Mechanism of asymmetric block of K channels by tetraalkylammonium ions in mouse neuroblastoma cells. Experiments were performed to compare the mechanism of block of voltage-dependent K channels by various short and long alkyl chain tetraalkylammonium (TAA) ions at internal and external sites. Current through single channels was recorded from excised membrane patches of cultured neuroblastoma cells using the patch-clamp technique. All of the TAA derivatives tested blocked the open channel when applied to either side of the membrane. Tetraethylammonium (TEA) reduced the amplitude of current through the open channel. Tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) reduced the open time as a function of the concentration. An additional nonconducting state was observed when TBA or TPeA was applied internally or externally, due to the presence of a drug-bound and blocked state of the channel. The closing rate under control conditions was similar to that in the presence of external tetramethylammonium (TMA), suggesting that channel closing is independent of external drug binding. The concentration for half maximal block of the channel by external TEA was 80 microM. The channel was less sensitive to internal TEA, which half blocked the channel at 27 mM. The dissociation rate of long alkyl chain TAA ions from the channel was slower when applied to the inside, compared to external application, suggesting the presence of distinct internal and external receptors. Long alkyl chain TAA derivatives, such as TBA had a faster association rate with the open channel when applied to the inside of the membrane than when applied to the outside.


Opposite effects of angiotensin II and the protein kinase C activator OAG on cardiac Na+ channels. Elementary Na+ currents were recorded at 19 degrees C in cell-attached and inside-out patch-clamp experiments to study the influence of the vasoactive peptide angiotensin II (A II) and of the diacylglycerol analogue OAG (1-oleoyl-2-acetyl-sn-glycerol) on open probability and gating properties of single cardiac Na+ channels from cultured neonatal rat cardiocytes. Treating the cardiocytes with A II caused Na+ channel activation: reconstructed peak INa increased to 137 +/- 17.5% of control at 3 mumol/liters and to 176 +/- 42% at 30 mumol/liter. This NPo increase developed without major changes in open state and burst activity, even at 30 mumol/liter. OAG (6 mumol/liter) did not mimic this A II action. By contrast, OAG treatment of the cardiocytes had the opposite effect on NPo and diminished reconstructed peak INa to 67 +/- 4.9% of the control. The putative protein kinase C inhibitor staurosporine (0.2 mumol/liter) abolished this INa depression and led to a normalization of NPo. OAG had the same effect on isolated Na+ channels. Exposure of the cytoplasmic surface of inside-out patches to 1 mumol/liter OAG reversibly depressed, in the simultaneous presence of 50 mumol/liter Mg-ATP, the reconstructed peak INa to 40 +/- 9.7% of the control but left unit, tau open and burst activity unaffected. No NPo depression was obtained in the absence of Mg-ATP indicating that Mg-ATP may serve as phosphate donor. Obviously, after phosphorylation by protein kinase C, cardiac Na+ channels attain a reduced open probability but appear to preserve their kinetic properties.(ABSTRACT TRUNCATED AT 250 WORDS)


Assessment of posttraumatic polymorphonuclear leukocyte accumulation in rat brain using tissue myeloperoxidase assay and vinblastine treatment. Polymorphonuclear leukocytes (PMN) are implicated in the pathogenesis of traumatic brain injury. We tested the following hypotheses: (1) leukocyte accumulation is present in brain tissue 24 h posttrauma, (2) leukocyte accumulation represents PMN, and (3) prior systemic PMN depletion attenuates brain tissue PMN accumulation. Trauma was induced in exposed right parietal cortex by weightdrop in anesthetized Wistar rats (n = 24). Of the traumatized rats, 12 were PMN-depleted with vinblastine sulfate i.v. Controls were 12 normal rats and 5 sham-operated rats (craniotomy). Sections of traumatized and contralateral hemispheres were analyzed for myeloperoxidase (MPO) activity. Brain MPO activity was increased fivefold at 24 h posttrauma, but only in the traumatized hemisphere (0.448 +/- 0.133 U/g vs 0.090 +/- 0.022 U/g in trauma vs normal, respectively, p < 0.05, mean +/- SEM). PMN depletion attenuated this increase in MPO activity and decreased circulating PMN counts (0.07 +/- 0.032 x 10(9)/L vs 0.894 +/- 0.294 x 10(9)/L PMN-depleted-trauma vs trauma rats, respectively, p < 0.05). Leukocyte accumulation in the brain posttrauma was confirmed by MPO assay. Inhibition of MPO activity in the PMN-depleted group and the specificity of vinblastine treatment for depletion of circulating PMN suggest that leukocyte accumulation in the brain at 24 h posttrauma is largely due to PMN.


Enteroviral aseptic meningitis in Japan, 1981-1991. A report of the National Epidemiological Surveillance of Infectious Agents in Japan. In Japan, aseptic meningitis cases due to enterovirus infections increase every summer in various degrees with an incidence peak usually in July. During the past 11 years from 1981 through 1991, a total of 8,595 enterovirus isolations from aseptic meningitis cases were reported from 54 participating laboratories. Eight enterovirus types caused large epidemics; more than 100 isolations of each type from aseptic meningitis cases were reported for every epidemic year of the respective type. They were coxsackievirus (C) types B3 and B5, echovirus (E) types 4, 6, 7, 9, 18 and 30. Among these, the highest meningitis-associating frequency was reported for E30, representing 82.6% of the total isolations reported for the type during this period, followed by E4, 71.1%. The frequencies of E9, E7, E6 and CB5 were in a range from 54.5% to 44.4%, while that of E18 was 37.7% and that of CB3 21.0%. During the epidemics, enterovirus-associated meningitis was most frequently reported among children of 4-7 years of age. High frequencies were also shown in infants less than 1-year of age in some types. A total of 4,240 enteroviruses were isolated from cerebrospinal fluid of aseptic meningitis cases, representing 49.3% of the cases with enterovirus isolation.


Organization and expression of the gene encoding chick kainate binding protein, a member of the glutamate receptor family. The gene encoding chick cerebellar Bergmann glia-specific kainate binding protein (chKBP), has been isolated, characterized and expressed in heterologous systems. The structural gene spans 11.2 kb and contains 11 exons and 10 introns. Several of the exons encode specific receptor domains, including each of the predicted transmembrane regions. Exon/intron boundaries flanking the second, putative channel-forming transmembrane domain are conserved between chKBP and other glutamate/kainate receptor subunits. The putative promoter region 5' to the first exon displays high GC content and TATA, CAAT and AP1 consensus sequences. Transcription of the chKBP gene is evident prior to full cerebellar cortical maturation. Transcripts are abundant in cells consistent with Bergmann glia, as revealed by in situ hybridization. Transfection of 293 kidney cell cultures with chKBP cDNA or chKBP gene expression constructs confers CNQX-sensitive kainate binding with the pharmacological specificity displayed by both chKBP and kainate receptors. However, expression of the same constructs in Xenopus oocytes fails to yield detectable agonist-activated currents.


Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat. Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Postnatal ontogenesis of neurons containing GABAA alpha 1 subunit mRNA in the rat forebrain. The expression of GABAA receptor alpha 1 subunit mRNA in the postnatal rat forebrain was examined by in situ hybridization histochemistry. In most regions, including the isocortex, olfactory bulb, amygdala, septum, nucleus of the diagonal band, bed nucleus of the stria terminalis, basal ganglia, thalamus, and hypothalamus, the expression of alpha 1 subunit mRNA was low at birth but showed a dramatic increase during the early postnatal period. Adult levels of expression were reached at around the second or third week of life in these regions. However, in the caudate-putamen, and the nucleus accumbens, the expression of this subunit was only transient.


Sensitivity of Xenopus oocytes to changes in extracellular pH: possible relevance to proposed expression of atypical mammalian GABAB receptors. Electrical recordings were made to characterize the sensitivity of Xenopus oocytes to changes in extracellular pH, and to determine whether rat cerebral cortex poly(A)+ RNA (mRNA) expressed GABAB receptors with atypical electrical properties. All oocytes showed some sensitivity to changes in pH, and those from a small fraction (< 10%) of frogs were found to be highly responsive to acidification of bathing Ringer. In these oocytes, reduction in extracellular pH elicited membrane current responses with two components: (1) Smooth, maintained currents, primarily associated with a decrease in K+ conductance. (2) Oscillatory Cl- currents, elicited through activation of the phosphoinositide/Ca2+ messenger pathway. Oocytes with highest levels of sensitivity responded to decreases as low as 0.1 pH unit (Ringer pH 7.0 lowered to pH 6.9). Rat cortex mRNA consistently showed strong expression of membrane current responses mediated by GABAA, glutamate, kainate, serotonin and acetylcholine (muscarinic) receptors, together with responses mediated by a variety of neuropeptide receptors. In these oocytes, enantiomers of the GABAB receptor agonist baclofen, at concentrations ranging between 1 microM and 10 mM (pH 7.0), activated no significant membrane current responses. However, at concentrations > 0.1 mM hydrochloride salts of baclofen caused appreciable acidification of Ringer solutions; for example, 1 mM baclofen lowered pH from 7.0 to 6.0. Thus, when assaying oocytes with high sensitivity to pH, failure to make the necessary re-adjustment could result in apparent baclofen responses that, in reality, are simply due to pH effects alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Plasma membrane Ca(2+)-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization. Several mRNAs which encode for isoforms of the plasma membrane Ca(2+)-transport ATPase (PMCA) are present in adult rat brain. Using in situ hybridization with antisense oligonucleotide probes we found complex patterns of specific hybridization for three isoforms (PMCA1-3). Each rat brain region studied exhibited a distinct pattern of expression of isoforms. PMCA1 mRNA, which is widely distributed in rat tissues, was highest in CA1 pyramidal cells of hippocampus and very low in hypothalamic nuclei, cerebellum and choroid plexus. PMCA2 mRNA was highest in Purkinje cells of cerebellum and low in caudate-putamen, hypothalamic nuclei, habenula and choroid plexus. The highest levels of PMCA3 mRNA were found in habenula and choroid plexus. The PMCA1-3 isoforms appeared to be expressed primarily in neurons since hybridization was detected neither in white matter nor in regions rich in astrocytes. In different regions, different levels of expression of each PMCA mRNA may underlie specialized requirements for calcium homeostasis in specific neurons.


Characterization of bovine aromatic L-amino acid decarboxylase expressed in a mouse cell line: comparison with native enzyme. Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.


Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain. The effects of peripherally administered thyroid hormone (TH; 500 micrograms/kg; i.p.; q.d.) on the relative abundances of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) RNA were determined by rtPCR in the cortex and hippocampus of young adult rats. Corresponding changes in choline acetyltransferase (ChAT) activity were measured since NGF and BDNF have been shown to enhance the expression of this marker enzyme of central cholinergic pathways. Abundance levels of NGF and NT-3, relative to cyclophilin (cycl), were increased significantly (+50%, P < 0.05) in the hippocampus following TH treatment. Despite enhanced abundance of NGF in the hippocampus, ChAT activity was unchanged, whereas ChAT activity was modestly increased by 28% in the cortex without corresponding changes in NGF, NT-3 or BDNF. These results demonstrate that TH administration is capable of inducing the accumulation of NT-3, in addition to NGF but that the induction levels of RNA cannot be directly correlated with responsivity of the cholinergic system as measured by ChAT activity.


Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of beta 3 subunit mRNA and [3H]muscimol binding sites. The GABAA/benzodiazepine receptor consists of at least four subunits, alpha, beta, gamma and delta, each comprised of several variants. The developmental expression of the alpha 1, beta 1-3, gamma 2 and delta subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [3H]flunitrazepam and [3H]muscimol binding sites, alpha and beta subunit-specific ligands respectively, were also studied autoradiographically. The beta 3 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week 1 and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [3H]muscimol. Detectable levels of alpha 1 subunit mRNA hybridization signal and [3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Low to moderate levels of beta 1, beta 2, and gamma 2 subunit mRNAs were present in olivary neurons throughout postnatal development, while delta mRNAs were largely absent. It has been reported previously that, during the 2nd postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3:1, as observed in neonates, to the 1:1 relationship observed in the adult cerebellar cortex. Our results raise the possibility that the subunit composition of the GABAA/benzodiazepine receptor in inferior olivary neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.


Regulation of gene expression in astrocytes by excitatory amino acids. We have studied the effect of excitatory amino acids on the expression of mRNA for the immediate early genes c-fos, c-jun, jun-B, and NGF-1A in isolated cortical astrocytes. The expression of the different genes was induced by 100 microM kainate, quisqualate, AMPA and high concentrations of K+ (140 mM). NMDA did not induce the expression of any of the genes studied. The effect of quisqualate stimulation was not inhibited by the antagonist CNQX or by withdrawal of external Ca2+. In contrast the kainate effect was abolished by CNQX but not by the removal of external Ca2+. However, elevated K+ induced c-fos only when calcium was present in the external medium. These findings suggest that type-1 astrocytes lack NMDA receptors and that the induction of genes by quisqualate and kainate is in part independent of the presence of calcium in the external medium and may be mediated through second messenger pathways.


Nerve growth factor (NGF)-mediated up-regulation of low-affinity NGF receptor gene expression in cultured basal forebrain cholinergic neurons from postnatal 3-day-old rats. We examined the effect of nerve growth factor (NGF) on the expression of low-affinity NGF receptor (LNGFR) in cultured P3 basal forebrain cholinergic neurons, on which NGF acts to promote differentiation. Based on the results of the RT-PCR (reverse transcriptase-polymerase chain reaction) method, over a 2-fold increase in the LNGFR mRNA level was found in NGF-treated cultures compared with control cultures at one day after the addition of 100 ng/ml NGF. This increase was maintained for up to 3 days after the addition of NGF. The increase in LNGFR mRNA was found even at 1 ng/ml NGF (= 40 pM), indicating that the up-regulation of the LNGFR mRNA level in cultured cholinergic neurons occurred at an NGF concentration near the Kd value for the high-affinity NGF receptor. In addition, immunohistochemical staining showed stronger staining with a monoclonal anti-LNGFR antibody of the cholinergic neurons in NGF-treated cultures than those in control cultures. Our results suggest that NGF can up-regulate LNGFR expression at the mRNA and protein levels in cultured P3 basal forebrain cholinergic neurons and that this mechanism may play an important role in potentiating the effect of NGF on these neurons.


A complementary DNA for human choline acetyltransferase induces two forms of enzyme with different molecular weights in cultured cells. Complementary DNA (cDNA) clones containing the entire coding region of human choline acetyltransferase (ChAT) were isolated from cDNA libraries prepared from the autopsied spinal cord. In the human cDNA, the ATG codon assigned to the putative initiation codon for pig, rat and mouse ChAT cDNAs was replaced by ACG. The human cDNA contained an in-frame ATG codon 324 nucleotides upstream of the ACG codon. Therefore, human ChAT cDNA should code for a 748 amino acid polypeptide of 82.6 kDa. This deduced molecular weight was larger than that of ChAT protein purified from the human brain and placenta (64-70 kDa). The human ChAT cDNA containing the entire coding region was ligated to an expression vector and introduced into African green monkey kidney (COS) cells and Chinese hamster ovary (CHO) cells. The cells expressed high ChAT activity and produced two protein bands immunostained with an antibody to monkey ChAT. The molecular weight of the proteins was estimated to be approximately 70 and 80 kDa by polyacrylamide-SDS gel electrophoresis. When partial cDNAs that lacked the first ATG but contained the replaced ACG codon were introduced into COS cells, the cells expressed moderate ChAT activity and an immunoreactive protein band of 70 kDa. These results indicate that translation of human ChAT mRNA starts at two sites and produces two enzyme proteins with different molecular weights. It might be that the larger form of ChAT molecule is an enzyme precursor for processing or that the N-terminal extrapeptide is needed for subcellular localization of the enzyme.


Antisense proenkephalin cDNA transfection decreases opioid binding in NG 108-15 cells. The proenkephalin A (PENK) gene codes for several opioid peptides, including Met-enkephalin, an endogenous ligand of opioid receptors. These peptides are thought to play an important role in a variety of neural processes. To study the role of the PENK gene in opioid related processes, an antisense sequence of PENK gene was subcloned into pSVL SV 40 late promoter expression vector and stably transfected into NG 108-15 cells, which contain opioid receptors. The sense orientation of the same fragment was also cloned and transfected, serving as a control. The presence and expression of transfected recombinant plasmids in NG 108 cells were confirmed by DNA and RNA PCR and by subsequent sequencing. Surprisingly, the endogeneous PENK message level was found to be 3 times higher in antisense cells than in sense or NG 108 cells. This high steady-state mRNA level seemed to be due to the increased stability of PENK mRNA rather than to an elevated transcription rate. Nevertheless, the level of total Met-enkephalin was found to be reduced in antisense-transfected cells, though free Met-enkephalin content did not differ from sense-transfected or non-transfected cells. We suggest that both the increased PENK message and the unchanged levels of free Met-enkephalin may be the result of compensatory mechanisms induced by translational inhibition by antisense, although the underlying processes remain to be determined. Binding of the opioid ligand [3H]diprenorphine was significantly reduced by 50-80% in the antisense-transfected cell lines, but not in the sense cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Specific expressions of Fyn and Lyn, lymphocyte antigen receptor-associated tyrosine kinases, in the central nervous system. The Src-like protein-tyrosine kinases Fyn and Lyn are expressed in lymphocytes. Fyn is expressed in T cells at elevated levels and is associated with the T cell antigen receptor complex, whereas Lyn is expressed in B cells and is associated with membrane-bound immunoglobulin. Thus, these kinases are suggested to participate in antigen-mediated signal transduction in lymphocytes. Previous report showed that fyn was also expressed in brain, but its cellular distribution was not examined. Expression of Lyn in neural tissues was not previously reported. Here we report that both fyn and lyn are expressed in discrete regions of the brain. To throw light on their functions in the brain, we investigated their expressions during brain ontogenesis in mice. In situ hybridization analysis showed that Fyn mRNA was specifically expressed in neurons of embryos and newborn mice. In adult animals, fyn mRNA was expressed in oligodendrocytes as well as neurons. In contrast, the expression of lyn mRNA was relatively low in brains of embryos and newborn mice, but in adults the transcript was specifically expressed in the granular layer of the cerebellum. Therefore, the Fyn and Lyn kinases may regulate distinct functions of specific cells during brain development. The specific expressions of Fyn and Lyn in both lymphatic and neural tissues could suggest common signalling mechanisms in the immune system and central nervous system.


Authentic and artifactual detection of the E. coli lacZ gene product in the rat brain by histochemical methods. The accurate detection of a marker gene is fundamental to the assessment of any gene delivery protocol. The use of E. coli lacZ as such a marker gene has become common in studies on gene transfer to the central nervous system. The straightforward histochemical assay that is available to detect the gene product, beta-galactosidase; has made it an attractive system. However, using standard protocols, we have found dramatic non-E. coli lacZ staining in cells with neuronal, glial and endothelial morphology in the normal, adult rat brain. This false staining is primarily in the brainstem, but is evident in cortical and subcortical regions as well. This endogenous reactivity is independent of substrate concentration within the range tested, but is exquisitely sensitive to even small fluctuations in pH. In light of these findings, one must carefully examine any findings of E. coli lacZ gene expression in the rat brain based solely on histochemical analysis of tissue sections.


Comparison between epidermal growth factor, transforming growth factor-alpha and EGF receptor levels in regions of adult rat brain. Examination of adult rat brain regions by specific radioimmunoassays revealed a widespread distribution of transforming growth factor-alpha (TGF-alpha), but not epidermal growth factor (EGF), the peptide that had previously been reported to be present in rodent brain. Polyadenylated RNA samples from the different regions of rat brain were analyzed by Northern blot to identify mRNA species encoding precursor proteins for EGF (preproEGF), TGF-alpha (preproTGF-alpha), and the EGF/TGF-alpha receptor. The results indicate that TGF-alpha is the most abundant ligand for the EGF/TGF-alpha receptor in most parts of the brain analyzed. Message for preproEGF was only detectable after prolonged autoradiographic exposure; levels of preproEGF mRNA were between two and three orders of magnitude lower in brain than those expressed in control tissue (kidney), and one to two orders of magnitude lower than preproTGF-alpha mRNA levels in all brain regions. These results were confirmed by analysis of mRNA by RT/PCR, and support the hypothesis that expression of preproEGF mRNA in the brain is limited to smaller discrete areas, whereas preproTGF-alpha gene expression is almost ubiquitous.


Desensitization of junctional and extrajunctional nicotinic ACh receptors expressed in Xenopus oocytes. Desensitization of nicotinic acetylcholine receptors (AChRs) was studied using the Xenopus oocyte expression system. Mouse and cat extrajunctional AChRs expressed in oocytes desensitized more slowly than Torpedo AChRs. Substitution of the mouse gamma subunit into the Torpedo AChR reduced the rate of desensitization, making it similar to the mouse AChR. Likewise, substitution of the Torpedo gamma subunit into the mouse extrajunctional AChR increased the rate of desensitization, making it similar to the Torpedo AChR. Furthermore, mouse junctional AChRs in which the gamma subunit was replaced by the epsilon subunit desensitized more rapidly than either mouse or cat extrajunctional AChRs, and resembled Torpedo AChRs. Thus, in addition to other properties, junctional and extrajunctional AChRs differ with respect to desensitization, suggesting a possible role of desensitization in normal development of neuromuscular junction.


Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system. Previous in situ hybridization experiments reported that beta4 (beta 4) neuronal nicotinic acetylcholine receptor (nAChR) transcripts were found only in the medial habenula (MHB). Co-expression in Xenopus oocytes of the beta 4 subunit and any one of three ligand-binding or alpha subunits results in the formation of functional nAChRs. Comparisons between the pharmacology of nAChRs expressed in oocytes and the pharmacology of nAChRs found in the rat CNS prompted a further investigation of the localization of transcripts encoding the beta 4 nAChR subunit. Using two beta 4-specific cRNA probes, in situ hybridization was performed in rat brain. beta 4 mRNA was detected at high levels in the presubiculum, parasubiculum, subiculum and dentate gyrus of the hippocampal formation, in layer IV of the isocortex, in the medial habenula, in the interpeduncular nucleus, and in the trigeminal motor nerve nucleus. Moderate hybridization signals were seen in the isocortex (layers I-III), in olfactory regions, in fields CA1 through CA4 of Ammon's horn and the entorhinal cortex of the hippocampal formation, in the supramammillary nucleus, in the pontine nucleus, in the cerebellum, and in the locus coeruleus. No hybridization above background was detected in the septum, basal ganglia, sensory portions of the brainstem, or spinal cord.


Regulation of G-PROTEIN mRNA abundancy and cAMP accumulation after long-term opioid incubation in primary cultures of astroglia from the rat cerebral cortex. Primary astroglial cultures were incubated with delta (10(-6) M DPDPE) or kappa (10(-5) M U-50,488H) receptor agonists for 5 days. Thereafter, the acute inhibitory actions of delta or kappa receptor agonists on forskolin stimulated cAMP accumulation were assayed. The G alpha s, G alpha i-1 and G alpha i-2 mRNA levels were quantified after 5 days of either delta or kappa receptor agonist treatment using a solution hybridization, RNase protection assay. Pronounced effects were observed after 5 days of kappa receptor agonist [10(-5) M U-50,488H] incubation. This treatment resulted in an attenuation in the acute inhibitory action of delta and kappa receptor agonists. Furthermore, a decreased stimulatory action of forskolin was seen. Similar effects were also seen after delta receptor stimulation. We also investigated the effects after 24 h and 3 days of incubation with the kappa receptor agonist (10(-5) M) U-50,488H. The 24 h incubation resulted in a decreased sensitivity to the acute inhibitory action of delta and kappa receptor agonists in the astroglial cultures. This effect was further accentuated after the 3 days of incubation with 10(-5) M U-50,488H. No significant change was seen in the basal accumulation of cAMP after incubation with the kappa agonist U-50,488H. However, after 5 days of incubation with the delta agonist DPDPE, a significantly increased basal accumulation of cAMP was seen in the astroglial cultures. After 5 days of delta or kappa agonist incubation, an increase in G alpha s mRNA level and a decrease in G alpha i-2 mRNA level was seen compared with controls. No statistically significant alterations in the amount of G alpha i-1 mRNA were seen. The data obtained in the present study indicate that the effects of long-term opioid treatment alters the sensitivity of glial cell opioid receptors. Furthermore, long term opioid treatment induces alterations in glial G-protein mRNA levels.


Protein kinase C in the rat retina: immunocharacterization of calcium-independent delta, epsilon and zeta isoenzymes. Using isoenzyme-specific antibodies, subtypes of protein kinase C were determined in isolated retinae of dark adapted and light-exposed rats by SDS-PAGE-Western blotting. In addition to the previously observed alpha- and beta-subspecies, the rat retina also expressed the delta-, epsilon- and zeta-isoenzymes of protein kinase C. Exposure of the animals to physiological or high levels of light does not elicit changes in the pattern or in the distribution of the different isoenzymes in the cytosolic or particulate fractions. This study demonstrates, for the first time, the presence of three calcium-independent protein kinase C isoenzymes in the rat retina.


Anion channel in basolateral cortical membranes of the rabbit kidney. The presence of chloride conductance in basolateral membranes of proximal tubule is controversial. We measured 36Cl uptake in basolateral membranes loaded with KCl and suspended in a K(+)-free solution to create a positive intravesicular potential difference. Under these conditions, 36Cl uptake was maximal at 1 min, remained stable for at least 10 min and decreased to equilibrium levels by 60-120 min. Collapse of the voltage by valinomycin decreased 36Cl uptake by 46%, indicating the presence of K(+)-gradient-dependent chloride uptake. The chloride channel inhibitor diphenylamine-2-carboxylic acid inhibited 36Cl uptake in a dose-dependent fashion. 36Cl uptake was inhibited equally by unlabeled chloride, iodide and nitrate but not by sulfate or gluconate, indicating that the basolateral anion conductance is relatively selective. 36Cl uptake was pH independent but was calcium dependent. Phosphorylation of basolateral membranes with ATP significantly decreased 36Cl uptake, but the inhibitory effect of ATP was not further altered by exogenous cyclic AMP or the active phorbol ester PMA. These data demonstrate the presence of a relatively selective basolateral anion conductance which is regulated by pH, calcium and ATP.


Brain cancer and farming in western Canada. A cohort study of the mortality experience (1971-1987) of male Canadian prairie farmers has been conducted. This involved linking the records of 156,242 male Alberta, Saskatchewan and Manitoba farmers identified on the 1971 Census of Agriculture and the corresponding Census of Population to mortality records. Exposure indices for individual farm operators were derived from 1971 Census of Agriculture records. Cancer histologies for brain cancer cases were obtained from the Canadian National Cancer Incidence Database and from Provincial Cancer Registries. A statistically significant association was noted between risk of dying of glioblastomas and increasing fuel/oil expenditures (test for trend p = 0.03, top quartile relative risk = 2.11, 95% confidence interval = 0.89-5.01). No significant association was found between brain cancer and either education or mother tongue. However, low income was associated with a significantly reduced risk of brain cancer mortality.


Is multiple sclerosis caused by a dual infection with retrovirus and Epstein-Barr virus? Although the etiology of multiple sclerosis is as yet unknown, epidemiological observations strongly point toward one or more infectious agent(s) being involved in the disease. In recent years some studies have indicated involvement of retrovirus in multiple sclerosis (MS). However, an intrafamilial epidemiological study revealed that MS and the known human retroviruses had a divergent epidemiology. Some studies have shown the association of Epstein-Barr virus (EBV) with MS and one recent study revealed dual infection by retrovirus and EBV in a cell line established from a patient with an MS-like disease. Our hypothesis for the development of MS and MS-like diseases is that a hitherto uncharacterized retrovirus is the etiological agent, but development of neurologic disease is related to or even dependent on a delayed EBV infection. The dual infection hypothesis is analyzed and found to be consistent with the epidemiological characteristics of MS.


Urethral reconstruction with a new synthetic absorbable device. An experimental study. Four centimeters of the canine urethra was replaced by a graft consisting of a polyglactin fiber mesh tube, coated with polyhydroxybutyric acid. Microscopic examination 8-12 months later showed almost complete regeneration of the urethral epithelium and the adjacent connective tissue. The neo-urethra was patent and there were no anastomotic strictures, nor was there any inflammatory reaction around the urethra. The validity of this concept in humans remains to be proved.


Erythropoietin treatment and plasma levels of corticotropin-releasing hormone, delta sleep-inducing peptide and opioid peptides in hemodialysis patients. An improvement of quality of life and objective brain function has been reported in patients receiving regular hemodialysis treatment (RDT) during treatment with recombinant human erythropoietin (r-huEPO). The mechanisms explaining this improvement are unknown. In this study the plasma levels of peptides known to be involved in CNS functions, namely corticotropin-releasing hormone, delta sleep-inducing peptide, beta-endorphin, methionine-enkephalin, beta-lipotropin and alpha-melanocyte-stimulating hormone, were measured by radioimmunoassay in seven stable RDT patients before the start of r-huEPO therapy and during 28 weeks' treatment. All patients responded with significantly increased hemoglobin concentrations. An improvement of well-being, state of mood and physical fitness was reported by the patients. There were no significant changes during the study in the plasma concentrations of any of the peptides measured. However, as the plasma levels of neuropeptides will not necessarily reflect the local concentrations in the vicinity of the nerve terminals, changes in the intracerebral concentrations of these peptides might occur in response to r-huEPO.


[Gelatinous cyst of the common peroneal nerve]. The authors report two cases of gelatinous cyst of the common peroneal nerve. They present two different approaches to surgical treatment used in their cases--decompression of the nerve trunk by simple drainage of the cyst in one case and total excision of the cyst and its wall with reconstruction of fascicles damaged by the lesion in another. Experiences and results of treatment by other authors are presented as well as their views on pathology and etiology of this rare entity.


State dental boards and mandatory HIV testing. Public and professional debate over what, if any, action should be taken by state regulatory agencies regarding AIDS and the HIV status of dentists has increased in recent months. To determine if state boards had workable policies in place or were considering such policies, a telephone survey of all state boards was undertaken. All boards participated, although three provided only limited information. As of early 1992, only two boards required evidence of immunity to HVB for relicensure and none require evidence of seronegativity to HIV for relicensure. Twelve percent of the boards anticipate such requirements within two years. None have mandatory HIV testing, but 13 percent anticipate such requirements in the near future. The authors conclude that while few agencies have taken more than tentative steps toward rigorous HIV testing or restrictions, most are aware of much closer public scrutiny. More restrictions seem likely as these agencies attempt to balance public and professional rights and responsibilities.


[The adaptation of the human immune system during an ecological disaster]. This article studies the adaptative mechanisms of the immune system of servicemen in the Aral Sea region in response to the influence of unfavourable xenobiotic factors of the environment. The greatest homeostatic shifts were disclosed in servicemen of the 2nd and the 4th periods of service (6 months and 2 years of active duty correspondingly). Precisely these periods of selective service have the highest infectivity index. It is recommended to measure the volume of catabolic products of cell receptors (P-proteins) in order to obtain an integral index which could characterize the adaptative mechanisms of the immune system during the screening of servicemen in the conditions of unfavourable environment.


Effect of total-body cold exposure on plasma concentrations of von Willebrand factor, endothelin-1 and thrombomodulin in systemic lupus erythematosus patients with or without Raynaud's phenomenon. The effect of total-body cold exposure on plasma concentrations of von Willebrand factor (vWF), endothelin-1 (ET) and thrombomodulin (TM), all of which are considered to be generated from the endothelium, was studied in systemic lupus erythematosus (SLE) patients with and without Raynaud's phenomenon. The plasma levels of vWF, ET and TM in SLE patients, irrespective of the presence of Raynaud's phenomenon, were significantly higher than in normal controls even before the cold provocation test. After the cold provocation test, plasma levels of vWF and ET were significantly higher in SLE patients with Raynaud's phenomenon than in those without and in normal controls. No significant increase in TM was observed in either the SLE patients or the controls. These results suggest that SLE patients, regardless of the presence of Raynaud's phenomenon, are in a hypercoagulable state and that this state may be further intensified by cold exposure. Hence, it is concluded that we should consider antithrombotic therapy for SLE patients, especially those with Raynaud's phenomenon, to prevent unwanted activation of the coagulation system and possible endothelial damage.


[Isolation and study of biological properties of non-oncogenic Marek's disease herpesviruses in chickens. 2. Characterization in vivo]. In a previous paper (Jurajda and Halouzka, 1992) the in vitro isolation of two chicken herpesviruses of Marek's disease (M and K strains) was described and results of their characterization were presented. The present paper deals with the in vivo characterization of both isolates: pathogenicity and immunosuppressive characteristics of isolates were observed in a five-week test period, along with the development and production dynamics of antibodies and viral antigen in the feathers of experimentally infected chickens of the Brown Leghorn breed. A technique of double immunodiffusion in agar gel according to Ouchterlony, modified by Woernl (1966), was used to determine the presence of antibodies to Marek's disease virus (MDV) in blood serum and of precipitating MDV-antigen in feather quills of tested chickens. Isolate multiplication and titration were performed in a system of chicken embryonal fibroblasts (CEF) (Jurajda et al., 1984). Chickens were infected i.m. with three virus doses - 10(2) to 10(4) PFU per chicken while the dose 10(4) corresponded to the titre of 10,800 PFU/0.2 ml for M isolate and to the titre of 8,600 PFU/0.2 ml for K isolate. The nature and rate of regressive changes in lymphatic organs were determined according to criteria described by Halouzka and Jurajda (1991). The results are summarized in Tabs. I and II. Neither of the isolates evoked clinical or pathomorphological macroscopic symptoms of the disease. M isolate induced microscopic MD-specific changes in the peripheral nerves (of C type) and only moderate and transient signs of immunosuppression in 11% of infected chickens.(ABSTRACT TRUNCATED AT 250 WORDS)


[Isolation and study of biological properties of non-oncogenic Marek's disease herpesviruses in chickens. 1. Characterization in vitro]. The present paper deals with isolation of nonpathogenic chicken herpesviruses of Marek's disease (MD) and their in vitro characterization. Two strains of herpesviruses (denoted as VUB-M and -K isolates) were isolated from leucocytes separated from the blood of fattening chickens without clinical and pathomorphological symptoms of Marek's disease and from the blood of laying hens of a laboratory nonvaccinated flock without clinical symptoms, but with almost 100% incidence of precipitating MD antibodies. After in vitro adaptation, they were cytopathic for chicken embryonal fibroblasts in three days after infection. Isolates were identified as chicken herpesviruses of Marek's disease on the basis of the origin and nature of cytopathic effect in CEF (Biggs and Milne, 1972), lack of infectivity of culture medium and loss of infectivity of cells after their destruction through repeated cycles of freezing and thawing (Biggs and Payne, 1967), then serologically by agar gel immunodiffusion against specific serum (Chubb and Churchill, 1968) and electronoptically.


Effects of cyclosporine on tubular acidification function in patients with idiopathic uveitis. Renal tubular acidification function was studied in 12 patients treated with cyclosporine (Cy) for idiopathic uveitis (IU) and in 5 patients with IU not treated with Cy. After intravenous bicarbonate loading fractional bicarbonate excretion was similar in both groups indicating normal proximal tubular acidification function. Plasma renin activity, plasma aldosterone and transtubular potassium gradient were similar in both groups. Distal hydrogen ion secretion evaluated by the ability to increase urine-blood (U-B) pCO2 in a highly alkaline urine was impaired in Cy-treated patients (31.8 +/- 3.2 mm Hg) as compared to controls (47.9 +/- 0.5 mm Hg) (p < 0.005). We conclude that Cy therapy is associated with a distal acidification defect with a low U-B pCO2 gradient during sodium bicarbonate loading. Because none of our Cy-treated patients spontaneously developed over metabolic acidosis one could classify them as having an incomplete form of distal tubular acidosis.


Surgical experience with juvenile nasopharyngeal angiofibroma. A retrospective analysis was made of 24 cases of nasopharyngeal angiofibroma treated by the ENT Department of the Hospital de Clínicas de Porto Alegre between 1975 and 1989. All the patients were male with an average age of 16. All were treated by surgery alone, with an average peri-operative blood loss of 1,784 ml; pre-operative embolization made no significant difference to the blood loss. No other operative complications were encountered. Five patients (21%) had a recurrence, one of which was intracranial and required further surgery to effect a cure. The average length of follow-up was 19 months. We believe that surgical excision must be the treatment of choice for nasopharyngeal angiofibroma.


[The use of lithium electrophoresis combined with balneotherapy in hypertension depending on the type of central hemodynamics]. Central chest rheography was used to study the central hemodynamics in 62 patients with hypertensive disease (grades I-II) before and after treatment. 23 patients (hyperkinetic hemodynamics) were treated with oxygen baths and endonasal lithium chloride electrophoresis; 20 with en--and hypokinetic types of hemodynamics received CO2 baths and endonasal lithium chloride electrophoresis. It is concluded that lithium chloride electrophoresis in association with balneotherapy increases the therapeutic effect in hypertensive disease, especially in patients with the hyperkinetic type of blood circulation.


Serotonin-stimulated phosphoinositide hydrolysis in platelets from post-withdrawal alcoholics. It has previously been reported that serotonin-stimulated second messenger formation is inhibited in platelets from alcoholics undergoing detoxification. Serotonin-stimulated signal transduction was therefore analysed in platelets from young post-withdrawal alcoholics to elucidate whether the previously reported inhibition is a state or trait marker of alcoholism. No difference between post-withdrawal alcoholics and controls was found with regard to serotonin-stimulated [32P]-phosphatidylinositol 4,5-bisphosphate hydrolysis or [32P]-phosphatidic acid formation. The results indicate that the inhibited serotonin2 receptor function seen in alcoholics undergoing detoxification is a state-dependent rather than a trait-dependent marker.


DNA cytophotometric analysis of breast cancer. Follow-up for 10 years. Forty-four cases of newly diagnosed invasive ductal breast cancer were studied by static cytometry at the time of diagnosis and were followed for 10 years to determine if the survival rate correlated with the nuclear DNA measurements. In 22 cases the stem line was in the 2c range, in 15 cases in the 3c range and in 7 cases in the 4c range. Thirty-four percent (n = 15) of the patients died of metastases during the first five years, 11% (n = 5) died of metastases between 5 and 10 years, and 55% (n = 24) survived for > 10 years without metastases. No correlation between increased tumor cell proliferation and recurrence of tumors could be found. In this study all patients with diploid tumors, tumor size T1 and negative lymph node status survived for > 10 years. In the presence of node metastasis (T1N1M0) all patients (n = 4) having tumors with the stem line in the 2c range survived for 5 years but only 2/4 for > 10 years. The percentage of patients surviving for 10 years showed no significant differences between the groups: in the 2c range, 59% (13/22); in the 3c range, 53% (8/15); and in the 4c range, 43% (3/7). The small number of cases does not allow definitive statistical conclusions, although the logistic regression analyses showed that stem line, pT and pN were the important prognostic factors for five-year survival. However, only pT and especially pN were of prognostic value for 10-year survival.


Mathematical morphologic analysis of liver cirrhosis. Correlation with etiology, clinical score and hepatocellular carcinoma. Remodeling of the cirrhotic liver was studied retrospectively by mathematical morphologic methods in 75 autopsy cases (40 alcoholic, 17 hepatitis B virus (HBV)-related and 18 cryptogenetic cirrhosis), including 28 hepatocellular carcinomas. The aim was to obtain objective measurements of cirrhotic patterns that could be correlated with liver function evaluated by the Pugh-Child score, establish the relationship among different morphogenetic features and evaluate the implications of an objective classification of cases by numerical taxonomy in terms of their etiology, liver function and malignant transformation. The results indicate that the Pugh-Child score was closely related to the global amount of fibrosis or to the percentage of regenerative nodules < 0.8 mm in diameter. In contrast, the higher the percentage of lobular-sized regenerative nodules (0.8-1.6 mm), the better the functional score, suggesting that they are probably residual lobules, albeit completely surrounded by fibrous tissue, rather than true regenerative pseudolobules. The four groups of cases obtained by numerical taxonomy (cluster analysis) showed different distributions for alcoholic and HBV-related cirrhosis. The pattern of the latter was practically analogous to that in classically labeled cryptogenetic cirrhosis, suggesting its viral etiology. Taxonomic classification had functional implications. The Pugh-Child score showed a definite relationship with the different clusters obtained. The incidence of malignant transformation gradually decreased from group G1 to G4, with a steeper descent between G2 and G3. These results might contribute to a more dynamic concept of morphologic changes in liver biopsies from patients with cirrhosis.


Side-effects of dental ceramics.

Side-effects of dental ceramics. Evaluation of side-effects to low-dose exposure of any agent is difficult, especially if the agent exhibits a low toxicity. The most common way to approach such evaluation is to define special groups which are exposed more than others. Studies of such risk groups may facilitate interpretation of information related to those exposed to a low dose. For dental materials, dentists, dental assistants, and laboratory technicians represent typical risk groups. In addition to receiving dental treatments and having restorations like anyone else, they handle the materials in their daily work. The exposure to the materials occurs more frequently and at a higher dose for these groups than for the patient receiving dental treatment. Thus, the possibilities for side-effects are greater. Some materials are handled more closely by laboratory technicians than by other members of the dental team, e.g., dental ceramics.


Anti-HIV plant proteins catalyze topological changes of DNA into inactive forms. GAP 31, DAP 32 and DAP 30 comprise a new class of plant proteins with potent anti-HIV activity and insignificant cytotoxicity. We report here the identification and characterization of a new DNA enzyme activity in these three proteins. They irreversibly relax and decatenate supercoiled DNA, as well as catalyze double-stranded breakage to form linear DNA. The relaxed molecules are topologically inactive and no longer serve as substrates for DNA gyrase to form supercoils, phenomena similar to those of cellular topoisomerases in the presence of topoisomerase poisons. The ability of these anti-HIV agents to interrupt essential topological interconversions of DNA may provide a novel mechanism for their antiviral and antitumor actions. The presence of this new DNA topological enzyme activity in these plant proteins also suggests that their anti-HIV activity may not be merely a consequence of ribosome inactivation previously recognized.


Fast quantitative assay of sequence-specific endonuclease activity based on DNA sequencer technology. A quantitative assay of the sequence-specific endonuclease activity of Vsr DNA mismatch endonuclease is described. The procedure rests on fluorescently labelled oligonucleotide substrates and an automated DNA sequencer to determine amounts of both educt and product of the reaction; thus each individual measurement is internally standardized. The assay achieves high sample throughput by parallel measurement of multiple samples. Because of its capacity to produce and process large sets of experimental data, the system is particularly well suited for the determination of reaction kinetics. The procedure lends itself to further simplification by implementing software additions for direct peak integration. Obviously, the principle of the assay can be extended to the study of other enzymes, such as restriction endonucleases or sequence-specific proteases.


An optical biosensor for lysine based on the use of lysine decarboxylase and a cadaverine-sensitive membrane. We describe an optical biosensor for lysine based on the use of lysine decarboxylase and an optical transducer for detection of cadaverine which is formed as a result of enzymatic action. A plasticized PVC (polyvinyl chloride) membrane containing a lipophilic tartrate as the amine carrier acts as the optical cadaverine sensor. The transport of the cadaverine cation into the membrane is coupled to a transport of a proton (of the indicator dye) out of the membrane. This causes a spectral change of the indicator dye which can be related to the cadaverine concentration, provided the pH is kept constant. The enzymatic reaction is performed in an enzyme reactor which is part of a flow-through system. The dynamic range is from 0.1 to 100 mM for both cadaverine and lysine. While the cadaverine sensor is moderately selective (ethylamines, for example, interfere), the whole sensor system is highly specific for lysine, nicotine being the only major interferent. Unlike other enzyme-based detection schemes where the production of CO2 (in case of decarboxylases) or consumption of oxygen (in case of oxidases) is measured, this scheme is based on the measurement of the organic ammonium ion (cadaverin cation) formed in the enzymatic reaction. The major advantage of this approach is that in many real samples there is a rather low and fairly constant background of organic amines.(ABSTRACT TRUNCATED AT 250 WORDS)


Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode. An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.


Granulocyte infiltration in experimental colitis in the rat is interleukin-1 dependent and leukotriene independent. Intracolonic administration of trinitrobenzene sulfonic acid in ethanol results in the development of colitis in the rat. Previous studies have demonstrated that the severity of this colitis can be markedly reduced by repeated treatment with inhibitors of leukotriene synthesis, although such treatment does not appear to affect the initial migration of granulocytes into the colon. The present study evaluated the contribution of leukotrienes and interleukin-1 to the recruitment of granulocytes into the colon during the first 12 h after induction of colitis. Rats were treated with a leukotriene synthesis inhibitor (PF-5901), a leukotriene B4 receptor antagonist (SC-41930), an IL-1 receptor antagonist or a corticosteroid (prednisolone) prior to induction of colitis. Granulocyte infiltration was assessed by measurement of colonic myeloperoxidase activity and severity of colonic damage was blindly scored. Despite significant inhibition of leukotriene synthesis, PF-5901 did not affect colonic myeloperoxidase activity or the severity of colonic injury at any time point. Similarly, SC-41930 was without significant effect. However, both the interleukin-1 receptor antagonist and prednisolone significantly reduced colonic myeloperoxidase activity (by approximately 50%) and severity of colonic damage at 6 h after induction of colitis, without significantly affecting colonic leukotriene synthesis. These beneficial effects were no longer apparent at 12 h after induction of colitis. This study demonstrates that the infiltration of granulocytes into the colon during the acute phase of colitis in the rat occurs independent of leukotriene synthesis and appears to be at least in part attributable to interleukin-1.


Prolactin secretion in anterior pituitary cells: effect of eicosanoids. Ovariectomized Fischer 344 rats were implanted with silastic capsules containing estradiol for approximately 4 weeks to increase the number of lactotrophs in the anterior pituitary gland. Anterior pituitary cells were enzymatically dispersed and cultured for 1 day and challenged with eicosanoids or other prolactin (PRL) secretagogues. The isolated pituitary cells from estradiol-pretreated animals exhibited an increase in the ability to secrete PRL in the presence of maximally effective concentrations of thyrotropin releasing hormone (TRH), arachidonic acid or 5,6-epoxyeicosatrienoic acid (5,6-EET). Anterior pituitary cells from estradiol-pretreated animals also showed an increased activity of cytochrome P-450. The possible involvement of cytochrome P-450 in PRL secretion from anterior pituitary animal cells isolated from animals pretreated with estradiol was shown by the fact that these anterior pituitary cells increased the synthesis of 5,6-EET, a potent PRL releasing agent. TRH and 5,6-EET increased the mobilization of both cyclic AMP and cytoplasmic calcium to about the same extent. The data suggest that cytochrome P-450 is important in the release of PRL from anterior pituitary cells isolated from estradiol-pretreated Fischer 344 rats and that a product of cytochrome P-450-catalyzed epoxidation of arachidonic acid, 5,6-EET, plays a significant role in the release of PRL.


Adrenaline stimulates thromboxane and inhibits leukotriene synthesis in man. Catecholamines and other catecholic compounds have opposite effects on the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism in human polymorphonuclear leukocytes and whole blood in vitro. The hypothesis that high levels of adrenaline, found e.g. in myocardial infarction, are involved in the regulation of arachidonic acid metabolism was tested. Adrenaline (0.1 micrograms/kg per min for 45 min and thereafter 0.2 micrograms/kg per min for 15 min) was infused to healthy male volunteers to mimic relationships between high levels of adrenaline and arachidonic acid metabolism in myocardial infarction. Adrenaline infusion increased Ca ionophore A23187-induced TXB2 formation in whole blood. The effect was smaller when spontaneous clotting was used as a stimulus. Urinary 11-dehydro-TXB2 excretion, an indicator of total in vivo thromboxane synthesis, increased twofold. Adrenaline infusion decreased both LTB4 and LTE4 synthesis in A23187-stimulated whole blood. These results demonstrate that high levels of adrenaline influence the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism differentially in man.


Modulation of arachidonic acid metabolism and cyclic AMP content of human alveolar macrophages. The exposure of human alveolar macrophages to inflammatory mediators such as PAF (1 microM), fMLP (1 microM) or the calcium ionophore A23187 (1 microM) induced a rapid decrease in their intracellular concentration of cAMP. Inhibition of TXA2 synthesis by the specific thromboxane synthase inhibitor ridogrel (1-10 microM) or by non-specific inhibitors as indomethacin (1-10 microM), the cyclooxygenase inhibitor, or BW A4C (1-10 microM), the 5-lipoxygenase inhibitor, partially prevented the decrease in cAMP induced by the different inflammatory stimuli. Evidence for an indirect control of cAMP levels in human alveolar macrophages by inflammatory mediators through the production of arachidonic acid derivatives from the cyclooxygenase pathway is supported by this study.


Formation of lipoxins and leukotrienes by human alveolar macrophages incubated with 15(S)-HETE: a model for cellular cooperation between macrophages and airway epithelial cells. Human alveolar macrophages (AM) from bronchoalveolar lavage of asthmatic patients (AP) and healthy volunteers (HS) were compared for their respective capacities to produce lipoxins and leukotrienes when stimulated by calcium ionophore A23187 with or without 15(S)-HETE. The metabolites were analyzed using an isocratic RP-HPLC system and their formation profiles evaluated on the basis of chromatographic behaviour, UV spectral characteristics and co-elution with synthetic standards. Without 15-HETE, AM from AP produced more LTB4 and 5-HETE than those from HS. In the presence of 15-HETE, human AM were able to produce 5,15-diHETE and lipoxins. Moreover, the total amount of lipoxins synthesized by AM from AP was 2 fold higher than that synthesized by AM from HS, thus showing an enhanced cell activation via the 5-lipoxygenase (5-LO) pathway. These results presented AM as in vitro 15-HETE metabolizing cells and suggested some hypothesis about human AM 5-LO regulation mechanism. The enhanced 5-LO activity in AM from AP suggested that in vivo they could participate in cell to cell interaction mechanisms involved in inflammatory lung diseases and might also take up and transform 15-HETE predominantly released by airway epithelial cells.


[Effect of cefepime on adhesion and phagocytosis of Pseudomonas aeruginosa]. We studied the effect of subinhibitory concentrations of cefepime on cell surface hydrophobicity expression and adherence to epithelial cells of Pseudomonas aeruginosa. We also evaluated the effect of this drug on the interaction between Pseudomonas aeruginosa and human polymorphonuclear leukocytes. The bacterial cell surface hydrophobicity was tested on a biphasic system (water/p-xylene). The bacterial adherence to epithelial cells as well as the phagocytosis of Pseudomonas aeruginosa was studied using radiometric techniques. The superoxide production was evaluated by means of cytochrome C reduction method. Pre-incubation of a non mucoid Pseudomonas aeruginosa strain with a 0.25 MIC value of cefepime produces a reduction in the adherence of this microorganism to epithelial cells, but without modifying the surface hydrophobicity. Pre-incubation of a mucoid and a non mucoid P. aeruginosa strain with a 0.25 MIC value of cefepime reduces its phagocytosis by polymorphonuclear leukocytes. The preincubation polymorphonuclear leukocytes with cefepime (1, 10, and 100 mg/ml) did not influence the phagocytosis of Pseudomonas aeruginosa nor the bactericidal mechanisms of PMN (superoxide production). At therapeutic concentrations, cefepime showed intracellular activity against P. aeruginosa in human polymorphonuclear leukocytes. The incubation of Pseudomonas aeruginosa with sub-MIC inhibit its adherence to epithelial cells and its intake by human polymorphonuclear leukocytes. At therapeutic concentrations, cefepime did not affect the bactericidal mechanisms of polymorphonuclear leukocytes, and shows a significant intracellular activity against P. aeruginosa.


Effects of the superoxide radical scavenger superoxide dismutase, and of the hydroxyl radical scavenger mannitol, on reperfusion injury in isolated rabbit hearts. Hydroxyl radical formation, secondary to superoxide radical generation, has been advocated as the actual mechanism of oxygen radical-mediated damage in biological systems. The present study was designed to compare the efficacy of administration of the hydroxyl radical scavenger mannitol vs. that of the superoxide radical scavenger superoxide dismutase (SOD) in reducing myocardial reperfusion injury, and to test whether combined treatment with both agents would confer better tissue protection compared with either intervention alone. Rabbit hearts perfused within a 31P nuclear magnetic resonance (31P-NMR) spectrometer were subjected to 30 minutes of total global ischemia at 37 degrees C. At reflow, 12 hearts in each group received either (a) a bolus of standard perfusion buffer, followed by 45 minutes of reperfusion (controls); (b) the superoxide radical scavenger recombinant human SOD (h-SOD, as a 60,000 U bolus followed by a 100 U/ml infusion for 15 minutes); (c) the hydroxyl radical scavenger mannitol (50 mM bolus followed by 15 minutes of 50 mM infusion; or (d) a combination of both agents. All treated hearts were switched to standard buffer for the remaining 30 minutes of reperfusion. Treatment with h-SOD alone was associated with a significant improvement in the recovery of cardiac contractility and coronary flow, as well as of ATP content, compared to control hearts. In contrast, mannitol treatment resulted in a small, nonsignificant improvement in these parameters. The addition of mannitol to h-SOD did not result in further significant improvement of contractility and ATP recovery compared to h-SOD alone. These data demonstrate that under our experimental conditions significant protection against reperfusion injury can be achieved by the administration of h-SOD alone, without the need for additional hydroxyl radical scavenger therapy with mannitol. These results do not exclude that significant tissue protection may be achieved by different doses of mannitol or by other agents. However, they suggest that under definite experimental conditions prevention of hydroxyl radical formation, rather than attempts to minimize hydroxyl radical toxicity, might be a more efficient method to prevent oxygen radical-mediated reperfusion injury in isolated hearts.


Protein IIIa of Rhizobium leguminosarum is probably a porin. The cloning, sequencing and expression of the gene encoding the 36-kilodalton (kDa) outer membrane protein of Rhizobium leguminosarum has been recently described in the literature (De Maagd RA et al (1992) J Bacteriol 174, 214-221). We present evidence that this protein is a porin from a sub-type covalently bound to the peptidoglycan.


Inhibition of plant and animal cytochrome oxidases by nitrous oxide as a function of cytochrome c concentration. Cytochrome oxidase from both pea leaves and bovine heart shows lower activity under a mixture of 79% N2O/21% O2 than under ambient air. This inhibition is not detectable below 5 microM cytochrome c but appears with increasing concentrations of cytochrome c. These results suggest that the N2O-induced inhibition of cytochrome c oxidase is modulated by cytochrome c concentration. This seems to concern only the lowest affinity site of the oxidase. Apparently, N2O and cytochrome c do not share the same site of fixation on the oxidase.


Evidence that MAP (mitogen-activated protein) kinase activation may be a necessary but not sufficient signal for a restricted subset of responses in IL-1-treated epidermoid cells. We have investigated the activation of mitogen-activated protein kinase (MAP-kinase) in KB human epidermoid carcinoma cells treated with interleukin 1 (IL-1). MAP-kinase activity was transient; the time required for activity to reach a maximal level was dependent upon the dose of IL-1, ranging from 15 minutes to 45 minutes. The level of kinase induction correlated well with dose-response curves for two characteristic IL-1-induced responses, PGE2 and IL-6 production. MAP-kinase activity returned to basal levels within 2 hours regardless of the amount of IL-1 added to the system. Exposure of KB cells to free IL-1 was accordingly restricted to periods of 2 hours or less, by replacing IL-1 with an excess of IL-1 receptor antagonist. Even after 2 hours exposure, the ability of IL-1 to induce IL-6 or PGE2 was still IL-1ra-inhibitable by more than 80%, suggesting that events downstream of, or parallel to MAP-kinase activation, requiring the continual formation of new IL-1 receptor complexes, are needed to fully elicit these responses. Two general serine/threonine kinase inhibitors, K252a and quercetin, were found to strongly inhibit MAP kinase in vivo with ED50s of c. 100 nM and 30 microM, respectively. At these concentrations, both compounds effectively inhibited IL-1-driven PGE2 and IL-6 induction without affecting general protein synthesis or secretion. Other non-selective kinase inhibitors had less effect on MAP-kinase activation or IL-1-induced biological responses. The transient activation of MAP-kinase induction correlated strikingly with activation of the transcription factor NF-kappa B. IL-1-induced NF-kappa B activation was, however, relatively insensitive to inhibition by K252a or quercetin. We suggest that MAP-kinase is likely to be a necessary, but not sufficient, intermediate in some (IL-6, PGE2 induction) but not all (NF-kappa B activation) IL-1 responses in these cells.


IL-1 synergy with IL-2 in the generation of lymphokine activated killer cells is mediated by TNF-alpha and beta (lymphotoxin). Interleukin 1 is a pleuripotent cytokine shown to synergize with IL-2 in the generation of lymphokine-activated killer (LAK) cells, when cultured with human peripheral blood mononuclear cells (PBMC) or peripheral blood lymphocytes (PBL). When IL-1 and low dose IL-2 are added in combination, both LAK cytotoxicity and proliferation are increased in short-term (5-6 day) and long-term (12-14 day) cultures compared with cells activated with IL-2 alone. The purpose of this study was to examine the contribution of tumor necrosis factor (TNF-alpha), lymphotoxin (LT, or TNF-beta) and the TNF receptor in the observed IL-1/IL-2 mediated synergy. Analysis of lymphocyte culture supernatants using the L929 bioassay and by specific ELISAs demonstrated an increased production of both TNF and LT in those cells cultured with IL-1 and IL-2. Utilizing specific neutralizing antisera, our experiments demonstrated the biologic activity of both cytokines, with LT-specific antibodies producing the greatest diminution of IL-1/IL-2 stimulated cell proliferation and cytotoxicity. The addition of IL-1 and IL-2 in combination markedly upregulated TNF-receptor expression (measured by Scatchard analysis) in comparison with cells stimulated with IL-2 alone. Characterization of the TNF-R by flow cytometric analysis revealed increased membrane expression of the 75 kDa, but not the 55 kDa, TNF binding protein as a result of IL-1 costimulation.


Regulation of IL-6 receptor and gp130 expression on human cell lines of lymphoid and myeloid origin. The expression of 80 kDa interleukin-6 receptor (IL-6R) and the associated molecule gp130 has been studied on human cell lines by FACS- and Northern blot analysis. The effects of dexamethasone, dibutyric-(DB)-cAMP and phorbol-12-myristate-13-acetate (TPA) have been studied on plasmacytoma cell line U266, B cell line BMNH and monocytoid cell line U937. Our data show a definite downregulation of IL-6R and gp130 expression by TPA in U266 and BMNH at both mRNA and cell surface protein levels. In U937 TPA inhibits only the IL-6R expression, without affecting that of gp130. DB-cAMP decreases the expression of both proteins in U937, slightly inhibits the IL-6R expression in U266, but is uneffective in BMNH. Dexamethasone induces considerable upregulation of gp130 only in U266. Our findings suggest separate regulation of IL-6R and gp130 on U266, BMNH and U937 cell lines.


1,25-Dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits lymphocyte proliferation and production of antibodies and lymphokines such as interleukin (IL)-2 and interferon gamma. These lymphocyte functions are dependent upon cytokines, including IL-1 alpha, IL-1 beta, IL-6 and tumour necrosis factor alpha (TNF-alpha), produced by the antigen presenting cells. In the present study we examined the effect of 1,25-(OH)2D3 on the production of these cytokines, as well as superoxide generation by freshly isolated mononuclear cells and partially purified monocytes. The immediate precursor of 1,25(OH)2D3, 25-OH D3, and the synthetic analogue MC 903 ('Calcipotriol') were examined in parallel. 1,25-(OH)2D3 dose-dependently inhibited the production of IL-alpha, IL-6 and TNF-alpha by Escherichia coli lipopolysaccharide (LPS)-stimulated monocytes, without affecting superoxide production. MC 903 had comparable effects while 25-OH D3 was ineffective. The inhibition caused by 1,25-(OH)2D3 was not abolished by supraoptimal concentrations of LPS or indomethacin. 1,25-(OH)2D3 had similar effects on secreted and cell-associated IL-alpha. Nuclear run-off analysis indicated that inhibition of these cytokines was not caused by impaired production of mRNA. Taken together, the study demonstrates a vitamin D-induced inhibitory effect of LPS-driven monokine production, which is most likely a vitamin D-receptor mediated phenomenon exerted at a post-transcriptional, presecretory level. Impaired monokine production may be of importance in 1,25-(OH)2D3-mediated inhibition of lymphocyte functions in vitro.


Changes in IL-6, IL-8, C-reactive protein and pancreatic secretory trypsin inhibitor after transcatheter arterial chemo-embolization therapy for hepato-cellular carcinoma. In an attempt to investigate the interaction between the changes of cytokines and acute phase reactants after transcatheter arterial chemoembolization therapy (TACE), the levels of interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein (CRP) and pancreatic secretory trypsin inhibitor (PSTI) in the blood of patients with unresectable hepatocellular carcinoma (HCC) were measured. Before the therapy, serum IL-6 and plasma IL-8 levels were detectable in 77.8% and 28.5%, respectively, of patients with HCC. Levels of serum IL-6 and plasma IL-8 increased after TACE and reached a peak on day 3 in all patients (18/18) and in 87.5% of patients (12/14), respectively. Both blood levels of IL-6 and IL-8 reached a peak earlier than those of CRP and PSTI did after the therapy. When the maximal values of IL-6 were compared with those of CRP and PSTI, there were significant positive correlations (r = 0.63, P < 0.01 and r = 0.81, P < 0.01, respectively). Similarly, comparisons of the maximal values of IL-8 with those of CRP and PSTI gave a significant correlation (r = 0.68, P < 0.01 and r = 0.67, P < 0.05, respectively). However, no significant correlation was found between the elevation of IL-6 and IL-8.


The nature of the last universal ancestor and the root of the tree of life, still open questions. The nature of the last universal ancestor to all extent cellular organisms and the rooting of the universal tree of life are fundamental questions which can now be addressed by molecular evolutionists. Several scenarios have been proposed during the last years, based on the phylogenies of ribosomal RNA and of duplicated proteins, which suggest that the last universal ancestor was either an RNA progenote or an hyperthermophilic prokaryote. We discuss these hypotheses in the light of new data on the evolution of DNA metabolizing enzymes and of contradictions between different protein phylogenies. We conclude that the last universal ancestor was a member of the DNA world already containing several DNA polymerases and DNA topoisomerases. Furthermore, we criticize current data which suggest that the rooting of the universal tree of life is located in the eubacterial branch and we conclude that both rooting the universal tree and the nature of the last universal ancestor are still open questions.


The evolution of RNA editing in kinetoplastid protozoa. RNA editing alters messenger RNA transcripts by the addition and deletion of uridine residues. This remarkable process corrects frameshift mutations and replaces AUG initiation codons in most trypanosomatid mitochondrial RNA transcripts. Sometimes, however, RNA editing results in open reading frames whose sequences differ by greater than 50% from the original RNA sequences. In this paper, we will explore two models for the evolution of RNA editing in kinetoplastid protoza.


[The characteristics of the opioid regulation of the afferent reactions of the stomach and duodenum to the early stages of their acute damage]. Acute cat experiments were conducted to study the effect of naloxone, moradol and dalargin on the formation of afferent reactions of gastroduodenal complex components by registration of cortical and subcortical evoked potentials during electrostimulation of the stomach and duodenum at an early stage of their acute lesion. It is found that agonists and antagonists of opiate receptors prevent the depression of afferentation of gastroduodenal complex components early after acute lesion. It is suggested that the main mechanism underlying the depression of gastric and duodenal afferent reactions at early post-alteration period may be related to activation of intraorganic opioid systems.


[Fibrinolytic and anticoagulant complexes of low-molecular heparin with tuftsin]. The complex of immunopeptide taftsin with low-molecular heparin has been obtained. The complex has fibrinolytic and anticoagulant activities in vitro and in vivo after the injection to albino rats.


Treatment of tuberculosis in HIV infection. Whether tuberculosis patients received short-course chemotherapy with treatment of isoniazid (INH) and rifampicin (RIF), combined or not with pyrazinamide (PZA) and ethambutol (EMB) or streptomycin (SM), or long term chemotherapy with INH, SM and thiacetazone (Tb1), the rate of sputum culture conversion was similar in HIV-positive and HIV-negative patients. To prevent relapses it was recommended to treat patients for a minimum of 9 months and for at least 6 months after culture conversion, or even to administer INH for life after the end of treatment. However, no difference was observed in the percentage of relapses between HIV-positive and HIV-negative patients. Side-effects were observed in approximately 20% of HIV-positive patients treated with INH + RIF + PZA + EMB (or SM) or with INH + SM + Tb1, Tb1 being responsible for epidermal necrolysis, in some cases fatal. The mean survival of HIV-patients with tuberculosis was from 10 to 18 months after the diagnosis of tuberculosis. Other opportunistic infections could have been the main cause of death. Acquired drug resistance is not a common complication of tuberculosis treatment in HIV-positive patients, but several epidemics of nosocomial transmission of multiple drug-resistant tuberculosis have recently been observed in the USA. Sparfloxacin, a new fluoroquinolone with a long half-life and low MIC (0.25-0.50 mg/l) against Mycobacterium tuberculosis, is a promising drug against tuberculosis.


Expression of neuroendocrine and epithelial markers in an adherent subline derived from a classic small cell lung cancer cell line. Small cell lung cancer (SCLC) cell lines usually grow as floating aggregates, in contrast to the adherent monolayers formed by non small cell lung cancer (NSCLC). Induction of an adherent phenotype by a variety of methods has been the subject of a number of recent publications. In this study, cultivation of the classic SCLC cell line, NCI-H69, on a substratum provided by the pretreatment of tissue culture dishes with medium conditioned by the growth of a well differentiated squamous carcinoma cell line, HN5, induced an adherent phenotype with a variety of epithelioid morphologies, commencing within 24 hr of plating. From such cultures an adherent subline, H69A, has been established, which differs in its growth, morphological characteristics, and immunocytochemical marker expression from the parent NCI-H69 cells, and in its marker expression from other adherent SCLC cell lines. H69A retained expression of neural cell adhesion molecule (NCAM) and the neuroendocrine markers neuron specific enoclase, chromogranin A, and synaptophysin, but showed diminished expression of the epithelial cell surface markers AUA1, Ber-EP4, epithelial membrane antigen (EMA), and desmosomal protein. Compared to NCI-H69 cells, the amounts of cytokeratin 18 detected were elevated, while those of cytokeratin 19 were diminished in H69A cells. Focal expression of cytokeratin 4 was found in some H69A cells, indicative of a capacity for partial squamous differentiation. The expression of the cell surface glycoproteins detected by AUA1 and Ber-EP4 was reduced throughout cultivation of the H69A subline, while that of EMA and desmosomal protein was further diminished with continued passage. Changes in the expression of these markers and NCAM were evident in NCI-H69 cells grown on an HN5-derived substratum.


[Malignant fibrous histiocytoma: productive use of imaging procedures]. Malignant fibrous histiocytoma (MFH) of soft tissue is a rare tumor. We present a case of MFH in the bicipital muscle. Local muscle hypertrophy or lesions of the long tendon of the bicipital muscle may be a difficult differential diagnosis. This case is used for outlining advantages of ultrasound, computed tomography and magnetic resonance imaging. In conventional radiography MFH is seen as a soft-tissue tumor sometimes involving the bone. CT is the best way to test bone involvement. Tumor extension will be assessed with great accuracy by means of ultrasonics and magnetic resonance imaging (MRI).


Reactivity of orthoquinones involved in tyrosinase-dependent cytotoxicity: differences between alkylthio- and alkoxy-substituents. It may be possible to use the melanogenic pathway as a therapeutic targeting strategy for melanoma, and encouraging clinical pilot studies of 4-hydroxyanisole have led to the search for more active analogue substrates of tyrosinase. A recent study of a range of alkoxy- and alkylthio-phenol analogues of tyrosine has shown that sulphur-containing compounds exhibit different behaviour to that of similar oxygen-containing compounds, indicating modified reactivities of their corresponding tyrosinase-induced o-quinones towards crucial cellular targets, in particular, thiols. We have therefore examined by pulse radiolysis the reactivities of a group of unstable alkylthio- and alkoxy-substituted o-quinones towards the biologically relevant thiols, cysteine and glutathione. The o-quinones were generated by rapid (microsecond) one-electron oxidation of the corresponding stable synthesized catechols, forming semiquinones which disproportionated over milliseconds to o-quinones. The latter reacted with the thiols in a pH-dependent manner, indicative of increased nucleophilicity of the thiolate anions as compared with their protonated forms, with rate constants in the region of 10(5)-10(6) M-1s-1. At pH 7.2, within the physiological range, the alkylthio-substituted o-quinones reacted with the thiols approximately 5-10 times faster than the alkoxy-substituted o-quinones. The corresponding alkylthio-substituted phenols might, therefore, in principle, be expected to be more effective targeted anti-melanoma drugs than their alkoxy-substituted counterparts. NMR studies of the reactions of several of the quinones with cysteine indicate that, where addition occurs, the product is exclusively the 6-S-cysteinyl-4-substituted-catechol.


Relationship between melanogenesis, glutathione levels and melphalan toxicity in human melanoma cells. Resistance to alkylating agents has been correlated with cellular levels of reduced glutathione (GSH) and glutathione-S-transferase (GST). GSH is also involved in regulation of melanin synthesis. Therefore, we examined sensitivity to melphalan as a function of differentiation and GSH/GST levels in three human melanoma cell lines. The Me8 cell line, classified as undifferentiated on the basis of cell shape, absence of pigment, insignificant dopa oxidase activity and presence of inhibitors of dopa-melanin formation, showed the lowest GST activity among the cell lines investigated. GLL19 cells exhibited normal differentiation as indicated by the presence of dendrites, typical eumelanosomes, melanin granules and dopa oxidase activity. These cells showed the highest GSH content and the highest GST activity. The JUSO cell line showed incomplete differentiation, and its dopa oxidase and GST activities were intermediate between the Me8 and GLL19 cell lines. The sensitivity of melanoma cell lines to melphalan increased with their degree of differentiation; it was lowest for Me8, intermediate for JUSO and highest for GLL19. Dibutyryl cyclic AMP (dbcAMP) enhanced melphalan toxicity against Me8 cells. Depletion of intracellular GSH with buthionine sulphoximine (BSO) resulted in a three-fold increase in melphalan sensitivity in all three cell lines. Our results indicate that melphalan toxicity is related to cell differentiation and GSH status of melanoma cells. Based on the observed relationship between dopa oxidase, GSH/GST levels and drug toxicity, it is proposed that competition for the GSH pool between quinonoid melanin intermediates and melphalan could diminish drug conjugation and increase cytotoxicity.


Modulators of intracellular Ca2+ and the calmodulin inhibitor W-7 alter the expression of metastasis-associated genes MTS1 and NM23 in metastatic variants of the B16 murine melanoma. MTS1 is a metastasis-associated gene highly expressed in highly metastatic tumours. NM23 has been described as a putative metastasis suppressor gene. Here we show that thapsigargin (which raises intracellular calcium [Ca2+]i from intracellular stores) and verapamil (which blocks Ca2+ influx) both down-regulate MTS1 and NM23 gene expression in the poorly metastatic F1 and highly metastatic ML8 variants of the B16 murine melanoma without altering their metastatic behaviour. The data presented here suggest that Ca2+ released from intracellular stores could be functionally differentiated from influxed Ca2+ and could be activating different components of the Ca2+ signalling system. Many of the cellular responses to calcium are mediated through calmodulin. We have therefore further investigated the role of Ca2+ in the regulation of the MTS1 and NM23 genes using the calmodulin inhibitor W-7. Both these genes were down-regulated after treatment of the F1 and ML8 cell variants. We have shown previously that retinoic acid reduces lung colonization by the highly metastatic variant ML8 and that melanocyte stimulating hormone (MSH) enhances lung colonization by the poorly metastatic variant F1, with corresponding changes in the relative expression of NM23 and MTS1. Here we have found that verapamil and thapsigargin have no effect on lung colonization, possibly due to both genes being down-regulated. These data support the concept that NM23 and MTS1 gene expression is linked and that metastatic potential may be determined by their relative expression.


Intracellular regulation of cell adhesion to extracellular matrix components in murine B16 melanoma cells. The involvement of signal transduction systems in the initial attachment of two murine B16 melanoma clones of differing metastatic potential to extracellular matrix components was examined to learn more of the early events in cell-matrix interaction. Clones of high and low metastatic capacity attached similarly in the absence of any stimulators, exhibiting a two phase time course of attachment with 100% attachment by 60 min. A slight difference in attachment characteristics between the clones was seen in response to phorbol ester stimulation, which significantly inhibited attachment of the low metastatic clone but which had no effect on the highly metastatic clone. Total protein kinase C activity and distribution was similar for both clones. Attachment of both clones was severely reduced, however, if intracellular calcium was elevated or intracellular calmodulin inhibited. This study suggests that signal transduction mechanisms are involved in melanoma cell attachment to matrix proteins and offers an approach to pharmacological manipulation of these cell-matrix interactions which may be relevant to reducing metastatic spread.


Ribonucleotide diphosphate reductase from human metastatic melanoma. Cell free extracts from metastases of human melanoma contain a highly active ribonucleoside diphosphate reductase (RR) which uses guanosine diphosphate (GDP) as substrate and deoxythymidine triphosphate (dTTP) as effector. No activity could be detected in these extracts when cytidine diphosphate (CDP) was used as the substrate with adenosine triphosphate (ATP) as effector. The activity of this RR required the presence of either magnesium or calcium: there was a time lag before cell extracts from melanotic melanoma metastases showed full activities, but extracts from amelanotic tumors showed normal kinetics in the presence of these divalent cations. By contrast to other RRs, the activities in cell-free extracts could not be inhibited by hydroxyurea (10(-2) M). Even though an activity related free radical could be detected by electron paramagnetic resonance spectroscopy at 77 degrees K, the signal could not be quenched by 10(-2) M of this free radical trap. However, after ammonium sulphate fractionation, enzyme activity from melanotic melanoma was inhibited by 66% in 1 h. In the presence of substrates, effector and cofactors, the radical signal at g = 2.009 was also quenched by 60%; in the absence of substrate, effectors and cofactors, this signal was unaffected. These results indicate that two different free radicals must be present on melanoma RR. One is present in the resting enzyme, and the other is used during catalytic activity. The thiolate-active site of RR from melanoma was inhibited by the new nitrosourea anti-tumour drug fotemustine (IC50 = 10(-4) M as determined from a dose-response study).(ABSTRACT TRUNCATED AT 250 WORDS)


[Treatment of Buruli's ulcers by excision-graft]. Buruli's ulcer is a real disease of public health. In Ivory Coast, 50 patients were treated from February 1987 to August 1991. It realizes a real destruction of skin and subcutaneous tissues. The germ is Mycobacterium ulcerans. The preferential localisation is the limbs in children of 14 years old. Large ulcer with polycyclic margin are treated by extended excision and thin skin grafts. But deformities persist after the surgical treatment. Hospitalisation is long and drugs are expensive.


The feasibility of castable ceramic material in dental bridge construction. This study was to investigate the feasibility of dental bridges constructed with castable ceramic material (Dicor). A dental bridge model was designed similar to the clinical situation. The marginal fit of Dicor crown and a three-unit bridge was evaluated and no statistically significant differences in marginal openings between Dicor crowns and bridges were determined except the mesial and distal end surfaces of the bridge works. The fracture strengths of different thicknesses of Dicor test bars were measured with a three-point bending technique in a jig mounted in a universal testing machine. The results revealed that the bridge design with the thickest (6mm) connector and the shortest (8 mm) width of the pontic had the highest fracture resistance. In addition, pores with size up to 50 microns were disclosed in the fractured interface. The optimal design of Dicor bridge is determined according to the results of this study along with the clinical trial.


Spectral analysis of bubble sounds in decompressed guinea pigs. Doppler ultrasonic monitoring was used to detect bubbles in sixteen guinea pigs subjected to a simulated air-dive profile of 9 ATA for 25 minutes. After completion of the decompression, eight subjects did not demonstrate any signs of decompression sickness (DCS), two developed paraplegia and six died. Under sedation, the Doppler ultrasonic bubble detector was placed precordially to record the bubble signals in both pre-dive and post-dive. The recorded signals were analyzed with a high resolution signal analyzer to compare changes between pre-dive and post-dive spectrograms. Bubble signals appeared in the frequency range between 0.64 +/- 0.02 KHz and 6.08 +/- 0.30 KHz. In terms of the net spectral level versus frequency areas (difference between pre-dive and post-dive spectrograms), the mean areas in the paraplegia group and the death group were significantly larger than those in the group without DCS. Therefore, we concluded that the spectral analysis may be an objective and quantitative adjunctive method to the interpretation of Doppler bubble signals.


Surgical treatment of metastatic lung tumor. Since the first pulmonary resection for metastatic cancer of the lung in 1939, the following procedure can now be offered to any patient who meets these criteria: 1. a controlled primary tumor, 2. resectable lung lesion, 3. unavailability of a better treatment and 4. a good surgical candidate. From 1982 to 1992, there were 42 patients who underwent an operation for pulmonary metastasis at the Taichung Veterans General Hospital. Their cumulative 3-year and 5-year survival rates were 35% and 25% respectively. The overall estimated median survival time was 23.1 months. The overall operative mortality was 2.4% (1/42). We analyzed the effect of different types of primary tumors, the disease-free interval, and the number of lesions on patient survival. In our series, we concluded that none of the above had an effect on the therapeutic results.


[The level of double bonds in the blood lipids of lung cancer patients]. The total unsaturation of lipids in plasma and erythrocytes of patients with lung carcinoma was studied. It was demonstrated that the level of unsaturation of blood lipids had a definite prognostic significance for patients with malignant tumors. More favourable prognosis for patients with lung carcinoma was connected with a decline of double-bond level and its invariability (or even its reduction) after chemotherapy.


Follow-up of a modified catheter ablation of the atrioventricular junction in patients with atrial tachyarrhythmias. In order to reduce cumulative energy, minimize barotrauma and infranodal injury, a modified catheter ablation technique was performed on seventeen patients. These patients had drug refractory atrial tachyarrhythmias (AT) consisting of thirteen with paroxysmal atrial fibrillation (PAf), three with sick sinus syndrome (SSS) with PAf, and one having SSS with rapid left atrial tachycardia. This technique, using the femoral approach, consisted of delivering a direct-current (DC) shock using a dual electrode configuration, to the ablation site adjacent the atrioventricular (AV) junction just before the disappearance of His deflection on the His bundle electrogram. After delivering 1-4 DV shock (mean 2.8) (cumulative energy 556 +/- 260 joules), 9 pts had first degree AV block (1st AVB) and 8 pts had complete AV block (CAVB). Only two pts had a right bundle branch block after ablation. During the clinical follow-up (15.4 +/- 2.7 months), sixteen pts were asymptomatic and were free of antiarrhythmic drugs. One pt was asymptomatic with quinidine which was ineffective before ablation. Pacemaker implantation was performed in 10 pts as a back-up for symptomatic SSS and CAVB. The results show this modified technique is relatively safe and effective.


[Comparative analysis of MDP bone imaging agents used in Taiwan]. Bone scans have been established as a major investigative procedure for various bone, muscle and joint diseases, and for the detection of bony metastases. MDP (methylene diphosphate) labeled with Tc-99m is currently used for bone imaging. The quality of bone imaging agents can affect the quality of the bone image. In this work we compared the quality control for three commercially available MDP kits (Daiichi, Amersham, NEN) in Taiwan, and the imaging quality among the various MDP kits. Our results revealed a slight difference of quality control in various brands of Tc-99m MDP. However, there was no significant difference in the imaging quality among the various MDP kits.