Modulatory role of glutathione on mu-opioid, substance P/neurokinin-1, and kainic acid receptor binding sites. Reduced glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) is an endogenous tripeptide involved in the formation and maintenance of protein thiol groups as well as in various detoxification reactions. Because multiple receptor types contain thiol groups or disulfide bridges, effects of GSH treatments on mu-opioid, neurokinin-1/substance P, and kainic acid receptor binding sites were investigated and compared with those produced by dithiothreitol (DTT), a potent synthetic reducing agent. GSH inhibited binding more potently than did DTT at all three receptor types in porcine striatal membrane homogenates as well as in CHAPS-solubilized preparations of the mu and neurokinin-1 sites. GSH-induced inhibitory effects were associated with decreases in maximal binding capacity (Bmax) without significant alteration in apparent affinity (KD). Cysteine, the functional moiety of GSH, mimicked GSH effects albeit with lower potencies, whereas oxidized glutathione had no effects at similar concentrations. In CHAPS-solubilized preparations, the combination of low concentrations of GSH and guanylylimidodiphosphate markedly decreased the Bmax values of the binding of [3H][D-Ala2,Gly-ol5]enkephalin and [3H]substance P. This GSH-mediated mechanism may be important to prevent cell overstimulation by accelerating receptor uncoupling, desensitization, and/or internalization. This is in keeping with purported roles of GSH related to the maintenance of cellular integrity.


Hippocampal 8-[3H]hydroxy-2-(di-n-propylamino) tetralin binding site densities, serotonin receptor (5-HT1A) messenger ribonucleic acid abundance, and serotonin levels parallel the activity of the hypothalamopituitary-adrenal axis in rat. We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared with the relatively resistant Fischer F344/N rat, is related to a hyporesponsive hypothalamopituitary-adrenal axis to inflammatory and other stress mediators. Because serotonin (5-HT) and the 5-HT1A receptor are important stimulators of this axis, we have investigated the levels of 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites, 5-HT1A mRNA, 5-HT, and 5-hydroxyindoleacetic acid in various brain regions of Lewis, outbred Harlan Sprague Dawley, and Fischer F344/N rats. Lewis rats expressed significantly fewer hippocampal and frontal cortical 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites and less 5-HT1A mRNA than Harlan Sprague Dawley and Fischer F344/N rats. Adrenalectomy increased the number of 8-[3H]hydroxy-2,3-(di-n-propylamino)tetralin binding sites and 5-HT1A mRNA expression in the hippocampus of all three strains. Levels of hippocampal 5-HT in Fischer F344/N rats were significantly greater than levels detected in the same regions from Lewis and Harlan Sprague Dawley rats. Hypothalamic 5-HT and 5-hydroxyindoleacetic acid levels in Harlan Sprague Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindoleacetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the activity and responsiveness of the hypothalamopituitary-adrenal axis.


Simultaneous effects of p-chloroamphetamine, d-fenfluramine, and reserpine on free and stored 5-hydroxytryptamine in brain and blood. The effects of acute treatment with p-chloramphetamine, d-fenfluramine, and reserpine on intracellular (brain tissue and whole blood) and extracellular (CSF and platelet-free plasma) compartments of 5-hydroxytryptamine (5-HT) in the brain and blood of the same rats have been examined. These treatments affected 5-HT in brain tissue and whole blood similarly (r = 0.823). Reserpine significantly reduced both intracellular pools at 2 and 24 h. p-Chloroamphetamine and d-fenfluramine were more effective on brain tissue 5-HT. The concentration of 5-HT in CSF was significantly increased by all treatments. p-Chloroamphetamine induced a dramatic 70-fold increase of CSF 5-HT, paralleling a 42% decrease in brain tissue. d-Fenfluramine significantly increased CSF 5-HT to 212% of controls and reduced whole brain 5-HT (-23%). The effects of p-chloroamphetamine and d-fenfluramine on 5-HIAA in brain, CSF, and plasma were nonsignificant. Individual values of 5-hydroxyindoleacetic acid (5-HIAA) in CSF and brain were highly correlated (r = 0.855), indicating that CSF 5-HIAA reflects well the concentration of 5-HIAA in brain tissue. Yet the intra- and extracellular concentrations of 5-HIAA were unrelated to the 5-HT changes. This indicates that CSF 5-HIAA does not reflect the active (extracellular) compartment of 5-HT in brain.


Alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding to human cerebral cortical membranes: minimal changes in postmortem brains of chronic schizophrenics. The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)


Depolarization-dependent tyrosine phosphorylation in rat brain synaptosomes. Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine-containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10-11 phosphotyrosine-containing proteins were detected. Depolarization of synaptosomes by transfer to a high (41 mM) K+ medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of M(r) 117,000 (ptp117). Additional proteins exhibiting depolarization-dependent increases in phosphotyrosine content had molecular weights of 39,000, 104,000, 135,000, and 160,000. The depolarization-dependent increase in the phosphotyrosine content of ptp117 was apparent within 30 s of the onset of depolarization, reached a maximum between 3 and 5 min, and then decreased to near control values by 30 min. The increase in tyrosine phosphorylation of ptp117 was dependent on the concentration of K+ in the depolarizing medium and was maximal with [K+] in excess of 50 mM. It was also calcium dependent and did not occur in the absence of extracellular calcium. The addition of veratridine to the incubation medium also resulted in an increase in the tyrosine phosphorylation of ptp117. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity.


Induction of nitric oxide synthase in glial cells. Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOS-like activity that was L-arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%, with a loss of 44% of NOS-like activity. C6 cells treated with LPS also showed Ca(2+)-independent and L-arginine-dependent NOS-like activity. N18 cells demonstrated constitutive Ca(2+)-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells.


Desensitization of GABA-activated currents and channels in cultured cortical neurons. Application of GABA to rat neocortical neurons maintained in cell culture produced a response that declined over several seconds, even in the continued presence of agonist. The decrement could be attributed to both a redistribution of Cl- and a true decline in GABA-induced membrane conductance, or desensitization. The extent and rate of desensitization were dose dependent in a manner similar to the dose dependence of the GABA-induced current, but were not related to the absolute magnitude of the current or to the charge transfer. Bicuculline slowed desensitization while diazepam enhanced the rate of desensitization, consistent with a localization of desensitization to the agonist-receptor binding site. When measured in the whole-cell recording mode, desensitization was voltage dependent, becoming much slower as the membrane was depolarized. Changes in extracellular or intracellular [Ca2+] did not appear to grossly affect the desensitization process or its voltage dependence. GABA-activated single channels, recorded in the outside-out configuration, also desensitized in the continued presence of agonist. However, desensitization differed from that seen in the same neurons in the whole-cell mode. Desensitization was considerably more rapid and did not show any voltage sensitivity. Moreover, single-channel responses often failed to recover after only a few exposures to agonist. Desensitization of GABA responses may play a role in the regulation of cortical inhibition, especially under conditions of intense excitatory and inhibitory synaptic activation.


Robust enkephalin innervation of the locus coeruleus from the rostral medulla. Substantial evidence indicates that the noradrenergic nucleus locus coeruleus (LC) is a key target of endogenous opioid neurons, and an important structure in mediating opiate effects. However, the detailed distribution of opioid fibers and terminals in the LC, and the sources of its opioid innervation are unknown. In the present study, the enkephalin innervation of the LC was investigated in the rat using an antibody directed against the extended enkephalin peptide Met-enkephalin-Arg6-Gly7-Leu8 (ENK), which is derived exclusively from the enkephalin precursor proenkephalin A. An antibody directed against tyrosine hydroxylase (TH), the synthetic enzyme for catecholaminergic neurons, was also applied to the same tissue sections to delineate LC neurons and their dendrites. Enkephalin fibers in the LC were dense and highly varicose. In horizontal sections, ENK-like-immunoreactive (ENK-ir) fibers of considerable length coursed throughout the rostrocaudal orientation of the LC proper, whereas in frontal sections ENK-ir processes appeared punctate, suggesting a rostrocaudal orientation. Dense ENK-ir fibers were also identified in the rostromedial and caudal juxtaependymal pericoerulear regions where extranuclear dendrites of LC neurons are extensive. As previously reported, there were no ENK-ir neurons in the LC nucleus proper, but such cells were present in neighboring structures such as the parabrachial, sphenoid, and Barrington's nuclei as well as in the central gray and in the subcoeruleus area. ENK-ir neurons were also present in nuclei of the rostral medulla reported to be major afferents of the LC, the nucleus prepositus hypoglossi (PrH), and the nucleus paragigantocellularis (PGi). In the dorsomedial medulla, numerous ENK-ir neurons were identified in the medial aspect of the PrH and along the medial longitudinal fasciculus in the perifascicular reticular formation. In the ventrolateral medulla, ENK-ir neurons were distributed in a conical caudorostral column throughout the PGi. Retrograde transport of a WGA-colloidal gold conjugate (WGA-apoHRP-Au) from LC, combined with immunohistochemistry for ENK in the same tissue sections, revealed that LC afferents in the PGi and PrH were interdigitated with ENK-ir neurons. Furthermore, an unexpectedly high incidence of doubly labeled neurons were identified in both PGi and PrH. Overall, 57% and 56% of the LC-projecting neurons in PGi and PrH, respectively, were also immunoreactive for ENK, suggesting that enkephalinergic neurons of PGi and PrH are major afferents to noradrenergic LC neurons.


Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression. Phototransduction in retinal rods involves a G-protein-mediated signaling cascade that leads to cGMP hydrolysis and the closure of a cGMP-gated channel. This channel has recently been purified from bovine retina and molecularly cloned (Kaupp et al., 1989). We report here the cloning of cDNA and genomic DNA encoding the human rod cGMP-gated channel, based upon its homology to the bovine counterpart. The human mRNA structure differs from the bovine in containing an Alu repetitive element spliced into the 5' untranslated region. The human cGMP-gated channel gene (CNCG) is located on chromosome 4 and contains at least 10 exons. One large exon encodes the carboxy-terminal two-thirds of the protein, whereas seven small exons encode the amino-terminal one-third of the protein. Alternative splicing removes one of the small exons in a subset of transcripts in the human retina, producing an internal in-frame deletion of 36 codons. When expressed in a human embryonic kidney cell line (293S), the full-length cDNA clone, but not the differentially spliced variant, produced functional ion channels broadly similar to the native channels in vertebrate rods.


Antitumor agents. 134. New shiraiachrome-A- and calphostin-C-related perylene derivatives as cytotoxic and antiviral agents and inhibitors of protein kinase C. Shiraiachrome-A and -B have been isolated from the mycelium of the Chinese bamboo fungus Shiraia bambusicola as the cytotoxic principles. A series of new perylene derivatives (7-27) related to Shiraiachrome-A and -B as well as Calphostin-C have been synthesized and evaluated for their cytotoxicity, antiviral activity, and inhibitory activity against protein kinase C. The results indicated that 11 and 12 are potent cytotoxic agents against HCT-8, RPMI-7951, and TE-671 solid tumor cells, whereas 24 and 26 demonstrated strong antiviral activity against HSV-1 and HSV-2. Compound 10 is an inhibitor of protein kinase C.


The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum. Eight new coumarin compounds (1-8) were isolated by anti-HIV bioassay-guided fractionation of an extract of Calophyllum lanigerum. The structures of calanolide A (1), 12-acetoxycalanolide A (2), 12-methoxycalanolide A (3), calanolide B (4), 12-methoxycalanolide B (5), calanolide C (6) and related derivatives 7 and 8 were solved by extensive spectroscopic analyses, particularly HMQC, HMBC, and difference NOE NMR experiments. The absolute stereochemistry of calanolide A (1) and calanolide B (4) was established by a modified Mosher's method. Calanolides A (1) and B (4) were completely protective against HIV-1 replication and cytopathicity (EC50 values of 0.1 microM and 0.4 microM, respectively), but were inactive against HIV-2. Some of the related compounds also showed evidence of anti-HIV-1 activity. Studies with purified bacterial recombinant reverse transcriptases (RT) revealed that the calanolides are HIV-1 specific RT inhibitors. Moreover, calanolide A was active not only against the AZT-resistant G-9106 strain of HIV-1 but also against the pyridinone-resistant A17 strain. This was of particular interest since the A17 virus is highly resistant to previously known HIV-1 specific, non-nucleoside RT inhibitors (e.g., TIBO; BI-RG-587; L693,593) which comprise a structurally diverse but apparently common pharmacologic class. The calanolides represent a substantial departure from the known class and therefore provide a novel new anti-HIV chemotype for drug development.


Amide, urea, and carbamate analogues of the muscarinic agent [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride. A series of amide, urea, and carbamate analogues of the muscarinic (M1) ganglionic stimulant [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343; 1) was prepared. The C1-methyl-substituted carbamates 8-11 were resolved into the enantiomers. In order to investigate the ganglionic stimulant activity and affinity of the new compounds we studied their ability to increase mean arterial blood pressure (MAP) in the pithed rat and their ability to displace the M1 receptor selective antagonist [3H]pirenzepine from rabbit sympathetic ganglia. The quaternary ammonium derivatives of 1, but not their corresponding tertiary amines, displayed ganglionic stimulant properties. The urea derivative 14 and the acetamide derivative 18 were almost equipotent to 1 as ganglionic agonists. In addition, 14 and 18 showed only 2- to 3-fold less affinity to ganglionic muscarinic receptors than 1. Introduction of a methyl group in the 1 position of the butynyl chain of 1 and its 4-chlorophenyl analogue increased ganglionic stimulant potency. The resulting (+/-)-9 and (+/-)-11 were the most potent analogues in this study. They were found to be partial agonists and showed 5- and 16-fold higher potency than 1, respectively, in increasing the MAP. They also displayed 6- and 18-fold higher affinity than 1 for ganglionic M1 receptors. The (S)-enantiomers of 9 and 11 were 1.5- and 4.9-fold more potent, respectively, than their antipodes as ganglionic muscarinic stimulants. The C1-methyl-substituted urea and acetamide derivatives (15 and 19) were 1.5- and 3-fold less potent than 1 and displayed several-fold lower affinity for ganglionic M1 receptors. The new quaternary analogues retained the selectivity for ganglionic muscarinic receptors since they produced weak partial agonist effects on the guinea pig ileum and showed several-fold lower nicotinic activity than 1 in the frog rectus abdominis assay.


Clinical profile of apparently healthy neonates with in utero drug exposure. To identify the characteristics of neonates exposed to drugs in utero but admitted to the normal newborn nursery because of apparent good health. Retrospective evaluation with chart review of toxicology screening tests sent from a normal newborn nursery. Newborn service in an urban hospital. Fifty newborns with positive drug screening results. Frequency of positive results and assessment of demographic features and neonates' clinical features. 43 (86%) of the apparently healthy newborns tested positive for cocaine. The results of simultaneous or proximate drug testing of mothers and their newborns were discordant in 11 (22%) cases. Neonates with in utero drug exposure often are born with few or no signs of abnormality and are admitted to the normal newborn nursery.


Structural requirements for neuropeptide Y18-36-evoked hypotension: a systematic study. It has been shown that NPY and select C-terminal fragments of NPY that evoke a hypotensive response upon intraarterial administration in the rat also cause mast cell degranulation and histamine release in vitro. Additionally, elevation of plasma histamine levels has been observed concomitant with the hypotensive effect of NPY and various C-terminal fragments. In order to investigate whether the hypotensive response to NPY18-36 is correlated to this observed elevation of histamine in vivo, we sought to characterize the structural requirements for each activity. We conducted a systematic replacement of each amino acid in NPY18-36 by its D-isomer. Additionally, various modifications were made to the N- or C-terminii of NPY18-36. The following rank order of potency was obtained for the hypotensive action of these analogues of NPY18-36 relative to NPY18-36. Only one analogue ([D-Tyr21]NPY18-36) exhibited significantly enhanced potency. Eleven analogues of NPY18-36, ([D-Thr32]-, [D-Arg35]-, [D-Ile31]-, [D-Leu30]-, [D-Tyr27]-, [D-Ser22]-, [D-Tyr36]-, [D-Gln34]-, [D-Asn29]-, [D-Ala23]-, and [D-Arg33]NPY18-36) were equipotent with NPY18-36. Four analogues ([D-His26]-, [D-Ile28]-, and [D-Ala18]NPY18-36 and -NPY18-27) had reduced potency (10-80%) while eight analogues ([D-Arg19]-, [D-Tyr20], [D-Leu24]-, [D-Arg25]-, [Ac-Ala18]-, [Me-Ala18]-, [desamino-Ala18]NPY18-36 and NPY18-36 free acid) failed to produce a significant hypotensive response (less than 10%) at the doses tested. The sensitivity of NPY18-36 to chiral inversion of single residues or other modifications at the N-terminus suggested the presence of a conformationally well defined N-terminal pharmacophore. Additionally, five NPY18-36 analogues were tested for elevation of plasma histamine levels. The rank order of potency ([D-Thr32]NPY18-36 = [D-Tyr21]NPY18-36 much greater than NPY18-36 greater than [D-Ala18]NPY18-36 greater than [Ac-Ala18]NPY18-36) was correlated with each analogue's potency at evoking a hypotensive response. In contrast, NPY1-36 failed to evoke an elevation in plasma histamine levels despite its hypotensive effects. Hence, we conclude that the magnitude of the hypotensive response evoked by an NPY18-36 analogue is primarily a function of its ability to elevate plasma histamine levels. However, the mechanism underlying NPY1-36-evoked hypotension appears to be different.


Mitochondrial transfer RNA genes in a black fly, Simulium vittatum (Diptera: Simuliidae), indicate long divergence from mosquito (Diptera: Culicidae) and fruit fly (Diptera: Drosophilidae). Sequences are given for nine complete genes and one partial mitochondrial tRNA gene of the black fly, Simulium vittatum (Zetterstedt). Sequenced tRNA genes were for alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, leucine(CUN), lysine, serine(AGN), and valine. Nucleotides were aligned with the same previously sequenced genes in Aedes albopictus Skuse and Drosophila yakuba Burla. A cluster of six tRNA genes, which differ in arrangement in Ae. albopictus and D. yakuba, was amplified by PCR and found to have the same position and orientation in S. vittatum as in D. yakuba. Overall, similarity with either D. yakuba or Ae. albopictus was 86%. Sequences that were common to the three insects suggest that black flies and mosquitoes are as divergent from each other as either is from Drosophila. Sequences for nine species of black flies were obtained for tRNA leucine(CUN) from DNA amplified with another primer set. Little variation occurred within the tRNA gene but, by including the flanking regions to provide 175 base pairs, a phylogeny of the nine species was obtained that was largely consistent with current classification.


IP3- and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish. Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP3 or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP3 were blocked by ruthenium red, a blocker of an IP3-gated Ca(2+)-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by L-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP3 in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP3 or cAMP when the buffering capacity for Ca2+ of the pipette solution was increased, when extracellular Ca2+ was removed or after addition of 20-200 nM charybdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Cai] that opened Ca(2+)-activated K+ channels. The results suggest that different conductances modulated by either IP3 or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca(2+)-activated K+ channels contribute to the termination of the IP3 and cAMP responses.


Differential sensitivity of pneumolysin-induced channels to gating by divalent cations. The induction of channels across planar lipid bilayers by purified, recombinant pneumolysin (a hemolytic protein from Streptococcus pneumoniae) has been studied by measuring increases in electrical conductivity. Pneumolysin-induced channels exhibit a wide range of single channel conductances (less than 50 pS to greater than 1 nS at 0.1 M KCl). Channels can be categorized on the basis of their K+:Cl- selectivity: the smallest channels are strongly cation selective, with t+ (the cation transference number) approaching 1.0; the largest channels are unselective (t+ approximately 0.5). Channels tend to remain open at all voltages (-150 to 150 mV); only the smallest channels exhibit any rectification. In the presence of divalent cations (1-5 mM Zn2+; 10-20 mM Ca2+), small (less than 50 pS) and medium-sized (50 pS to 1 nS) channels are closed in a voltage-dependent manner (more closure at higher voltages); at 0 voltage channels reopen. Overall selectivity is reduced by divalent cations, compatible with small, selective channels being closed preferentially to large, nonselective ones. It is concluded that a single molecular species (pneumolysin) induces multiple-sized channels that can be categorized by cation:anion selectivity and by their sensitivity to closure by divalent cations.


Regulation of insulin-like growth factor-binding protein (IGFBP) expression by breast cancer cells: use of IGFBP-1 as an inhibitor of insulin-like growth factor action. The insulin-like growth factors (IGFs) play an important role in normal growth and development. Evidence suggests they may also regulate the growth of several cancer cell types. This regulation is mediated by interactions between the receptors and ligands. There is now ample evidence to suggest that these interactions are also influenced by extracellular IGF-binding proteins (IGFBPs). Six different IGFBPs have been cloned. Some species may act to inhibit the mitogenic effects of the IGFs. Since breast cancer cells are responsive to the IGFs, it is possible that regulated expression of the IGFBPs affects tumor growth. Furthermore, inhibitory binding proteins could be used as neutralizers of IGF action. We conducted this study to fully characterize the expression and hormonal regulation of IGF-binding protein expression in human MCF-7 breast cancer cells and to test the ability of purified IGFBP-1 to inhibit IGF-I action. We used ribonuclease protection assays and Western ligand blotting to examine IGFBP expression in MCF-7 cells. The effect of IGF-I, IGFBP-1, and 17 beta-estradiol on serum-free cell growth was also studied. MCF-7 cells expressed IGFBP-2, IGFBP-4, and IGFBP-5 RNA and protein. These cells are dependent on estrogen for growth. In short-term culture, IGF-I can substitute for estrogen. Concomitant addition of IGF-I and estrogen enhanced stimulation above the level achieved by either factor alone. Estrogen also increased IGFBP production, making it unlikely that the IGFBPs induced by estrogen in MCF-7 cells could function as major inhibitors of IGF action. In contrast, exogenous addition of IGFBP-1 could block IGF-I-induced mitogenesis; this effect was reversible by excess IGF-I. The studies suggest that cancer cell growth may be regulated by endogenous IGFBP expression. Furthermore, the exogenous addition of the IGFBP-1 blocked IGF-I action and potentially could be used as a pharmacologic inhibitor of IGF action.


Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. The purpose of this study was to evaluate the effects of exogenous recombinant basic fibroblast growth factor (bFGF) on angiogenesis in severely ischemic tissue beds. We used a two-stage procedure to produce severe ischemia of the hindlimb of 34 New Zealand rabbits. The ischemic hindlimb received intramuscular injection of saline (group A), 1 microgram bFGF (group B), or 3 micrograms bFGF (group C), daily for 2 weeks. Tissue perfusion, skeletal muscle infarction, angiogenesis, and collateral growth were assessed by angiography, transcutaneous oximetry (TcPO2), quantitative spectrophotometric assay of triphenyltetrazolium chloride reduction in muscle, capillary density (capillaries per square millimeter), and capillary per muscle fiber ratio. There were no significant differences in baseline TcPO2 among the three groups for both thigh and calf measurements. Angiography revealed extensive perfusion of the left hindlimb in all the assessed bFGF treated animals. Both thigh and calf TcPO2 values showed a significant increase in all groups over the 14 days ischemia was induced (p less than 0.0001), but the two treatment groups exhibited a much more rapid rise in TcPO2 than the control group (p less than 0.0001). The capillaries per square millimeter and capillaries per muscle fiber ratios were significantly increased in all posttreatment measurements for all animals that received bFGF. The treatment groups with bFGF had a significant (p = 0.025) increase in thigh muscle viability compared with controls based on triphenyltetrazolium chloride reduction. Whereas there was evidence of muscle infarction in both the thighs of groups A and B, there was none in group C.(ABSTRACT TRUNCATED AT 250 WORDS)


The value and limitation of iloprost infusion in decreasing skeletal muscle necrosis. Iloprost has been shown to minimize skeletal muscle necrosis when administered before the onset of ischemia in animal experiments, possibly by preventing neutrophil activation. Since patients with acute limb ischemia are seen after the process has begun, we investigated whether iloprost can be protective when given only during reperfusion. After anesthesia, 18 adult mongrel dogs underwent a standard isolated gracilis muscle preparation. In six control animals (group I) the gracilis muscle was subjected to 6 hours of ischemia followed by 48 hours of reperfusion. Group II animals (n = 6) received intravenous infusion of iloprost at a dose of 0.45 microgram/kg/hr beginning 1 hour before the onset of muscle ischemia and throughout the experiment (6 hours of ischemia and 1 hour of reperfusion). In addition to the continuous infusion, they received 0.45 microgram/kg intravenous boluses of iloprost 10 minutes before the induction of ischemia and 10 minutes before reperfusion. Group III animals (n = 6) had a similar ischemic interval, but were given a bolus of iloprost of 0.45 microgram/kg at end ischemia followed by continuous infusion of 0.45 microgram/kg/hr for 48 hours during reperfusion. Muscle biopsies were obtained at baseline and after 1 hour of reperfusion in all groups. Additional biopsies were obtained at 48 hours of reperfusion in groups I and III. Myeloperoxidase activity, a marker of neutrophil activation, was measured in all muscle biopsies. At the end of reperfusion, the gracilis muscle was harvested in all animals and weighed. Muscle necrosis was estimated by serial transection, nitroblue tetrazolium histochemical staining followed by computerized planimetry.(ABSTRACT TRUNCATED AT 250 WORDS)


Serial cultivation of adult human endothelium from the great saphenous vein. The growth of endothelial cells from small segments of human great saphenous vein was investigated for possible functional studies of endothelial cell properties and endothelialization of cardiovascular prosthetic graft materials. Growth in medium containing fetal calf serum or human serum was compared, and the effects of adding compounds that increase intracellular cyclic adenosine monophosphate levels, that is, cholera toxin and isobutylmethylxanthine, were also examined. It was shown that human serum was more efficient in stimulating cell growth than fetal calf serum at all concentrations tested. It was also shown that the addition of cholera toxin and isobutylmethylxanthine significantly potentiated growth. Minimal Essential Medium with the addition of 40% human serum and cholera toxin (1 nmol/L) and isobutylmethylxanthine (33 mumol/L) was shown to be optimal. From a single segment (3 to 5 cm), 20 x 10(6) human saphenous vein endothelial cells corresponding to 2000 cm2 or more could be achieved after 3 to 4 weeks in culture. The human saphenous vein endothelial cell cultures retained their cobblestone appearance, expression of von Willebrand Factor (vWF)-antigen, and capacity for prostacyclin production after six passages. We suggest that this provides a practically useful method for studies of cultured endothelium and possibly for pre-endothelialization of cardiovascular prosthetic materials.


[Combination effect of KW-2228 and cephem antibiotics in a systemic infection model in neutropenic mice]. A modified recombinant human granulocyte colony-stimulating factor (rhG-CSF), KW-2228, has some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. Recently, the application of G-CSF against infectious diseases has been considered, and some animal experiments have been carried out to support its clinical applications. In this paper, we investigated protective effects of KW-2228 against systemic infections caused by Klebsiella pneumoniae in mice with leukopenia induced by the administration of cyclophosphamide. KW-2228 (1 microgram/mouse) was administered (s.c.) once a day for 4 days following cyclophosphamide administration, then mice were challenged with K. pneumoniae (i.p.) 4 hours after the last administration of KW-2228. An antibiotic was administered (s.c., p.o.) 2 hours after the bacterial challenge. Combination effects of KW-2228 with cefazoline, cefmetazole, ceftazidime or cefaclor were evaluated in the systemic infection with K. pneumoniae. Each combination therapy using KW-2228 with each of the cephems exhibited an excellent protective effect in comparison to the therapy with a cephem alone. These results show the possibility that KW-2228 could be of use in treating obstinate infections not successfully treated with an antimicrobial agent alone.


[Effects of KW-2228 on the function of polymorphonuclear neutrophils in rats for the phagocytosis and killing activity and the production of active oxygen]. KW-2228 is a novel derivative of a recombinant human granulocyte colony-stimulating factor (rhG-CSF) with a modification. KW-2228 has some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. In the present study, we investigated the effect of KW-2228 on phagocytic capacity and killing action of polymorphonuclear neutrophils (PMNs) obtained from normal rats which had been treated with KW-2228 using various microorganisms, such as Candida albicans, Escherichia coli and Pseudomonas aeruginosa. We also investigated the effect of KW-2228 on the production of superoxide anion using a luminol-chemiluminescence method. KW-2228 enhanced the phagocytosis and killing activity of PMNs of rats against C. albicans and E. coli. PMNs treated with KW-2228 showed some bactericidal activity against P. aeruginosa. These data obtained with PMNs treated with KW-2228 show a correlation between the bacterial susceptibility to phagocytosis and killing action and the lunimol-dependent chemiluminescence response. These results suggest the significance and the efficacy of KW-2228 used in an additional therapy to that of antibiotics in the treatment of infections diseases. KW-2228 is currently in Phase III clinical trials in Japan.


[Protective effect of KW-2228 in a systemic infection model of CPA-treated tumor-bearing mice]. A modified recombinant human granulocyte colony-stimulating factor (rhG-CSF), KW-2228, has some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. Recently, the application of G-CSF against infectious diseases has been considered, and some animal experiments have been carried out to support its in clinical applications. Patients with underlying diseases such as leukemia or cancer often have recurrent infections because of reduced number and functions of neutrophils, which mediate an early stage of host defense. We investigated the prophylactic effect of KW-2228 against an experimental systemic infection with Pseudomonas aeruginosa in tumor-bearing mice (colon 26: BALB/c) treated with cyclophosphamide. KW-2228 (0.25-2.0 micrograms/mouse) was administered (s.c.) once a day for 4 days before the experimental bacterial infection. As a result of KW-2228 administration, the reduction in peripheral blood neutrophils usually caused by the injection with cyclophosphamide was prevented markedly. KW-2228 displayed excellent protective potency dose-dependently against the infection with P. aeruginosa in tumor-bearing mice. These data show the possibility that prophylactic therapy with KW-2228 may augment the host defense of immunocompromised patients to infections. It present, clinical efficacy studies on KW-2228 are under way.


[Combination effects between KW-2228 and antibiotics against systemic infections in normal and neutropenic mice]. KW-2228, a mutationally modified recombinant human granulocyte colony-stimulating factor (rhG-CSF), possesses some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. Recently, the application of G-CSF against infectious diseases has been considered, and some animal experiments have been carried out to support such an application on human infectious diseases. In this paper, we examined combination effect of KW-2228 with various chemotherapeutic drugs in experimental infectious in mice. A combination effect of KW-2228 with ceftazidime (CAZ) was evaluated in a systemic infection with Pseudomonas aeruginosa in normal mice. Combination effects of KW-2228 with CAZ, astromicin and amphotericin B were also evaluated in experimental systemic infections caused by P. aeruginosa, Serratia marcescens and Candida albicans in immunosuppressed mice treated with cyclophosphamide. Synergistic effects were generally observed at KW-2228 doses from 1 to 5 micrograms per mouse with all combinations. We concluded that combination therapies of KW-2228 with various chemotherapeutic drugs in experimental infections in mice showed that it should be effective in normal and immunosuppressed host. These results of our laboratory studies suggest that KW-2228 in combination with antibiotics would be useful in the clinical treatment of microbial infections. Recently, clinical efficacy studies of KW-2228 have been initiated in Japan.


[Combination effect of KW-2228 and aminoglycoside antibiotics on systemic infection in cyclophosphamide-treated tumor-bearing mice]. A modified recombinant human granulocyte colony-stimulating factor (rhG-CSF), KW-2228, has some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. Recently, the application of G-CSF against infectious diseases has been considered, and some animal experiments have been carried out to support its clinical applications. Patients with underlying diseases such as leukemia and cancer often have recurrent infections because of reduced numbers or functions of neutrophils, which mediate an early stage of host defense. In out present study, we established a new method to evaluate in vivo potency of G-CSF in colon 26 tumor-bearing mice. By using the method, we examined combination effects of KW-2228 with aminoglycoside antibiotics against a systemic infection caused by Pseudomonas aeruginosa. KW-2228 (1 microgram/mouse/day) was administered (s.c.) once a day for 4 days before the bacterial infection was introduced in colon 26 tumor-bearing mice receiving cyclophosphamide 3 days after the transplantation of tumor. Antibiotics were administered (s.c.) 2 hours after the introduction of the bacterial infection. ED50 of gentamicin (GM) alone and that of the combination with KW-2228 were 40.7 mg/kg and 3.6 mg/kg, respectively. ED50 of astromicin (ASTM) alone and that of the combination with KW-2228 were 386 mg/kg and 17.8 mg/kg, respectively. Thus the combination therapy of KW-2228 with GM or ASTM exhibited excellent protective effects in comparison to the treatment with antibiotic alone.(ABSTRACT TRUNCATED AT 250 WORDS)


[Basic and clinical studies of local hyperthermia for prostatic cancer]. The effect of local hyperthermia on the prostate using 13.56 MHz radio frequency wave (RF wave) was reported. Firstly, temperature and blood flow of the prostate in normal dogs were measured during local hyperthermia. In most part of the prostate, the temperature reached over 42 degrees C, which was considered as favorable for the hyperthermia therapy. Blood flow of the prostatic tissue rose more slowly than that of muscle tissue. Secondly, the tissue concentration of anticancer agents after local hyperthermia was measured. There was a tendency that drug concentration in the prostate tissue after local hyperthermia was higher than that without local hyperthermia. Histological findings showed interstitial edema and congestion. As a clinical trial, 14 cases of prostatic cancer were treated with local hyperthermia after the administration of anticancer agents. Seven of them were fresh cases and the others were relapsed cases. After treatment, tumor size was reduced in 13 cases. According to "The Response Criteria for Urologic Tumor", one Complete Response, 3 Partial Response and 10 No Change cases were obtained. There was no tumor progression. As for side effects, bone marrow suppression, loss of appetite, diarrhea and skin burns were noted. However, these side effects were mild, and did not interrupt the treatment. Local hyperthermia of the prostate after systemic chemotherapy could be carried out safely and effectively in patients with prostatic cancer.


[The effect of intra-urethral catheter for prostatic hypertrophy patients who are unfit for operation and suffer from urinary retention]. The effect of double Malecot type polyurethane intraurethral catheter (IUC) was examined in 17 benign prostatic hypertrophy patients who were unfit for operation and suffered from urinary retention. Patients were aged 68 to 90 (mean 80.5) years old and the causes of IUC insertion were cardiac, cerebrovascular, respiratory and gastrointestinal diseases, diabetes mellitus and aging. IUC was selected among three types (55, 60, 65 mm) according to the length of prostatic urethra. Insertion of IUC was carried out easily under fluoroscopic guidance without endoscopy. All patients could void by themselves just after insertion of IUC and the longest indwelling period was 10 months. The length of IUC need not be longer than that of prostatic urethra and patients with normal or hypertonic bladder could void better than those with atonic bladder. Urinary tract infection did not get worse in any patients with indwelling IUC. Double Malecot type polyurethane IUC is a safe and an effective alternative method in place of urethral balloon catheter for inoperable prostatic hypertrophy patients in urinary retention.


[Adjuvant chemotherapy (MVP-CAB; methotrexate, vincristine, cisplatinum, cyclophosphamide, adriamycin, and bleomycin) for bladder cancer]. Twenty-three patients with bladder cancer (TCC; 18 patients, SCC; 5 patients) were treated with adjuvant chemotherapy (day 1: methotrexate 20 mg/m2, vincristine 0.6 mg/m2, cyclophosphamide 500 mg/m2, adriamycin 20 mg/m2, bleomycin 30 mg, day 2: cisplatinum 50 mg/m2; MVP-CAB). A total of 3 cycles of MVP-CAB were given as preoperative or postoperative therapy. The following results were obtained. Group 1 (purpose to preserve the bladder, preoperative MVP-CAB): Four of 7 patients achieved a partial response. It was possible to perform bladder preservation surgery in 3 of these 4 patients. All 3 patients had pedunculated, solitary tumors, and there was no carcinoma in situ. Group 2 (purpose to improve the prognosis; preoperative MVP-CAB): Hydronephrosis did not resolve in the 4 patients with this complication. They received total cystectomy, and 2 patients died of cancer 23 months later. Group 3 (purpose to improve the prognosis; postoperative MVP-CAB): Ten of 12 patients (total cystectomy; 10 patients, partial cystectomy; 2 patients) survived disease-free for an average of 17 months (5-44 months), 1 patient developed recurrence 12 months later, and 1 patient died of cancer 6 months later. The 1-year survival rate in Group 3 was 86% for TCC, 100% for SCC, 80% for grade 3 TCC, and 89% for pT2 or more advanced cancer.


[Functional and morphological changes in rat kidney induced by FK506 and its reversal by various vasodilators]. FK506, a newly developed immunosuppressive agent, has recently been used for human liver and kidney transplantation. The present study was carried out to assess the functional and morphological changes by acute or chronic administration of FK506 in heminephrectomized rats. FK506 was given intravenously at the dose of 0.384 mg/kg/hr for 90 min in acute experiment. FK506 was administered by gastric gavage at doses of 1, 2.5, 5 mg/kg for 21 days. Blood and urinary biochemistry were monitored every week. Inulin and PAH clearance studies were conducted during the infusion of FK506 for acute study, and at day 21 for chronic study. Some of the rats were treated with diltiazem (Dilt), captopril or prazosine for 21 days to prevent FK506 nephrotoxicity. Acute infusion of FK506 did not change renal and systemic hemodynamics. Creatinine clearance showed a dose dependent decrease by 10-20% in the rats with chronic administration of FK506. Inulin/PAH clearance indicated a decrease in glomerular filtration rate and renal plasma flow with an increase in renal vascular resistance. The renal histology showed vacuolization in proximal tubuli and media of smooth muscle cells of arterioles. The administration of Dilt functionally and morphologically improved renal impairment induced by FK506. In conclusion, FK506 induces a dose dependent decrease in renal function with significant histological changes in tubuli and arterioles in rat kidney. Intracellular calcium deregulation seems to be involved in FK506-induced nephrotoxicity.


[Endocrine environment of benign prostatic hyperplasia--relationships of sex steroid hormone levels with age and the size of the prostate]. To determine the influence of endocrine factors on benign prostatic hyperplasia (BHP), the levels of three sex steroid hormones i.e., total testosterone (Total-T), free testosterone (Free-T) and estradiol (E2), were measured in serum of healthy 154 men. Their ages ranged from 18 to 91 years old. In 59 men, prostatic size was estimated by digital examination and was subdivided into three groups: smaller than or equal to walnut size, small hen's egg size and equal to or larger than hen's egg size. Firstly, relationships of sex hormone levels with age were studied. There was a slight decrease in Total-T over 60 years old, a significant decrease in Free-T, and no change in E2 with age. Thus, E2/Total-T and E2/Free-T ratio increased significantly after middle-age. Secondly, relationships of hormone levels with prostatic size were studied. In the larger prostate group, a significantly lower level of Total-T and significantly higher level of E2 were detected. But there was no difference in Free-T. Thus, the prostatic size was correlated positively with E2 level, E2/Total-T and E2/Free-T ratio. These suggest that the endocrine environment tended to be estrogens-dominant with age, in particular, after middle-age, and that patients with large prostates have more estrogens-dominant environments. We conclude that estrogens are key hormones for the induction and the development of BPH.


[The status of biological risk factors and the optimality concept in diagnosis of early childhood developmental disorders]. In a prospective study the psychomotor development up to the end of the second year of life of 409 preterm and term newborn infants was examined in order to identify which optimality score might be associated with disturbances of normal childlike development. Significant connections between 24 and 55 risk factors and the mortality could be demonstrated, but only 3 factors (sex, apgar, acidosis) exert influence on psychomotor development. The risk loading of the collective was high, not one of the children showed an optimal score. Decreased patients possessed a stronger reduced optimality than survivors. The optimality concept is not suitable for the prediction of later disturbed development of children, because the items are unspecific. The marker of hypoxia, the erythrocytic-density-test, as well as the neuron-specific enolase showed better diagnostic values than the optimality concept.


The safety and efficacy of ten percent pentastarch as a cardiopulmonary bypass priming solution. A randomized clinical trial. Ten percent pentastarch is a low-molecular-weight hydroxyethyl starch with greater oncotic pressure and shorter intravascular persistence than 6% hetastarch. To evaluate its safety and efficacy as a component of cardiopulmonary bypass priming solution, we prospectively studied 90 patients undergoing coronary artery bypass grafting or valve replacement necessitating cardiopulmonary bypass (bubble oxygenator and moderate systemic hypothermia). Sixty patients were randomized to receive 75 gm of either 10% pentastarch (group P) or 25% albumin (group A), and 30 patients received lactated Ringer's solution alone (group C). Intravascular colloid osmotic pressure during cardiopulmonary bypass was highest with either of the colloid primes (15-minute measurement: group P, 15.7 +/- 2.2 mm Hg (mean +/- standard deviation); group A, 15.2 +/- 2.0 mm Hg; group C, 11.3 +/- 1.7 mm Hg; p less than 0.05, groups P and A compared with group C). This was associated with a lower volume requirement during cardiopulmonary bypass to maintain the venous reservoir (group P, 333 +/- 318 ml; group A, 483 +/- 472 ml; group C, 1332 +/- 1013 ml; p less than 0.05, groups P and A compared with group C). Urine output during cardiopulmonary bypass was similar in each group. Net intraoperative fluid balance was lowest in the colloid groups (groups P and A, 5.7 +/- 1.4 L; group C, 6.9 +/- 1.3 L; p less than 0.05, groups P and A compared with group C). Cardiac index shortly after weaning from cardiopulmonary bypass was greatest in group P (group P, 3.2 +/- 0.9; group A, 2.8 +/- 0.8; group C, 2.7 +/- 0.6 dyne.sec.cm-5; p less than 0.05, group P compared with group C). Changes in alveolar-arterial oxygen gradients, shunt fraction, and effective compliance were similar in all groups. During cardiopulmonary bypass, pentastarch appeared to cause the greatest degree of hemodilution, as suggested by the lowest hemoglobin, factor VII and IX levels and platelet count. The activated partial thromboplastin time was significantly prolonged during and immediately after cardiopulmonary bypass in group P relative to groups A and C (p less than 0.05), although there were no significant differences in the activated clotting time before cardiopulmonary bypass, during cardiopulmonary bypass, or after heparin neutralization. As well, clinical indices of hemostasis, including mediastinal drainage, red cell, platelet, and fresh frozen plasma requirements, and reoperation for excessive postoperative bleeding, were similar. We conclude that pentastarch, when used in cardiopulmonary bypass prime, is as safe as either albumin or Ringer's solution alone.(ABSTRACT TRUNCATED AT 400 WORDS)


Late sudden cardiac death in the follow-up of patients having a heart valve prosthesis. Eight hundred thirty-one patients with Björk-Shiley prostheses (341 aortic, 345 mitral, and 145 double) had a mean follow-up time of 68.2 months per patient. Of these, 24 (16% of all deaths) died suddenly: six of 42 with aortic valve replacement (14.3%), 12 of 56 with mitral valve replacement (21.4%), and six of 36 with double valve replacement (16.6%). This correlated with evidence of premature ventricular complexes detected in multiple routine 12-lead follow-up electrocardiograms (p less than 0.001 for simple aortic or mitral valve replacements and p less than 0.01 for combined aortic and mitral valve replacements). Premature ventricular complexes were significantly more frequent among those who died suddenly than among survivors and those who died of other causes (p less than 0.001 in both cases); there were no significant differences between the latter two groups. The actuarial risk of sudden death was significantly greater among those patients who had premature ventricular complexes than among those who did not have this disorder (p = 0.0002). We conclude that the presence of premature ventricular complexes, as an independent variable, was correlated with the risk of sudden death.


Treatment of recurrent head and neck cancer with cisplatin and 5-fluorouracil vs. the same plus bleomycin and methotrexate. A prospective randomized trial of 62 patients with recurrent squamous cell carcinoma of the head and neck was conducted to compare the effectiveness of our standard chemotherapy program with that of our test regimen. The standard chemotherapy regimen consisted of cisplatin 80 mg/M2 on day 1 followed by 5-fluorouracil 800 mg/M2 days 2 through 6. Our test regimen consisted of the same two drugs plus 15 U bleomycin on day 1 and methotrexate 100 mg/M2 on day 16 followed in 24 hours with 15 mg leucovorin every 6 hours for six doses. One patient in each arm of the study was not evaluated. Among 29 patients receiving the two-drug regimen, there was 1 complete response and 10 partial responses (38% response rate). Among 31 patients receiving the four-drug regimen, there were 3 complete responses and 16 partial responses (61% response rate; two vs. four-drug regimen, P = .06). The failure-free survival in the four-drug group was better than the two-drug group, median 4.5 vs. 2.3 months (P = .02). The overall survival for both groups was the same (median of 7.8 months). A detailed analysis of toxicity did not reveal any important differences between the two regimens. The addition of bleomycin and methotrexate to our cisplatin and 5-fluorouracil regimen resulted in an increase in effectiveness without adding toxicity.


Demonstration of synergistic effects of hyperthermia and photodynamic therapy using the chick chorioallantoic membrane model. The chick chorioallantoic membrane (CAM) model was used to study vascular effects of photodynamic therapy (PDT) and hyperthermia (HPT) and the synergism of these modalities. The CAM is a convenient medium for monitoring the modifications of the vasculature. It is possible to view the CAM and to examine structural changes of individual blood vessels in real time. Moreover, the CAM is a closed system which lends itself to mathematical modeling of the temporal and spatial temperature profile and in which HPT can be performed quantitatively and to a selected depth, using different lasers. A porphyrin-type photosensitizer solution was applied to areas of the CAM, defined by teflon O-rings placed on the surface. Uptake dynamics of the sensitizer into the CAM was determined by analyzing its fluorescence in vivo. The CAM area was irradiated with a dual-wavelength laser system composed of a dye laser at 644 nm (to induce PDT) and a CO2 laser at 10.6 microns (to bring about HPT). Damage to the CAM vasculature, due to combined PDT+HPT, was compared to the outcome of the separate modalities, and a synergistic effect of about 40% was observed.


Cloning and functional expression of a cDNA encoding the mouse beta 2 subunit of the kainate-selective glutamate receptor channel. The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.


Synthesis and transport of GAP-43 in entorhinal cortex neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis. Metabolic labeling and quantitative 2D gel autoradiography were used to assess changes in the synthesis and transport of GAP-43 in entorhinal cortex (EC) neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis. In normal adult rats, there is a high constitutive level of GAP-43 synthesis and transport in EC neurons projecting to the hippocampus. Following unilateral EC lesions, there is a 2-fold (100%) increase in the transport of newly synthesized GAP-43 to the contralateral or 'sprouting' hippocampus. The timing of this upregulation (between 6 and 15 days) suggests that changes in GAP-43 expression occur in response to the growth of presynaptic terminals during sprouting.


PCR-amplified length polymorphisms in tRNA intergenic spacers for categorizing staphylococci. The intergenic spacers between some adjacent tRNA genes were shown to be polymorphic in length when closely related Staphylococcus species were compared. A simple procedure was developed to detect and sequence these tRNA intergenic length polymorphisms (tRNA-ILPs). A comparison of homologous tRNA gene sequences flanking these ILPs in three Staphylococcus species was used to derive primers for high-stringency amplification of the ILPs by the polymerase chain reaction (PCR). The detection of tRNA-ILPs by PCR allowed the classification of virtually all strains from the five species of Staphylococcus that were examined. The procedure used to identify, sequence and derive primers for PCR detection of tRNA-ILPs in Staphylococcus should be applicable to many other genera of eubacteria. These primers could be used on uncultured material such as clinical samples.


In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501. Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of pIP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, pI, pII and pIII within this region. A putative fourth promoter (pIV) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter pI while the repR gene is transcribed from promoter pII located just downstream of copR. The pII transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter pIII. The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR expression is proposed. Comparative analysis of the replication regions of pAM beta 1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.


Localised mutagenesis of the fts YEX operon: conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients. After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX, whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.


Structure and functional expression of the acid-labile subunit of the insulin-like growth factor-binding protein complex. Nearly all of the insulin-like growth factor (IGF) in the circulation is bound in a heterotrimeric complex composed of IGF, IGF-binding protein-3, and the acid-labile subunit (ALS). Full-length clones encoding ALS have been isolated from human liver cDNA libraries by using probes based on amino acid sequence data from the purified protein. These clones encode a mature protein of 578 amino acids preceded by a 27-amino acid hydrophobic sequence indicative of a secretion signal. Expression of the cDNA clones in mammalian tissue culture cells results in the secretion into the culture medium of ALS activity that can form the expected complex with IGF-I and IGF-binding protein-3. The amino acid sequence of ALS is largely composed of 18-20 leucine-rich repeats of 24 amino acids. These repeats are found in a number of diverse proteins that, like ALS, participate in protein-protein interactions.


Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene. Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone alpha-subunit gene that are required for tissue-specific expression. In the initial phase of these studies, a variant mouse alpha gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse alpha gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Transfection experiments using both mouse alpha gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human alpha gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse alpha-subunit genes has been examined in cells of the gonadotrope, thyrotrope, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the alpha gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific alpha expression in gonadotropes and thyrotropes.


The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin. The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. It has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10.(ABSTRACT TRUNCATED AT 250 WORDS)


A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin. Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the beta-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG beta gene(s). Data from Southern analyses suggest that the eCG beta and eLH beta subunits are products of the same single copy gene (eLH/CG beta). Overlapping fragments of the eLH/CG beta gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG beta gene spans less than 1.2 kilobase-pairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG beta subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG beta gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CG beta genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression.


Expression of hepatic insulin-like growth factor-I and insulin-like growth factor-binding protein-1 genes is transcriptionally regulated in streptozotocin-diabetic rats. Insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (BP-1) are critical cell regulators, with regulation and action in endocrine, paracrine, and autocrine modes. Although IGF-I and BP-1 are thought to be modulated mainly at the level of synthesis, underlying molecular mechanisms are poorly understood. To examine regulation by insulin, we used run-on assays to measure IGF-I and BP-1 gene transcription rates in nuclei isolated from the livers of normal and diabetic rats. Streptozotocin (STZ)-treated rats exhibited 20-25% weight loss, a 2.5- to 3-fold increase in serum glucose, and a 50-60% fall in circulating IGF-I levels (all P less than 0.001). Diabetic animals also had a 45% reduction in hepatic IGF-I mRNA and over 400% increases in BP-1 mRNA (both P less than 0.005); all parameters were restored toward normal after treatment with insulin. Metabolically responsive IGF-I gene transcription was evaluated effectively with a 3.2-kilobase BglII/EcoRI genomic probe located down-stream from all initiation sites in exon 1, while BP-1 gene transcription was studied with a cDNA probe. Animals treated with 144 mg/kg STZ exhibited 50-97% decreases in IGF-I gene transcription (P less than 0.05), while insulin treatment raised IGF-I gene transcription to control levels (P less than 0.02). IGF-I gene transcription appeared to be more sensitive to metabolic status than IGF-I mRNA levels, resulting in a modest correlation between transcription rates and mRNA levels (r = 0.68; P less than 0.001). In contrast, changes in BP-1 mRNA and gene transcription appeared to be exquisitely sensitive to metabolic status.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural origin of the immunological diversity of two closely related tetrapeptides: CIDNP study of TyrTyrGluGlu and TyrGluTyrGlu epitopes. Photochemically induced dynamic nuclear polarization (photoCIDNP) measurements, specific for exposed tyrosine residues, have been applied to elucidate conformational differences responsible for the immunological diversity of the synthetic multichain copolymers, Tyr1Tyr2Glu3Glu4-poly-DL-Ala-poly-Lys and Tyr1Glu2Tyr3Glu4-poly-DL-Ala-poly-LS. These two copolymers are essentially identical in their molecular weight, size, shape and composition, and differ only in the order of the two internal amino acid residues within the sequence of the tetrapeptide epitopes. Nonetheless, previous studies have shown that the two macromolecules behave differently, as evidenced by their immunological and immunogenic properties. As immunogens they act under different genetic control mechanisms, and differ in their interactions with antigen presenting cells, T cells and B cells. Antibodies elicited against these two antigens do not cross react. The photoCIDNP measurements of these two polymers, intended to elucidate discrete structural differences controlling immune recognition, showed that in the TyrTyrGluGlu polymer, Tyr1 and Tyr2 rings are free, non-interacting and undergo fast internal rotation. Computed minimum energy conformations confirm these conclusions and indicate that Tyr1 and Tyr2 point to different regions in space. In TyrGluTyrGlu, however, CIDNP measurements give rise to one broad tyrosine 3,5 proton signal, the result of a strong Tyr1-Tyr3 hydrophobic interaction. These two tyrosine residues are thus close in space, and undergo slow internal rotation. These results are in agreement with the computed minimum energy conformations.


Recognition of multiple epitopes in the coiled-coil domain of lamin B by human autoantibodies. The nuclear lamina of mammalian cells consists of three major proteins, lamins A, B and C, which form a fibrous meshwork interposed between the inner nuclear membrane and the chromatin. Sera from certain patients with systemic lupus erythematosus (SLE) and autoimmune liver disease contain high titers of autoantibodies against lamin B. We have shown previously that anti-lamin B autoantibodies in SLE recognize epitopes highly specific for lamin B, even though lamin B and lamins A/C are highly homologous proteins. To further characterize the specificities of these autoantibodies, fusion proteins carrying fragments of lamins B and C were tested for reactivity with SLE sera by immunoblotting. Five distinct epitopes of lamin B were identified, at least four of which were located in the highly conserved coiled-coil rod domain. Epitopes located on amino acids (AA) 80-193 and 245-303 were recognized by 4/10 and 8/10 anti-lamin B positive sera, respectively. Affinity purified anti-lamin B autoantibodies reacted preferentially with lamin B, indicating that they recognized mainly portions of lamin B that differ from lamins A and C. On the contrary, most of the affinity-purified anti-lamin C autoantibodies from SLE sera cross-reacted with lamin B, suggesting that the anti-nuclear lamina immune response in these patients is directed primarily against lamin B. The preferential reactivity of these sera with multiple epitopes specific to lamin B, and the finding that the autoantibodies to lamins A and C present in some of these sera cross-react with lamin B suggest that autoantibodies to lamin B are generated in response to the authentic lamin B protein rather than a cross-reactive foreign protein.


At least five antigenic epitopes on the streptokinase molecule are recognized by human CD4+ TCR alpha beta+ T cells. The streptokinase molecule (415 AA) was cleaved at methionine 237, 347 and 370 yielding four polypeptide fragments. Human HLA-class II restricted streptokinase-specific T cell clones and cell lines (CD2+, CD3+, CD4+, CD8-, TCR alpha beta+, TCR gamma delta-) recognized antigenic epitopes on all four fragments AA 1-236, AA 238-346, AA 348-369 and AA 371-415. T cell clones recognizing fragment AA 1-236 were restricted by at least two different HLA-class II elements, this indicating that more than one antigenic epitope can be recognized on this fragment. In addition, two streptokinase-specific T cell clones recognized only the intact molecule and none of the molecular fragments. These two clones probably recognized an antigenic epitope including one of the methionine residues used for molecular cleavage. We conclude that T cell proliferative responses to streptokinase are determined by recognition of at least five different antigenic epitopes distributed along the entire streptokinase polypeptide chain.


Demethylation of the constant region genes of immunoglobulins reflects the differentiation state of the B cell. Previous results showed a developmentally regulated, strong linkage between demethylation and transcriptional activity for the light chain kappa locus in the mouse (Kelley et al., Molec. cell. Biol. 8, 930-937, 1988). These results indicate the existence of a stage of development of the B cell in which permanent expression (which may be enhancer independent) of a gene is associated with its demethylation. According to this result, demethylation could mirror terminal differentiation of a cell. We tested this hypothesis by analyzing the methylation status of immunoglobulin (Ig) genes in normal B cells before and after their activation with lipopolysaccharide (LPS) to induce IgM secretion and an immunoglobulin class switch. This pattern of methylation has been compared with that of Ig genes in nonlymphoid tissues and in transformed cell lines. In general, transformed cells are terminally differentiated cells. Our results show, that in normal splenic B cells only regions proximal to the heavy chain enhancer are demethylated. The coding regions of the c mu, c delta and the c gamma 1 genes remain methylated regardless of transcription. Demethylation of the coding regions is only detectable in transformed cell lines. Hence demethylation of immunoglobulin genes may reflect a stage of terminal differentiation in which the transcription pattern of the cell is fixed. Methylation of the genes before terminal differentiation may be necessary to allow controlled expression of genes on the transcriptional level, such as by splicing and differential termination.


Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-alpha(1----6)dextrans with groove- or cavity-type combining sites. The variables regions of three syngeneic anti-idiotypic antibodies (Ab2s) were cloned and sequenced. They are encoded by different VL genes, two are from different members of V kappa-Ox1 superfamily. The H chains are encoded by VH genes belonging to three different VH families, J558, Q52 and 7183. Together with a previous report from this laboratory, the nucleotide sequences of four Ab2s to anti-alpha(1----6)dextrans have been presented. They are derived from a number of unrelated germline genes, and differ from similar studies in anti-NP, anti-GAT and anti-Ars systems. Three of four Ab2s in the anti-alpha(1----6)dextran system appear to have D-D fusions, which has also been reported in several other Ab2s.


The peptide binding specificity of HLA class I molecules is largely allele-specific and non-overlapping. To understand better the specificity of peptide binding by MHC class I molecules, we have evaluated the capacity of a panel of unrelated peptides to compete for the presentation of viral peptides presented by HLA-A3 and HLA-B27. The HIV-Nef7F peptide (74-82) was presented by HLA-A3 to Nef-specific HLA-A3-restricted CTL lines, and the influenza nucleoprotein peptide NP(380-393) was presented by HLA-B27 to NP(380-393)-specific HLA-B27-restricted CTL lines. In addition, we have extended studies from our group that have evaluated the capacity of a similar panel of peptides to inhibit presentation of an influenza nucleoprotein peptide NP (335-349) by HLA-B37 and a matrix peptide, M1 (57-68), by HLA-A2 to the appropriate peptide-specific CTL lines. Out of 41 peptides tested, only five bound to more than one of the MHC molecules analyzed. Pairwise comparisons of the peptide binding specificities among these four different class I molecules revealed no common competitor peptides in four of the six possible comparisons. Thus, each class I molecule appears to have a functionally distinct peptide binding site, as reflected by the ability to bind largely non-overlapping sets of peptides.


Localisation of linear epitopes at the carboxy-terminal end of the mycobacterial 71 kDa heat shock protein. Four distinct linear epitopes localized within species-specific sequences at the carboxy-terminal end of the 71 kDa heat shock protein of M. tuberculosis have been identified by scanning 94 overlapping peptides with 13 human sera. One epitope ("C") of entirely M. tuberculosis-specific core sequence (GEAGPG) has been found immunogenic in smear-negative tuberculosis, but not in non-tuberculous mycobacterial diseases. This peptide appears to be a valuable candidate for further serodiagnostic evaluation.


Clastogenicity of 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells. The clastogenicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated at various exposure conditions. CS efficiently induced chromatid-type aberrations in a dose-dependent manner provided the cells could run through at least one or two S-phases during a 20-h exposure over a 3-h exposure followed by a 20-h recovery period (cell cycle time 8-10 h). The induction of SCEs indicates an S-dependent mechanism. The hydrolysis products o-chlorobenzaldehyde and malonitrile were inactive in these experiments.


Mutagenicity of methyl methanesulfonate and cyclophosphamide in resting and growing Saccharomyces cerevisiae D7 cells. In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp+ convertants) and point reverse mutation (ilv+ revertants) in log or stationary phase cells after either 4 or 16 h of treatment. The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells. The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase cells. This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells. Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.


Decreased electrophilicity of chemicals carcinogenic only at the maximum tolerated dose. While there was no significant difference between the actual or predicted mutagenicity and clastogenicity of a group of chemicals carcinogenic only at the maximum tolerated dose (MTD) and a group of chemicals carcinogenic below the MTD, as a group, the chemicals carcinogenic below the MTD exhibited a significantly decreased LUMO (Lowest Unoccupied Molecular Orbital) energy, indicative of increased electrophilicity (i.e. DNA reactivity). These findings suggest that chemicals carcinogenic only at the MTD either require increased doses of "weak" electrophiles to be carcinogenic or that they may act by a "non-genotoxic" mechanism.


UV resistance of E. coli K-12 deficient in cAMP/CRP regulation. Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.


Inhibition of sulfotransferase affecting unscheduled DNA synthesis induced by 2-acetylaminofluorene: an in vivo and in vitro comparison. The unscheduled DNA repair (UDS) assay was conducted using the in vivo and in vitro procedures to investigate the role of arylsulfotransferases (AST) in the genotoxicity of 2-acetylaminofluorene (AAF). The in vivo assay had 4 groups of rats that consisted of those treated with pentachlorophenol (PCP), PCP and AAF, or AAF and an untreated control. The in vitro assay used hepatocytes from 3-methylcholanthrene or corn oil (control) treated rats. In both the in vivo and in vitro UDS assays AAF induced DNA damage. PCP, an inhibitor of arylsulfotransferase, significantly decreased AAF induced DNA damage. In the in vivo assay, PCP induced a significant increase in UDS and confounded an investigation of the role of sulfotransferase. The in vitro UDS assay more clearly defined the effect of PCP on AAF genotoxicity.


Chromosomal analysis of Chinese hamster V79 cells exposed to multiple gamma-ray fractions: induction of adaptive response to mitomycin C. Chinese hamster V79 cells were irradiated daily with 0.3 Gy of gamma-rays 5 times per week for 12 weeks (total 18 Gy). These cells were challenged with an additional dose of 15. Gy gamma-rays or treated with 5 micrograms/ml of mitomycin C (MMC) for 2 h. In spite of the high total accumulated dose of gamma-rays, the number of chromosomal aberrations and sister-chromatid exchanges (SCEs) did not significantly increase in the preirradiated cells, as compared to control cells. If preirradiated cells were challenged with an additional 1.5 Gy of gamma-rays, an insignificant decrease in the yield of chromatid aberrations was observed. In contrast, preirradiated cells became significantly more resistant to the induction of chromosomal damage when challenged with mitomycin C. Our results suggest that multiple fractions of gamma-rays can induce the adaptive response to mitomycin C in preirradiated cells.


X-rays, microwaves and vinyl chloride monomer: their clastogenic and aneugenic activity, using the micronucleus assay on human lymphocytes. Chromosome aberration assays, sister-chromatid exchange techniques and micronucleus assays are commonly used methods for biomonitoring genetic material damaged by chemical or physical agents. On the other hand, their aneugenic activity, which can lead to hypoploidy and may also be associated with carcinogenesis, has not been thoroughly investigated. In our study we chose the micronucleus assay with a new mathematical approach to separate clastogenic from aneugenic activity of three well-known mutagens (vinyl chloride monomer, X-rays and microwaves) on the genome of human somatic cells. The comparison of frequencies of size distribution of micronuclei in the lymphocytes of humans exposed to each of these three mutagens showed that X-rays and microwaves were preferentially clastogens while vinyl chloride monomer showed aneugenic activity as well. Microwaves possess some mutagenic characteristics typical of chemical mutagens.


Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium. Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens. It may therefore play a key role in the metabolic activation of xenobiotics. The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays. The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested. When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activation by this mammalian peroxidase. The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet. Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens. Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes. Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems.


Are mitotic index and lymphocyte proliferation kinetics reproducible endpoints in genetic toxicology testing? Lymphocyte proliferation kinetics is an endpoint used in genetic toxicology which has recently been proposed as an alternative for the screening of new cytostatic drugs. Although great variability for this parameter has been reported, there are few reports about the intra- and inter-individual variation of the effects of chemicals on this endpoint. For this reason, experiments were conducted to evaluate the reproducibility of the effects of a well-known cytostatic, mitomycin C (MMC), on the proliferation of PHA-stimulated human lymphocytes, both over time and among samples from several donors. Although inter-individual variability was shown in both parameters in untreated and treated cultures, this variation was not significant. Intra-individual variation was significantly detected only in cultures treated with 0.1 microM MMC.


Dextrans as markers for endocytosis in innervated and denervated skeletal muscle. Fluorescence-labeled dextrans were evaluated as markers for endocytosis in skeletal muscle. Fluorescein isothiocyanate (FITC)-labeled dextrans (average molecular weight 3900 to 71200) showed a higher uptake in denervated than in innervated muscle both in vitro and in vivo. The in vitro uptake of FITC-dextran (35.600) increased linearly with time at 37 degrees C, and was almost completely inhibited by low temperature (4 degrees C). The uptake was not a pure bulk uptake, because a saturable component was evident from the concentration dependence and from competition experiments with unlabeled dextran. The uptake of FITC-labeled or rhodamine B isothiocyanate (RITC)-labeled dextrans in denervated muscle occurred mainly in small segments of the fibers centered around the denervated endplate region. However, not all denervated fibers showed such segments. Periodic acid Schiff's base staining for carbohydrates stained dextrans in denervated muscle fibers. Some staining, probably of lysosomes, was also observed in denervated muscle not exposed to dextran.


Vasculitic neuropathy in a patient with cryoglobulinemia and anti-MAG IGM monoclonal gammopathy. A patient with sensorimotor mononeuritis multiplex had a type II cryoglobulin with an IgM kappa M-protein that appeared to contain monoclonal anti-MAG antibodies of the same isotype. A sural nerve biopsy demonstrated necrotizing arteritis and features of both axonal degeneration and demyelination. IgM kappa and C3 deposits were present on the myelin sheath of some residual nerve fibers. The findings suggest that the anti-MAG antibodies contributed to the myelin damage, while cryoprecipitates may have caused the vasculitis and axonal degeneration.


Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.


Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis. Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.


Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis.


Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell growth and differentiation. We have shown that integrin/ligand interactions can trigger tyrosine phosphorylation of a protein of M(r) 120,000 (pp120), so it is possible that signal transduction by integrins might involve activation of intracellular protein tyrosine kinases as an early event in cell binding to the extracellular matrix. Here we report that pp120 is identical to the focal adhesion-associated protein tyrosine kinase pp125FAK (refs 3, 4). We show that tyrosine phosphorylation of this protein is modulated both by cell adhesion and transformation by pp60v-src, and that these changes in phosphorylation are correlated with increased pp125FAK tyrosine kinase activity. A model is proposed to relate these findings to the molecular basis of anchorage-independent growth of transformed cells.


Maternal serum screening for fetal Down syndrome in women less than 35 years of age using alpha-fetoprotein, hCG, and unconjugated estriol: a prospective 2-year study. To evaluate prospectively maternal serum screening with alpha-fetoprotein (AFP), hCG, and unconjugated estriol (uE3) as a screen for fetal Down syndrome. Women less than 35 years of age were offered screening between 15-20 weeks' gestation. Screening results calculated by an algorithm to be equal to or greater than 1:274 (the risk of a 35-year-old for fetal Down syndrome at the second trimester) were considered positive. If gestational age was confirmed by ultrasonography, genetic counseling and amniocentesis were offered. In the first 2 years of our program, 9530 women were screened, of which 686 (7.2%) were found to be screen-positive. Ultrasonographic examination explained the abnormal values in 379 (4.0%). The remaining 307 (3.2%) received genetic counseling and 214 (2.2%) elected amniocentesis or CVS. Four cases of fetal Down syndrome and one de novo chromosomal marker were detected. In three additional cases of fetal Down syndrome, triple-analyte screening failed to identify the pregnancies to be at increased risk. None of the seven cases of fetal Down syndrome would have been detected through screening with maternal serum alpha-fetoprotein (MSAFP) and age alone. Measurement of MSAFP, hCG, and uE3 in women less than 35 years old is an effective screening test for fetal Down syndrome, with a sensitivity of 57% in our study and an amniocentesis rate (false-positive rate) of 3.2%.


Risk factors for developmental disorders in infants born to women with Graves disease. To identify risk factors for disorders of fetal growth and thyroid function in the presence of maternal Graves disease. Two hundred thirty pregnancies in gravidas with Graves disease were analyzed. Maternal thyroid status was evaluated by serum free thyroxine (T4) or free T4 index, TSH, and TSH-receptor antibody; personal history of thyrotoxicosis and total dose of antithyroid drugs during pregnancy were also noted. Neonatal thyroid function was assessed at birth and on the fifth day after birth. Fifteen neonates (6.5%) were small for gestational age (SGA), and this occurrence was significantly associated with thyrotoxicosis lasting for 30 weeks or more of pregnancy, TSH-receptor antibody level of 30% or more at delivery, history of Graves disease of 10 years or longer, and onset of Graves disease before 20 years of age. However, no significant correlation was found between maternal thyroid hormone level and SGA neonates. Thyroid dysfunction developed in 38 infants (16.5%), of whom only four were SGA; development of this dysfunction was significantly related to the mother's total dose of antithyroid drugs, duration of thyrotoxicosis in pregnancy, and/or TSH-receptor antibody level at delivery. Duration of maternal Graves disease or thyrotoxicosis, either mild chemical or overt, in pregnancy is significantly associated with SGA neonates. Neonatal thyroid dysfunction is associated with the maternal thyroid condition, especially the serum TSH-receptor antibody level.