Neuroprotective effects of phenyl-t-butyl-nitrone in gerbil global brain ischemia and in cultured rat cerebellar neurons.

We examined the ability of phenyl-t-butyl-nitrone (PBN), an electron spin trapper, to attenuate ischemia-induced forebrain edema and hippocampal CA1 neuronal loss in gerbils, and to protect rat cerebellar neurons in primary culture from glutamate-induced toxicity. PBN, given i.p. at 75 or 150 mg/kg 30 min before ischemia (5 min occlusion), increased survival (at 7 days) of CA1 neurons from 60 +/- 14 (vehicle-treated, n = 17) to 95 +/- 15 (P less than 0.05, n = 15) and 145 +/- 3 (P less than 0.01, n = 15), respectively. When gerbils were treated with PBN (50 mg/kg, i.p.) immediately and 6 h after reperfusion, followed by b.i.d. for an additional 2 days, CA1 neurons survival improved from 35 +/- 9 (vehicle, n = 20, 6 min occlusion) to 106 +/- 17 (P less than 0.01, n = 13). In gerbils exposed to a more severe ischemia (10 min), pretreatment with 150 mg/kg PBN increased the survival of CA1 neurons from 6 +/- 6 (vehicle) to 27 +/- 10 (P less than 0.05, n = 11). Pretreatment with PBN, at 150 mg/kg, reduced forebrain edema (following 15 min ischemia) by 24.7% (P less than 0.01, n = 16). PBN at 50 mg/kg, i.p. had no hypothermic effect and at 75 or 150 mg/kg caused a transient hypothermia. The presence of PBN in the brain was confirmed in microdialysis samples and brain tissue extract using HPLC. In vitro, PBN protected rat cerebellar neurons against 100 microM glutamate-induced toxicity with an EC50 value of 2.7 mM. Our results further support the concept that free radicals contribute to brain injury following ischemia and suggest the potential therapeutic application of electron spin trappers in stroke.


Activation of kappa-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca2+ in secretory nerve terminals of the neurohypophysis.

Nerve endings of the magnocellular neurohypophysial neurones possess kappa-opioid receptors. Using a preparation of isolated terminals from the neurohypophysis we studied kappa-opioid effects on secretion of oxytocin and vasopressin and on intracellular Ca2+ concentration ([Ca2+]i) measured fluorimetrically or using digital video imaging with Fura-2. The dihydropyridine Ca(2+)-channel antagonist nicardipine reduced [Ca2+]i responses to K(+)-depolarisation (30-40 mM K+) by 55-75% and inhibited evoked secretion of oxytocin and vasopressin to a similar extent. The selective kappa-receptor agonist D-Pro10 Dynorphin A 1-11 (DPDYN) substantially inhibited K+ evoked secretion of oxytocin by 40-90% and secretion of arginine vasopressin (AVP) by 20-50%. DPDYN caused only a 10% reduction in the average total population [Ca2+]i response to K+ depolarisation. No sub-population of inhibitory responses was observed when samples of individual terminal [Ca2+]i responses were examined with imaging. Although kappa-receptors are coupled to Ca(2+)-channels at neuronal somata our data suggest that alternative effector mechanisms operate in these secretory nerve endings.


Glycine response in isolated dorsal cochlear nucleus of C57BL/6J mouse.

Pharmacological properties of glycine (Gly)-induced Cl- current (ICl) in the dorsal cochlear nucleus (DCN) neurons acutely dissociated from C57BL/6J mouse were investigated in the whole-cell configuration of the patch-clamp technique. Gly-induced ICl increased in a sigmoidal manner with higher Gly concentrations. Strychnine blocked the Gly response competitively at low and non-competitively at high concentrations. Both glutamate (Glu) and N-methyl-D-aspartate (NMDA) responses were augmented by adding 10(-6) M Gly, at which concentration Gly did not induce any ICl. This facilitation was not affected by strychnine. Our results clearly show the existence of strychnine-sensitive and -insensitive glycine receptors in the DCN neurons.


Voltage-gated potassium current and resonance in the toadfish saccular hair cell.

Resonance of the membrane potential in response to a perturbing current has been demonstrated in sensory hair cells of many acoustico-lateralis systems and modelled as the result of the interaction of passive membrane properties and the magnitude and kinetics of activation and deactivation of an outward calcium-activated potassium current (IKCa) and an inward calcium current (ICa). However, the majority of the hair cells of the toadfish saccule have, in addition to IKCa, a voltage-gated potassium current (IK) active in the same membrane potential range as IKCa but with considerably slower activation and deactivation kinetics. Additionally, some of these cells have an A current (IA). In the present work, the resonance of cells with these three outward potassium currents were compared with those from cells containing only IKCa. Hair cells with only IKCa produced a high-quality factor (Q) resonance with symmetrical ringing at current onset and termination. In many cells having the IK, resonance could be evoked as a high Q ringing only at the onset of the current pulse. The resonance at command onset was dependent on the presence of IKCa and could be converted into a spike by blocking the IKCa with TEA. Some hair cells with IKCa and IK produced spikes rather than resonance at all holding potentials tested. This spiking was seen in cells with low levels of IKCa or slowly activating IKCa and with cells with IA. The presence of cells with such different response modes implies a difference between hair cells in their role in sensory coding.


Chronic exposure to alcohol during development alters the responses to excitatory amino acids in cultured Purkinje neurons.

The effects of chronic alcohol exposure during development on the responses evoked by glutamate and the selective excitatory amino acid receptor agonists quisqualate (Quis) and kainate were studied in cultured cerebellar Purkinje neurons. The cultures were treated with 22 mM or 44 mM ethanol continuously for one or two weeks during the main period of morphological and physiological development. Extracellular recordings used for most studies characterized the responses to all 3 agonists as initial increase in simple spike firing, usually including a period of burst activity, followed by reduced activity or total inhibition, then return to control firing pattern. Analysis of these responses and background spontaneous activity showed several significant differences between control and ethanol treated Purkinje neurons. Background spontaneous firing, agonist evoked firing, the initial period of activity of the response to Quis, and the inhibitory period of the response to glutamate were all significantly reduced in the chronically treated neurons; the inhibitory period of the response to kainate was significantly increased. In contrast to the effects of chronic ethanol exposure, acutely administered ethanol significantly increased background spontaneous firing and the inhibitory period of the response to Quis. Thus, administering both acute and chronic ethanol altered the responses evoked by excitatory amino acids in the developing Purkinje neurons. The effect of chronic ethanol exposure on some response components was similar for all agonists tested and may be linked to changes in intrinsic membrane properties. However, alterations in the inhibitory component of the agonist responses were agonist specific, indicating that receptor-linked actions of ethanol were involved.(ABSTRACT TRUNCATED AT 250 WORDS)


In vivo modulation of excitatory amino acid receptors: microdialysis studies on N-methyl-D-aspartate-evoked striatal dopamine release and effects of antagonists.

Striatal dopamine (DA) release was measured following intrastriatal (i.s.) administration of N-methyl-D-aspartate (NMDA) to unanesthetized, freely-moving rats. One hour after insertion of a removable microdialysis probe and perfusion with normal Ringer's solution, a modified Ringer's solution containing 100 mM potassium (high-K+ Ringer's) was used to standardize the preparation. DA release following i.s. administration of NMDA (12.5 mM in normal Ringer's) was dose-dependent. When NMDA (12.5 mM) was administered in high-K+ Ringer's, DA release was greatly potentiated. Administration of the competitive NMDA receptor antagonist aminophosphonovalerate (APV) in normal Ringer's prior to treatment with NMDA in high-K+ Ringer's resulted in a significant reduction of DA release compared to control animals. In contrast, administration of APV priot to treatment with NMDA in normal Ringer's resulted in a significantly increased release of DA compared to controls. Administration of the non-competitive NMDA antagonist, dextromethorphan (DXT) prior to treatment with NMDA in normal Ringer's or NMDA in high-K+ Ringer's caused significant reductions of DA release compared to controls. Intrastriatal DXT also caused dose-dependent inhibition of high-K+ Ringer's-induced DA release. Similarly, administration of the non-specific calcium channel blocker, cadmium, prior to treatment with NMDA resulted in a significant decrease when compared to control values. Results of this study indicate that dose-dependent NMDA-induced striatal DA release is greatly potentiated by potassium suggesting that under physiological conditions in vivo, striatal NMDA receptors are mostly inactivated.(ABSTRACT TRUNCATED AT 250 WORDS)


Differences in neuronal and glial cell phenotypic expression in neuron-glia cocultures: influence of glia-conditioned media and living glial cell substrata.

Neuron-glia cocultures were prepared using, as a source for glial cells, either C6 glia (2B clone) of early (2B23) or late (2B111) passages or advanced passages of glial cells derived from primary cultures prepared from aged mouse cerebral hemispheres (MACH). Six-day-old chick embryo cerebral hemispheres (E6CH) were the source of neuron-enriched cultures. Glutamine synthetase (GS) activity was used as a marker for astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was used as a marker for oligodendrocytes. GS activity was markedly enhanced in cocultures of E6CH neurons and 2B23 glioblastic cells, whereas GS activity was reduced in cocultures of E6CH neurons and 2B111 astrocytic glia. In contrast, CNP activity was enhanced in cocultures of C6 glial cells with E6CH neurons. Glial cells from aged mouse brain did not respond to coculturing with E6CH neurons. It appears from these findings that neuronal input enhances the differentiation of glioblastic cells to either astrocytic or oligodendrocytic expression, whereas it decreases the activity of committed astrocytes. In contrast, glial cells from aged mouse brain do not respond to neuronal input. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, was enhanced only when E6CH cultures were grown in conditioned medium (CM) from 2B23 glioblastic cells. In contrast, ChAT activity was markedly diminished when E6CH neurons were cocultured with MACH glial cells but not when grown in CM from MACH glial cells. Thus, humoral factors from immature glial cells appear to enhance cholinergic neuronal phenotypic expression whereas cell-cell membrane contacts with aged glial cells diminish cholinergic phenotypic expression. The findings present supportive evidence that neuron-glia interrelationships are age dependent.


[Schizophrenia and Wilson's disease].

An 18 year-old male first presented a clinical picture of acute psychosis with two recurrences at ages 22 and 23. The diagnosis made at that time was paranoid schizophrenia. Twelve years after his first psychiatric hospitalization, it was discovered that he was suffering from Wilson's disease. In retrospect, the clinical picture was atypical, notably with an important neurologic involvement mainly parkinsonism almost uncontrollable and aggravated with neuroleptics. The chelating treatment with d-penicillamine resulted in partial improvement of the neurological involvement because the extrapyramidal and neurovegetative symptoms persisted. The psychiatric symptoms improved with fewer neuroleptics than during the 12 previous years. However, neuroleptics had to be continued because of the delay in diagnosing the illness, which diminished the efficiency of the single chelating treatment. The clinical presentation and therapeutic response of this patient strongly suggest a link between the cerebral intoxication by copper and the psychiatric symptoms.


A new chromosomal breakpoint in Ph positive, bcr negative chronic myelogenous leukemia. Report of a case.

We report a new case of Ph positive chronic myeloid leukemia (CML) without the classical rearrangement in Mbcr. By Southern blot analysis the molecular breakpoint was mapped 3 to 8 kb upstream of Mbcr. This region has not been shown to be rearranged in any other described case of CML. We did not detect any specific abnormal BCR-ABL transcript even with the use of the very sensitive RNA-PCR technique.


Loss of heterozygosity at D3S2 locus of short arm of chromosome 3 in chronic myelogenous leukemia.

Loss of heterozygosity (LOH) on the short arm of chromosome 3 was studied in four patients with chronic myelogenous leukemia (CML). The bcr gene rearrangement-negative spleen cells and a B-cell line were used as normal tissue controls. Five probes showing restriction fragment length polymorphisms (RFLP) and a variable number of tandem repeats on chromosome 3 were used. DNA patterns in Southern blotting were compared between normal cells and leukemic cells. One of the four patients had LOH at the D3S2 locus mapped to 3p14.3-3p21.3. The LOH was detected in the blastic phase, but not in the chronic phase. This patient showed normal chromosomes 3 in the blastic phase. These data suggest the possibility of the existence of LOH in CML, occurring as a secondary event in the blastic phase, and which might have been induced by submicroscopic deletion or somatic recombination.


Detection of DNA alterations in human bladder tumors by DNA fingerprint analyses.

DNA fingerprint analyses were used to examine the constitutional and tumor DNA from 22 bladder tumor patients. DNA alterations, such as loss of bands, new bands, and intensity shifts were observed in 10 of the 22 patients. The most frequent DNA alteration, occurring in 80% of the patients, was a complete loss of one or several bands. Fingerprint abnormalities were present both in low-malignant superficial tumors and in high-malignant invasive tumors, but were also lacking in the latter group. Apparently no relationship exists between fingerprint abnormalities and gross chromosomal aberrations or the proportion of S-phase cells as measured by flow cytometry or development of recurrent tumors during a limited observation period. Thus, whether fingerprint aberrations express genetic alterations directly involved in the malignancy potential of bladder carcinoma remains an open question.


Difference in the response of neu and ras oncogene-induced rat mammary carcinomas to early and late ovariectomy.

Rat mammary carcinomas were induced by directly inserting activated neu or ras genes into in situ rat mammary ductal cells using replication-defective retroviral vectors. neu was over 200 times more potent than ras in inducing rat mammary carcinomas. Ovariectomy 2 days postinfection dramatically reduced the occurrence of carcinomas induced by neu and extended their latency. In general, early ovariectomy had much less effect on the occurrence of carcinomas induced by ras and had no significant effect on their latency. Carcinomas induced by neu in ovariectomized rats had down-regulated estrogen receptor and progesterone receptor, while those induced by ras had only down-regulated progesterone receptor. Fully progressed mammary carcinomas in intact rats induced by both neu and ras had a similar response to ovariectomy, with an approximate regression rate of 60%. These data suggest that the activation of ras, but not neu, can replace at least some functions performed by ovarian hormones in the early phases of mammary carcinogenesis. These data also suggest a role for antiestrogen drug therapy in the prevention of neu-associated breast cancer.


Evidence for a tumor suppressor gene on chromosome 19q associated with human astrocytomas, oligodendrogliomas, and mixed gliomas.

Previous studies have shown frequent allelic losses of chromosomes 9p, 10, 17p, and 22q in glial tumors. Other researchers have briefly reported that glial tumors may also show allelic losses of chromosome 19, suggesting a putative tumor suppressor gene locus on this chromosome (D. T. Ransom et al., Proc. Am. Assoc. Cancer Res., 32:302, 1991). To evaluate whether loss of chromosome 19 alleles is common in glial tumors of different types and grades, we performed Southern blot restriction fragment length polymorphism analysis for multiple chromosome 19 loci in 122 gliomas from 116 patients. Twenty-nine tumors had loss of constitutional heterozygosity of 19q, and four tumors had partial deletions of 19q. Allelic losses on 19q were restricted to grade III anaplastic astrocytomas (4/9) and grade IV glioblastomas (11/46), grade II oligodendrogliomas (2/5) and grade III anaplastic oligodendrogliomas (2/2), and grade II (5/8) and grade III (5/7) mixed oligoastrocytomas. These data demonstrate genetic similarities between astrocytomas, oligodendrogliomas, and mixed glial tumors and indicate the presence of a glial tumor suppressor gene on chromosome 19q.


Adenovirus E1A targets key regulators of cell proliferation.

Studies of E1A support the notion that small DNA tumour viruses target cellular pathways at key points that are amenable to regulation. In the case of E1A, these targets appear to be points of control of cellular proliferation and, in particular, proteins that regulate the progression of cells from G0 and G1 phases of the cell cycle into the S phase. In several cases, recent studies have identified complexes between the viral targets and other cellular proteins. These interactions may provide insight not only into the mechanism of E1A mediated transformation but also into the control of proliferation in normal cells.


Functional evidence for human tumour suppressor genes: chromosome and molecular genetic studies.

There is now ample genetic and some functional evidence for the existence of tumour suppressor genes. Although much of the functional evidence has been derived from somatic cell hybrid and chromosome transfer studies, it is critical that cloned candidate tumour suppressor genes be used in such functional assays. Our experience with RB and p53 indicates that much will be learned about the control of the cell cycle from studies of tumour suppressor genes. However, the handful of candidate genes cloned to date also indicates a variety of cellular localizations and cellular functions. Thus, just as oncogenes seem to act to promote growth at many levels of metabolic control, it would seem that tumour suppressor genes act in complementary ways to control cell proliferation. The molecular genetic study of cancer has truly entered an exciting phase.


Cardiovascular mass and ventricular function after celiprolol in Wistar-Kyoto and spontaneously hypertensive rats.

The effects of a new beta 1 adrenergic receptor blocking agent with beta 2 receptor agonistic properties on cardiovascular mass, left ventricular function, and aortic distensibility were studied in Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 20 male SHR and 20 male WKY rats (10 treated and 10 untreated) aged 22 weeks were studied after three weeks of treatment. Cardiovascular mass was measured and left ventricular function was assessed using electromagnetic flowmetry while rapidly infusing whole blood at pharmacologically reduced mean arterial pressure and at pretreatment arterial pressure levels. Aortic distensibility was assessed by obtaining pressure-volume relationships in isolated aortic segments. Mean arterial pressure was reduced without changing cardiac output in SHR (p less than 0.01); it remained unchanged in WKY despite reduced cardiac output. Most noteworthy, and like no other agent studied to date, celiprolol significantly reduced both left and right ventricular as well as aortic mass in both WKY and SHR. Despite these similar mass reductions, celiprolol improved left ventricular function (p less than 0.01) and aortic distensibility (p less than 0.05) only in the SHR, a function maintained even when mean arterial pressure was increased abruptly to pretreatment levels. Unlike other beta receptor blockers (or any other agent studied in the SHR), celiprolol was effective in reducing mass of right and left ventricles and of aorta; decreasing mean arterial pressure through a fall in total peripheral resistance; and improving left ventricular function and aortic distensibility in the SHR. In contrast, while these structural changes were also produced in WKY, they were not associated with similar functional responses. These findings provide further support for the thesis of a structural and haemodynamic dissociation in antihypertensive therapy.


Effects of alpha and beta adrenergic blockade on coronary arterial microvessels in the beating canine heart.

The aim was to clarify the effects of alpha and beta adrenergic blockade on coronary arterial microvessels and to assess the role of alpha and beta adrenergic tone in normally beating hearts. 47 anaesthetised open chest dogs were studied. The diameters of epicardial arterial microvessels were measured in beating hearts using an incident light fluorescence microscope equipped with a floating objective. Drugs were infused into the left anterior descending coronary artery keeping the heart rate and aortic pressure at control levels. To examine the effect of alpha adrenergic blockade, phentolamine (100 micrograms.kg-1) was given in the absence or presence of beta adrenergic blockade (propranolol 50 micrograms.kg-1). To examine the effect of beta adrenergic blockade, propranolol (50 micrograms.kg-1) or three doses of ICI 118,551 (a selective beta 2 antagonist, 0.1, 0.5, and 1.0 microgram.kg-1.min-1) was given. Coronary arterial microvessels were divided into three groups according to the control diameters (D) of small (D less than 100 microns), medium (100 less than or equal to D less than 200 microns) and large (D greater than or equal to 200 microns) groups. In the absence of beta adrenergic blockade, phentolamine significantly dilated all vessel groups: small +19.6 (SEM 5.6)%, medium +5.8(2.3)%, large +5.3(0.9)%. In the presence of beta adrenergic blockade, the vasodilator effect of phentolamine was completely abolished. Propranolol constricted all vessel groups: small -3.6(1.1)%, medium -4.8(1.0)%, large -3.5(1.0)%. ICI 118,551 significantly constricted the large vessel group [-2.5(0.6)%] at the mid dose, and the medium and large vessel groups [medium -3.1(0.8)%, large -3.5(1.3)%] at the highest dose. These data indicate that (1) the vasodilator effect of phentolamine is induced by beta adrenergic stimulation; (2) resting alpha adrenergic tone of coronary arterial microvessels is minimal in normally beating hearts, and (3) resting beta adrenergic tone may play a physiological role in coronary arterial microvessels, and beta 2 adrenergic tone predominates in arterial microvessels greater than 100 microns in diameter.


[Subdural empyema. Present possibilities of diagnosis and therapy].

The authors give an account of their experience with the diagnosis and treatment of subdural empyema. In 1953-1991 in the neurosurgical department in Olomouc a total of five patients with this diagnosis were treated. None of them died. In all patients before operation symptoms of meningeal irritation, fever and in four patients a focal neurological symptomatology was observed. The authors reached the conclusion that the best surgical approach is craniotomy or craniectomy which should be preferred to minor surgical operations. Treatment of the primary inflammatory focus leading to the development of subdural empyema must be part of the intracranial operation. Regular postoperative follow-up of the patient by means of CT makes it possible to detect in time relapses and leads to early surgical operation. The authors mention experience focused on possible errors in the CT diagnosis. The creation of the picture of subdural collection precedes oedema of the hemisphere with a shift of the structures in the median line. The subdural collection occurs in the subsequent stage of development of the disease.


H-2M3 presents a Listeria monocytogenes peptide to cytotoxic T lymphocytes.

We report evidence that a major histocompatibility complex-encoded nonclassic class I molecule presents a foreign peptide to cytotoxic T lymphocytes (CTL) during an infection. Mice immunized with virulent Listeria monocytogenes generate CD8+ CTL with alpha beta receptors specific for a bacterial peptide presented by a conserved class I molecule encoded in the M region of the major histocompatibility complex. The Listeria peptide is digested by carboxypeptidase Y but resists aminopeptidase M, and only peptides with N-formyl methionine competitively block its presentation to CTL. Transfection with the H-2M3d gene enables a negative (H-2w17) cell line to present the bacterial peptide. One function, therefore, of H-2M3 is to present bacterial peptides to CTL during infection.


Inhibition of smooth muscle cell proliferation by an antisense oligodeoxynucleotide targeting the messenger RNA encoding proliferating cell nuclear antigen.

The process by which normally quiescent vascular smooth muscle cells (SMCs) change into proliferating cells, which express and respond to multiple growth factors, plays a major role in restenosis after coronary angioplasty. We are attempting to inhibit SMC proliferation by interventions that inhibit specific factors involved in signal transduction pathways leading to cell division. To date, all studies taking this approach have attempted to block the effects of mitogens acting on the cell surface. In contrast, we have focused on a strategy that bypasses cell surface-mediated events by directly inhibiting the expression of proliferating cell nuclear antigen (PCNA), an intranuclear protein that functions in a final common pathway shared by diverse mitogen-induced signals. In the present investigation, we determined whether antisense oligodeoxynucleotides (ODNs) complementary to the messenger RNA of PCNA will inhibit PCNA expression and thereby reduce SMC proliferation. When antisense ODNs (15- or 18-mer), modified to inhibit their degradation, are introduced into the medium of rat aortic SMCs in concentrations ranging from 10 to 100 microM, the 18-mer ODN, in a concentration-related manner, decreases SMC growth (as assessed by cell counting) by more than 50%. This effect persists for at least 9 days. An ODN with the same nucleotides but a scrambled sequence has little effect. Western blots and immunocytochemistry indicate that the antisense ODN reduces expression of PCNA protein. Our results demonstrate that an antisense ODN directed at the messenger RNA of PCNA decreases expression of the PCNA gene product and reduces SMC proliferation. In addition, these results provide an important impetus to initiating in vivo studies to determine the feasibility of antisense strategies in the prevention of coronary restenosis.


Enhancement of the force-frequency effect on myocardial contractility by adrenergic stimulation in conscious dogs.

The influence of changes in heart rate on myocardial contractility (the force-frequency effect) differs under various experimental conditions, including the anesthetized versus the conscious state. To assess the influence of beta-adrenergic stimulation on force-frequency effects on myocardial contraction and relaxation, seven instrumented conscious dogs were studied in which heart rate could be controlled by atrial pacing after the intrinsic rate was slowed with a bradycardiac agent (UL-FS 49 0.5-0.75 mg/kg). Left ventricular (LV) pressure was measured with a micromanometer under resting conditions and during dobutamine infusion at low, intermediate, and high doses (2.7, 5.4, and 10.7 micrograms/kg/min). At each dose, heart rate was progressively increased from 100 to 210 beats per minute. In the absence of dobutamine (control), no significant positive force-frequency effect was detected on LV dP/dtmax; this was probably due to the known effect of the observed decrease in preload to reduce LV dP/dtmax, thereby offsetting an effect of the force-frequency response to increased dP/dt. However, during dobutamine infusions, the force-frequency effect was observed to increase significantly in a dose-dependent manner with increases in heart rate. An increase in heart rate from 100 to 210 beats per minute increased LV dP/dtmax by 12.4 +/- 12.5% with low-dose, 22.7 +/- 13.1% with intermediate-dose, and 27.5 +/- 8.9% with high-dose dobutamine. Changes in preload and aortic pressure were within the same ranges under control conditions and at each of the three dobutamine doses. The time constant of LV pressure fall (tau) was significantly shorter with increases in heart rate during control, but only the highest dobutamine dose caused further significant shortening in tau with increased heart rate. These data indicate that there is a pronounced dose-dependent action of beta-adrenergic stimulation to enhance force-frequency-induced contractile responses in normal conscious dogs.


Hemodynamic and reflex sympathetic control of transmural activation and rate of ventricular tachycardia in ischemic and hypertrophic ventricular myocardium of the dog.

A previous study found that the electrophysiological response to ischemia is altered in hypertrophic myocardium, resulting in prolonged transmural activation time (TAT) associated with induction of sustained monomorphic ventricular tachycardia. This study investigated the role of hemodynamics in modulating TAT and the cycle length of induced ventricular tachycardia (VT) in dogs with left ventricular hypertrophy (LVH). Anesthetized open-chest dogs underwent 3 hours of uninterrupted circumflex coronary occlusion. During atrial drive, TAT was recorded between endocardial and epicardial bipolar pairs on the same multipolar plunge needle placed in nonischemic and ischemic zones, documented by triphenyltetrazolium chloride staining. TAT and VT induced by up to three extrastimuli were studied during hypertension (control), during normotension produced most frequently by nitroprusside infusion (3-6 micrograms/kg/min), and during further hypertension most frequently produced by phenylephrine infusion (1-5 micrograms/kg/min). Twenty-five dogs with chronic hypertension and LVH (group 1) produced by a single-kidney renal clamp mechanism and 15 control dogs were studied. In the latter, neither intervention altered TAT, and no VT was inducible. In group 1, however, nitroprusside reversibly prolonged TAT within the ischemic zone (mean +/- SEM, 31 +/- 3 to 34 +/- 3 msec, p less than 0.005) and cycle length of induced VT (204 +/- 19 to 240 +/- 17 msec, p less than 0.01). Phenylephrine reversibly shortened both TAT in the ischemic zone (33 +/- 2 to 28 +/- 2 msec, p less than 0.05) and cycle length of VT (219 +/- 17 to 165 +/- 11 msec, p less than 0.025). Cycle length of VT and TAT were dissociated from blood pressure elevation in two dogs with LVH; when blood pressure was elevated by sympathetic nerve stimulation, cycle length of VT and TAT were prolonged. In 11 dogs with LVH (group 2), prolongation of TAT with nitroprusside infusion was prevented by intravenous metoprolol (1.0 mg/kg). Of 12 dogs with LVH and inducible VT (group 3), seven still had VT inducible after metoprolol, but the cycle length of VT was still prolonged with nitroprusside infusion. These results suggest that 1) TAT in acutely ischemic LVH was uniquely responsive to hemodynamic influences, an effect prevented by beta-blockade with metoprolol, and 2) the cycle length of VT was also uniquely regulated by hemodynamic influences but not blocked by metoprolol.


N-methyl-D-aspartate antagonists suppress the development of frog symmetric monocular optokynetic nystagmus observed after unilateral visual deprivation.

In monocular vision, frogs display a unidirectional optokinetic nystagmus (OKN), reacting only to temporal-nasal (T-N) stimulation. The OKN N-T component is almost absent. However, prolonged monocular visual deprivation by unilateral eyelid suture provoked the appearance of the N-T component. The analysis of search coil recordings showed that the slow phase velocity gain of both T-N and N-T components became similar. Chronic administration of N-methyl-D-aspartate (NMDA) antagonists for the duration of deprivation prevented the appearance of a symmetrical monocular OKN in frogs: following repeated intraperitoneal injections of either MK 801, CGS 19755 or intrapretectal microinjections of 2-amino-5-phosphonovalerate (APV), the N-T component did not appear, and OKN remained asymmetrical. Thus NMDA receptors appear to be involved in the control of the plasticity process which allows monocular OKN of adult lower vertebrates to become symmetrical.


Involvement of dipeptidyl peptidase IV in an in vivo immune response.

Dipeptidyl peptidase IV (DP IV) is a serine protease that selectively cleaves X-Pro dipeptides from polypeptides and proteins. Among blood cells, this enzyme occurs preferentially on the surface of CD4+ T cells and the amount of enzyme activity increases with T cell activation. In previous work, two potent and specific peptidyl-boronic acid inhibitors of DP IV, Ala-boroPro and Pro-boroPro, were synthesized and Pro-boroPro was shown to suppress antigen-specific proliferative responses of T cells in vitro. In this study, we tested the in vivo effects of these inhibitors. Subcutaneous injection of Ala-boroPro or Pro-boroPro into BALB/c mice inhibited DP IV activity in serum and spleen cell suspensions. Repeated injections of more than 10 micrograms of Ala-boroPro or Pro-boroPro at 12 h intervals maintained in vivo DP IV activity at less than 30% of the normal level. Repeated injections of the inhibitors during the primary, secondary or tertiary immune response to bovine serum albumin (BSA) reduced anti-BSA antibody production. Without inhibitor, immunization with BSA was followed by a temporary decrease in serum DP IV activity and then by enhanced serum enzyme activity after several days. These results provide the first direct evidence that DP IV plays an important role in immune response in vivo.


Scintigraphic study of extra-adrenal ganglioneuroma in a patient with overlap between multiple endocrine neoplasia types 1 and 2.

A 27-year-old woman was diagnosed with a pituitary prolactinoma. Seven years later, when she was 34, an abdominal mass was incidentally discovered and ascribed to the right adrenal gland on the basis of evidence from ultrasonography, computed tomography, and arteriography. Adrenal scintigraphy with Se-75 selenomethylcholesterol imaged both adrenal glands, but the right gland was distorted, suggesting external compression. I-131 MIBG was not taken up by the mass. At surgery, an extra-adrenal ganglioneuroma was found and excised. This case represents an overlap between multiple endocrine neoplasia types 1 and 2. The failure of the ganglioneuroma to concentrate MIBG was likely caused by secretory inactivity of a biologically mature tumor.


[Effects of beta blocking agent iontophoresis by modulated sinusoidal current].

The action of the transcutaneous application of propranolol using modulated sinusoidal current iontophoresis was studied. When this way of administration is used, the drug, while maintaining its negative chronotropic effect, loses the negative inotropic one. Investigations were performed on 58 men aged 32 to 62 who underwent an aortocoronary bypass for myocardial ischemia. Thirty-four patients had already had myocardial infarction. The drug reduced the heart rate and increased cardiac contractility. Moreover, the study showed that only the levogyral isomer of propranolol (the only active one) passes through the skin.


The prion protein gene: a role in mouse embryogenesis?

The neural membrane glycoprotein PrP (prion protein) has a key role in the development of scrapie and related neurodegenerative diseases. During pathogenesis, PrP accumulates in and around cells of the brain from which it can be isolated in a disease-specific, protease-resistant form. Although the involvement of PrP in the pathology of these diseases has long been known, the normal function of PrP remains unknown. Previous studies have shown that the PrP gene is expressed tissue specifically in adult animals, the highest levels in the brain, with intermediate levels in heart and lung and low levels in spleen. Prenatally, PrP mRNA has been detected in the brain of rat and hamster just prior to birth. In this study we have examined the expression of the PrP gene during mouse embryonic development by in situ hybridisation and observed dramatic regional and temporal gene expression in the embryo. Transcripts were detected in developing brain and spinal cord by 13.5 days. In addition, PrP gene expression was detected in the peripheral nervous system, in ganglia and nerve trunks of the sympathetic nervous system and neural cell populations of sensory organs. Expression of the PrP gene was not limited to neuronal cells, but was also detected in specific non-neuronal cell populations of the 13.5 and 16.5 day embryos and in extra-embryonic tissues from 6.5 days. This cell-specific expression suggests a pleiotropic role for PrP during development.


Engrailed expression in the anterior lineage compartment of the developing wing blade of Drosophila.

The developing wing of Drosophila melanogaster was examined at larval and pupal stages of development to determine whether the anterior-posterior lineage boundary, as identified by lineage restrictions, was congruent with the boundaries defined by the expression of posterior-specific (engrailed, invected), and anterior-specific (cubitus interruptus-D) genes. The lineage boundary was identified by marking mitotic recombinant clones, using an enhancer trap line with ubiquitous beta-gal expression in imaginal tissues; clones of +/+ cells were identified by their lack of beta-gal expression. Domains of gene expression were localized using antibodies and gene specific lacZ constructs. Surprisingly, it was found that engrailed expression extended a small distance into the anterior lineage compartment of the wing blade, as identified with anti-en/inv mAb, anti-en polyclonal antiserum, or an en-promoter-lacZ insert, ryxho25. This anterior expression was not present in early third instar discs, but appeared during subsequent larval and pupal development. In contrast, the expression of cubitus interruptus-D, as identified using the ci-Dplac insert, appeared to be limited to the anterior lineage compartment. Thus, en expression is not limited to cells from the posterior lineage compartment, and en and ci-D activities can overlap in a region just anterior to the lineage compartment boundary in the developing wing. The lineage boundary could also be identified by a line of aligned cells in the prospective wing blade region of wandering third instar discs. A decapentaplegic-lacZ construct was expressed in a stripe several cells anterior to the lineage boundary, and did not define or overlap into the posterior lineage compartment.


Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system.

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677-2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.


Transplantation of encapsulated canine islets into spontaneously diabetic BB/Wor rats without immunosuppression.

Extended survival of canine islet xenografts implanted in spontaneously diabetic BB/Wor rats has been achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes. Intraperitoneal implantation of the encapsulated islets reversed the diabetic state of the 10 recipients within 24 h. Plasma glucose levels declined from a preimplantation level of 459 +/- 30 to 102 +/- 14 mg/dl during the first 10 days. All of the animals sustained these levels for at least 1 month, and 2 animals for at least 2 and 8 months, respectively. To confirm that glucose homeostasis resulted from the encapsulated islet grafts, the implants were removed from 2 rats 1 month postimplantation, whereas a third was removed at 2 months. Hyperglycemia was observed immediately in all 3 animals, with glucose levels rising from 100 +/- 3 to 510 +/- 43 mg/dl within 1 day. In contrast, diabetic control rats (n = 4) receiving nonencapsulated islets became hyperglycemic in less than 1 week. The iv glucose tolerance test K value (decline in glucose levels, percent per min) at 10 days was 2.3 +/- 0.4 compared with 0.6 +/- 0.1 (P less than 0.005) and 3.1 +/- 0.1 (P less than 0.02) for untreated diabetic (n = 4) and normal control (n = 4) groups. Histological analyses and electron microscopy of long term functioning grafts revealed well preserved islets, with hormone-producing alpha-, beta-, and delta-cells; the membranes were generally free of fibrosis and host cell adherence. These results demonstrate that permselective artificial membranes can protect discordant islet xenografts from both graft rejection and autoimmune destruction for more than 1 month in an animal model that is similar in several respects to human type I diabetes.


Estradiol selectively regulates agonist binding sites on the N-methyl-D-aspartate receptor complex in the CA1 region of the hippocampus.

Estradiol alters cognitive function and lowers the threshold for seizures in women and laboratory animals. Both of these activities are modulated by the excitatory neurotransmitter glutamate in the hippocampus. To assess the hypothesis that estradiol increases the sensitivity of the hippocampus to glutamate activation by increasing glutamate binding sites, the densities of N-methyl-D-aspartate (NMDA) agonist sites (determined by NMDA displaced glutamate), competitive antagonist sites (CGP 39653), noncompetitive antagonist sites (MK801) as well as the non-NMDA glutamate receptors for kainate and AMPA (using kainate and CNQX, respectively) were measured using autoradiographic procedures. Two days of estradiol treatment increased the density of NMDA agonist, but not of competitive nor noncompetitive NMDA antagonist binding sites exclusively in the CA1 region of the hippocampus. The density of noncompetitive NMDA antagonist sites, however, was decreased in the dentate gyrus by estradiol treatment. Ovarian steroids had no effect on the density of kainate or AMPA receptors in any region of the hippocampus examined. These data indicate that the agonist and antagonist binding sites on the NMDA receptor/ion channel complex are regulated independently by an as yet unidentified mechanism, and that this regulation exhibits regional specificity in the hippocampus. The increase in NMDA agonist sites with ovarian hormone treatment should result in an increase in the sensitivity of the hippocampus to glutamate activation which may mediate some of the effects of estradiol on learning and epileptic seizure activity.


PC12 rat pheochromocytoma cells synthesize dynorphin. Its secretion is modulated by nicotine and nerve growth factor.

The PC12 is a cloned rat pheochromocytoma cell line that retains a number of chromaffin cell characteristics, such as the presence of nicotinic cholinergic receptors, the synthesis and secretion of catecholamines, and the expression of a number of neuropeptide genes. The PC12 cell line is a useful model for the study of neuronal development, since PC12 cells can be induced to differentiate toward sympathetic neurons after exposure to nerve growth factor (NGF). PC12 cells can also be induced to differentiate toward the opposite direction, i.e. toward mature chromaffin cells. Morphological and biochemical changes mark the differentiation of PC12 cells toward either direction. Among the substances proposed as biochemical markers of PC12 cell differentiation toward chromaffin cells is the endogenous opioid precursor proenkephalin and its posttranslational peptide products. Indeed, the proenkephalin gene is expressed in both adrenal chromaffin and PC12 cells. The secretion of enkephalins from PC12 cells increases by several-fold after differentiation toward chromaffin cells. On the other hand, prodynorphin (another endogenous opioid precursor) is not present in normal adrenal chromaffin cells, but it is synthesized by human pheochromocytomas. It, thus, appears that dedifferentiation of chromaffin cells induces expression of the prodynorphin gene. Consequently, the aim of this study was to determine whether the rat pheochromocytoma-derived PC12 cell line expresses the prodynorphin gene, if it secretes dynorphins, and if the NGF-induced differentiation of PC12 cells toward sympathetic neurons affects the secretion of the latter. We found the following. 1) PC12 cells synthesize prodynorphin and secrete its peptide products. 2) The size of the prodynorphin transcript and the mol wt of its dominant form of dynorphin appear to be similar or identical to those of prodynorphin in rat anterior pituitary. 3) PC12 dynorphin secretion is increased, in a dose-dependent manner, after nicotinic cholinergic stimulation, an effect blocked by the specific nicotine antagonist hexamethonium. Thus, it appears that after cholinergic stimulation, PC12 dynorphin is cosecreted with catecholamines, a phenomenon described for a number of neuropeptides, including the proenkephalin-derived opioids. 4) The NGF-mediated differentiation of PC12 cells into sympathetic neurons exerted a stimulatory effect on basal, nicotine-induced, and depolarization-dependent dynorphin secretion. However, NGF did not shift the nicotine dose-response curve of dynorphin secretion. In conclusion, it appears that while changes in the secretion of proenkephalin-derived peptides may serve as a marker of PC12 cell differentiation toward chromaffin cells, an increase in the secretion of prodynorphin-derived peptides may represent a marker of NGF-induced differentiation of PC12 cells toward sympathetic neurons.


Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons.

The activity of both somatostatin (SS) and GH-releasing hormone (GHRH) neurons within several hypothalamic nuclei is regulated, in part, by the feedback effects of GH. However, whether GH, or its intermediate, insulin-like growth factor I, acts on these neurons to alter the synthesis and release of SS and GHRH is unknown. We argued that if GH itself acts directly on the brain to govern its own secretion, then regions of the brain containing SS and GHRH neurons may express the GH receptor gene. We tested this hypothesis by performing in situ hybridization for GH receptor messenger RNA (mRNA) and mapping its distribution in the brain. We observed GH receptor mRNA-containing cells in various brain regions including the thalamus, septal region, hippocampus, dentate gyrus, amygdala, and hypothalamus. Next we sought evidence for expression of the GH receptor mRNA by SS neurons in the hypothalamus. We addressed this by performing a double-label in situ hybridization to identify neurons expressing both SS mRNA and GH receptor mRNA. We report that SS neurons in the periventricular nucleus and in the paraventricular nucleus coexpress the GH receptor gene, whereas few, if any, of the SS neurons in the cortex express detectable amounts of the GH receptor mRNA. These findings suggest that GH acts directly on the brain and participates in the regulation of its own secretion through a direct action on hypothalamic SS neurons.


Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.

The polycomb group (Pc-G) genes are responsible for maintaining the repressed state of homeotic genes during development. It has been suggested that the Pc-G exerts its transcriptional control by regulating higher order chromatin structure. In particular, the finding of genetic and molecular similarities to components involved in heterochromatin formation, led to the proposal that homeotic genes are permanently repressed by mechanisms similar to those responsible for heterochromatin compaction. Because of synergistic effects, Pc-G gene products are thought to act in a multimeric complex. Using immunoprecipitation we show that two members of the Pc-G, Polycomb and polyhomeotic, are constituents of a soluble multimeric protein complex. Size fractionation indicates that a large portion of the two proteins are found in a distinct complex of molecular weight 2-5 x 10(6) Da. During embryogenesis the two proteins show the same spatial distribution. In addition, by double-immunofluorescence labelling we can demonstrate that Polycomb and polyhomeotic have exactly the same binding patterns on polytene chromosomes of larval salivary glands. We propose that some Pc-G proteins act in multimeric complexes to compact the chromatin of stably repressed genes like the homeotic regulators.


Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.


Presentation of superantigen by human T cell clones: a model of T-T cell interaction.

Superantigens (SAg) interact with T lymphocytes bearing particular V beta sequences as part of their T cell receptor (TcR). The interaction, however, requires the presence of major histocompatibility complex (MHC) class II molecules on antigen-presenting cell (APC). In peculiar circumstances, MHC class II+ T cell clones (TCC) have been shown to present peptides and selected antigens interacting with antigen-specific TCC in the absence of APC. In this report we studied the capacity of SAg to mediate a T-T cell interaction, investigating the TCC ability to present a panel of staphylococcal enteroxins (SE) independently of the presence of added APC. Upon exposure to a broad range of SE concentrations, MHC class II+ TCC showed an intense proliferative response even in the absence of professional APC. Diverse SE optimally stimulated responder TCC at different concentrations. The proliferation was inhibited by anti-DR monoclonal antibodies, both in the presence and in the absence of APC. The SE activation of TCC in the absence of APC induced the same series of phenotypic variations as that observed following the TCC stimulation with APC. Irradiated TCC efficiently presented membrane-bound SE to responder TCC as well as professional APC. These results show that a single cell of a given clone effectively presents the SE to other cells of the same clone, and provide evidence that SAg can efficiently mediate T-T cell interaction. In addition, the possibility also exists that one cell of the clone can actually undergo an auto-stimulation via SAg-mediated interactions between its own TcR and MHC class II molecule. It has recently been suggested that the V beta-selective depletion of T cells observed in acquired immunodeficiency syndrome (AIDS) patients might be a consequence of the interaction between a human immunodeficiency virus (HIV)-encoded SAg and T cells expressing a SAg complementary V beta. We suggest that the hypothesized HIV-encoded SAg might mediate T-T cell interactions that could play a relevant role in the V beta-selective depletion of T lymphocytes observed in HIV-infected patients.


Alternative polyadenylation of the amyloid protein precursor mRNA regulates translation.

The sequence of several cDNAs encoding the amyloid protein precursor showed that two polyadenylation sites of the mRNA are utilized; RNA blot analysis with different riboprobes indicated that this explains the difference between the two major 3.2 and 3.4 kb mRNAs found in the human brain. These two mRNAs, which contain the whole sequence of the natural molecules, were synthesized by in vitro transcription and translated in Xenopus oocytes. The long mRNA using the second polyadenylation site produced more protein than the short mRNA. The sequence contained within the two polyadenylation sites used in the 3' untranslated region of the amyloid protein precursor mRNA was also able to increase the production of the chicken lysozyme or the chloramphenicol acetyl transferase, as demonstrated by in vivo translation of different chimeric mRNAs obtained by in vitro transcription. This difference in protein production was also observed when chimeric cDNA constructs were transfected into Chinese hamster ovary cells. Since long mRNAs are not more stable than short mRNAs, the sequence contained within the two polyadenylation sites of the amyloid protein precursor mRNA increases the translation.


Synthesis and characterization of biotinylated and photoactivatable neuroleptics. Novel bifunctional probes for dopamine receptors.

We have synthesized and characterized a series of novel derivatives of established antagonists of the neurotransmitter dopamine, i.e. butyrophenones, hexahydrocarbolines and phenothiazines. All derivatives were biotinylated, some of them carried an additional (photoactivatable) azido group. In the case of butyrophenones, the structural modifications were introduced at the aliphatic keto group and/or the heterocyclic ring system, both modifications resulting in significant decreases in binding affinity to dopamine D2 and dopamine D1 receptor subtypes. Biotinylation of hexahydrocarbolines significantly increased their binding affinity to D1 receptors, with the affinity for D2 receptors increasing only slightly, or remaining approximately the same, as compared to the parent compound. As a consequence, the derivatized hexahydrocarbolines behaved as nonselective antagonists of dopamine. Biotinylation of phenothiazines increased their binding affinity to both main subtypes of dopamine receptors by at least one order of magnitude, resulting in binding affinities in the nM range. These derivatives bound to both D1 and D2 receptor subtypes. In three of the biotinylated derivatives the photoactivatable azido group was introduced. These compounds bound to synaptosomal membranes from bovine caudate nuclei with similar affinity and subtype specificity as the biotinylated derivatives, and photoaffinity labelling was shown to proceed under mild conditions and selectively. These novel bifunctional ligands may become useful tools in the purification and characterization of dopamine receptors including their visualization and localization in the central nervous system and in tissue culture.


Alpha 1-adrenoceptors in parotid cells: age does not alter the ratio of alpha 1A and alpha 1B subtypes.

Epinephrine stimulation of 45Ca2+ efflux and inositol 1,4,5-trisphosphate ((1,4,5)IP3) production in parotid cell aggregates from mature rats was greatly inhibited (approximately 70%) by WB 4101 and 5-methylurapidil as compared to a small decrease by chloroethylclonidine (approximately 30%). The combination of WB 4101 or 5-methylurapidil and chloroethylclonidine completely blocked the action of epinephrine. The same relative inhibition was observed with senescent animals. The results suggest (1) that rat parotids contain both alpha 1-adrenoceptor subtypes, i.e., alpha 1A and alpha 1B, in an approximate functional ratio of 70:30, (2) that this relative ratio is not altered during aging, and (3) that both receptors partially mediate 45Ca2+ efflux and (1,4,5)IP3 production in this system.


Subtypes of alpha 1-adrenoceptors in DDT1 MF-2 and BC3H-1 clonal cell lines.

We examined which subtype(s) of alpha 1-adrenoceptors are expressed in the widely used DDT1 MF-2 and BC3H-1 cell lines. Pretreatment with chloroethylclonidine (CEC) inactivated 76-85% of the specific [125I]BE 2254 binding sites in membrane preparations from both cell lines. Competition with subtype-selective competitive antagonists showed primarily the alpha 1B subtype in both cell lines. However, in BC3H-1 cells 5-methyl-urapidil showed complex behavior suggesting that about half of the binding sites had a lower affinity. Chloroethylclonidine pretreatment eliminated [3H]inositol phosphate responses to norepinephrine in both cell lines. Measurement of intracellular Ca2+ with fura-2 in DDT1 MF-2 cells showed that norepinephrine induced a complex response involving both transient and sustained components. Chloroethylclonidine pretreatment blocked both responses, while chelation of extracellular Ca2+ left the transient response intact but eliminated the sustained component. These results support previous work that these cell lines contain alpha 1B-adrenoceptors linked to inositol phosphate formation and mobilization of intracellular Ca2+. However, these results show that alpha 1B-adrenoceptors can be linked to Ca2+ influx as well as intracellular mobilization, and support the existence of pharmacologically distinct alpha 1B variants.


Subchronic treatment of rats with remoxipride fails to modify sigma binding sites in the brain.

The effects of 14 days' treatment of rats with haloperidol, remoxipride or both combined on the sigma binding sites in whole brain and liver were determined. The compounds were given subcutaneously via osmotic minipumps at a dose of 0.25 mg/rat/day (corresponding to about 1 mg/kg body weight/day at start) for haloperidol and 2.5 mg/rat/day (10 mg/kg/day) for remoxipride hydrochloride. The sigma recognition sites were determined after a washout period of 2 days with the radioligand [3H](+)-N-propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-3-PPP). It was found that the haloperidol treatment but not the remoxipride treatment decreased the density of the sigma sites in the brain, without any effect on the affinities of the ligands. Haloperidol had no effect on the binding of [3H](+)-3-PPP to the sigma recognition sites in the liver.


Bilateral distribution of aminopeptidase activities in selected structures of a photoneuroendocrine circuit and other rat brain areas.

Provided that soluble aminopeptidases, the most abundant proteolytic enzymes found in brain, are involved in the metabolism of several neuropeptides, their activity could be a reflect of neuropeptide function. Therefore, in order to analyze their rate of participation, we have measured 4 soluble aminopeptidase activities: leucine aminopeptidase, arginine aminopeptidase, aspartate aminopeptidase, and pyroglutamate aminopeptidase, using arylamide derivatives as substrates, in selected structures integrating the photoneuroendocrine circuit related to the melatonin rhythm generating system and other rat brain areas. The regional distribution of all the activities was heterogenous: a 3-fold (leucine-, arginine- and aspartate-aminopeptidase) and 5-fold (pyroglutamate aminopeptidase) difference was observed between the regions with the highest and lowest activity. Significant differences were displayed between the left and right retina for pyroglutamate aminopeptidase and arginine aminopeptidase activities. High levels of pyroglutamate aminopeptidase were evident in the retina and adenohypophysis, which is consistent with a role for thyrotropin releasing hormone in photoreceptive mechanisms, and support its well established role in controlling thyrotropin releasing hormone in anterior pituitary. The presence of a high activity rate of aspartate aminopeptidase in adenohypophysis implies an active participation of angiotensin peptides at this level.


Inhibitory effect of somatostatin on abnormal GH response to TRH in primary hypothyroidism.

Thyrotropin releasing hormone (TRH) does not promote GH secretion in normal subjects but it stimulates GH in a proportion of hypothyroid patients. In this study the response of GH to thyrotropin releasing hormone (TRH) was evaluated in 21 patients with primary hypothyroidism of different origin: 12 with autoimmune thyroiditis, 3 idiopathic, 3 congenital, 3 iatrogenic. 11 of these patients had never been treated, the others were tested after a drug-free period of at least two weeks. Basal plasma concentration of GH was normal in all patients; after TRH administration, a significant increase in plasma GH was observed in 4 patients. In these responsive patients, somatostatin infusion inhibited the abnormal GH response to TRH. It is suggested that the abnormal GH response to TRH in primary hypothyroidism might be caused by a relative deficiency of somatostatinergic control, which is corrected by exogenous somatostatin administration.


Cycloheximide can rescue heat-shocked L cells from death by blocking stress-induced apoptosis.

Cultured mouse L cells undergo apoptosis upon 1 h heat shock at 43 and 45 degrees C. Morphologically characteristic apoptotic cells begin to appear soon after the shock. Immunohistochemistry with anti-transglutaminase antibody shows that in most treated cells the enzyme is induced. Its activation results in the formation of highly cross-linked detergent-resistant apoptotic bodies during recovery. Cycloheximide added during hyperthermic stress inhibits the appearance of apoptotic bodies, showing that heat-shock-induced apoptosis is dependent on protein neosynthesis. The analysis of colony-forming ability of heat-shocked L cells shows a survival of 5% at 43 degrees C and less than 0.02% at 45 degrees C. When protein synthesis is inhibited during heat shock the fraction of surviving cells increases to 23% at 43 degrees C and 0.9% at 45 degrees C. This suggest that part of the cells that die upon heat shock are not heavily damaged and would have survived in the presence of a block in protein synthesis.


Structure and identity of a late-replicating and transcriptionally active gene.

Eukaryotic genes are usually replicated early during S-phase in the cell lineages in which they are expressed. Using partially characterized cDNA probes, we recently established two exceptions to this rule in the slime mold Physarum polycephalum. In this paper, we analyzed the structure and the identity of one of these two genes. By genomic cloning and Southern analysis we demonstrate that it is a single-copy gene and decipher the structure of the two alleles by taking advantage of a restriction fragment length polymorphism. By cDNA cloning and sequencing, we deduced the amino acid coding capacity of the mRNA. Finally, we confirmed the late replication of this abundantly expressed gene by "gene dosage" analysis, an experiment that did not require any drug treatment of the cell. Our results provide for the characterization and the structure of the first developmentally regulated gene known to be replicated late in S-phase and abundantly expressed within a eukaryotic cell.


Effects of glucagon and an analogue of cAMP on tyrosine aminotransferase in isolated chick embryo hepatocytes.

Hepatocytes were isolated from 17-day-old chick embryos by the use of collagenase. Glucagon and dibutyryl cAMP (bt2cAMP), individually or in combination, stimulated tyrosine aminotransferase (TAT) activity and synthesis in the isolated hepatocytes; maximal stimulation occurred 4 h after exposure of hepatocytes to the inducers. The stimulatory effects produced by glucagon and bt2cAMP were abolished by treatment of hepatocytes with cordycepin or cycloheximide. The effects of the hormone and the cyclic nucleotide were not additive. The induction of the enzyme by glucagon suggests a physiological role for the hormone in the expression of TAT activity during chick embryonic development.


Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.


Theileria parva: CD4+ helper and cytotoxic T-cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes.

Theileria parva is a protozoan parasite which infects and transforms bovine lymphocytes, resulting in a fatal lymphoproliferative disease. There is evidence that immunity to the intralymphocytic schizont stage is mediated by T cells. We have previously reported derivation of CD4+ T-cell clones which recognize parasite-derived antigens presented on the surface of infected cells in conjunction with MHC molecules and partial characterization of the antigens. The present study further evaluated one of these antigens, demonstrating that it could be derived from cells infected with different parasite stocks as well as from purified theilerial schizonts and that it was recognized by primed, but not unprimed, bovine lymphocytes including cytolytic CD4+ T cells. Using a cloned CD4+ cytolytic cell line, lysis of schizont-infected cells was shown to be MHC-restricted but not parasite-strain restricted. In addition we demonstrated that T cells which respond to the HSS antigen preparation were generated in cattle immunized with parasites from any of the three subspecies of T. parva. The antigenic material was fractionated by sequential subjection to anion-exchange chromatography, hydroxylapatite chromatography, and gel filtration using HPLC, which resulted in recovery of approximately 20% of the antigenic material with more than 10(6)-fold purification in selected fractions. To assess the molecular size of the proteins in the highly purified antigenic fractions, the T. parva-infected lymphocytes were metabolically labeled before fractionation with 3H-amino acids and the material was analyzed by SDS-polyacrylamide gel electrophoresis and liquid scintillation counting of gel slices. The major protein in these fractions had a molecular mass of 9-10 kDa.


A farnesyl arabinoside as an enhancer of glucose transport in rat adipocytes from a soft coral, Sinularia sp.

Separation of a lipophilic extract of a soft coral, Sinularia sp., assayed by enhancement of glucose transport in rat adipocytes, gave farnesyl 4-O-beta-D-arabinopyranosyl-beta-D-arabinopyranoside-2,2',3- triacetate (1a) whose structure was determined by spectroscopy. Enhancers of glucose transport may be useful for the prevention and treatment of diabetic disorders.


NO., CO and .OH. Endogenous soluble guanylyl cyclase-activating factors.

Several low molecular weight compounds are capable of activating soluble guanylyl cyclase. Recent evidence suggests that some of these are formed under physiological conditions: the nitric oxide radical, carbon monoxide and the hydroxyl radical. Thus, multiple signal transduction pathways appear to exist that form a family of guanylyl cyclase activating factors and thereby regulate the intracellular cyclic guanosine 3',5'-monophosphate level.


Neurotransmitter transporters. A novel family of integral plasma membrane proteins.

The re-uptake of neurotransmitters into the nerve terminal terminates synaptic transmission at most central synapses and constitutes a key step in the modulation of synaptic efficacy. Recently, the cloning of several Na(+)-driven neurotransmitter transporters has resulted in the description of a novel family of homologous membrane proteins, each with 12 transmembrane segments. These transporters constitute major targets of widely used drugs, and modulation of transporter gene expression and/or activity may represent an important substrate for plasticity in the nervous system.


Expression of the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript.

Luteinizing hormone/chorionic gonadotropin (LH/CG) receptor complementary DNA (cDNA) isoforms were amplified using pseudopregnant rat ovarian total RNA as a template and the primers reaching over the coding regions at both ends in a reverse transcriptase-polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products revealed three bands corresponding to about 2.1, 2.0 and 1.8 kilobases (kb). Subcloning of pooled PCR products into EcoRI site of pUCBM20 resulted in 167 clones, from which five different restriction patterns were obtained by digestion with EcoRI and HaeIII. One clone of each was further characterized. It could be predicted from the nucleotide sequences that the clone rLHR2100 encoded a full-length receptor (a 674 amino acid mature protein), the clone rLHR2075 lacked part of exon IX (nucleotides 693-717) and encoded a truncated 225 amino acid mature protein, the clone rLHR1950 lacked exons III and IV (nucleotides 246-395) and encoded a nearly full-length protein (a 624 amino acid mature protein), and the clones rLHR1834 and rLHR1759 lacked the same part of exon XI (nucleotides 960-1225), with exon V (nucleotides 396-470) also absent in the latter, the deletion in exon XI leading both these clones to premature termination. The clone rLHR1834 encoded a 316 amino acid mature protein and rLHR1759 a 291 amino acid mature protein, respectively. The sequence data suggest that all of these isoforms contain the putative signal sequence and are derived from a single copy gene via alternative splicing. These results point further to the fact that the expression of the 90 kDa LH/CG receptor is regulated via an extensive alternative splicing of the receptor gene primary transcript.


The effects of peptide histidine isoleucine on antral gastrin and somatostatin.

The present studies were directed to determine whether peptide histidine isoleucine (PHI) affects expression of the gastrin and somatostatin genes and whether such effects may be functionally linked. In separate experiments, the effects of PHI on medium gastrin and somatostatin concentrations, the incorporation of 35S-labelled amino acids into newly synthesized gastrin and somatostatin, and steady state gastrin and somatostatin mRNA were determined. PHI inhibited basal expression of the gastrin gene at all levels examined, while no significant effect on basal somatostatin gene expression could be detected. PHI also decreased carbachol-stimulated antral gastrin release and simultaneously increased somatostatin release. However, in contrast to its structural analogues, secretin and gastric inhibitory peptide, the immunoneutralization of endogenous somatostatin by the administration of specific antibodies did not affect significantly the capacity of PHI to inhibit gastrin release into the culture medium stimulated by carbachol. The results of these studies indicate that PHI exerts a physiological inhibitory effect on antral gastrin cells and that this inhibition may occur at several steps along the biosynthetic pathway. In addition, unlike its structural analogues, PHI inhibition of carbachol-stimulated gastrin release is not functionally linked to its stimulatory effects on somatostatin release.


Gastroprotective and ulcer-healing activities of a new H2-receptor antagonist: ebrotidine.

Ebrotidine is a novel H2-receptor antagonist that exhibits both gastroprotective and ulcer-healing properties. Gastroprotection afforded by ebrotidine against ethanol damage was observed only after intragastric, but not parenteral administration, and it was accompanied by an increase in the mucosal blood flow. Ranitidine given at the same dose (100 mg/kg i.g. or s.c.) did not show any protective activity. When administered twice daily at various doses (1-100 mg/kg) for 10 days, ebrotidine reduced dose dependently the area of chronic gastric ulcers, and it was accompanied by significantly higher contents of epidermal growth factor (EGF) in the ulcer bed than in the intact mucosa. Administration of ranitidine resulted in a similar rate of ulcer healing and in a similar accumulation of EGF in the ulcer area to that observed after ebrotidine, but the increments in plasma gastrin levels in rats treated with ranitidine were observed at lower doses than in tests with ebrotidine. Concurrent administration of indomethacin delayed ulcer healing and reduced the accumulation of EGF in the ulcer area, but did not affect the ulcer healing by ebrotidine or ranitidine. We conclude that ebrotidine but not ranitidine shows gastroprotective activity, but it enhances the healing of chronic ulcerations in a similar manner to ranitidine.


Effect of aspirin and indomethacin on epidermal growth factor secretion in duodenal tissue fragments cultivated in vitro.

The aim of the present study was to determine the effect of aspirin and indomethacin on epidermal growth factor (EGF) secretion in duodenal tissue fragments cultivated in vitro. The fragments were obtained from healthy subjects by gastroscopy, cultured in McCoy's medium and gassed with 95% O2 and 5% CO2 at 37 degrees C. After an incubation of 30 min, the culture medium was decanted, and the quantity of hormone determined by radioimmunoassay. The mean EGF level detected in the medium was 10.94 ng/mg protein tissue. The addition of aspirin (final concentration 10(-7) M) to the medium reduced mean EGF levels to 7.5 ng/mg (p less than 0.05), whereas aspirin 10(-8) M did not produce such a modification. The addition of indomethacin (final concentration 10(-8) M) decreased mean EGF levels to 5.37 ng/mg (p less than 0.001). In all experimental conditions, the addition of anti-somatostatin (SRIF) antibodies determined a remarkable increase in EGF (p less than 0.01). The results of this study show aspirin and indomethacin to be direct, not SRIF-mediated inhibitors of EGF release.


Diversity among mouse motor nerve terminals with respect to release transmitter quanta.

1. The aim of this work was to reexamine whether a positive correlation exists between the frequency (F, sec-1) of miniature endplate potentials (m.e.p.ps) and the quantal content (m) of endplate potentials (e.p.ps) or between quantal content, frequency and twin-pulse facilitation of transmitter release at a large number of neuromuscular junctions in the mouse. 2. The values of F and m were both measured intracellularly at endplates of mouse diaphragm in a high Mg2+/low Ca2+ bathing solution. 3. Values of both F and m varied from junction to junction. Smaller values of F were correlated with smaller values of m, and vice versa, resulting in a linear relationships. Histograms of F and m were skewed towards smaller values. 4. E.p.ps evoked by twin pulses gave the quantal contents of the first (m1) and second (m2) responses. 5. The ratio of m2 to m1 varied from junction to junction. A histogram of this ratio was skewed towards smaller values. 6. The ratio of m2 to m1 showed larger fluctuations at junctions with smaller values of F or m1 but was focused around 1 at junctions with larger values of F or m1. 7. The skewed parts of the histograms of F, m and m2/m1 accounted for the major population of junctions. 8. These results support the hypothesis that an intrinsic ability to release transmitter plays a role in regulation of the evoked output of transmitter at neuromuscular junctions in the mouse. 9. Such an ability is not correlated with the twin-pulse facilitation of transmitter release.


Interaction of tertatolol at the "atypical" or beta 3-adrenoceptor in guinea-pig ileum.

1. Experiments were performed to investigate the possible interaction between tertatolol and the "atypical" beta-adrenoceptor present in guinea-pig ileum. 2. In the electrically field stimulated guinea-pig ileum, both isoprenaline and BRL-37344 produce concentration-dependent inhibition of the "twitch" response. 3. Schild regression analysis of the interaction between isoprenaline and tertatolol indicated two sites of action and yielded an apparent pA2 value for tertatolol of 6.8 at the "atypical" beta-adrenoceptor. 4. BRL-37344 selectively activated the "atypical" beta-adrenoceptor and tertatolol again yielded an apparent pA2 value of 6.7 and 6.8.


Vasodilatation of canine cerebral arteries by nicorandil, pinacidil and lemakalim.

1. Nicorandil, pinacidil and lemakalim relaxed precontracted rings of canine cerebral artery. 2. The order of potency was lemakalim greater than nicorandil approximately equal to pinacidil, but all these agents were less effective than nimodipine. 3. The effects of nicorandil were inhibited by methylene blue but not by glibenclamide, while the effects of pinacidil and lemakalim were inhibited by glibenclamide but not by methylene blue. 4. Thus nicorandil probably causes relaxation mostly by effects on guanylate cyclase while lemakalim and pinacidil produce the same effect by action at ATP-dependent potassium channels.


[Restriction polymorphism of mitochondrial DNA among Russian residents of Western Siberia].

Hereditary polymorphism of mitochondrial DNA is described for 5 restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI in 60 Russian individuals living in Tomsk. 13 morphotypes of mitochondrial DNA were revealed as a whole for the enzymes analysed. Comparative analysis of mitochondrial DNA morphs and morphotypes with the literature data was conducted.


[DNA polymorphism in the Russian population. Analysis of dna restriction fragment length polymorphism in seven loci of the nuclear genome].

Using the method for polymerase chain reaction the polymorphism of eight markers of the nuclear DNA was studied. In a sample of Russians taken at random (N = 118) from predominantly southern and central regions of Russia, allele frequencies were determined for restriction sites HindIII at HBG-2 and PAH loci, AvaII at the HBB locus, MspI at the ApoB locus, PstI at D7S8, HincII at LDLR, TaqI and BamHI at the DSX164. Comparative data for different world regions are presented.


Structure and lens expression of the gene encoding chicken beta A3/A1-crystallin.

The beta A1- and beta A3-crystallins are major polypeptides in the lenses of vertebrates. We present evidence that a single beta A3/A1 gene encodes these two proteins in the chicken. The beta A3/A1 gene has been sequenced and its functional promoter identified in transfection experiments. The chicken beta A3/A1 gene has the same structure as the human orthologue: six exons with standard splice sites and two alternative start codons from which the protein products are apparently translated. Northern analysis revealed an abundant 0.9-kb transcript in the lenses of 1-2-day-old chickens and no detectable transcripts in the rest of the eye, brain, heart, kidney, liver or skeletal muscle. The 5'-flanking sequence of the chicken beta A3/A1 gene is very similar to that of the human and mouse genes, suggesting conservation of important putative regulatory sequences in addition to the TATA box. A thymidine-rich element (bp -218 to -163) and a potential AP-1-binding site (bp -264 to -258) are present within the chicken 5'-flanking region. A DNA fragment from -382 to +22 of the chicken beta A3/A1 gene is sufficient to promote expression of the bacterial cat gene in transfected chicken primary lens epithelial cells, but not in transfected dermal fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification and partial characterization of a somatostatin-14 binding protein on rat liver plasma membranes.

Binding of somatostatin-14 to rat liver plasma membranes was characterized with 125-labeled[tyr11] somatostatin-14. Binding at 24 degrees C reached a plateau at 50 min and was reversible by synthetic somatostatin-14. Scatchard analysis revealed a single class of binding sites (affinity constant = 2.4 +/- 0.2 nmol/L, binding capacity = 148 +/- 0.02 fmol/mg protein). Specificity for somatostatin-14 was demonstrated by the inhibition of 125I-[tyr11]somatostatin-14 binding by biologically active somatostatin analogs but not by a biologically inactive somatostatin analog or unrelated peptides. The radioiodinated binding site complex could be cross-linked with disuccinimidyl suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel autoradiography revealed a 70,000-Da band. Dithiothreitol, a reducing reagent, did not alter the mobility of the band, and the band could be abolished in the presence of 10 mumol/L synthetic somatostatin-14. Covalently cross-linked, iodinated binding protein complexes could be solubilized by the nonreducing detergents Zwittergent 3-12 and 3-([3-cholamidopropyl] diethylammonio)-1-propanesulfonic acid (CHAPS). Solubilized complex bound to wheat-germ agglutinin-agarose columns and was eluted by N,N',N"-triacetylchitotriose. Binding to wheat-germ agglutinin agarose columns was lost after pretreatment with endo-beta-N-acetylglucosaminidase F. Binding studies with liver plasma membranes, 125I-labeled[tyrosine11]somatostatin-14 and guanine nucleotides showed inhibition of binding in the presence of guanine nucleotides. These results indicate that the purified rat liver plasma membranes contain a specific binding protein for somatostatin-14, the binding protein appears to be glycosylated and somatostatin-14 binding to rat liver plasma membranes may be regulated by G proteins.


Isolation and high-resolution mapping of new DNA markers from the pericentromeric region of chromosome 10.

The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including D10Z1, D10S94, D10S97, and D10S102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small subchromosomal fragments that include both D10S94 and D10Z1. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a lambda library made with pp11A DNA. We have completed regional mapping of 22 of those clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between D10S34 and RBP3. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A.


A high-resolution meiotic mapping panel for the pericentromeric region of chromosome 10.

Familial multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome characterized by tumors in tissues derived from the neural crest. The disease manifests as medullary carcinoma of the thyroid, pheochromocytoma, and hyperparathyroidism. The MEN2A locus has been mapped near the centromere of chromosome 10 by linkage analysis. Statistical analyses have not resolved the location of MEN2A among several close markers. We have used our family material to refine the positions of 36 identified and confirmed crossovers among the markers most closely linked to MEN2A. This high-resolution meiotic mapping panel will help order loci in this pericentromeric region and narrow the region in which MEN2A maps.


A preliminary linkage map of the chicken genome.

We have used backcross progeny from a cross between two inbred lines of chickens to construct a linkage map of the chicken. The map currently consists of 100 loci, identified using either anonymous cloned fragments of genomic DNA or sequences corresponding to cloned genes. Parent birds were derived from two lines of White Leghorn chickens, which differ in susceptibility to a number of diseases. Restriction fragment length variants were identified by comparison of the DNA of these two parent birds using a panel of seven restriction enzyme digests and the segregation pattern observed in progeny of these two birds. Restriction fragment length variants were detected for approximately 41% of the clones tested, whether these were known genes or random genomic fragments. This high level of variability was also reflected in the presence of variation within the parental lines for some clones. The overall size of the linkage groups and the progressively higher incidence of linkage as further clones were added suggests that the map covers the majority of the genome, although it is unlikely that there are marker loci on all the microchromosomes. The present map will be of use in locating genes affecting disease resistance, but also illustrates the relative ease with which such maps for the chicken can be constructed.


Genomic organization of a polymorphic duplicated region centromeric of HLA-B.

The region between tumor necrosis factor (TNF) and HLA-B in the central major histocompatibility complex (MHC) is polymorphic and associated with several autoimmune diseases. The polymorphisms are haplospecific or haplotypic and retained within the same MHC ancestral haplotype (AH). We have cloned this region from four AHs into lambda bacteriophage and found that a highly polymorphic region in the TNF-HLA-B interval is duplicated. Clones from this region isolated from three MHC AHs show two populations. The regions, designated CL1 and CL2, have different sizes of Bam HI fragments carrying the duplicated sequences. These fragments correspond to those seen after Bam HI restriction fragment length polymorphism (RFLP) analysis of genomic DNA from the same cell lines. Pulsed field gel electrophoresis analysis shows that both CL1 and CL2 are in the central MHC and are about 16 kilobases apart. DNA cloning and RFLP analysis demonstrate that the CL region is highly polymorphic but retained within an MHC AH. Polymorphism and duplication are common characteristics of the genes found in the MHC and therefore the CL sequences have the potential to be interesting in this respect.


Interaction of H-2Eb with an IAP retrotransposon in the A20/2J B cell lymphoma.

In the A20/2J BALB/c B cell lymphoma, Southern analysis revealed an insertion of approximately 6 kilobases of DNA into the first intron of one Ebd-allele. Two observations suggest that the rearrangement did not occur recently in the A20/2J subline. Firstly, normal and altered Ebd-alleles are present in equal numbers, and secondly, the LB 27.4 and LS 102.9 somatic cell hybrids formed at an earlier date both possess the rearrangement. Sequences of two cDNA clones, lambda Eb-7 and lambda Eb-125, selected from an A20/2J cDNA library prepared from poly [A+] RNA indicate that the rearranged Ebd-allele directs the synthesis of atypical Eb transcripts. The clones contain Eb sequence linked to a portion of retroviral-like intracisternal A-particle (IAP) genomic sequence, and they appear to be copies of mRNA produced by splicing between a 5' donor site in the retroviral transcript and the 3' acceptor site of the Eb gene's first intron. The longer lambda Eb-125 insert corresponds to RNA that initiated in the 5'-untranslated region of the Eb gene. The 3'-end of the first Eb exon joins to long terminal repeat sequence, and retroviral sequence extends up to the splice junction with the second Eb exon; 3' of the junction, the lambda Eb-125 sequence corresponds to that of a correctly spliced Eb transcript. It seems feasible that the cDNA clones represent hybrid RNA synthesized by read-through transcription of the Eb coding region and an IAP element inserted into the first intron of the rearranged Ebd-allele.


Interaction of capsulate Haemophilus influenzae with human airway mucosa in vitro.

Two pairs of isogenic capsulate and noncapsulate and one pair of capsulate fimbriate and nonfimbriate strains of Haemophilus influenzae type b were studied in an organ culture of human respiratory mucosa. Over 24 h, the numbers of recovered bacteria increased from the original inoculum size of 10(5) to 10(8) CFU/ml. Transmission electron microscopy and scanning electron microscopy showed that noncapsulate organisms caused significant epithelial damage, whereas capsulate strains did not. Association of noncapsulate bacteria with damaged epithelial cells was observed by 14 h of incubation. In contrast, capsulate organisms were associated with a dense, thick, gel-like matrix which was observed above the epithelial surface. These capsulate organisms were not seen to associate with the epithelial surface (by transmission electron microscopy), though they were occasionally seen adhering to cells by scanning electron microscopy. Fimbriate capsulate H. influenzae showed increased adherence to buccal cells compared with nonfimbriate capsulate organisms. There was also association of fimbriate capsulate bacteria with damaged organ culture epithelium in one of four experiments. It is concluded that both capsule and fimbriae affect the interaction of H. influenzae with human airway mucosa in vitro by influencing adherence to and damage of the epithelium.


Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages.

Pertussis toxin (PT) and filamentous hemagglutinin can each mediate the association of Bordetella pertussis with human macrophages. Adherence via filamentous hemagglutinin leads to integrin-mediated entry and survival of the bacteria within the human cell. We determined the contribution of PT to bacterial adherence to human macrophages. Plating macrophages on wells coated with recombinant PT subunit 2 (S2) or S3 decreased PT-dependent bacterial binding by greater than 60%; S1, S4, and S5 were ineffective. S3-dependent adherence was reduced 63% +/- 8% by sialic acid, while S2-dependent adherence was reduced 53% +/- 11% by galactose. Loss of the carbohydrate recognition properties of S2 by deletion of residues 40 to 54 or site-specific mutations at Asn-93, His-47, or Arg-50 eliminated the ability of the subunit protein to competitively inhibit bacterial binding. Peptides corresponding to residues 28 to 45 of S2 and S3 competitively inhibited adherence. Treatment of macrophages with antibodies to Le(a) or Le(x) but not CD14, CD15, CD18, or HLA interfered with PT-mediated binding. Exposure of the macrophages to the B oligomer, S2, or S3 increased binding to the CD11b/CD18 integrin. These results indicate that the carbohydrate recognition domains of both S2 and S3 participate in adherence of B. pertussis to human macrophages. The PT receptor(s), as yet unidentified, appears to carry the Le(a) or Le(x) determinants and is functionally capable of modulating integrin-mediated binding to the macrophage.


Roles of the pap- and prs-encoded adhesins in Escherichia coli adherence to human uroepithelial cells.

In this study, we reexamined the structural prerequisites for the attachment of P-fimbriated Escherichia coli to human urinary tract epithelial cells. The epithelial cells were obtained from A1P1 nonsecretor individuals, who express the globoseries of glycolipids without the ABH blood group determinants, and from A1P1 secretor individuals, who in addition express globo-A, a receptor for the prsJ96 adhesin. The wild-type E. coli strains J96, AD110, and IA2 and the recombinant clones HB101 papJ96, HB101 prsJ96, HB101 papIA2, and HB101 papAD110 were tested for binding. They expressed P fimbriae, as defined by P blood group-dependent agglutination of human erythrocytes of the globoseries, but differed in reactivity with galactose alpha 1-4galactose beta (Gal alpha 1-4Gal beta)-latex beads, isolated glycolipids of the globoseries, sheep erythrocytes, and uroepithelial cells. Three different patterns of binding were represented among the recombinant clones. HB101 papIA2 and HB101 papAD110 agglutinated sheep erythrocytes and Gal alpha 1-4Gal beta-latex beads and attached to both secretor and nonsecretor epithelial cells. HB101 prsJ96 agglutinated sheep erythrocytes, reacted poorly with Gal alpha 1-4Gal beta-latex beads, and attached to A1 secretor but not to A1 nonsecretor epithelial cells. HB101 papJ96 agglutinated Gal alpha 1-4Gal beta-latex beads but not sheep erythrocytes and attached poorly to human uroepithelial cells. The receptors relevant for adhesion were analyzed by inhibition with glycolipids in suspension. The sheep erythrocyte agglutination and attachment to secretor and nonsecretor epithelial cells of HB101 papIA2 and HB101 papAD110 were inhibited by globotetraosylceramide, while the Forssman glycolipid had no effect. The sheep erythrocyte reactivity and attachment to secretor epithelial cells of HB101 prsJ96 were inhibited by the Forssman glycolipid. These results permitted three conclusions. First, the expression of functionally active Gal alpha 1-4Gal beta-specific adhesins, as in HB101 papJ96, was not sufficient to make E. coli competent to attach to human uroepithelial cells. Attachment required P fimbriae of the papIA2 or papAD110 type. Second, the sheep erythrocyte reactivity of P-fimbriated strains could not be attributed solely to recognition of the Forssman glycolipid and may not be used to define the prsJ96-encoded phenotype. Third, the P-fimbrial adhesins which mediate secretor state-independent attachment to human uroepithelial cells recognized receptor epitopes provided by globotetraosylceramide.


Identification of type 4 pili in Kingella denitrificans.

Kingella denitrificans is an occasionally pathogenic member of the family Neisseriaceae and is a member of the normal respiratory flora. Electron microscopy, colony morphology types, DNA transformation patterns, and immunoblots suggest that K. denitrificans and K. kingae have type 4 pili. This was confirmed by N-terminal amino acid sequencing for K. denitrificans.


Expression of c-sis and c-fos genes in human meningiomas and neurinomas.

The HindIII c-sis RFLP was analyzed in constitutional and tumor DNAs of 19 meningioma patients. Loss of a c-sis allele (allele I in all cases) was observed in 6 out of 10 tumors from heterozygous patients. No alteration in the structure or dosage of the c-sis gene was detected in any tumor sample. Northern analysis of c-sis evidenced sis transcripts in 7 out of 8 meningiomas which were amenable to analysis. All tumors expressing c-sis also expressed c-fos, suggesting that the autocrine loop elicited by sis activation may have c-fos as its nuclear target. Co-expression of c-sis and c-fos was also observed in 5 neuromas out of 7 analyzed. Very high levels of fos-mRNA were observed in 2 neuromas, but were not found to be caused by apparent alteration or amplification of the c-fos gene.