hepatocytes of the chick (Gallus domesticus).

1. Isolated hepatocytes from chicks were used to study the effects of pantethine supplementation to incubation medium on in vitro lipogenesis, CO2 production and beta-oxidation of fatty acid. 2. In vitro lipogenesis, determined by the incorporation of 1-[14C]acetate into total lipid and various lipid fractions, as depressed in concordance with the increase of pantethine concentration in the medium. 3. Incubation of isolated hepatocytes with pantethine resulted in a significant decrease (P < 0.01) in the activities of acetyl-CoA carboxylase and fatty acid synthetase. 4. The results suggest that in vitro fatty acid synthesis from 1-[14C]acetate was depressed and CO2 production was elevated in hepatocytes of chicks through pantethine addition to the medium at a low level.


Size distribution of adipocytes and variation in adipocyte number in lines of mice selected for high or low body fat.

1. Adipocyte size and number were measured in epididymal fat pads from lines of mice selected for high or low body fat. 2. Epididymal fat pad weight at 10 weeks of age was 4-fold greater in the high fat than the low fat mice, accompanied by a 7-fold greater adipocyte volume but there was no difference in adipocyte number. Smaller differences in fatness and cell volume were observed at younger ages. 3. The high fat mice showed one or two populations of cells up to 5 weeks of age and at 7 weeks 3 days and 10 weeks showed two or more populations. In contrast, the low fat animals at all ages showed either one or two populations. 4. These findings imply that greater lipogenesis and cell filling, not cell proliferation, is the primary mechanism of increased fatness in these selected lines of mice.


Effects of exercise and food restriction in pregnant and newborn rats. Pre-pregnancy maximum oxygen consumption.

1. In the present study, the effects of exercise and food restriction in pregnant and newborn rats were investigated. 2. The following groups were formed: adequate food supply, with and without exercise (AE and AN) and 30% food restriction, with and without exercise (RE and RN). 3. Exercise was performed throughout the pregnancy on a treadmill at a speed of 18 m/min for 30 min/day, which represented 84% of maximum VO2. 4. The results show that food restriction affected body weight gain while exercise only affected the RE group (P < 0.05). 5. Body temperature was increased by exercise. The initial temperature was lower in group RE. 6. No differences were obtained in average offspring number but reabsorption, preterm and natimortality were observed in group RE. 7. Newborn body weight was lowered by food restriction rather than by exercise. 8. Newborn brain and heart weights were not affected but lung and liver weights were significantly affected by the nutritional factor (P < 0.05).


Presynaptic modulation of sensory afferents in the invertebrate and vertebrate nervous system.

1. Ultrastructural examination of the central terminals of sensory afferent neurons in both invertebrates and vertebrates demonstrates that the synapses that form the substrate for presynaptic inhibition and facilitation are almost universally present. 2. Presynaptic modulation of afferent input acts in many ways which tailor the inflow of sensory information to the behaviour of the animal, effectively providing a means of turning this on and off, or of combining information of the same or different modalities to refine responsiveness or clarify ambiguity. 3. Presynaptic modulation may act in several different roles on the same afferent. 4. A comparison of the mechanisms of presynaptic inhibition in different animals demonstrates the likelihood of a variety of common mechanisms, several of which may act simultaneously on the same terminal. These include changes in the conductance of the afferent membrane to Cl-, K+ and Ca2+ ions, in addition to less well understood mechanisms that directly affect transmitter release. 5. A single transmitter can produce several effects on a terminal through the same or different receptors. 6. Ultrastructural studies of afferent terminals reveal that only a proportion of boutons on a given afferent may receive presynaptic input and that this may depend on the region of the nervous system in which these are found or on the identity of the postsynaptic neurons contacted. 7. The synaptic relationships of afferent terminals can be complex. In invertebrates different types of presynaptic neuron may interact synaptically, as may postsynaptic dendrites in vertebrates. 8. Axons presynaptic to afferent terminals in vertebrates frequently synapse also with dendrites postsynaptic to the afferents. 9. In both invertebrates and vertebrates reciprocal interactions between afferents and postsynaptic neurons are seen. 10. Ultrastructural immunocytochemistry reveals the likely dominance of GABA as an agent of presynaptic inhibition but also demonstrates the possible presence of other transmitters some of whose roles are less completely understood.


Periodicity and diversity in ant mating flights.

1. Flight hours and seasons are alike within species, but differ among species and among genera. 2. Laboratory studies indicate internal circadian control of the phase relation to the light-dark cycle, with field evidence for modification by temperature (or other environmental variables). 3. In at least two species there is abrupt loss of obvious rhythm after mating, suggesting some special function before loss, e.g. to facilitate cross-breeding within species or reproductive isolation between species.


Aldosterone increases the amiloride-sensitivity of the rat gustatory neural response to NaCl.

1. The percentage inhibition of the chorda tympani neural response to NaCl by topical application of amiloride to the tongue was significantly larger in rats pretreated with aldosterone than in control animals. 2. Adrenalectomized rats pretreated with aldosterone had significantly larger amiloride-induced inhibitions to a NaCl stimulus than did adrenalectomized control animals. 3. These data suggest that aldosterone may increase the number of active amiloride-sensitive sodium channels in the apical membrane of taste cells, as is known to occur in sodium transporting tissues of amphibians and mammals. They additionally represent a previously unnoticed hormonal influence over the gustatory system.


Monoamines and their metabolites in the amphibian (Ambystoma tigrinum) brain: quantitative changes during metamorphosis and captivity.

1. Monoamine neurotransmitters (epinephrine, norepinephrine, dopamine, serotonin and some of their metabolites (DOPEG, MHPG, DOPAC, 5-HIAA) were measured by HPLC in extracts from telencephalon (TEL) and diencephalon-midbrain (DM) before, during at the end of metamorphosis. 2. During metamorphosis MHPG increased and 5-HIAA decreased in TEL and DM while DOPEG decreased only in DM. 3. Monoamine levels were greater in the TEL and a larger increase in MHPG occurred there. 4. Captivity without metamorphosis also caused a significant depression of 5-HIAA in TEL and depression of DOPEG, MHPG and DOPAC in DM.


Neutral amino acid transport in an androgen-dependent epithelium.

1. Transport of neutral amino acids by the isolated seminal vesicle epithelium of normal and gonadectomized guinea pigs has been investigated by measurement of the uptake of 2-amino[1-14C]-isobutyric acid and 2-methylamino[1-14C]isobutyric acid. 2. The Vmax for Na-dependent and -independent transport of both amino acids was reduced by gonadectomy but the general transport characteristics appeared to be unchanged by this treatment. 3. The most likely explanation of the decreased transport is the loss of transporter molecules associated with the tissue regression that follows rapidly on gonadectomy.


The metabolic output of avian (Sturnus vulgaris, Calidris alpina) adipose tissue liver and skeletal muscle: implications for BMR/body mass relationships.

1. The oxygen uptake rate of avian adipose tissue, liver and skeletal muscle slices were measured. 2. The energy consumption of fat was less than one tenth that of liver and muscle. 3. Thus, interspecific allometric equations for the prediction of basal metabolic rate from body mass will not be accurate throughout the avian annual cycle unless changes in body composition are taken into account.


Sex-specific androgen binding in spotted hyaena (Crocuta crocuta) plasma.

1. A charcoal adsorption assay demonstrated a large variance in androgen binding ability in female spotted hyaenas. 2. A positive correlation between plasma androgen binding ability and ovarian steroid concentrations was demonstrated in adult females. 3. The strong plasma binding affinity for testosterone and dihydrotestosterone (DHT) (nM) together with the lack of cortisol and weaker oestradiol-17 beta binding suggests that a specific androgen binding substance, possibly a protein, is present in adult females of this species. 4. The lack of high affinity binding in male spotted hyaenas is unusual and deserves further investigation. 5. Some androgen binding in all, including males and immature animals suggests that albumin may bind some plasma androgens in this species.


Core and periphery concentrations of short-chain fatty acids in luminal contents of the rat colon.

1. The regional concentration of short-chain fatty acids was higher in the core than in the periphery of the round slices of contents. 2. Marked circadian and segmental fluctuations existed in their concentrations.


Adaptive changes in total pyruvate dehydrogenase activity in lipogenic tissues of rats fed high-sucrose or high-fat diets.

1. Pyruvate dehydrogenase complex (PDC) activity was measured in several tissues of rats fed for 7 or 15 days on control, or high-sucrose or high-fat diets. 2. Total activity in adipose tissue increased in the three groups 3-4 fold as compared with chow-fed animals in the first week. Total activity was 60% lower in rats fed the diet containing 22% corn oil for 2 weeks. 3. Hepatic total and PDCa activities were 50-80% higher in rats fed the sucrose diet for 7 or 15 days and decreased 30-40% in those fed on the high-fat diet for 2 weeks.


Development of hepatic microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase in the young chick.

1. The 3-hydroxy-3-methylglutaryl-CoA reductase activity increased from 1 to 4 weeks of age, but decreased from 4 to 8 weeks of age. 2. Cholesterol 7 alpha-hydroxylase activity increased from 1 to 4 weeks, decreased from 4 to 6 weeks, and increased again from 6 to 8 weeks of age. 3. Serum total and free cholesterol concentrations decreased from 1 to 6 weeks of age, but increased from 6 to 8 weeks of age.


Sonographic measurement of diaphragmatic motion after coronary artery bypass surgery.

Forty-eight patients were prospectively evaluated following coronary artery bypass grafting (CABG) in order to determine values for diaphragmatic mobility by sonography, to compare diaphragmatic motion to chest x-ray findings, to relate diaphragmatic motion to pulmonary function tests, and to determine whether use of the left internal mammary artery (LIMA), aortic cross-clamp time, or other clinical variables were predictive of diaphragmatic dysfunction. Mean left diaphragmatic motion was 2.8 +/- 1.1 cm (range, 1.0 to 5.7 cm), mean right diaphragmatic motion was 3.9 +/- 1.1 cm (range, 1.8 to 6.4 cm), and ratio of left to right motion was 0.74 +/- 0.27 (range, 0.19 to 1.4). Forty-one patients had normally positioned diaphragms on the chest x-ray film; four of these had poor mobility by ultrasonography (< 1.6 cm). Of the seven elevated left hemidiaphragms on chest x-ray films, three had an excursion of 1.6 cm or more by ultrasonography. The mean FVC for all patients was 59 +/- 13 percent of predicted. There was no relationship between diaphragmatic mobility and FVC or negative inspiratory pressure. The diaphragmatic motion in 36 patients having LIMA grafting was similar to those without (2.7 +/- 1.2 cm [n = 36] vs 2.8 +/- 0.8 cm [n = 12], respectively). Aortic cross-clamp time and respiratory symptoms also did not correlate with diaphragmatic mobility. Sonography can be used in the evaluation of diaphragmatic motion after CABG and may be more accurate in detecting a poorly mobile diaphragm than is the chest x-ray film.


The islet amyloid polypeptide gene and non-insulin-dependent diabetes mellitus in south Indians.

Islet amyloid polypeptide (IAPP), otherwise called amylin, is the monomeric component of islet amyloid. Deposition of this amyloid is a characteristic feature of non-insulin-dependent diabetes mellitus in humans and may play a role in the pathogenesis of the disease. As such, abnormalities in the structure or expression of the IAPP gene might contribute to the inheritance of this condition. The IAPP gene was studied in a well-characterised population of 62 unrelated Dravidian subjects with non-insulin-dependent diabetes mellitus and 56 normal Dravidian controls, using a restriction fragment length polymorphism generated by PvuII digestion. Genotype and allele frequencies did not differ between diabetic subjects and controls. Taken together with recent findings in Europid and other racial groups, an abnormality of the IAPP gene is highly unlikely to represent a major gene for the development of non-insulin-dependent diabetes mellitus.


Serotonin-induced decrease in hypothalamic tyrosine hydroxylase activity and corresponding increase in prolactin release are abolished at midpregnancy and by transplants of rat choriocarcinoma cells.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating PRL levels in two physiological models: 1) pregnant rats expressing (day 8) or not expressing (days 11 and 16) PRL surges, and 2) ovariectomized rats transplanted with rat choriocarcinoma cells, which secrete functional placental lactogen-I. Over a 4-min period between 0900 and 1400 h, rats were administered either vehicle or 5-HT (20 micrograms/6 microliters) through lateral ventricular cannulae. Plasma PRL levels were determined by RIA. NSD 1015 (25 mg/kg intraarterial), a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, was injected 20 min after initiation of ventricular infusion. Ten min later, the stalk-median eminence (SME) was dissected. The rate of DOPA accumulation, determined by measuring DOPA levels in the SME by HPLC, was used as an index of tyrosine hydroxylase catalytic activity, indicating tubero infundibular dopamine neuronal activity. In day-8 pregnant rats 5-HT reduced DOPA accumulation to 57% of vehicle-injected controls and increased circulating PRL levels 13-fold. In contrast, on days 11 and 16 of pregnancy 5-HT did not alter DOPA accumulation in the SME or plasma PRL levels. In nonpregnant rats ovariectomized for 24 h, 5-HT decreased DOPA accumulation in the SME to 43% of vehicle-infused controls and increased PRL levels approximately 26-fold. However, in nonpregnant rats with rat choriocarcinoma cells, 5-HT produced no changes in either DOPA accumulation in the SME or in circulating PRL levels. The inability of 5-HT to reduce tyrosine hydroxylase activity after mid-pregnancy may account for the lack of a PRL response. Placental lactogens secreted at midpregnancy, particularly placental lactogen-1, may induce this loss of 5-HT effect.


Differential effects of neuroexcitatory amino acids on corticotropin-releasing hormone-41 and vasopressin release from rat hypothalamic explants.

We have investigated the direct effects of different neuroexcitatory amino acids (EAA) on the secretion of CRH-41 and arginine vasopressin (AVP) from the rat hypothalamus maintained in vitro. CRH-41 and AVP released in the medium were assayed by RIA before and after incubation with N-methyl-D-aspartate (NMDA), N-methyl-D,L-aspartic acid, kainate (KA), and quisqualate in the concentration range 1 nM to 1 mM in either the absence or the presence of 1 mM Mg2+ in the medium. In the case of NMDA, the effect of the addition of glycine (1 and 10 microM) to the incubation medium was also studied. Finally, we investigated whether different periods of exposure (up to 100 min) of hypothalamic explants to NMDA and KA would affect CRH-41 release. While no EAA was able to induce CRH-41 release under any of the above conditions, 20-min incubations with NMDA in the dose range of 1 nM to 1 mM in the absence of added Mg2+ significantly stimulated AVP release in a dose-related fashion; the maximum effect occurred at a concentration of 1 mM [ratio of stimulated collection/basal collection: NMDA, 1.51 +/- 0.10, controls, 0.86 +/- 0.05 (mean +/- SEM); P < 0.001]. KA also showed a dose-related stimulatory effect in the dose range of 1 nM to 1 mM, with maximal AVP stimulation at 10 microM (KA, 1.91 +/- 0.28; controls, 0.90 +/- 0.03; P < 0.01). The effects of both NMDA and KA on AVP were completely reversed by the competitive antagonists D,L-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3 dione, respectively, at doses 10 times higher than those of the agonists. N-Methyl-D,L-aspartic acid stimulated AVP secretion only at a dose of 10 mM (P < 0.01), whereas quisqualate was ineffective at any concentration. The addition of 1 mM Mg2+ to the medium blocked the effect of NMDA, while attenuating AVP stimulation induced by KA. The stimulatory effect of KA on AVP was significantly reduced by D-L-2-amino-5-phosphonovaleric acid (P < 0.05), suggesting that KA may also act through NMDA receptors. Moreover, the presence of glycine in the incubation medium did not result in any effect of NMDA on CRH-41 secretion, nor did it appear to potentiate NMDA-induced AVP release.(ABSTRACT TRUNCATED AT 400 WORDS)


Inhibitory effects of ethanol on the growth hormone (GH)-releasing hormone-GH-insulin-like growth factor-I axis in the rat.

Ethanol administration decreases GH secretion in humans and experimental animals. The mechanism of these inhibitory effects was investigated by evaluating the spontaneous secretory pattern of GH in chronically cannulated unanesthetized rats, plasma insulin-like growth factor-I (IGF-I) concentrations, and hypothalamic GH-releasing hormone (GHRH) and somatostatin, and pituitary GH mRNA levels. Body weight gain was reduced in ethanol (5%)-liquid diet-fed rats (n = 6) for 6 days compared to that in both isocalorically pair-fed controls (n = 6) and ad libitum-fed animals (n = 6). Spontaneous GH secretion was markedly decreased (by 75-90%) in ethanol-fed rats compared to that in pair-fed and ad libitum-fed groups, while pulsatile pattern of GH release was preserved, with secretory bursts occurring every 180-220 min in all groups. Mean 6-h plasma GH levels in ethanol-, pair-, and ad libitum-fed animals were: 18.8 +/- 4.5, 113.3 +/- 14.9, and 179.6 +/- 30.1 ng/ml, respectively (P < 0.01, ethanol vs. each control). Plasma IGF-I concentrations were decreased in the ethanol-fed rats (338 +/- 16 ng/ml) compared to those in pair-fed (427 +/- 39 ng/ml; P < 0.05) and ad libitum-fed (769 +/- 25 ng/ml; P < 0.01) rats. Ethanol treatment decreased GHRH mRNA levels to 9% of those in ad libitum-fed (P < 0.01) and 20% of those in pair-fed (P < 0.05) animals, whereas it did not significantly alter somatostatin or GH mRNA levels. The results indicate that the effects of ethanol inhibit GH secretion primarily at the hypothalamic level, resulting in impaired GHRH gene expression. Since the GHRH-GH-IGF-I axis has an important role in growth regulation, the growth retardation seen in experimental models of alcohol abuse may be a consequence at least in part of the suppressive effects of ethanol on this axis.


Ontogeny of insulin-induced hypoglycemia stimulation of adrenocorticotropin secretion in the rat: role of catecholamines.

We previously reported that insulin-induced hypoglycemia (IIH) induces a large increase in plasma ACTH and corticosterone levels in the developing rat during the stress hyporesponsive period and that this effect is mediated, at least partially, by arginine vasopressin (AVP), but not corticotropin-releasing factor. Nevertheless, ACTH secretion in response to IIH in rats immunoneutralized against AVP was still stimulated, suggesting that other regulatory factors participate in the stimulation of ACTH secretion during IIH. It has been suggested that, in the adult rat, during profound hypoglycemia, epinephrine may act at the pituitary level through beta 2-adrenergic receptors to stimulate ACTH secretion. In this report, we studied the effect of the blockade of beta-adrenergic receptors on the pituitary-adrenal axis response to IIH. Rats (20 or 8 day old) were pretreated with saline or 2.5 mg/kg propranolol (a beta-adrenergic receptors antagonist) and subsequently injected with 3 IU/kg insulin. In 20-day-old rats, insulin injection induced a large increase of plasma ACTH concentrations that were unaffected by propranolol pretreatment. In 8-day-old rats, the IIH-induced increase of plasma ACTH levels was significantly reduced by propranolol pretreatment. Pretreatment of 8-day-old rats with 5 mg/kg CGP 20712A (a selective beta 1-adrenergic receptor antagonist) did not change the plasma ACTH response to insulin injection, while pretreatment with 2.5 mg/kg ICI 118551 (a selective beta 2-adrenergic receptor antagonist) resulted in a significant decrease of the IIH-induced stimulation of ACTH secretion. We next studied the effect of the blockade of circulating AVP and/or beta-adrenergic receptors on the pituitary response to IIH. Pretreatment of 8-day-old rats with antiserum anti-AVP or propranolol was followed by a significant reduction of IIH-induced increase of plasma ACTH concentrations. No additive effect was found after pretreatment with both antiserum anti-AVP and propranolol, suggesting that the stimulatory effect of catecholamines during IIH in 8-day-old rats is mediated through a modulation of hypothalamic AVP secretion.


Effects of cysteamine administration on somatostatin biosynthesis and levels in rat hypothalamus.

A single injection of cysteamine (CSH; 2-aminoethanethiol; 300 mg/kg, sc) into male rats produced a rapid decline in immunoreactive somatostatin (IR-SRIF) levels in the hypothalamus (to 20% of preinjection values within 12 h) which persisted for several days. The levels of both somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28) were reduced. In contrast, the levels of somatostatin-28(1-12) were unaffected. Most (80-90%) of the lost SRIF molecules (both SRIF-14 and SRIF-28) could be recovered from CSH-injected rats by subjecting hypothalamic samples to denaturing, reducing, and reoxidizing conditions. These results suggest that CSH does not deplete the hypothalamus of SRIF molecules, but, instead, alters their chemical structures, rendering them undetectable using a SRIF-14-directed RIA. CSH injection also caused a rapid and complete suppression (within 1 h) of [35S]cysteine incorporation into SRIF-14 and SRIF-28. This reduction, however, was short-lived; normal incorporation rates returned within 10 h of drug administration. CSH did not influence [35S] cysteine incorporation into acid-precipitable protein or [35S]cysteine specific activity in the hypothalamus. In addition, [35S]SRIF molecules were not recovered from hypothalami of CSH-treated rats by subjecting samples to denaturing, reducing, and then reoxidizing conditions. These findings indicate that CSH injection causes a true, but short-lived (1- to 10-h), suppression of hypothalamic SRIF-14 and SRIF-28 formation. Finally, biosynthesis studies of longer duration revealed no prolonged effects of CSH. The drug produced no changes 4, 24, or 72 h postinjection in hypothalamic levels of the prepro-SRIF mRNA. Moreover, two injections of CSH, separated by 3 days, which continuously suppressed IR-SRIF levels for almost 1 week, caused only a transient suppression of [35S]SRIF-14 and [35S]SRIF-28 synthesis after each injection. These results indicate that the SRIF biosynthetic pathway is not activated by the prolonged CSH-induced depletion of IR-SRIF stores.


Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells.

N-Glycosylation, biosynthesis and degradation of dipeptidylpeptidase IV (EC 3.4.14.5) (DPP IV) were comparatively studied in primary cultured rat hepatocytes and Morris hepatoma 7777 cells (MH 7777 cells). DPP IV had a molecular mass of 105 kDa in rat hepatocytes and of 103 kDa in MH 7777 cells as assessed by SDS/PAGE under reducing conditions. This difference in molecular mass was caused by differences in covalently attached N-glycans. DPP IV from hepatoma cells contained a higher proportion of N-glycans of the oligomannosidic or hybrid type and therefore migrated at a slightly lower molecular mass. In both cell types DPP IV was initially synthesized as a 97-kDa precursor which was completely susceptible to digestion with endo-beta-N-acetylglucosaminidase H converting the molecular mass to 84 kDa. The precursor was processed to the mature forms of DPP IV, glycosylated with N-glycans mainly of the complex type with a half-life of 20-25 min. The transit of newly synthesized DPP IV to the cell surface displayed identical or very similar kinetics in both cell types with the major portion of DPP IV appearing at the cell surface after 60 min. DPP IV molecules were very slowly degraded in hepatocytes as well as in hepatoma cells with half-lives of approximately 45 h. Inhibition of oligosaccharide processing with 1-deoxymannojirimycin led to the formation of DPP IV molecules containing N-glycans of the oligomannosidic type. This glycosylation variant was degraded with the same half-life as complex-type glycosylated DPP IV. By contrast, inhibition of N-glycosylation with tunicamycin resulted into rapid degradation of non-N-glycosylated DPP IV molecules in both cell types. Non-N-glycosylated DPP IV could not be detected at the cell surface indicating an intracellular proteolytic process soon after biosynthesis.


The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.


Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria. Structure/function relationship involving one lysine and two cysteine residues.

Treatment of the reconstituted aspartate/glutamate carrier from mitochondria with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl) led to complete inactivation of carrier function. Inhibition could be attributed to chemical modification of one single cysteine in the active site. This residue was specifically protected in the presence of aspartate or glutamate, 50% substrate protection being observed at half-saturation of the external binding site. The bifunctional reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) also modified the same cysteine and, in addition, an active-site lysine identified previously [Dierks, T., Stappen, R., Salentin, A. & Krämer, R. (1992) Biochim. Biophys. Acta 1103, 13-24]. The proximity of the cysteine [Cys(a)] and the lysine residue was confirmed by a mutual exclusion of the respective reagents when added consecutively. By using a variety of reagents a further cysteine [Cys(b)] and probably a histidine residue could be discriminated from Cys(a) and the lysine. The applied reagents were classified according to functional and structural criteria. Class A reagents, like Nbd-Cl, modified the active-site Cys(a) thereby inhibiting the antiport function. Class B reagents, like HgCl2, reacted with both Cys(a) and Cys(b) leading to a conversion of the carrier from antiport to uniport function [Dierks, T., Salentin, A., Heberger, C. & Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268-280]. DIDS at relatively high concentration (60 microM) also acted as a uniport inducer. Class C reagents finally, like pyridoxal phosphate or diethyl pyrocarbonate, modified the active-site lysine or histidine, respectively, and blocked antiport and uniport activity. By testing the accessibility of the mentioned residues to the various reagents, when applied in different order, topological relationships could be elaborated indicating the location of these amino acids with respect to the exofacial active site of the carrier protein.


Afferent lymph veiled cells prime CD4+ T cell responses in vivo.

The interdigitating cell (IDC) population of the lymph node paracortex is believed to be responsible for the induction of CD4+ T cell responses to soluble antigens. We have examined the role of afferent lymph veiled cells (ALVC), the putative precursors of IDC, in the induction of primary bovine CD4+ T cell responses in vivo. ALVC prepared from lymph draining an antigen inoculation site stimulated maximal responses in antigen-specific T cell clones as soon as 30 min after inoculation. In addition, antigen-pulsed ALVC were shown to induce primary antigen-specific T cell responses when administered in vivo. Observed influences of fixation and the addition of chloroquine or class II major histocompatibility complex-specific monoclonal antibodies on presenting function confirmed that ALVC process and present antigens using the endosomal pathway. We conclude that ALVC rapidly internalize antigens deposited in the periphery, and process them for presentation to naive T cells in the draining lymph node. Their function is, therefore, likely to be an important factor in the induction of primary T cell responses to soluble antigens in vivo.


Role of T cell subsets during the recall of immunologic memory to Leishmania major.

The contributions of different T cell subpopulations to the maintenance of immunity during secondary Leishmania major infections were analyzed in healed, resistant animals by depletion of T cell subsets in vivo. The strong delayed-type hypersensitivity mounted in immune genetically resistant mice upon challenge with viable promastigotes was mediated by both CD4+ and CD8+ T cells. Each T cell subpopulation alone contributes, although to a different extent, to the resolution of secondary lesions; both subsets, however, are required for an efficient and rapid healing of the secondary lesions and the decrease in the parasite burden in infected tissues. The results indicate that in immune, genetically resistant CBA mice, the activity of both T cell subsets is required for successful resistance to reinfection and an efficient maintenance of immunity.


Regulation of tumor necrosis factor (TNF)-alpha synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway.

The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-alpha/TNF receptor system in human T lymphocytes has been investigated. It has been found that both TNF-alpha synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover, TNF-alpha secretion was also observed by activation of T lymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2-activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75-kDa receptor form, whereas they lacked detectable levels of the 55-kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75-kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55-kDa TNF receptor. In addition, we have found that TNF-alpha enhanced the proliferative response triggered by the mixture of anti-CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF-alpha/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.


Peritoneal sanctuary for human lymphopoiesis in SCID mice injected with human peripheral blood lymphocytes from Epstein-Barr virus-negative donors.

The successful engraftment in SCID mice of intraperitoneally (i.p.) injected human lymphocytes (hu-PBL-SCID) and the failure of intravenously injected peripheral blood lymphocytes (PBL) directed the present study to investigate the early events of donor cell proliferation in the peritoneal cavity. We found focal lymphocyte engraftment together with histio-monocytic interleukin (IL)-6+ cell phenotypes which must have been transferred with the human cell inoculum, which could explain certain immune functions observed in hu-PBL-SCID chimeras. Following i.p. injection of 10(8) PBL, human cells suspended in peritoneal fluid as well as those adherent to the serosal peritoneum and abdominal organs were investigated by immunocytology and immunohistology. Human cells were found to form foci consisting predominantly of proliferating human lymphoblastoid CD3+ cells, which were mostly activated HLA-DR+ CD8+ lymphocytes. Among the lymphoid cells larger epithelioid-like cells were found to belong to the monocytic series and to stain strongly with anti-HLA-DR and anti-CD11c antibodies. Some of these cells were also positive with anti-ICAM and anti-IL-6. Congenic as well as allogeneic mouse PBL, injected i.p. into SCID mice, temporarily produced analogous foci, which shifted later on to foci similar in appearance to milky spots. However, the human cell foci appeared less compact, more closely resembling in vitro-culture soft agar colonies. It is possible that cytokines in the human histio-monocytic cells of the foci may have a feeder effect on the human lymphocytes and be a prerequisite for proliferation of human PBL in SCID mice. The observed early HLA-DR activation of human lymphocytes in the peritoneal foci could reflect triggering of immune reactions like xenogeneic graft-versus-host reactions in the peritoneal site, where the human CD11c+ HLA-DR+ histio-monocytic cells may act as antigen-presenting cells.


An activated murine B cell lymphoma line (A-20) produces a factor-like activity which is functionally related to human natural killer cell stimulatory factor.

In the present article we show that supernatants derived from lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA)-stimulated A-20 B cell lymphoma are able to induce polyclonal immunoglobulin (Ig) secretion by normal B cells in a T-cell-dependent manner. This activity could be blocked by neutralizing monoclonal antibodies against interferon-gamma, but not by monoclonal antibodies against interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10 and granulocyte macrophage colony-stimulating factor (GM-CSF) or even a polyclonal antibody against tumor necrosis factor (TNF)-alpha. Furthermore, A-20 supernatants induced the production of measurable amounts of interferon-gamma by normal murine spleen cells and activates natural killer (NK) cells. Fractionation of factor-rich supernatants on a Sephacryl S-200 column revealed that the factor activity is located in the fractions corresponding to a molecular mass of 160-150 kDa and 80-70 kDa. The biological activities found in the A-20 supernatant are very similar to the ones described for the recently cloned human IL-12/NK cell stimulatory factor. These results suggest the existence of a murine analogous factor for the human IL-12 produced by A-20 B cell lymphoma.


Characterization of the binding of [3H](+)-pentazocine to sigma recognition sites in guinea pig brain.

The selective sigma compound (+)-pentazocine was radiolabeled and its binding characteristics in guinea pig brain membranes were investigated. [3H](+)-Pentazocine bound to a single high-affinity site with a KD of 2.9 nM and a Bmax of 1998 fmol/mg protein. Saturation was achieved at a ligand concentration of 15 nM. Maximal specific binding was observed at 37 degrees C and was greater than 90% of total binding. Equilibrium was reached by 120 min and dissociation was complete by 420 min, with a t1/2 of 121 min. Li+, Ca2+ and Mg2+ inhibited binding at high concentrations, and binding was insensitive to adenyl and guanyl nucleotides. Stereoselectivity was observed for the inhibition of binding by benzomorphans, 3-(3-hydroxyphenyl)-N-propylpiperidine and butaclamol, and the (+) enantiomers and alpha diastereomers of pentazocine and cyclazocine were more potent than their corresponding (-) enantiomers and beta diastereomers. The rank order of potency for the sigma reference agents to displace [3H](+)-pentazocine binding was similar to that reported using the [3H]sigma ligands dextromethorphan, 1,3-di(2-tolyl)guanidine and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine. Haloperidol, (+)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine and rimcazole were competitive inhibitors of binding to the [3H](+)-pentazocine-defined sigma recognition site, suggesting that these different structural classes of compounds all bind to a single molecular entity.


Atypical molecular pharmacology of a new long-acting beta 2-adrenoceptor agonist, TA 2005.

The molecular pharmacology of a new putative long-acting bronchodilator TA 2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxy-phenyl)- 1-methylethyl]amino]ethyl]carbostyril hydrochloride) has been compared with that of the reference compounds isoprenaline and salbutamol in both methacholine (3 x 10(-6) M) precontracted guinea pig tracheal smooth muscle relaxation and in bovine trapezium muscle binding experiments. TA 2005 appeared very potent compared with isoprenaline and salbutamol (pD2 values of 9.29 vs. 7.65 and 7.10 respectively). For isoprenaline and salbutamol a shallow displacement curve was observed and addition of the non-hydrolysable GTP analogue guanylyl-imidodiphosphate (GppNHp) gave a rightward shift (pKd,high and pKd,low values of 7.3 and 6.1 vs. 7.0 and 5.4 respectively). For TA 2005 a steep displacement curve was found with only one binding state even without GppNHp (pKd,high value of 8.2). The long duration of action of TA 2005 might be explained by tight binding of this compound to the beta 2-adrenoceptor. The extent of tight binding for TA 2005 was extremely large. The molecular basis of the tight agonist binding phenomenon for TA 2005 seems to be of different origin than for isoprenaline. It is hypothesized that a different mechanism of activation of the beta 2-adrenoceptor may be involved for TA 2005.


Pharmacologic characterization of cloned alpha 1-adrenoceptor subtypes: selective antagonists suggest the existence of a fourth subtype.

In membranes prepared from Cos-7 or HeLa cells expressing one of three individual cloned alpha 1-adrenoceptor subtypes, competition with 2-[(beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-tetralone ([125I]HEAT) by the selective compounds [+]-niguldipine, 5-methyl-urapidil, and benoxathian reveals high affinity for the cloned alpha 1C-adrenoceptor subtype and low affinity for both the cloned alpha 1A-adrenoceptor and alpha 1B-adrenoceptor. Competition with [125I]HEAT by spiperone revealed high affinity for the cloned alpha 1C-adrenoceptor, intermediate affinity for the cloned alpha 1B-adrenoceptor, and low affinity for the cloned alpha 1A-adrenoceptor. Combining pharmacological properties previously described for alpha 1-adrenoceptor subtypes in rat membranes and here described from cloned receptors, these data suggest the existence of a fourth distinct alpha 1-adrenoceptor subtype.


Visualization of dopamine D3-like receptors in human brain with [125I]epidepride.

In sections of human brain containing the striatum (caudate, nucleus, putamen, nucleus accumbens) the competition for binding of [125I]epidepride by compounds with differing selectivity for dopamine D2 and D3 receptors was examined. Domperidone showed higher affinity for D2-like than D3-like sites whereas 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) and quinpirole demonstrated the reverse selectivity. The pattern of [125I]epidepride binding in the presence of a high concentration of domperidone was negligible in the dorsal striatum but indicated islands of dense binding to D3-like receptors in the nucleus accumbens and ventral putamen.


The glutamate- and glycine-induced reduction of [3H]MK-801 binding in the presence of Mg2+ is reversed at low pH.

At normal pH, glutamate and glycine are known to inhibit [3H]MK-801 binding in the presence of Mg2+. We found, however, that at pH 5.4 glutamate and glycine enhance [3H]MK-801 binding in rat cerebrocortical membranes. Furthermore, H+, similarly to Mg2+, inhibited basal [3H]MK-801 binding. These results indicate that H+ competes with binding sites for Mg2+ and [3H]MK-801, and may be correlated to the depression in neuronal activity observed during acidosis.


Metipranolol-induced adverse reactions: I. The rechallenge study.

Previously unreported adverse drug reactions can be difficult to detect and it may be even more difficult to establish a cause and effect relationship, particularly if the adverse reactions mimic naturally occurring disease. In a previous paper we reported 29 patients with granulomatous anterior uveitis, blepharoconjunctivitis, periorbital dermatitis, marginal keratitis and elevation in intraocular pressure (IOP), suspected to be caused by metipranolol (Glauline). With the approval of the District Ethics Committee 7 of those patients were rechallenged with metipranolol 0.3% compared to timolol maleate 0.5% in a double blind trial. The 7 metipranolol treated eyes developed an adverse reaction within 14 days. Metipranolol (Glauline) has been conclusively proven to cause granulomatous anterior uveitis, blepharoconjunctivitis and elevation in IOP, adverse effects never previously reported with any of the ophthalmic topical beta-blockers. The multidose preparations of metipranolol (Glauline) in all three strengths 0.1%, 0.3% and 0.6% and the single dose minim preparation of metipranolol 0.6% have now been withdrawn from clinical use in the United Kingdom.


Metipranolol-induced adverse reactions: II. Loss of intraocular pressure control.

This paper reviews the behaviour of intraocular pressure (IOP) in glaucomatous eyes treated with metipranolol with and without drug-induced adverse reactions (ADRs). Two hundred and forty seven patients with open angle glaucoma who were receiving the three different strengths of metipranolol (0.1%, 0.3%, and 0.6%) in our Department and the 7 patients who participated in the metipranolol rechallenge trial were included in this study. Out of the 247 patients, there were 52 eyes of 29 patients who showed 78 episodes of ADRs associated with metipranolol. Forty five of these 78 episodes (57.6%) were associated with loss of IOP control. Two of the 7 eyes treated with metipranolol in the rechallenge trial showed loss of IOP control, 1 of them without any signs of ocular inflammation. We further studied all the glaucomatous eyes controlled with metipranolol 0.6% only and 22 eyes were identified with loss of IOP control but without recognisable signs or symptoms of ADR. Five other eyes in this group later developed metipranolol-induced ADRs. The possible pathophysiological mechanisms for the loss of IOP control are discussed and it is suggested that the active drug, metipranolol, could be directly implicated.


Prolactin gene expression in human thymocytes.

Recent evidence suggests that lymphocytes produce prolactin (PRL). Here, we report the cDNA cloning and expression of PRL from normal human thymocytes. Sequence analysis showed that the thymocyte cDNA encodes a 23 kDa protein which is identical to pituitary PRL. RNA blot analysis showed that the thymocyte PRL mRNA is approximately 170 nucleotides larger than the pituitary PRL message. PRL message was also detected in several non-pituitary human cell lines including Jurkat T, HeLa, and JEG cells. Furthermore, PRL gene expression in JEG cells was inhibited by glucocorticoid treatment. Our data support the hypothesis that PRL is a T cell-derived cytokine.


Developmental expression of the growth hormone gene in the gilthead sea bream Sparus aurata.

Expression of growth hormone (GH) gene during early stages of larval development of the teleost Sparus aurata was determined by Northern blot analysis. Poly(A+) RNA was prepared from a pool of larvae collected on different days after hatching. When hybridized to Sparus aurata GH cDNA, GH specific mRNA was first seen on day 6 post-hatching. In contrast, the levels of beta-actin mRNA, which was used to normalize for RNA amounts, were already high on the day of hatching. Our results suggest that expression of the GH gene is very low immediately after hatching, and increases dramatically within 6 days.


Characterisation of adult Sertoli cell cultures from cryptorchid rats: inhibin secretion in response to follicle-stimulating hormone stimulation.

Testes from adult (90-120-day-old) rats, which had been made cryptorchid 28 days previously, were dispersed by successive treatment with trypsin, collagenase and hyaluronidase. The resulting crude cell suspension was fractionated on discontinuous Percoll density gradients to yield five distinct cell bands (1-5), at the interface between successive layers of Percoll. Crude cells and purified fractions were cultured for up to 7 days, and inhibin was subsequently measured in the media by radioimmunoassay and in vitro bioassay. Sertoli cells from density gradient bands 2 (1.03-1.04 g/ml) and 3 (1.04-1.05 g/ml) showed minimal germ cell or peritubular cell contamination, as determined by morphological and histochemical techniques. Cells from these bands secreted significantly higher levels of immunoactive inhibin/microgram DNA/48 h under both basal and either follicle-stimulating hormone (FSH)- (100 ng/ml) or dibutyryl cAMP-stimulated (100 micrograms/ml) conditions than did cells from the other bands. While there was a decline in basal secretion of inhibin with increasing duration of culture, the capacity of the purified Sertoli cells (bands 2 and 3) to respond to both FSH and dibutyryl cAMP increased over the culture period. The addition of dibutyryl cAMP (31.25-500 micrograms/ml) to the purified Sertoli cells also caused a stimulation of bioactive inhibin. Immunoactive inhibin production by purified Sertoli cells was unaffected by the addition of either rat LH (8 ng/ml) or testosterone (10(-6) M). The data describe a method for the isolation of adult Sertoli cells from cryptorchid testes, and demonstrate their responsiveness to both FSH and dibutyryl cAMP in vitro using the measurement of immunoactive inhibin as a marker of Sertoli cell function.


Stabilization of follicle-stimulating hormone-receptor complexes may involve calcium-dependent transglutaminase activation.

Calcium-dependent transglutaminase (TGase) activity, determined by incorporation of [1,4-14C]diaminobutane dihydrochloride (putrescine) into casein, was demonstrated in a light membrane fraction prepared from bovine calf testicular homogenates. Purification of these membranes by sucrose density gradient centrifugation produced a follicle-stimulating hormone (FSH) receptor-enriched fraction containing TGase activity which cosolubilized with the FSH receptor and could be incorporated with detergent-solubilized receptor into liposomes. In the present study, we show that calcium increases specific binding of FSH to receptor in a concentration-related manner, and is associated with an increase (13.2-fold at 20 mM) in the affinity (Ka) of the receptor with no significant (P greater than 0.05) change in receptor concentration. Treatment of the light membrane fraction with monodansylcadaverine (MDC, 1 mM), a specific inhibitor of TGase, did not affect specific binding of FSH, but resulted in only a 3.9-fold increase in Ka at 20 mM calcium with no change in receptor concentration. Specific binding of FSH to receptor at 4 degrees C was also enhanced by calcium. Scatchard analysis of competitive binding inhibition data showed a Ka at 20 mM calcium similar to that observed with MDC. Dissociation of [125I]hFSH-receptor complexes formed at 30 degrees C in the presence of calcium was significantly less than dissociation of complexes formed at 30 degrees C in the absence of calcium. When [125I]hFSH-receptor complexes were formed at 30 degrees C in the presence of calcium and dissociated in calcium-deficient buffer, dissociation increased 3-fold. Similar results were obtained in the presence of MDC.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of gene transcription and proliferation by parathyroid hormone is blocked in mutant osteoblastic cells resistant to cyclic AMP.

We employed a cyclic AMP-resistant subclone of UMR 106-01 osteoblastic osteosarcoma cells (UMR 4-7) with a regulated, dominant-negative mutation of cyclic AMP-dependent protein kinase (PK-A), to examine the mechanism(s) whereby parathyroid hormone (PTH) regulates growth of these cells. Expression of a transiently transfected CAT reporter gene controlled by the cAMP response element of the rat somatostatin gene ('SST-CAT') was used to monitor PK-A activation in intact cells. Agonist-stimulated SST-CAT expression was specific for agents known to activate adenylate cyclase, required an intact cAMP response element and was specifically blocked following induction of the mutant cAMP-resistant phenotype in UMR 4-7 cells. Inhibition of the proliferation of UMR 106-01 cells by PTH, which is mimicked by forskolin and 8-bromo-cAMP, was blocked completely in mutant cyclic AMP-resistant UMR 4-7 cells. We conclude that control of proliferation in UMR 106-01 cells by PTH involves the cAMP messenger system and requires activation of PK-A.


Lack of feedback inhibition of insulin secretion in denervated human pancreas.

In this study, pancreas transplantation is used as a clinical model of pancreas denervation in humans. To assess the role of innervation on the feedback autoinhibition of insulin secretion, we studied four groups of subjects--group 1: 16 patients with combined pancreas and kidney transplantation (plasma glucose = 5.1 mM, HbA1c = 6.4%, creatinine = 86 mM); group 2: 8 patients with chronic uveitis on the same immunosuppressive therapy as transplanted patients (12 mg/day prednisone, 5 mg.kg-1.day-1 CsA); group 3: 4 uremic, nondiabetic patients in chronic hemodialysis; group 4: 7 normal, nondiabetic control subjects. The following means were used to study the groups: 1) a two-step hyperinsulinemic euglycemic clamp (insulin infusion rate = 1 mU and 5 mU.kg-1.min-1); and 2) a 0.3 mU.kg-1.min-1 hypoglycemic clamp (steady-state plasma glucose = 3.1 mM). Basal plasma-free IRI (84 +/- 6, 42 +/- 12, 72 +/- 12, and 30 +/- 6 pM in groups 1, 2, 3, and 4, respectively), basal C-peptide (0.79 +/- 0.05, 0.66 +/- 0.05, 3.04 +/- 0.20, and 0.59 +/- 0.06 nM in groups 1, 2, 3, and 4, respectively), and glucagon (105 +/- 13, 69 +/- 4, 171 +/- 10, and 71 +/- 5 pg/ml in groups 1, 2, 3, and 4, respectively) were increased in groups 1 and 3 with respect to groups 2 and 4 (P < 0.01). During euglycemic hyperinsulinemia, plasma C-peptide decreased by 45, 20, and 44% in groups 2, 3, and 4, respectively, but showed no significant change from the basal in patients with transplanted pancreases.(ABSTRACT TRUNCATED AT 250 WORDS)


Linkage analysis of GLUT1 (HepG2) and GLUT2 (liver/islet) genes in familial NIDDM.

Familial NIDDM probably results from combined inherited defects of insulin secretion and action. Members of the facilitative glucose transporter family are strong candidates for both defects, and RFLPs for both GLUT1 (erythrocyte) and GLUT2 (liver/islet) genes have been associated with NIDDM in some populations. To test the hypothesis that GLUT1 and GLUT2 mutations contribute to the inherited predisposition to NIDDM, we examined linkage of these loci with NIDDM in 18 large Utah white pedigrees (two and three generation) ascertained for > or = 2 NIDDM siblings. We used two RFLPs detected with Xba1 and Stu1 for the GLUT1 transporter. For the GLUT2 (liver/beta-cell) transporter gene, we used an RFLP detected with EcoR1 and a highly polymorphic (6-allele) dinucleotide (microsatellite) repeat. Analysis was performed with the MLINK program of the LINKAGE package. We tested four models for each locus: dominant and recessive, with IGT alternately considered as unknown affection status, or affected if IGT was diagnosed < or = 45 yr of age and unknown if > 45 yr. Disease gene frequencies were chosen to give approximate disease prevalence in American whites (q = 0.03, dominant; q = 0.25, recessive). Linkage of GLUT1 and NIDDM was strongly and significantly rejected under all models, with total (pooled) LOD scores of -5.7 to -8.9, indicating > 500,000:1 odds against linkage. Pooled LOD scores were significantly negative (< -2.0, or 100:1 odds against linkage) to a recombination fraction of > 5%. No heterogeneity was apparent. Analysis of GLUT2 gave similar results, with LOD scores of < -4.0 under each model, indicating at least 10,000:1 odds against linkage.(ABSTRACT TRUNCATED AT 250 WORDS)


Homeoboxes in flatworms.

A search for homeobox-containing genes was done in the genome of a primitive metazoan, the parasitic tapeworm Echinococcus granulosus. Five different homeoboxes were isolated, none of them belonging to the classical Antennapedia-type. Three of the homeodomains are similar to those from the Drosophila melanogaster NK-type genes. The fourth homeodomain shares extensive identity with that of the recently reported homeobox-containing gene goosecoid from Xenopus laevis. The third helix (the recognition helix) of the fifth isolated homeodomain is identical to that of the Xlim-1 gene of X. laevis and the lin11 gene of Caenorharbditis elegans. Using PCR, some Antennapedia-type homeoboxes were cloned from the genome of two other Platyhelminthes, Dugesia tigrina (planaria) and Fasciola hepatica. These data suggest that, contrary to what is found for the majority of the more complex metazoans, Platyhelminthes contain few homeobox genes belonging to the Antennapedia-type.


The 28S ribosomal RNA-encoding gene of Hymenoptera: inserted sequences in the retrotransposon-rich regions.

The genomes of two parasitoid wasps, Diadromus pulchellus and Eupelmus vuilleti, and the honey bee, Apis mellifera, contain few interspersed repeated sequences corresponding to transposons (Tn). This suggests that the genomic organisation of Hymenoptera could be due to the elimination of deleterious Tn in haploid males. We have used restriction-fragment length polymorphism analysis to show that nondeleterious Tn are present in the DNA (rDNA) encoding ribosomal RNA of twelve species of Hymenoptera. Sequence analysis of the 28S rDNA type-I and type-II insertion-rich regions of 80 species showed that this region is very highly conserved (95.8%). A consensus sequence and restriction map of the rDNA region were established. These sequence data were used to develop a strategy for detecting inserted elements in the rDNA fragments containing type-I or type-II insertion sites, and this strategy was used to screen twelve hymenopteran species and four non-Hymenoptera control species. The rDNA fragments from the Hymenoptera and control species contained inserted sequences in the area where type-I and type-II elements are inserted in the 28S rDNA retrotransposon-rich region of Diptera and Lepidoptera. The hymenopteran genomes therefore appear to contain repeated elements, the mobility and nature of which remain to be determined.


Sequence of a novel chicken genomic DNA fragment that hybridizes to the murine Hox-3.1 homeobox.

A chicken genomic library was screened for novel homeobox-like elements. We describe a chicken genomic DNA fragment which hybridizes to the murine Hox-3.1 homeobox. The fragment is a single-copy sequence which seems to be transcribed without spacial or temporal restrictions. The sequence presented here is not representative of anything yet in the databases; however, it contains a very probable open reading frame which would encode a peptide slightly homologous to the Hox-3.1 protein.


Different DNA changes in primary and recurrent hepatocellular carcinoma.

DNA restriction fragment length polymorphism analysis was carried out on a primary and recurrent hepatocellular carcinoma in a hepatitis B virus negative patient. For the primary tumour, allele losses were found on the short arm of chromosome 17 (probe: p144-D6, 17p13) and the long arm of chromosome 5 with the probe Lambda MS8 (5q35-qter); other probes showed either no allele loss or a non-informative pattern. The recurrent cancer also showed allele loss with p144-D6, but not with Lambda MS8. In addition, the recurrent tumour had allele losses with Lambda MS43 (12q24.3-qter), pYNZ22 (17p13), and DNA rearrangement revealed by the probe Lambda MS32 (1q42-43), a pattern not seen in the primary lesion. These results indicate that the second hepatocellular carcinoma was of independent clonality and probably represents a de novo neoplasm rather than a recurrence.


[Modification of classical drug receptor mechanisms].

It is generally accepted that full and partial agonists interact with the same receptors according to the classical receptor mechanisms. We have modified the drug receptor mechanisms of M3-, alpha 1- and beta-receptors. Among the muscarinic receptors, there are two subtypes of M3-cholinoceptors, propylbenzilylcholine mustard (PrBCM)-sensitive receptors and (PrBCM)-resistant ones. Full agonists contract the guinea pig ileum through both types of cholinoceptors, while the partial agonists produce contractions through only the PrBCM-sensitive receptors. Two subtypes of alpha 1-adrenoceptors, alpha 1A and alpha 1B, were demonstrated in some arteries. Full agonists contracted the rabbit aorta through both the alpha 1A- and alpha 1B-adrenoceptors, while the partial agonists mediated contraction through only the alpha 1A-adrenoceptors. beta-Chloroethylamines (PrBCM and chloroethylclonidine) can discriminate the subtype of M3- or alpha 1-receptors in the presence of GTP. beta-Adrenoceptors have two different types of binding sites, high and low affinity sites. The competitive antagonistic effect of the partial agonist is due to their ability to compete with the full agonists for the high affinity site, while the partial agonists interact with the low affinity site to induce the beta-adrenergic effect. A regional difference in alpha 1-adrenoceptor mechanisms was discussed. The potency of norepinephrine in veins is related to alpha 1-adrenoceptor densities. In contrast, the potency of norepinephrine is linearly related to the agonist dissociation constant. This discrepancy suggests a qualitative difference between alpha 1-adrenoceptor mechanisms in the veins and arteries.


Hepatitis C virus and porphyria cutanea tarda: evidence of a strong association.

Porphyria cutanea tarda in human beings is believed to be due to reduced hepatic uroporphyrinogen decarboxylase activity. However, extrinsic factors such as alcohol abuse and drug intake are required for clinical manifestation of the disease. In addition to typical cutaneous lesions, patients with porphyria cutanea tarda usually have chronic liver disease and moderate iron overload. Of 74 Italian patients with porphyria cutanea tarda, hepatitis C virus antibodies were detected in 76% by enzyme-linked immunoassay and in 82% by recombinant immunoblot assay. Viral genome, studied with nested polymerase chain reaction, was found in the sera of 49 subjects--47 positive and 2 indeterminate on recombinant immunoblot assay. Five percent of the patients were HBsAg-positive, and about 40% had had past hepatitis B contacts. Alcohol abuse was present in 38%. Liver biopsies performed in 42 patients showed chronic persistent hepatitis in 7 patients, chronic active hepatitis in 22 patients, fibrosis in three patients and cirrhosis in 10 patients. Hepatitis C virus antibody was detected in 100% of patients with chronic active hepatitis and in about 80% of all other groups. Alcohol abuse was more frequent in patients with cirrhosis (80%) than in the other groups. In Italian patients with porphyria cutanea tarda, the prevalence of hepatitis C virus infection was very high, comparable to that in non-A, non-B hepatitis and high-risk patient groups. Hepatitis C virus is probably the main pathogenetic factor of the liver disease of patients with porphyria cutanea tarda.


Susceptibility to primary biliary cirrhosis is associated with the HLA-DR8-DQB1*0402 haplotype.

In studies to date seeking associations between human leukocyte antigens (HLA) and primary biliary cirrhosis, no class I association but several different class II associations have been described. The aims of this study were to reassess the DR associations in primary biliary cirrhosis and to examine for the first time the role of DQB. DRB genotypes were determined on standard Taq1 restriction-fragment-length polymorphism analysis in 159 white northern European patients with the disease and 162 racially matched local controls. Polymerase chain reaction gene amplification and sequence-specific oligonucleotide analysis were used to determine DQB genotypes in 89 patients and 181 controls. An increased frequency of human leukocyte antigen DR8 was observed in the patient group (11% vs 4%; relative risk = 3.3; p < 0.01). Although we saw an increased frequency of the DQB1*0402 allele (11% vs. 3%; relative risk = 3.5; p < 0.025), this was not significant after correction for multiple testing. The strongest association was with the two-locus haplotype DR8-DQB1*0402 (11% vs. 2.2%; relative risk 5.5; p < 0.001). The DRB data reported here confirm the findings of previous studies, although the described association with DR8 is considerably weaker. The weak genetic contribution of human leukocyte antigen in the susceptibility to primary biliary cirrhosis is in contrast to its role in other autoimmune liver diseases.


Cytoprotective effect of somatostatin in a rat model of hepatic ischemic reperfusion.

To evaluate the possible cytoprotective effect of somatostatin in hepatic ischemic reperfusion injury we used 75 adult male Wistar rats randomly separated into four groups. The rats in group 1 underwent sham operations, and those in group 2 underwent resection of the median and left lateral lobes. The rats in group 3 underwent a 90-min period of ischemia of the right lateral lobe, which we induced by temporarily occluding the right portal vein and the hepatic artery. On restoration of flow to the right lateral lobe, the median and left lateral lobes (about 80% of total liver mass) were excised (and later assayed for thymidine kinase basal activity). The rats in group 4 were given the same treatment as group 3 rats except that a saline solution of somatostatin was infused at a rate of 2 micrograms/min starting at laparotomy and lasting 24 hr. The rats in groups 1, 2 and 3 were infused with saline. Rats in groups 2, 3 and 4 were randomly assigned to two subgroups; one of these subgroups was observed until spontaneous death, and rats in the other group were killed 24 hr after the procedure for obtaining peripheral blood and liver samples. Somatostatin infusion improved the animals' survival rates from 0% (group 3) to 60% (group 4) (p < 0.05) and decreased bilirubin levels (0.78 +/- 0.17 mg/dl, n = 15 [group 4] vs. 1.69 +/- 0.04 mg/dl, n = 15 [group 3]; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Valid estimation of IL2 secretion by PHA-stimulated T-cell clones absolutely requires the use of anti-CD25 monoclonal antibody to prevent IL2 consumption.

A major problem encountered for quantification of IL2 production by stimulated T cells is its simultaneous consumption by these activated cells. In the present study, 40 T-cell clones (TCC) derived from normal peripheral blood, hyperplastic lymph nodes (LN) or lymph nodes involved by malignant lymphomas, were studied for their ability to produce IL2. When supernatants were generated in the presence of 20% fetal calf serum (FCS), no IL2 could be detected for 22 of the 40 TCC, whereas very low levels were found for the 18 other TCC (mean value 31 pg/ml; range from 10 pg/ml to 114 pg/ml); in contrast, when conditioned media were produced with reduced amounts of FCS (final concentration, 1%) as well as in the presence of an anti-CD25 monoclonal antibody (final concentration, 50 micrograms/ml), all TCC were found to release IL2, and very high quantities of this lymphokine were measured (mean value: 11,387 pg/ml; range, from 250 pg/ml to 37,000 pg/ml). Consequently, inhibition of IL2 consumption by PHA-stimulated TCC seems to be an absolute requirement for estimating the true capacity of T cells to produce this lymphokine.


Bronchodilation and beta 2-selectivity of broxaterol in asthmatics: a crossover, single-blind, terbutaline- and placebo-controlled study.

To investigate the beta 2-receptor selectivity of a new beta 2-adrenoceptor agonist, broxaterol, we compared the respiratory and cardiovascular effects of this compound with those of terbutaline and placebo. Twelve asthmatic patients were evaluated in a randomized, single blind, crossover study. A single dose of each study treatment (broxaterol 400 micrograms and terbutaline 500 micrograms was administered with metered dose inhalers. Measurements of lung function (vital capacity, forced expiratory volume in one second, airway resistance and specific airway conductance), heart rate and systolic/diastolic blood pressure were performed before and at 15, 30, 60, 120, 240 and 360 min after each study treatment. No significant difference was observed between broxaterol and terbutaline in VC, FEV1 and Raw changes, although a greater and significant increase in sGaw was found only with broxaterol. Significant increases in heart rate and systolic blood pressure were observed only with terbutaline. The results of this study suggest that broxaterol can promote a greater bronchodilator effect with less cardiovascular side effects than terbutaline, probably through a greater beta 2-receptor selectivity.


Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.


Skeletal muscle and liver glutathione homeostasis in response to training, exercise, and immobilization.

Female beagle dogs were treadmill trained 40 km/day at 5.5-6.8 km/h, 15% upgrade, 5 days/wk for 55 wk. With training, hepatic and red gastrocnemius (RG) total glutathione increased, glutathione peroxidase (GPX) and glutathione reductase (GRD) increased in all the leg muscles studied, and hepatic glutathione S-transferase (GST) activity increased. Joint immobilization (11 wk) did not affect GPX, GRD, and GST of RG, but total glutathione decreased. Male Han Wistar rats were treadmill trained 2 h/day at 2.1 km/h, 5 days/wk for 8 wk. With training, hepatic total glutathione and leg muscle GPX increased but GRD of RG decreased, perhaps because of an increased muscle flavo-protein breakdown during exhaustive training. gamma-Glutamyl transpeptidase was higher in the trained leg muscles. Exhaustive exercise decreased muscle gamma-glutamyl transpeptidase of only control leg muscle, depleted muscle (lesser extent in trained rats) and liver total glutathione of both groups, decreased GRD only in untrained RG, and increased hepatic GST. Endurance training elevated the antioxidant and detoxicant status of muscle and liver, respectively.


Role of leukocyte CD11/CD18 complex in endotoxic and septic shock in rabbits.

Two models of sepsis were investigated using rabbits. In the first model, rabbits given lipopolysaccharide (LPS) were treated with saline (group II) or CD18 monoclonal antibody (MAb) 60.3 (group III). Group I animals received no LPS. Cardiac output was maintained by infusion of lactated Ringer solution with group II (95 +/- 68 ml/kg) requiring significantly more than group I (0 +/- 0 ml/kg) or group III (39 +/- 27 ml/kg). Lung permeability indexes in groups II (median 0.002, range 0.023) and III (median 0.0035, range 0.053) were not different but were significantly greater than group I (median 0.0007, range 0.001). In the second model, peritonitis was produced by devascularizing the appendix, leaving it in situ for 19 h, and then performing an appendectomy. Saline or MAb 60.3 treatment was at appendectomy and every 12 h for 3 days. Survival was significantly greater in the MAb 60.3-treated group at day 10 (90 vs. 40%). Lung permeability was increased at day 2 and was not different between groups. Day 1 fluid requirements were greater in the saline-treated group. These data are consistent with MAb 60.3 protection of systemic but not pulmonary circulation in two models of sepsis.


Cardiac secretion of atrial natriuretic factor with exercise in chronic congestive heart failure patients.

We have previously reported a fivefold increase of plasma atrial natriuretic factor (ANF) in patients with congestive heart failure (CHF) compared with normal subjects. However, given the marked increase of ANF under basal conditions, the extent to which ANF secretion can further increase under physiological stress is not been clarified in CHF. We therefore evaluated ANF secretion during ergometric exercise in 11 patients with CHF, with peripheral venous ANF samples obtained at rest and peak exercise. In seven patients, simultaneous peripheral venous and right ventricular ANF samples were obtained to estimate myocardial ANF secretion. Hemodynamic characteristics of exercise included a significant increase of heart rate, mean arterial pressure, and cardiac output (all P < 0.01); reduction of systemic vascular resistance (P < 0.001); and increase of right atrial and pulmonary wedge pressures (P < 0.001). ANF was abnormally elevated at baseline (108 +/- 58 fmol/ml) yet increased further to 183 +/- 86 fmol/ml with exercise (P < 0.003). A step-up of right ventricular ANF, particularly during exercise, was consistent with active myocardial secretion, despite elevated baseline ANF levels.


Differential expression of a Clostridium acetobutylicum antisense RNA: implications for regulation of glutamine synthetase.

The Clostridium acetobutylicum glutamine synthetase (GS) DNA region is characterized by a downstream promoter, P3, oriented toward the glnA gene, which controls the transcription of an RNA complementary to the start of the glnA mRNA. Expression of the predicted 43-base antisense RNA was demonstrated in C. acetobutylicum and Escherichia coli cells containing the cloned glnA DNA. Antisense RNA transcription from P3 was not regulated by nitrogen in E. coli cells, but the expression of antisense RNA was associated with decreased levels of GS activity. In C. acetobutylicum, GS activity and the transcription of glnA mRNA and antisense RNA were regulated by nitrogen. GS activity and glnA mRNA were repressed in cells grown in nitrogen-rich medium. Repression ratios for GS activity varied from 1.6 to 9.0, depending on the sampling time. The relative number of glnA transcripts was approximately 25% lower in cells grown for 72 h in nitrogen-rich medium than in cells grown in nitrogen-limiting medium. This finding contrasted with the expression of antisense RNA, which was repressed in nitrogen-limiting medium but induced in nitrogen-rich medium. The relative number of antisense RNA transcripts was increased approximately sixfold in cells grown in nitrogen-rich medium. There was a 1.6-fold excess of antisense RNA over glnA mRNA under conditions that repressed GS activity. Under conditions that induced GS activity, glnA mRNA transcripts exceeded antisense RNA transcripts by fivefold.