Organization of the pontine nuclei.

Organization of the pontine nuclei. The pontine nuclei provide the cerebellar hemispheres with the majority of their mossy fiber afferents, and receive their main input from the cerebral cortex. Even though the vast majority of pontine neurons send their axons to the cerebellar cortex, and are contacted monosynaptically by (glutamatergic) corticopontine fibers, the information-processing taking place is not well understood. In addition to typical projection neurons, the pontine nuclei contain putative GABA-ergic interneurons and complex synaptic arrangements. The corticopontine projection is characterized by a precise but highly divergent terminal pattern. Large and functionally diverse parts of the cerebral cortex contribute; in the monkey the most notable exception is the almost total lack of projections from large parts of the prefrontal and temporal cortices. Within corticopontine projections from visual and somatosensory areas there is a de-emphasis of central vision and distal parts of the extremities as compared with other connections of these sensory areas. Subcorticopontine projections provide only a few percent of the total input to the pontine nuclei. Certain cell groups, such as the reticular formation, project in a diffuse manner whereas other nuclei, such as the mammillary nucleus, project to restricted pontine regions only, partially converging with functionally related corticopontine connections. The pontocerebellar projection is characterized by a highly convergent pattern, even though there is also marked divergence. Neurons projecting to a single cerebellar folium appear to be confined to a lamella-shaped volume in the pontine nuclei. The organization of the pontine nuclei suggests that they ensure that information from various, functionally diverse, parts of the cerebral cortex and subcortical nuclei are brought together and integrated in the cerebellar cortex.


Adult extrarenal rhabdoid tumor of the lacrimal gland. A 50-year-old man presented with a rapidly growing mass in the area of the right lacrimal gland. An initial erroneous histopathologic diagnosis of a pleomorphic adenoma made on a small-incisional biopsy was later corrected to a malignant rhabdoid tumor when a wide local excision of the tumor was performed. The tumor was composed predominantly of dyscohesive, globoid, and eosinophilic cells, which frequently contained cytoplasmic inclusions. These were demonstrated to be composed of whorls of intermediate vimentin filaments. The tumor cells expressed epithelial membrane antigen as well as cytokeratin. Ultrastructurally, they displayed intercellular junctions and interrupted segments of linear basement membrane material. These findings, together with the development of the lesion within the parenchyma of the lacrimal gland, are suggestive of an epithelial origin. The patient was treated with radical surgery and adjunctive radiotherapy and chemotherapy, which are the recommended treatment modalities for this highly malignant tumor.


Comparison of fluorescein and rose bengal staining. The authors have recently reported that rose bengal is not a vital dye, and stains whenever the cultured cells are not covered by such components as albumin and mucin, and such a tear substitute as carboxycellulose. In this report, using cultured cells as well as normal rabbit corneas, they characterize and correlate the staining differences between rose bengal and fluorescein with the differences in their chemical structures. Fluorescein differs from rose bengal in its lack of intrinsic toxicity, photodynamic action, and ability to be blocked by the above-mentioned substances. Fluorescein staining is increased by rapid stromal diffusion and hence can manifest whenever there is disruption of cell-cell junctions. In contrast, rose bengal staining ensues whenever there is deficiency of preocular tear film protection. These experimental data may help interpret the clinical staining properties of these two dyes and enhance the understanding of the pathogenesis of various ocular surface disorders.


Studies on the regranulation of salivary gland serous cells. An electron microscopical study in the rat. The degranulation and regranulation process was investigated after alpha-adrenergic stimulation on the rat submandibular gland. The submandibular gland in rat contains both serous and mucous cells. It has earlier been shown that serous cells filled with heavy-metal granules, are markedly more radiosensitive than cells without granules. In experiments with emptying the serous cell of their content of granules by administering an alpha-adrenergic stimulant, cyclocytidine, there has been found a decrease in irradiation damage in salivary gland tissue after irradiation. Injection of cyclocytidine, 150 mg/kg, was given i.p. to the rat. After 1 h there was almost complete depletion of granules in the serous cells, no morphological aberration was seen in the mucous cells. This effect still remained after 6 h. A beginning of regranulation with apical granules was seen 12 h after injection. After 24 h an almost complete regranulation had occurred in the salivary gland serous cells. The mucous cells did not show any morphological aberration. Our intent is to reduce unwanted salivary gland damage in patients with head and neck cancer when treated with radiotherapy. Depletion of heavy-metal granules in serous cells, before irradiation may diminish morphological destruction in the salivary glands. As a nearly total degranulation is present between 1-6 h after stimulation, this should be the optimal time for radiotherapy.


Implantation of a cardioverter/defibrillator in the subpectoral region combined with a nonthoracotomy lead system. An implantable cardioverter defibrillator was placed into a subpectoral pocket via the incision for cephalic venotomy during implantation of a nonthoracotomy lead system. The approach obviated another incision and subcutaneous tunneling of the leads. There were no perioperative complications and after 6 months of follow-up, the patient continues to tolerate the device satisfactorily.


Seven years of left ventricular pacing due to malposition of pacing electrode. The case report is presented of a patient in whom an uncomplicated left ventricular transvenous pacing produced right bundle branch block (RBBB). A diagnostic echocardiography, confirmed by cine cardiovascular computed tomography, showed that there was no rupture of the right ventricle and diagnosed a left ventricular pacing, due to malposition of the pacing electrode. The patient was treated with aspirin and dipyridamole during the last 6 years of follow-up, without any complications, including 1 year of pacing, prior to admission.


Proarrhythmic effects of reactive hypoglycemia. A patient with refractory atrioventricular nodal reentry tachycardia is reported in whom it was possible to document that reactive hypoglycemia was the trigger for aggravation of arrhythmia. Over a period of 6 years, a series of electrophysiological studies revealed that, when the patient was in a hypoglycemic state, initiation of tachycardia was easy and most importantly that tachycardla termination by extra-stimulus pacing always failed. Furthermore, atrial fibrillation was inducible or spontaneously occurred only when the blood glucose level was reduced by IV insulin administration.


Inverted pacemaker mediated tachycardia induced during noninvasive electrophysiology testing. This report describes a case in which an implanted pacemaker programmed to perform noninvasive electrophysiology testing resulted in an unusual form of pacemaker mediated tachycardia. The method of chest wall stimulation was used by programming a unipolar, triggered pacing mode with a short refractory period. In the AAT mode, far-field R wave sensing occurred beyond the physiological atrial refractory period. The triggered atrial response resulted in a single chamber, pacemaker mediated tachycardia.


Propafenone associated agranulocytosis.

Propafenone associated agranulocytosis. Propafenone hydrochloride was approved for marketing by the United States (U.S.) Food and Drug Administration (FDA) in November 1989. During U.S. clinical trials of propafenone, one case of agranulocytosis was seen. Seven additional cases have been reported outside the U.S. One German report of profound but reversible granulocytopenia appeared in 1982. In January 1991, the FDA reviewed adverse events reported with propafenone. Four reports of agranulocytosis were identified and are described. The reporting rate of approximately one case of agranulocytosis per 10,000 propafenone prescriptions per year likely underestimates the true incidence of this adverse event.


Hemodynamic effects of different temporary pacing modes for the management of bradycardias complicating acute myocardial infarction. Twelve patients requiring temporary pacing following acute myocardial infarction (AMI) (10 heart block, 2 junctional bradycardia) had hemodynamic measurements taken with ventricular demand pacing at 80 ppm (VVI80), ventricular demand pacing at the atrial rate (VVIa), physiological pacing (DDD), and spontaneous (intrinsic) rhythm. VVI80 mode did not improve any hemodynamic parameter compared with spontaneous rhythm. VVIa mode improved diastolic and mean arterial pressures only. DDD mode improved most hemodynamic parameters compared with spontaneous rhythm (cardiac output by 29% [P less than 0.0001]; blood pressure: diastolic by 24% [P less than 0.01], systolic by 19% [P less than 0.01], mean by 21% [P less than 0.005]; pulmonary wedge pressure by 10% [P = 0.057] and right atrial pressure by 24% [P less than 0.005]) and also significantly improved some parameters compared with VVIa (cardiac output by 20% [P less than 0.001], systolic blood pressure by 11% [P less than 0.01] and right atrial pressure by 15% [P less than 0.01]). Physiological pacing is hemodynamically superior both to ventricular pacing and spontaneous rhythm for patients requiring temporary pacing following AMI. Ventricular pacing at 80 ppm has little hemodynamic advantage over spontaneous rhythm.


Atrial natriuretic factor release during exercise in patients successively paced in DDD and rate matched ventricular pacing. Dual chamber pacemakers were implanted in nine patients with permanent second or third degree AV block (eight had complete retrograde block). Two identical exercise tests were performed after at least 1 month after implantation. During the first test (T1) the pacemaker was programmed to the DDD mode and heart rates were recorded every 15 to 30 seconds during exercise and 30 minutes after exercise. Following 30 minutes of rest, the implanted pacemaker was programmed to the VVT mode and driven by an external pacemaker via a skin electrode. The second exercise test (T2) was then performed and the rate of the external pacemaker was progressively changed to reproduce exactly the rate observed during T1 at the same exercise stress. Atrial natriuretic factor (ANF) levels were determined at rest, at regular intervals during exercise, and 30 minutes after exercise. ANF levels and release were statistically higher during rate matched ventricular, than DDD pacing. It is concluded that preservation of AV synchrony reduces ANF release induced by heart rate acceleration during exercise.


Quantitative relationship between atrial refractoriness and the dispersion of refractoriness in atrial vulnerability. We investigated the quantitative relationship between the atrial refractory period and the dispersion of refractoriness with respect to atrial vulnerability in 19 adult mongrel dogs. The atrial effective refractory period (AERP) was measured at the sinus node area (SNA), the low posterior right atrium (LRA), and the distal coronary sinus. The study was performed under the following conditions: (1) control status; (2) hypothermia (30 degrees C); (3) vagus nerve stimulation; and (4) a combination of (2) and (3). The subjects were separated into two groups: atrial fibrillation (AF) (+) group (n = 23), which developed AF by atrial extrastimulus due to increased vulnerability, and AF (-) group (n = 39), which did not develop AF. The mean AERP was 97 +/- 21 msec (mean +/- SD) in the AF (+) group and 124 +/- 23 msec in the AF (-) group, with a significantly shorter refractory period seen in the former (P less than 0.001). The dispersion of refractoriness was 59 +/- 24 msec in the AF (+) group and 29 +/- 18 msec in the AF (-) group, with a significant increase noted in the former (P less than 0.001). On X-Y coordinates (where X denotes the AERP, and Y denotes the dispersion of refractoriness) the data from the AF (+) group were clustered in the upper left region of the graph while the data from the AF (-) group were clustered in the lower right region. These two groups were separated by a linear equation of Y = 0.86X - 57 with a predictability of 90.3%.(ABSTRACT TRUNCATED AT 250 WORDS)


Catheter ablation of ventricular tachycardia: role of the underlying etiology and the site of energy delivery. The role of DC catheter ablation (CA) to treat patients with sustained monomorphic ventricular tachycardia (VT) is still debated. To assess the efficacy of VT CA, we studied the follow-up of 49 patients with VT who underwent CA. There were 33 patients with an old myocardial infarction (MI) (group G I) and 16 patients had noncoronary VT (group G II): CA was performed at the earliest endocardial activation (EEA) (20 patients in G I, 14 patients in G II) or at the area of slow conduction (ASC) (13 patients in G I, 2 patients in G II). During the mean follow-up of 35 +/- 25 (1-79) months, there were 17 patients in G I (52%) and 12 patients in G II (75%) with VT recurrences (P less than 0.05). Recurrences of VT was observed in 4 of 15 patients (27%) when CA was performed at the ASC, compared to 25 of 34 patients (74%) with CA at the EEA (P less than 0.01). These data show that DC CA is more successful in patients with coronary artery disease, particularly when CA is performed at the ASC.


Is DDD pacing superior to VVI,R? A study on cardiac sympathetic nerve activity and myocardial oxygen consumption at rest and during exercise. Rate responsive ventricular pacing (VVI,R) has been demonstrated to equal atrial synchronous ventricular pacing (DDD) with regard to hemodynamics and exercise tolerance. Whether the two modes are also comparable, with regard to cardiac metabolic effects, is not yet clear. We assessed central hemodynamics, cardiac sympathetic nerve activity (cardiac norepinephrine overflow), and myocardial oxygen consumption in 16 patients treated with rate responsive atrial synchronous ventricular pacemakers (DDD,R) due to high degree AV block. The study was performed at rest and during supine exercise at two workloads (30 +/- 12 and 68 +/- 24 watts, respectively) during VDD and rate matched VVI pacing (VVIm). Ventricular rates at rest and during both workloads were almost identical. Cardiac output at rest tended to be higher in the VDD mode, due to a slightly higher stroke volume. Central pressures including right atrial pressure and pulmonary capillary wedge pressure were similar in the pacing modes. The coronary sinus blood flow, the coronary sinus arteriovenous oxygen difference, and the myocardial oxygen consumption did not differ between the two pacing modes. Cardiac norepinephrine overflow was similar in the two pacing modes, at rest or during exercise. Thus, we found no significant differences between VDD and VVIm pacing with regard to central hemodynamics, cardiac sympathetic nerve activity (cardiac norepinephrine overflow), or myocardial oxygen consumption either at rest or during moderate exercise.


Programmed electrical stimulation of the ventricle: an efficient, sensitive, and specific protocol. A relatively simple and efficient ventricular programmed electrical stimulation (PES) protocol was developed, capable of achieving high degrees of sensitivity and specificity. In a series of 481 subjects, 1, 2, and 3 extrastimuli (ES) were used successively during sinus rhythm and ventricular pacing at two drive cycle lengths, at one or more ventricular sites, together with rapid ventricular pacing, and other maneuvers such as isoproterenol infusion. Three ES were used immediately after two ES at each drive rate, rather than returning after completion of the protocol with two ES. Using the protocol, appropriate arrhythmias could be induced in 88% of all patients with ventricular fibrillation, 84% of all patients with sustained ventricular tachycardia (91% with underlying coronary disease), and 58% of patients with severe nonsustained ventricular tachycardia. There were significant differences in inducibility between patients whose ventricular arrhythmias were due to coronary artery disease and other causes. In contrast, sustained ventricular arrhythmias (all ventricular fibrillation) could be induced in only 5% of a control group of control patients, for a specificity of 95%. The protocol described is simpler and more efficient than those that use exhaustive testing of two ES before going to three ES. Three ES during sinus rhythm proved to be the most productive step, with a higher yield ratio (true: false-positives) than two ES or three ES during pacing, especially at faster rates. Greater efficiency is also achieved by leaving the timing of an extrastimulus just beyond its effective refractory period when an additional extrastimulus is to be added, compared to protocols in which the extrastimulus is moved later in the cycle and then decremented in tandem with the additional extrastimulus. Coupling intervals less than 200 msec produced some false-positives, but fewer overall than intervals greater than or equal to 200 msec, and with yield ratios comparable to other protocol steps. The protocol described meets NASPE standards for ventricular programmed stimulation protocols, and with its demonstrated specificity and relative simplicity and efficiency may be useful as a model for groups not yet committed to an alternative protocol.


Sudden death in patients with congestive heart failure: future directions. Sudden, unexpected cardiac death continues to be a major clinical problem in patients with congestive heat failure. This review summarizes the current state of knowledge regarding the identification and management of these patients. The roles of ambulatory ECG monitoring, electrophysiological testing, signal-averaged ECG, and other methods of predicting increased risk of sudden death are discussed. The modes of sudden cardiac death and the potential mechanisms of ventricular arrhythmias in congestive heart failure are reviewed. Current therapeutic options including antiarrhythmic drugs, neurohormonal blockade, and automatic implantable cardioverter defibrillators are discussed. Finally, future directions and ongoing clinical investigations of the management of these complex patients are considered.


Ontogeny of the secretory immune system: maturation of a functional polymeric immunoglobulin receptor regulated by gene expression. In the rat, secretion of polymeric IgA from serum into bile is dependent upon the presence of a functional polymeric immunoglobulin receptor (pIgR) that acts as a hepatocyte plasma membrane receptor for ligand binding and as a transcellular transport molecule. The objective of this study was to document the developmental maturation and regulation of functionally intact rat liver pIgR. An adult pattern of IgA secretion was not detected until after day 23 postpartum (dPP), by using intravenously injected 125I-labeled dimeric IgA. Radioactive dimeric IgA was not detectable in hepatocyte transport vesicles until 21 dPP by electron microscopy autoradiographic analysis. By using a rabbit polyclonal antibody against the rat secretory component domain of the pIgR, Western blot analysis demonstrated that the plasma-membrane-bound pIgR levels in hepatocytes from rats aged 19-22 dPP increased 10-fold during this period. To determine whether or not this increase in membrane-bound pIgR reflected increased pIgR gene expression, we probed Northern blots of total cellular RNA extracted from neonatal rat liver with pIgR cDNA [GORF-1; Banting, G., Brake, B., Braghetta, P., Luzio, J.P. & Stanley, K. K. (1989) FEBS Lett. 254, 177-183]. The pIgR RNA levels between 19 and 22 dPP rose more than 20-fold and paralleled the increased membrane-bound pIgR protein during this same interval. These data demonstrate a developmentally regulated process that controls the ontogeny of biliary dimeric IgA secretion at the termination of the third week postpartum. The process appears to depend on the up-regulation of pIgR gene expression.


Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues. Three inositol 1,4,5-trisphosphate receptor (IP3R) cDNAs, designated IP3R-II, -III, and -IV, were cloned from a mouse placenta cDNA library. All three display strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains. Levels of mRNA for the three additional IP3Rs in general are substantially lower than for IP3R-I, though in the gastrointestinal tract the levels of IP3R-III may be comparable to IP3R-I. Cerebellar Purkinje cells express at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell.


Differential expression of c-fos and zif268 in rat striatum after haloperidol, clozapine, and amphetamine. Antipsychotic drugs are monoamine receptor antagonists. However, the mechanisms by which these direct actions are translated into therapeutic effects are unknown. Candidate mechanisms include receptor-mediated regulation of gene expression in target neurons. Inducible transcription factors, including certain immediate early genes (IEGs), may mediate between receptor-activated second messenger systems and expression of genes involved in the differentiated functions of neurons. We examined the specificity of induction of the IEGs c-fos and zif268 after acute administration of several antipsychotic drugs and, for comparison, the stimulant amphetamine, which has pharmacologic effects relatively opposite to those of antipsychotics. Antipsychotic drugs with potent dopamine D2 receptor antagonist properties, such as haloperidol, induced both c-fos and zif268 mRNA in the caudate-putamen; however, the atypical antipsychotic drug clozapine induced zif268 but not c-fos mRNA in that region. Similarly, haloperidol, but not clozapine, induced c-Fos-like immunoreactivity in the caudate-putamen. In contrast, both drugs induced c-Fos-like immunoreactivity in the nucleus accumbens. Like haloperidol, amphetamine induced both c-fos and zif268 mRNA in the caudate-putamen, but the anatomic patterns of induction of c-Fos-like immunoreactivity by the two drugs were dramatically different. Haloperidol and amphetamine induced AP-1 binding activity in cell extracts from the caudate-putamen, indicating that drug-induced IEG expression results in protein products that may function in the regulation of target gene expression. Thus these data demonstrate that inductions of IEG expression by haloperidol, clozapine, and amphetamine are specific, may be biologically relevant, and suggest avenues for further investigation.


Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Follitropin (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of anovulatory women. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, we constructed chimeric genes containing the sequence encoding the C-terminal peptide of the chorionic gonadotropin beta subunit (CG beta) fused to the translated sequence of the human FSH beta subunit (FSH beta). This region of CG beta is important for maintaining the prolonged plasma half-life of human CG dimer. The presence of the C-terminal peptide sequence did not significantly affect assembly of FSH beta with the alpha subunit or secretion of the dimer. In vitro receptor binding and steroidogenic activity of dimer bearing the FSH beta-C-terminal peptide chimera were the same as wild-type FSH. However, both the in vivo potency and half-life in circulation of the dimer bearing either one or two C-terminal peptide units were enhanced. Dimers containing FSH beta-CG beta chimeras could serve as potent FSH agonists for clinical use, and the present strategy may have wide applications for enhancing the in vivo half-life of diverse proteins.


Two mutations in the beta-globin polyadenylylation signal reveal extended transcripts and new RNA polyadenylylation sites. Two mutations in the beta-globin poly(A) signal were identified in Israeli patients with beta +-thalassemia by sequence analysis following PCR. One is a point mutation (AATAAA----AATAAG) and the other is a 5-base-pair deletion (AATAAA----A----). The mutant genes were used to investigate the function of the poly(A) signal in vivo and to evaluate the mechanism whereby these mutations lead to a thalassemic phenotype. Analysis of RNA derived from peripheral blood demonstrated the presence of elongated RNA species in patients carrying either mutation. Other aspects of RNA processing (initiation, splicing) were unimpaired. RNA obtained from the patients carrying the point mutation contained four discrete, extended RNA species, 1500-2900 nucleotides long, which were found to be polyadenylated. Some normal cleavage-polyadenylylation was also observed. The 5-base-pair deletion completely abolished cleavage at the normal site. This deletion mutation resulted in a phenotype of beta +-thalassemia, thus providing evidence that the extended mRNAs are translatable in vivo. Furthermore, additional transcripts, greater than 5 kilobases, presumably mRNA precursors, were found in all RNA samples, including those of nonthalassemic controls. The extended transcripts of the poly(A) mutants, together with the high molecular weight precursors, suggest that the human beta-globin gene transcription unit is significantly longer than previously recognized.


A 33-kDa polypeptide with homology to the laminin receptor: component of translation machinery. A 33-kDa polypeptide (termed p40), which shares an antigenic determinant with a laminin receptor and is under translational control, is believed to serve as a precursor to the receptor and to be related to the neoplastic state. The present study of subcellular localization of this protein shows it to be a cytoplasmic component not associated with the plasma membrane. Most of the cellular p40 was found to be associated with polyribosomes as well as with 40S to 60S cytoplasmic particles. Conditions that lead to polysome disruption also caused release of the polysomal form of p40 as smaller particles, and polysome reconstitution was accompanied by uptake of p40 into these structures. Because of the large abundance of this protein in the cells (six to eight copies per ribosome), it is unlikely that it represents a factor that associates with the 40S preinitiation complex. The p40-containing particles appear to represent a newly discovered structure involved in the process of polysome formation.


Primary structure of high molecular weight tau present in the peripheral nervous system. The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.


A leucine-to-proline mutation in the putative first transmembrane domain of the 22-kDa peripheral myelin protein in the trembler-J mouse. Peripheral myelin protein PMP-22 is a potential growth-regulating myelin protein that is expressed by Schwann cells and predominantly localized in compact peripheral myelin. A point mutation in the Pmp-22 gene of inbred trembler (Tr) mice was identified and proposed to be responsible for the Tr phenotype, which is characterized by paralysis of the limbs as well as tremors and transient seizures. In support of this hypothesis, we now report the fine mapping of the Pmp-22 gene to the immediate vicinity of the Tr locus on mouse chromosome 11. Furthermore, we have found a second point mutation in the Pmp-22 gene of trembler-J (TrJ) mice, which results in the substitution of a leucine residue by a proline residue in the putative first transmembrane region of the PMP-22 polypeptide. Tr and TrJ were previously mapped genetically as possible allelic mutations giving rise to similar, but not identical, phenotypes. This finding is consistent with the discovery of two different mutations in physicochemically similar domains of the PMP-22 protein. Our results strengthen the hypothesis that mutations in the Pmp-22 gene can lead to heterogeneous forms of peripheral neuropathies and offer clues toward possible explanations for the dominant inheritance of these disorders.


2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2'-dioxide)pyrimidine (TSAO) nucleoside analogues: highlyselective inhibitors of human immunodeficiency virus type 1 that are targeted at the viral reverse transcriptase. A series of pyrimidine nucleoside analogues containing [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino- 1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-ribofuranose as the pentose were found to inhibit human immunodeficiency virus type 1 [HIV-1(IIIB)] replication at a concentration of 0.06-0.8 microM but were not cytotoxic at a 1000- to 10,000-fold higher concentration. These nucleoside derivatives were also effective against various other HIV-1 strains, including those resistant to 3'-azido-3'-deoxythymidine, but not against HIV-2, simian immunodeficiency virus, Moloney murine sarcoma virus, or other RNA or DNA viruses. They proved to be highly specific inhibitors of the RNA-dependent DNA polymerase function of the HIV-1 reverse transcriptase, showing no marked inhibition of the HIV-1 reverse transcriptase-associated DNA-dependent DNA polymerase activity, HIV-2 reverse transcriptase, DNA polymerase alpha, herpes simplex virus 1 DNA polymerase, or Thermus aquaticus DNA polymerase.


Reduced transcription of the ferredoxin gene in metronidazole-resistant Trichomonas vaginalis. Metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] is used to treat infections caused by Trichomonas vaginalis, a sexually transmitted human parasite. This drug is administered in an inactive form and is reduced to its cytotoxic form within the hydrogenosome, an unusual organelle found in trichomonads. Metronidazole reduction occurs via ferredoxin-mediated electron transport. We have investigated the role of ferredoxin in metronidazole resistance. Immunoblot analysis of drug-resistant and -sensitive T. vaginalis strains shows that intracellular levels of ferredoxin are invariably reduced in the resistant strains relative to a sensitive strain. Similarly, Northern blot analysis shows that ferredoxin mRNA levels are reduced 50-65% in resistant strains. Using nuclear run-on assays, we show that ferredoxin gene transcription is reduced 40-65% in resistant strains. Sequence comparison of the region 5' of the ferredoxin gene among drug-sensitive and -resistant strains reveals two point mutations, at -178 and -239 nucleotides relative to the start of transcription, in a resistant strain. Interestingly, a protein of approximately 23 kDa binds to a 28-base-pair region that encompasses the mutation at -239 nucleotides. The binding affinity of this protein appears to be reduced in the mutant. These data strongly correlate drug resistance with altered regulation of ferredoxin gene transcription. A reduction in gene transcription results in decreased intracellular levels of ferredoxin. This, in turn, may play a role in metronidazole resistance by decreasing the ability of the cell to activate the drug.


Modulation of L-type calcium channels by sodium ions. It is universally believed that the removal of external sodium ions is without effect on calcium current. We now report that in enzymatically isolated guinea pig ventricular cells, the replacement of external sodium ions with certain other cations causes a 3- to 6-fold increase in peak L-type calcium current. The increase in current is reversibly blocked by L-type calcium-channel antagonists, not mediated by changes in internal calcium, and is inhibited by intracellular 5'-adenylyl imidodiphosphate, a nonhydrolyzable ATP analogue. The effects of sodium removal (and isoproterenol) were almost completely blocked by intracellular application of a specific (peptide) inhibitor of cAMP-dependent protein kinase. These experiments demonstrate a previously unknown effect of sodium ions to modulate calcium-channel phosphorylation via cAMP-dependent protein kinase.


Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope. An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.


Long-term agonist exposure induces upregulation of beta 3-adrenergic receptor expression via multiple cAMP response elements. During continuous stimulation by agonist, beta 1- and beta 2-adrenergic receptors (ARs) undergo processes that lead to decreases in receptor expression. This receptor down-regulation serves to limit the cellular cAMP response during chronic agonist exposure. In the recently described third subtype of the beta AR, denoted beta 3AR, we found four potential cAMP response elements in the 5' flanking region, suggesting that expression of this receptor might be positively regulated by agonists. These elements were cloned into the vector pA10CAT2, which contains a chloramphenicol acetyltransferase reporter gene, and transiently expressed in VERO cells. Three of these elements, TGACTCCA, TGAGGTCT, and CGAGGTCA (located 518, 622, and 1125 bases upstream of the beta 3AR coding block, respectively) were found to increase transcription of the chloramphenicol acetyltransferase gene in response to cAMP analogues and agents that increase intracellular cAMP. 3T3-F442A cells, when differentiated into the adipocyte phenotype by insulin, expressed beta 3AR, and nuclear runoff studies from such cells confirmed cAMP enhancement of beta 3AR mRNA transcription. In these cells, beta 3AR mRNA increased in response to exposure to the beta 3AR agonist isoproterenol and remained elevated during exposures of up to 24-30 hr. During prolonged exposure to agonist, no downregulation of beta 3AR expression in 3T3-F442A cells occurred. Indeed, beta 3AR expression increased during agonist exposure to approximately 165% of basal expression. In marked contrast, beta 1AR expression declined by approximately 70% in response to chronic agonist exposure. These studies reveal a subtype-specific prolonged transcriptional regulation of a beta AR gene by the end product of its signal transduction pathway. Thus, the beta 3AR undergoes a paradoxical increase in receptor expression during chronic agonist exposure.


Evidence that Mos protein may not act directly on cyclin. Using affinity-purified antiserum we have examined cyclin B2 levels in Xenopus oocytes at various stages of oogenesis. We found that cyclin B2 is detected from stage 2 to stage 6 as two bands, one of which is phosphorylated, and that cyclin B2 mass increases about 28-fold between stage 2 and stage 6. To examine the effect of Mos protein on cyclin phosphorylation, we microinjected synthetic Xenopus c-mos (c-mosxe) RNA into stages 4, 5, and 6 Xenopus oocytes. In stage 6 oocytes, maturation was induced by c-mosxe RNA, and, as is the case with progesterone treatment, all cyclin B2 was shifted to the phosphorylated form. However, c-mosxe RNA injected into stage 4 or 5 oocytes did not induce maturation or cause a shift in the relative proportion of the two cyclin B2 bands. These data suggest that Mos does not act directly to phosphorylate cyclin B2, causing the band shift during maturation. Cyclin B2 synthesis increases about 2-fold during maturation, in concert with total protein synthesis. Data from experiments on cyclin B2 stability indicate that the half-life of cyclin B2 is about 85 hr in stage 6. This suggests that if Mos protein has a direct effect on cyclin stability, it does so only at a later stage in oocyte maturation, but not at the onset.


Characterization of a type II collagen gene (COL2A1) mutation identified in cultured chondrocytes from human hypochondrogenesis. A subtle mutation in the type II collagen gene COL2A1 was detected in a case of human hypochondrogenesis by using a chondrocyte culture system and PCR-cDNA scanning analysis. Chondrocytes obtained from cartilage biopsies were dedifferentiated and expanded in monolayer culture and then redifferentiated by culture over agarose. Single-strand conformation polymorphism and direct sequencing analysis identified a G----A transition, resulting in a glycine substitution at amino acid 574 of the pro alpha 1(II) collagen triple-helical domain. Morphologic assessment of cartilage-like structures produced in culture and electrophoretic analysis of collagens synthesized by the cultured chondrocytes suggested that the glycine substitution interferes with conversion of type II procollagen to collagen, impairs intracellular transport and secretion of the molecule, and disrupts collagen fibril assembly. This experimental approach has broad implications for the investigation of human chondrodysplasias as well as human chondrocyte biology.


Gamma delta T-cell receptor repertoire in acute multiple sclerosis lesions. Gamma delta T cells are a distinct lymphocyte population that can exhibit reactivity with heat shock proteins over-expressed at inflammatory sites. As gamma delta T cells may be involved in the central nervous system (CNS) inflammatory process in multiple sclerosis (MS), we examined T-cell populations in MS plaque tissue by quantitative immunohistochemistry and sequence analysis of T-cell antigen receptor (TCR) delta chains. Gamma delta T cells that express the variable (V) gene segments V delta 1, V delta 2, and V gamma 2 (V gamma 9) were found to accumulate in acute, demyelinating MS plaques and appeared to have undergone clonal expansion, most likely because of recognition of a specific CNS ligand. Further, 60-kDa and 90-kDa heat shock proteins (hsp60 and hsp90), which may be target antigens for autoreactive gamma delta T cells, were found to be expressed in normal CNS tissue and overexpressed in acute MS plaques. In acute plaques, hsp60 was found in foamy macrophages, while hsp90 was detected in reactive astrocytes. These results provide evidence for a role of gamma delta T cells in active stages of MS.


Region-specific expression of a K+ channel gene in brain. Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions.


Expression cloning of a rat brain somatostatin receptor cDNA. We have used an expression-cloning strategy to isolate a cDNA encoding a somatostatin (somatotropin release-inhibiting factor, SRIF) receptor from rat cortex and hippocampus. A positive clone was identified by autoradiography after binding of radiolabeled SRIF to COS-1 cells previously transfected with pools of cDNA clones. The deduced amino acid sequence of the receptor displays sequence and structural homology to the family of G-protein-coupled receptors. The affinity of various SRIF analogs to the expressed receptor resembles their effects on growth hormone release from pituitary cells. In addition, the distribution of the mRNA in various tissues corresponds to that described for native SRIF receptors. Therefore, we conclude that we have isolated a rat brain SRIF receptor cDNA.


How cytotoxic T cells manage to discriminate nonself from self at the nonapeptide level. Class I major histocompatibility complex (MHC) antigens are confronted with an apparently insurmountable dilemma. Each should show a binding preference to a common enough variety of nonapeptides, so that one relevant nonapeptide can be found in at least every other viral protein to provoke a cytotoxic T-cell response. By so doing, however, the chance of that viral T epitope being self is greatly increased. Examination of human and viral nonapeptides preferred by HLA-B27 led to the following conclusions. (i) In normal cells, peptide fragments originating from 5000 or more diverse proteins vie for a finite number of class I MHC sites. Consequently, only those nonapeptides having the optimal binding affinity to a given class I MHC antigen can gain access to the plasma membrane. (ii) Tolerance is rendered only to those host nonapeptides with the optimal binding affinity. (iii) Because of the above noted tolerance, viral nonapeptides with the optimal binding affinity are invariably ignored. (iv) Viral T epitopes actually chosen are always second-echelon nonapeptides that are endowed with slightly less than the optimal binding affinity to a given class I MHC antigen. (v) Since such second-echelon nonapeptides would not gain access to the plasma membrane in normal cells, the issue of self or nonself is rendered irrelevant by this choice. (vi) Since viral T epitopes are of this type, cytotoxic T-cell responses against infected cells are expected to be effective only when a few viral proteins are made in large amounts at the expense of host proteins.


Induction by fungal elicitor of S-adenosyl-L-methionine synthetase and S-adenosyl-L-homocysteine hydrolase mRNAs in cultured cells and leaves of Petroselinum crispum. Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates transcription of many genes encoding specific steps in pathogen defense-related pathways. We report evidence that three cDNAs corresponding to such genes represent two key enzymes of the activated methyl cycle. Two cDNAs are derived from distinct members of the S-adenosyl-L-methionine synthetase gene family, based on extensive similarity of the deduced polypeptides with authentic enzymes from Arabidopsis thaliana, rat, yeast, and Escherichia coli. The third cDNA exhibits large similarity with a functionally related gene, encoding S-adenosyl-L-homocysteine hydrolase, from rat and a slime mold. Marked differences in the mRNA levels occurred in different organs of parsley plants. Elicitor treatment strongly induced both mRNAs in cultured cells as well as intact leaves and led to marked increases in S-adenosyl-L-homocysteine hydrolase enzyme activity. These results suggest a close metabolic link between pathogen defense and an increased turnover of activated methyl groups.


Tumor necrosis factor alpha is an autocrine growth regulator during macrophage differentiation. Previous experiments have revealed the expression of tumor necrosis factor alpha (TNF-alpha) transcripts in all murine bone marrow-derived macrophage colonies isolated from days 5 through 9 of differentiation in vitro. These results implicated a role for TNF-alpha gene expression during macrophage differentiation. Antisense oligomers to the initiation region of the TNF-alpha message were used to inhibit its expression, thus allowing the role of TNF-alpha gene expression in controlling the differentiation of macrophages to be determined. Results showed that TNF-alpha regulated the proliferation of macrophages during differentiation. Cells isolated on day 3 were exclusively vulnerable to the effects of blocking TNF-alpha gene expression, displaying a 30% increase in proliferation over control cells or sense oligomer-treated cells. Thus, in the absence of TNF-alpha gene expression, cells maintained proliferation instead of undergoing terminal differentiation. Exogenous TNF-alpha was capable of rescuing day 3 antisense-treated cells, therefore maintaining normal levels of proliferation. In contrast, blocking interleukin 1 beta gene expression by antisense oligonucleotide treatment had no effect on proliferation. Addition of exogenous recombinant murine or human TNF-alpha decreased the total cell number 25-50% regardless of whether cells were grown in medium containing colony-stimulating factor 1 (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These results suggested that exogenous TNF-alpha suppressed proliferation of early hematopoietic progenitors, whereas endogenous TNF-alpha regulated proliferation of macrophage progenitors. The number of differentiated, adherent macrophages on day 5 of differentiation in vitro was increased by TNF-alpha treatment of GM-CSF-induced macrophages but was suppressed in CSF-1-induced macrophages. These findings suggest that distinct TNF receptor expression and/or signaling is induced in differentiating macrophages stimulated with either growth factor.


Molecular cloning and characterization of the human cardiac Na+/Ca2+ exchanger cDNA. The Na+/Ca2+ exchanger plays important roles in Ca2+ handling in many excitable cells. In particular, the Na+/Ca2+ exchanger is expressed at high levels in the cardiac sarcolemma and is the dominant mechanism of Ca2+ extrusion from the cells. In addition, the exchanger has been suggested to play key roles in digitalis action and in postischemic reperfusion injury of cardiac myocytes. We report here the isolation and characterization of the cDNA encoding the human cardiac Na+/Ca2+ exchanger. Twelve overlapping clones corresponding to 5.6 kilobases of the exchanger cDNA sequence were isolated from 5 x 10(5) phage plaques screened. The sequence predicted a 973-amino acid polypeptide with a putative leader peptide, 11 potential membrane-spanning regions, and one large putative cytoplasmic loop between the fifth and sixth transmembrane helices. When RNA was synthesized in vitro from the cloned cDNA and injected into Xenopus oocytes, it induced expression of Na+/Ca2+ exchange activity at high levels, confirming that this clone encodes the functional Na+/Ca2+ exchanger. Southern blot analysis indicated that the cardiac exchanger gene exists as a single copy in the human genome, although existence of other related genes cannot be ruled out. Northern blot and S1 mapping analyses revealed that the cardiac type exchanger mRNA is expressed most abundantly in the heart and next in the brain. The cardiac-type exchanger mRNA was also expressed in the retina and in skeletal and smooth muscles at very low levels. The levels of mRNA encoding the exchanger were significantly lower in fetal hearts than in adult hearts but were unchanged in the myocardium from patients with end-stage heart failure.


Impairment of albuterol-induced suppression of food intake in diabetes mellitus. Albuterol (salbutamol), a beta 2 adrenoreceptor agonist, produced a dose-dependent decrease in food intake in Sprague-Dawley male control rats. This phenomenon appeared to be impaired in streptozotocin (STZ) diabetic rats. The density of beta 2 adrenoreceptors in the ventromedial hypothalamic nucleus was increased as a function of diabetes. In contrast, a decrease in the ventromedial hypothalamic 5-hydroxyindoleacetic acid (5-HIAA) concentration, an indicator of serotonin (5-hydroxytryptamine; 5-HT) release or turnover rate, was observed in this disease state. Neither the beta 2 adrenoreceptor level nor 5-HT turnover rate was altered in the periventricular hypothalamic nucleus of STZ diabetic rats. The concentrations of 5-HT in both hypothalamic nuclei were unchanged in these animals. Neurochemical and behavioral abnormalities featured in the diabetic state were reversed with institution of insulin therapy. These data conclude that diabetes-related impairment in the anorexic action of albuterol may be due to derangements in ventromedial hypothalamic beta 2 adrenoreceptor function.


[Histochemical representation of acetylcholinesterase in Alzheimer's disease]. Senile plaques in the cerebral neocortex, entorhinal cortex and hippocampus are reactive for Acetylcholinesterase (AChE). The same types of plaques are observed, as with immunostains for beta-protein, including the very simple ones, consisting nearly exclusively of loose deposits of beta-protein. If there are many plaques, the normal network of AChE-positive axons disappears. Explanations for both, the apparent shift of AChE from the axons to the plaques on the one hand and the very early development of AChE positive deposits during the plaque development on the other hand are sought.


[Changes of microenvironment and tumor cell heterogeneity--consequences for bioptic diagnosis]. During the last several decades, immunohistochemical studies of tumors, along with other approaches, have suggested that the clinical and biological progression results, at least in part, from the sequential appearance within the neoplasm of cellular subpopulations whose new characteristics reflect specific somatic genetic changes. However, CNS may provide a different microenvironment for activation and proliferation than other tissues. The tissue-specific distribution of intermediate filament proteins, in particular the keratins, permits their use as marker in histopathology, but several important exceptions are recognized. In this connection, it is of interest that, according to the other reports, glial tumors may be positive for different anti-keratin antibodies. However, the gliomas did not show an immunoreaction in any of the cases when HEA-125 and Ber-EP4 were applied. The great number of multihormonal pituitary adenomas and possible change of the immunohistochemically detectable hormone status in cases of recurrent tumors have particularly re-emphasized the need for new thinking about patterns of classification. The diagnosis of malignant melanoma has been considerably facilitated recently by the introduction of immunohistological labelling with antibodies selective against melanoma antigen (HMB-45). Our results confirmed the necessity of cautious interpretation of HMB-45 immunoreactivity because a HMB-45 expression can be observed in several non-melanotic tumors.


Type VIII collagen in the normal and diseased human brain. Type VIII collagen has been localized to specialized extracellular matrices in fetal tissues and has been suggested to be associated with cellular proliferation and angiogenesis (Sage and Iruela-Arispe 1990). In view of this hypothesis we studied its distribution in the normal and diseased human brain. Focal immunoreactivity was seen in histologically abnormal vessels of all 10 angiomas and 40 of 52 brain tumors. Staining was very weak in 3 embryonal and fetal brains, and it was absent in 20 normal adult brains and in 15 adult brains showing various cerebrovascular abnormalities. Our results provide additional evidence for the participation of type VIII collagen in some types of angiogenesis.


[Immunohistochemical studies of intermediate filament type in the human hypophysis and in adenoma]. The paper describes the value of immunohistologically estimated differences in the intermediate filament protein composition of the different cell types of human adenohypophysis and the corresponding pituitary adenomas. Interestingly, some tumors failed to express any type of intermediate filament proteins, whereas other coexpress cytokeratins and vimentin/or neurofilament protein.


Rapid development of giant aneurysm at the base of the brain in an 8-year-old boy with perinatal HIV infection. An 8-year-old boy with perinatal HIV infection developed a large fusiform aneurysm in the circle of Willis two years prior to death which was confirmed by radiological studies. The postmortem examinations revealed a predominantly intimal, proliferative lesion, and partial destruction of the internal elastic lamina in the involved arteries. Within the intima hyperplasia of fibroblasts and smooth muscle cells was observed. No inflammatory alterations, no granulomas and no multinucleated giant cells could be noted in the vascular walls and in the cerebral parenchyma. A small ischemic infarct was present in the left thalamus. Cerebellum, brainstem and medulla showed multiple areas of progressive multifocal leukoencephalopathy (PML). Immunohistochemistry with anti-gp41, a monoclonal antibody against HIV envelope did not exhibit any positive results. These findings implicate that the vascular lesion might be attributed to primary infection of the brain by HIV which led to a defect of elastic lamina and consecutive intimal hyperplasia. A second hypothesis could be based on the effect of extremely high dose AZT therapy avoiding inflammatory reaction after HIV infection.


Azelastine potentiates the prostaglandin-induced increase of cyclic AMP content in human platelets and in guinea-pig alveolar macrophages. The effect of azelastine on intracellular cyclic AMP concentration and on various indexes of cell activation was evaluated in guinea-pig alveolar macrophages and in human platelets. The effect of azelastine was further investigated on adenylate cyclase activity using membranes and homogenates from guinea-pig alveolar macrophages. Pretreatment of alveolar macrophages with azelastine prevented the activation induced by PAF-acether and by the chemotactic peptide fMLP as estimated by the reduced liberation of arachidonic acid metabolites formed by the cyclooxygenase and the lipoxygenase pathways. The effect of azelastine was concentration-dependent (50 to 500 microM) and reversible. Similarly, a short pretreatment with azelastine (100 microM) prevented arachidonic acid-induced platelet aggregation. This effect was also reversible after washing the platelets. In guinea-pig alveolar macrophages, azelastine induced a concentration-dependent (10 to 500 microM) increase in intracellular cyclic AMP and markedly potentiated the increase induced by PGE2. In human platelets, azelastine alone increased intracellular cyclic AMP concentration marginally only but, as in the case of macrophages, synergized with PGI2. Azelastine did not activate significantly adenylate cyclase unless a cytosolic factor was included within the membrane fraction. This effect of azelastine was not due to Ca2+ movements and was not modified by GTP. Our findings show that azelastine interferes with cell activation through a mechanism related to an increase in intracellular cyclic AMP concentration. The increase in cyclic AMP was induced by azelastine in intact cells and in homogenates but not in a crude membrane fraction. Those results indicate that azelastine modifies a cytosolic factor that may be phosphodiesterase. In addition, similarities between the effects of azelastine and those of reference phosphodiesterase inhibitors (theophylline, isobutyl-methyl-xanthine) are shown in this study, suggesting that azelastine might behave as a phosphodiesterase inhibitor.


Time-course of capsaicin-evoked release of calcitonin gene-related peptide from rat spinal cord in vitro. Effect of concentration and modulation by Ruthenium Red. The capsaicin-evoked release of calcitonin gene-related peptide (CGRP) from rat superfused dorsal spinal cord slices was investigated during sustained capsaicin exposure thought to represent equilibrium conditions. The dose-effect relationship for total peptide release evoked by single capsaicin doses (26 min exposure) was very steep with a threshold at 0.06 microM and a maximum at 0.3 microM capsaicin. With concentrations of capsaicin within this range the slow decline of the peptide release in the presence of capsaicin was not a consequence of exhaustion of an available peptide pool nor of neuronal impairment because potassium depolarization was still able to release CGRP. In contrast, with concentrations of capsaicin at 1.5 microM and above, there was a much faster decay of the release after the peak, most probably due to a loss of the secretion capacity caused by neuronal inactivation. When cumulative dose regimens for capsaicin were employed, release of CGRP could be stimulated only up to a dose of 1-1.5 microM capsaicin; further increase in capsaicin concentration was ineffective. This was also most probably due to a loss of the secretion capacity caused by neuronal inactivation and not caused by depletion of a releaseable peptide pool. Release of CGRP evoked by capsaicin concentrations in the range of 0.1-0.3 microM in either dosage protocol was reduced in the presence of Ruthenium Red (RR, 2.5 microM). RR did not reduce neuropeptide release evoked by capsaicin concentrations at or above 1-1.5 microM, nor did it affect the inactivation of the release process at such high capsaicin concentrations. The results demonstrate that, upon sustained exposure to capsaicin, different ranges of concentration can be established at which either only stimulatory or a mixture of stimulatory and inhibitory effects determine the amount of neuropeptides released.


Specificity of oligonucleotide probes complementary to evolutionarily variable regions of 16S rRNA from Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs.


Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-antitrypsin in carcinomas of the exocrine pancreas of the dog. The immunocytochemical detection of amylase, carboxypeptidase A, alpha 1-antitrypsin, carcinoembryonic antigen (CEA) and keratin in normal canine pancreatic tissue and in carcinomas of the exocrine pancreas of the dog is described. In the normal pancreas, the acinar cells contain amylase, carboxypeptidase and alpha 1-antitrypsin. The pancreatic ducts react with the antikeratin antibody. Twelve out of 14 pancreatic exocrine carcinomas showed immunoreaction with antiamylase antibody, and 10 with anticarboxypeptidase antibody. Five neoplasms reacted with anti-CEA antibody and three with the anti-alpha 1-antitrypsin antibody. It was not possible to find any systematic difference in the immunocytochemical profiles of acinar, tubular and undifferentiated carcinomas. These results indicate that immunocytochemical marking of amylase and carboxypeptidase is of value in the diagnosis of pancreatic neoplasms in the dog, especially if metastasis is the only material available for study and the tumour does not show any diagnostic feature on routine light microscope preparations.


In vivo assessment of drug sensitivity of African trypanosomes using the akinetoplastic induction test. Following treatment of mice infected with Trypanosoma congolense or T brucei brucei with various doses of isometamidium chloride or diminazene aceturate, the induction of akinetoplastic (AK) forms was observed in the trypomastigotes of both species within 10 hours of drug administration. The levels of AK-induction were closely correlated with the levels of resistance to each compound found using a standard in vivo drug assay in mice. In general, ineffective doses of either compound conferred AK-induction rates of less than 30 per cent; relapsing cases had between 30 and 50 per cent while curative doses had AK-induction rates of 50 per cent or more. In vivo determination of AK-induction rates using ordinary light microscopy is thus a potentially feasible alternative indicator to the conventional use of mice infection and treatment methods for assessing drug sensitivity in African trypanosomes.


Intraarterial injection therapy of newly developed cisplatin-phosphatidyl choline-lipiodol suspension for hepatocellular carcinoma. Twenty-seven patients with hepatocellular carcinoma were treated by intraarterial injection of a suspension of cisplatin powder and iodized oil using phosphatidyl choline as a dispersing stabilizer. A reduction in tumor size of over 25% was obtained in 23 patients (85%) and a reduction of more than 50% was obtained in 17 patients (63%). In all of 14 patients with a high serum alpha-fetoprotein level (more than 200 ng/ml), 27% to 99% reduction in the level was obtained. The one-, two-, and three-year survival rates were 74%, 50%, and 35%, respectively. As for side effects, digestive symptoms were rather frequently observed. Liver abscess and cholangitis were observed in one patient each in patients combined with embolization using gelatin sponge particles. Injection of the suspension with embolization was superior to injection only in therapeutic effect, but was associated with a higher frequency of side effects. The therapeutic effect was better in cases of higher Lipiodol retention in the tumor on follow-up CT.


Isolation and preliminary characterisation of a novel retrovirus isolated from a leukaemic dog. A novel canine retrovirus was isolated from mononuclear cells of the peripheral blood of a leukaemic dog. The main clinical and pathological findings in this dog were lethargy, anorexia, weakness, dyspnoea, severe anaemia, thrombocytopenia and a high white blood cell count, practically all of which were lymphoblasts. The virus was isolated from mononuclear cells obtained from the blood, cocultivated with indicator cells. The virus particles encode a reverse transcriptase with Mg++ preference, have a density in sucrose gradients of 1.16 g ml-1, and induce syncytia in permissive cell cultures such as Himalayan tahr ovary and canine fetal thymus lines. This agent replicates to high titres. The virus exhibits a morphogenesis and morphology typical of lentiviruses. Immunoblotting and competitive radioimmunoassays failed to detect immunological crossreactivity with other representative lentiviruses and oncoviruses of the retrovirus family.


Analysis of patients treated with radiotherapy in the year 1990. We analyzed the patients treated with radiotherapy in Gunma University Hospital in 1990, to determine the characteristics of the practice of radiotherapy. During this period, 540 patients with various diseases received radiation therapy. Five hundred thirty-one (98.3%) of them had malignant tumors, and the remaining nine had benign diseases. Of the patients with malignant tumors, primary tumor of the head and neck (21.7%), and lung and mediastinum (13.0%) were most common. Four of nine patients with benign diseases had pterygium. Three hundred thirty-two patients (63%) had previously untreated malignant tumors. Curative radiotherapy was performed in 296 patients (56%), and palliative radiotherapy in 235 (44%). Of CRG patients, 43% were treated with radiation alone and 40% with surgery followed by radiotherapy. The proportion who received curative radiotherapy was the highest in patients with cervical cancer (80%). Twenty-four of 34 patients treated with intracavitary irradiation had cervical cancer. Interstitial irradiation was performed in nine patients. Four patients with pterygium were treated postoperatively with surface irradiation using an Sr-90 contact device. Radioactive iodine was administered to ten patients: eight thyroid cancer patients and two hyperthyroid patients.


Single lung transplantation for eosinophilic granulomatosis. A 26-year-old woman with end-stage lung disease due to eosinophilic granulomatosis had single right lung transplantation with an excellent function result that persists beyond 9 months of follow-up. Single lung transplantation offers excellent palliation to selected patients with end-stage lung disease.


[Hodgkin's disease and infection with human immunodeficiency virus]. Three patients with Hodgkin's disease, mixed cellularity subtype, plus infection by human immunodeficiency virus are presented. Two of them were intravenous drug abusers, and one had promiscuous heterosexual behaviour; they all presented B-type symptoms. One patient died because of infection, whereas the other two persisted in complete remission after treatment at 4 and 5 years of follow-up, respectively. None of the patients still alive has developed AIDS. The criteria for considering Hodgkin's disease as an AIDS-related lymphoproliferative disorder are discussed.


Growth cones and structural variation of synaptic end-bulbs in the cochlear nucleus of the adult cat brain. To explore the potential for structural variation and new growth at the synapse, we studied the morphological patterns of the end-bulbs of cochlear nerve axons in adult cats by using rapid Golgi, reduced silver, and electron microscopic methods. Horseradish peroxidase labeling of these endings in the anterior division of the antroventral cochlear nucleus was produced by anterograde transport following injection into the cochlea. Three types of end-bulbs were distinguished, regardless of method: reticular, coalescent, and ringed forms, all synapsing on spherical bushy cells. The reticular variety corresponds to the classically described end-bulb and constitutes the majority in all regions of the tonotopic map. The ringed end-bulb, described here for the first time, forms an excitatory synaptic cuff around the base of a bushy cell's main dendrite; these endings were localized to the region receiving cochlear input in the 1-6 kHz range, which is used in vocalization. The coalescent ending forms a small fraction of the end-bulb population throughout the region studied. The findings raise the possibility of functional differences between these synaptic types. Growth cones and retraction clubs were present on most, if not all, of the end-bulbs in every adult cat studied. A systematic survey of the end-bulb patterns revealed a continuous gradient of variation, in which each synaptic type forms a distinct mode. These findings lead us to hypothesize that the end-bulbs are in a continual state of structural and functional flux. These endings should prove useful for studies on the modifiable properties of central synapses.


Effect of thiol group modification on ion flux and ligand binding properties of the GABAA-benzodiazepine receptor chloride channel complex. Agents that modify thiol groups have been shown to alter ligand binding at a variety of receptor sites. In addition, alkylation of sulfhydryls has been shown to block ion channel conductance. We studied the effects of thiol reagents on gamma-aminobutyric acid (GABA)-activated chloride flux (36Cl-) and [3H]-diazepam binding in mouse brain membrane preparation (microsacs). Incubation of microsacs in the presence of: mercuric chloride (HgCl2), p-chloromercuriphenylsulfonic acid (pCMBS), hydroxymercuribenzoate (HMB), n-ethylmaleimide (NEM), or iodoacetic acid (IAA) attenuated GABA-stimulated Cl- uptake. The thiol reagents reduced both maximal stimulation and the potency of GABA to induce Cl- uptake. Thiol reagent treatment decreased the affinity of high-affinity [3H]-muscimol equilibrium binding. Supernatant prepared from microsacs treated with pCMBS stimulated Cl- uptake in the absence of GABA agonist in microsacs unexposed to thiol reagents. The supernatant taken from pCMBS-treated microsacs also stimulated [3H]-diazepam binding. This effect was blocked by the addition of the GABA receptor antagonist bicuculline. The concentration of endogenous GABA in supernatant from pCMBS-treated microsacs was sixfold greater than that in supernatant from control microsacs. This increase in levels of endogenous GABA by thiol reagents was due to both an increase in GABA release and a decrease in high-affinity GABA uptake.


Selective subregional dopamine depletions in the rat caudate-putamen following nigrostriatal lesions. Previous work has demonstrated a complex neurochemical and neuroanatomical heterogeneity of the striatum in normal brains. The present research investigated whether the heterogeneous distribution of dopamine would be altered following unilateral injections of the neurotoxin 6-hydroxydopamine into the substantia nigra of the rat. Four weeks following injection, the nucleus accumbens and subregions of the caudate-putamen and substantia nigra were dissected and analyzed by HPLC with electrochemical detection for dopamine, 5-hydroxytryptamine, and their respective metabolites. Levels of dopamine and its metabolites in the anterodorsolateral caudate-putamen were depleted more than medial, posterior, and ventral, striatal areas in partially lesioned animals (less than 90% dopamine depletion). This resulted in an alteration of striatal heterogeneity such that a mediolateral gradient of dopamine tissue content was now superimposed on the normal rostrocaudal gradient observed in controls. Paralleling these findings, dopamine was more depleted in the lateral, as opposed to the medial, substantia nigra. These results indicate that the nigrostriatal dopamine system degenerates in a heterogeneous fashion following 6-hydroxydopamine administration. It is speculated that the differential loss of dopamine neurons observed in the nigra of Parkinson's patients may be due to a differential sensitivity to toxins within the nigra.


Acute toxicity of tri-n-butyltin chloride (TBTC) in the Syrian golden hamster. The effects of tri-n-butyltin chloride (TBTC) on Syrian golden hamsters were studied. TBTC in single doses of 0, 29.6, 44.4, 66.7, 100 or 150 mg/kg were given to 10 male hamsters in each group and the mortality rates were determined two weeks thereafter. They were 0% (0/10), 0% (0/10), 10% (1/10), 10% (1/10), 30% (3/10) and 70% (7/10) for 0, 29.6, 44.4, 66.7, 100 and 150 mg/kg groups, respectively. In the case of females, seven groups consisting of 10 animals each were given TBTC at doses of 0, 29.6, 44.4, 66.7, 100, 150, or 225 mg/kg. The mortality rates determined two weeks after the TBTC treatment were 0% (0/10), 10% (1/10), 10% (1/10), 0% (0/10), 40% (4/10), 30% (3/10) and 90% (9/10) for 0, 29.6, 44.4, 66.7, 100, and 225 mg/kg groups, respectively. Based on these mortality data, the LD50s via oral administration were determined as 146.9 mg/kg (95% C.I. 111.8-193.3 mg/kg) for the male and 172 mg/kg (95% C.I. 127.2-233.4 mg/kg) for the female. Regarding pathological changes, animals experienced lesions in the bile duct, such as the dilatation of the common bile duct and cholestasis, and/or adhesion of the bile duct to the liver, gallbladder, pancreas and duodenum. In a separate experiment, a single dose of 44.4 mg/kg TBTC was administered orally to 12 male hamsters, then the concentrations of TBTC and its metabolite, di-n-butyltin chloride (DBTC), in the liver were analyzed by means of gas chromatography for 14 days after the treatment. The maximum concentrations of TBTC and DBTC appeared one day after the administration, and decreased rapidly thereafter. The concentration of DBTC was found to be higher than that of TBTC throughout the experimental period.


Parasympathetic innervation of the rat thymus during first life period: histochemical and biochemical study. In the present study we analyzed development of the rat thymus parasympathetic innervation using histochemical determination of distribution and density of acetylcholinesterase (AChE) positive nerve profiles, as well as biochemical measuring of the activity of this enzyme. Rat thymuses from late embryonal to adult periods of life were analyzed. The AChE-positive nerve profiles were found, for the first time, on day 18 of fetal life in capsule and interlobulary septae, but also in the subcapsulary and cortico-medullary areas. The density of these profiles increased during the thymic development. The AChE positive nerve profiles in subcapsulary region were observed mainly in close proximity to the thymic epithelial cells, while in the cortico-medullary region they were found in apposition to the thymocytes. The biochemical analysis showed that the specific AChE activity in rat thymus was high on day 19 of gestation. A significant increase in the activity of this enzyme was measured by the third day of postnatal development, and its activity remained approximately at the same level up to the day 90. The present results suggest that thymus receives parasympathetic innervation relatively early in ontogeny; in addition, these nerve fibers could be involved in the regulation of the organ activity, at least, through action upon the thymocytes and/or by modulation of the thymic epithelial cell activity.


Anti-hepatitis C virus (HCV) screening at a Canadian Red Cross center: significance of a positive c100 HCV enzyme-linked immunosorbent assay. The c100 hepatitis C virus (HCV) enzyme-linked immunosorbent assay (ELISA) has been used to screen blood donors to prevent transfusion-associated non-A,non-B hepatitis. This test is not specific, and only about 25 percent of c100 HCV ELISA-positive blood samples appear to transmit hepatitis C. However, the intensity of the ELISA (sample/cutoff ratio [S/C], greater than 2) could identify a subpopulation of donors that are at high risk for transmitting hepatitis. Blood samples from 20,186 volunteer blood donors at a Canadian Red Cross blood transfusion center were screened for antibodies to HCV using the c100 HCV ELISA. Fifty-nine (0.3%) of these donors were repeatably reactive on ELISA. When their samples were tested with the c100 recombinant immunoblot assay (RIBA) and second-generation RIBA (RIBA-2), 26 (44%) and 31 (52%) samples, respectively, were found to be positive. Thirty-three of the 59 ELISA-reactive donors had an S/C greater than 2. Of these 33 donors, 30 (91%) had elevated alanine aminotransferase (ALT), 27 (82%) were RIBA-2 positive, and 22 (67%) had risk factors for hepatitis. In contrast, of the 26 ELISA-reactive donors with S/C less than 2, only 7 (27%) had elevated ALT, and 4 (15%) were RIBA-2 positive and also had high risk factors for hepatitis. Thus, while the HCV ELISA may lack specificity, its intensity can serve to identify a subgroup of donors that are at high risk for transmitting hepatitis.


Is the multidrug transporter a flippase? The phenomenon of multidrug resistance is correlated with the presence of a membrane protein, P-glycoprotein, which pumps a wide variety of drugs out of cells thus reducing their toxicity. However, the mechanism of this pumping action remains unclear. In this article, we suggest that several properties of the multidrug transporter may be explained if it acts as a 'flippase' to transport drugs from the inner leaflet of the lipid bilayer to the outer or to the external medium.


Rapid isolation of human CD34 hematopoietic stem cells--purging of human tumor cells. Human CD34+ hematopoietic stem cells were purified using a new technology in which monoclonal antibodies are covalently immobilized on polystyrene surfaces. The CD34+ cell isolation scheme involved three sequential processes: (1) purification of bone marrow mononuclear cells; (2) enrichment of CD34+ cells using covalently immobilized soybean agglutinin; and (3) positive selection of CD34+ cells using polystyrene surfaces coated with the anti-CD34 monoclonal antibody ICH3. CD34+ cells purified by this process have both low-to-medium forward light scatter and low 90 degrees light-scatter properties. Moreover, the purified CD34+ cells are greater than 85% viable, express appropriate characteristic surface antigens, and are 10-50-fold enriched in short- and long-term hematopoietic activity. CD34+ cells collected in this manner from bone marrow samples contaminated with radiolabeled breast carcinoma, neuroblastoma, acute myelogenous leukemia, or small cell lung carcinoma cells were 99.9% depleted of the tumor cells. The CD34+ cell selection devices are sterile and are easily scaled-up to process clinical scale bone marrow samples.


Conversion of liver allograft recipients from cyclosporine to FK506 immunosuppressive therapy--a clinicopathologic study of 96 patients. The effect of conversion from cyclosporine-steroid immunosuppression to the new agent FK506 was studied in 96 liver allograft recipients who were experiencing graft dysfunction or cyclosporine toxicity. Patients were stratified according to the cause of graft dysfunction that ultimately led to conversion to FK506. Response to FK506 introduction was monitored pathologically and biochemically. The outcome of a switch from CsA to FK506 was highly favorable in patients experiencing acute and the early stages of chronic rejection, despite optimal conventional therapy. Patients with later stages of chronic rejection did not respond to conversion to FK506 and most eventually lost their liver grafts in this process. Patients in whom we had difficulty separating chronic rejection from chronic persistent or low-grade chronic active hepatitis were mostly unaffected by conversion to FK506. Active hepatitis was a poor indication for conversion, because most of the patients experienced graft failure or died from liver failure. As a group, there was no statistically significant change in renal function 180 days after conversion to FK506. These findings expand the experience with FK506 in human liver allograft recipients.


A polymorphic human kidney-specific non-MHC alloantigen. Its possible role in tissue-specific allograft immunity. Tissue specific non-MHC alloantigens play a crucial role in allograft immunity. However, their structural properties have remained elusive, largely due to their inability to induce a strong antibody response. We report the characterization of a monkey heteroantiserum, MHK-I, raised against human kidney cells, that serologically reacts specifically with kidney cells after extensive absorptions of anti-HLA class I and II reactivities. The non-MHC MHK-I-binding molecule(s) is expressed only in the renal cortex on the glomerulus, peritubular capillaries, venous endothelium, and tubular epithelium. Immunochemically, MHK-I recognizes a kidney-specific non-MHC alloantigen of Mr 90,000 to 100,000 (90 kD). These properties of MHK-I are similar to those of the previously characterized alloantibodies eluted from rejected kidneys. These alloantibodies bind to the kidney from which the antibody was eluted and to a few others but are unlike MHK-I, which binds to extracts prepared from all human kidneys. Biochemical analysis by two-dimensional electrophoresis (pI ranging between 4.5 and 5.5) and peptide fingerprinting provide further evidence that the alloantigen is polymorphic. These findings imply that the non-MHC kidney-specific molecule(s) may function as target(s) for immune destruction of renal allografts.


Evidence that umbilical cord blood contains a higher frequency of HLA class II-specific alloreactive T cells than adult peripheral blood. A limiting dilution analysis. Umbilical cord blood has been used to effect hematological reconstitution and there are sufficient stem cells available in the cord blood obtainable from a single placenta to reconstitute an adult patient. Umbilical cord blood might therefore, have widespread potential use in the field of bone marrow transplantation. We compared alloreactivity of paired samples of adult and cord peripheral blood mononuclear cells, by measuring frequencies of both alloreactive T helper cells and cytotoxic T cell precursors (CTLp), using limiting dilution analysis. In addition we compared the phenotype of adult and neonatal PBMC, using monoclonal antibody staining. Cord PBMC in general showed higher frequencies of alloreactive Th than adult PBMC, with statistically significant differences in 6 out of 10 experiments. There was no statistically significant difference between adult and cord CTLp frequencies. Adult and cord PBMC surface phenotype was similar, except that cord blood contained fewer lymphocyte function-associated-3 (LFA-3) positive (memory) cells.


The protective effect of FK506 pretreatment against renal ischemia/reperfusion injury in rats. The effect of pretreatment with FK506 on renal ischemia and reperfusion (I/R) injury was investigated using a rat model. Animals were assigned to one of two groups (20 rats each). Group 1 animals (controls) received 0.5 ml saline while group 2 animals received FK506 (0.3 mg/kg), administered intravenously 24 hr prior to the induction of renal ischemia. A 60-min period of ischemia of the right kidney was induced, and upon reperfusion a left nephrectomy was performed. Blood samples for estimation of BUN, creatinine, and tumor necrosis factor were collected on days 0 (preischemia), 1, 2, 3, 5, 7, and 10 (postischemia). Rats were sacrificed after day 10 and renal tissue was examined histologically. All animals survived the ischemic episode. FK506 pretreatment significantly reduced the serum levels of BUN (P less than 0.02), creatinine (P less than 0.02), and TNF (P less than 0.05) as compared with that seen in controls. Histologically, at day 10, the kidneys showed the expected sequelae of prior renal I/R with various degrees of tubular damage. However, no objective differences were evident between the two groups. Based upon these data, it can be concluded that (1) FK506 pretreatment ameliorates the functional renal injury associated with I/R, (2) renal ischemia induces the release of TNF, and (3) FK506 pretreatment results in a significant inhibition of TNF production. These data suggest that the release of TNF may be responsible for the increasing of BUN and creatinine levels seen after renal I/R and that pretreatment of renal donors with FK506 may improve renal function in the immediate post-transplant period.


Developmental and abnormal cell death in C. elegans. Genetic analysis in Caenorhabditis elegans has identified several genes that function in normal developmental death as well as genes that can mutate to cause inappropriate cell death. The processes whereby some of these abnormal deaths occur depend on genes that participate in normal programmed cell death; others occur by an independent mechanism whereby mutation of members of a gene family leads to cell lysis. Molecular characterization of these 'death' genes in C. elegans is beginning to provide insight into the normal and aberrant mechanisms of cell death.


Separate visual pathways for perception and action. Accumulating neuropsychological, electrophysiological and behavioural evidence suggests that the neural substrates of visual perception may be quite distinct from those underlying the visual control of actions. In other words, the set of object descriptions that permit identification and recognition may be computed independently of the set of descriptions that allow an observer to shape the hand appropriately to pick up an object. We propose that the ventral stream of projections from the striate cortex to the inferotemporal cortex plays the major role in the perceptual identification of objects, while the dorsal stream projecting from the striate cortex to the posterior parietal region mediates the required sensorimotor transformations for visually guided actions directed at such objects.


Control of neuronal excitability by corticosteroid hormones. The rat adrenal hormone corticosterone can cross the blood-brain barrier and bind to two intracellular receptor populations in the brain--the mineralocorticoid and glucocorticoid receptors. Recent studies have revealed that the corticosteroid hormones are able to restore changes in neuronal membrane properties induced by current or neurotransmitters, probably through a genomic action. In general, mineralocorticoid receptors mediate steroid actions that enhance cellular excitability, whereas activated glucocorticoid receptors can suppress temporarily raised neuronal activity. The steroid-mediated control of excitability and the implications for information processing in the brain are reviewed in this article.


Intrinsic neuronal determinants of regeneration. Axon growth and axon regeneration are co-operative processes; the speed and extent of axon growth are influenced both by the properties of the environment surrounding the axon growth cone, and the properties of the neuron itself. In recent years, the environmental influences on axon growth have received most of the attention directed towards this area of research, but the properties of the neurons themselves are likely to be just as important. Within both adults and embryos there are differences in the growth potential of different neuronal types, and there is also evidence for an overall decrease in the vigour of axon growth with neuronal age.


A consideration of neural counting methods. It is often necessary to obtain unbiased estimates of neuronal or synaptic numbers. In the past, estimates were almost always done by counting profiles of these structures in single histological sections. Assumptions were then made and calculations were done to determine particle numbers or ratios. To the extent that the assumptions deviated from reality, the conclusions will be biased. That these biases are, in fact, serious has recently become apparent. To obtain unbiased particle counts, the presently available methods are serial-section reconstructions (which are accurate but cumbersome), and the recently developed disector method. The disector method, because it is unbiased and easy to use, is becoming the method of choice. The goals of this paper are to show why previous methods are biased and to describe the rationale behind the disector method so that neuroscientists can consider its appropriateness for their work.


Transmission at autonomic neuroeffector junctions. For organs innervated by the autonomic nervous system, it is generally held that neuroeffector transmission is achieved by varicosities releasing transmitters some distance from the membranes of target cells. Transmitters are thought to diffuse through the extracellular space and interact with post-junctional receptors that are widely distributed over the cell membranes. This article presents an alternative view, suggesting that transmission can occur at organized neuroeffector contacts, that transmitters interact with restricted pools of specialized junctional receptors, and that many receptors on target cells are not involved in neuroeffector transmission.


Activation and modulation of neuronal K+ channels by GABA. Although the concept of GABAB receptors was introduced only ten years ago, several actions of GABAB agonists are already well established. They cause depression of transmitter release, a decrease in voltage-dependent Ca2+ conductance and an increase in K+ conductance. It has recently been reported that GABA also changes the voltage dependence of the transient ('A' type) K+ channel. Depression of transmitter release by GABAB agonists may be caused by a decrease in Ca2+ conductance, an increase in K+ conductance or a modulation of A channels in presynaptic nerve terminals. Slow IPSPs in some neurons are generated by an increase in K+ conductance that can be blocked by GABAB antagonists and pertussis toxin. K+ channels of variable amplitude that are blocked by pertussis toxin are activated by GABAB agonists in cultured hippocampal neurons. Since arachidonic acid activates similar channels in excised patches of membrane, it may form part of a normal second messenger system linking GABAB receptors to K+ channels.


Australian marsupials as models for the developing mammalian visual system. This article makes two points. First, the diprotodont marsupials, including the kangaroos, wallabies and the Australian possum are not primitive mammals, and their brains make as good a general model of the higher mammals such as monkeys and humans as do those of the more common laboratory mammals such as cats and rats. Second, the peculiarities of marsupial reproduction, which comprises a very short period of intrauterine development, followed by a relatively protracted period of development in the pouch, provide unparalleled advantages for research into mammalian neuroembryology. Examples will be provided of how such research has made a contribution to our understanding of neural development, concentrating primarily on the visual system.


Mechanical preprocessing in the mammalian cochlea. The mammalian cochlea responds with exquisite sensitivity to the small fluctuations in air pressure that make up the stimulus of sound. Moreover, it responds to pressure fluctuations that occur extremely rapidly and that vary over a wide range of intensities--in both cases, to an extent outside the capabilities of unaided nerve fibres. Research performed during the past decade has shown that these properties are dependent on a physiological source of mechanical energy that operates probably within the outer hair cells of the organ of Corti. These cells, which are anatomically and functionally similar to the primary receptor cells, the inner hair cells, are believed to function as a source of mechanical power to assist the mechanical sensitivity of the cochlea, by mechanisms that currently are not understood. Several possible mechanisms have been proposed, but each has limitations that may make it an unsuitable candidate. Recent work has also demonstrated the likely role of mechanoelectrical transduction in outer hair cells in controlling the power source and thereby influencing the sensitivity and amplitude range of the cochlea.


Kinaesthetic signals and muscle contraction. Signals generated both peripherally and centrally contribute to the group of sensations termed kinaesthesia. Many experiments report sensations of position and movement under passive relaxed conditions without muscle contraction. However, kinaesthetic acuity is probably of greater functional value when subjects are active rather than passive and, accordingly, movement detection is markedly improved by muscular contraction. One mechanism contributing to this enhancement is likely to involve muscle spindle volleys. When identical microstimulation techniques are applied to skin, joint and muscle spindle endings innervating the hand, some cutaneous afferents and some joint afferents elicit a sensation, but activation of certain other cutaneous afferents and muscle spindle afferents rarely does. Activity in more than one muscle spindle afferent may be required for kinaesthetic sensations, whereas some single cutaneous and joint afferents may have a more 'secure' central projection.


Roles of peptides in transmission in the enteric nervous system. Studies of the enteric nervous system have proved to be important in the development of new concepts of the chemical nature of transmission from neurons. In particular, they have revealed the multiplicity of influences that peptides can have on transmission, such as their action as primary transmitters, and the fact that they often act as co-transmitters in enteric neurons. However, in other cases no roles can be attributed to neuropeptides in enteric neurons, and their involvement in short-term changes in excitability seems minor.


Choline metabolism as a basis for the selective vulnerability of cholinergic neurons. The unique propensity of cholinergic neurons to use choline for two purposes--ACh and membrane phosphatidylcholine synthesis--may contribute to their selective vulnerability in Alzheimer's disease and other cholinergic neurodegenerative disorders. When physiologically active, the neurons use free choline taken from the 'reservoir' in membrane phosphatidylcholine to synthesize ACh; this can lead to an actual decrease in the quantity of membrane per cell. Alzheimer's disease (but not Down's syndrome, or other neurodegenerative disorders) is associated with characteristic neurochemical lesions involving choline and ethanolamine: brain levels of these compounds are diminished, while those of glycerophosphocholine and glycerophosphoethanolamine (breakdown products of their respective membrane phosphatides) are increased, both in cholinergic and noncholinergic brain regions. Perhaps this metabolic disturbance and the tendency of cholinergic neurons to 'export' choline--in the form of ACh--underlie the selective vulnerability of the neurons. Resulting changes in membrane composition could abnormally expose intramembraneous proteins such as amyloid precursor protein to proteases.


Axonal trees and cortical architecture.

Axonal trees and cortical architecture. In modern computer design considerable care is taken to arrange the components in such a way that wiring is kept to a minimum. Certain features of cortical structure--the mappings, stripes and blobs within areas, and areas themselves--are somewhat reminiscent of the layout of computer components, and suggest that the cortex may also be organized so as to economize on neuronal 'wiring'. One important difference between the brain and a computer is that the wiring in the brain takes the form of elaborate branched structures, namely axonal trees. In this article, it is argued that an assessment of the efficiency of cortical wiring must take account of the branching rules of these trees.


The pulvinar and visual salience.

The pulvinar and visual salience. One of the major problems of living in a rich visual environment is deciding which particular object or location should be chosen for complete processing or attention; that is, deciding which object is most salient at any particular time. The pulvinar has enlarged substantially during evolution, although little has previously been known about its function. Recent studies suggest that the pulvinar contains neurons that generate signals related to the salience of visual objects. This evidence includes: (1) anatomical and physiological observations of visual function; (2) augmented responses in the pulvinar for visual stimuli presented in important contexts; (3) suppression of activity for stimuli presented in irrelevant conditions; (4) thalamic modulation producing behavioral changes in cued attention paradigms; and (5) similar changes with visual distracter tasks.


The neostriatal mosaic: multiple levels of compartmental organization. The striatum, which is the major component of the basal ganglia, displays a complex mosaic organization of neurochemical systems that are related to its neuroanatomical connections. This mosaic organization reflects multiple levels of functional compartments. The first level is determined by the segregation of two major striatal output systems, one to the globus pallidus (external segment) and the other to the entopeduncular nucleus and substantia nigra. The second level segregates the cortical outputs of sublaminae of layer V between the patch and matrix compartments of the striatum, which project to the dopaminergic and GABAergic neurons in the substantia nigra, respectively. The third level is related to the topography of cortical inputs by which regions of the striatum may be functionally defined on the basis of the cortical areas with which they are connected. Neurochemical markers display complex mosaic patterns in the striatum that, when examined in the context of the multi-level compartmental organization of the striatum, reveal the highly organized manner by which the striatum processes cortical information.