Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts. Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.


High affinity binding of the leucocyte adhesion molecule L-selectin to 3'-sulphated-Le(a) and -Le(x) oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides. The binding of the leucocyte adhesion molecule L-selectin has been investigated toward several structurally defined lipid-linked oligosaccharides immobilized on silica gel chromatograms or plastic wells. In both assay systems the 3'-sulphated Le(a)/Le(x) type tetrasaccharides [formula: see text] were more strongly bound than 3'-sialyl analogues. A considerable binding was observed to the 3'-sulphated oligosaccharide backbone in the absence of fucose but not to a 3'-sialyl analogue or fuco-oligosaccharide analogues lacking sulphate or sialic acid. Affinity for other sulphated saccharides: 3'-sulphoglucuronyl neolactotetraosyl ceramide and glycolipids with sulphate 3'-linked to terminal or sub-terminal galactose or N-acetylgalactosamine was detected in the chromatogram assay only. These studies, together with earlier reports that L-selectin binding to endothelium is inhibited by sulphatide, highlight the relative importance of sulphate in the adhesive specificity of this protein.


Dityrosine formation is impaired by tyrosine phosphorylation. Using pure tyrosine and phosphotyrosine we have recently shown that phosphotyrosine is unable to form peroxidase catalyzed dimers (1989, FEBS Lett. 255, 395-397). In the present report, the effect of phosphotyrosine residues within a protein structure on dityrosine formation was studied using casein as a model protein. Dephosphorylation of casein resulted in a dose and time dependent increased synthesis of dityrosines following treatment with peroxidase/H2O2. The extent of crosslink formation was inversely related to the amount of phosphorylated tyrosine residues as quantitated by immunoblotting. Thus, phosphorylation of tyrosine residues could play a regulatory role in protein-crosslinking where dityrosine bonds are involved.


Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Vasoconstrictors such as angiotensin II (ang II) stimulate vascular smooth muscle cell growth and share many signal transduction mechanisms with growth factors. Recently, growth factors have been shown to stimulate mitogen-activated protein (MAP) kinases, a family of serine/threonine protein kinases which phosphorylate pp90rsk, a cytosolic kinase that phosphorylates ribosomal S6 protein. We examined the effect of ang II on MAP kinase activity and phosphorylation. Ang II stimulated MAP kinase activity by 4-fold after 5 min exposure and also increased tyrosine phosphorylation of 42 kDa (74 +/- 41%) and 44 kDa (263 +/- 85%) proteins, shown to be pp42mapk and pp44mapk by Western blot analysis using a MAP kinase antibody. These results suggest that ang II-stimulated protein synthesis is mediated by a MAP kinase dependent pathway.


Graves' IgG recognizes linear epitopes in the human thyrotropin receptor. Twenty-nine peptides covering the full extracellular domain of the human thyrotropin receptor have been synthesized and tested for reactivity with Graves' patients' and normal sera in ELISA. Two peptides, amino acids 331-350 and the second extracellular loop of the transmembrane segment, bound IgG-s from 5 and 4 of 10 Graves' disease patients' sera, respectively. Neither of these two peptides showed enhanced binding to normal IgG. There were no apparent differences between the Graves' disease and normal group with respect to the other 27 peptides. We conclude that peptide 331-350 and the second extracellular loop carry important linear epitopes which may contribute to the disease process in selected Graves' patients.


Deletion mutagenesis of stem cell factor defines the C-terminal sequences essential for its biological activity. We constructed a series of murine stem cell factor (mSCF) cDNAs which were sequentially truncated at the 3' termini. The resultant six mutant cDNA encode N-terminal 183, 179, 162, 149, 142 and 133 amino acid residues of the mature mSCF protein fused to the heterogeneous C-terminal peptides derived from the linker sequences. Each mutant cDNA was transiently expressed in COS cells, and the cultured supernatant was assayed for its ability to support the growth of a human factor-dependent cell line, TF-1 and to enhance colony formation by murine hematopoietic progenitor cells. The results showed that as few as N-terminal 142 but not 133 amino acid residues of mSCF remained biologically active in vitro, suggesting that the region of 9 amino acids from Asp134 to Ser142 containing a Cys138-mediated disulfide bond may contribute to the C-terminal end of the active subdomain of mSCF.


Molecular cloning of the acid-labile subunit of the rat insulin-like growth factor binding protein complex. The insulin-like growth factors, IGF-I and IGF-II, circulate in both humans and rats as part of a 125-150 kDa complex comprising IGFs, the IGF binding protein IGFBP-3, and an acid-labile subunit. Clones encoding rat acid-labile subunit have been isolated from a rat liver cDNA library probed with a human acid-labile subunit cDNA. Two overlapping clones encode a leucine-rich protein of 576 amino acids preceded by a 27-residue signal sequence, with 78% homology to the human acid-labile subunit. Northern analysis of mRNA from adult rat brain, kidney, heart, lung, spleen, muscle and liver shows a major species of about 4.4 kb and minor bands of about 2 kb, 1.4 kb and 1 kb. The tissue distribution of this protein may therefore be wider than previously recognized.


Interleukin-2 (IL-2) induces erythroid differentiation and tyrosine phosphorylation in ELM-I-1 cells transfected with a human IL-2 receptor beta chain cDNA. The molecular mechanism of erythroid differentiation has been still ill-defined. In this study, we introduced a human interleukin-2 receptor (IL-2R) beta chain cDNA into ELM-I-1 cells which differentiated into hemoglobin-positive cells in the presence of erythropoietin (Epo), and established the transformant which expressed IL-2R beta chain. In this transformant, we revealed that IL-2 induced erythroid differentiation and the same pattern of tyrosine phosphorylation as Epo. These data suggest that tyrosine phosphorylation is involved in signal transduction pathway of erythroid differentiation. It is also implicated that the Epo and IL-2 receptor system share a common signal transduction pathway.


Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation. Interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) and increased the cyclic GMP (cGMP) concentration in the cells. The dose dependencies of the two effects were similar (IC50 = 4 U/ml for the anti-proliferation and EC50 = 3 U/ml for cGMP formation) and the effect of IFN-gamma was enhanced by tumor necrosis factor-alpha treatment. Furthermore, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, inhibited both activities induced by IFN-gamma. These findings show that the anti-proliferation and cGMP formation are closely related and that IFN-gamma inhibits the proliferation of rat VSMC by generation of NO through the induction of an NO synthase.


Differential gene expression and regulation of type-1 angiotensin II receptor subtypes in the rat. A simplified and sensitive method for measuring expression levels of type-1 angiotensin II (AT1) receptor subtypes, AT1A and AT1B, was established. The two receptor cDNAs were co-amplified and measured by polymerase chain reaction using primers based on the corresponding receptor subtype genes. Both AT1A and AT1B mRNAs were widely expressed in the rat tissues including adrenal gland, kidney, heart, aorta, lung, liver, testis, pituitary gland, cerebrum and cerebellum. AT1A mRNA was predominantly expressed in the rat tissues examined except adrenal gland and pituitary gland where AT1B mRNA was predominantly expressed. Sodium depletion did not change mRNA levels of AT1A and AT1B in the all tissues. However, both AT1A and AT1B mRNA levels in the heart and aorta were down-regulated by treatment with AT1 specific antagonist, TCV 116. In contrast, AT1B mRNA in the adrenal gland was mainly reduced by the treatment. These results suggest that the expression level of AT1B mRNA in the adrenal gland depends on the activity of the renin-angiotensin-aldosterone system (RAAS) and both receptor subtypes mediate contraction and hypertrophy of the smooth and cardiac muscles via the RAAS.


Regulation of manganese superoxide dismutase: IL-1 and TNF induction in pulmonary artery and microvascular endothelial cells. IL-1 and TNF are important mediators in the inflammatory response, and have been associated with endothelial cell damage in the lung. TNF and IL-1 cell-mediated injury has been proposed to occur through an increase in intracellular oxygen free radical production. However, these cytokines have also been shown to protect the lung from hyperoxia-mediated oxidant injury. In this paper we evaluated the response of the antioxidant enzymes, MnSOD and Cu/ZnSOD to IL-1, TNF, and LPS in both rat pulmonary artery and microvascular endothelial cells. These mediators produced an increase in MnSOD but not Cu/ZnSOD expression in both rat pulmonary endothelial cells. An additive effect was observed with co-treatment by the cytokines with LPS. The MnSOD mRNA induction is dependent upon a transcriptional event, but did not require de novo protein synthesis.


Gamma-interferon counteracts interleukin-1 alpha stimulated expression of urokinase-type plasminogen activator in human endothelial cells in vitro. The effect of gamma-interferon (gamma-IFN) on the interleukin-1 alpha (IL-1 alpha) induced stimulation of urokinase-type plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and in human umbilical vein endothelial cells (HUVEC) was investigated. When gamma-IFN and IL-1 alpha were added to the cells simultaneously, gamma-IFN inhibited the IL-1 alpha induced increase in u-PA antigen production in both HFMEC and HUVEC in a dose dependent fashion, with a maximum inhibitory effect achieved between 2.0 and 20.0 U/ml of gamma-IFN. Pretreatment of HFMEC with gamma-IFN for 1 hour before addition of IL-1 alpha resulted in a significant reduction in u-PA synthesis. However, when HFMEC were pretreated for 8 hours with gamma-IFN before the addition of IL-1 alpha the reduction in u-PA production was even more significant. When gamma-IFN was added to HFMEC 1 hour after IL-1 alpha, a significant inhibition in u-PA synthesis was seen. In contrast only a slight inhibition in IL-1 alpha induced u-PA production was seen when gamma-IFN was added to the cells 8 hours after IL-1 alpha. gamma-IFN also inhibited significantly the IL-1 alpha induced increase in u-PA specific mRNA in HUVEC and HFMEC.


Roles of the aromatic residues in cobrotoxin in antigenicity and binding activity to nicotinic acetylcholine receptor. The single Trp-29 in cobrotoxin was modified with 2-hydroxy-5-nitrobenzyl bromide, and Tyr-25 and -35, with tetranitromethane. Although the binding activity of cobrotoxin to the nicotinic acetylcholine receptor (nAChR) was decreased after modification of Trp-29, the antigenicity remained essentially unchanged as measured by a competitive RIA. Tyr-35-nitrated cobrotoxin still retained relatively high antigenicity and potent binding activity to nAChR. However, modification of both Tyr-25 and Tyr-35 led to an altered secondary structure, with greatly reduced binding to antibody and receptor. Hence, Tyr-25 in cobrotoxin is essential for the maintenance of the active conformation for the bindings. Although the non-precipitating antibody against cobrotoxin exhibiting a higher neutralizing capacity than did the precipitating antibody, the binding affinity of the former to cobrotoxin and its modified derivatives was lower than that of the latter.


Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36). We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAPp37) from the plasma of a patient with Thrombotic Thrombocytopenic purpura (TTP), and have shown recently that p37 causes platelet agglutination through its binding to membrane glycoprotein IV (GPIV). To gain further insight into the mechanism of p37 binding to GPIV, we have studied the interaction between p37 and GPIV. We now demonstrate specific complex formation of p37 with GPIV. In Western immunoblotting p37 binds to purified GPIV and the complex formed between the two proteins was detected by polyclonal antibody to p37 and peroxidase conjugated second antibody. No binding of p37 was noticed with the purified GPIIIa. A solid phase binding assay was developed to study the complex formation. Microtiter wells were coated with GPIV and the control proteins; 125I-p37 was added, allowed to bind and bound radioactivity was measured. Several lines of evidence indicate that the binding of p37 to GPIV was specific. a) GPIV immobilized on Immulon-2 wells bound 10-30 fold more p37 than immobilized fibrinogen, GPIIIa, and BSA. b) Polyclonal antibodies against p37 and GPIV inhibited the binding by 39-68% as compared with control IgG. c) GPIIIa antibody did not inhibit the binding. Molecular sieve chromatography of a mixture of 125I-p37 and GPIV also revealed the fluid phase complex formation ranging in molecular weight from 132,000 to over 350,000 daltons. These results show the specific interaction between p37 and GPIV and suggest that GPIV functions as a p37 receptor during platelet agglutination.


Lack of effect of hypothyroidism on rat liver regeneration. Thyroid hormone has been postulated to be important for liver regeneration. In the present studies liver regeneration in hypothyroid male rats treated with methimazole was compared to euthyroid rats. At 5 days after 70% partial hepatectomy liver weight was significantly less in the hypothyroid rats, but total hepatic DNA and protein were the same in the hypothyroid and euthyroid rats and RNA was significantly greater in the hypothyroid rats. These results indicate that liver regeneration is nearly normal in hypothyroid rats, despite the fact that whole body growth has ceased due to the hypothyroidism. Since both thyroid and growth hormones are low in hypothyroid rats, these results suggest that neither thyroid nor growth hormones are required for liver regeneration.


Influence of immunogen modification of the gonadotropin releasing hormone antibody reactivity. The binding of MoAbs and PoAbs to native GnRH, azo-GnRH, GnRH-BSA and azo-GnRH-BSA conjugates was studied using a solid phase ELISA test. The use of carbonate/bicarbonate buffer (pH 9.2) significantly affected the coating of peptide to the ELISA plates. Azo-GnRH coated in PBS showed seven times less binding to MoAb than the native GnRH. Saturation analysis studies indicated comparable binding to the antibodies to the coated GnRH and GnRH-BSA but binding to azo-GnRH-BSA was higher than to azo-GnRH. The epitope recognition ability of MoAbs was therefore investigated. Blocking ELISA additivity tests were performed to analyse whether MoAbs recognised a common epitope involving a conformation or sequence of the modified peptide (azo-GnRH). The plates coated with azo-GnRH or azo-GnRH-BSA and saturated with MoAbs did not bind PoAbs. The inhibition activity was similar when GnRH or GnRH-BSA was used instead of azo-GnRH or azo-GnRH-BSA thus suggesting that the MoAbs were directed against a conformational epitope of the GnRH molecule.


Peptide fragments of myelin basic protein as substrates of protein kinase C. A set of peptides derived from myelin basic protein was synthesized and the kinetics of their phosphorylation by protein kinase C was studied. The replacement or the removal of the N-terminal Gln had no effect on the activity of the parent peptide. The removal of the following Lys or Arg led to a systematic decrease in substrate activity. The modifications in the C-terminal part of the peptide had a weaker influence on the parameters Vmax and KM than those in the N-terminal. The rather regular dependence of the activity of substrates upon their structure does not allow the strict definition of a minimum substrate for protein kinase C.


Senescent human fibroblasts are more sensitive to the effects of a phorbol ester on macromolecular synthesis and growth characteristics. 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) alters cellular growth properties by modulating gene expression in a wide variety of cell types. Human diploid fibroblasts MRC-5 were treated with PMA at different phases of their lifespan in vitro and the alterations of their short-term growth characteristics and macromolecular synthesis in response to PMA were analysed. PMA stimulates DNA and RNA synthesis in both Phase II (young) and Phase III (senescent) MRC-5 cells. Treatment of senescent cells with various PMA concentrations results in a greater stimulation of DNA and RNA synthesis than that in young cells. Senescent cells are also more sensitive to the PMA-induced alterations of growth characteristics and higher concentrations of PMA become toxic for them. The age-related alterations of cellular responsiveness are also apparent in the gradual loss of responsiveness to serum, observed in parallel with the increased sensitivity to PMA. Furthermore, serum-induced stimulation of macromolecular synthesis is inhibited by PMA. Since it is known that serum and PMA elicit their effects via different signal transduction pathways, our results point to suggest the differential regulation of the signalling mechanisms during cellular ageing.


Neutrophil priming by granulocyte colony stimulating factor and its modulation by protein kinase inhibitors. Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-.) generation, phagocytosis enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca(2+)-and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2-. generation in PMN was enhanced by the priming of recombinant human granulocyte colony stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2-. by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2-. generation from G-CSF were 0.5 and 5 microM, respectively. In contrast, O2-. generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genistein and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2-. might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2-. generation of neutrophils.


Bleomycin-induced unscheduled DNA synthesis in non-permeabilized human and rat hepatocytes is not paralleled by 8-oxo-7,8-dihydrodeoxyguanosine formation. The genetic toxicity of the antitumour antibiotic bleomycin (BLM) is thought to involve the formation of a reactive oxygen intermediate. 8-Oxo-7,8-dihydrodeoxyguanosine (oxo8dG), an oxidation product of deoxyguanosine, is one of the major products formed when isolated DNA is exposed to oxygen radical generating systems. Gamma-irradiation (10-500 Gy 60Co; 10 Gy/min) or BLM and Fe2+ (37.5-150 U/L and 0.5 mM, respectively) treatment of isolated DNA (0.25 mg/mL) increased oxo8dG above background. In the latter case, the effect was greater than that with Fe2+ (0.5 mM) alone and was dependent on the dose of BLM. When DNA was irradiated with 500 Gy60Co, deoxyguanosine oxidation was inhibited by antioxidants (ethanol: 37.5 and 98% inhibition at 2 and 20 mM, respectively; mannitol: 20.5, 60 and 92% inhibition at 0.1, 1.0 and 10 mM, respectively). Similarly the BLM-induced production of oxo8dG was inhibited (64%) by mannitol (10 mM). BLM also caused production of base propenals on interaction with isolated DNA. In contrast, oxo8dG was not induced above background concentration (27 mol oxo8dG/10(6) mol dG) in permeabilized (37 degrees) and non-permeabilized (4 degrees and 37 degrees) rat hepatocytes treated with BLM (260 U/L). Despite this, there was extensive BLM-induced unscheduled DNA synthesis (10 and 100 U/L) in non-permeabilized rat and human hepatocytes in the absence of hydroxyurea. These findings, in accord with other observations, draw into question the role of .OH in BLM-induced DNA damage and the mimicry of ionizing radiation in cellular systems.


Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes. Thirty-one flavonoids were tested for their effects on low Km phosphodiesterase with cyclic AMP as the substrate. Quercetin, luteolin, scutellarein, phloretin and genistein showed inhibitory potencies comparable to or greater than 3-isobutyl-2-methylxanthine (EC50 30-50 microM). Only four compounds namely, catechin, epicatechin, taxifolin and fustin stimulated the enzyme activity (stimulatory EC50 130-240 microM). The most potent phosphodiesterase (PDE) inhibitors were aglycones that had a C2.3 double bond, a keto group at C4 and hydroxyls at C3' and/or C4'. However, when the C-ring is opened then the requirement for the C2.3 double bond is eliminated. The same series of flavonoids were also tested for their lipolytic activity. The structural features required for effective synergistic lipolysis (with epinephrine) were generally similar to that required for potent PDE inhibition except that, for lipolytic activity, an intact C-ring was necessary. Fisetin and quercetin having the above-mentioned structure showed a dose- and time-dependent increase in lipolysis which was synergistic with epinephrine. Only butein and hesperetin showed inhibition of epinephrine-induced lipolysis, and their effect was dose-dependent. A time-course study indicated that hesperetin was able to delay the lipolytic action of epinephrine. It is most likely that the lipolytic effects of these compounds were not a result of PDE inhibition, as the orders of potency for the two activities had poor correlation. Apparently, the effective lipolytic flavonoids were also potent PDE inhibitors but not all the PDE inhibitors were able to induce lipolysis.


de novo synthesis of calmodulin binding protein in substance P-induced steroidogenesis in bovine adrenocortical cells. In order to clarify the mechanism of substance P (SP)-induced cortisol secretion from bovine adrenocortical (BAC) cells, protein synthesis at the early stage of SP-stimulation in BAC cells was investigated. Both SP and adrenocorticotropic hormone (ACTH) increased [3H]leucine uptake into BAC cells in a dose-dependent fashion. Although the SP-induced [3H]leucine uptake precedes the cortisol secretion, ACTH was slower in inducing [3H]leucine uptake and cortisol secretion. Protein synthesis inhibitors, actinomycin D and cycloheximide, were potent in inhibiting the SP-induced cortisol secretion. SDS-PAGE analysis, revealed that a 240 kDa protein is newly synthesized in BAC cells in response to SP but not ACTH. It was also indicated that the production of this 240 kDa protein was elicited about 30 min after stimulation by SP. Moreover, A23187 and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also caused a rapid [3H]leucine uptake and production of 240 kDa protein. In contrast, dibutyryl cAMP did not induce the synthesis of this 240 kDa protein. Calmidazolium, a calmodulin inhibitor, effectively inhibited not only [3H]leucine uptake but also 240 kDa protein production due to SP. On the other hand, KT-5720, an inhibitor of protein kinase A, had no effect on [3H]leucine uptake or 240 kDa production. Using the [125I]calmodulin-membrane overlay method, it was found that the 240 kDa protein was a newly synthesized calmodulin binding protein. From the present study, it was concluded that the de novo synthesis of this 240 kDa protein may be intimately related to the cortisol secretion in SP-stimulated BAC cells associated with an activation of the Ca-calmodulin pathway.


Inhibition of reverse transcriptase from feline immunodeficiency virus by analogs of 2'-deoxyadenosine-5'-triphosphate. The replication of feline immunodeficiency virus (FIV) in cultured cells was inhibited by 2',3'-dideoxyadenosine (ddA) and by 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with IC50 values of 0.98 and 0.95 microM, respectively. The effects of the presumed active forms of these inhibitors, ddATP and PMEA-diphosphate (PMEApp), upon the FIV reverse transcriptase (RT) were examined with two different template-primer systems. Both of these compounds were potent inhibitors of the FIV RT in reactions with primed phi X-174 DNA, yielding Ki values of 8.8 nM for ddATP and 5.0 nM for PMEApp. However, they were both poor inhibitors of the reaction with poly(rU)-oligo(dA); concentrations of ddATP or PMEApp greater than 10 microM were required to inhibit this reaction by 50%. Further analysis of the reaction with poly(rU)-oligo(dA) revealed that even in the absence of inhibitors the primers were extended by less than 20 nucleotides. In contrast, high molecular weight products were obtained in reactions with phi X-174 DNA. These results suggest that the reaction of FIV RT with poly(rU)-oligo(dA) is not highly processive. The high degree of termination encountered during this reaction with poly(rU)-oligo(dA) may be responsible for the low inhibitory potential of ddATP and PMEApp.


Practical usage concentrations of monensin have non-specific actions other than as a sodium ionophore in rat parotid acinar cells. Monensin is used as a sodium ionophore to examine the effect of Na+ on cellular function in a variety of cell types. In the present study, we investigated the effects of different concentrations of monensin on the signal transduction system in exocrine parotid acinar cells. Monensin increased cytosolic free Na+ concentration, measured by the Na+ indicator sodium-binding benzofuran isophthalate in a concentration-dependent manner (0.01 to 100 microM). Likewise, monensin concentration-dependently increased amylase release and intracellular Ca2+ concentration in the presence and the absence of extracellular Ca2+. Low concentrations (0.01 to 1 microM) of monensin did not release Ca2+ from non-mitochondrial intracellular pools in permeabilized cells with saponin but high concentrations (10 and 100 microM) of monensin which are of practical usage did. Monensin itself did not change the cyclic AMP accumulation, whereas high concentrations (10 and 100 microM) but not low concentrations (0.01 to 1 microM) of monensin inhibited cyclic AMP accumulation elevated by isoproterenol in the presence and absence of extracellular Na+. These results indicate that high concentrations of monensin, which are practically used, have nonspecific actions in rat parotid acinar cells, and lower concentrations of monensin are recommended for use as a sodium ionophore.


The chondroprotective drugs, Arteparon and sodium pentosan polysulphate, increase collagenase activity and inhibit stromelysin activity in vitro. The effects of the chondroprotective drugs, sodium pentosan polysulphate (SP54) and Arteparon (glycosaminoglycan polysulphate), on the in vitro activities of the purified matrix metalloproteinases interstitial collagenase (matrix metalloproteinase 1, MMP1) and stromelysin (MMP3) were examined. Both drugs produced concentration-dependent enhancement of the degradation of type I collagen fibrils by purified human fibroblast collagenase and rat tumour collagenase. Rat collagenase activity was increased by drug concentrations above 0.5 microgram/mL, whereas human collagenase activity was only increased by higher drug concentrations, above 5 micrograms/mL. The concentration dependence of the increase in rat collagenase activity was similar for both drugs, with a maximal 3-fold increase at 50 micrograms/mL. In contrast, human collagenase activity was increased to a greater extent by SP 54 compared to Arteparon, with maximal increases at 5000 micrograms/mL of 6-fold and 2-4-fold, respectively. Both drugs produced concentration-dependent inhibition of the proteoglycan-degrading activity of both human fibroblast stromelysin and rat tumour stromelysin. Rat and human stromelysin activities were inhibited at drug concentrations above 0.005 microgram/mL, with a similar concentration dependence for both drugs. Fifty percent inhibition of rat stromelysin was produced by concentrations of each drug in the 0.5-5 microgram/mL range. The pattern of inhibition of human stromelysin was similar, except that drug concentrations in the 500-5000 micrograms/mL range produced 50% inhibition. The possible modes of action for these drug effects and their possible pharmacological significance are discussed.


Anti-human immunodeficiency virus effects of cationic metalloporphyrin-ellipticine complexes. A series of cationic metalloporphyrin-ellipticine complexes were found to inhibit the cytopathicity of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus in MT-4 cells at concentrations ranging from 1.4 to 17 micrograms/mL, i.e. at a concentration that was 2.5-30-fold below the cytotoxicity threshold. These compounds were also found to inhibit syncytium formation between persistently HIV-1-infected HUT-78 and uninfected Molt/4 cells, to interfere with HIV-1 binding to the cells, and to suppress HIV-1-associated reverse transcriptase activity.


Absence of a direct coupling of a G protein to dihydropyridine binding sites in rat heart. In rat heart membranes, the addition of guanine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), a stable GTP analogue, did not significantly modify the displacement of [3H]PN 200-110 binding by the 1,4-dihydropyridine (DHP) agonist Bay K 8644 and antagonists, nifedipine and nicardipine. These results are in agreement with some previously reported electrophysiological and pharmacological data, and they suggest that there is no direct involvement of a G protein in the modulation of DHP sensitive Ca channels in cardiac cells.


[Effect of toxic components of snake venoms on binding of substance P with rat brain membranes]. Ion-exchange HPLC is used for purification of the snake venom alpha-neurotoxins, chi-bungarotoxin, cytotoxins, and phospholipases A2. Among these purified polypeptides, phospholipases A2 are found to be the most potent in inhibiting the substance P binding to rat brain membranes, Ki approximately 10(-8) M. Other toxins are weak inhibitors (Ki greater than or equal to 10(-4)-10(-5) M), earlier data on the inhibiting activity of alpha-bungarotoxin being caused by the commercial preparations' contamination with phospholipase A2.


[Mono- and polyspecific glycoconjugates based on synthetic O-antigen determinants of Salmonella and their serological characteristics]. Mono- and polydeterminant synthetic antigens have been prepared via copolymerisation of acrylamide with allyl-, 2-acrylamidoethyl, and p-acrylamidophenyl glycosides of di- and trisaccharides representative of the group-specific Salmonella O-antigenic determinants (0:2, 0:3, 0:4, and 0:9). Serological specificity of the glycoconjugates obtained has been studied in enzyme immunoassay (EIA) using monoreceptor antisera.


[Modulation of membrane activity of an enzyme in reversed micelle system with a change of media pH (using alkaline phosphatase as an example)]. Regulation of the membrane active properties of alkaline phosphatase from calf intestinal mucosa in reversed micelles of Aerosol OT (AOT) in octane was studied. The dependence of the catalytic activity on the surfactant concentration at the constant hydration degree, which characterises the membrane activity of enzymes, is modulated through pH variation. The variation may cause conformational changes of the protein molecule, resulting in exposition of anchor groups which provide the interaction of the enzyme with the micellar matrix.


[Study of the antigenic structure of human immunodeficiency virus using synthetic peptides]. In a search for synthetic peptide antigens fit to detect anti-HIV antibodies, a set of algorithms were used to predict the probable antigenic determinants of gag, pol, env and nef proteins of HIV-1 and HIV-2. Over forty peptides were synthesized by the solid-phase method. The reactivity of the peptide antigens was evaluated in ELISA on panels of HIV-1/2-positive sera. Application of the synthetic peptides for the early HIV diagnostics was examined.


Expression of human tenascin in synovitis and its regulation by interleukin-1. Tenascin is an extracellular matrix glycoprotein with effects on cell adhesion, cell migration, and lymphocyte activation. We proposed to identify the expression of human tenascin messenger RNA (mRNA) and protein in inflammatory synovitis and in normal synovium, and to identify potential regulatory cytokines. Immunohistochemistry and in situ hybridization were used to identify the expression of tenascin in synovium. Northern blot analysis of RNA and both immunoblot analysis and enzyme-linked immunosorbent assay of proteins were used to identify tenascin in synovial cell cultures. Tenascin was found along the synovial lining layer and in perivascular areas of normal synovium. In inflammatory synovitis, tenascin protein and mRNA expression were shown to be increased in the synovial lining layer, in perivascular areas, in lymphoid aggregates, and in areas of fibrosis. Interleukin-1, a major mediator of tissue injury in inflammatory synovitis, induced tenascin expression and deposition in primary synovial fibroblast cultures. Tenascin mRNA and protein are increased in inflammatory synovitis, and interleukin-1 is an inducer of tenascin in synovial fibroblasts. This identifies a new pathway by which interleukin-1 alters the extracellular matrix composition in synovitis. Since tenascin has effects on lymphocyte activation and cell adhesion, the induction of tenascin in inflammatory synovitis may play a role in the pathophysiology of arthritis.


Changes in anti-U1 RNA antibody levels correlate with disease activity in patients with systemic lupus erythematosus overlap syndrome. To evaluate correlations between changes in anti-U1 RNA antibody levels and disease activity in 9 patients with systemic lupus erythematosus (SLE) overlap syndrome who were prospectively followed up for at least 3 years. Anti-U1 RNA antibody levels were measured quantitatively, using a nitrocellulose filter binding assay. Disease activity was measured with a validated SLE activity index. All 9 major disease exacerbations were associated with peaks in anti-U1 RNA antibody level. These results seem to indicate that measuring anti-U1 RNA antibody levels can be useful for monitoring disease activity.


Antibodies in rabbits immunized with cationized IgG react with histones H3 and H4. Rabbits immunized with cationized rabbit IgG develop antinuclear antibodies. This study was aimed at identifying the reactive antigens. Rabbits were immunized with homotypic or allotypic IgG that were physicochemically altered to produce positively charged dimethylpropanamide (polycationic) side chains. Seven of 11 rabbits injected with cationized IgG produced antinuclear antibodies detected by indirect immunofluorescence. By enzyme-linked immunosorbent assay, these were IgG antibodies reacting with histone (H3-H4)2 tetramer and with individual histone polypeptides H3 and H4. The antihistone antibodies were absorbed by cationized IgG but not by normal IgG. Cationized IgG appears to possess antigenic determinants of sufficient similarity to produce antibody responses cross-reactive with histones H3 and H4.


Selection of T cell receptor V beta elements by HLA-DR determinants predisposing to rheumatoid arthritis. Rheumatoid arthritis (RA) is genetically linked to a sequence motif encoding for the middle portion of the alpha-helical loop, which is adjacent to the antigen-binding groove of the HLA-DR molecule. The disease-associated element might be involved in binding the antigen or in interacting with the T cell receptor (TCR). To investigate the contribution of the disease-associated element in T cell recognition events, we studied structural requirements in the interaction of T cell clones with HLA-DR determinants. T cell clones restricted to disease-associated HLA-DR determinants were established by allogeneic stimulation of HLA-DRB1*0401+ or *0401- responders with HLA-DRB1*0404/8+ stimulators. Allele specific primer sets were used to identify the V beta gene segment expressed by individual clones. Sequence analysis was applied to study the diversity of the TCR beta-chain junctional regions. The repertoire of TCR V beta elements was strongly biased toward the usage of V beta 6. HLA-DRB1*0401+ and *0401- donors preferentially recruited V beta 6+ T cells to recognize the disease-associated HLA-DR determinant. Sequence data revealed that the V beta 6.6/7 and V beta 6.8/9 subtypes of the V beta 6 multigene family were overrepresented. The TCR beta chains were characterized by a high degree of junctional diversity, supporting the view that a multitude of peptide-DR complexes were recognized and that the preferential use of V beta 6 was dictated by the TCR beta chain-DR beta 1 chain contact. T cells reactive with the disease-associated HLA-DR structure are nonrandomly selected. The HLA-DR component predisposing to RA might define molecular requirements that restrict the TCR-HLA interaction. Thus, the phenomenon of HLA association in RA might reflect a genetic control of T cell recognition, through the selection of the TCR repertoire.


[The effect of indomethacin and iloprost on the cardiodepressive actions of various anesthetics on isolated atrial preparations from guinea pigs]. Effects of Indomethacin and Iloprost on the Cardiodepressant Properties of Various Narcotics on Isolated Auricle Preparations from Guinea Pigs. In isolated auricle preparations from guinea pigs several narcotics (hexobarbital, pentobarbital, ketamine, etomidate, urethane) dose-dependently showed cardiodepressive effects. Premedication of the preparations with indomethacin significantly reduced the narcotics-induced changes of the cardiac action. Iloprost, a stable prostacyclin analogon, had the same efficacy. The influence of both substances could be due to an unspecific membrane effect or a positive inotropic action.


Effect of gyrase inhibitors on some eukaryotic short-term test systems. DNase I in vitro nucleic acid synthesis and DNA repair in primary cultures of chicken embryo and rat cells. DNase I activity was diminished by ciprofloxacin (CFL), nalidixic acid, norfloxacin, and ofloxacin in a dose-dependent manner, the MIC's (minimal significantly inhibiting concentrations) being 3.2, 2.8, 2.4, and 7.6 micrograms/ml, resp., in phase I-reaction (increase in DNA hyperchromicity) and 21, 20, 55, and 56 micrograms/ml, resp., in phase II-reaction (formation of acid-soluble products). The Line-weaver-Burk plots indicated inhibition by substrate (phase I) and uncompetitive inhibition (phase II). The decrease in scheduled DNA synthesis by CFL showed MIC's of 270, 100, 1000, and 850 micrograms/ml in chicken embryo brain (B) and liver (L) cells and in rat thymic (T) and splenic (S) cells, resp. With regard to ribonucleic acid synthesis, MIC values of 82, 82, 12.5, and 48 micrograms/ml CFL were determined, resp. Within a concentration range of 25-1600 micrograms/ml, no principal differences existed between the 4-quinolones used. In T-cells, DNA repair as induced by X-irradiation or UV-light and determined by nucleoid sedimentation was inhibited by CFL (greater than 100 micrograms/ml). The present results demonstrate biological effects of 4-quinolones on eukaryotic systems at remarkably low concentrations. In this context, the possibility of interactions with DNA catabolizing enzyme systems and synergistic effects with DNA/chromatin-damaging agents should be considered further.


Large-vessel endothelium switches to a microvascular phenotype during angiogenesis in collagen gel culture of rat aorta. The purpose of this study was to investigate the vasoformative behavior in vitro of the native intimal endothelium of the rat aorta. To visualize the intimal surface directly, thoracic aortas were everted using a procedure that sequestered adventitial cells and possible remnant microvessels of periaortic soft tissues inside the aortic tube. Everted aortas embedded in collagen gel and cultured under serum-free conditions generated branching microvessels by a process of sprouting from the aortic intima. The newly formed microvessels originated from patches of activated intimal endothelial cells, which had survived the mechanical damage of the eversion procedure. Activated endothelial cells crawled over each other and engaged in lumen formation forming bilayers or multilayers of cells which became the source of sprouting histotypic microvessels. The endothelium of the newly formed microvessels was positive for factor VIII-related antigen and was partially surrounded by periendothelial cells which expressed alpha-smooth muscle actin. The results of this study indicate that the intimal endothelium of the rat aorta has considerable functional plasticity and can switch to a vasoformative phenotype in response to changes in the surrounding extracellular matrix environment.


[Analytical study of the Abbott and Ortho tests for screening and confirmation of anti-HCV antibodies. Le groupe de Travail "Hépatites virales" de la Société Française de Transfusion Sanguine]. Anti-HCV systematic screening on blood donation was mandatory in France since first of March 1991. Two laboratories (Ortho-Chiron and Abbott) have introduced in Europe successively two kinds of hepatitis C positive diagnosis with 1st and 2nd generation ELISA screening and confirmatory assays. The aim of this multicentric study was to evaluated the sensibility and specificity of these tests. For that, they used 10,090 blood sera. As a result we have seen that the new "second generation" screening assays have a higher sensitivity without less of specificity for the confirmatory tests.


[Screening tests for anti-HCV antibodies: comparative results]. Comparative results between anti-HCV screening tests have shown better performances with 2nd generation tests than with 1st generation assays, since anti-C100-3 antibodies are less reliable and less durable than anti-C33 c and anti-C22-3 antibodies. Comparison between the four 2nd generation commercialized assays (Ortho, Abbott, Pasteur, Ubi-Organon) done on the hepatitis study group panel and on 109 samples from 18 patients collected before, during and after anti-HCV seroconversion, has shown similar results for most of the samples but some discordances on samples with isolated anti-C100-3 or anti-C33 c antibodies. For the 18 patients, 3 discordances were observed between the four assays. These three discordances concerned 3 of the 6 seroconversions with anti-C33-c as the first detectable antibody. In two cases, Ubi-Organon assay did not recognize the seroconversion as early as the 3 other assays; in another case, it was the Pasteur assay which answered later than the other assays. As for the 15 other patients, no difference was observed between the four assays, in particular when anti-C22-3 was one of the first detectable antibodies.


[Anti-HCV and recipients: situation in hemophiliacs in 1991]. Sera from 273 French haemophiliacs who had received non viral inactivated concentrates, were tested for antibodies to HCV by first and second generation assays. Antibodies to HCV were detected in 66% of the sera by the first generation assays (anti-C 100-3) reaching 100% by the second generation assays. None of the 53 patients only exposed to solvent-detergent treated Factor VIII or IX concentrates had HCV seroconversion. HCV core protein antibody was always detectable often as a single antibody in seropositive hemophilic patients.


Relatively reduced values of plasma alpha-fetoprotein at early second trimester of pregnancies with hypertensive disorders. Hypertensive disorders during pregnancy implicate placental pathologic conditions, which may interfere with the normal passage of alpha-fetoprotein (AFP) to the maternal blood. We compared the levels of maternal serum (MS) in early second trimester of pregnancies complicated by hypertensive disorders with those of matched controls. The distribution in the study group of MS-AFP multiple of median values was significantly different from the distribution in the control group. Moreover, up to multiple of the median 1.00, the number of hypertensive patients was larger than the number of normotensive pregnant women. The mean level of multiples of the median in the study group was significantly lower than that of the control group (p value = 0.003, 95% confidence interval: -0.30, -0.05). In the analysis of the distinct types of hypertension, the difference remained significant for 85 women in the moderate hypertension subgroup (p value = 0.032, confidence interval: -0.34, -0.02) and was not significant for the severe hypertension subgroup of 22 women (p value = 0.24, 95% confidence interval: -0.57, 0.15) and chronic hypertension subgroup of women (p value = 0.52, 95% confidence interval: -0.44, 0.00). The trend was consistent in all the subgroups. Relatively low values of maternal serum AFP at early second trimester of pregnancies with hypertensive disorders may be a result of placental pathologic involvement and can help in the identification of the women at risk.


Monoclonal antibodies to malignant human gliomas. Operationally specific monoclonal antibodies (MAbs) reactive with tumor but not normal adult tissues offer great potential for diagnosis and therapy of CNS neoplasms. Two targets for specific MAb localization were chosen for this study: (1) glioma-associated gangliosides GM2 [II3NeuAc-GgOse3Cer], GD2 [II3(NeuAc)2-GgOse3Cer], GD3[II3(NeuAc)2-LacCer], 3'-isoLM1 [IV3NeuAc-LcOse4Cer], and 3',6'-isoLD1 [IV3NeuAc,III6NeuAc-LcOse4Cer] and (2) epidermal growth factor receptor (EGFR) variant molecules. Epitopic specificity of isolated ganglioside hybridomas was determined with FAB-MS defined ganglioside standards. All MAb are IgM. Assay of 14 cytologic specimens and 31 frozen sections of primary CNS neoplasms revealed staining with anti-GD3 (14/14, 31/31), anti-GM2 (9/14, 26/31), and anti-GD2 (6/14, 24/30), respectively. 3'-isoLM1 and 3',6' isoLD1, which exhibit a restricted oncofetal expression pattern and are not detectable in adult human brain, are present in 15/31 primary CNS neoplasms and in 1/8 human glioma xenografts, as detected by MAbs SL-50 and DMAb-14, respectively. EGFR proteins, the second target, have unique amino acid spans resulting from gene deletion in the amplified EGFR gene present in subsets of malignant human gliomas. Antibodies against EGFR deletion-mutant Type III show highly restricted activity with a subset of glioma biopsies (6/35) expressing the mutant EGFR. These reagents should be useful for in vitro and in vivo diagnosis and, potentially, for treatment of malignant brain tumors.


Report of a case with aplasia cutis congenita, elevated amniotic fluid alpha-fetoprotein, and positive acetylcholinesterase band. A case report of a pregnant mother with elevated midtrimester maternal serum alpha-fetoprotein, an elevated amniotic fluid alpha-fetoprotein with a positive acetylcholinesterase band, and grossly normal fetal anatomy is presented. A premature female infant was delivered with bilateral symmetrical flank skin lesions consistent with the diagnosis of aplasia cutis congenita.


Apoptosis of alpha beta T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: its possible implications for recovery and acquired tolerance. We have recently shown that apoptosis, an active process of cellular self-destruction, occurs in the central nervous system in Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Conventional light and electron microscopic studies suggested that some of the apoptotic cells were oligodendrocytes and that others were hematogenous mononuclear cells. To determine whether any of the apoptotic cells were T lymphocytes, we used the technique of pre-embedding immunolabelling which allows sufficient preservation of the ultrastructure to permit recognition of apoptotic changes while at the same time preserving surface antigens so that the identity of the apoptotic cells can be determined by immunocytochemistry. Light microscopic immunocytochemistry using the monoclonal antibodies OX-34 (CD2) and R73 (alpha beta T-cell receptor) revealed that 10% of the CD2+ cells and 5% of the alpha beta T lymphocytes in the parenchyma of the spinal cord were dying by apoptosis. The presence of apoptotic alpha beta T cells was confirmed by electron microscopy. About half of all the apoptotic cells within the spinal cord were labelled by these antibodies. It is possible that some of the unlabelled apoptotic cells were also T lymphocytes but that others were glial cells such as oligodendrocytes. One possible interpretation of this T-cell apoptosis is that it represents activation-induced cell death, which has recently been shown to provide a mechanism of clonal elimination of mature as well as immature autoreactive T cells. Another possible interpretation is that it is a result of corticosterone released during the course of EAE. The apoptotic elimination of target-antigen-specific lymphocytes within the target organ in this autoimmune disease may contribute to the subsidence of inflammation and, if ongoing, to the development of tolerance.


Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). I. Cell detachment and lysis induced by encephalitogenic CD4+ T lymphocytes. Myelin basic protein (MBP) reactive CD4+ T lymphocytes, capable of inducing experimental autoimmune encephalomyelitis (EAE), were examined for their ability to damage target cells of central nervous system (CNS) origin. Damage was assessed by monitoring detachment of adherent astrocytes from substratum and astrocyte lysis. MBP-specific, but non-encephalitogenic CD4+ T cells mediated astrocyte detachment but not lysis. However, encephalitogenic CD4+ T cell lines were more efficient in causing astrocyte detachment and could also cause astrocyte lysis. The detachment and lytic activities of the MBP-reactive T cell lines tested were often independent of the presence of specific antigen, and were not restricted to syngeneic major histocompatibility (MHC) antigens. MBP often augmented the detaching and, if lytic, lytic activities of these T cells. The encephalitogenic CD4+ T cells also detached and lysed allogeneic 'bystander' fibroblasts in the presence of unlabelled syngeneic astrocytes, suggesting the involvement of a soluble mediator(s). Although MBP is essential for the initiation of EAE, the presence of MBP on cells of CNS origin, such as astrocytes and oligodendrocytes, does not appear to be necessary for their damage by MBP-specific CD4+ T cells. Immune CD4+ T cells, which penetrate the CNS, may disorganize brain tissue structure by lysing astrocytes directly and by damaging other brain cells indirectly by soluble mediators. Thus cellular detachment, in addition to cell lysis, mediated by MBP-specific CD4+ cells may contribute to EAE pathogenesis.


Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). II. Characteristics of astrocyte detachment mediated by myelin basic protein (MBP) specific CD4+ T lymphocytes. We have shown that encephalitogenic, myelin basic protein (MBP)-specific CD4+ T cells can cause astrocyte and oligodendrocyte detachment in vitro. Similar processes may damage the central nervous system (CNS) in vivo by causing disorganization and destruction of brain tissue structure. The finding that 'bystander' allogeneic fibrosarcoma cells were detached by MBP-specific CD4+ T cells only when syngeneic astrocytes were present, suggested that a soluble cell-detaching factor (CDF) is released during the specific astrocyte-CD4+ effector interaction. In this study, CDF activity was detected in the supernatants of MBP-reactive CD4+ T cells incubated with concanavalin A or astrocytes. Lymphocyte-induced astrocyte lysis, but not detachment, was inhibited by the protein synthesis inhibitors, cycloheximide and puromycin, indicating that de novo protein synthesis is required for this type of lysis, but not for detachment. Astrocyte detachment was not inhibited, but rather augmented, by the trypsin inhibitors, soybean trypsin inhibitor (SBTI) and alpha-1-antitrypsin (alpha 1), suggesting that the CDF activity is not due to tryptic serine proteases, although it may be protease susceptible. The heparanase inhibitor, heparin, inhibited CD4+ T cell-mediated astrocyte detachment at low doses, but augmented detachment at higher doses, indicating that detaching activity is not due to heparanases. The actin microfilament disrupting agent, cytochalasin B (CB), inhibited astrocyte detachment induced by MBP-specific CD4+ T cells. CB pretreatment of the target astrocytes, but not of the effector CD4+ T cells, inhibited astrocyte detachment, suggesting that the integrity of the target's, but not the effector's, cytoskeleton is required for astrocyte detachment. The results herein suggest that during astrocyte interaction with MBP-specific CD4+ T cells, soluble factors are released that trigger an intrinsic astrocyte detachment mechanism.


Anti-CD4 monoclonal antibody therapy in severe psoriasis. We report here the treatment of psoriasis, a chronic inflammatory skin disease characterized by uncontrolled keratinocyte proliferation, with BB14, a CD4 murine IgG1 antibody. Three patients with severe psoriasis were treated with anti-CD4 mAb infusions (0.2 mg/kg/day for the first patient, 0.4 mg/kg/day for 2 days and 0.8 mg/kg/day during the following days for the 2 others) for 7 or 8 days, without other therapy. Rapid clinical improvement, with major reduction of the Psoriasis Area Severity Index, was observed during 1 month after treatment. Moderate decreases in CD4+ blood cells occurred in the last two patients but not in the first one. Circulating T cells coated with anti-CD4 mAb were detectable during the first 48 h in the first patient and from day 1/2 to day 7/8 in the two others. The density of CD4 molecules on the surfaces of peripheral blood lymphocytes was decreased in all patients and remained low as long as anti-CD4 mAb was detectable in patient serum. The maximal 24 h residual mAb levels ranged from 0.3 microgram/ml in the first patient to 3.8 and 7.0 microgram/ml in the two others. The three patients produced IgM antibodies against the anti-CD4 mAb at day 7/8 or 15 and two patients had IgG antibodies at day 15. Lesional skin samples demonstrated (1) gradual improvement in parakeratosis, papillomatosis and acanthosis, (2) decreased expression of ICAM-1 and HLA-DR by keratinocytes, (3) an increase in CD1a+ Langerhans cell number, (4) partial decrease in epidermal T cell infiltrate and (5) no major change in the dermal infiltrate composed of CD3+, TcR alpha beta+, CD45Ro+, HLA-DR+ T cells. We conclude that anti-CD4 mAb administration can induce a rapid and major improvement in psoriatic lesions, with immunohistochemical changes different from those induced by cyclosporin A or 8-methoxypsoralen plus long wave UV light (PUVA) therapy. Our data provide strong evidence for a critical role of CD4+ lymphocytes in psoriasis.


Cross-reactive maternal autoantibodies and congenital heart block. Epitopes with linear sequences recognized by anti-La autoantibodies from seven mothers of children with congenital heart block were recently defined. Eight of these epitopes share sequence identity with three other proteins in addition to the original autoantigen, La. The three proteins are human cardiac myosin beta heavy chain, laminin B1 chain and the M6 protein of Streptococcus pyogenes. Affinity purified anti-La antibodies from a further three mothers bound to the La antigen and also to human cardiac myosin and mouse laminin. Affinity purified antibodies from three mothers of healthy children bound to the La antigen but showed minimal binding to either human cardiac myosin or mouse laminin. Cardiac myosin inhibited the binding of CHB-related anti-La antibodies to both La and myosin. These data support a role for maternal autoantibodies crossing the placenta and recognizing foetal cardiac antigens accessible at a critical developmental stage during gestation. We suggest that this would lead to complement fixation, inflammation and the subsequent pathology associated with congenital heart block.


The beta-adrenoceptor-coupled adenylate cyclase system in rat C6 glioma cells. Deamplification by isoproterenol and oxaprotiline. The beta-adrenoceptor-coupled adenylate cyclase system in rat C6 glioma cells displays many characteristics observed in brain tissue: using nonlinear regression analysis of agonist competition binding curves, we demonstrated that the bulk of beta-adrenoceptors show high nanomolar affinity for isoproterenol; like in brain tissue, Gpp(NH)p does not shift agonist competition binding curves to the right; and the agonist isoproterenol rapidly downregulates the number of beta-adrenoceptors and deamplifies the norepinephrine signal. However, unlike in brain tissue, where (-)-oxaprotiline fails to decrease the number of beta-adrenoceptors and to desensitize the cyclic adenosine monophosphate generating system, it desensitizes the beta-adrenoceptor-coupled adenylate cyclase system in C6 glioma cells. Determination of the relative steady-state levels of beta-adrenoceptor messenger ribonucleic acid (mRNA) by Northern blot analysis showed a twofold increase in the steady-state levels of the mRNA at 30 minutes following exposure to (-)-isoproterenol or (-)-oxaprotiline. At 48 hours, basal values of mRNA were observed at a time when beta-adrenoceptors were maximally decreased. Further experiments on transcriptional activation, and mRNA stability and translation will be required to unravel the complexity of agonist-dependent and agonist-independent regulation of beta-adrenoceptor density and function.


Detection of interstitial increase in macrophages, characteristic of acute interstitial rejection, in routinely processed renal allograft biopsies using the monoclonal antibody KP1. Acute interstitial rejection (AIR) of renal allografts is accompanied by a characteristic peritubular increase in macrophages, which can be identified with the CD14 monoclonal antibody (mAb) WT14 in cryostat sections. Since frozen tissue is not always available, we tested whether this increase can also be demonstrated in Bouin-fixed, paraffin-embedded biopsies, using the CD68 antimacrophage mAb KP1, which can also be applied to paraffin sections. Sections of 16 biopsies with AIR and 11 controls were stained with KP1. In 25 of the 27 biopsies, macrophages were strongly positive for KP1. Two AIR biopsies were completely negative, probably due to prolonged fixation. In the remaining 14 AIR biopsies, the number of KP1-positive cells was significantly higher than in the controls [1184 +/- 410 per mm2 (mean +/- SD) vs 112 +/- 126 per mm2]. We conclude that, especially in cases in which frozen tissue is not available, the demonstration of increased numbers of monocytes/macrophages with mAb KP1 can be a helpful adjunct in the histological diagnosis of AIR in routinely processed renal biopsies.


Recognition of synthetic peptides with sequences of rubella virus E1 polypeptide by antibodies and T lymphocytes. Antigenicity of rubella virus E1 polypeptide was analyzed using synthetic peptides with predicted amino acid sequences. Overlapping solid-phase bound peptides were used to define antibody binding domains and a panel of free peptides to study T-cell responsiveness. Several antibody-binding areas including those earlier described to contain major neutralizing epitopes were recognized by human sera positive for rubella antibodies. T-cell lines specific for rubella virus were established from 14 rubella immune subjects. All cell lines responded to rubella virion-derived antigen but only eight (57%) responded to one or more of the synthetic peptides. Individual patterns of peptide recognition were found but peptide 8 representing amino acids 402-422 was most often stimulatory to T-cells lines, either alone (3 subjects) or in combination with peptide 3 (amino acids 245-269) or 3 and 4 (amino acids 269-287). The response was HLA restricted but no single DR specificity for this restriction was identified.


[A familial case of metabisulfite intolerance. Clinical and pharmacological study]. Within a context of a familial case of severe respiratory signs, with intolerance of sulfites and aspirin, we decided to study the clinical reliability of "in vitro" activation of human basophils in the presence of sulfites. Positivity of these tests for the three patients in the study (60, 73, and 66%) is confirmed by passive sensitization of donor basophils, which was destroyed when the serum was heated. This argument in favour of IgE dependence was strengthened by observation of an "in vitro" desensitization by an anti-IgE and not by an anti-IgG. These "in vitro" data are in agreement with the clinical history, daily routine of the patient, skin tests and where done, oral provocation tests.


[In vitro simulation of rabbit cecal fermentation in a semi- continuous flow fermentor. II. Effect of inoculum type]. Two Rusitec fermentors were operated under identical conditions. One was seeded with an inoculum of rabbit caecal contents, and the other with bovine rumen contents. The fermentation substrate was rabbit feed that had been digested with amylase and pepsin. The substrate constituents (organic matter, OM and NDF) were lost in 48 h at a significantly higher rate in the presence of rumen inoculum (OM: +10%, NDF: +15%). The pHs of the 2 fermentors were similar at pH 6.6. The fermentors produced similar amounts of protein nitrogen per 24 h, after 6 d of adaptation. Volatile fatty acid production was slightly higher in the presence of rumen inoculum. The fermentor inoculated with rumen contents produced a higher percentage of propionic acid (25%) than of butyric acid (7%), while fermentation with rabbit caecal contents gave the opposite ratio (C3/C4 = 0.81). Consequently, only the rabbit caecal inoculum provided the fermentation profile characteristic of the species.


The complement system in human reproduction. Regulation of the complement system in reproduction is unique inasmuch as reproductive tissues represent the only condition where allogeneic interactions occur naturally. Both allogeneic extraembryonic membranes and semen that contact and interact with maternal cells and tissues must avert complement-mediated damage to ensure reproductive success. Several regulators of complement activation exist. Membrane cofactor protein (MCP) and decay accelerating factor (DAF) inactivate C3 and C5 convertases on cell surfaces. In addition, CD59 inhibits the membrane attack complex (MAC) of the complement cascade. Strong expression of these membrane glycoproteins by trophoblast and amniotic epithelium has been observed. MCP, DAF, and CD59 likely safeguard extraembryonic tissues from complement damage originating from maternal and fetal blood or amniotic fluid. Different reproductive tract fluids vary in complement levels. With the exception of ovarian follicular fluid, these levels are generally much less than those in blood. Endometrial and cervical content of C3 appear to be regulated by hormones. These observations suggest that the effects of complement activation may vary in reproductive tissues. MCP is absent from the surfaces of oocytes. Sperm express MCP and DAF in discrete areas that would not be associated with the known complement-regulatory functions of these proteins. Seminal plasma contains MCP and the MAC inhibitor SP-40,40 but not DAF.SP-40,40 may exemplify how complement-regulatory proteins perform alternative functions as it interacts with molecules other than complement components. We have reviewed aspects of the complement system that relate to allogeneic interactions in reproduction and that suggest fruitful areas for further research.


[Chemical constituents of Rhodiola kirilowii (Reg.) Reg]. Three compounds were isolated from the water-soluble part of alcohol extracts of rhizomes of Rhodiola kirilowii. Two of them were identified as salidroside and tyrosol, respectively by chemical and spectral analysis. beta-sitosterol was obtained from the petroleum extracts of the plant.


Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis. Except under certain pathological conditions such as wound healing and solid tumor growth, angiogenesis is a relatively rare event in the adult. One exception, however, is the angiogenesis that occurs during the cyclical changes in the female reproductive tract. Many factors, chemical as well as mechanical, have been shown to be capable of promoting or inhibiting angiogenesis in vivo and in vitro. However, despite intense research efforts, the mechanisms involved in the regulation of angiogenesis in vivo are not fully understood. In this article we briefly review the basic steps involved in angiogenesis and present examples of factors and conditions that may serve as potential regulators of angiogenesis in the nonpregnant uterus. Finally, we discuss some of the architectural, anatomical, and physiological differences between the microcirculatory beds established during normal, self-limited vessel growth and that associated with the uncontrolled, pathological vascular growth that accompanies tumor growth and metastasis.


[Influences of naomaitong upon cerebral blood flow in dogs]. This experiment has proven that the drug naomaitong can synchronously increase the blood flow of both dog's internal carotid artery and vertebral artery and also can antagonize the decrease of blood flow of both arteries caused by pituitrin. The increase of blood flow of both internal carotid artery and vertebral artery due to administration of naomaitong (1.2 g/kg) through the dog's duodenum is approximately equal to the increase of blood flow of both arteries resulting from the intramuscular injection of papaverine (1 mg/kg). Besides, the dosage of naomaitong is positively correlated to the effect and action of the drug.


[Mechanisms of protective action of radix Salviae miltiorrhizae (RSM) against experimental hepatic injury in rats]. The results indicated that RSM could significantly inhibit the lipid peroxidation of normal livers and cultured hepatocytes of rats, induce liver microsomal cytochrome P450 in normal rats, increase nucleic acids, proteins, urea and cerolloplasmin of damaged cultured hepatocytes of rats, relieve ultrastructural damage of cultured hepatocytes induced by CCl4. The pharmacological actions mentioned above should be considered as important mechanisms of RSM against liver injury.


HLA class I binding regions of HIV-1 proteins. To identify HIV peptides containing HLA class I binding regions, different studies have been performed. These include the detection of interactions between HIV peptides and purified HLA molecules in solid-phase assays, the measurement of HLA molecule assembly in the presence of peptide added to cell lysates, and the detection of inhibition of CTL-mediated cytolysis by competition between peptides on target cells. To date, the HIV epitopes recognized by anti-HIV CTL are from the Env, Gag, Nef, and Pol proteins and they are identified using synthetic peptides of 12 to 20 amino acids. The search for a correlation between known HIV CTL epitopes and the results of HLA/peptide interaction assays reveals that: (1) most of the peptides that are positive in the assembly assay contain a HLA-A2 peptide motif but the correlation between these positive peptides and the CTL epitopes is not obvious; (2) a high proportion of HIV epitopes are included in the peptides positive in solid-phase binding and in inhibition of cytolysis assays, although these tests do not allow us to predict HLA restriction; (3) the HLA-A2 peptide motif is not systematically included in HLA-A2-restricted CTL epitopes, this observation raising the possibility that other sequences are involved in HLA binding.


The heavy chains of human plasma inter-alpha-trypsin inhibitor: their isolation, their identification by electrophoresis and partial sequencing. Differential reactivity with concanavalin A. Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.


Electrophoretic pattern of the inter-alpha-inhibitor family proteins in human serum, characterized by chain-specific antibodies. The inter-alpha-inhibitor (I alpha I) family encompasses four plasma proteins, namely free bikunin as well as I alpha I, pre-alpha-inhibitor (P alpha I) and inter-alpha-like inhibitor (I alpha LI). Each of the last three proteins is a distinct assembly of one bikunin chain with one or two out of three distinct, heavy (H) chains designated H1, H2 and H3. I alpha I is made of [H1 + H2 + bikunin], P alpha I is made of [H3 + bikunin] and I alpha LI is made of [H2 + bikunin]. We characterized various I alpha I family-related antisera/antibodies, including a reagent recognizing bikunin, another directed against the four H1, H2, H3 and bikunin chains, and a third one directed against both H1 and H2 chains. By a simplified absorption procedure of the latter two antisera onto solid-phase immunoadsorbents made with i) purified I alpha I, or ii) a recombinant H1 polypeptide, we also obtained specific anti-H3 or anti-H2 antibodies, respectively. This series of new antibodies was used to identify each component within the pattern of bands corresponding to I alpha I family in human serum, as separated by SDS-PAGE and western blotting.


Isolation of a high-molecular mass glycoprotein from culture supernatant of an arthritogenic strain of the bacteria Erysipelothrix rhusiopathiae reacting with "inductive" monoclonal antibodies derived from rats with erysipelas polyarthritis. A glycoprotein exhibiting a relative molecular mass of about 1000 kDa was purified to homogeneity from culture supernatant of arthritogenic bacteria (Erysipelothrix rhusiopathiae, strain T28) by ultrafiltration, ammonium sulfate precipitation, molecular mass exclusion, and ion exchange chromatography. Fractions obtained were analysed for their antigenic content by an enzyme linked immunosorbent assay (ELISA) using rabbit immune serum raised against this strain of Erysipelothrix rhusiopathiae. Distinct monoclonal antibodies obtained from rats suffering from erysipelas polyarthritis display a unique property by inducing very efficiently protective and regulatory mechanisms while being unable to generate classical "passive immunity". These "inductive" monoclonal antibodies recognize most likely linear epitopes on the purified glycoprotein. This makes it a prime source for analysing the target structure of these in vivo "inductive" antibodies.


FK-506 and cyclosporin A: immunosuppressive mechanism of action and beyond. Cyclosporin A and FK-506 are important therapeutic agents that have found widespread use in preventing graft rejection during tissue transplantation. Research efforts aimed at elucidating the molecular mechanism of action of these drugs have, in addition to defining their immunosuppressive functions, led to the identification of two new gene families whose products may function as components of several diverse signal transduction pathways. In the presence of the immunosuppressive drugs, some members of the receptor families interact with the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin. Inhibition of phosphatase activity may effect several downstream biochemical processes. In this way, cyclosporin A and FK-506 have proved to be useful probes of signaling events in both lymphocytic and other cell types.


The influence of HIV status on single and multiple drug reactions to antituberculous therapy in Africa. To document the influence of HIV status on drug reactions occurring in patients on antituberculous therapy in Harare, Zimbabwe. Retrospective cohort study. City of Harare Tuberculosis Unit. Records of 906 patients with tuberculosis, of whom 162 reacted to antituberculous therapy, were analysed. Reactions to antituberculous drugs were more frequent in HIV-positive (98 out of 363) than in HIV-negative (64 out of 543; P less than 0.0001) patients. The most common drug reaction was cutaneous hypersensitivity, occurring in 139 patients, 89 (64%) of whom were HIV-positive. Thiacetazone was implicated in 115 (82.7%) of the 139 cutaneous reactions and streptomycin in 10 (7.2%). Almost all cutaneous reactions occurred within 8 weeks of beginning treatment. Severe cutaneous reactions occurred more often in HIV-positive patients (P less than 0.001) and the only two deaths occurred in this group. Reactions to multiple drugs occurred in 18 HIV-positive and three HIV-negative patients (P = 0.017). The use of thiacetazone and streptomycin in antituberculous drug regimens should be reassessed in those countries where coinfection with HIV and tuberculosis is common.


The semi-quantitative distribution and cellular localization of angiotensinogen mRNA in the rat brain. The present study describes the regional distribution and cellular localization of angiotensinogen-mRNA in the rat brain as investigated by means of in situ hybridization also in combination with immunocytochemistry for glial fibrillary acidic protein. The angiotensinogen gene expression seemed to be restricted to astroglia and showed marked regional differences. In some areas angiotensinogen-mRNA was present in almost all astrocytes with a strong signal (e.g. hypoglossal nucleus), whereas in other areas the angiotensinogen gene was expressed only in a certain population of glial cells. Some areas like the lateral septum were devoid of any detectable angiotensinogen-mRNA. A semi-quantitative atlas of the regional distribution of brain angiotensinogen-mRNA was obtained by using computer-assisted microdensitometry and revealed considerable rostro-caudal fluctuations of the angiotensinogen-mRNA content of certain regions (e.g. the subfornical organ). Furthermore, a semi-quantitative analysis on the cellular level of angiotensinogen gene expression was performed showing a correlation of the angiotensinogen gene expression to the glia content of the regions examined. It was also demonstrated that the angiotensinogen gene expression had its highest levels in several distinct areas of the brain (e.g. the preoptic region and the hypothalamus), whereas other areas showed only low to moderate levels (e.g. the thalamus). The expression of the angiotensinogen gene in the rat brain was not only restricted to areas involved in cardiovascular and neuroendocrine control, but was also present in functionally different regions. Our data thus indicate that, based on the regional distribution of angiotensinogen-mRNA, angiotensin peptides may have other functions besides participation in cardiovascular and neuroendocrine control.


Fine distribution of substance P-like immunoreactivity in the dorsal nucleus of the vagus nerve in cats. The ultrastructure of substance P (SP)-immunoreactive elements in the cat dorsal motor nucleus of the vagus nerve was examined using pre- and post-embedding immunocytochemical procedures. Substance P-like immunoreactivity was observed in axon terminals and axon fibres which were mostly unmyelinated. Quantitative data showed that at least 16% of axon terminals contained SP. Their mean diameter was larger than that of their non-immunoreactive counterparts. Most (83%) SP-containing terminals were seen to contact dendrites but some were observed adjoining soma or entirely embedded in the cytoplasm of vagal neurons (4.5%). Only 0.5% were observed to contact soma of internuerons. A few immunoreactive axon terminals (4%) were observed in contact with non-immunoreactive axon terminals. Round agranular vesicles and numerous dense core vesicles were visible in most SP-containing axon terminals (84.6%). The immunogold procedure showed the preferential subcellular location of SP to be dense core vesicles. In 32.4% of cases, SP-containing terminals were involved in synaptic contacts that were generally of the asymmetrical Gray type 1 and mainly apposed dendrites. The theoretical total of synaptic contacts was 74.5% and this suggests the existence of weak non-synaptic SP innervation involving approximately 25% of SP-containing axon terminals. No axo-axonic synapses were observed in the dorsal vagal nucleus. These results support the hypothesis that SP found in the dorsal vagal nucleus originates partly from vagal afferents and is involved in direct modulation of visceral functions mediated by vagal preganglionic neurons.


Sympathetic denervation and chronic serotonin uptake blockade by fluoxetine do not affect pineal gland 5-hydroxyindole acetic acid: evidence that oxidative deamination of pineal serotonin is a property of the pinealocyte. This investigation tested the hypothesis that oxidative deamination of 5HT in the pineal gland occurs primarily in cellular compartments other than the pinealocyte (i.e., noradrenergic nerve terminals and glia). Following sympathetic denervation of the pineal gland by bilateral superior cervical sympathectomy, pineal levels of 5HT and its oxidative metabolite, 5HIAA, were measured by HPLC from samples collected at six time points over the 24 h photoperiod. The role of glia in 5HT deamination was further examined by chronic treatment with the 5HT uptake blocker, fluoxetine (10 mg/kg). Sympathectomy abolished the circadian rhythms of both 5HT and 5HIAA, but had no statistically significant effect on the ratio of 5HIAA/5HT compared to sham-operated and intact controls over the 24 h period. Pineal daytime levels of both 5HT and 5HIAA were unaffected by fluoxetine treatment. These findings indicate that the pinealocyte is an important cellular compartment for 5HT oxidative metabolism.


Comparison of the effects of bifemelane hydrochloride, idebenone and indeloxazine hydrochloride on ischemia-induced changes in brain monoamines and their metabolites in gerbils. Bifemelane hydrochloride (bifemelane), idebenone and indeloxazine hydrochloride (indeloxazine) are used clinically to reduce apathy and other emotional disturbances in patients with cerebrovascular disease. In gerbil brains, ischemia affects many monoaminergic neurotransmitters and their metabolites. In the present study, the effects of treatment with bifemelane, idebenone and indeloxazine on ischemia-induced changes in monoamines and their metabolites were studied in ischemic gerbil brains. Although these drugs had no effect on the monoaminergic neurotransmitters or their metabolites in sham-operated animals, in the ischemic brains both dopamine and serotonin turnovers were abnormal after idebenone or indeloxazine treatment. Bifemelane, in contrast, tended to correct the ischemia-induced changes in the dopaminergic and serotonergic systems in the cerebral cortex, hippocampus and thalamus + midbrain. From the present results and those in previous reports, we conclude that bifemelane is more appropriate than idebenone or indeloxazine as a treatment for the ischemia-induced changes in monoaminergic neurotransmitter systems.


Multiple putative neuromessenger inputs to the phrenic nucleus in rat. Immunohistochemical reactions for 12 putative neuromessengers combined with retrograde labeling of phrenic motoneurons identified seven neuromessengers (5-hydroxytryptamine, substance P, thyrotropin-releasing hormone, methionine enkephalin, cholecystokinin, galanin, neuropeptide Y) located within terminal varicosities in the phrenic nucleus. The degree of terminal labeling in the phrenic nucleus varied depending on the peptide. Substance P, thyrotropin-releasing hormone and methionine enkephalin were each tested for colocalization with 5-hydroxytryptamine within terminal varicosities in the phrenic nucleus, and the coincidence of double-labeling varied for each peptide. These results indicate that phrenic motoneurons are subject to modulation by many peptide neuromessengers that may alter their responsiveness to primary excitatory and inhibitory inputs.


Characterization of peptidergic efferents from the lateral parabrachial nucleus to identified neurons in the rat dorsal raphe nucleus. The peptidergic content of the lateral parabrachial nucleus (LPB) efferents to the dorsal raphe nucleus (DRN) was studied by combining visualization of the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin within fibers that were immunocytochemically stained for neurotensin (NT), calcitonin gene-related peptide (CGRP) or galanin (GAL). The identity of DRN target neurons was determined with simultaneous immunocytochemical labelling for serotonin, the major transmitter within the nucleus. Within the DRN, we estimated that about two-thirds of the anterogradely labelled fibers arising from the LPB also showed peptidergic immunoreactivity. NT was the most commonly observed neuropeptide in LPB neuronal efferents directed to the DRN, followed by CGRP and GAL. The peptidergic afferents in the DRN were oriented preferentially in the dorsoventral plane. Peptidergic fibers from the LPB possessed varicosities (diameters not exceeding 3 microns) and were apposed on serotoninergic neuronal somata. Some of the anterogradely labelled peptidergic fibers were not associated with cells showing immunoreactivity for serotonin. The present results suggest that NT-ergic, CGRP-ergic and GAL-ergic neurons within the LPB are in contact with serotoninergic and non-serotoninergic neurons within the DRN. Since the DRN is known to project to the LPB, it is likely that bi-directional interconnections between these nuclei exist. Such linkages may provide anatomical substrates for coordinated autonomic responses.


[Effects of administration of three kinds of catecholamine on mitral complex]. To evaluate cardiac injury induced by injection of 3 kinds of catecholamine, 219 male rabbits were examined. Eighty-five to 100 percent of the animals having high doses of adrenaline and noradrenaline showed ventricular premature contraction, and 65 to 75 percent of them showed mitral complex injury; whereas, these effects were identified in none or only in 10 percent of the animals with isoproterenol injection. The cardiac injury was similar to that of vagal stimulation, which was reported previously. These results indicate that ventricular premature contractions are important for developing the adrenaline- and noradrenaline-induced mitral complex injury.


Subpopulations of neurons in the guinea-pig inferior mesenteric ganglia distinguished by the differential distribution of neurofilament triplet epitopes. The distribution of the neurofilament protein triplet was examined in neurochemically identified subpopulations of neurons in the guinea-pig inferior mesenteric ganglion. A majority of the catecholamine-containing nerve cell bodies also contained the neurofilament protein triplet. However, a major proportion of the noradrenergic, neuropeptide Y-immunoreactive neurons did not contain neurofilament protein triplet immunoreactivity. Furthermore, a specific subpopulation of neurons that lacked catecholamines and were associated with the hypogastric nerve could be distinguished by the unusual feature of cell body content of post-translationally modified neurofilament protein triplet epitopes. These studies indicate that neurons in the inferior mesenteric ganglia can be distinguished by the presence of specific neurofilament protein triplet epitopes, and thus this class of intermediate filament proteins may confer specific properties to the neurons in which it is contained.


Chronic ear pathology in a model of neonatal amniotic fluid ear inoculation. Human histopathologic studies have demonstrated that amniotic fluid cellular contents, keratinized squamous epithelial cells and lanugo hair, induce an intense inflammatory reaction including granulation tissue in the neonatal temporal bone. To investigate this reaction over a prolonged period, an animal model was studied. An aliquot of sterilized autologous hair and keratinized epithelial cells was placed into 14 gerbil bullae; saline was used as a contralateral control. The animals were sacrificed at intervals up to 6 months and the temporal bones were studied by light microscopy. All animals demonstrated nonpurulent inflammatory changes on the experimental side including granulation tissue, osteoneogenesis, tympanosclerosis, and cholesteatoma; the control side demonstrated minimal middle ear changes. We conclude that in this model autologous keratinized tissue provokes a foreign body response similar to the granulation tissue observed in human infants and, further, that over a prolonged period the middle ear demonstrates more severe pathologic consequences.


Reflectance method for simple determination of proteinase activity in microliter samples of a complex serum-like fluid. A technique using an optical instrument, a reflectometer, for quantitative determination of proteinase activity in microliter samples of complex serum-like fluids, e.g., crevicular exudate from single sites, was developed. The technique allowed the use of various proteins as enzyme substrate. The reflectometer measures the mass of a layer, such as protein, adsorbed to a reflecting surface. This is done by measuring the reflected light intensity of the p-polarized light beam on a surface. We used methylized silicon surfaces that were coated with fibrinogen, alpha 2-macroglobulin, or hemoglobin as enzyme substrates. The test solution was incubated overnight in a basin made in an agar gel applied on the top of the protein-coated surface. In 82 exudates from periodontitis sites, with pocket depths greater than or equal to 6 mm, fibrinogenolytic activity corresponding to 1 microgram ml-1 of trypsin and pronase P was found in 20% of the samples.


The application of aqueous two-phase systems to oligosaccharide synthesis by alpha-mannosidase catalysed glycosyl transfer reactions. Aqueous two-phase systems formed from PEG and dextran have been applied to the synthesis of oligosaccharide by Jack bean alpha-mannosidase in reverse. Whilst rates of synthesis and percentage yields were similar in two-phase systems and one-phase aqueous buffer systems, a ten-fold increase in yield of product per unit of enzyme was seen. In addition, the use of aqueous two-phase systems offers potential process advantages over one-phase systems for the synthesis of oligosaccharides.


Distribution of an enzyme in porous polymer beads. Trypsin has been immobilized by adsorption onto Amberlite XAD-7 beads. The Michaelis constant (Km) of the enzyme was increased about sevenfold following the immobilization. Its rate of penetration into the porous beads was determined by staining the beads, which had been split, with naphthol blue black. The extent of diffusional rate limitation of immobilized trypsin was related to the penetration depth of the enzyme into the beads. This can be controlled by manipulating the conditions during the preparation of the immobilized enzyme.


DNA polymorphism studies. Approaches to elucidating multifactorial ischaemic heart disease: the apo B gene as an example. It is well established that elevated plasma levels of low-density lipoprotein (LDL) particles are a risk factor for ischaemic heart disease with the distribution in LDL levels seen in the general population being the result of interaction between environmental factors, such as dietary fat intake, and genetic variation that is present in different individuals. One of the candidate genes where such variation is likely to occur, is the gene coding for apolipoprotein B (apo B). Many studies have reported an association between a common polymorphism of the apo B gene, detected using the restriction enzyme XbaI, and differences in plasma lipid levels, explaining 3-5% of the variance in LDL-cholesterol levels in samples representative of the healthy population. It has been proposed that the mechanism of this association is due to functional amino acid changes within the apo B protein, that affect LDL catabolism by altering binding affinity to the LDL-receptor. Several amino acid substitutions in the apo B gene have now been characterized, and these form the basis of the different epitopes that create the Ag marker system. Previous studies have reported that the Ag(x) epitope is associated with lower plasma lipid levels, and until recently the molecular basis for this association has been unclear. We have determined that the Ag(x) epitope is associated with both a Pro-Leu2712, and Asn-Ser4311 substitution, with the Leu-Ser allele being associated with significantly lower levels of plasma lipids in a sample of healthy individuals from Sweden.(ABSTRACT TRUNCATED AT 250 WORDS)


Language intervention with children who have developmental delays: effects of an interactive approach. The interactive model of language intervention instructs parents to use techniques that promote reciprocal social interactions and facilitate the development of communication and language abilities. In this evaluation study, 32 mothers and their preschool-age children with developmental delays were randomly assigned to treatment and control (delayed treatment) groups. Consistent with the interactive model, mothers in the treatment group became more responsive, less directive, and provided clearer linguistic models. Furthermore, these changes were maintained for at least 4 months after intervention, and involvement in the parent-centered intervention program did not increase maternal stress. More important, these changes were accompanied by concomitant increases in children's use of vocal turns. Contrary to predictions, developmental improvements in children's communicative and linguistic abilities were comparable in both groups. Findings suggest that an interactive model may afford a useful adjunct to other intervention approaches by instructing parents on how to promote children's use of existing abilities, but an interactive model may have no effect on language acquisition of at least some children with developmental delays.


Do children with developmental delays use more frequent and diverse language in verbal routines? The current study is the first to test the hypotheses that children with developmental delays use more frequent language and more diverse vocabulary in routines than in nonroutines. The 19 child participants were in Brown's (1973) first stage of language learning. Using a novel method of measuring routines, 18 of the parents identified at least one routine in a videotaped play session with their children. Results support both hypotheses and provide descriptive information about the content of the routines displayed by the parents and children in the free-play context. The importance of replicating the findings in the context of an experimental design before concluding that "routineness" caused the children to talk more often and with more diverse vocabulary was emphasized.


Early language and communicative abilities of children with periventricular leukomalacia. Ten 2-year-old children with periventricular leukomalacia (PVL), a brain injury associated with prematurity, were evaluated using language samples. Five children scored less than 80 on cognitive testing (delayed). Five children with this disorder and normal cognitive scores were assessed at two ages, matched with the delayed group on CA and developmental level. The delayed group produced significantly fewer lexical tokens and spontaneous verbal utterances than did the CA-matched group. No significant differences were observed between the delayed group and either comparison group on other measures of lexicon, grammar, or communication. The data demonstrate a relation between cognitive abilities and measures of verbal productivity in children with PVL.


Vocal development in infants with Down syndrome and infants who are developing normally. The development of early vocalizations was investigated with 13 infants who had Down syndrome and 27 infants who were developing normally at bimonthly intervals from 4 to 18 months of age. A perceptually based framework was used to categorize utterances according to their developmental relations with adult, well-formed, or "canonical" syllables. Over time, both groups demonstrated increased production of mature vowel and canonical consonant-vowel syllables (characterized by full vowels, consonants, and rapid, well-coordinated articulatory movements) and decreased production of less mature "quasi-vowel" and marginal syllables. Infants in both groups were also quite variable in vocal development, both within group and across time.


Micrometastases from squamous cell carcinoma in neck dissection specimens. The incidence of micrometastases in cervical lymph nodes from squamous cell carcinomas of the head and neck was studied using routine histopathological examination. Micrometastases were found in 66 lymph nodes in 41 of the 92 tumor-positive neck dissection specimens. The detection of these micrometastases influenced postoperative treatment in 3 of the 77 patients with neck node metastases. The value of additional sectioning for detecting micrometastases was thus assessed. Sectioning at a deeper level in 600 originally histopathologically negative lymph nodes from 64 patients revealed 7 additional micrometastases in 5 patients. Antikeratin staining with a mixture of two monoclonal antibodies (AE1 and AE3) revealed 4 micrometastases in 739 originally histopathologically negative lymph nodes in 3 of 13 patients studied. Because of the unknown prognostic significance of micrometastases and the consequent arbitrary consequences for postoperative treatment, present findings show that the extra workload of immunostaining and deeper sectioning does not warrant their routine use in clinical practise.


Semi-dry electroblotting of DNA and RNA from agarose and polyacrylamide gels. Semi-dry electroblotting of nucleic acids was originally of interest for its speed and convenience, but it had significant limitations in reliability of transfer. This work describes optimalization of the buffer systems and general conditions for use that enable the semi-dry method of nucleic acids transfer to yield quantitative, fast and reliable results.


Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning. The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.