Analysis of drug action at single-channel level.

Many drugs interact directly with ion channel proteins to alter gating and permeation functions. Single-channel recording affords resolution of drug-induced functional changes in channel behavior at the molecular level. Drug and toxin molecules that block ion channels are useful probes of channel mechanisms because blocking sites are often coupled to other pharmacologically relevant binding sites. Simple kinetic schemes describing fast block, slow block, and binding competition between two blocking molecules provide useful models of drug-induced blocking processes. From a careful perspective, a single channel is best approached as the analog of a purified enzyme preparation in the hands of an enzymologist. The confidence gained by knowing that one is viewing a single subtype must be weighed against the possibility that the channel could have been altered in the process of patch isolation or bilayer reconstitution. As in all kinetic studies, a curve fit to a two-state scheme is contingent on the possibility that a more complex multi-state system can masquerade as the simple cartoon one would like to put forward.


[Prevalence of hepatitis C virus antibodies in hospital personnel].

In this study, 416 hospital staffs from different departments and profession were screened for Hepatitis C virus (HCV) antibody by Enzyme Immune Assay method. Two hundred fifty blood bank donors were tested as a control group. Anti HCV positivity were found in four of hospital staffs (0.9%) and in four of donors (1.6%). According to our results, generally we didn't find any data about transmission of hepatitis C virus from hospital but it is interesting that all of the positive cases were from intensive care units.


Characterization of the 5'-flanking transcriptional regulatory region of the human Fc gamma receptor gene, Fc gamma RIIA.

The human Fc gamma receptor gene Fc gamma RIIA is expressed in platelets, neutrophils, monocytes and macrophages. Understanding the regulation of expression of Fc gamma RIIA will enhance our knowledge of regulated gene expression and immune function in these cells. We cloned a 3.65 kb region of the 5' end of the Fc gamma RIIA gene and characterized 3.4 kb of previously unreported sequence of the 5'-flanking region. Primer extension studies and RNase protection analyses of mRNA from HEL, K562 and U937 cells revealed multiple transcription start sites. One transcription start site mapped to a 5'-untranslated (5'UT) exon approximately 1 kb 5' to the ATG translation initiation codon, while a second start site mapped near the ATG codon. Reverse transcription combined with PCR (RT-PCR) employing an oligonucleotide in the putative 5'UT exon and an antisense oligonucleotide in the translated region yielded products which confirm that transcription starts in this 5'UT exon 881 bp upstream of the ATG codon. Sequence analysis of the RT-PCR products showed two related RNA splice products which use alternative 3'-consensus AG splice acceptor sites. Fc gamma RIIA mRNA thus has three distinct potential 5'UT regions, two alternatively spliced forms from the start site in the 5'UT exon and the third from the start site near the ATG codon. Comparisons of the human Fc gamma RIIA 5'-flanking region with human Fc gamma RI and mouse Fc gamma RII beta genes as well as with other genes expressed in megakaryocytes, neutrophils and monocytes reveal structural similarities and shared promoter elements.


Subtyping of human immunodeficiency virus isolates with a panel of monoclonal antibodies: identification of conserved and divergent epitopes on p17 and p25 core proteins.

We have investigated the feasibility and significance of subtyping of human immunodeficiency virus (HIV) isolates with monoclonal antibodies (mAb) raised against the core proteins of HIV. A panel of 37 mAb tested for reactivity with HIV1 oligopeptides was used to analyse the antigenic relatedness among 14 HIV isolates which included 12 isolates of HIV1 from different geographical origins and 2 isolates of HIV2. Three out of these 37 mAb reacted with conserved epitopes expressed by all 14 HIV isolates tested. These reagents which included 2 mAb reacting with the 285-310 amino acid sequence of p25 and 1 mAb reacting with an epitope of p25 not mapped by the peptides' approach, also reacted with a non-human primate lentivirus. Five mAb reacting either with the 11-25 or 121-132 amino acid sequences of p17 or the 302-320 amino acid sequence of p25 reacted with strain-specific epitopes. The other 29 mAb reacted with polymorphic epitopes and thereby define subfamily and subtype-specific markers.


Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.

Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.


DNA probes for Bordetella species and a colorimetric reverse hybridization assay for the detection of Bordetella pertussis.

Three oligonucleotide probe sequences were inferred from the 16S ribosomal ribonucleic acid (rRNA) and the 16S-23S rRNA spacer sequences of Bordetella pertussis ATCC 10380. These probes were used in hybridization tests with deoxyribonucleic acid from Bordetella species and other relevant bacterial taxa. A probe from the spacer region hybridized exclusively to the B. pertussis strains tested and not to strains from other species. Using a combination of three probes, B. pertussis, B. parapertussis/B. bronchiseptica and B. avium could be specifically identified and differentiated from other taxa. Differentiation between B. parapertussis and B. bronchiseptica was not possible with the probes used. Using the spacer probe, a colorimetric hybridization assay specific for B. pertussis was developed based on enzymatic amplification of the 16S-23S rRNA spacer and reverse hybridization in microtitre wells. As compared with results using agarose gel electrophoresis, and Southern and dot-spot hybridization with a 32P-labelled probe, this assay proved to be faster and easier to perform and was found to be at least as sensitive and specific.


Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the delta F508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis (CF) is a common genetic disorder in Caucasians, and in some populations 70% of cases are associated with a 3 base pair (bp) deletion (delta F508) in the CFTR gene. We have implemented a fluorescence-based, multiplex allele-specific polymerase chain reaction (MASPCR) assay for deletion of the delta F508 mutation. Different allele-specific fluorescently-tagged primers are used in the PCR reaction to distinguish between normal and delta F508 alleles. Fluorescent PCR products are then visualized in a single lane on an agarose gel following electrophoresis combined with real-time multicolour fluorescence detection. The approach simplifies diagnosis of the most common mutation in the CFTR gene, and holds promise for a multiplex allele-specific, fluorescence-tagged gene amplification strategy for detection of additional CF mutations which may result in more cost-effective testing without increasing the risk of missed or erroneous diagnoses.


[An epidemiological study of the health conditions of Milan traffic police with respect to pollution from vehicular traffic].

An investigation on the health effects of occupational exposure to motor vehicle exhaust and environmental pollutants was carried out on traffic wardens in Milan (Italy). Randomized samples of 292 traffic wardens (exposed group) and 60 hospital staff members (control group) underwent a physical examination and laboratory tests. No significant difference was observed between the exposed and control groups as regards general morbidity, apart from musculo-skeletal disorders in females. The mean blood lead level (PbB) among traffic wardens was 15.2 micrograms/dl compared with 11.7 in control (p less than 0.01). The carboxyhaemoglobin concentration (COHb) in traffic wardens at the beginning of the shift was 2.8% for smokers and 1.2% for non-smokers (3.0% and 0.9% respectively in controls). At the end of the shift COHb in the exposed group was 4.3% for smokers and 2.5% for non-smokers (p less than 0.01). PbB was significantly correlated (r = 0.17) with Median Nerve Motor Conduction Velocity (NCV) in the exposed but not in the control group. The same pattern was observed for the correlation of PbB and Systolic Blood Pressure (SBP) (r = -0.24). COHb was significantly correlated with HDL cholesterol (r = -0.20) in the exposed group only. It is questionable whether very low PbB levels can affect NCV and SBP directly or rather whether PbB, as well as COHb, should be regarded as tracers of exposure to those urban pollutants leading also to cardiovascular and nervous disorders.


Structure-mutagenicity relationships in a series of indolo[3,2-c]quinoline-1,4-diones that have shown cytotoxic properties on leukemia cells.

A series of seven 6-methylindolo[3,2-c]quinoline-1,4-diones substituted either in the 2 position or in 3 position by various groups were examined for their ability to induce mutation in the Ames test at several concentrations in four strains of Salmonella typhimurium (TA97, TA98, TA100, and TA102). First, relationships were established between their mutagenic activities and either the nature or the position of the substituent on the quinonic nucleus. Compounds substituted in the 2 position were less mutagenic than the 3 isomers. In the second study, the mutagenic properties were compared to the in vitro antitumor activity. Interestingly, some very cytotoxic quinones were only weak mutagens. So where the cytotoxicity is similar, the less mutagenic compounds may be suitable for clinical use as antitumor drugs, in order to avoid important side effects; the Ames test can then be used guide the selection of molecules for further in vivo antitumor screening. It can also be very helpful in selecting the best candidate molecules to be synthesized.


Mutagenic activities of azido analogues of amsacrine and other 9-anilinoacridines in Salmonella typhimurium and their enhancement by photoirradiation.

Analogues of amsacrine and other 9-anilinoacridines, bearing azide groups at various positions, were tested for their mutagenic activity in five strains of Salmonella typhimurium, both before and after photoirradiation. Azido substitution at the 2- or 3-position of the acridine or the 1'-position of the aniline led to compounds which were active frameshift mutagens, as detected in strain TA1537. Photoirradiation enhanced both the mutagenicity and the cytotoxicity of the azido compounds. Analogues bearing two azido groups at either the 2,6- or 3,6-positions were less strongly mutagenic in the dark, and light activation led to a toxic but only weakly mutagenic product. The effects of photoirradiation were decreased by aniline ring substitution, and were essentially eliminated by additional methyl substitution in the acridine ring. Comparison of events in TA1537 and TA98 suggested that photoirradiation of the 2- or 3-azido compounds gave a product which was capable of forming covalent bonds with DNA. The azide-containing analogues readily formed single strand DNA breaks on irradiation in the presence of DNA, but the efficiency of this reaction varied considerably.


1,4-Dioxane: prediction of in vivo clastogenicity.

1,4-Dioxane was analyzed with the CASE program to determine the structural basis of its potential genotoxicity and carcinogenicity. These investigations led to the prediction that while 1,4-dioxane was not genotoxic in vitro, it was an inducer of micronuclei in the bone marrow of rats and a carcinogen for both rats and mice. If it is assumed that the induction of micronuclei is the result of DNA damage, then this potential and the previous report of the in vivo induction of DNA strand breaks in rat liver raise the possibility of a genotoxic action for 1,4-dioxane. However it is also conceivable that we have identified a structural feature which contributes to the induction of micronuclei by a non-genotoxic mechanism.


A cytogenetic study of men environmentally and occupationally exposed to airborne pollutants.

The level of sister-chromatid exchanges (SCE), high-frequency cells (HFC), chromosomal aberrations (CA) as well as the proliferation rate index (PRI) were measured in peripheral blood lymphocytes from three groups of volunteers. The environmentally exposed donors were residents from the vicinity of a coke factory; the occupationally exposed persons were cokery workers, while rural region inhabitants served as a control group. Compared with the control group, statistically significant increases of SCE and HFC, as well as decreased cell kinetics (PRI) were observed for both occupationally and environmentally exposed groups. The effect was especially pronounced when only smokers were taken into account. A statistically significant increase of CA was observed in the environmentally exposed group when CA including gaps (CA + G) were evaluated. The proportion of HFC was found to be the most sensitive method to detect genetic effects on the tested human population. This study demonstrates the usefulness of all 4 biomarkers (SCE, HFC, CA and PRI) in monitoring populations exposed to ambient pollution and clearly indicates effects from residential as well as occupational exposure to industrial air pollutants.


Cytogenetics study on coke oven workers with abnormal blood counts.

Thirty steelworkers with abnormal blood counts and 22 controls were studied cytogenetically. Fifty percent of the workers had a combination of leukopenia, neutropenia and lymphocytosis, and the remaining 50% showed different combinations or even a single type of alteration. The frequency of chromosome and chromatid gaps and chromosome deletions was significantly higher among workers than among controls, and the same was true for the number of individuals with some type of chromosome alteration. We also noted that the major factor related to the production of chromosome breaks is not the total time of work in the steel mill, but specifically the time of work at the coke oven.


Cytogenetic study in peripheral blood lymphocytes from asthmatic patients receiving continued therapy with theophylline.

Possible cytogenetic effects of theophylline have been investigated in asthmatic patients undergoing continuous therapy with this drug. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and proliferating rate indices (PRI) were evaluated in cultured peripheral blood lymphocytes from patients receiving theophylline alone, theophylline plus inhaled beta 2 adrenergic drugs or theophylline in combination with beta 2 adrenergic agents and corticoids. Two samples from each individual were obtained in order to perform a prospective study: before the theophylline medication (sample A) and at a time after the beginning of treatment (sample B). After treatment (66.3 +/- 37.8 days), an increase in SCE was observed without modifications either in PRI or in CA. Patients receiving beta 2 adrenergics or beta 2 adrenergics plus glucocorticoids before and during theophylline treatment, did not respond differently than those on theophylline alone.


Effects of amino acids on sister-chromatid exchanges.

The effects of 10 amino acids on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) and six amino acids on the SCE frequency in root tip cells of Hordeum vulgare were studied. Alanine (Ala), glycine (Gly), phenylalanine (Phe), valine (Val), histidine (His) and serine (Ser) induced a significant increase in SCE in PBL but threonine (Thr), isoleucine (Ile), lysine (Lys) and arginine (Arg) did not. Ala, Gly, Thr, Ile and Val induced a significant increase in SCE in root tip cells of Hordeum vulgare but Lys did not. The effect of Lys and bromodeoxyuridine (BrdU) on SCE levels in PBL and the interaction between them were also studied. The results show that Lys can inhibit the SCE induced by BrdU.


Cytogenetic studies on gas station attendants.

Forty-nine gas station attendants employed to pump fuel and 24 controls were studied cytogenetically. This type of worker is occupationally exposed to fuel fumes and to automotive vehicle emissions. Chromosome analysis showed a significantly higher frequency of chromosome deletions among the gas station attendants than a control group. Taking into account the relationship between clastogenicity and increased cancer risk, we may consider these workers to form a risk group.


Genotoxicity of four herbicides in the Drosophila wing spot test.

The herbicides alachlor, atrazine, maleic hydrazide and paraquat were evaluated for genotoxicity in the Drosophila melanogaster wing spot test. Third-instar larvae trans-heterozygous for two recessive mutations of wing trichomes, multiple wing hairs (mwh) and flare (flr3), were treated by chronic feeding with different concentrations of the four herbicides. Feeding ended with pupation of the surviving larvae. The genotoxic effects were determined from the appearance of clones of cells with mwh, flr3 or mwh-flr3 phenotypes. Exposure to maleic hydrazide resulted in a significant increase in the frequency of the three categories of spots recorded (small single, large single and twin spots) in a dose-related fashion. Exposure to alachlor induced significant increases in both small and total spots at the four concentrations assayed and in the frequency of twin spots at the highest concentration tested (10 mM). Atrazine and paraquat also induced significant increases in both small and total spots at three of the four concentrations tested, without indication of a direct dose-effect relationship.


Cystic fibrosis in the mouse by targeted insertional mutagenesis.

Cystic fibrosis is a fatal genetic disorder which afflicts 50,000 people worldwide. A viable animal model would be invaluable for investigating and combating this disease. The mouse cystic fibrosis transmembrane conductance regulator gene was disrupted in embryonal stem cells using an insertional gene targeting vector. Germ-line chimaeras were derived and the offspring of heterozygous crosses studied. These homozygous mutant mice survive beyond weaning. In vivo electrophysiology demonstrates the predicted defect in chloride ion transport in these mice and can distinguish between each genotype. Histological analysis detects important hallmarks of human disease pathology, including abnormalities of the colon, lung and vas deferens. This insertional mouse mutation provides a valid model system for the development and testing of therapies for cystic fibrosis patients.


Synthesis of nucleic acids in blood cells in mice after irradiation with gamma rays.

In the blood of mice irradiated with a single total-body dose of 8 Gy gamma rays we examined the concentration of nucleic acids and incorporation of 3H-thymidine and 14C-orotic acid. The concentration of nucleic acids, above all that of DNA in the so-called blood leukocyte mass dropped significantly in the course of the first three days after irradiation. Recovery began in the course of the second week following irradiation. On day 21 after irradiation, DNA concentration was found to be recovered temporarily and RNA concentration to exceed the control values. The increase of incorporation of 3H-thymidine confirmed an increased incidence of DNA synthesizing cells in the blood within days 12-15 after irradiation. Since the increase of incorporation of 3H-thymidine into DNA was not preceded by an increase of incorporation of 14C-orotic acid into RNA, we presume that the DNA synthesizing cells in the blood of irradiated animals do not represent the stimulated lymphocytes but circulating hemopoietic cells. Therefore, the examination of DNA synthesis in blood cells enables to record the course of bone marrow regeneration after irradiation or after application of hemopoiesis depressing substances.


Intensive combination chemotherapy (TTL-I protocol) of large cell and immunoblastic lymphomas--long-term observation.

Fifty patients with advanced (Stage III and IV) large cell and immunoblastic lymphoma were treated with eight 4-week courses of chemotherapy. The first two identical A courses were composed of high dose cyclophosphamide, vincristine, 5-day administration of bleomycin, 2-week prednisone, and methotrexate with calcium leucovorin. The next two "B" courses were composed of vincristine, 3-day administration of doxorubicin together with bleomycin, and prednisone. The next two "C" courses were composed of cyclophosphamide, vincristine, bleomycin, prednisone, methotrexate, and calcium leucovorin. The last two "D" courses were the same as "B" courses. CNS prophylaxis was done with intrathecal methotrexate. Fourty-two patients (84%) achieved complete remission, 7 patients entered partial remission, and 1 patient failed to respond. The median survival of all groups was 80 + months (range 2-181 + months). Nine patients relapsed (21%), and seven patients died in complete remission, three of them died of toxicity. The most frequent toxicity was myelosuppression, mostly leukopenia, frequently followed by infection, sometimes severe. Neurotoxicity and stomatitis were frequent, but usually not severe. Two patients developed secondary malignancies. Most of the patients (54%) are alive without evidence of disease at present.


Quality control program of steroid receptor assays: an international study.

Lyophilized calf uterine cytosol standards were prepared for quality control of estrogen receptor (ER) determination, and lyophilized cytosols and tissue powders were used for quality control of progesterone receptor (PR) analysis. Two series of four samples were analyzed either for ER or PR contents, twice within one month, by 7 laboratories in 5 countries. Coefficient of variation (CV) of the between-laboratory averages assayed in a single run of ER-positive (ER+) and PR-positive (PR+) standards varied from 29.6 to 61.8% and from 32.4 to 76.2%, respectively. All laboratories, with the exception of a single value, could recognize samples of low, medium, an high ER level, as well as a negative sample. Most laboratories evaluated properly also the level of PR samples. The average between-laboratory CV values of protein determination in the relevant standards were 23%.


Renal involvement in sickle cell-beta thalassemia.

Renal function studies were performed in 41 patients with sickle cell-beta thalassaemia (S/b thal) and compared to 14 normal controls and 8 sickle cell (SS) patients. Polyuria, hyposthenuria and mild proteinuria were common in both S/b thal and SS patients. A renal concentrating defect was manifest in all patients studied, and in 4 of the 7 S/b that patients tested, an abnormal acidification test was found. A statistically significant negative correlation (n = 19, r = -0.48, p less than 0.05) was noted between creatinine clearance (CCr) and age for the patients over 30 years. There was no correlation between hemoglobin and CCr; on the contrary, a statistically significant negative correlation was found between CCr and hemoglobin F (n = 29, r = -0.428, p less than 0.05) Our S/b thal and SS patients showed a decreased daily excretion of sodium, calcium, phosphate and magnesium and lower serum magnesium levels than the controls. One third of the S/b thal patients showed hyperuricosuria, and a statistically significant negative correlation was noted between serum uric acid and its fractional excretion in all S/b thal patients (n = 41, r = -0.450, p less than 0.01). Serum phosphate levels were independent of age. A statistically significant positive correlation was found between the tubular reabsorptive capacity for phosphate and the number of painful crises per year (n = 33, r = 0.836, p less than 0.001). We conclude that renal involvement in the double heterozygous state is as severe as in homozygous sickle cell disease.


Some regional anatomical relationships of TRH to 5-HT in rat limbic forebrain.

It is now a recognized principle that various neuropeptides are neuronally co-localized with biogenic amine or aminoacid neurotransmitters. In the rat CNS it has previously been shown that TRH is co-localized with 5-HT (and also with substance P) in cell bodies of the posterior raphe that project to the spinal cord. Although TRH cell bodies are known to be widely distributed throughout the forebrain there is no other known co-localization with 5-HT. In this study we further specify the forebrain there is no other known co-localization with 5-HT. In this study we further specify the anatomical relationship of TRH with 5-HT by use of surgical and neurotoxic lesioning with reference to limbic forebrain regions wherein TRH is greatly increased following seizures. In groups of rats, the fimbria-fornix was lesioned alone, or combined with a lesion of the dorsal perforant path or the ventral perforant path. There was a sham lesioned control group. Additional groups were lesioned with 5,7 dihydroxytryptamine, 100 micrograms i.v.t., 45 min. after i.p. desipramine, 25 mg/kg. All rats were sacrificed three weeks after lesions. Indoleamines were determined by HPLC in left anterior cortex, left pyriform/olfactory cortex, left dorsal hippocampus and left ventral hippocampus. TRH was determined by specific RIA in the corresponding right brain regions. The modal n was 7 rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative immunohistochemical study on the expression of alpha B crystallin, ubiquitin and stress-response protein 27 in ballooned neurons in various disorders.

This report deals with a comparative study on the expression of alpha B crystallin, ubiquitin, stress-response protein 27 (srp 27), srp 72 and phosphorylated neurofilament protein (pNFP) by ballooned neurons in Pick's disease, Creutzfeldt-Jakob disease (CJD), amyotrophic lateral sclerosis (ALS), leptomeningeal carcinomatosis, anterior spinal artery syndrome and pellagra. Immunohistochemical techniques were used. alpha B Crystallin was expressed by the majority of ballooned neurons of Pick's disease and CJD, but not by those of the other disorders. Ubiquitin and srp 27 expression was also restricted to abnormal neurons of Pick's disease and CJD, but the proportion of stained cells was less than that expressing alpha B-crystallin. There was no evidence of ballooned neurons expressing srp 72. Except for those of pellagra patients, phosphorylated neurofilament protein (pNFP) was detected in most abnormal neurons. Our results suggest that the mechanisms involved in formation and maintenance of swollen neurons in Pick's disease and CJD may be different than those of ballooned neurons in the other entities studied.


Ubiquitin, PGP 9.5 and dense body formation in trimethyltin intoxication: differential neuronal responses to chemically induced cell damage.

Ubiquitin in normal cells may be important in degrading or transferring short-lived or aberrant proteins to lysosomal dense bodies. To examine its role in degrading proteins produced by a chemical insult, changes in the distribution of ubiquitin and the carboxy-terminal hydrolase, PGP 9.5, have been studied in rat hippocampal neurons and cerebellar Purkinje cells in trimethyltin intoxication. Here tubulovesicular dense bodies (TVBs) form from 12h onwards associated with vacuolation of the Golgi apparatus. Striking accumulations of lysosomal dense bodies follow in hippocampal pyramidal cells but not in cerebellar Purkinje cells; many of the hippocampal neurons later die, while the Purkinje cells generally survive. Ubiquitin immunoreactivity was diffusely increased in hippocampal pyramidal and Purkinje cells 6 h after dosing. By 12 h both diffuse and granular ubiquitin immunoreactivity was present that intensified over 24 and 48 h. Both by light and electron microscopy TVBs showed ubiquitin immunoreactivity, but dense bodies in hippocampal perikarya did not stain with an anti-ubiquitin antibody. PGP 9.5 immunoreactivity was not altered in hippocampal cells at any time, while Purkinje and Golgi cell dendrites and perikarya showed intensified labelling at 3 h that reached a peak of 12 h. At 48 h Western blot analysis of hippocampal homogenates showed significant increases in high molecular weight (HMW) ubiquitin conjugates, while cerebellar homogenates showed an increase in ubiquitin-histone conjugates. Northern blot analyses showed no change in ubiquitin or PGP9.5 gene expression in hippocampus or cerebellum. These findings suggest that the material in the TVBs in hippocampal cells is not being degraded by the ubiquitin system but passes ubiquitinated into the lysosomal system, while material in Purkinje cell TVBs is degraded by the ubiquitin system, suggesting it may have a different composition in each type of neuron.


Experimental traction injuries of cervical spinal nerve roots: a scanning EM study of rupture patterns in fresh tissue.

This study examines the levels at which fresh rat ventral and dorsal C4 to T1 spinal roots rupture under traction stress. Rupture rarely occurs at the CNS-PNS transitional zone. Despite its delicate appearance this is strengthened in a number of ways, so that it is less vulnerable than the roots and their constituent rootlets and aggregated rootlet bundles (ARBs). Ventral roots commonly rupture either where the rootlets join to form the ARBs, along the course of the latter, or where these in turn join to form the ventral root. Dorsal root rupture shows a similar pattern but has a higher frequency of rupture within the root proper than is the case with the ventral roots. The level of rupture also varies systematically over the series of roots examined: rupture points tend to be located at more distal levels in caudal roots. This may occur because of mechanical differences in the distribution of traction stress along roots, related to their courses through the vertebral canal. Upper roots run transversely and the traction force is transmitted directly to rootlet levels, while more caudal roots run obliquely and tend to rupture where they are drawn taut against the pedicle. Many of the morphological features of ruptured roots would tend to inhibit regeneration of fibres distally. Differences in the pattern of rupture between fresh and fixed roots are examined. The latter tend to rupture at levels closer to the CNS than the former.


The effects of rubidium, caesium and quinine on 5-HT-mediated behaviour in rat and mouse--1. Rubidium.

The administration of TCP (15 mg/kg, i.p.) to rats pretreated with either intraperitoneal RbCl (3 mmol/kg, twice daily for 5 days) or dietary RbCl (30 mmol/kg diet, for 14 days), resulted in the complete 5-HT behavioural syndrome. Pretreatment with p-chlorophenylalanine (i.p. 300 mg/kg x2) or (-)-propranolol (20 mg/kg, i.p.), pindolol (4 mg/kg, i.p.) and ritanserin (0.4 mg/kg, s.c.) prevented the occurrence of the 5-HT syndrome, produced by dietary RbCl plus TCP. Intraperitoneal administration of RbCl had no effect upon the 5-HT behavioural syndrome, produced by 8-OH-DPAT (0.5 mg/kg, s.c.) or 5-MeODMT (2 mg/kg, i.p.) but enhanced the 5-HT syndrome produced by quipazine (20 mg/kg, i.p.), DOI (8 mg/kg, s.c.), p-chloramphetamine (4 mg/kg, i.p.) or by TCP plus L-tryptophan (50 mg/kg, i.p.) in rats. Dietary administration of RbCl resulted in the enhancement of the 5-HT2-mediated head-twitches in the mouse and the attenuation of hypothermia in the mouse, induced by 8-OH-DPAT (0.5 mg/kg, s.c.). The accumulation of 5-HT (after inhibition of monoamine oxidase) and the rate of synthesis of 5-HT in the whole brain (minus cerebellum) were enhanced by dietary and intraperitoneal administration of RbCl, respectively. The effects of lithium and rubidium, respectively, on 5HT function in brain are compared.


5-HT3 receptors are not involved in the modulation of the K(+)-evoked release of [3H]5-HT from spinal cord synaptosomes of rat.

The ability of 5-HT3 receptor agonists to modulate the resting efflux or K(+)-evoked release of [3H]5-HT from superfused synaptosomes from the spinal cord of the rat was investigated. Phenylbiguanide did not alter the resting efflux of [3H]5-HIAA or [3H]5-HT or modify the K(+)-evoked release of [3H]5-HT. 2-Methyl-5-HT (10 microM) caused an increase in resting efflux of [3H]5-HIAA, an effect that was blocked by the inhibitor of the uptake of 5-HT fluoxetine. No effect on K(+)-evoked release of tritium was observed. Bufotenine (100-1000 nM) increased the resting efflux of [3H]5-HT and [3H]5-HIAA. These effects were not antagonized by the 5-HT3 antagonist ICS 205-930 but were antagonized by fluoxetine. The drug ICS 205-930 (1 microM) did not alter resting efflux or block the ability of serotonin (30 and 100 nM) to decrease K(+)-evoked release of tritium. Quipazine, a potent antagonist of peripheral 5-HT3 receptors (subnanomolar concentrations), was also unable to alter resting or K(+)-evoked release of [3H]5-HT. It did, however, attenuate the inhibitory effect 5-HT on K(+)-evoked release. The concentrations required were in the micromolar range, consistent with the ability of the drug to antagonize the 5-HT1B autoreceptor. These results support the idea that 5-HT3 receptors do not act as nerve terminal autoreceptors in the spinal cord of the rat.


Behavioural consequences following infusion of selective neurokinin agonists into the median raphe nucleus of the rat.

The locomotor activity induced after infusion of selective neurokinin (NK) agonists into the median raphé nucleus of rats was investigated. In photocell cages, the NK-2-agonist, GR64349, and the NK-3 agonist, senktide, both increased motor activity in a dose-dependent manner. However, the NK-1 agonist, GR73632, had little effect over a range of doses. In the open field, the motor effect of all three NK agonists was identical to that observed in the photocell cages. In addition, senktide induced straub-tail, hind-limb splaying and various oral movements. Such effects were not noted with the other two agonists. These results suggest that activation of NK-2 or NK-3 receptors by the neurokinins, in the median raphe nucleus of the rat, leads to an increase in locomotor activity.


The management of symptomatic benign prostatic hyperplasia with the once-daily alpha 1 blocker, terazosin.

The once-daily alpha 1 blocker terazosin was administered to 36 men with symptomatic benign prostatic hyperplasia (BPH) who had previously been scheduled for transurethral resection of the prostate (TURP). At 3 months, terazosin at 5 mg q.d. produced improvements in symptom scores, peak urinary flow rates, mean urinary flow rates, and residual urine. These improvements were maintained at 6 and 9 months. Terazosin also proved to be well tolerated, with no cases of hypotension or erectile dysfunction. We conclude that terazosin is effective in relieving obstructive urinary symptoms of BPH and that it allows many patients to be placed in a "holding pattern," allowing surgery to be delayed until the disease progresses. The use of terazosin also increased the capabilities of our urologic clinic. By reducing the urgency of surgery, it allowed us to schedule TURPs more conveniently and thereby accommodate more prostate surgery within the limited resources of our clinic. As a result, the use of terazosin saved considerable CHAMPUS dollars that would otherwise have been lost to the system.


Unnecessary mastectomy for gynecomastia in testicular cancer patient.

A young man presented with a 4-month history of progressive, bilateral tender gynecomastia and underwent a bilateral subcutaneous mastectomy without regard for a possible occult testicular tumor. Following the mastectomy, metastatic testicular cancer was discovered; the patient had an overlooked scrotal mass and a very elevated serum human chorionic gonadotropin tumor marker. The possibility of testicular tumor must be considered in any male, especially those in the younger age group, who present with gynecomastia.


[Reducing bleeding during extracorporeal circulation interventions by high doses of aprotinin].

The great risk of severe infectious diseases transmission through blood transfusion, has increased during the last years the effort to reduce the bank blood and its derivates use. Many techniques have proposed to achieve this purpose during and post cardiopulmonary bypass: normovolemic hemodilution, perioperative blood autotransfusion, postoperative return of extra corporeal circuit and chest drains blood and the particular use of some drugs. In the last few years several reports have been presented in the literature concerning the reduction of intra and postoperative bleeding in cardiac surgery by high dose Aprotinin administration. A randomized study with the use of this pharmacologic agent is presented: a group of patients was treated with Aprotinin (shared in two subgroups receiving respectively a different dose of the drug) and a control group. The results were highly encouraging both because of the reduction of peri and postoperative bleeding and because of the bank blood use important reduction.


Delta sleep-inducing peptide as a factor increasing the content of substance P in the hypothalamus and the resistance of rats to emotional stress.

The influence of delta sleep-inducing peptide (DSIP, 60 and 120 nmole/kg, intraperitoneally) on the content of substance P (SP) in the hypothalamus of rats was studied in male rats of the August line. It was demonstrated that the administration of DSIP significantly increases the average content of SP in the hypothalamus, as well as its content in animals resistant to and predisposed to emotional stress. A daily one-time administration of DSIP before placing the rats in conditions of stress increases the content of SP in the hypothalamus which was decreased during emotional stress. The preliminary one-time administration of DSIP to animals subjected to a stressor influence also increases the SP content in the hypothalamus. It was established that a one-time administration of DSIP in a dose of 60 nmole/kg sharply decreases the classical manifestations of stress such as the hypertrophy of the adrenals and involution of the thymus.


Fast and slow depolarizations produced by substance P and other tachykinins in sympathetic neurons of rat prevertebral ganglia.

Using intracellular recording, we examined the effects of three mammalian tachykinins, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), on sympathetic neurons of isolated rat coeliac-superior mesenteric ganglia (C-SMG). The 3 tachykinins elicited two distinct depolarizing responses in ganglion cells: fast depolarization with time-to-peak of 1-2 sec and duration of 5-10 sec, and slow depolarization with time-to-peak of about 20 sec and duration of 120-140 sec. Both fast and slow responses persisted in a solution containing low Ca2+ and high Mg2+ or tetrodotoxin, which indicates that the tachykinins directly act on ganglion cells to produce fast and slow depolarizations. The two types of tachykinin-induced responses exhibited clearly distinguishable properties. The membrane conductance was increased during the fast response, but not significantly changed, slightly decreased or sometimes increased during the slow response. Within certain range of membrane potential, the amplitude of fast response increased upon membrane hyperpolarization and decreased upon depolarization of ganglion cells. In contrast, the amplitude of slow response associated with membrane conductance decrease was increased with membrane depolarization and decreased with hyperpolarization. The fast response was markedly suppressed in a Na(+)-deficient solution, a solution containing nominally zero Ca2+ (plus 0.1 mM EGTA in some cases), and in a solution containing Cd2+ or Mn2+, whereas the slow response was not affected in these solutions and was augmented in some cells in K(+)-free solution. Thus it seems that the increase in Ca(2+)-dependent cationic conductance underlies the fast response and that the slow response is produced at least in part by suppression of certain K+ channels. The fast response progressively decreased in amplitude upon repeated application of the peptides with short intervals, whereas the slow response was rather augmented by repeated application. Lowering the temperature markedly depressed the slow response, while the fast response remained almost unaffected. It is therefore likely that the fast and slow depolarizations are mediated by two different subtypes of tachykinin receptors or a single class of receptors linked with two different intracellular mechanisms. Measurement of tachykinins in several sympathetic ganglia by combined use of HPLC and radioimmunoassay revealed that the highest amount of SP occurs in the C-SMG where the content of SP (136.0 pmol/g protein) was higher than those of NKA (44.3) and NKB (18.7). SP thus appears to function as a major tachykinin in rat C-SMG.


Ascending projections from the gigantocellular reticular and dorsal paragigantocellular nuclei of the medulla oblongata in the rat: an anterograde PHA-L tracing study.

Ascending projections from the gigantocellular reticular (NGc) and dorsal paragigantocellular (DPG) nuclei of the medulla oblongata were examined in the rat by utilizing anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L). New observations of fiber connections could be made by the merits of the PHA-L. PHA-L-labeled axon terminals were observed in the dorsal part of the nucleus of the solitary tract, the rostrolateral subnucleus of the interpeduncular nucleus, bed nucleus of the stria terminalis, lateral septal nucleus and subfornical organ, in which no previous authors had found projections from the NGc or DPG. The strongest projections were observed in the motor nucleus of the trigeminal nerve, lateral and dorsolateral subnuclei of the facial nucleus, and prepositus hypoglossal nucleus, which suggested that both NGc and DPG had some relation to the movements around the mouth and to the eyeball movements. The projections to the dorsal part of the nucleus of the solitary tract and to the subfornical organ from the NGc and DPG are considered to reveal certain roles in cardiovascular regulation. Mainly, the facial nucleus received projections from the NGc, the subfornical organ from caudal NGc, the red nucleus from the DPG, and the rostrolateral subnucleus of the interpeduncular nucleus from the rostral DPG, respectively. The NGc and DPG would have partially different roles with regard to the ascending innervation. The present PHA-L study was partially confirmed using wheat germ agglutinin conjugated to horseradish peroxidase which was injected into the bed nucleus of the stria terminalis and subfornical organ.


Influence of taurine on the electrically regulated ionic channels of the somatic membrane of neurons of the pond snail.

The influence of taurine (in concentrations of 10(-8) to 10(-2) mole/liter) on the fast sodium, calcium, and potassium, the slow potassium, and the leakage currents of the somatic membrane of the neurons of the pond snail was investigated. A monotonic decrease was demonstrated in the calcium and the slow potassium currents with an increase in the concentration of taurine. The sodium currents increased at taurine concentrations of 10(-8)-10(-4) mole/liter, while at concentrations of 10(-3)-10(-2) mole/liter they decreased. The fast potassium currents remained essentially unchanged at all concentrations of taurine. The leakage currents of the membrane at small concentrations of taurine decreased somewhat, and increased insignificantly at higher concentrations of it. All of the effects of taurine were reversible. It is hypothesized that the mechanism of the neuromodulatory, anticonvulsant, antihypoxic effects of taurine is at its base associated with changes in the electrically regulated ionic channels of the cell membrane.


Substance P and neuropsychiatric disorders: an overview.

Substance P (SP) is a naturally-occurring tachykinin peptide isolated from brain tissues and gastrointestinal tract. In the brain, substantia nigra and basal ganglia contain relatively high amounts of substance P. There is evidence suggesting that substance P functions as a neurotransmitter. It has been implicated in the pathophysiology of several neuropsychiatric disorders. Substance P may also serve as a useful tool in studying the effects of antidepressant drugs and electroconvulsive therapy. However, the contribution of substance P to the understanding of neuropsychiatric disorders is far from clear. Future studies should focus on the interactions and coexistence of substance P with other neurotransmitters and neuropeptides.


Combined chemotherapy and radiotherapy versus best supportive care in the treatment of inoperable non-small-cell lung cancer.

Between October 1984 and July 1988, 119 patients with limited-stage inoperable non-small-cell lung cancer (NSCLC) were randomized to receive either active treatment (arm 1) or best supportive treatment (arm 2). Arm 1 patients received 3 courses of chemotherapy with cisplatin (100 mg/m2, day 1) and etoposide (125 mg/m2 i.v., day 1; 250 mg/m2 p.o., day 2-3), followed by radiotherapy (4,000 cGy/20 fractions/4 weeks). Arm 2 patients only received best supportive care. Fifty-three and 66 patients were randomized to arms 1 and 2, respectively. Thirty-eight patients in arm 1 and 57 in arm 2 were evaluable for survival. Median survivals of arms 1 and 2 were 12.4 and 8.7 months, respectively (p = 0.047). In the multivariate analysis, only age and histology were independent prognostic variables in predicting survival. The overall response rate after chemotherapy was 20.6% (complete remission 5.9%, partial remission 14.7%). Toxicities were mainly anemia, leukopenia, vomiting and alopecia. This study suggests that active treatment has marginal survival benefit in NSCLC though with considerable toxicities.


Randomized study with the pineal hormone melatonin versus supportive care alone in advanced nonsmall cell lung cancer resistant to a first-line chemotherapy containing cisplatin.

At present, there is no effective medical therapy in metastatic nonsmall cell (NSC) lung cancer patients who progressed under a first-line chemotherapy containing cisplatin. Since recent data have demonstrated the antineoplastic properties and the lack of toxicity of the pineal hormone melatonin (MLT), a randomized study was designed to evaluate the influence of an MLT treatment (10 mg/day orally at 7.00 p.m.) on the survival time at 1 year from the progression under chemotherapy in respect to supportive care alone in a group of metastatic NSC lung cancer patients, who did not respond to a first-line chemotherapy containing cisplatin. The study includes 63 consecutive metastatic NSC lung cancer patients, who were randomized to receive MLT (n = 31) or supportive care alone (n = 32). The percentage of both stabilizations of disease and survival at 1 year was significantly higher in patients treated with MLT than in those treated only with supportive care. No drug-related toxicity was seen in patients treated with MLT, who, on the contrary, showed a significant improvement in performance status. This randomized study shows that the pineal hormone MLT may be successfully administered to prolong the survival time in metastatic NSC lung cancer patients who progressed under a first-line chemotherapy with cisplatin, for whom no other effective therapy is available up to now.


Subclinical hepatocellular carcinoma in Hong Kong Chinese.

Of the 208 Chinese patients with histologically proven hepatocellular carcinoma (HCC) seen during a 5-year period, 191 patients presented with symptomatic HCC and 17 patients with asymptomatic HCC (subclinical HCC, SCHCC) being picked up by alpha-fetoprotein (AFP) screening. Compared with the patients with symptomatic HCC, patients with SCHCC had a better performance status (p less than 0.01), higher serum albumin levels (p less than 0.05) and lower alkaline phosphatase levels (p less than 0.01). In those patients with symptomatic HCC, 4.7% were operable and only 2 patients had a tumour diameter of less than 5 cm. In contrast, patients with SCHCC had a higher operability rate (76.5%, p less than 0.0001) and all had a tumour of less than 5 cm in diameter (p less than 0.0001). Patients with SCHCC, most of whom had their tumour resected, had a better long-term survival (p less than 0.0001). We conclude that patients with SCHCC picked up by AFP serosurveillance have a better performance status, higher operability and better prognosis.


Nasal patency, aerobic capacity, and athletic performance.

The patency of the nasal airway may directly affect pulmonary ventilation, with obstruction and increased nasal resistance resulting in increased pulmonary resistance, hypoxia, and hypercapnea. Nine aerobic athletes were evaluated to assess the role of the nasal airway on aerobic capacity and athletic performance. A step-ladder graded maximal aerobic capacity test was performed under three test conditions: obstructed, decongested with oxymetazoline hydrochloride, and saline control. No differences in maximum VO2, work load, oxygen saturation, maximal blood pressure, heart rate, or respiratory rate were noted between test conditions. Pre-exercise nasal resistance was lower in the decongested compared to control conditions, but no differences were found after exercise. Athletic performance was not influenced by nasal patency in this model.


Use of a replica-plate assay for the rapid assessment of salivary protein-bacteria interactions.

A replica-plate assay was used to screen for the interaction of salivary molecules with dental plaque bacteria. Bacterial colonies cultured from supragingival plaque on sheep-blood (SB) agar were replica-plated onto nitrocellulose membranes overlaying SB or mitis-salivarius agar. Membranes with attached colonies were removed and incubated with 125I-amylase or 125I-proline-rich glycoprotein (PRG). Positive interactions were detected by autoradiography. Only strains of Streptococcus gordonii and Actinomyces viscosus bound amylase, and strains of A. viscosus bound PRG. The results suggest that amylase and PRG bind to selected species of aerobic dental plaque bacteria.


Whole-cell recordings of chloride currents in cultured human skeletal muscle.

Chloride currents in human myoballs were investigated with the tight-seal whole-cell recording method in a wide range of membrane voltages (-125 to +145 mV). Two current components having different kinetics could be distinguished. In more than 90% of the myoballs the following results were obtained. At negative potentials, the amplitude of the Cl- current was small and independent of time. The amplitude of the current increased as the membrane potential was made more positive. At potentials positive to +75 mV, the current increased monoexponentially with time. Inactivation occurred only during very long (greater than 3 s) pulses. When such a test pulse was preceded by a conditioning pulse to +60 mV, the current at potentials more than +90 mV was markedly smaller than in the absence of a prepulse, and no activation was provoked by strongly pulses. Recovery from inactivation could only be measured at potentials negative to -40 mV. The Cl- conductance at -85 mV was 5.9 +/- 3.64 microS/cm2 (SD; n = 10). In about 5% of the myoballs a kinetically different current was visible, characterized by fast inactivation at highly positive potentials. The current amplitudes were substantially larger in such cases, the Cl- conductance at -85 mV being 12.2 +/- 9.02 microS/cm2 (n = 4).


Single-channel recordings of chloride currents in cultured human skeletal muscle.

The Cl- channels in human myoballs were investigated with several recording techniques. Three types of channels were found and dubbed "small", "intermediate", and "large", according to their different conductance. The intermediate Cl- channel was observed most frequently. It was active at the resting potential immediately after seal formation in cell-attached as well as in excised patches. Its Cl- selectivity was rather high (PCl/PNa = 9.46; PCl/PMeSO4 = 7.85 where P denotes permeability) and the slope conductance at the reversal potential with [Cl-]o/[Cl-]i equal to 160 mM/42 mM was 31 pS. The channel showed an open-channel substructure with two subconductance levels having equal amplitudes. It can conduct two kinetically different currents that correspond to the activating and the inactivating Cl- current components described by Zachar et al. (1992). The small Cl- channel had a conductance of 10 pS at the reversal potential, a PCl/PNa of 2.7, and a PCl/PMeSO4 of 22.6. Its open probability was biggest negative to -85 mV, resulting in an inactivating whole-cell Cl- current component. Because of the small channel density and conductance the contribution of this channel type to the whole-cell current seems to be small. Patches with only one small channel were never observed which suggests that this channel type occurs in clusters. A third type of channel with very large conductance (250 pS) was seen only four times.


Properties of the inactivating outward current in single smooth muscle cells isolated from the rat anococcygeus.

The properties of the voltage- and time-dependent outward current in single smooth muscle cells isolated from the rat anococcygeus were studied. The outward current was activated by depolarizations to membrane potentials positive to -40 mV. Activation followed third order kinetics; at +20 mV, the time for the current to reach half its maximal amplitude was around 55 ms. The current inactivated with a time course that could best be described by a single exponential with a time constant around 1500 ms. The steady-state inactivation curve was voltage dependent over the range -110 to -30 mV, with a half-inactivation point of -67 mV. Recovery from inactivation followed an exponential time course with a time constant of around 770 ms at -90 mV. Deactivating tail current analysis revealed that a 10-fold change in the extracellular potassium ion concentration resulted in a 42 mV change in the reversal potential of the current. The current was blocked by 4-aminopyridine, tetraethylammonium, quinine and verapamil with IC50's--the concentrations producing 50% inhibition of the peak current--of 2 mM, 4 mM, 12 microM and 20 microM respectively. The current was not blocked by Toxin I (100 nM) or glibenclamide (10 microM). The current was still present in cells containing 5 mM EGTA; in these cells, replacing extracellular calcium with cadmium depressed the peak current by around 12%. This could be explained, at least in part, by a negative shift in the voltage dependence of inactivation.


Calcium-dependence of the calcium-activated chloride current in smooth muscle cells of rat portal vein.

Ca(2+)-activated Cl- current in freshly isolated smooth muscle cells from rat portal vein was studied using the whole-cell patch-clamp technique. Simultaneously, the free-cytosolic Ca2+ concentration (Cai) was estimated using emission from the dye Indo-1. Pretreatment of the cells with amytal and carbonyl-cyanide-m-chlorophenylhydrazone, which reduced the intracellular adenosine triphosphate concentration, was used to weaken the cellular Ca2+ homeostatic system. Cai of treated cells slowly increased during perfusion with an external Ca(2+)-containing solution. This rise in Cai gradually activated a Ca(2+)-dependent Cl- current which allowed the study of the relationship between activation of this current and Cai levels. The threshold Cai for activation of Cl- channels was around 180 nM and full activation occurred at 600 nM. The Cai dependence of the Cl- channels was not changed during application of noradrenaline and did not depend on the membrane potential. The gating of Ca(2+)-dependent Cl- channels of rat portal vein myocytes seems to be mainly controlled by intracellular Ca2+.


Disparate effects of calcium channel blockers on pressure dependence of renin secretion and flow in the isolated perfused rat kidney.

Using the model of isolated perfused rat kidneys this study was performed to investigate whether or not voltage-operated calcium channels are essentially involved in the pressure control of renin secretion from the kidneys. At a perfusion pressure of 100 mm Hg (13.3 kPa) renin secretory rates were 4.2 +/- 0.7 (ng angiotensin I h-1) min-1 g-1. Stepwise reduction of renal perfusion pressure to 80, 60, and 40 mm Hg (10.6, 8.0, 5.3 kPa) resulted in an increase of renin release yielding a 30-fold stimulation at 40 mm Hg vs 100 mm Hg. Increasing the perfusion pressure above 100 mm Hg did not further significantly decrease renin secretion. The perfusate flow rate was also pressure-dependent. Flow rates increased linearly with pressure and reached a plateau at 100 mm Hg, which was maintained up to 160 mm Hg (21.3 kPa). The averaged flow rate at the plateau was 15.5 ml min-1 g-1. In the presence of the three different calcium antagonists nifedipine (5 microM), nitrendipine (3 microM) and verapamil (5 microM), myogenic autoregulation of flow was abolished as indicated by the rise of the pressure/flow curve between 40 and 160 mm Hg. At the same time, however, these calcium channel blockers did not alter the relationship between perfusion pressure and renin secretion. Moreover, the calcium channel agonist Bay K 8644 (5 microM) caused a strong and long-lasting vasoconstriction, without changing renin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)


Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells.

Single, large-conductance chloride-selective channels were studied in the membrane of pig aortic endothelial cells. These channels were usually inactive in cell-attached recordings and activated spontaneously upon formation of inside-out patches or amphotericin B-perforated vesicles. Channel activity was voltage dependent, with a maximum open probability within the range of -20 mV to + 20 mV. Addition of 1 mM Zn2+ to either the cytoplasmic or extracellular side blocked channel activity reversibly. Extracellular 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) blocked the channels; the concentration necessary for half-maximum blockade was 100 mumol/l. The frequency of observing channels in cell-attached patches increased from less than 5% to 27% when cells were treated for several minutes with 1 mumol/l bradykinin and to 80% in the presence of the calcium ionophore A23187 (1 mumol/l). Both agents increase the cytoplasmic Ca2+ concentration, thereby stimulating nitric oxide (NO) synthesis and cGMP formation in endothelial cells. Sodium nitroprusside (100 mumol/l), which spontaneously releases NO, did not increase Cl- channel activity in intact cells. Polymyxin B (100 mumol/l), an inhibitor of protein kinase C, clearly enhanced Cl- channel activity in intact cells, resulting in the observation of Cl- channels in 70% of cell-attached patches. Our results demonstrate the existence of a large-conductance (LC-type) Cl- channel in vascular endothelium which is subject to a complex cellular regulation, possibly involving inhibition via phosphorylation by protein kinase C, and activation by a Ca2(+)-dependent process which is different from the NO/cGMP pathway.


Transmitter-induced changes of the membrane voltage of HT29 cells.

The colonic carcinoma cell line HT29 was used to examine the influence of agonists increasing cytosolic cAMP and Ca2+ activity on the conductances and the cell membrane voltage (Vm). HT29 cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4-10 days after seeding, when they had formed large plaques. In 181 impalements Vm was -51 +/- 1 mV. An increase in bath K+ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba2+ depolarized the cells by 10 +/- 1 mV (n = 49) or by 9 +/- 2 mV (n = 3), respectively. A decrease of bath Cl- concentration from 145 to 30 mmol/l depolarized the cells by 11 +/- 1 mV (n = 24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 mumol/l) or isoprenaline (10 mumol/l) depolarized the cells by 6 +/- 1 (n = 13), 15 +/- 3 (n = 5) and 6 +/- 2 (n = 3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9 +/- 1 mV (n = 6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP greater than ATP greater than ITP greater than GTP greater than TTP greater than CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27 +/- 6 mV (n = 5) at 0.1 mmol/l, and to a transient depolarization by 12 +/- 4 mV (n = 5) at 10 mumol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)


cAMP-dependent activation of small-conductance Cl- channels in HT29 colon carcinoma cells.

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT29 and the activation of Cl- channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was -56 +/- 0.4 mV (n = 294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K+ conductance and a smaller Cl- conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K+ conductance was reversibly inhibited by 1 - 5 mmol/l Ba2+. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl- conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n = 10) and isoproterenol (1 mumol/l, n = 12) depolarized the cells dose-dependently and reversibly by 12 +/- 2 mV and 13 +/- 2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl- conductance. Simultaneously the conductance of the cell-attached membrane increased from 155 +/- 31 pS to 253 +/- 40 pS (VIP, n = 4) and from 170 +/- 43 pS to 268 +/- 56 pS (isoproterenol, n = 11), reflecting the gating of Cl- channels in the cell-attached membrane.(ABSTRACT TRUNCATED AT 250 WORDS)


Small-conductance Cl- channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP.

The present study demonstrates the activation of Cl- channels in HT29 cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca2+, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast whole-cell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with half-maximal effects at 0.4 mumol/l for ATP, 60 pmol/l for neurotensin and 0.8 mumol/l for carbachol. The depolarization, which was caused by Cl- conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295 +/- 32 pS to 1180 +/- 271 pS (ATP; 10 mumol/l, n = 21) and 192 +/- 37 pS to 443 +/- 128 pS (neurotensin; 1 nmol/l, n = 8). The effects of the agonists could be mimicked by ionomycin (0.2 mumol/l), suggesting that an increase in intracellular Ca2+ was responsible for the activation of Cl- channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10 +/- 0.8 mV and the cell-attached conductance increased from 228 +/- 29 pS to 410 +/- 65 (n = 26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mumol/l) did not prevent the effects of Ca(2+)-increasing hormones and of hypotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)


Left ventricular free-wall rupture occurring during programmed ventricular stimulation in a patient with recent myocardial infarction.

In this report we describe a patient who died during programmed ventricular stimulation due to a rupture of the left ventricular free wall at the site of a recent myocardial infarction. The patient was a 75-year-old male who presented with an extensive anterior wall myocardial infarction complicated by sustained ventricular tachycardia occurring 8 days after admission. Cardiac catheterization revealed total occlusion of left anterior descending coronary artery and an anteroapical aneurysm. The patient died due to electromechanical dissociation during electrophysiological testing 11 days after myocardial infarction. Postmortem examination showed a rupture of the left ventricular free wall at the site of the myocardial infarction and distant from the site of catheter placement. It is suggested that caution be taken in choosing patients for electrophysiological studies who have had recent large myocardial infarctions with ventricular aneurysm.


The performance of the probability density function in differentiating supraventricular from ventricular rhythms.

The ability of the probability density function (PDF) of an automatic implantable cardioverter defibrillator (AICD) to reject supraventricular arrhythmias being recognized as ventricular was evaluated in 12 patients who were treated with an AICD (Ventak P 1600). The PDF criterion was monitored via telemetry with the sinus rate during exercise test. PDF was satisfied in seven patients at a rate of 75-144/min (mean 109/min), and not in the remaining five patients (mean rate 141/min). PDF was fulfilled in five of ten patients at a lower heart rate than predicted by the duty cycle index, derived from the ventricular patch lead electrogram at the implantation. Thus PDF is often fulfilled already at a moderately elevated sinus rate. If used to prevent inadvertent AICD discharges during rapid supraventricular rhythms, its performance should be tested in the individual patient.


Sinus node dysfunction after orthotopic cardiac transplantation: postoperative incidence and long-term implications.

We investigated incidence, normalization rates, and clinical significance of sinus node (SN) dysfunction after cardiac transplantation. Thirty-nine of 90 patients systematically evaluated presented with impaired SN function in the postoperative period. Of these, 22 normalized their SN function during follow-up while 17 remained impaired after 3 months. The proportion normalized was higher in patients with prolonged SN recovery time (11/16, 68.8%) and those reverting from escape rhythm to sinus rhythm until discharge (5/8, 62.5%) when compared to patients in escape rhythm throughout the postoperative period (5/11, 45.5%) or pacemaker dependence (1/4, 25%). There was no reliable postoperative predictor of normalization and necessity of long-term pacing. A postoperative heart rate consistently less than 70 beats/min (irrespective of the native rhythm) was significantly related to outcome (P = 0.01), but revealed unacceptable sensitivity (61.5%) and specificity (81%). After all, both patients with severe symptoms (near syncopes and syncope), were derived from this group. These two patients, who had been discharged in sinus rhythm, had late pacemakers implanted. An additional 17 patients had early pacemaker placement, only seven of which were constantly paced during follow-up. Given the low normalization rates, patients with postoperative escape rhythm are those most likely to benefit from pacemaker therapy over the long term. Even those in, or reverting back to, sinus rhythm until discharge may experience severe symptoms, particularly in the setting of a postoperative rate consistently less than 70 beats/min since this indicates a relatively lower probability of recovery.


Radiofrequency ablation of symptomatic but benign ventricular arrhythmias.

Two cases are presented where ablation of severely symptomatic ventricular arrhythmias not responding to medical therapy was accomplished with radiofrequency current application. After a routine programmed stimulation protocol, a quadripolar ablation catheter with a 4-mm tip was advanced percutaneously into the left ventricle in one case and into the right ventricle in the second case; and after precise pace mapping, the arrhythmogenic focus was successfully ablated using radiofrequency current. The postablation ambulatory recording revealed virtual eradication of ventricular ectopy in both cases. In conclusion, in severely symptomatic cases of "benign" ventricular arrhythmias, radiofrequency ablation offers an effective therapeutic alternative.


The use of head-upright tilt table testing in the evaluation and management of syncope in children and adolescents.

Recurrent syncope in an otherwise healthy child or adolescent is a common anxiety provoking disorder. Vasovagally mediated hypotension and bradycardia are believed common, yet difficult to diagnose, causes of syncope in this age group. Upright tilt table testing has been suggested as a potential method to test for vasovagal episodes. This study evaluated the utility of this technique in the evaluation and management of recurrent syncope in children and adolescents. Thirty patients with recurrent unexplained syncope were evaluated by use of an upright tilt table test for 30 minutes, with or without an infusion of isoproterenol (1 to 3 micrograms/min given intravenously), in an attempt to produce hypotension, bradycardia, or both. There were 15 males and 15 females, mean age 14 +/- 6 years. Each of the tilt positive patients received therapy with either fluorohydrocortisone, beta blockers, or transdermal scopolamine. Syncope occurred in six patients (20%) during the base line tilt and in 15 patients (50%) during isoproterenol infusion (total positives 70%). All initially positive patients were rendered tilt negative by therapy. Over a mean follow-up period of 20 months, no further episodes have occurred. We conclude that tilt table testing is a useful and effective test in the evaluation of unexplained syncope in childhood.


Failure of a second and third generation implantable cardioverter defibrillator to sense ventricular tachycardia: implications for fixed-gain sensing devices.

Failure to sense ventricular tachycardia and/or ventricular fibrillation by implantable cardioverter defibrillators (ICDs) is rare. We report a case in which persistent undersensing of monomorphic and polymorphic ventricular tachycardia occurred with a second and third generation ICD using fixed-gain sensing. This occurred despite adequate R wave sensing during sinus rhythm. The use of an endocardial sensing lead did not correct the problem. Failure to sense ventricular tachycardia in the third generation device with fixed-gain sensing occurred late after implantation and was discovered only at follow-up electrophysiology testing of the ICD. This problem could not be corrected by reprogramming of the device, and was not related to lead dislodgement. Placement of a new device with an automatic-gain sensing algorithm and use of previously implanted epicardial leads with better sensing characteristics provided appropriate sensing of ventricular tachyarrhythmias. The case illustrates the importance of testing the sensing of all ventricular arrhythmias in patients with fixed-gain ICD's. Follow-up electrophysiology testing and evaluation of epicardial and endocardial leads may be necessary in certain cases to ensure adequate sensing of ventricular tachyarrhythmias late after implantation.


Frequency and output-dependent change in conduction over slow pathways in a patient with sustained ventricular tachycardia unrelated to coronary artery disease.

In a patient with sustained ventricular tachycardia, we obtained two different paced QRS morphologies from a single pacing site. In one QRS morphology the stimulus to the QRS complex was long, 150 msec, and in the other it was 100 msec. At the paced cycle length of 600 msec and the stimulus output of 4 V, one QRS morphology with the stimulus to the onset of QRS activation (St-QRS) interval of 150 msec was observed. At the paced cycle length of 400 msec, the other QRS morphology with a St-QRS interval of 100 msec was observed alternatively with the former. At the paced cycle length of 353 msec or 316 msec, the latter with a shorter St-QRS interval was exclusively observed. When the stimulus output was increased from 4 to 10 V, keeping with the paced cycle length at 400 msec, the St-QRS interval was shortened from 100 to 80 msec. For the two QRS morphologies with two St-QRS intervals, two slowly conducting pathways would be responsible. The site of the block in the faster pathway must be located at the proximity of the pacing site and the conduction at a shorter paced cycle length would be explained by "supernormal conduction."


Incidence and timing of activity parameter changes in activity responsive pacing systems.

The incidence and timing of rate response parameter reprogramming in activity responsive pacing systems during the year after implantation was evaluated in two groups of patients: 24 patients in whom a VVI,R system was implanted (Activitrax, Medtronic, Inc.), and 21 patients in whom a DDD,R system was implanted (Synchrony, Siemens Pacesetter, Inc.). Activity parameter changes in Activitrax patients were made based on the presence of symptoms, while in Synchrony patients, changes were based on objective data obtained using a sensor indicated rate histogram with a slow and fast walk protocol. No significant difference in the incidence of activity parameter reprogramming was noted at various time intervals during the first year in Activitrax patients; in Synchrony patients a greater incidence of reprogramming changes was noted at the 1-month follow-up visit compared to later follow-up visits (P less than 0.02). Further, the incidence of changes at 1 month was greater for Synchrony compared to Activitrax patients (P less than 0.001), while no difference was detected between groups at subsequent follow-up intervals. Use of the slow and fast walk protocol, by permitting serial evaluation of sensor response, demonstrated alterations in sensor drive rates with similar levels of activity over the initial 4 to 6 postimplant weeks. This may result from postoperative changes at the pacemaker insertion site. Based on this experience, predischarge programming may not predict long-term rate response requirements. We recommend evaluation of sensor function using an exercise protocol performed at 4 to 6 postimplant weeks in all rate responsive pacing systems that utilize a piezoelectric crystal.


Effect of the atrioventricular relationship on atrial refractoriness in humans.

Atrial arrhythmias occur frequently in the setting of increased atrial size and pressure. This may result from contraction-excitation feedback. The objective of this study was to investigate the effect of alterations in atrial pressure, induced by varying the atrioventricular (AV) interval, on atrial refractoriness, and on the frequency of induction of atrial fibrillation. Twenty-seven patients without structural heart disease participated in the study. In each patient the atrial effective (ERP) and absolute refractory period (ARP) were measured during AV pacing at a cycle length of 400 msec and AV intervals of 0, 120, and 160 msec. The ERP was defined as the longest extrastimulus coupling interval that failed to capture with an extrastimulus current strength of twice the stimulation threshold. The ARP was defined in a similar manner with an extrastimulus current strength of 10 mA. The ERP and ARP were determined during continuous pacing using the incremental extrastimulus technique. A subset of patients had the pacing protocol performed during autonomic blockade. As the AV interval was increased from 0 to 160 msec, the peak right atrial pressure decreased from 16 +/- 4 mmHg to 7 +/- 3 mmHg and the mean right atrial pressure decreased from 7 +/- 3 mmHg to 3 +/- 22 mmHg (P less than 0.001). The atrial ERP and ARP did not change with alterations in the AV interval. There was no difference in the frequency of induction of atrial fibrillation. Similar results were obtained during autonomic blockade. These findings suggests that the phenomenon of contraction-excitation feedback may not be of importance in the development of atrial arrhythmias in patients without structural heart disease.


Permanent AV block or modification of AV nodal function by selective AV nodal artery ethanol infusion.

The acute and chronic effects of selective AV nodal artery ethanol infusion on AV nodal function was studied in 9 closed chest anesthetized dogs. Using standard percutaneous techniques of arterial catheterization, a 2.2 French infusion catheter was positioned in the AV nodal artery. Ten minute infusions into the AV nodal artery of 25%, 50%, or 100% ethanol in normal saline at rates of 0.5 mL/min acutely resulted in complete AV nodal block (AVB) in 5 dogs, 2:1 AV nodal block in 1 dog, and prolongation of AV nodal effective refractory period and/or Wenckebach cycle length in the remaining 3 dogs. One dog died with persistent complete AV block 1 week after the ethanol infusion. When restudied 4 weeks later, 7 of the 8 surviving dogs had persistent modification of AV nodal function, including complete AV block in 5 dogs and lengthening of AV nodal effective refractory period and/or Wenckebach cycle length without AV block in 2 dogs. Pathologic examination of the animals exhibiting chronic modification or ablation of AV nodal function revealed healing infarction of the AV node or its approaches. Distant myocardial necrosis was not observed and left ventricular function was normal. Slow infusion of low concentrations of ethanol into the AV nodal artery results in AV nodal modification or ablation due to localized necrosis in or around the AV node. This technique may have a role in AV nodal modification or ablation, particularly in patients who have failed DC shock or radiofrequency ablation.


Stimulation hierarchy: optimal sequence for double and triple extrastimuli during electrophysiological studies.

To determine the optimal ventricular stimulation sequence, an 11-step programmed electrical stimulation (PES) protocol was completed, even if a ventricular arrhythmia (VA) was induced with earlier steps. The protocol consisted of one, two, and three extrastimuli during sinus rhythm (SR), and at two drive pacing rates (VP1 and VP2) plus rapid burst and ramp pacing. By analyzing the 79 completed protocols that induced the clinical arrhythmia, the following were determined: (1) the frequency of induced clinical and nonclinical VA with each stimulation step; (2) the yield ratio (YR) of each step, defined as the probability of inducing clinical versus nonclinical arrhythmia; (3) the cumulative yield of induced clinical and nonclinical arrhythmia with two widely used stimulation sequences, i.e., triple extrastimuli delivered early in the stimulation protocol (MMC sequence) and triple extrastimuli delayed until after double extrastimuli failed to induce the clinical arrhythmia (B sequence); (4) the relative efficiency of these sequences were determined. The percentage of induced clinical and nonclinical arrhythmia with SR + 3 extrastimuli, VP1 + 2 extrastimuli, and VP2 + 2 extrastimuli were (53%, 5%), (36%, 5%), and (41%, 9%), respectively. The cumulative yield of induced clinical VA with the MMC-type sequence reached 55% by the third step of the protocol, whereas 50% was attained only at the eighth step of the B-type sequence. The cumulative percentage of induced nonclinical VA with either sequence was similar during the early steps of the protocol. The MMC sequence was more efficient, requiring overall 36% of potential steps for clinical arrhythmia induction, compared with 48% for the B sequence (P less than 0.001). For questionable arrhythmia states, e.g., syncope of unknown origin and nonsustained VT, a modified sequence is proposed that may further reduce the induction of uninterpretable arrhythmias.


Diagnosis and localization of accessory pathways.

The WPW syndrome is a curable disease. The evolution of nonpharmacological methods of accessory pathway ablation has had a significant impact on management strategies in patients with arrhythmias mediated by accessory pathways. Despite an incidence of preexcitation in the general population of 0.1% to 0.3%, curative therapy is underutilized. This review has highlighted the traditional and newer methods of diagnosing and localizing accessory pathways. The number of patients benefiting from definitive therapy will parallel increased physician awareness of these methods.


Demonstration of the feasibility of implantation of a skeletal muscle pulse generator for fecal incontinence in a patient with an implanted unipolar DDD pacemaker.

Electromagnetic fields and myopotentials from skeletal muscle may interfere with the function of a cardiac pacemaker. A 65-year-old woman with a unipolar DDD cardiac pacemaker underwent dynamic graciloplasty (transposition of the gracilis muscle around the anal canal and subsequent implantation of a bipolar pulse generator to stimulate the gracilis muscle), for the treatment of fecal incontinence. This gracilis pulse generator is turned "off" with an external magnet to allow defecation. Appropriate functioning of these two pulse generators (the cardiac pacemaker and the gracilis pulse generator) was tested during implantation of the gracilis pulse generator and afterwards. It was demonstrated that the combination could be used safely in this patient.


Bidirectional tachycardia induced by herbal aconite poisoning.

This report details the clinical, electrocardiographic, and electropharmacological characteristics of an unusual case of bidirectional tachycardia induced by aconites present in a Chinese herbal decoction consumed by a previously healthy subject. The tachycardia showed marked susceptibility to vagotonic maneuvers, cholinesterase inhibition, and adenosine triphosphate. The incessant nature of the tachycardia, rapid recurrence after transient suppression, and failure to respond to direct current cardioversion suggested an automatic tachycardia mechanism consistent with known data on the cellular electrophysiological mechanism of aconitine-mediated arrhythmogenesis. A fascicular or ventricular myocardial origin of the tachycardia with alternating activation patterns, or dual foci with alternate discharge, appeared most plausible. The rootstocks of aconitum plants have been commonly employed in traditional Chinese herbal recipes for "cardiotonic" actions and for relieving "rheumatism." Multiple pitfalls could occur during the processing of these herbs that might have predisposed to aconite poisoning. The need for strict control and surveillance of herbal substances with low margins of safety is highlighted.


Chronic hepatitis B and C. What is the status of drug therapy?

Chronic hepatitis remains difficult to treat. Use of interferon has been successful against both hepatitis B and C viruses, but the outcome of long-term administration has yet to be determined. Not all patients respond to interferon, however, and some have side effects that cause them to discontinue therapy. Dr Wright discusses the results of studies to evaluate therapy with alpha, beta, and gamma interferon as well as with other agents, such as ribavirin, thymosin, and ursodeoxycholic acid.


Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen.

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.


Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.

Administration of epidermal growth factor (EGF) to neonatal mice resulted in rapid tyrosine phosphorylation of a number of specific substrates in liver, kidney, lung, bladder, skin, and brain as detected by Western blot analysis of tissue homogenates with anti-phosphotyrosine antibodies. In the liver, three prominent EGF-dependent substrates of M(r) approximately 170,000, 120,000, and 55,000 were detected. A number of less prominent EGF-dependent substrates also were noted. Maximal tyrosine phosphorylation of pp170, pp120, and pp55 occurred within 5 min of subcutaneous injection and the levels of these phosphoproteins remained elevated for at least 45 min. Direct hepatic injection of EGF resulted in the tyrosine phosphorylation of these substrates within 60 sec of treatment. Tyrosine-phosphorylated pp170 was identified as the EGF receptor (EGFR). The tyrosine-phosphorylated pp55 substrate appeared to be associated with EGFR; both pp55 and EGFR were adsorbed to EGF-Affi-Gel, wheat germ lectin-Sepharose, and anti-EGFR antibodies bound to protein A-Sepharose. pp55 was not immunoreactive with anti-EGFR antiserum by Western blot analysis, indicating that it was not a fragment of the receptor. These results were confirmed by repeating the liver experiments using 32P-labeled neonatal mice. Increased amounts of 32P-labeled pp170 and pp55 were detected in anti-EGFR immunoprecipitates from liver extracts of EGF-treated animals as compared with controls. Phospho amino acid analysis of the 32P-labeled phosphoproteins revealed that EGF stimulated both serine and tyrosine phosphorylation in pp55 as well as in EGFR. The neonatal mouse may be a useful model for the study of signal transduction mediated by a variety of growth factors.


UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: relationship to dose, CD1a-DR+ epidermal macrophage induction, and Langerhans cell depletion.

Increasing UVB radiation at the earth's surface might have adverse effects on in vivo immunologic responses in humans. We prospectively randomized subjects to test whether epicutaneous immunization is altered by prior administration of biologically equalized doses of UV radiation. Multiple doses of antigens on upper inner arm skin (UV protected) were used to elicit contact sensitivity responses, which were quantitated by measuring increases in skin thickness. If a dose of UVB sufficient to induce redness (erythemagenic) was administered to the immunization site prior to sensitization with dinitrochlorobenzene (DNCB), we noted a marked reduction in the degree of sensitization (P less than 0.0006) that was highly dose responsive (r = 0.98). Even suberythemagenic UV (less than a visible sunburn) resulted in a decreased frequency of strongly positive responses (32%) as compared to controls (73%) (P = 0.019). The rate of immunologic tolerance to DNCB (active suppression of a subsequent repeat immunization) in the groups that were initially sensitized on skin receiving erythemagenic doses of UV was 31% (P = 0.0003). In addition, a localized moderate sunburn appeared to modulate immunization with diphenylcyclopropenone through a distant, unirradiated site (41% weak responses) as compared to the control group (9%) (P = 0.05). Monitoring antigen presenting cell content in the epidermis revealed that erythemagenic regimens induced CD1a-DR+ macrophages and depleted Langerhans cells. In conclusion, relevant and even subclinical levels of UV exposure have significant down modulatory effects on the ability of humans to generate a T-cell-mediated response to antigens introduced through irradiated skin.


Heterogeneity of immunoglobulin-associated molecules on human B cells identified by monoclonal antibodies.

Two covalently linked transmembrane molecules, encoded in mice by the mb-1 and B29 genes, have been defined as integral components of the antibody receptor units expressed on B cells. We have produced monoclonal antibodies against an exposed extracellular epitope on the putative human equivalent of the mouse B29 product. These antibodies, CB3-1 and -2, were used to show that cytoplasmic expression of this molecule begins in human pro-B cells (terminal deoxynucleotidyltransferase-positive, mu chain-negative), whereas surface expression coincides strictly with surface immunoglobulin expression of all isotypes. Immunochemical analysis of the human immunoglobulin-associated molecules revealed greater molecular heterogeneity than has been noted for the murine analogues. This molecular heterogeneity of immunoglobulin-associated molecules varied as a function of differentiation stage and the immunoglobulin isotypes expressed by B-lineage cells. Our data support the hypothesis that biochemical heterogeneity of the surface immunoglobulin-associated molecules may contribute to the variability in biological effects of antigen receptor crosslinkage on B cells of different maturational stages. Because the CB3 antibodies are capable of down-modulating the antigen receptors on all B cells, they may prove therapeutically useful as universal B-cell suppressants.


Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12.

The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.


Adenosine decreases neurotransmitter release at central synapses.

Adenosine, at concentrations ranging from 5 to 100 microM, decreases the efficacy of transmission at the perforant path synapses on dentate granule cells. We have used whole cell recording from these cells in slices to determine the mechanism of the reduced synaptic strength. We find that size of miniature excitatory postsynaptic currents (mepscs) is unaffected by adenosine at concentrations up to 100 microM, an observation that indicates adenosine's mode of action is not through a decreased postsynaptic sensitivity to neurotransmitter. A quantal analysis indicates, however, that the quantity of neurotransmitter released is sufficiently diminished by adenosine to account entirely for the adenosine-produced decrease in synaptic strength. Application of 3-isobutyl-1-methylxanthine (IBMX), a drug that antagonizes the effects of endogenous adenosine, produces an increase in synaptic strength. This observation suggests that the resting level of adenosine in our slices is appreciable, and an analysis of the adenosine dose-response relation is consistent with endogenous adenosine levels of about 10 microM. IBMX application produces only slight changes in the amplitude of mepscs, whereas a quantal analysis demonstrates that the drug significantly increases the amount of neurotransmitter released. Thus IBMX acts as an "anti-adenosine" in our experiments. In some experiments we have been able to record excitatory and inhibitory synaptic currents produced by the same perforant path stimulus. In these instances we find that inhibitory transmission is unaffected by concentrations of adenosine that produce a marked decrease in the strength of excitatory synapses.


Molecular organization of Leishmania RNA virus 1.

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.