Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction. Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.


Characterisation by restriction mapping of three subtypes of molluscum contagiosum virus. DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease digestion, identifying three virus subtypes. The structural features of MCV DNA are typical of poxviral DNA. Physical maps of cleavage sites for BamHI, CIaI, and HindIII were constructed for single isolates of each subtype. These differ extensively, indicating the independence of the three subtypes. However, they are closely related, as determined by molecular hybridisation and nucleotide sequence analysis, and their genomes are essentially colinear. There is marked geographical variation in the relative incidence of MCV I and II, whilst MCV III is uniformly rare.


Genomic variation among herpes simplex virus type 1 strains: virus DNA analysis of isolates from Saudi patients. Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes HindIII and BamHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with HindIII restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BamHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise to the observed DNA polymorphism were mapped to the unique region of the long and short segments of the genome.


Further characterisation of human rotaviruses isolated from asymptomatically infected neonates in South Africa. Stool specimens were collected from healthy neonates at Ga-Rankuwa Hospital in the winters of 1984 and 1986 and tested for the presence of rotavirus infection. Asymptomatic excretion was found to occur in 25% of the newborn babies analysed. Gel electrophoresis of the rotavirus RNA genome revealed that a genomically stable strain of rotavirus was endemic in the ward at the time intervals examined. Hybridisation analysis of the VP4 and VP7 rotavirus genes, which encode the outer capsid neutralization proteins of the virus, was conducted. These results showed the presence of a serotype 4 rotavirus strain with an M37-like VP4 gene allele, which remained conserved in the nursery over the time period examined. Partial nucleotide sequences were obtained for a variable region of the VP7 gene and for the hyperdivergent region of the VP4 gene from 8 of these viruses and showed that remarkable conservation of these regions in the genes of the viruses occurred over time.


Serum IgA, IgG, and IgM responses to different enteroviruses as measured by a coxsackie B5-based indirect ELISA. An enterovirus-specific indirect ELISA, based on a single local isolate of coxsackie B5 as antigen, was used to study the IgA, IgG, and IgM responses in 19 patients with a recent or current enterovirus infection. Twelve different enterovirus serotypes were isolated from 15 patients. Paired serum samples were available from 10 and a single serum from 5 of these 15 patients. In addition, 4 patients diagnosed by a significant titer rise of complement fixing antibodies to enterovirus were included. A serological diagnosis, defined as an increase in titer of enterovirus IgG and/or presence of enterovirus IgM, were established in all 14 patients with paired sera. Enterovirus IgM was present in either a single serum or in both sera in 13 of them. Out of 5 patients with a single serum sample only, enterovirus IgA or enterovirus IgM was found in 4. Specific IgA was present in either a single serum or in both sera in 14 of the 19 patients. Seven of the 10 enterovirus isolate patients with paired sera had a significant titer rise of complement fixing antibodies; however, all 10 were diagnosed by ELISA. Among 64 healthy controls 2 had enterovirus IgA and none had enterovirus IgM. In conclusion, the use of a single antigen-based ELISA was found to be reliable for the diagnosis of recent and current enterovirus infections.


Improved detection of cytomegalovirus viremia in AIDS patients using shell vial and indirect immunoperoxidase methodologies. One hundred twelve peripheral blood specimens were tested for the presence of cytomegalovirus (CMV) by the tube culture indirect immunoperoxidase (TC-IPA) procedure, the shell vial assay [shell vials were pre- and postinoculation treated with medium containing 2 of 10% fetal bovine serum (FBS) or 100 micrograms% cortisol] (SV-IFA), and conventional (MRC-5) tube cultures (TC-CPE). CMV was detected in 25 (22%) of the 112 specimens tested by at least one of these methods. The detection/isolation of CMV among the 25 positive specimens in shell vials maintained with 2% FBS, 100 micrograms% cortisol + 2% FBS, and 10% FBS was 36, 44, and 52%, respectively. Detection/isolation of the virus from blood by TC-IPA and TC-CPE was 52% and 76%, respectively. A significantly greater CMV detection rate occurred using TC-CPE compared to SV-IFA treated with medium supplemented with an FBS concentration of 2% (P = .0132), but not medium containing the higher serum supplement or the glucocorticoid (P greater than .05). Differences in the identification of a CMV viremia were observed by IPA, SV-IFA, and TC-CPE methodologies on a patient-to-patient basis, denoting the necessity of incorporating each methodology into the CMV screening panel. Demographic analysis of 82 AIDS patients showed a CMV viremia prevalence of 9% (2/28) in intravenous drug users, 57% (27/47) in homosexual patients, and 22% (2/9) in heterosexual and transfusion patients. Overnight (24 hr) storage of whole blood at 4 or 24 degrees C, respectively, reduced CMV recovery by 40% and 65%, when tested by TC-CPE.(ABSTRACT TRUNCATED AT 250 WORDS)


Detection of enteroviral RNA by polymerase chain reaction in faecal samples from patients with aseptic meningitis. An assay based on the polymerase chain reaction (PCR) for detection of enteroviral RNA in stool samples was carried out using specimens from 74 patients with aseptic meningitis. The primer pair and probe were derived from the highly conserved 5' non-coding enterovirus genomic region. Enteroviral RNA was detected in faeces of all 36 patients in whom an enterovirus was isolated from stool. The PCR assay yielded positive results in additionally 3/6 cases where enterovirus diagnoses were obtained by virus isolation from cerebrospinal fluid and/or serological tests. Thus, the positive outcome of the PCR assay was 39 (93%) among the 42 patients with enterovirus diagnoses. Furthermore, 7/19 (37%) cases with an etiology that was not established by other means were positive in the test indicating that the PCR assay may give considerable additional etiological information in patients with aseptic meningitis. The limit of RNA detectability in the PCR assay was about 100 TCID50 when highly cytopathogenic enterovirus types (coxsackievirus type B5 and echovirus type 11) were tested. The PCR was negative in all 13 patients with non-enterovirus diagnoses except in one case with a herpes simplex virus type 2 infection. Since enterovirus-specific IgM antibodies could be detected in this case a dual infection seemed probable. All the negative controls, included in the study, were PCR-negative and no contamination was encountered. This study proves the usefulness of the PCR assay for detection of enteroviral RNA in stool samples and suggests that the test may be an alternative to virus isolation for rapid enterovirus diagnosis in patients with aseptic meningitis.


Detection of human herpesvirus-6 DNA by polymerase chain reaction in serum or plasma. Human herpesvirus-6 (HHV-6) is a newly identified human pathogen. Currently clinicians rely mainly on blood lymphocyte culture and serological tests to diagnose HHV-6 infection. The polymerase chain reaction (PCR) was carried out on the plasma or sera of patients to determine the value of PCR in the diagnosis of HHV-6 infection. A total of 30 patients entered the study; 10 were experiencing acute HHV-6 infections and 20 were healthy and served as controls. HHV-6 DNA was detected by PCR in the serum or plasma of the 10 cases with acute HHV-6 infections. All 20 controls had no HHV-6 DNA in their sera. The time for serum to become PCR-positive coincided with the appearance of IgG HHV-6 antibody. The relatively late presence of HHV-6 DNA in serum might result from late lysis of infected cells by immune responses. It is concluded that detection of HHV-6 DNA by PCR in the serum is a valuable tool for the diagnosis of acute and/or active viral infection.


Toga virus-like particles in acute liver failure attributed to sporadic non-A, non-B hepatitis and recurrence after liver transplantation. Toga virus-like particles (typically 60-70 nm: enveloped with small surface spikes) were detected in the native hepatectomy specimens in 7 of 18 patients grafted for acute liver failure attributed to sporadic non-A, non-B hepatitis and in 2 patients grafted for fulminant hepatitis attributed to anti-epileptic drug hepatotoxicity. These particles were not detected in the hepatectomies from 12 other patients grafted for other causes of acute liver failure, 12 for various chronic liver diseases, and 2 histologically normal livers. Acute hepatic failure, characterized histologically by severe haemorrhagic necrosis, developed 7 days after grafting in 5 patients, all in the non-A, non-B group with toga virus-like particles in native liver. Similar virus-like particles were detected in all grafts and were in greater abundance than in the native livers. The agent may be novel because pre- and post-grafting sera were negative for antibodies against representative panels of arboviruses and in first and second generation antibody tests for hepatitis C virus.


Global forebrain ischemia induces a posttranslational modification of multifunctional calcium- and calmodulin-dependent kinase II. The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.


Altered microviscosity at brain membrane surface induces distinct and reversible inhibition of opioid receptor binding. In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis-delta 6) acid, oleic (18:1, cis-delta 9) acid, and vaccenic (18:1, cis-delta 11) acid or their corresponding methyl esters at 0.5 mumol/mg of membrane protein caused a similar 7-10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5-7% by the fatty acids but only 1% by their methyl esters. Concomitantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated mu-, delta-, and kappa-opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order delta greater than mu much greater than kappa, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax and affinity of opioid binding. Cholesteryl hemisuccinate (0.5-3 mumol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited mu-, delta-, and kappa-opioid binding toward control values. In particular, the Bmax of [3H]-DPDPE binding completely recovered after being undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)


Chromatographic resolution and characterization of a nerve growth factor-dependent kinase that phosphorylates microtubule-associated proteins 1 and 2 in PC12 cells. When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.


Ethanol reduces norepinephrine-stimulated melatonin synthesis in rat pinealocytes. In this study, the in vitro effects of ethanol on norepinephrine-stimulated cyclic AMP (cAMP), N-acetyltransferase (NAT), and melatonin (MT) production were examined in dispersed rat pinealocytes. Cellular cAMP content was determined 15 min after treatment; whereas NAT activity and MT release in the medium were determined 4.5 h after treatment. It was found that ethanol less than or equal to 200 mM had no effect on norepinephrine-stimulated cAMP response, whereas 25 mM ethanol resulted in a significant inhibition of norepinephrine-stimulated NAT and MT levels. Furthermore, ethanol was equally effective in inhibiting the dibutyryl cAMP-stimulated NAT and MT levels. The inhibitory action of ethanol was not due to a direct effect or a delay in the onset of NAT activity. When alcohols with different chain lengths were used, it was found that their inhibitory potencies were related to their chain lengths with butanol greater than propanol greater than ethanol greater than methanol. Taken together, these findings indicate that (1) ethanol has an inhibitory action on norepinephrine-stimulated MT synthesis, (2) one site of ethanol action is distal to cAMP elevation, and (3) the inhibitory effect of ethanol on pineal MT synthesis appears to be secondary to its hydrophobic action.


Involvement of gamma-aminobutyric acid and N-methyl-D-aspartate receptors in the inhibitory effect of ethanol on pentylenetetrazole-induced c-fos expression in rat brain. The expression of c-fos mRNA in rat brain was induced by intraperitoneal administration of pentylenetetrazole (PTZ) and picrotoxin, which act on the picrotoxin binding site of the gamma-aminobutyric acid-benzodiazepine (GABA-BZ) receptor complex, by N-methyl-D-aspartate (NMDA) and kainic acid, agonists of different classes of glutamate receptors and by caffeine, an antagonist of adenosine receptors. The actions of PTZ and picrotoxin but not that of NMDA were blocked by ethanol and the NMDA-receptor antagonist, MK-801. Ro 15-4513 partially reversed the inhibitory effect of ethanol on PTZ-induced c-fos mRNA synthesis. Acute ethanol administration blocked the actions of PTZ and NMDA without affecting the response to kainic acid or caffeine. Taken together, these data suggest that ethanol blocks c-fos gene activation by noncompetitive antagonists of the GABA-BZ receptor via actions on both the NMDA and GABA receptors.


Tetanus toxin inhibits depolarization-stimulated protein phosphorylation in rat cortical synaptosomes: effect on synapsin I phosphorylation and translocation. Synapsin I, a prominent phosphoprotein in nerve terminals, is proposed to modulate exocytosis by interaction with the cytoplasmic surface of small synaptic vesicles and cytoskeletal elements in a phosphorylation-dependent manner. Tetanus toxin (TeTx), a potent inhibitor of neurotransmitter release, attenuated the depolarization-stimulated increase in synapsin I phosphorylation in rat cortical particles and in synaptosomes. TeTx also markedly decreased the translocation of synapsin I from the small synaptic vesicles and the cytoskeleton into the cytosol, on depolarization of synaptosomes. The effect of TeTx on synapsin I phosphorylation was both time and TeTx concentration dependent and required active toxin. One- and two-dimensional peptide maps of synapsin I with V8 proteinase and trypsin, respectively, showed no differences in the relative phosphorylation of peptides for the control and TeTx-treated synaptosomes, suggesting that both the calmodulin- and the cyclic AMP-dependent kinases that label this protein are equally affected. Phosphorylation of synapsin IIb and the B-50 protein (GAP43), a known substrate of protein kinase C, was also inhibited by TeTx. TeTx affected only a limited number of phosphoproteins and the calcium-dependent decrease in dephosphin phosphorylation remained unaffected. In vitro phosphorylation of proteins in lysed synaptosomes was not influenced by prior TeTx treatment of the intact synaptosomes or by the addition of TeTx to lysates, suggesting that the effect of TeTx on protein phosphorylation was indirect. Our data demonstrate that TeTx inhibits neurotransmitter release, the phosphorylation of a select group of phosphoproteins in nerve terminals, and the translocation of synapsin I. These findings contribute to our understanding of the basic mechanism of TeTx action.


Preparation of antibodies to beta subunits of gamma-aminobutyric acidA receptors. Antisera were produced in rabbits against synthetic peptides based on two regions of the cDNA sequence of the beta 1 subunit of bovine gamma-aminobutyric acidA (GABAA) receptors. The deduced amino acid sequences were similar in other beta subunits of bovine, rat, and chick receptors, predicting cross-reactability with all beta subunits. One antiserum (anti-beta e) was raised against an extracellular moiety near the invariant disulfide loop thought to be located near the neurotransmitter binding domain; the other (anti-beta c) was raised against an intracellular moiety containing a consensus sequence for cyclic AMP-dependent protein kinase phosphorylation of a serine residue. Predicted secondary structures suggested high potential immunogenicity for the chosen antigen peptides. Both antisera at high dilutions recognized the same polypeptide bands on western blots of GABAA receptors purified from three regions of bovine brain (four bands at 57, 54, 53, and 52 kDa in cerebral cortex) but fewer bands (57, 54, and 52 kDa) in hippocampus and cerebellum (one major band at 54 kDa, traces at 57 and 53 kDa). This is consistent with the presence of multiple beta subunits whose expression varies with brain region, as shown by molecular cloning. The anti-beta c antibody was able to immunoprecipitate purified GABAA receptor [3H]-muscimol binding, 87% in bovine cortex and 75% in total rat brain; the anti-beta e was unable to immunoprecipitate any antigen. These antibodies indicate a region-dependent heterogeneity of beta subunits and should be useful for analyzing structure, function, and localization of GABAA receptor subtypes in brain.


Amino acid neurotransmitter receptor changes in cerebral cortex in alcoholism: effect of cirrhosis of the liver. Gamma-aminobutyric acidA/benzodiazepine receptor binding sites and the N-methyl-D-aspartate subclass of glutamate receptor sites were assessed in synaptic plasma membrane homogenates of cerebral cortex tissue obtained at autopsy from cirrhotic and noncirrhotic alcoholic patients and matched control subjects. The alcoholic patients consumed an average of greater than 80 g of ethanol/day, the control subjects less than 20 g/day. Postmortem delays up to approximately 100 h caused no significant loss of any of the binding sites; the patient and subject groups were closely matched for age. The affinities (KD) of the receptor sites did not differ between the patient and subject groups, nor between cortical regions. Using three different radioligands ([3H]muscimol, [3H]flunitrazepam, and [3H]diazepam), the gamma-aminobutyric acidA/benzodiazepine receptor complex was found to have greater density (Bmax) in superior frontal gyrus in alcoholic patients (which selectively shows morphological change in alcoholic patients), but was unchanged in motor cortex. Alcoholic patients with cirrhosis had much less pronounced changes. The density of the N-methyl-D-aspartate subclass of glutamate receptors, assessed with [3H]MK-801, did not vary across patient and subject groups.


Noncompetitive inhibition of N-methyl-D-aspartate by conantokin-G: evidence for an allosteric interaction at polyamine sites. Conantokins T and G are polypeptide toxins present in snails of the genus Conus. These substances were recently reported to act as N-methyl-D-aspartate (NMDA) antagonists. In the present study, we examined the possible mechanisms producing this antagonism. Conantokin-G inhibited spermine- and spermidine-stimulated [3H]MK-801 binding to extensively washed rat forebrain membranes in a noncompetitive manner with IC50 values of approximately 507 and approximately 946 nM, respectively. In contrast, glutamate-enhanced [3H]MK-801 binding was unaffected by conantokin-G concentrations of less than or equal to 20 microM. At concentrations greater than or equal to 5 microM, conantokin-G effected a modest, noncompetitive inhibition of glycine-stimulated [3H]MK-801 binding and also produced a small enhancement of basal [3H]MK-801 binding. Conantokin-G reduced (IC50 approximately 1.08 microM) the NMDA-stimulated accumulation of cyclic GMP in cerebellar granule cell cultures to basal values, but did not affect kainate-mediated increases in cyclic GMP. These findings indicate that conantokin-G acts as a noncompetitive NMDA antagonist through an allosteric inhibition of polyamine responses. The neurochemical profile of this polypeptide is distinct from previously described noncompetitive NMDA antagonists.


Increased neopterin levels in brains of patients with human immunodeficiency virus type 1 infection. Postmortem levels of native neopterin (D-erythro-neopterin) were measured in cerebral cortical samples from 44 human immunodeficiency virus type 1-infected and eight uninfected, nonneurological control patients. Cerebral cortical gray and white matter neopterin levels for the controls ranged from 0.5 to 7.2 pmol/mg of protein in contrast to neopterin levels in brains of the virus-infected patients, which frequently were more than threefold and occasionally more than 30-fold higher than mean control levels. Cortical neopterin levels did not correlate with severity of the acquired immunodeficiency syndrome dementia complex, but subcortical levels correlated with the presence of active human immunodeficiency virus type 1 infection, as reflected by pathological evidence of multinucleated giant cell encephalitis. Evidence of opportunistic cytomegalovirus infections in approximately 25% of the human immunodeficiency virus type 1-infected patients was associated with enhanced levels of neopterin in frontal cortex.


Drosophila cyclodiene resistance gene shows conserved genomic organization with vertebrate gamma-aminobutyric acidA receptors. Genomic clones from the Rdl locus of Drosophila, whose mutant phenotype is resistant to cyclodiene insecticides and picrotoxin, were characterized by restriction mapping and partial sequencing to determine intron/exon structure. The coding region of the gene comprises nine identified exons and spans greater than 25 kb of genomic DNA. The structure of the Drosophila Rdl receptor subunit was compared with those of vertebrate gamma-aminobutyric acid subtype A (GABAA) receptors and nicotinic acetylcholine receptors (nAChRs). The first six introns in Rdl show positions similar to those in vertebrate GABAA receptors, whereas the last two differ. It is interesting that the last intron appears to be in a position similar to that in nAChRs. These results are examined in relation to the proposal, based on amino acid identities, that Rdl codes for a novel class of GABAA receptor subunit more closely related to glycine receptors, and the possible place of Rdl in the lineage of the receptor superfamily is discussed.


Expression of plasma membrane calcium-pumping ATPase mRNAs in developing rat brain and adult brain subregions: evidence for stage-specific expression. The plasma membrane calcium-pumping ATPases (Ca(2+)-ATPases) maintain resting free cytosolic calcium concentrations in cells at the submicromolar level. These Ca(2+)-ATPases are encoded by four genes that can be alternately spliced to produce nine different mRNAs, each of which has a unique tissue-specific distribution. Examination of the expression of these mRNAs in rat brain during development revealed that transcripts from three of the four known genes are expressed by the end of gestation. However, the stage of transcription induction varies among the isoforms. The mRNA encoding plasma membrane Ca(2+)-ATPase (PMCA) 1b, the isoform though to maintain a housekeeping function, was present from embryonic day 10. The other alternatively spliced PMCA1 mRNAs, PMCA1a and c, which are preferentially expressed in the brain, did not appear until embryonic day 14. PMCA2a mRNA and the alternatively spliced PMCA2b and c transcripts were coordinately induced on embryonic day 18. The PMCA3a transcript first appeared on embryonic day 18 but did not reach steady-state levels until postnatal day 3, whereas production of PMCA3b mRNA first occurred on embryonic day 10 and reached steady-state expression by embryonic day 18. Several PMCA mRNAs tested varied in expression in specific regions of the brain that were examined at three postnatal time points.


Ischemia-induced changes in extracellular levels of striatal cyclic AMP: role of dopamine neurotransmission. Dopamine has been demonstrated to be involved in the development of ischemic neuronal damage in the striatum. This detrimental effect of dopamine may involve activation of second messenger systems, such as the cyclic AMP (cAMP) cascade, which may enhance the susceptibility of striatal neurons to ischemia. In the present study, we have evaluated the relationship between ischemia-induced changes in cAMP and dopamine neurotransmission. Microdialysis probes were implanted in both striata, and a D1 antagonist (SCH-23390, 100 microM) was administered through one probe and modified Ringer's solution through the other. After a stabilization period, rats (n = 6) were subjected to 20 min of ischemia by two-vessel occlusion plus hypotension. Extracellular samples were collected from both striata, before, during, and after ischemia, and analyzed for cAMP by radioimmunoassay. Ischemia induced a significant increase in extracellular cAMP (means +/- SE, fmol/microliter; baseline: 4.35 +/- 1.1, ischemia: 12.2 +/- 1.98), which was also observed at 4 h of recirculation (mean level of 8.45 +/- 1.14). Treatment with the D1 antagonist significantly inhibited the rise in extracellular cAMP during ischemia and recirculation. These results indicate that an ischemia-induced surge in dopamine and activation of D1 receptors are involved in the generation of cAMP during ischemia and recirculation. Because activation of the adenylate cyclase cascade may modulate the effects of glutamate, generation of cAMP through this pathway may play a role in facilitating the injurious effects of dopamine during ischemia.


Inhibition of cyclic AMP accumulation in intact NCB-20 cells as a direct result of elevation of cytosolic Ca2+. Earlier studies established that adenylyl cyclase in NCB-20 cell plasma membranes is inhibited by concentrations of Ca2+ that are achieved in intact cells. The present studies were undertaken to prove that agents such as bradykinin and ATP, which elevate the cytosolic Ca2+ concentration ([Ca2+]i) from internal stores in NCB-20 cells, could inhibit cyclic AMP (cAMP) accumulation as a result of their mobilization of [Ca2+]i and not by other mechanisms. Both bradykinin and ATP transiently inhibited [3H]cAMP accumulation in parallel with their transient mobilization of [Ca2+]i. The [Ca2+]i rise stimulated by bradykinin could be blocked by treatment with thapsigargin; this thapsigargin treatment precluded the inhibition of cAMP accumulation mediated by bradykinin (and ATP). A rapid rise in [Ca2+]i, as elicited by bradykinin, rather than the slow rise evoked by thapsigargin was required for inhibition of [3H]cAMP accumulation. Desensitization of protein kinase C did not modify the inhibitory action of bradykinin on [3H]cAMP. Effects of Ca2+ on phosphodiesterase were also excluded in the present studies. The accumulated data are consistent with the hypothesis that hormonal mobilization of [Ca2+]i leads directly to the inhibition of cAMP accumulation in these cells and presumably in other cells that express the Ca(2+)-inhibitable form of adenylyl cyclase.


Characterization of a digitonin-solubilized bovine brain H3 histamine receptor coupled to a guanine nucleotide-binding protein. The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.


Generation of arachidonic acid and diacylglycerol second messengers from polyphosphoinositides in ischemic fetal brain. Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.


Multiple mechanisms for inhibition of excitatory amino acid receptors coupled to phosphoinositide hydrolysis. Excitatory amino acid (EAA) analogues activate receptors that are coupled to the increased hydrolysis of phosphoinositides (PIs). In these studies, hippocampal slices were prepared from neonatal rats (6-11 days old) to characterize the effects of EAA analogues on these receptors. The concentrations of ibotenate and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate (trans-ACPD) required to evoke half-maximal stimulation (EC50 values) were 28 and 51 microM, respectively. Although the data for stimulation of PI hydrolysis by ibotenate and trans-ACPD were best fit to theoretical curves that had Hill slopes of 1, data for stimulation of PI hydrolysis by quisqualate were best fit to two sites. The EC50 values were 0.43 and 44 microM. The high-affinity sites were 70% of the total. A number of EAA analogues were tested for inhibition of PI metabolism. One of these, L-aspartate-beta-hydroxamate (L-A beta HA), was identified as a novel inhibitor of this response. L-A beta HA was equipotent as an inhibitor of PI metabolism stimulated by ibotenate, quisqualate, and trans-ACPD. The data for this inhibition were best fit to two sites. Between 32 and 48% of the total sites had high affinity with IC50 values in the range of 1.2-6.3 microM. The low-affinity sites had IC50 values between 610 and 2,700 microM. DL-2-Amino-3-phosphonopropionate (DL-AP3) was also equipotent as an inhibitor of PI hydrolysis stimulated by ibotenate, quisqualate, and trans-ACPD (IC50 values were 480-850 microM). In contrast to the data for L-A beta HA, the data for DL-AP3 were best fit to a single site. Both of these inhibitors reduced the maximal response caused by the agonists, consistent with noncompetitive mechanisms of action. Several experiments were designed to examine potential mechanisms for these noncompetitive effects. These studies suggest that either L-A beta HA and DL-AP3 bind to a site on the receptor and irreversibly block activation of the receptor, or that these inhibitors act via a distinct site that specifically regulates EAA receptors coupled to PI hydrolysis.


Investigations about a direct neurotensin-dopamine interaction by nuclear magnetic resonance study, synaptosomal uptake of dopamine, and binding of neurotensin to its receptors. Interactions between dopamine and neurotensin can occur at various levels of the dopaminergic pathways. By using different approaches in vitro, we investigated the proposed hypothesis that neurotensin might bind to dopamine in the synaptic cleft. Nuclear magnetic resonance spectra of neurotensin were not modified by the addition of dopamine, and no nuclear Overhauser effect was detected. Synaptosomal uptake of [3H]dopamine in the presence of neurotensin did not lead to any modifications of the kinetic constants of the uptake. Neurotensin binding was not modified by the addition of dopamine. These results did not confirm the suggestion that neurotensin can form a complex with dopamine.


Measurement of lithium-induced changes in mouse inositol(1)phosphate levels in vivo. An anion-exchange HPLC mass assay was used to characterize Swiss-Webster mouse brain and peripheral tissue inositol(1)phosphate [Ins(1)P]levels. Ins(1)P was identified in all tissues studied but Ins(4)P could be identified only in brain, and then only as a part of a peak containing an additional, unidentified component. As a result, it was not possible to quantify Ins(4)P levels. Following a single subcutaneous dose of lithium (10 mmol/kg), brain Ins(1)P levels were maximally elevated after 6 h (corresponding to peak brain lithium concentrations) and were increased to levels 35- and 20-fold higher than in saline-treated animals in cholinergic agonist (pilocarpine)-stimulated and unstimulated animals, respectively. The ED50 for the lithium-induced accumulation of brain Ins(1)P 6 h after administration was 4-6 mmol/kg. The pilocarpine stimulation of lithium-induced brain Ins(1)P accumulation had an ED50 of 22 mg/kg, with maximal accumulation occurring 120 min after pilocarpine administration. Atropine reduced Ins(1)P levels, in both the absence and the presence of lithium, by 40%, indicating that cholinergic systems contribute a large (40%) component of basal brain phosphatidylinositol (PI) cycle activity. In peripheral tissues, there were lithium-induced accumulations of Ins(1)P in kidney, heart, and liver (but not testes) but these were less than that seen in the brain, suggesting that under basal (and pilocarpine-stimulated) conditions, the brain has a higher turnover of the PI cycle than the various peripheral tissues studied. These data support the hypothesis that lithium exerts its effects in vivo via modulation of the PI cycle.(ABSTRACT TRUNCATED AT 250 WORDS)


Tyrosine phosphorylation of microtubule-associated protein kinase after transient ischemia in the gerbil brain. The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.


Okadaic acid, a protein phosphatase inhibitor, inhibits nerve growth factor-directed neurite outgrowth in PC12 cells. The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.


The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.


In vivo activation of kainate receptors induces dephosphorylation of the heavy neurofilament subunit. Injection of kainic acid (KA) into the rat hippocampus reduced the phosphorylation-related immunoreactivity of the heavy subunit of neurofilament proteins (NF-H). The effect was demonstrated quantitatively with a dot-immunobinding assay and qualitatively by immunoblotting with monoclonal antibodies against phosphorylation-dependent and nonphosphorylation-related epitopes of NF-H. The KA-induced reduction affected 50% of the phosphorylated NF-H in half of the hippocampus after 48 h. At the same time, the nonphosphorylation-related NF-H immunoreactivity increased as revealed by immunoblotting, indicating a shift from phosphorylated to nonphosphorylated NF-H. The effects on NF-H preceded a decrease in content of the neuron-specific enolase, a soluble neuronal cytoplasmic protein. No alterations of the light subunit of neurofilament proteins occurred, suggesting that KA has a preferential effect on NF-H phosphorylation. N-Methyl-D-aspartate administered similarly did not lead to a rapid dephosphorylation of NF-H. We propose that kainate receptor-mediated dephosphorylation in NF-H is involved in the signal transduction of excitatory amino acids with consequences for neuronal functions dependent on intermediary filament phosphorylation.


Effect of vigabatrin on striatal dopamine receptors: evidence in humans for interactions of GABA and dopamine systems. Vigabatrin is a specific gamma-aminobutyric acid transaminase inhibitor. The clinical use of this drug in the treatment of epilepsy has been sporadically linked to the development of psychosis. Using 123I-IBZM, a specific dopamine D2 receptor ligand and single photon emission tomography (SPET), one month of treatment with vigabatrin was associated with a decrease in specific binding of 123I-IBZM to D2 receptors in the left hemisphere basal ganglia. This change may provide one explanation for the development of psychosis in vulnerable patients.


Gating of trigemino-facial reflex from low-threshold trigeminal and extratrigeminal cutaneous fibres in humans. Changes in the size of the test components (R1 and R2) of the trigemino-facial reflex were studied after electrical subliminal conditioning stimulation were applied to the trigeminal, median and sural nerves. After conditioning activation of the trigeminal nerve (below the reflex threshold), the early R1 reflex component showed phasic facilitation, peaking at about 50 ms of interstimulus delay, followed by a long-lasting inhibition recovering at 300-400 ms. The same conditioning stimulation resulted in a monotonic inhibition of the late R2, starting at 15-20 ms, with a maximum at 100-150 ms and lasting 300-400 ms. Intensity threshold for both the R1 and R2 changes ranged from 0.90 to 0.95 times the perception threshold. A similar longlasting inhibition of the R2 reflex response was also seen after conditioning stimulation applied to low-threshold cutaneous afferents of the median and sural nerves. The minimum effective conditioning-test interval was 25-30 ms and 40-45 ms respectively and lasted 600-700 ms. By contrast the early R1 reflex response exhibited a slight long-lasting facilitation with a time course similar to that of the R2 inhibition. The threshold intensity to obtain facilitation of the R1 and inhibition of the R2 test responses after conditioning volley in the median and sural nerves was similar and ranged from 0.9 to 1.2 times the perception threshold. These results demonstrate that low-threshold cutaneous afferents from trigeminal and limb nerves exert powerful control on trigeminal reflex pathways, probably via a common neural substrate. There is evidence that, in addition to any post-synaptic mechanism which might be operating, presynaptic control is a primary factor contributing to these changes.


Botulinum toxin therapy: distant effects on neuromuscular transmission and autonomic nervous system. To evaluate distant effects of botulinum toxin, single fibre electromyography on the extensor digitorum communis muscle and six tests of cardiovascular reflexes were performed in five patients injected with BoTox (Oculinum(R) 20-130 units) for craniocervical dystonia and hemifacial spasm. Patients underwent two sessions of treatment and the second time the dosage was doubled. Botulinum toxin injection induced an increase of mean jitter value above normal limits in all cases. An increase of fibre density was recorded six weeks after the treatment. Cardiovascular reflexes showed mild abnormalities in four patients. The data confirm distant effects of botulinum toxin on neuromuscular transmission and on autonomic function.


The indusium griseum in Alzheimer's disease: an immunocytochemical study. The immunocytochemical features of the indusium griseum (IG) were compared with the corresponding hippocampus in 5 patients with Alzheimer's disease (AD) and 5 age-matched nondemented individuals using antibodies against beta-amyloid, the A68 protein (Alz-50 antibody), tau, ubiquitin and synapsin I. beta-Amyloid-positive plaques were prominent in the AD hippocampus but were not present in the IG. Numerous Alz-50, tau and ubiquitin-positive neurofibrillary tangles and dystrophic neurites were observed in the AD hippocampus but were infrequent in the IG. Synapsin I immunoreactivity was significantly reduced in both the AD hippocampus and the AD IG when compared to age-matched patients. These findings suggest that the IG may be resistant to factors that trigger production of abnormal AD-associated proteins. Loss of synaptic input alone may not account for the AD-associated changes in the hippocampus since synaptic depletion was seen in both the hippocampus and the unaffected AD IG.


Stereotactic brain biopsy in AIDS.

Stereotactic brain biopsy in AIDS. In the hope of finding a treatable condition, the need for rapid diagnosis in HIV-seropositive patients with brain lesions is apparent. In order to evaluate the efficacy of stereotactic brain biopsy in AIDS patients, we retrospectively studied 25 HIV-infected patients undergoing stereotactic biopsy. Brain lesions were identified with gadolinium-enhanced MRI and/or contrast CT. Brain biopsy was performed using the system of Riechert. From 8 up to 15 small tissue samples from one or two targets were obtained in every patient. The biopsy material was examined cytologically, histologically (including electron microscopy), immunohistochemically and, in part, by animal test and polymerase chain reaction (PCR). A definite diagnosis was achieved in 92%. Diagnosis included primary central nervous system lymphoma (PCNSL) (10), toxoplasmosis (10), progressive multifocal leukoencephalopathy (2) and one case of co-existing toxoplasmosis and cytomegalovirus infection. Two biopsies were non-diagnostic. All PCNSLs showed polymorphic B-cell populations of high malignancy; accurate classification according to the Kiel classification was not possible. In 3 lymphomas Epstein-Barr nuclear antigen (EBNA) 2-mRNA could be detected by PCR and confirmed immunohistochemically by EBNA 2 expression. In 6 cases autopsy confirmed the biopsy diagnosis. Conventional histology was not sufficiently decisive for toxoplasmosis and progressive multifocal leukoencephalopathy, so that immunohistochemistry and animal tests became very important for a final diagnosis. With the help of different morphological and molecular biological techniques stereotactic brain biopsy appears to be an effective method in the diagnosis of HIV-associated brain lesions. In view of the marked radio- and chemosensitivity of PCNSLs it is mandatory to establish an early and accurate histological diagnosis for adequate treatment.


Circulating antineuronal antibodies reach neurons in vivo: an autopsy study. In serum and cerebrospinal fluid of 6 out of 15 patients with small cell lung cancer circulating antineuronal antibodies could be detected by indirect immunofluorescence. None of the patients showed signs of a neurological paraneoplastic syndrome, usually associated with the presence of these antibodies. On sections of frontal brain, cerebellum, dorsal root ganglia and peripheral nerve, obtained at autopsy, a direct immunofluorescence test was performed. Only in antibody-positive individuals were membrane-bound immunoglobulin deposits detected on neurons of dorsal root ganglia and Purkinje cells. The present study showed that circulating antineuronal antibodies reach the central and peripheral nervous system in vivo. These findings support the theory that these antibodies might be directly involved in the pathogenesis of neurological paraneoplastic syndromes.


Conformational behaviour and molecular similarity of some beta 1-adrenergic ligands. The conformational behaviour of a series of aryloxypropanolamines was investigated by means of a new procedure which allows the sampling of the molecular torsional surface in a very efficient way. The combination of such a procedure with the standard molecular mechanics algorithms for the geometry optimization gives, as a result, the definition of a powerful computational scheme for the detailed analysis of the potential energy surface of complex molecules. The compounds studied show a remarkable tendency to form intra-molecular hydrogen bonds, which seem to play a key role in determining the lowest energy structures. The indices of molecular similarity proposed by Carbó, computed for the most stable conformers, do not account for differences between diastereoisomers, and, as a consequence, can hardly be used to attempt a structure-activity correlation.


The incidence of second primary tumors in long-term survivors of small-cell lung cancer. A review of 446 patients who were enrolled consecutively in small-cell lung cancer (SCLC) protocols was performed to identify in long-term survivors the frequency of new primary tumors and their clinical impact. Forty-seven patients (10.5%) were identified to be free of disease at 2 years. Second primary tumors (SPTs) were diagnosed in 14 patients. The overall risk for developing an SPT was 10.3% per person-year. Actuarial risk at 8 years was 50.3% for an SPT. In this review, SCLC showed one of the highest incidences of SPTs reported in aerodigestive tract malignancies. A long-term survivor was more likely to have an SPT than a relapse of SCLC. Consequently, the odds of death from an SPT compared with that from a relapse increased sharply from 1:13 within 4 years from diagnosis to 8:1 afterwards. Long-term survivors of SCLC would be excellent candidates for chemoprevention trials.


Second primary malignancies following diagnosis of small-cell lung cancer. The records of 800 patients with small-cell carcinoma of the lung (SCLC) treated between 1971 and 1985 at University of Toronto-affiliated hospitals were reviewed for the occurrence and relative risk of second primary malignancies (SPMs). Almost all patients who developed a SPM were treated previously with chemotherapy and radiation therapy. Nineteen metachronous SPMs (MSPMs) and 11 synchronous SPMs (SSPMs) were identified. SSPMs were detected between 1 and 12 months after the diagnosis of SCLC. The MSPMs were identified between 1 and 10 years after the diagnosis of SCLC. MSPMs included non-small-cell lung cancer (NSCLC) (four patients), hematologic malignancies (HM) (three patients), and 12 with other solid tumors (OST). The median survival times after the diagnosis of MSPM was 33 months, 10 months, and 1 month, respectively, for those with NSCLC, OST, and HM. Expected cancer incidence rates were used to compute a relative risk rate for developing a MSPM in a subset of 392 patients on whom accurate follow-up information was available. The calculated relative risk for all tumors was 3.73. The relative risk for the development of secondary NSCLC was 6.83. We suggest that increased predisposition to SPM may relate to secondary effects of multimodality treatment and biologic considerations.


Fasting plasma lipid measurements following cisplatin chemotherapy in patients with germ cell tumors. Elevated total serum cholesterol levels have been reported recently in a group of patients with metastatic testicular cancer after treatment with cisplatin combination chemotherapy. We have studied the lipid profile of a similar group of patients in an attempt to confirm this observation. Fasting plasma lipid concentrations were measured in 47 patients with advanced germ cell tumors who were previously treated with a cisplatin combination chemotherapy. The values obtained for mean total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, apolipoprotein A1, and apolipoprotein B concentrations were compared with those obtained from a control group of 59 patients with germ cell tumors who were not treated with chemotherapy and with data from the New Zealand male population. Median time from the completion of chemotherapy to lipid measurement in the treated group was 50 months (range, 2 to 138 months). The median total dose of cisplatin given was 720 mg (range, 300 to 1,625 mg). Mean total plasma cholesterol concentrations in the cisplatin group (5.87 mol/L) and the control group (5.70 mmol/L) did not differ significantly (P > .4). There was no significant difference for any of the variables between the chemotherapy and control groups and those of the New Zealand male population. There was a trend toward higher mean triglyceride concentrations in the chemotherapy group, but this did not reach significance. We have not demonstrated an elevation in total plasma cholesterol after cisplatin chemotherapy as has been reported by previous investigators. Our results suggest that in these patients, cisplatin-containing combination chemotherapy is not associated with a significant adverse effect on plasma lipid profile.


CPT-11 in combination with cisplatin for advanced non-small-cell lung cancer. The purpose of this study was to determine the maximum-tolerated dose and the dose-limiting toxicities of CPT-11, a new derivative of camptothecin, in combination with a fixed dose of cisplatin in patients with non-small-cell lung cancer (NSCLC). Twenty-seven previously untreated patients with stage IIIB or IV NSCLC were assessable for toxicity, and 26 were assessable for response. The initial dose of CPT-11 was 30 mg/m2 given as a 90-minute intravenous (IV) infusion on days 1, 8, and 15 in combination with cisplatin (80 mg/m2 IV on day 1) given every 4 weeks. The dose of CPT-11 was escalated in increments of 10 mg/m2 until severe or life-threatening toxic effects were observed. Significant toxicity was infrequent up to 60 mg/m2 of CPT-11. The maximum-tolerated toxicity was reached at a dose of 70 mg/m2. Three of six patients either had leukocyte count nadirs of less than 2,000/microL or experienced grade 4 diarrhea during the first cycle of therapy at 70 mg/m2. The major toxic effects were leukopenia and diarrhea. There were 14 partial responses (54%) among the 26 patients. A combination of CPT-11 and cisplatin seems to be effective against NSCLC with acceptable toxicities. The recommended dose for phase II studies is 60 mg/m2 of CPT-11 on days 1, 8, and 15, and 80 mg/m2 of cisplatin on day 1 every 4 weeks.


Metastatic spinal cord compression secondary to lung cancer. Metastatic spinal cord compression (MSCC) is a disabling complication to cancer, the optimal treatment for which is not settled. An analysis was performed for all patients with MSCC secondary to lung cancer in East Denmark from 1979 to 1988. The total series included 102 cases with small-cell carcinoma (SCLC; 40%), adenocarcinoma (ACL; 26%), squamous cell carcinoma (SQLC; 18%) and large-cell carcinoma (LCC; 9%). Symptoms, clinical presentations, and therapeutic results are described. The outcome of treatment depended fundamentally on the patient's neurologic condition at the time of the diagnosis. All patients with SCLC who were able to walk at the time of MSCC remained ambulatory, whereas 15% of the nonambulatory SCLC patients regained walking ability. In non-SCLC, 95% of patients continued to be able to walk, whereas 22% regained the ability to walk. No major differences in the immediate outcome of treatment between the various histologic types of lung cancer and the different treatment modalities were observed; however, 82% of the patients with non-SCLC benefited from treatment with laminectomy followed by radiotherapy (RT) compared with either laminectomy (47%) or RT (39%) alone (P = .03, chi 2 test). The group of patients who were treated with laminectomy followed by RT had a better survival (median value, 3.5; range, 0 to 132 months) than patients who were treated with either laminectomy (median value, 1.5; range, 0 to 32 months) or RT (median value, 1; range, 0 to 59 months) alone (P = .03, log-rank test). No significant difference was observed in survival between the various histologic types of lung cancer (P = .18, log-rank test). Despite a short survival, early diagnosis and immediate treatment is crucial because it may preserve the gait function in 97% of lung cancer patients who develop malignant spinal cord compression.


Comparative in vitro activity of sparfloxacin (AT 4140, RP 64206--SPFX) against 275 multiresistant clinical isolates. The in-vitro activity of sparfloxacin was compared with that of pefloxacin, ofloxacin, ciprofloxacin, imipenem, ceftazidime, gentamicin and amikacin against 275 multiresistant nosocomial clinical isolates. They consisted of Pseudomonas aeruginosa (37), Enterobacter cloacae (42), Acinetobacter anitratus (60), Klebsiella pneumoniae (37) and Staphylococcus sp (99). Minimum inhibitory concentrations (MIC90) and geometric mean MICs for sparfloxacin were as follows (mg/l): P. aeruginosa 128-23.7, E. cloacae 1-0.13, A. anitratus 2-0.14, K. pneumoniae 1-0.08, MRSA 16-0.98, MSSA 0.12-0.03, MRSE 0.25-0.12 and MSSE 0.12-0.05. It is concluded that sparfloxacin was the most potent agent against staphylococci and A. anitratus including strains resistant to the other quinolones while ciprofloxacin was the most potent agent against P. aeruginosa, E. cloacae and K. pneumoniae.


Effect of biological and synthetic polymers on BK virus infectivity and hemagglutination. The effect of several biological and synthetic polymers, chosen on the basis of different physical and chemical properties, was investigated on BK virus infectivity and hemagglutination. It was observed that polyanions like mucin, dextran sulfate and heparin depressed the viral binding, whereas polycations had no significant activity, with the exception of poly-L-lysine, which enhanced it. The effect of the active polymers was studied in different experimental conditions and the results obtained suggested that polyanions may act directly on the virus particle, whereas the target of polycations could be at the level of cell membranes. However, the effect shown by the active compounds did not appear to be simply related to the electric charge since neutral compounds, such as tamarind gum and locust bean gum, showed a marked inhibitory effect on BK virus binding to the cells.


Generation of superoxide anion and candidacidal activity by lipopolysaccharide-treated macrophages from patients affected by neoplasia. Macrophages, derived from in vitro cultured monocytes from both healthy donors and patients affected by metastatic breast cancer, treated or not with Escherichia coli lipopolysaccharide (LPS), were tested for phagocytosis and intracellular killing of Candida albicans and superoxide anion release. We found a marked impairment in intracellular killing closely linked to the lack of superoxide production in macrophages from patients affected by neoplasia treated or not with LPS. On the other hand, the LPS treatment significantly enhanced the phagocytic activity of all the macrophage populations tested, except for phagocytes obtained from patients affected by neoplasia and differentiated in autologous serum.


Epidemiological survey of genes encoding aminoglycoside phosphotransferases APH (3') I and APH (3') II using DNA probes. The epidemiological survey of APH (3') I and APH (3') II genes, at a time when the specific antibiotic pressure was very low, was carried out by DNA-DNA hybridization. The sample included 334 aminoglycoside resistant Gram-negative bacteria collected from patients of a General Hospital. Of these, 251 hybridized with the APH (3') I-probe and 19 with the APH (3') II-probe but only 190 strains showed high resistance levels (CIM greater than 64 micrograms/ml) for kanamycin, neomycin and paromomycin. These strains were isolated both from inpatients and outpatients with different infectious diseases. The APH (3') I-gene was dispersed among all the bacterial species and clinical specimens tested but the APH (3') II-gene was not found in Pseudomonas spp, Escherichia coli, Citrobacter freundii and Enterobacter cloacae, nor in infected catheters. Several plasmids of different sizes carrying APH (3') genes were detected among different bacteria. Plasmids along with transposable elements (the probes used in this work were developed from Tn906 and Tn5) and the high consumption of other antibiotics whose resistance is carried by these bacteria might be playing an important role in the maintenance and dispersion of APH (3') genes.


Presynaptic inhibition of excitatory synaptic transmission by adenosine in rat hippocampus: analysis of unitary EPSP variance measured by whole-cell recording. We have utilized the favorable signal-to-noise ratios provided by whole-cell recording, combined with variance analysis, to determine the pre- or postsynaptic actions of a variety of manipulations on unitary EPSPs evoked by low-intensity stimulation of afferents to CA1 pyramidal neurons in slices of hippocampus. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared average unitary EPSP amplitude (determined from 150-275 responses) to the variance of these responses (M2/sigma 2), while quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSP size (sigma 2/M). Estimates of mcv were highly correlated with those determined using the method of failures (mf). With paired stimulation (50 msec interpulse interval) there was a significant facilitation of the second unitary EPSP, accompanied by an increase in mcv, but not qcv, suggesting that this facilitation was of presynaptic origin. Superfusion of hippocampal slices with various concentrations of adenosine, the A1-selective adenosine receptor agonist cyclohexyladenosine, or the Ca2+ channel blocker cadmium significantly reduced average unitary EPSP amplitudes and mcv, without significantly altering qcv, suggesting a presynaptic locus for this inhibition. The 50% effective concentration for the apparent presynaptic action of adenosine on mcv in the present study (5.7 microM; 95% confidence limits = 4.2-7.7 microM) was significantly lower than its EC50 for reducing conventional, large EPSPs (33 microM; recorded with high-resistance microelectrodes), or extracellular field EPSPs (29 microM), as previously reported by this laboratory. The glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) reduced average unitary EPSP amplitudes; in contrast to the above manipulations, it had no effect on mcv, but significantly altered qcv, which is consistent with its presumed postsynaptic mechanism of action. We conclude from these data that adenosine presynaptically reduces synaptic strength at Schaffer collateral-commissural synapses in the hippocampus by diminishing the number of quanta released, not by reducing the size of these individual quanta or postsynaptic sensitivity to excitatory neurotransmitter. These results suggest that the mechanism by which adenosine inhibits synaptic transmission in the hippocampus is similar, if not identical, to the mechanism by which it inhibits synaptic transmission at the neuromuscular junction.


Contribution of polysynaptic pathways in the mediation and plasticity of Aplysia gill and siphon withdrawal reflex: evidence for differential modulation. The gill and siphon withdrawal (GSW) reflex of Aplysia is centrally mediated by a monosynaptic and a polysynaptic pathway between sensory and motor neurons. The first objective of this article was to evaluate quantitatively the relative importance of these two components in the mediation of the GSW reflex. We have used an artificial sea water (ASW) solution containing a high concentration of divalent cations to raise the action potential threshold of the interneurons without affecting the monosynaptic component of the reflex (2:1 ASW). Compound EPSPs induced in gill or siphon motor neurons by direct stimulation of the siphon nerve or by tactile stimulation of the siphon skin were reduced by more than 75% in 2:1 ASW. These results indicate that interneurons intercalated between sensory and motor neurons are responsible for a considerable proportion of the afferent input to the motor neurons of the reflex. The second objective of this article was to compare the modulation of the monosynaptic and polysynaptic pathways. We have evaluated their respective contribution in sensitization of the GSW reflex by testing the effects of two neuromodulators of the reflex, 5-HT and small cardioactive peptide B (SCPB). We found that these two neuromodulators have a differential action on the two components of the GSW neuronal network. The polysynaptic pathway was more facilitated than the monosynaptic pathway by the neuropeptide SCPB. By contrast, 5-HT displayed an opposite selectivity. These results suggest that the polysynaptic component of the neuronal network underlying the GSW reflex is very important for its mediation. The data also indicate that the monosynaptic and polysynaptic components of the reflex can be differentially modulated. The diversity of modulatory actions at various sites of the GSW network should be relevant for learning-associated modifications in the intact animal.


The downregulation of growth-associated proteins in motoneurons at the onset of synapse elimination is controlled by muscle activity and IGF1. Axonal growth during development and regeneration coincides with the expression of growth-associated proteins (GAPs), including GAP-43 and tubulin-alpha 1. Following contact with the target region and synaptogenesis, GAPs are downregulated. However, high levels of GAP-43 are expressed in some neurons in the adult CNS, indicating that its function may not be restricted to axonal growth. To define the type of signals that regulate GAP levels during development, we have determined whether GAP downregulation coincides with a defined phase of synapse development and whether it is controlled by the postsynaptic target. Levels of GAP-43 mRNA in spinal motoneurons and protein at the neuromuscular junction were analyzed in the developing neuromuscular system of chick and rat. In both species, GAP-43 mRNA declined rapidly at a time corresponding to the onset of synapse elimination, and nerve terminal GAP-43 immunoreactivity became undetectable with a delay of 2-3 d. In rat motoneurons, GAP-43 and tubulin-alpha 1 mRNA levels declined with a similar time course, indicating that at least two GAPs are downregulated at the same time. Blockade of neuromuscular transmission with either a pre- or a postsynaptically acting toxin prevented GAP downregulation, indicating that target activity affects GAP mRNA levels in motoneurons. Finally, counteracting the activity-dependent decline of muscle insulin-like growth factors during synapse elimination by local subcutaneous injections prevented motoneuron GAP downregulation, suggesting that these growth factors may be involved in the regulation of motoneuron GAPs by the muscle. These results demonstrate that motoneurons undergo target-sensitive changes in GAPs at the onset of synapse elimination. In addition, these results raise the possibility that termination of a presynaptic growth mode may be a prerequisite for synapse elimination.


Estradiol selectively regulates alpha 1B-noradrenergic receptors in the hypothalamus and preoptic area. We previously demonstrated that estradiol administered in vivo elevates the number of alpha 1-adrenoceptors in preoptic area (POA) and hypothalamic membranes from ovariectomized female rats and potentiates alpha 1 receptor augmentation of beta-adrenoceptor-stimulated cAMP formation in slices from these brain regions. Present studies examined (1) if estradiol selectively regulates any alpha 1-adrenoceptor subtype, and (2) which alpha 1 receptor subtype mediates the augmentation of cAMP synthesis. Hypothalamic and POA membranes from estradiol-treated rats, when compared to ovariectomized rats, had modestly (30-50%) but significantly elevated numbers of 3H-prazosin (alpha 1) binding sites. Estradiol affected neither the number of alpha 1 receptor sites in frontal cortex nor the affinity of 3H-prazosin binding in any brain region examined. Results of binding studies conducted in the presence of chlorethylclonidine, a selective, irreversible inactivator of the alpha 1B receptor subtype, indicated that the estrogen-dependent increase in total alpha 1 binding sites in POA and hypothalamic membranes was attributable to a selective, five- to sixfold increase in alpha 1B receptor number. Progesterone had no measurable effects on alpha 1 receptor binding. Blockade of alpha 1B receptors with chlorethylclonidine eliminated phenylephrine augmentation of isoproterenol-stimulated cAMP formation in slices, whereas the alpha 1A antagonist 5-methyl-urapadil did not. This suggests that the alpha 1B receptor subtype potentiates cAMP formation. Thus, the increased alpha 1 receptor augmentation of cAMP formation seen in slices from estradiol-treated rats is correlated with increased alpha 1B receptor number.


Noradrenergic inhibition of synaptic transmission between mitral and granule cells in mammalian olfactory bulb cultures. Noradrenergic modulation of the glutamatergic-GABAergic synapses between mitral/tufted (M/T) and granule cells has been implicated in some forms of olfactory learning (Brennan et al., 1990). Norepinephrine (NE) has been shown to disinhibit mitral cells (Jahr and Nicoll, 1982), but its site of action is not well defined. The effects of NE on synaptic transmission between monosynaptically coupled pairs of mitral and granule cells have been examined using primary culture and whole-cell recording techniques. Intracellular stimulation of M/T cells evoked dual-component EPSPs in granule cells consisting of both NMDA and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) receptor-mediated mechanisms. The EPSPs were reversibly inhibited by approximately 50% during application of 30 microM NE. NE had no effect, however, on the membrane current evoked by exogenous application of glutamate, indicating a presynaptic site of action. The effect of NE on EPSPs was mimicked by the alpha-adrenergic agonist clonidine but not by the beta-adrenergic agonist isoproterenol. NE had no significant effect on either accommodation or macroscopic currents in either M/T or granule cells. NE also inhibited spontaneous GABAergic IPSPs recorded in M/T cells, by a presynaptic alpha-adrenergic-mediated mechanism. These results support previous results suggesting a disinhibitory role for NE in the olfactory bulb. This action, however, is at least in part mediated by a reduction in mitral cell-mediated granule cell excitation.


Calbindin and parvalbumin cells in monkey VPL thalamic nucleus: distribution, laminar cortical projections, and relations to spinothalamic terminations. The ventral posterior lateral nucleus (VPL) of the monkey thalamus was investigated by histochemical staining for cytochrome oxidase (CO) activity and by immunocytochemical staining for the calcium-binding proteins parvalbumin and 28 kDa calbindin. Anterograde and retrograde tracing experiments were used to correlate patterns of differential distribution of CO activity and of parvalbumin and calbindin cells with the terminations of spinothalamic tract fibers and with the types of cells projecting differentially to superficial and deeper layers of primary somatosensory cortex (SI). VPL is composed of CO-rich and CO-weak compartments. Cells are generally smaller in the CO-weak compartment. Parvalbumin-immunoreactive cells and parvalbumin-immunoreactive medial lemniscal fiber terminations are confined to the CO-rich compartment. Calbindin-immunoreactive cells are found in both the CO-rich and CO-weak compartments. The CO-weak compartment, containing only calbindin cells, forms isolated zones throughout VPL and expands as a cap covering the posterior surface of the ventral posterior medial nucleus (VPM). Spinothalamic tract terminations tend to be concentrated in the CO-weak compartment, especially in the posterior cap. Other CO-weak, parvalbumin-negative, calbindin-positive nuclei, including the posterior, ventral posterior inferior, and anterior pulvinar and the small-celled matrix of VPM are also associated with concentrations of spinothalamic and caudal trigeminothalamic terminations. Parvalbumin cells are consistently larger than calbindin cells and are retrogradely labeled only after injections of tracers in middle and deep layers of SI. The smaller calbindin cells are the only cells retrogradely labeled after placement of retrograde tracers that primarily involve layer I of SI. The compartmental organization of VPL is similar to but less rigid than that previously reported in VPM. VPL and VPM relay cells projecting to different layers of SI cortex can be distinguished by differential immunoreactivity for the two calcium-binding proteins. The small-celled, CO-weak, calbindin-positive zones of VPL and VPM appear to form part of a wider system of smaller thalamic neurons unconstrained by traditional nuclear boundaries that are preferentially the targets of spinothalamic and caudal trigeminal inputs, and that may have preferential access to layer I of SI.


Detection and localization of parathyroid adenomas in patients with hyperparathyroidism using a single radionuclide imaging procedure with technetium-99m-sestamibi (double-phase study). Dual radionuclide imaging using a combination of 201Tl with either 99mTcO4- or 123I is recognized as a useful procedure in the preoperative localization of parathyroid adenomas. Recently, 99mTc-sestamibi (MIBI) has been introduced for myocardial perfusion imaging as an alternative to 201Tl. The purpose of this prospective study was to evaluate parathyroid scan using early and late imaging following MIBI injection. Twenty-three patients (21 F, 2 M, mean age: 57 yr) with a clinical and biologic diagnosis of hyperparathyroidism were submitted to a MIBI study prior to surgical exploration of the neck. Cervico-thoracic planar imaging (anterior view, 10 min/view) was performed at 15 min and at 2-3 hr after an intravenous injection of 20-25 mCi of MIBI. A positive MIBI scan for parathyroid adenoma was defined as an area of increased focal uptake which persisted on late imaging, contrary to the uptake in the normal thyroid tissue which progressively decreases over time (differential washout). Surgical exploration of the neck, performed between 1 day and 72 days (average: 16 days) after the MIBI study, showed a parathyroid adenoma in 21 patients and hyperplasia in two patients. MIBI scan correctly detected and localized 19/21 adenomas (90%). In conclusion, parathyroid imaging using a single radionuclide with MIBI (early and late study with differential washout analysis) is a promising procedure in the preoperative detection and localization of parathyroid adenomas in patients with primary hyperparathyroidism.


Scintigraphy of torsion in triorchidism. A 24-yr-old male who experienced fleeting episodes of left scrotal pain since the age of 13 had an ultrasound that suggested testicular duplication on the side (triorchidism). Five months later he had acute left scrotal pain lasting for 12 hr. At that time routine scrotal scintigraphy revealed late torsion and an additional sitting view demonstrated triorchidism. Literature review indicates that in this rare condition there is an increased risk of torsion. In only two prior reports did an ultrasound examination suggest triorchidism preoperatively.


Vascular endothelium, mechanical properties of the arterial wall and local angiotensin converting enzyme inhibition. An experimental model of in situ isolated carotid arteries was used to study the contribution made by angiotensin II (Ang II) towards the mechanical properties of the arterial wall in 12-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The effects of local incubation with saralasin (Sar1-Thr8-Ang II, 10(-6) mol/l) and with lisinopril (5 x 10(-6) mol/l) on carotid compliance were compared to the effects of removing the endothelium and the effects of totally abolishing vasomotor tone with potassium cyanide (0.1 mg/ml). With an intact endothelium, local incubation with lisinopril increased carotid compliance by 23% in WKY rats (P less than 0.05) and by 14% in SHR (P less than 0.01). Under the same experimental conditions, saralasin increased carotid compliance by 24% in WKY rats and 23% in SHR relative to control values (P less than 0.05 and P less than 0.001, respectively). Removal of the endothelium induced significant increases in carotid compliance in WKY rats (17%, P less than 0.01) and in SHR (33%, P less than 0.001). After removal of the endothelium, there was no further increase in carotid compliance with lisinopril in either strain. In contrast, saralasin induced further significant compliance increases in both strains (+18%, P less than 0.001, and +11%, P less than 0.01, in WKY and SHR, respectively). After the artery had been poisoned with potassium cyanide, there was no further increase in compliance relative to values obtained with saralasin in either strain with or without the endothelium. These findings suggest that Ang II receptors have a major effect on the control of basal vasomotor tone of large arteries in both normotensive and hypertensive rats. Furthermore, the increase in carotid compliance induced by local incubation with saralasin and with angiotensin converting enzyme (ACE) inhibitors may involve similar mechanisms acting on smooth muscle angiotensin receptors.


Non-invasive determination of arterial diameter and distensibility by echo-tracking techniques in hypertension. A new non-invasive ultrasonic device was developed to characterize the biomechanical properties of medium and large peripheral arteries. Simultaneous recordings of internal diameter and blood pressure over the whole cardiac cycle are used to establish compliance-pressure curves. Since blood pressure, which is an inherent co-determinant of arterial compliance, is taken into account, the comparison of arteries from patients with markedly different blood pressures has become possible. In a first study, the effects of three different antihypertensive drugs (20 mg lisinopril, 100 mg atenolol, 20 mg nitrendipine administered once a day) on arterial compliance and distensibility were investigated in young healthy volunteers. After 8 days of treatment, lisinopril induced a significant increase in arterial compliance. Subsequently, we compared the mechanical behaviour of arteries from newly diagnosed hypertensive patients (radial artery) or the carotid artery from spontaneously hypertensive rats (SHR) with that of corresponding arteries in normotensive counterparts. No decrease in arterial distensibility was found in the hypertensive groups over the measured blood pressure range. This result is not totally consistent with previous in vitro or in situ localized studies. Methodological differences, the absence of blood flow and/or denervation may partly explain these contradictory results. Finally, we tested the effects of hydralazine (5 mg/day) and captopril (25 mg/day), administered for 6 weeks in drinking water, on the behaviour of the carotid arteries of 16-week-old SHR. The two drugs effectively reduced blood pressure while shifting the distensibility-pressure curves upward in comparison to the placebo-treated animals, suggesting an improvement in arterial compliance. While hypertension does not itself appear to alter the elastic behaviour of large peripheral arteries, antihypertensive treatment may increase the compliance of these blood vessels.


Type III acromioclavicular joint separation associated with late brachial-plexus neurapraxia. We report the case of a 28-year-old woman who developed signs and symptoms of brachial-plexus neurapraxia eight years after a type III acromioclavicular (AC) joint separation. Stabilization of the AC joint resulted in resolution of the symptoms.


[Determination of trigeminal sensory evoked potentials in Bell's palsy]. Trigeminal sensory evoked potentials (TSEP) were determined in Bell's palsy cases of recent onset, and the following findings were observed: 1. TSEP was recorded with an exploring electrode at C5 or C6 on the scalp on the side opposite the Bell's palsy and with a reference electrode at Fz by stimulation with a bipolar stimulating electrode at the middle point of the inside of the upper lip. A tetra-phasic (N12, P20, N29 and P41) stable normal wave pattern was achieved. 2. TSEP was evaluated from the right-left difference for each case on the basis of a decrease (by less than 50%) in amplitude and an increase (by more than 10%) in the latency. 3. Ten of 20 patients with Bell's palsy showed abnormal TSEP. 4. Attempts at evaluation of the severity, course and prognosis of facial palsy by TSEP were unsuccessful. 5. All of the patients with an antibody titer for herpes simplex virus (HSV) of less than 4-fold showed normal TSEP. 6. The majority of patients with abnormal TSEP were considered to have been suffering from HSV infection. 7. Some patients with HSV infection showed normal TSEP. 8. Taking into consideration that patients with abnormal TSEP were infected with HSV, acyclovir was administered.


[A retrospective analysis of surgical operations on malignancies of the ear]. In the past 20 years from 1970 to 1990, 21 patients with malignancies of the ear were treated at Cancer Institute Hospital. The primary sites were the auricle and cartilaginous external auditory canal (group 1: n = 8), the bony external auditory canal (group 2: n = 6) and the middle ear (group 3:n = 7). In group 1, pathological types were squamous cell carcinoma in four patients and basal cell carcinoma in the other four patients. Seven patients were treated by surgery alone, and the remaining patient was treated by interstitial implant radiotherapy. No recurrence have been noted up to today in this group. In group 2, pathological types were squamous cell carcinoma in four patients and adenoid cystic carcinoma in the other two patients. All of the six patients underwent external canal resection or subtotal temporal resection. Two patients received postoperative radiotherapy. A five year survival rate of group 2 was 60%. In group 3, pathological types were squamous cell carcinoma in six patients and undifferentiated carcinoma in the remaining patient. Most of them underwent radical mastoidectomy followed by irradiation. However, all the patients died within two years after treatment. In group 2 and 3, cytodiagnosis was useful for early discovery. In group 2, temporomandibular joint seemed to be a key point in surgical treatment. In group 3, the combination of conservative surgery with radiotherapy was not a radical treatment. This suggested it necessary to perform subtotal temporal resection to treat malignancy of the middle ear.


Enzyme-linked immunoadsorbent assay for the detection of cytomegalovirus-IgM: comparison between eight commercial kits, immunofluorescence, and immunoblotting. Eight commercially available enzyme-linked immunoadsorbent assays (ELISA) for the detection of cytomegalovirus (CMV)-specific IgM were used in parallel to determine the presence of CMV-IgM in 123 serum samples from pregnant women. The results obtained with the eight kits were compared. Based on concordance of six or more of the eight kits, we assessed sensitivity, specificity, and overall agreement, as well as incidence of false-positive and -negative results for each kit. The results obtained by ELISA were then compared with those obtained by immunofluorescence (IF) and immunoblotting (IB). Our study did not single out one outstanding ELISA kit among the eight evaluated, nor did it suggest that IF or IB are better than ELISA. Furthermore our results indicate that IB might be useful in several cases as, beside its good sensitivity, most IB-false-positive sera are easily recognized as reacting exclusively with pp150, the unique reactivity to pp150 not being among the IB profiles of IB-true-positive sera. Nevertheless 14.6% of sera remained CMV-IgM-indeterminate.


Herpes simplex virus type 1 DNA is present in specific regions of brain from aged people with and without senile dementia of the Alzheimer type. We have investigated the possible involvement of viruses, specifically Herpes simplex virus type 1, in senile dementia of the Alzheimer type (SDAT). Using the highly sensitive polymerase chain reaction, we have detected the viral thymidine kinase gene in post-mortem brain from 14/21 cases of senile dementia of the Alzheimer type and 9/15 elderly normals. The temporal cortex and hippocampus were usually virus-positive; in contrast, the occipital cortex was virus-negative in 9/9 SDAT cases and 5/5 elderly normals. Temporal and frontal cortex from younger normals (five infants and five middle-aged) were negative. Thus, the presence of Herpes simplex virus type 1 DNA is a region-dependent feature of the aged brain.


Frequent expression of Epstein-Barr virus latent membrane protein-1 in tumour cells of Hodgkin's disease in HIV-positive patients. Epstein-Barr virus (EBV) is believed to be implicated in the aetiology of non-Hodgkin's lymphomas developing in immunodeficient individuals including AIDS patients. EBV has also been associated with Hodgkin's disease (HD), where the genomes have been demonstrated in the Hodgkin and Reed-Sternberg cells in some of the cases. Recent evidence has shown that EBV genomes are transcribed in these cells, because the EBV-encoded latent membrane protein-1 (LMP-1) can be demonstrated in the tumour cells in about half of the HD cases in HIV-negative patients using immunohistochemistry. LMP-1 is of special interest as a possible oncogenic agent because of its strong transforming capacity in vitro. In this study we have examined the expression of LMP-1 in HD of HIV-positive patients compared with HD in HIV-negative patients. We investigated 18 lymph nodes from 16 HIV-positive patients with HD (eight mixed cellularity, nine nodular sclerosis, one unclassified) using the CS.1-4 anti-LMP-1 monoclonal antibodies, which can usually be applied successfully to archival biopsy material. In each case, 50-90 per cent of the tumour cells were labelled. Staining was excellent for both fixatives used (4 per cent buffered formalin, Bouin's fluid). It is concluded that EBV-encoded LMP-1 is firmly associated with HD of HIV-positive patients. This is most conspicuous in the nodular sclerosing subtype HD in HIV-positive patients, in which 100 per cent were LMP-1 positive as compared with 32 per cent of nodular sclerosis HD in HIV-negative cases in a previously published series. This difference is statistically significant (P < 0.001). The possible biological and clinical significance of this difference should therefore be studied in larger series.


The extra-weak chemiluminescence generated during oxidation of some tetracycline antibiotics. 1. Autoxidation. Chemiluminescence (CL) appearing during autoxidation of tetracycline (TC) antibiotics has been studied. The CL spectrum consists of four emission bands with maxima at 520, 585, 640 and 700 nm. The bands at 585, 640 and 700 nm are similar to those observed for singlet molecular oxygen (1O2). The effect of 1O2 quenchers and free radical reaction inhibitors on the light emission is also reported. The data support the concept that, during the autoxidation of TCs, cytotoxic oxygen species such as HO., O2.-, H2O2 and 1O2 are formed.


Alpha-adrenergic bronchoprovocation in neonates with bronchopulmonary dysplasia. Ophthalmic administration of phenylephrine caused decreased pulmonary compliance, tidal volume, and peak airflow values in infants with bronchopulmonary dysplasia but not in control infants. The alpha-adrenergic effects of phenylephrine may aggravate the bronchospastic component of bronchopulmonary dysplasia.


Field trial of an infant formula containing anti-rotavirus and anti-Escherichia coli milk antibodies from hyperimmunized cows. Two groups of 124 and 108 children, respectively, living in urban Santiago, Chile in low socioeconomic conditions were prospectively followed for 6 months for their incidence of diarrhea. Each cohort was divided into two subgroups receiving either a commercial milk formula or the same formula containing 1% (wt/wt) bovine milk immunoglobulin concentrate from cows hyperimmunized with human rotaviruses and the major enteropathogenic Escherichia coli (EPEC) serogroups. Neither group differed with respect to incidence of diarrhea (98 episodes in 117 treated children versus 95 episodes in 115 control children), duration and clinical symptoms of diarrhea, and weight gain. Furthermore, neither group differed with respect to isolation of rotavirus (14 and 13 isolates in treatment and control groups, respectively) and isolation of enteropathogenic E. coli (14 and 15 isolates in treatment and control groups, respectively). The treatment but not the control formula contained neutralizing antibody against all human rotavirus serotypes. Titers were comparable to human breast milk samples. All isolated EPEC serogroups were included in the vaccine used for the immunization of the cows. The treatment, but not the control formula, protected mice against a lethal challenge with an EPEC strain. In conclusion, feeding an antibody-supplemented formula had no positive effect on diarrheal diseases under the conditions of a fairly well-controlled small-scale field trial.


Eicosanoids in hypoxic insult to neonatal rabbit bowel. Eicosanoids, derivatives of arachidonic acid, play a role in several inflammatory diseases of the bowel. To determine whether prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and leukotriene C4D4E4 (LTC4D4E4), have a role in hypoxic insult to the intestine, we examined the levels of these mediators in a hypoxic neonatal rabbit model. One group of animals underwent hypoxic insult postnatally, the second group did not undergo hypoxia and served as a control. The levels of PGE2, LTB4, and LTC4D4E4 were determined by radioimmunoassay. PGE2 in the hypoxic group was 1,779 +/- 142 pg/mg protein (mean +/- SD) as opposed to 2,380 +/- 197 pg/mg protein in the control group (p less than 0.02). LTB4 level was 5,446 +/- 3,492 pg/mg protein in the hypoxic rabbits and 3,362 +/- 2,570 pg/mg protein in the control group (p less than 0.03). There was no statistically significant difference in the level of LTC4D4E4 between the two groups. Our study shows that hypoxia shifts the arachidonic acid metabolism toward enhanced lipoxygenase activity with a resultant increase in LTB4 levels and a concomitant decrease in cyclooxygenase activity with reduced PGE2 levels in the bowel. The shift in the balance between these eicosanoids may play a role in the pathogenesis of ischemic-hypoxic bowel diseases by enhancing the inflammatory response in the intestine, and simultaneously, diminishing cytoprotection.


Unbalanced translocation of chromosome 3p in Wilms' tumor. Genetic studies in Wilms' tumor have most commonly shown a deletion involving band 13 on the p arm of chromosome 11 in association with aniridia. Structural rearrangements of chromosome 3p have been found in carcinoma of renal cell and lung origin but have not been previously reported in Wilms' tumors. We present two phenotypically normal, unrelated patients with Wilms' tumors, one of which was bilateral, in which cytogenetic analysis of the tumors showed an unbalanced translocation of the p arm of chromosome 3. Two biopsies were done in the patient with bilateral Wilms' tumor. The first biopsy specimen showed a translocation between chromosome 3 and 13 with partial trisomy of 3p and loss of material from 13q. The second biopsy three and a half months later again showed trisomy of chromosome 3p. The unilateral Wilms' tumor showed trisomy of 3p with partial loss of 7p. Neither patient showed a constitutional chromosomal abnormality and neither tumor showed any cytogenetic abnormality involving chromosome 11p. Quantitative DNA analysis was performed in the tumors of both patients. The bilateral Wilms' tumor was nearly diploid with a DNA index of 1.284 (mean ploidy, 2.45; SD, 0.854) while the unilateral Wilms' tumor was aneuploid with a DNA index of 1.531 (mean ploidy, 3.35; SD, 0.976). DNA analysis results are discussed in relationship to the chromosome abnormality seen on the karyotype analysis. These cytogenetic findings suggest that genetic oncogenesis in Wilms' tumor is heterogenous.