SCID-hu mice as a model to study tolerance after fetal stem cell transplantation. SCID-hu mice were constructed with human fetal liver and human fetal thymus, obtained from the same or from different donors. Hematopoietic cells originating from the fetal liver migrate to the fetal thymus and give rise to medullary and cortico-medullary macrophages and dendritic cells. Thymic epithelial cells remain of thymic donor origin. The fetal liver donor derived stem cells differentiate in this environment into double positive and finally single positive CD4 or CD8 expressing T cells. The TCR V beta repertoire generated at the double positive stage is identical to that generated in the thymus of the donor. Thymic selection induces changes in V beta usage comparable to those previously reported for normal human thymus. Single-positive, functionally mature, and mainly TCR alpha beta+ T cells reach the peripheral blood compartment of the SCID-hu mice. These T cells are able of specific proliferative and cytotoxic alloresponses. No autoreactivity is observed. Single positive T cells which differentiated in the thymus of SCID-hu mice transplanted with liver and thymus of two different donors are tolerant to the HLA antigens of both donors. By limiting dilution analysis, it could be demonstrated that tolerance to the fetal liver donor is due to clonal deletion whereas tolerance to the fetal thymus donor is not. These data show that human T cell development is progressing normally in these mice and gives rise to a mature, functional and polyclonal T cell repertoire which is comparable to that observed in normal individuals.(ABSTRACT TRUNCATED AT 250 WORDS)


Human-ovine xenogenic transplantation of stem cells in utero. The preimmune status of the early gestational age fetus provides a permissive environment that bypasses the immunological barrier and permits the engraftment and expression of hemopoietic stem cells (HSC). We used in utero approach to establish long term (greater than 2 years) engraftment and expression of human fetal liver HSC in sheep. Engraftment occurred in 40% (13 of 33) of the recipients. Of 5 live born sheep, all were chimeric. Engraftment was multilineage, involving lymphoid, myeloid and erythroid donor (human) cells. Interestingly, these progenitors have continued to exhibit responsiveness to human specific growth factors both in vitro and in vivo. Therefore, the integration of human HSC into the hemopoietic framework of the host appeared to be incomplete, with donor progenitors retaining certain phenotypic characteristics that may be exploited to preferentially manipulate the donor (human) cell population in these animals. Donor HSC primarily seeded the host bone marrow. Since the donor cells were of liver origin and the host liver at the time of transplantation was the major hemopoietic organ, this near exclusive seeding to the marrow indicates the greater affinity of marrow for the homing HSC. Nonetheless, no cells of donor origin appeared in the host circulation until the perinatal period, suggesting that donor HSC expand with the developing marrow spaces, but do not undergo terminal differentiation. The absence of a significant immunological barrier and the availability of expanding marrow homing sites render the fetus an excellent host (and donor) for HSC transplantation.


The child conceived to give life. The point of view of the hematologist. The different modalities of stem cell transplantation and the indication of each of them are briefly discussed. Transplantation can be successfully applied in hematologic diseases or in congenital disorders.


The child conceived to give life.

The child conceived to give life. Bone Marrow Transplantation (BMT) demands a living compatible donor. Some parents will conceive a child "for BMT". These pregnancies have three main characteristics. The traumatic situation of the impending death of a child, the inevitable uncertainty of their issue, of BMT results, the intrusion of biology into the family dynamics.


In utero transplantation of stem cells in humans: technical aspects and clinical experience during pregnancy. Four fetal patients have received fetal liver cell transplants in utero, at the fertilization ages of 12-28 weeks. Depending on the age, intraperitoneal injection or intravenous infusion into the umbilical vein was used, under ultrasonic guidance. In three of the four cases, engraftment has been obtained and has resulted in cure or significant improvement of the inherited disease.


Integrated multimodal therapy of children with attention-deficit hyperactivity disorder. Attention-deficit hyperactivity disorder (ADHD) affects about 3-9% of children in the United States. It is a complex, multidetermined ailment that causes profound difficulty not only for the children but also for their families and those in the broader social environment (e.g., schools). Although medication helps an ADHD child's day-to-day functioning, it is less effective in producing long-lasting improvement when used alone. The author therefore discusses the rationale and techniques for multimodal treatment, including the use of education, cognitive-behavioral therapy, behavior modification, and structural and dynamic therapy, as well as medication. He emphasizes that the use of multiple modalities produces therapeutic benefit greater than the sum of each modality's contribution. The author's basic premise is that psychodynamic understanding is crucial in determining how to combine various therapies and make them mutually facilitative.


Formoterol and salbutamol inhibit bradykinin- and histamine-induced airway microvascular leakage in guinea-pig. 1. The effects of the beta 2-adrenoceptor agonists, salbutamol and formoterol, on the increase of microvascular permeability induced by histamine or bradykinin in guinea-pig airways have been studied in vivo. Extravasation of intravenously injected Evans blue dye was used as an index of permeability. The effects of salbutamol and formoterol on the increase in pulmonary airway resistance induced by histamine or bradykinin have also been studied. 2. The increase in pulmonary airway resistance induced by histamine or bradykinin was totally inhibited by salbutamol and formoterol. The ED50 of the two mediators were 0.59 +/- 0.21 (n = 5) and 0.20 +/- 0.14 (n = 5) micrograms kg-1 respectively for salbutamol, and 0.13 +/- 0.12 (n = 6) and 0.02 +/- 0.01 (n = 6) micrograms kg-1 respectively for formoterol. 3. Salbutamol (10 and 30 micrograms kg-1) and formoterol (1 and 10 micrograms kg-1) inhibited the increase of microvascular permeability induced by histamine (30 micrograms kg-1) in the guinea-pig airways. The inhibitory effect was predominant in the trachea and the main bronchi, with a maximum inhibition of 20 to 50%. The two drugs had little or no inhibitory effect on the other structures studied, viz. nasal mucosa, larynx, proximal and distal intrapulmonary airways. 4. Salbutamol and formoterol (1 and 10 micrograms kg-1) abolished the increase in microvascular permeability induced by bradykinin (0.3 micrograms kg-1). This inhibitory effect of two beta-adrenoceptor stimulants was predominant in the trachea and the nasal mucosa where it was observed with 1 microgram kg-1 of the beta-adrenoceptor agonists.In the main bronchi, and in the proximal and distal intrapulmonary airways, the effects of bradykinin were abolished by 10 pg kg- of formoterol and salbutamol.5. The effects of bradykinin, but not those of histamine, were significantly reduced (nasal mucosa, main bronchi and distal intrapulmonary airways) or abolished (trachea, proximal intrapulmonary airways) by morphine 10mgkg-1, i.v. These results suggest that an indirect effect, through non-adrenergic noncholinergic (NANC) nerves is involved in the action of bradykinin on the microvascular permeability.6. In conclusion, intravenously injected beta-adrenoceptor stimulants can inhibit, partially or totally, the increase of airways microvascular permeability induced by intravenous histamine or bradykinin. However, these effects require doses that are higher than those that inhibit the increase in pulmonary airway resistance induced by these mediators. As suggested by the results obtained with morphine, the higher efficacy of beta2-adrenoceptor agonists versus bradykinin may occur through activation of presynaptic receptors of the non-adrenergic non-cholinergic (NANC) nerves preventing release of inflammatory neuropeptides such as substance P and neurokinin A.


Inhibition by salmeterol of increased vascular permeability and granulocyte accumulation in guinea-pig lung and skin. 1. The long-acting beta 2-adrenoceptor agonist, salmeterol has been evaluated for its anti-inflammatory effects in the guinea-pig lung and skin. 2. Salmeterol, administered in bronchodilator doses to conscious guinea-pigs by both oral (0.01-1.0 mg kg-1) and inhaled (nebulizer concentration, 0.001-1.0 mg ml-1) routes, inhibited histamine-induced plasma protein extravasation (PPE) into the airway lumen. 3. Inhibition of PPE by salmeterol was long-lasting (greater than 6 h) and was inhibited by prior administration of propranolol (1 mg kg-1, s.c.), indicating an effect mediated by beta-adrenoceptors. 4. Inhaled salbutamol (nebulizer concentration, 0.001-1.0 mg ml-1) also inhibited PPE in guinea-pig lung but, in contrast to salmeterol, this effect was short-lived with substantial loss of activity 2 h after administration. 5. Inhaled salmeterol (0.1 mg ml-1) and salbutamol (1.0 mg ml-1) inhibited the accumulation of neutrophils in guinea-pig lung in response to lipopolysaccharide (100 micrograms ml-1). Salmeterol, but not salbutamol, inhibited the infiltration of eosinophils into the airway lumen in response to platelet activating factor (100 micrograms ml-1). These effects of salmeterol were blocked by prior administration of propranolol (5 mg kg-1, s.c.), indicating that they were also beta-adrenoceptor-mediated. 6. Oral salmeterol (10 mg kg-1, p.o.), but not salbutamol (10 and 100 mg kg-1, p.o.), inhibited zymosan-induced granulocyte accumulation and PPE in guinea-pig skin. Lower doses of salmeterol (0.1 and 1 mg kg-1) inhibited PPE, but not granulocyte accumulation.The effects of salmeterol were blocked by prior administration of propranolol (1mgkg-', s.c.). Both salmeterol and salbutamol inhibited histamine-induced PPE in guinea-pig skin.7. Intradermal salmeterol (10-'mol per site), but not salbutamol, was also effective in inhibiting zymosan-induced granulocyte accumulation and PPE in guinea-pig skin.8. It is concluded that salmeterol, at bronchodilator doses in the guinea-pig, inhibits granulocyte accumulation and PPE, possibly by an action on the vasculature. As this profile of activity is not shared by the shorter-acting compound, salbutamol, it would seem that anti-inflammatory activity is associated with beta-adrenoceptor agonism of long duration. The implications of these findings for the use of salmeterol in the treatment of bronchial asthma are discussed.


Evidence for an atypical, or beta 3-adrenoceptor in ferret tracheal epithelium. 1. A preparation of the ferret trachea in vitro was used to examine the effects of three selective beta-adrenoceptor agonists on lysozyme secretion from submucosal gland serous cells and epithelial albumin transport into tracheal mucus following sustained, submaximal stimulation of mucus production with methacholine (20 microM). 2. Prenalterol, salbutamol and BRL 37344 all enhanced methacholine-induced albumin output. BRL 37344 was 10,000 times more potent than salbutamol, and salbutamol was slightly more potent than prenalterol. The concentrations required to increase albumin output by 100% (EC100%) were 1.4 nM, 0.7 mM and approximately 1.0 mM for BRL 37344, salbutamol and prenalterol, respectively. All three agonists inhibited methacholine-induced lysozyme output, with salbutamol being 60 times more potent than BRL 37344, and BRL 37344 being approximately 100 times more potent than prenalterol. 3. The selective beta 2-adrenoceptor antagonist, ICI 118551, inhibited the increase in albumin output produced by BRL 37344, but much more potent at inhibiting the response to salbutamol; the pA2 for ICI 118551 was 5.55 and 7.18 (P less than 0.001) when the agonist was BRL 37344 and salbutamol, respectively. ICI 118551 also attenuated the inhibition of lysozyme output produced by the two agonists, but was 10-30 times more potent at inhibiting this response than the albumin response to BRL 37344 and salbutamol. 4. The greater potency (4-5 orders of magnitude) of BRL 37344, compared to the beta 1- (prenalterol) and beta 2- (salbutamol) adrenoceptor selective agonists, in stimulating methacholine-induced albumin transport suggests that tracheal epithelium possess an atypical, or beta 3-adrenoceptor similar to that previously reported for adipocytes and gastrointestinal smooth muscle. The weak antagonism of the response to BRL 37344 by ICI 118551 would also be consistent with an atypical adrenoceptor mediating the albumin transport response. Inhibition of methacholine-induced serous cell lysozyme output would appear to be mediated predominantly by beta2-adrenoceptors.5. In view of the possible beneficial protective effects of albumin in airway surface liquid, selective beta3-agonists like BRL 37344 might have potential value in the prevention and/or treatment of inflammatory airway disease.


Alpha-adrenoceptor modulation of the efferent function of capsaicin-sensitive sensory neurones in guinea-pig isolated atria. 1. Transmural nerve stimulation of guinea-pig atria, obtained from animals pretreated with reserpine (5 mg kg-1, i.p.), in the presence of atropine 1 microM and of the beta-adrenoceptor blocker CGP 20712A 1 microM, induced a positive inotropic effect which was reduced by the calcitonin gene-related peptide (CGRP) antagonist hCGRP-(8-37) and abolished by pretreatment with capsaicin 1 microM. 2. Noradrenaline concentration-dependently (0.01-10 microM) reduced the increase in cardiac contractility induced by transmural nerve stimulation. The inhibitory effect of noradrenaline was antagonized by yohimbine (0.5-1 microM), in a dose-dependent manner. Prazosin (0.5-1 microM) antagonized the effect of noradrenaline and this effect was independent of concentration. 3. In the presence of yohimbine, the lower part of the inhibitory-response curve for noradrenaline was slightly but significantly shifted by prazosin. A similar degree of antagonism was observed in the presence of 1 microM phenoxybenzamine. 4. The selective alpha 2 agonists BHT 920 and clonidine reduced, in the same concentration-range (0.01-1 microM), the cardiac response to transmural nerve stimulation in a yohimbine-sensitive fashion. 5. Phenylephrine (0.1-100 microM) and methoxamine (1-300 microM) also induced an inhibitory effect on transmural nerve stimulation. The effect of phenylephrine was antagonized by yohimbine (1 microM) more efficiently than by prazosin (0.5 microM). 6. These results are in keeping with the presence of inhibitory prejunctional alpha 2-adrenoceptors on cardiac sensory nerve endings which modulate the efferent function of capsaicin-sensitive neurones.


Cardiovascular actions of a new selective postjunctional alpha-adrenoceptor antagonist, SK&F 104856, in normotensive and hypertensive dogs. 1. SK&F 104856 (2-vinyl-7-chloro-3,4,5,6-tetrahydro-4- methylthieno[4,3,2ef][3]benzazepine) is a novel postjunctional alpha 1- and alpha 2-adrenoceptor antagonist. 2. SK&F 104856 as well as prazosin and SK&F 86466 reduced blood pressure in the anaesthetized normotensive dog. 3. SK&F 86466 and rauwolscine but not SK&F 104856 or prazosin, produced a marked increase in myocardial contractility which corresponds with their ability to block prejunctional alpha 2-adrenoceptors. 4. Intravenous or oral administration of SK&F 104856 resulted in dose-dependent antihypertensive responses in 1-kidney, 1-clip (1-K, 1-C) Goldblatt hypertensive dogs with baseline blood pressure of approximately 140 mmHg. At 0.1 and 1 mg kg-1, i.v., mean arterial blood pressure fell by 11 +/- 5 and 23 +/- 5 mmHg, respectively. At 3 and 10 mg kg-1, p.o., blood pressure fell by 9 +/- 3 and 22 +/- 5 mmHg, respectively. At 10 mg kg-1, p.o., the antihypertensive effect of SK&F 104856 was still evident at 4 h. 5. The data indicate that SK&F 104856 shows selectivity in vivo for postjunctional versus prejunctional alpha-adrenoceptors and is a potent and long-acting antihypertensive agent in 1-K, 1-C Goldblatt hypertensive dogs.


Effects of peptidase inhibition on angiotensin receptor agonist and antagonist potency in rabbit isolated thoracic aorta. 1. Experiments were performed with peptidase inhibitors on rabbit aortic strip preparations, to determine whether endogenous peptidase activity can influence the potency estimates for angiotensin receptor agonists and antagonists in this tissue. 2. Angiotensin II (A II) and angiotensin III (A III) both induced concentration-related contractions of rabbit aortic strip preparations. A III was approximately 38 fold less potent than A II, and the gradient of the A III concentration-response curve (1.00 +/- 0.04) was significantly more shallow than that (1.76 +/- 0.05) of the A II curve. 3. Neither the aminopeptidase-A and -M inhibitor, amastatin, nor the aminopeptidase-B and -M inhibitor, bestatin, affected the potency of, or the maximum response to, A II. In contrast, the potency of A III was increased by both amastatin and bestatin. Amastatin had the most marked effect and at 10 microM caused approximately a 12 fold increase in the potency of A III (EC50 values, 102 nM and 8.6 nM in the absence and presence of amastatin, respectively), and also significantly steepened the gradient of the A III concentration-response curve. Amastatin did not affect the position or shape of the concentration-response curve to the alpha 1-adrenoceptor agonist, phenylephrine. Finally, the carboxypeptidase-N inhibitor, D-L-mercaptomethyl-3-guanidine-ethylpropanoic acid (MERGETPA) did not change the position or shape of the concentration-response curves to either A II or A III.4. In the presence of amastatin, the potency of the peptide angiotensin receptor antagonist, Ile7-A III (100nM-l microM ), was increased approximately 13 fold (pA2, with A II as the agonist, 7.0 +/- 0.1 and 8.1 +/- 0.1, in the absence and presence of amastatin, respectively). However, the potency of the nonpeptide angiotensin receptor antagonist, DuP 753 (30-300 nM), was little affected by amastatin (pA2, 8.2 +/- 0.1 and 8.1 +/- 0.1 in the absence and presence of amastatin, respectively).5. The results of this study suggest that endogenous aminopeptidase activity in the rabbit thoracic aorta can profoundly affect estimates of the potency of peptide angiotensin receptor agonists and antagonists.A suitable aminopeptidase inhibitor should therefore be included in studies, using this tissue, which aim to classify angiotensin receptor subtype(s) based on the rank order of peptide angiotensin receptor agonist and/or antagonist potencies.


Manifestations of acute opiate withdrawal contracture in rabbit jejunum after mu-, kappa- and delta-receptor agonist exposure. 1. Following a 5 min in vitro exposure to morphine (1.3 x 10(-7) M), U-50,488H (2.5 x 10(-8) M) and deltorphin (1.6 x 10(-8)-6.5 x 10(-9) M), the rabbit isolated jejunum exhibited a precipitated contracture after the addition of naloxone (2.75 x 10(-7) M). 2. The precipitated responses to U-50,488H and deltorphin but not to morphine were reproducible in the same tissue. 3. The precipitated contractures were blocked completely by tetrodotoxin (3 x 10(-7) M), partially by atropine (1.5 x 10(-7) M) and not affected by hexamethonium (1.4 x 10(-5) M). 4. Naloxone administration (2.75 x 10(-7) M) before the agonist prevented the development of the adaptive response to morphine and U-50,488H but not to deltorphin. 5. The selective antagonists norbinaltorphimine (2.7 x 10(-8)-2.7 x 10(-9) M) and naltrindole (1.1 x 10(-7) M) prevented the adaptive response development only to the respective agonists. 6. The opioid agonists partially inhibited the spontaneous activity of the tissue. This study has shown that independent activation of mu-, kappa- and delta-opioid receptors can induce dependence in this isolated tissue. Rabbit jejunum is a suitable tissue for studying the acute effects of opioids on the adaptative processes determined by their administration.


Beta-adrenoceptor agonist stimulation of acid secretion by rat stomach in vitro is mediated by 'atypical' beta-adrenoceptors. 1. A previous study showed beta-adrenoceptor agonists stimulated acid secretion by rat stomach in vitro. The receptors could not be classed as either the beta 1- or beta 2-subtype. This study examines the effect of 2 'atypical' beta-agonists on acid secretion. 2. Basal and isoprenaline-stimulated acid secretion were compared in tissues bathed either in HEPES/O2- or HCO3-/CO2-buffer. Basal secretion was underestimated in HCO3- by an amount equal to the rate of base section. Tissues responded well in HEPES buffer and there was no base secretion following acid inhibition with SCH 28080. HEPES was used for the study. 3. SR 58611A stimulated acid in a concentration-related way (0.1-5 microM). Maximum response at 1 microM was equal to the response to a maximal concentration of isoprenaline. BRL 37344 (1 microM) also stimulated to the same extent. 4. Responses to isoprenaline (5 microM) and SR 58611A (1 microM) were reduced by propranolol (10 microM) but not by alprenolol (10 microM) or by practolol (12.5 microM) plus ICI 118551 (1 microM). 5. Exposure to SR 58611A (1 microM) led to desensitization to isoprenaline but not to bethanechol (1 microM) or histamine (50 microM). 6. We conclude that a HEPES/O2-buffer is advantageous when measuring gastric acid secretion in vitro and the stimulatory effect of beta-adrenoceptor agonists is mediated by 'atypical' receptors.


The non-adrenergic, non-cholinergic response counteracts changes in guinea-pig airway tone with and without sympathetic activation. 1. We examined whether the non-adrenergic, non-cholinergic (NANC) neural response can counteract changes in smooth-muscle tone with and without simultaneous sympathetic activation in guinea-pig airways. 2. Isolated airway preparations were pretreated with indomethacin (10 microM) and incubated with either atropine (1 microM) and guanethidine (10 microM) or atropine (1 microM) alone. The response to electrical field stimulation (EFS: 1200 mA, 0.5 ms, 3 Hz for 240 s) was studied at various levels of tone prior to EFS: first without induced tone, then at a moderate tone induced by histamine (0.3 microM) and finally at a high tone induced by histamine (6 microM). 3. The response to EFS was a contraction when the tone prior to EFS was low and a relaxation when the tone prior to EFS was high. These responses converged towards a similar level of tone, in the distal trachea and in the main bronchus, with and without guanethidine. 4. The mean (s.e. mean) level of tone towards which the responses to EFS converged was lower after incubation with atropine alone compared with incubation with atropine and guanethidine, both in the distal trachea [8 (1)% compared with 30 (6)% of maximum tone] and in the main bronchus [28 (4)% compared with 57 (2)% of maximum tone]. In separate experiments, the guanethidine-induced effect on the responses to EFS was imitated by propranolol (1 microM) but not by prazosin (0.3 microM) and yohimbine (1 microM). 5. These findings indicate that the NANC neural response can counteract changes in airway smooth-muscle tone via a contraction or via a relaxation, depending on the tone prior to activation.This stabilizing effect on tone does not appear to depend upon adrenergic activation per se. The level of tone towards which the NANC responses converge can, however, be reduced by beta-adrenoceptor activation, thus suggesting an interaction which provides protection from severe airway smooth-muscle contraction.


Release of endogenous adenosine and its metabolites by the activation of NMDA receptors in the rat hippocampus in vivo. 1. The effects of N-methyl-D-aspartate (NMDA), KCl, and veratridine on the release of endogenous adenosine and its metabolites, inosine and hypoxanthine, from the rat hippocampus have been studied by in vivo microdialysis. 2. In the hippocampus of rats anaesthetized with urethane the adenosine level reached a stable state estimated at 0.93 microM during the first 2 h after the implantation of the dialysis probe. NMDA (50 microM to 25 mM) in the perfusate evoked a concentration-dependent release of adenosine, inosine and hypoxanthine with an EC50 of 180 microM. The release was reduced by 93% by the specific NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (2-AP5) at 200 microM, indicating an NMDA receptor-mediated process. In addition, the 100 mM KCl-evoked release of adenosine was also substantially reduced by 77% by 2-AP5, suggesting that a large component of the K(+)-evoked release is NMDA-receptor-mediated. 3. Perfusion with zero-Ca2+ artificial cerebrospinal fluid attenuated the NMDA-evoked release of adenosine only by 16% (not significant) but depressed the K(+)-evoked release by 62%, indicating that most of the NMDA-evoked release is directly receptor-mediated, whereas a large component of the K(+)-evoked release could be via the release of an excitatory amino acid acting at the NMDA receptors.


Cooling and response to adrenoceptor agonists of rabbit ear and femoral artery: role of the endothelium. 1. The effects of cooling on the response of the rabbit central ear (cutaneous) and femoral (non-cutaneous) arteries to stimulation of adrenoceptors and the role of the endothelium in these effects, were studied in 2 mm long cylindrical segments. 2. Concentration-response curves for noradrenaline (10(-9)-3 x 10(-4) M), phenylephrine (alpha 1-adrenoceptor agonist, 10(-9)-3 x 10(-4) M) and B-HT 920 (alpha 2-adrenoceptor agonist, 10(-7)-10(-3) M) were recorded isometrically in arteries with and without endothelium at 37 degrees C and at 24 degrees C (cooling). To analyze further the endothelial mechanisms in the responses to adrenoceptor stimulation during cooling, the effects of the adrenoceptor agonists on ear arteries in the presence of NG-nitro-L-arginine methyl esther (L-NAME) (10(-5) M) were also determined. 3. In every condition tested, the three adrenoceptor agonists produced a concentration-dependent arterial contraction and the order of potency in ear and femoral arteries was noradrenaline greater than or equal to phenylephrine greater than B-HT 920. The response of ear and femoral arteries to phenylephrine or B-HT 920 was blocked by prazosin (10(-6) M). Yohimbine (10(-6) M) decreased slightly the response of ear arteries and increased that of femoral arteries to B-HT 920. 4. The sensitivity of both ear and femoral arteries to the three adrenoceptor agonists was significantly lower at 24 degrees C than at 37 degrees C. 5. In ear arteries, endothelium removal or treatment with L-NAME did not influence the response at 37 degrees C, but did increase it during cooling to adrenoceptor stimulation.In femoral arteries, endothelium removal increased the sensitivity to noradrenaline and, especially, to B-HT 920 at 37 degrees C, but did not affect the response at 24 degrees C.6. The results suggest that: (a) rabbit ear and femoral arteries are equipped mainly with alpha 1-adrenoceptors;(b) at 37 degrees C, the contraction of the ear artery to adrenoceptor agonists is mostly endothelium-independent, and in the femoral artery the contraction to alpha 2-adrenoceptor activation is endothelium-dependent; (c) cooling inhibits the contraction to adrenoceptor agonists in both ear and femoral arteries: in the ear artery probably by increasing the availability of endothelial nitric oxide, but in the femoral artery by depressing the sensitivity of alpha-adrenoceptors in the smooth musculature.7. The results suggest that the endothelium may modulate the adrenoceptor response of cutaneous arteries during changes in temperature.


Characterization of muscarinic receptors that mediate contraction of guinea-pig isolated trachea to choline esters: effect of removing epithelium. 1. The muscarinic receptor subtype that mediates contraction of guinea-pig trachea, in the presence and absence of epithelium, to acetic and carbamic acid choline esters was determined by use of preferential muscarinic receptor antagonists: pirenzepine (M1 receptor), methoctramine (M2 receptor) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3 receptor). 2. Acetylcholine (ACh), methacholine (MeCh), carbachol (CCh), bethanechol (BeCh) and oxotremorine induced concentration-dependent contraction of guinea-pig isolated tracheal strips in the presence and absence of epithelium. Contraction to acetic choline esters (ACh and MeCh) was augmented by removal of the epithelium, whereas contraction to carbamic acid choline esters (CCh and BeCh) and oxotremorine was not influenced by removal of the epithelium. 3. Pirenzepine, methoctramine and 4-DAMP caused parallel rightward displacements of the concentration-contraction curves to the muscarinic agonists. The pA2 values (determined from Arunlakshana-Schild graphs) for pirenzepine and 4-DAMP in guinea-pig trachea in the presence of epithelium were: ACh as the agonist, 7.6 and 9.0, respectively; CCh as the agonist, 7.6 and 9.1, respectively. The apparent pKB values for methoctramine with the same system were: ACh as the agonist, 5.6; CCh as the agonist, 5.6. Similar values were obtained with MeCh, BeCh and oxotremorine as the agonists. These values were agonist- and epithelium-independent. 4. It is concluded from the pA2 and apparent pKB values obtained for the muscarinic receptor antagonists used in this study that contraction of guinea-pig isolated trachea, with and without epithelium, to both acetic and carbamic acid choline esters is mediated via the muscarinic M3 receptor subtype.Differential contractile responses of guinea-pig trachea to acetic and carbamic acid choline esters upon the mechanical removal of the epithelium may not be explained by activation of different muscarinic receptor subtypes by these agonists.


Selective reduction of glutamate in the rat superior colliculus and dorsal lateral geniculate nucleus after contralateral enucleation. The effects of afferent lesions on the levels of glutamate, aspartate and gamma-aminobutyric acid (GABA) in the laminae of the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN) of the rat were studied, using microassay methods for these amino acids. The analysis was performed 12-14 days after left eye enucleation, or ablation of right visual cortical area, or both left eye enucleation and ablation of right visual cortex. Superficial gray layer (SGL) and deep layers in the SC were dissected out from the thin-sectioned, freeze-dried sample. In the dLGN, the outer and inner laminae were separately dissected. The glutamate contents in the upper half of SGL and outer lamina of dLGN contralateral to eye enucleation decreased significantly (15%). Combination of eye enucleation and visual cortical ablation further decreased the glutamate content in the upper half of the right SGL (29.3%). On the other hand, aspartate and GABA concentrations in the SC and dLGN exhibited no significant reduction after deafferentations. These results indicate that the retino-tectal and retino-geniculate pathway of the rat may be glutamatergic in nature.


Somatostatin receptors in the human cerebellum during development. The ontogeny of somatostatin receptors (SRIF-R) was studied in the human cerebellum from mid-gestation to the 15th month postnatal. The brains were collected 3-26 h after death, from 18 fetuses and infants, and from 4 adults aged from 48 to 82. SRIF-R were characterized by membrane-binding assay and their localization was determined by in vitro autoradiography. Both techniques were conducted with two radio-ligands: [125I-Tyr0, DTrp8]S14 and D-Phe-Cys-125I-Tyr-DTrp-Lys-Thr- ol (125I-SMS 204-090). Membrane-binding studies carried out with each radioligand showed the presence of a single population of saturable, high affinity binding sites. Neither were the Kd values for either ligand (assessed by Scatchard analysis) changed appreciably during development, mean Kd values being 0.36 +/- 0.04 nM and 0.56 +/- 0.11 nM for [125I-Tyr0,DTrp8]S14 and 125I-SMS 204-090, respectively. Although inter-individual fluctuations of the Bmax were observed, the concentration of SRIF-R in the cerebellum of fetuses and infants up to 8 months appeared to be at least 2- to 10-fold higher than in the adult cerebellum. No appreciable differences in the Bmax values were found using either radioligand. The highest density of SRIF-R was observed in the cerebellar cortex of fetuses, in particular in the external granule cell layer (EGC), where stem cells of the granule cells are generated and enter the differentiation process. A high density of SRIF-R also occurred in the internal granule cell layer.(ABSTRACT TRUNCATED AT 250 WORDS)


Deficits in the somatostatin SS1 receptor sub-type in frontal and temporal cortices in Alzheimer's disease. The aim of this study was to evaluate the possible differential alterations of somatostatin (SRIF) receptor sub-types in Alzheimer's disease (AD). Consequently the binding profile of cortical SRIF receptors were examined in normal and AD brains using non-selective ([125I]Tyr0, D-Trp8-SRIF14) and SS1 receptor sub-type-selective ([125I]SMS204-090) radioligands. Maximal binding capacities, but not affinities, were reduced for both ligands in the temporal cortex. In contrast, only the maximal binding capacity of [125I]SMS204-090 was significantly reduced (68%) in the frontal cortex; no alterations were detected using the non-selective probe. This reveals that while the maximal binding capacity of the SS1 receptor sub-type is altered in frontal and temporal cortices in AD, other putative cortical SRIF receptor classes (such as SS2 sites) are not as broadly affected. This could be of significance for eventual therapeutic approaches using SRIF-related analogues.


NBQX, an AMPA antagonist, reduces glutamate-mediated brain edema. Glutamate (2.5 mg) was administered after the blood-brain barrier had been opened by a unilateral intracarotid infusion of 5 mg protamine sulfate in rats. Whereas the brain specific gravity, measured 24 h later, did not differ between the injected and non-injected side after protamine alone, glutamate significantly reduced the specific gravity in the right hemisphere indicating brain edema (P less than 0.01). NBQX, a potent AMPA receptor antagonist, significantly reduced the edema (P less than 0.01) and completely inhibited the glutamate mediated increase in albumin content in cerebrospinal fluid (P less than 0.01).


In vivo microdialysis of amino acid neurotransmitters in the hippocampus in amygdaloid kindled rat. Extracellular concentrations of gamma-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) were determined by microdialysis in rat hippocampus during various amygdaloid kindled stages. The values of GABA and Glu were increased 3-4 times in C2-C3 stages in comparison with the values in control animals. After reaching the C5 stages, these values were increased 3-7 times. However, the concentration of Asp decreased depending on the kindling stage, reaching the lowest value of 33% in comparison with the normal value. The observed changes may be related to kindling induced seizures.


G proteins are involved in the regulation of transmitter release at an Aplysia cholinergic synapse. At an identified cholinergic synapse of the Aplysia buccal ganglion, presynaptic injections of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) depressed the amplitude of evoked postsynaptic responses. This reduction of acetylcholine (ACh) release by GTP-gamma-S, prevented by pre-injection of guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) in the presynaptic neuron, was due to a reduction of the number of ACh quanta released. The mean amplitude of the evoked miniature postsynaptic current (MPSC) was unchanged. The presynaptic Ca2+ influx was lowered.


Potassium-induced long-term potentiation in rat hippocampal slices. We observed that a transient increase in extracellular potassium concentration (50 mM for 40 s) was sufficient to induce long-term potentiation (LTP) of synaptic transmission in area CA1 of the hippocampal slice. Potassium-induced potentiation of the Schaffer collateral/commissural synapses demonstrated several features characteristic of tetanus-induced LTP: (1) population excitatory post-synaptic potential (EPSP) amplitudes were enhanced to a similar magnitude (on average 70% above baseline) which (2) lasted for more than 20 min; (3) induction was blocked by bath application of the specific N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-APV), and (4) was attenuated by reduction of the concentration of calcium in the extracellular medium. Induction of either potassium-induced LTP or tetanus-induced LTP occluded the subsequent expression of the other. Finally, exposure to high potassium in the absence of electrical stimulation was sufficient to induce LTP. Taken together, these data indicate that brief depolarizing stimuli other than tetanus can induce LTP. Because potassium-induced LTP is not restricted to the subset of afferents examined electrophysiologically, such a method could facilitate analyses of the biochemical events underlying both the induction and expression of LTP.


The effect of glutamate antagonists on c-fos expression induced in spinal neurons by irritation of the lower urinary tract. Chemical irritation of the lower urinary tract (LUT) of the rat increases the expression of c-fos in neurons in the dorsal horn, dorsal commissure and intermediolateral region of the spinal cord. The role of glutamatergic synapses in this response was examined using two glutamate receptor antagonists, MK-801 (an NMDA antagonist) and CNQX (an AMPA antagonist). In rats with an intact spinal cord, MK-801 (3.5 mg/kg, i.v.) administered 15 min before bladder irritation decreased (50-60%) the number of c-fos-positive cells in all regions of the cord. A smaller dose of MK-801 (0.8 mg/kg, i.v.) was ineffective. In spinal transected rats (4-7 days prior to the experiment) MK-801 (3.5 mg/kg, i.v.) decreased c-fos expression only in the medial dorsal horn. CNQX (1.2 mg/kg, i.v.) was ineffective in both preparations. These results indicate that activation of NMDA receptors at glutamate synapses in the central nervous system may play a role in the processing of nociceptive input from the LUT and may also be involved in reflex pathways mediating micturition.


Asymmetric elevation of striatal dopamine D2 receptors in the chakragati mouse: neurobehavioral dysfunction in a transgenic insertional mutant. We have previously reported the discovery of a transgenic insertional mutant, recently named the chakragati (ckr) mouse, which displays lateralized circling, locomotor hyperactivity, hyperreactivity, as well as body weight deficits. Since lateralized dopamine function is associated with circling behavior we sought to determine whether dopamine (DA) D1 and D2 receptors were asymmetrically distributed in the striata of adolescent and adult ckr mice using receptor autoradiography. Stereotypic and rotational responses to quinpirole served as behavioral indices of D2 receptor function. The ckr mice showed hemispherically asymmetric elevations in DA D2 receptors in the lateral subregions of the striatum whereas medial regions of the striatum were symmetrically and bilaterally elevated (overall elevation = 30%). As a group, ckr mice had higher D2 receptor levels on the side which was contralateral to the preferred direction of spontaneous nocturnal rotation. Striatal D1 receptors and mesolimbic D2 and D1 receptors of ckr mice were neither elevated nor differentially asymmetric. Young adult ckr mice showed dose-dependent increases in net rotations in response to quinpirole whereas normal mice showed no change from baseline levels. Both groups showed similar stereotypic responses. Older adult ckr mice, however, showed dose-dependent reductions in rotation after quinpirole whereas normal mice turned at baseline levels. Older ckr mice also displayed significantly greater stereotyped sniffing behavior. This unique mutant provides a novel genetic model of basal ganglia dysfunction, and may be useful in studying aspects of neuropsychiatric disorders associated with dopaminergic abnormalities.


Dynorphin A-(1-17) and dynorphin B are released from in vitro superfused rat hypothalami. Effects of depolarizing agents and ovariectomy. We measured the release of immunoreactive (ir) dynorphin (dyn) A-(1-17) and dyn B from the rat hypothalamus by an in vitro superfusion technique. The system was validated on the basis of the recovery and stability of radiolabeled peptides added to the superfused hypothalami. These were detected as authentic peptides by reverse-phase high-performance liquid chromatography (rp-HPLC) only in the presence of a cocktail of peptidase inhibitors added to the superfusion medium. We observed spontaneous release of ir-dyn B, evaluated by a validated radioimmunoassay in the superfusates, that was increased by potassium and veratridine depolarization. It was calcium-dependent and tetrodotoxin-sensitive. We could not evaluate ir-dyn A-(1-17) directly in the superfusates, because the peptidase inhibitors added to the medium significantly altered the tracer-antibody reaction. To obviate this problem, pooled superfusate samples were purified on C18 cartridges and assayed by rp-HPLC. Rp-HPLC analysis of superfusates revealed two molecular forms with the same retention time as authentic dyn A-(1-17) and dyn B which were four times higher in K(+)-stimulated fractions. We could not detect dyn A-(1-32), comprising dyn A-(1-17) and dyn B, even though this peptide is recognized by the antibodies used in this study and is detected in acetic acid extracts of the rat hypothalamus. The spontaneous and K(+)-evoked release of ir-dyn A-(1-17) and ir-dyn B were significantly higher in 2-week ovariectomized rats, in parallel with the increase of their content in the anterior hypothalamus preoptic area.


Comparison of neuroendocrine and behavioral effects of ipsapirone, a 5-HT1A agonist, in three stress paradigms: immobilization, forced swim and conditioned fear. Ipsapirone is an anxiolytic drug and a serotonin1A (5-HT1A) agonist. The aim of the present study was to investigate the effects of low doses of ipsapirone on the hormonal and behavioral response to three stress procedures: immobilization, forced swim and conditioned emotional response (CER). We examined the effect of ipsapirone (0.1, 0.5 or 1.0 mg/kg) on plasma renin concentration (PRC), adrenal corticotropic hormone (ACTH), corticosterone, prolactin and defecation in rats exposed to immobilization, forced swim or CER stress. All three stressors significantly elevated all the hormone levels (P less than 0.01). Immobilization-induced elevations of PRC, and corticosterone were inhibited by the highest doses of ipsapirone (0.5 and 1 mg/kg, i.p.). However, ipsapirone did not modify the immobilization-induced elevations of plasma ACTH, prolactin or defecation. Ipsapirone was relatively ineffective at reducing the endocrine responses to forced swim. Ipsapirone reduced some, but not all of the hormonal responses to CER stress. CER-induced elevations of corticosterone and prolactin were not inhibited by ipsapirone. However, the ACTH response to CER was significantly (P less than 0.01) inhibited by all doses of ipsapirone and the highest dose of ipsapirone attenuated the renin response. In contrast with the hormonal responses, ipsapirone inhibited all of the behavioral responses to CER stress. Ipsapirone inhibited CER-induced freezing behavior and defecation, while dose-dependently reversing the suppressive effect of CER on exploring, grooming and rearing behaviors. In conclusion, there is a dissociation between the influence of ipsapirone on the endocrine and behavioral responses to CER stress. Ipsapirone also has differential effects on the neuroendocrine response to the three stressors studied. Ipsapirone was most effective in attenuating the hormonal responses to CER, followed by immobilization and swim stress. Of the hormones studied, the stimulation of renin secretion after exposure to the three stressors was most sensitive to ipsapirone, while corticosterone and prolactin were the least sensitive to ipsapirone.


Action potentials produce a long-term enhancement of M-current in frog sympathetic ganglion. M-current is a voltage-gated K+ current that can be turned off by the muscarinic action of acetylcholine. We examined the effects of postsynaptic action potential firing on the level of M-current in B-cells of the bullfrog sympathetic ganglion. High frequency stimulation of action potentials induced an approximately two-fold increase in the level of the M-current that could last up to 35 min. The 'enhanced' M-current was similar to the 'resting' one in its time-dependence, voltage-dependence and sensitivity to neurotransmitters. Experiments were undertaken to examine the functional consequences of the enhanced M-current. Following high frequency stimulation the number of spikes evoked by depolarizing current was reduced. In addition, the excitatory postsynaptic potential (EPSP) evoked by maximal input became subthreshold, thereby blocking information flow through the ganglion cell. These results indicate that the enhancement of M-current by spikes provides a negative feedback mechanism for the control of excitability. It has been reported that postsynaptic stimulation of ganglion cells also produces a long-term increase in the nicotinic EPSP, but we were unable to confirm this observation.


Insulin-specific sensitization of cultured cerebrocortical neurons to glutamate excitotoxicity. The effect of insulin on the sensitivity of neurons to excitatory amino acid-induced cytotoxic cell death was examined in primary cultures of the rat cerebral cortex. Cells developed for two weeks in serum supplemented medium in the presence or absence of insulin, insulin-like growth factor or b-fibroblast growth factor. Excitotoxic cell death was induced by 1 mmol/l glutamate, N-methyl-D-aspartate, kainate or quisqualate. The vulnerability of cells was evaluated by the measurement of lactate dehydrogenase release due to cytotoxic injury. In contrast to the moderate evaluation of protein content by all the 3 growth factors, only insulin increased the vulnerability of cells to the neurotoxic effects of glutamate and of the 3 excitatory amino acid receptor agonists examined. Our results show that the induction of vulnerability in cortical cultures is a specific action of insulin and not a general effect of growth factors. Moreover, the increased vulnerability to N-methyl-D-aspartate, quisqualate and kainate suggests that the effect of insulin is exerted through intracellular mechanisms other than a selective induction of one subpopulation of excitatory amino acid receptors.


Glutamate selectively increases the high-threshold Ca2+ channel current in sensory and hippocampal neurons. Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca2+ channels in hippocampal neurons. We studied whole-cell Ca2+ currents (ICa) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1-30 microM) increased high-threshold Ca2+ current. It was independent of the charge carriers, Ca2+ or Ba2+. Low-threshold Ca2+ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1-2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits 'sleepy' Ca2+ channels. Whatever its mechanism, overlasting increase of ICa by glutamate may be important in neuronal plasticity.


Presumptive adrenergic neurons containing phenylethanolamine N-methyltransferase immunoreactivity in the medulla oblongata of neonatal swine. Given the importance of the swine (Sus scrofa) as an animal model for human development, physiology and disease, neurons containing the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), were mapped in the medulla oblongata of neonatal swine as a first step in identifying their roles in central autonomic control. Neurons were labeled immunocytochemically by using an antiserum to PNMT raised in rabbits against trypsin-treated enzyme purified from the bovine adrenal gland. The general regional organization of neurons expressing PNMT (-like) immunoreactivity (ir) in the neonatal swine was similar to data obtained in other species and, in some aspects, more closely resembled the pattern observed in the primate brain. Immunolabeled cells appeared to be more abundant and caudally more extensive than observed in other adult animals. PNMT-immunoreactive (ir) neuronal somata, however, were largely confined to the reticular formation in the ventrolateral quadrant and the nucleus tractus solitarii (NTS) and more restricted in distribution than those expressing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H)-ir on serial transverse sections. A close correspondence was observed between the distributions of TH- and PNMT-ir neurons and processes throughout the C1 and C2 areas. However, in the C1 and C3 regions TH-ir neurons outnumbered those containing D beta H and PNMT-ir. In contrast, cell groups enriched in PNMT-ir neurons and processes were characterized by relatively weak D beta H-ir. In the ventrolateral medulla (VLM), PNMT-ir cell bodies were concentrated rostrally and extended from the caudal pole of the facial nucleus to a level posterior to the calamus scriptorius. The rostral VLM was characterized by an admixture of bipolar and multipolar primarily medium-diameter immunostained neurons. A prominent cell column (condensation) organized ventromedially to the nucleus ambiguus pars compactus (NAc). A loosely organized cluster bordered the lateral aspect of the special visceral efferent column; another smaller aggregate was located in the ventromedial reticular formation adjacent to the inferior olive. At middle medullary levels, PNMT-ir neurons formed two distinct subgroups (dorsal and ventral) interrupted by a band of precerebellar relay neurons that extended between the medial and lateral limbs of the lateral reticular nucleus of Walberg. At obex, the dorsal cell group formed a diagonal array and assumed a position dorsal and dorsolateral to the medial limb of LRN. This group was distinguished by bipolar neurons with axes of orientation directed perpendicularly to the majority of neurons in the rostal VLM or those lying near the caudal ventromedullary surface.(ABSTRACT TRUNCATED AT 400 WORDS)


The effect of hypoxia on neurotransmitter phenotype of forebrain cholinergic neurons. The effect of hypoxia on the neurotransmitter phenotype of rat forebrain cholinergic neurons was analyzed using a dissociated fetal rat culture system. The aims of this study were to examine the feasibility of using choline acetyltransferase (ChAT) activity as a measure of cell injury and/or recovery, to measure the time course of hypoxic effects on ChAT activity, to determine how changes in ChAT activity at 48 h post-injury relate to microscopic changes and LDH release into the medium during that time, and finally to explore the possible mechanisms of hypoxic injury in this model. At exposure to 0.5-1.5% O2 there was a time-dependent decrease in ChAT activity when cells were harvested 48 h after exposure. Forty-eight hours after 8-9 h hypoxic exposure ChAT activity was 50-60% that of controls without any alteration in morphology of neurons. An 8 h exposure to hypoxic conditions caused a post-exposure time-dependent decrease in ChAT activity to 20% of control level at 72 h. Thereafter there was spontaneous recovery of phenotype to 60% of control which remained stable between 5 and 7 days post-exposure. Loss of neurotransmitter phenotype was not well correlated with other measures of cytotoxicity including morphological changes and LDH release. The loss of phenotype observed with hypoxia was mimicked by glutamate and kainate but not by NMDA. Consistent with these observations, neither APV nor AP3 significantly altered the effect of hypoxia on forebrain cholinergic neurons, while the addition of APV and CNQX in combination protected the phenotype of these neurons only if there was 50% or less loss of phenotype following hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)


Activation of kappa-opioid receptors depresses electrically evoked excitatory postsynaptic potentials on 5-HT-sensitive neurones in the rat dorsal raphé nucleus in vitro. Intracellular recordings from dorsal raphé neurones in slices from rat brains were used to study the actions of kappa-opioid receptor agonists on an excitatory postsynaptic potential (epsp) evoked by local electrical stimulation of afferent terminals. The epsp was observed on all 5-HT-sensitive neurones and was blocked by 1 microM TTX. The epsp was reduced in a dose-dependent manner by the specific kappa-opioid receptor agonist [5R-(5 alpha,7 alpha,8 beta)]-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro[4.5]dec-8-yl]-4-benzofuranacetamide monohydrochloride (CI-977) (1-100 nM). The effects of CI-977 were blocked by the specific kappa-opioid receptor antagonist norbinaltorphimine (NorBNI) (0.1-1 microM). In the presence of the GABAA receptor antagonists picrotoxin and bicuculline (30 microM), CI-977 still had its depressant action on the epsp. Application of the excitatory amino acid receptor antagonists either kynurenic acid (0.5-1 mM) or 6-cyano-2,3-dihydro-7-nitro-quinoxaline-2,3-dione (CNQX) (30 microM) and DL-2-amino-5-phosphonovaleric acid (APV) reduced both the peak and area of the epsp suggesting that the main component of the epsp evoked by electrical stimulation was largely due to release of excitatory amino acids from afferent terminals. Using potassium chloride-filled recording electrodes an epsp which was only partially occluded by kynurenic acid or CNQX and APV was seen on some neurones, this residual epsp was insensitive to CI-977 but was blocked by 30 microM picrotoxin and bicuculline. The specific mu-opioid receptor agonist, DAGOL, had no consistent effect on the fast epsp. Longer duration electrical stimuli produced a slow inhibitory postsynaptic potential (ipsp) and a long duration increase in firing. CI-977 did not affect either the slow 5-HT-mediated ipsp which was blocked by spiperone or the slow noradrenaline-mediated increase in firing which was sensitive to prazosin. CI-977 did not change the depolarizing response to brief applications of either glutamic acid or N-methyl-D-aspartic acid (NMDA). CI-977, NorBNI, naloxone, DAGOL, picrotoxin, bicuculline and kynurenic acid had no consistent effects on the resting postsynaptic membrane potential or conductance. Under voltage-clamp conditions CI-977 had no effect on a membrane current resembling IA. These results suggest that kappa-opioid receptors are present on the terminals of afferents which release excitatory amino acids onto the 5-HT-sensitive neurones in the raphé.


Lateral hypothalamus and local stimulation induced postsynaptic responses in zona incerta neurons in an in vitro slice preparation of the rat. Postsynaptic potentials evoked in the zona incerta (ZI) neurons were studied in in vitro slice preparations. Lateral hypothalamus (LH) and local stimulation evoked fast IPSPs, fast EPSPs, and slow EPSPs. The amplitude of the slow EPSPs increased when the neuron was hyperpolarized by a low intensity current injection but was blocked when it was hyperpolarized with a strong current. The slow EPSPs were reversibly suppressed by an application of 50 microM DL-2-amino-5-phosphonovaleric acid (APV) and 20 microM 3-[(+/-)-2-carboxypiperazine-4-yl-]-propyl-1-phosphonic acid (CPP). The slow EPSPs were augmented in Mg-free medium and by train pulse stimulation. Pressure application of NMDA induced a depolarization similar to the slow EPSP. On the other hand, the fast EPSPs showed a conventional voltage dependency and were antagonized by kynurenic acid but not by APV or CPP. The fast IPSPs were completely blocked by 10 microM bicuculline methiodide. The results indicate that LH and local stimulation evoked monosynaptic fast EPSPs and slow EPSPs mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. The IPSPs appear to be mediated by GABAA receptors and regulate the expression of NMDA receptor-mediated slow EPSPs.


Dopamine microinjected into the nucleus ambiguus elicits vagal bradycardia in spinal rats. To investigate the effects of dopamine (DA) on vagal efferent activity, DA was microinjected into the right nucleus ambiguus (NA) in rats. Experiments were done in 19 urethane anaesthetized, artificially ventilated spinal (C1) rats. Sites in the right NA containing cardioinhibitory neurons were identified by observing a marked and reproducible decrease in heart rate (HR; 64.9 + 2.8 bpm; n = 36) elicited by microinjecting L-glutamate (GLU; 1.5. nmol in 10 nl). No decreases in arterial pressure (AP) were obtained at these sites. Microinjection of DA (1-15 nmol in 10 nl) into 24 of these 36 sites caused a dose-dependent decrease in HR. The responses to 1 nmol and 3 nmol DA were blocked by (+/-)-sulpiride, a specific D2 receptor antagonist (0.1 nmol in 10 nl). A higher dose of (+/-)-sulpiride (1 nmol in 10 nl) was required to block the responses to 15 nmol of DA. Bradycardia elicited by even the lowest amount of DA (1 nmol) was not blocked by SCH-23390, a specific D1 receptor antagonist. These experiments demonstrate that the bradycardia caused by microinjection of DA into the NA is due to the excitation of dopamine D2 receptors present on vagal preganglionic cardioinhibitory neurons controlling HR.


Comparative inhibition profiles of three non-sedating antihistamines assessed by an extended Lewis model. Antihistaminic drugs are widely prescribed across a multitude of medical specialties such as Allergy and Dermatology. The potentially serious sedative effect of these valuable agents has previously restricted their full use and the choice of drug has been dictated more by individual patient acceptability than by any laboratory demonstrations of comparative efficacy. Unsurprisingly therefore, there is a trend towards prescribing those newer preparations which leave the central nervous system unclouded. We have studied the most frequently prescribed non-sedating antihistamine preparations, terfenadine (Triludan, Triludan Forte), cetirizine (Zirtek) and loratadine (Clarityn) in pharmacodynamic and relative efficacy trials using a quantifiable and reproducible extension of the classic Lewis model. The results indicate that two preparations, terfenadine 120 mg (Triludan Forte) and cetirizine 10 mg (Zirtek) are superior to their immediate rivals in degree of efficacy and/or speed of action. These results should assist clinicians in the positioning of effective, rapidly acting antihistamines for the symptomatic treatment of immediate hypersensitivity reactions such as urticaria and rhinitis.


[Use of DNA analysis in medicine].

[Use of DNA analysis in medicine]. Advances in genetic engineering influence to an increasing extent a number of medical disciplines. DNA analysis can be used in the diagnosis of hereditary diseases, in investigations of malignant processes, in forensic medicine and for detection of infectious pathogens. Two main methodical approaches to DNA analysis, Southern's method and procedures based on primer directed enzymatic amplification of DNA by the PCR method, resolve the complicated detection of slight changes in the vast volume of human genetic information. Cystic fibrosis may serve as an example of a serious hereditary disease the diagnosis of which improved greatly after introduction of DNA analysis. The diagnosis of this disease is nowadays possible by direct analysis of mutations and indirectly by investigations of the link between the disease and DNA polymorphisms.


[Laboratory-confirmed cases of hemorrhagic fever with renal syndrome which occurred in Breclav 1989-1990]. The authors describe the first three cases with a serologically confirmed hantaviral aetiology in the Czech Republic. It is the causal agent of haemorrhagic fever with renal syndrome, western type (strain CG 18-20, identical with the Puumala prototype). All patients come from a locality where since 1984 very actively a natural focus of hantaviral infection was investigated in small mammals, and where formerly groups of farmers were found with a high herd immunity with hantavirus. The disease was present in a mild to medium severe form with some sequelae. In the discussion the authors draw attention to the necessity of wider use of aetiological diagnosis in suspect diseases.


[New findings on alpha-hemolytic strains of E. coli in urinary tract infections. I. alpha-hemolysin and adhesins]. Alphahaemolysin (AH) is one of the important factors of virulence of E. coli strains in urinary infections. The synthesis and secretion of AH is coded by four HLA genes which in the majority of strains are located on the chromosome. Alphahaemolytic strains possess fimbrial and non-fimbrial adhesins.


Changes in acute myocardial infarction risk and patterns of practice for patients older and younger than 70 years, 1987-90. To evaluate temporal changes in risk and patterns of hospital practice for acute myocardial infarction (AMI). Retrospective analysis of age-related medical therapy and outcome of 342 consecutive patients (132 at least 70 years old and 210 younger than 70) with AMI between July 1, 1989, and June 30, 1990, and comparison with data from two previous analyses of AMI practice in 1987 (n = 207) and 1988-89 (n = 402). Tertiary care medical centre. No direct interventions; results of the two previous AMI practice pattern analyses, however, were propagated during the practice time of the most recent analysis. In 1989-90, hospital mortality was higher (19%) among patients at least 70 years old compared with patients younger than 70 (8%) (P less than 0.01). Therapies proven by repeated clinical trials to be effective in reducing AMI risk were all used less frequently in patients aged at least 70 years: thrombolysis (20 versus 43%); beta-blockers (41 versus 62%); acetylsalicylic acid (71 versus 87%); and nitrates (86 versus 97%). Qualitatively, these age-specific patterns of AMI mortality and therapy were similar to previous studies. Quantitatively, however, comparing 1987 with 1989-90 demonstrated parallel and marked increases in the use of all proven medications in both age groups, ranging from 42 to 230% (P less than 0.01). There was also a significant overall decrease in mortality from the 1987 patient cohort (20%) to the 1989-90 cohort (13%) (P less than 0.05). The decrease in mortality was entirely due to decreased mortality within the group 70 years or older; 35% in 1987 versus 19% in 1989-90 (P less than 0.05). Mortality in the AMI patients younger than 70 years old remained unchanged from 1987 to 1989-90. Pattern of practice analyses were associated with, and may have contributed to, improved patient care and outcomes in AMI. Increased use of effective AMI medical therapy had a greater benefit in elderly higher risk AMI patients than lower risk younger patients. Persisting age-specific differences in AMI therapy may respond to more direct quality improvement measures, such as critical path management.


Octreotide decreased liver metabolic activity in patients with hepatitis B surface antigen-positive cirrhosis. The influence of octreotide and somatostatin on liver metabolic activity were studied in 16 patients with cirrhosis that was positive for hepatitis B surface antigen (HBsAg). In patients receiving a 50 micrograms bolus and a 50 micrograms/hr infusion of octreotide, the hepatic blood flow, hepatic clearance, and the maximum velocity/metabolic elimination rate constant (Vmax/km) were significantly reduced after octreotide infusion compared with basal values. Similarly, the hepatic blood flow, hepatic clearance, and Vmax/km were significantly decreased in patients receiving a 250 micrograms bolus and a 250 micrograms/hr infusion of somatostatin. The extraction ratio and the systemic hemodynamic values, including cardiac index, heart rate, mean arterial pressure, and systemic vascular resistance, showed no significant changes in patients receiving either octreotide or somatostatin. These findings suggest that, as with somatostatin, octreotide reduced hepatic blood flow and impaired liver metabolic activity in patients with HBsAg-positive cirrhosis. These effects may have important clinical implications in the management of bleeding esophageal varices in patients with cirrhosis.


Contemporary medical management of left ventricular dysfunction and congestive heart failure. The primary purpose of this review was to address the following question: based on the best available evidence, what should be the current medical management of congestive heart failure (CHF)? The major sources for this review were from searches of the English language literature, including computer and bibliography reviews, of all randomized, controlled clinical trials and overview analyses of positive inotropic agents, preload/afterload reduction agents and beta-blocker medications in CHF. The number of studies reviewed was approximately 40. The major criterion for selection was that the studies be of CHF patients in randomized controlled clinical trials, particularly with a mortality/survival endpoint. Additional clinical trials of nonmortality endpoints in CHF patients and mortality trials in non-CHF patients were also selected to support possible pathophysiological insights for future CHF trials. The data, particularly for the accompanying tables, were initially extracted by a single reviewer using common qualitative guidelines as far as was possible within the different temporal, etiological and geographic frameworks of the original component studies. Conclusions are drawn from this data synthesis and from published overviews. Angiotensin converting enzyme (ACE) inhibition therapy is effective in reducing mortality and morbidity in severe left ventricular dysfunction and CHF. Other systemic vasodilators may also be beneficial. The effects of digitalis on survival and morbidity in CHF are presently uncertain, but should be resolved in the near future. Other inotropic agents, at least in the long term, are clinically detrimental. Diuretics decrease morbidity, but their effect on mortality in CHF remains unknown. Beta-blocker and magnesium therapy offer promise in CHF, but await definitive clinical trials evaluation. The current medical therapy of CHF should definitely include ACE inhibitors, probably diuretics and possibly other vasodilators. Further viable trials of promising new, and older heretofore under-evaluated, CHF therapies are needed. Additionally, innovative strategies are needed to deal with this disease which has an increasing prevalence. Two strategies, primary prevention of CHF and a 'Heart Function Clinic', are discussed.


Comparison of insulin-like growth factor interaction with satellite cells and embryonic myoblasts derived from the turkey. 1. The interaction of insulin-like growth factors (IGFs) with receptors on clonal-derived turkey satellite cells and embryonic myoblasts was compared using competitive binding assays and affinity cross-linking analysis. 2. Although [125I]IGF-I and [125I]IGF-II were displaced similarly by IGF-I and IGF-II within cell lines (P greater than 0.05), displacement, and therefore dissociation constants, differed between cell lines (P less than 0.0001). 3. Receptor cross-linking analysis using iodinated IGFs suggests that both IGF-I and IGF-II interact with the type I receptor on turkey embryonic and posthatch myogenic cells.


Effect of triiodothyronine on cAMP-dependent and cAMP-independent protein kinase activities in developing chick embryo liver. 1. Changes in liver cytosol cAMP-dependent kinase and cAMP-independent growth-related quercetin-inhibited casein kinase activities during chick embryo development were studied. 2. Both kinase activities were found to increase continuously during the experimental period. 3. Upon treatment of embryos with triiodothyronine, an activation of cAMP-dependent kinase A and cAMP-independent casein kinase was observed which was most pronounced on days 12 and 14.


Adaptation effect of sucrose on the salt taste response. 1. Intracellular recordings from mouse taste receptor cells were made to study cellular adaptation properties. 2. The sugar and salt receptor mechanisms of mammalian taste cells were investigated with cross-adaptation experiments. 3. The responding of taste cells to sucrose as well as to NaCl does not contradict the independency of their binding mechanisms. 4. With a mixture of sucrose and NaCl, different adsorption mechanisms are observed. 5. From these observations, it was concluded that adaptation occurs in the taste receptor cell.


Oxygen dependency of synaptic transmission at the squid Loligo pealei giant synapse. 1. Synaptic transmission at the squid giant synapse was blocked in 45 min by exposure to 0.02 atm oxygen but was maintained for more than 90 min in air (0.21 atm oxygen) or pure oxygen (1 atm). 2. Excitatory post-synaptic potential amplitude decreased in 0.02 atm oxygen but did not change in either 1 or 0.21 atm oxygen. Fast facilitation was increased in 0.02 atm oxygen only. 3. Post-synaptic resting membrane potential (Vm) and input resistance (Ro) remained unchanged in 1 or 0.21 atm oxygen but Ro was increased in 0.02 atm oxygen. 4. Our results suggest that severe hypoxia decreased the release of transmitter from the pre-synaptic terminal.


D-glucose transport activities in erythrocytes and hepatocytes of dogs, cats and cattle. 1. The activities of D-glucose transport and hexokinase were investigated in erythrocytes or hepatocytes of dogs, cats and cattle. 2. The mean D-glucose transport activity in erythrocytes of dogs was 6.0 nmol/min/mg protein, half the value of hepatocytes. 3. The activities of D-glucose transport in erythrocytes and hepatocytes or hepatic hexokinase of cats were about one-third of those of dogs. 4. Cattle with low blood glucose concentrations showed considerably low activities of D-glucose transport and hexokinase, about one-third of those of dogs.


Seasonal temperature and its influence on plasma corticosterone, triiodothyronine, thyroxine, plasma protein and packed cell volume in mature male chickens. 1. The relationship between seasonal changes in environmental temperature and hematological parameters was investigated in mature, single comb white leghorn (SCWL) male chickens. 2. Samples of blood plasma, obtained monthly from two groups of birds over two separate 12 month periods, were analysed for corticosterone (CT), 3,5,3'-triiodothyronine (T3), thyroxine (T4), plasma protein (PP), and packed cell volume (PCV). 3. Statistical analyses revealed that blood plasma concentrations of T3 were significantly correlated negatively with monthly dry-bulb temperatures. 4. There were no consistent or significant relationships between monthly dry-bulb temperature and CT, T4, PP or PCV over the two 12 month periods. 5. The results of this study indicate that blood plasma concentrations of T3 are influenced by season of year in mature, male domestic fowl.


Comparative hematology in marine fish.

Comparative hematology in marine fish. 1. A comparative study involving 80 species (14 ray, 14 shark and 52 teleost species) of marine fish found at the southeastern Brazilian coast is presented. 2. Active species displayed higher values for all hematological parameters studied when compared to the less active forms. 3. Mean values of hematocrit, hemoglobin concentration and red blood cell counts increased according to the sequence: rays, sharks, teleosts. 4. As a group, cartilaginous fish blood displayed larger and fewer erythrocytes containing more hemoglobin than teleosts; mean cell hemoglobin concentration was significantly higher in rays and sharks than in teleosts. 5. For all but the hemoglobin concentration, the hematological values studied revealed a marked contrast between bony and cartilaginous fishes which suggests distinct ways to accomplish their oxygen demands.


Erythrocytic nucleoside triphosphates in marine fish. 1. Whole blood purine nucleotides were determined in 19 species of selachians and 27 species of marine teleosts. Concomitant ATP and GTP were revealed inside the erythrocytes of almost all species studied. 2. ATP seems to be the main potential modulator of oxygen affinity in rays, sharks and teleosts, and GTP was not detected in only two teleost species. 3. The mean erythrocytic NTP concentration and the ratio between NTP and intraerythrocytic Hb concentrations in rays were the lowest (2.3 mM and 0.6 respectively), increasing in sharks (3.8 mM and 1.0), and further in teleosts (5.9 mM and 2.1). 4. The intraerythrocytic phosphate contents probably reflect different adaptative strategies associated with the fish habits and habitats, and with the Root effect expression.


The effects of feeding triiodothyronine on reproductively inhibited prairie deermice (Peromyscus maniculatus bairdii) from laboratory populations. 1. Two reproductively inhibited populations were given 250 ng triiodothyronine (T3) per gram of food for 35 days to elevate the reduced serum thyroid hormone concentration previously demonstrated in reproductively inhibited animals and to determine if the treatment would promote recovery. 2. Per capita food intake was significantly increased but per cent body fat tended to decrease and body weight did not significantly increase during T3 treatment. 3. Mean testis and seminal vesicle weights of T3-treated males were significantly increased and testis histology indicated some increase in spermatogenesis. 4. Mean ovary weights of T3-treated females only tended to increase although the uterine weights were significantly increased and ovarian histology revealed a significant increase in preovulatory follicles. 5. The limited recovery of reproductively inhibited animals may indicate that thyroid activity per se is related to, but not the direct cause of, the reproductive inhibition observed in populations.


Oxytocin and vasopressin change the activity of the contractile vacuole in Tetrahymena: newer contributions to the phylogeny of hormones and hormone receptors. 1. Primary interaction with oxytocin accounted for a significant prolongation of the time interval between two systolic contractions of the contractile vacuole in Tetrahymena, whereas primary interaction with vasopressin had no appreciable influence on that functional parameter. 2. Primary treatment (imprinting) with vasopressin increased sensitivity to vasopressin and reduced responsiveness to oxytocin. 3. Primary treatment (imprinting) with oxytocin did not increase cellular response either to oxytocin or to vasopressin on second exposure. 4. Oxytocin, which is chemically related to the antidiuretic hormone vasopressin, influences the water metabolism in protozoa; vasopressin develops a similar effect after imprinting. 5. The experimental observations allow conclusions on certain events involved in the phylogenesis of hormones and receptors.


Temporal evaluation of fatty acid-binding protein (FABP) activity in association with the development of atherosclerosis in the rabbit. 1. The relationship between atherosclerosis development and changes in arterial fatty acid binding protein (FABP) activity was investigated in the aortas of New Zealand rabbits which were fed an atherogenic diet containing 1% cholesterol and 3% peanut oil for 16 weeks. 2. At 4-week intervals, FABP activity, cholesterol and microsomal acylCoA:cholesterol acyltransferase (ACAT) activity were determined in aortic tissue and serum cholesterol was measured; age-matched normal rabbits served as control comparators. 3. Serum cholesterol increased from 35 mg/dl in the normal rabbits to 2290 mg/dl in the 16-week cholesterol-fed rabbits. 4. The microsomal fraction isolated from cholesterol-fed rabbit aortas exhibited a progressive elevation in ACAT activity as time on the diet increased. By 12-16 weeks, ACAT activity had increased approximately 10-fold relative to normal activity. 5. Arterial cholesterol content of the cholesterol-fed animals increased from less than 2 mg/g wet weight to greater than 10 mg/g wet weight at 12 and 16 weeks. In contrast, arterial FABP activity gradually decreased with time on the cholesterol diet; a significant decrease (P less than 0.05) was observed at 16 weeks, where palmitoyl CoA binding was decreased from 61.0 to 36.3 pmol/mg protein. 6. In the cholesterol-fed rabbits, total arterial cholesterol and ACAT activity showed a significant (P less than 0.05) inverse correlation to FABP activity with correlation coefficients of -0.93 and -0.95, respectively. 7. Additionally, FABP activity increased significantly (P less than 0.05) in the 16-week normal rabbit as compared to the 4-week normal rabbit, suggesting an age-dependent interaction.


Effect of lithocholic acid feeding on plasma lipoproteins and binding of radioiodinated human lipoproteins to hepatic membranes in rats. 1. Male Sprague-Dawley rats fed diets containing 0.25% lithocholic acid for 6 weeks exhibited elevated serum cholesterol. 2. The rats were fed diets containing 5 or 20% fat with and without the lithocholate and/or oxytetracycline-HCl. 3. The cholesterol elevation was associated with high density lipoprotein (HDL) and not very low density lipoprotein (VLDL) or low density lipoprotein (LDL). 4. Specific binding of human [125I]HDL to hepatic membranes was lowered in lithocholate-fed rats, but binding of human [125I]LDL to these membranes was not affected.


The influence of dietary protein on carcass composition and sexual maturity in a randombred population of Japanese quail (R1) and a subline of R1 selected for increased body weight. 1. Selection for body weight in Japanese quail has altered the protein requirements for growth and reproductive development in the selected line and randombred control population from which it was developed. 2. In both the selected and randombred lines, the protein requirement for growth is different from that for maximal reproductive development.


Growth and development of lines of Japanese quail (Coturnix coturnix japonica) divergently selected for body weight at 4 weeks of age. 1. Divergent selection for body weight resulted in significant increases and decreases in body weight and the relative weight of the pectoralis major breast muscle in heavy (HW) and light weight (LW) strains of Japanese quail compared with a randombred control strain (R1). 2. The relative weights of the abdominal fat pad and total carcass lipid were increased in the HW strain, particularly in the females. 3. Sexual maturity was similar in HW and R1 hens but was delayed in the LW hens. HW hens laid significantly more double-yolked eggs.


Changes in plasma glucagon, insulin and tissue metabolites associated with prolonged fasting in brown trout (Salmo trutta fario) during two different seasons of the year. 1. Pyrenean brown trout juveniles (Salmo trutta fario) were fasted for 50 days in late winter (experiment 1) and summer (experiment 2). Plasma insulin, glucagon and glucose and some metabolites in plasma and in tissues were analysed. 2. Glucagon increased significantly on the 3rd day of fasting in the winter experiment (controls 653.7 +/- 92.4 pg/ml, fasted 912.7 +/- 135.2 pg/ml), and the same tendency was observed on the 5th day in the summer experiment (controls 430.5 +/- 56.2 pg/ml, fasted 555.5 +/- 95.3 pg/ml). During this initial period of fasting, plasma glucose was maintained in both experiments (75.5 +/- 4.7-67.6 +/- 4.1 mg/100 ml), but from day 8, glucose and glucagon decreased simultaneously. 3. Insulin decreased from the beginning of fasting, reaching lowest values after 50 days of fasting (winter experiment: controls 6.4 +/- 0.3 ng/ml, fasted 1.6 +/- 0.1 ng/ml; summer experiment: controls 4.8 +/- 0.1 ng/ml, fasted 1.2 +/- 0.2 ng/ml). Glucagon/insulin molar ratio (G/I) increased after 3 days in the winter experiment (controls 0.21 +/- 0.02, fasted 0.39 +/- 0.05), while in the summer experiment, the ratio rose from day 5 and reached a peak at day 30 (controls 0.16 +/- 0.02, fasted 0.48 +/- 0.07). 4. Muscle proteins were significantly mobilized after 50 days of fasting. Visceral index decreased significantly after day 15 while liver glycogen was already significantly lower at day 8. However, in the summer experiment, a transitory increase of liver glycogen was observed at day 30, coinciding with the peak of G/I.


The influence of high dietary protein, energy and mineral intake on deficient young camel (Camelus dromedarius)--I. Changes in metabolic profiles and growth performance. 1. The main forage for camels in northern Djibouti (mangrove with Avicennia marina) is very poor in nitrogen and energy. In a trial, 32 young camels (less than 2 years old) were used in four groups of eight each. 2. All the camels received mangrove as basal diet ad lib. 3. After 1 month, the camels received mineral supplementation in copper and zinc (groups 1 and 3) or/and a concentrate rich in protein and energy (groups 2 and 3) or continued with the basal diet (controls). 4. Any supplementation was stopped after 2 months for 1 month. 5. Growth performance was 550 g/day (concentrate-supplemented camels) and 570 g/day (concentrate+mineral-supplemented camels). 6. The growth was negative for the two others groups (-260 g/day). 7. Food intake of mangrove was slightly more important with mineral supplementation only and with mineral+concentrate supplementation. 8. The changes in metabolic profiles have shown an important catabolism in non-supplemented animals, an increase of urea and free fatty acid concentrations in plasma and a decrease of glucose concentrations. 9. Three camels died in the control group with symptoms of starvation and signs of liver damage (increase of liver enzymes glutamate dehydrogenase and gamma-glutamyl transferase).


The influence of high dietary protein, energy and mineral intake on deficient young camel (Camelus dromedarius)--II. Changes in mineral status. 1. Mangrove Avicennia marina is poor in some trace elements such as copper, zinc and manganese. In a trial we used 32 young camels divided into four groups. 2. Groups 1 and 3 were supplemented with copper and zinc in drinking water after 1 month of mangrove feeding. 3. Groups 2 and 3 received concentrate rich in protein and energy. The supplementation was stopped after 2 months. 4. All the camels were deficient in trace elements at the beginning of mineral supplementation. 5. The plasma concentration of copper increased significantly up to normal levels (less than 70 micrograms/100 ml) in energy protein supplemented groups, but the quantity supplied (100 mg of copper sulphate/day) was not sufficient to maintain this level after the end of supplementation. 6. The original zinc deficiency was too severe to observe a significant effect of the mineral supplementation. 7. Calcium, magnesium and phosphorus levels were improved during the supplementation period in protein-energy supplemented groups. 8. A high interaction between mineral absorption and quality of the diet was observed. A well-balanced diet seems essential to avoid deficient mineral status.


Third International Workshop on Human Chromosome 17 Mapping. Highlights of the meeting this year include progress in merging two independently derived genetic maps, expansion of the composite hybrid breakpoint map, and enhancements in working group communications through the chromosome 17 file server at Baylor. Progress is also being made in developing STS primers for framework markers and reference markers. The task remains of fully reconciling the framework map and composite breakpoint map with the list of chromosome 17 reference markers (Solomon and Ledbetter, 1991). There remain several gaps in the overall map, particularly near the distal end of the long arm, where there has been limited activity.


Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.


Localization of two new DNA markers on the linkage map of human chromosome 6q. Recently, an autosomal homolog of the dystrophin gene (DMDL) was identified on chromosome 6q24. As part of our analysis of the DMDL locus, we endeavoured to isolate DNA markers to further define the genetic map of this region. We have isolated and characterized two new genetic markers in the region of the DMDL locus, the RFLP D6S129 and a (CA)n dinucleotide repeat polymorphism within the DMDL gene itself and have positioned them on the existing genetic map of chromosome 6q. These markers will be important in testing the hypothesis that the DMDL gene is the locus responsible for autosomal forms of neuromuscular disease.


Localization of mouse parathyroid hormone-like peptide gene (Pthlh) to distal chromosome 6 using interspecific backcross mice and in situ hybridization. The single copy parathyroid hormone-like peptide gene (Pthlh) was mapped to distal mouse chromosome 6 using genetic linkage analysis with a panel of DNA samples from interspecific backcross mice. In all 114 meiotic events examined, the Pthlh locus cosegregated with the locus for the Kirsten ras-2 gene (Kras-2) which was previously localized to distal mouse chromosome 6. In addition, Pthlh was localized to chromosome 6 band F-G and the mouse parathyroid hormone Pth was localized to chromosome 7 band F, by in situ hybridization. These studies confirm the previous localization of Pthlh to mouse chromosome 6 using somatic cell hybrids and show that the Pthlh/PTHLH locus is a part of a conserved linkage group between distal mouse chromosome 6 and the proximal segment of the short arm of human chromosome 12.


Inguinal hernia in paediatric age-group: Ibadan experience. Ninety-nine children with inguinal hernia undergoing elective surgery over a 2-year period at the University College Hospital, Ibadan, were studied. These were all indirect inguinal hernias. There was a male-female ratio of 5.6:1. Fifty-six per cent of cases were located on the right side, twenty-eight per cent of cases on the left side, and fifteen per cent were bilateral. Ipsilateral hydrocoeles and ipsilateral undescended testes were in 8% and 19% of cases respectively. There was no significant morbidity, mortality or recurrence in this series.


Evidence for protein kinase-C mediation of the neurotensin-induced activation of tyrosine hydroxylase in tuberoinfundibular dopaminergic neurons. The purpose of the present study was to determine whether neurotensin acts within the arcuate nucleus/median eminence to activate tyrosine hydroxylase (TH) within tuberoinfundibular dopamine neurons. The role of Ca2+/phospholipid-dependent protein kinase (protein kinase-C) in the regulation of TH and its involvement in the neurotensin-induced activation of TH within tuberoinfundibular dopamine (TIDA) neurons also was investigated. The activity of TH within TIDA neurons was assessed by quantification of the formation of 3,4-dihydroxyphenylalanine in the arcuate nucleus/median eminence after inhibition of 3,4-dihydroxyphenylalanine decarboxylase. Neurotensin (0.1-10 nM) increased the activity of TH within the arcuate nucleus/median eminence under in vitro conditions by approximately 80%. The activity of TH in the arcuate nucleus/median eminence also was increased approximately 55% by the phorbol ester 12-O-tetradecanoyl(phorbol-13-acetate) (1-100 nM), which activates protein kinase-C. Sphingosine (10 microM), an inhibitor of protein kinase-C, attenuated the activation of TH within TIDA neurons that was induced by both 12-O-tetradecanoyl(phorbol-13-acetate) and neurotensin. Sphingosine alone did not alter the activity of TH, nor did it alter the (Bu)2cAMP-induced activation of TH in the arcuate nucleus/median eminence. It is concluded that neurotensin acts directly within the arcuate nucleus/median eminence to activate TIDA neurons. Furthermore, it is suggested that the activity of TH within these neurons is enhanced after the activation of protein kinase-C and that protein kinase-C may mediate the neurotensin-induced activation of TH within these hypothalamic dopamine neurons.


Beta 1-adrenergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via receptors positively coupled to adenylate cyclase. The release of GnRH evoked by norepinephrine (NE) was studied in GT1 GnRH neuronal cell lines in superfusion and static cultures. GnRH release from static cultured GT1-7 cells was stimulated by NE in a dose-dependent fashion. This effect was mimicked by the nonsubtype-selective beta-adrenergic agonist isoproterenol and blocked by the beta-adrenergic antagonist propranolol and the beta 1-adrenergic subtype-specific antagonist CGP 20712A. However, the stimulation of GnRH release by NE was not affected by the beta 2-, alpha-, alpha 1-, or alpha 2-adrenergic antagonists ICI 118.551, phentolamine, prazosin, or yohimbine, respectively. Superfusion of GT1-1 cells with NE for 60-100 min resulted in rapid and sustained increases in GnRH secretion. The NE-stimulated GnRH release showed a higher amplitude and longer duration than the spontaneous GnRH pulses characteristic of GT1-1 cells. In parallel to the stimulation of GnRH release, NE also rapidly increased (first observed at 60 sec) the intracellular concentration of cAMP in isobutylmethylxanthine-pretreated GT1-1 and GT1-7 cells in a dose-dependent fashion. The stimulation of intracellular cAMP concentration was also mimicked by isoproterenol and blocked by propranolol and CGP 20712A. In addition, GT1 cells express beta 1- but not beta 2-adrenergic receptor mRNA, as probed by Northern blot analysis. These results demonstrate a direct stimulatory effect of NE on GnRH neurons. The pharmacological evidence and the mRNA analysis are consistent with NE acting through a beta 1-adrenergic receptor positively coupled to adenylate cyclase.


Transcriptional antagonism of phorbol ester-mediated induction of plasminogen activator inhibitor types 1 and 2 by cyclic adenosine 3',5'-monophosphate. Gene expression of plasminogen activator inhibitor (PAI) types 1 and 2 is modulated by the protein kinase-C (PKC) and cAMP-dependent protein kinase-A (PKA) signal transduction pathways. To determine whether the PKC and PKA pathways functionally interact during modulation of PAI gene expression, we assessed changes in gene transcription rates, mRNA, and antigen levels of PAI-1 and PAI-2 in HT-1080 fibrosarcoma cells treated with the PKC activator phorbol 12-myristate 13-acetate (PMA), alone or in combination with cAMP agonists and analogs. PMA produced a transient increase in PAI-1 and a sustained increase in PAI-2, which was evident at the level of gene transcription and mRNA. Treatment with the cAMP agonist forskolin or the cAMP analog 8-bromo-cAMP decreased constitutive and PMA-mediated expression of PAI-1 mRNA. PAI-2 mRNA was below detection limits in nontreated and cAMP-treated cells. However, elevated levels of cAMP reduced the stimulatory effect of PMA on PAI-2 mRNA. The antagonism of the PMA effect by cAMP was evident at the level of gene transcription, suggesting that the end point of the functional interplay between the PKC and PKA pathways requires modulation of a nuclear transcription factor(s). Our results suggest that the PKC- and PKA-dependent signaling pathways have counteractive effects on transcriptional expression of the PAI-1 and PAI-2 genes in HT-1080 cells.


Influence of gastric acid on circulating somatostatin-14 and -28 released after insulin-induced hypoglycemia in conscious dogs. Insulin hypoglycemia is a potent mechanism for somatostatin secretion into the circulation. Whether the associated increase in gastric acid mediates the rise of one or both principle molecular forms of somatostatin, somatostatin-14 (S-14) and somatostatin-28 (S-28), was examined in four conscious dogs. Somatostatin molecular forms were separated by gel filtration chromatography after extraction of acidified plasma on octadecyl silyl cartridges and quantified by RIA. Basal plasma levels of S-14 and S-28 were 3.4 +/- 0.2 and 4.1 +/- 0.6 fmol/ml, respectively. After hypoglycemia induced by insulin, plasma S-14 increased by 29.5 +/- 3.9 fmol/ml (P less than 0.001), and plasma S-28 increased by 7.2 +/- 0.9 fmol/ml (P less than 0.01). Suppression of hypoglycemia-mediated gastric acid secretion after the administration of omeprazole or ranitidine inhibited elevations of S-14 by 82 +/- 6% (P less than 0.001) and 81 +/- 7% (P less than 0.001), respectively, but had no effect on the rise of S-28. Atropine (50 micrograms/kg, iv), which also suppresses gastric acid secretion after insulin hypoglycemia, decreased S-14 by 59 +/- 3% (P less than 0.01) without influencing S-28. Atropine given after omeprazole treatment, however, increased S-14 levels observed after atropine (P less than 0.001) or omeprazole (P less than 0.001) alone and was equivalent to control levels. S-28 remained unaltered after atropine and omeprazole treatment. These results in conscious dogs indicate that after vagal stimulation induced by insulin hypoglycemia 1) both S-14 and S-28 are released into the circulation, but S-14 predominates; 2) gastric acid contributes directly to the stimulation of S-14, but not S-28, secretion; 3) muscarinic inhibitory mechanisms participate in the regulation of S-14 secretion, and this mechanism is amplified when vagally stimulated gastric acid secretion is suppressed; and 4) nonmuscarinic mechanisms mediate in part S-28 secretion. This study suggests the presence of a reciprocal functional relationship between gastric acid secretion and circulating S-14 that is mediated by vagal muscarinic mechanisms.


Beta-endorphin innervation of dopamine neurons in the rat hypothalamus: a light and electron microscopic double immunostaining study. Pharmacological data suggest that opiates, acting indirectly via the catecholaminergic system, are involved in the inhibition of LH release and the stimulation of PRL secretion. The aim of this study was to demonstrate on the ultrastructural level whether beta-endorphin-immunoreactive fibers form synaptic contacts with hypothalamic dopaminergic neurons. Light and electron microscopic double immunostaining experiments were performed on vibratome sections prepared from the hypothalamus of acrolein-fixed female rat brains. Immunoreactivity for beta-endorphin was visualized by a dark blue to black nickel ammonium sulfate-intensified diaminobenzidine reaction, and in a consecutive immunostaining procedure, the tyrosine hydroxylase-immunoreactive dopamine cells were labeled with the brown diaminobenzidine reaction product. Under the light microscope, beta-endorphin axon terminals were found to contact dopamine cell bodies and dendrites throughout the hypothalamus. The majority of opiate target dopamine neurons were found in the periventricular area, retrochiasmatic area, and lateral part of the zona incerta. A much smaller number was observed in the dorsomedial hypothalamic nucleus and the anterior hypothalamus, and only a very few dopamine cells could be detected in contact with beta-endorphin axons in the arcuate nucleus (particularly in the posterior part where the beta-endorphin cells are located) and the medial part of the zona incerta. After light microscopic examination and color photography, the double immunostained sections were embedded for correlated electron microscopy to verify and characterize the putative synaptic connections. Electron microscopy revealed symmetric synaptic connections between beta-endorphin-immunoreactive boutons and tyrosine hydroxylase-immunopositive cell bodies and dendrites. These results together with the observation of dopamine innervation of LHRH-producing neurons and progesterone receptor-containing cells indicate that neurons of the hypothalamic dopaminergic system probably mediate opiate effects on hypophyseal hormone secretion.