Cytokines in the treatment of fungal infections

The incidence of invasive fungal infections in the immunocompromized host has increased during the past decade. Even the recently developed antifungal drugs are unable to cure these infections in patients with severely impaired host defense mechanisms. Cytokines have great potential to augment host resistance and as adjunctive therapy of invasive mycoses. We discuss the mechanisms of host defense against invasive candidiasis, aspergillosis, and cryptococcosis, and review the use of cytokines and growth factors in this setting. Interleukin-1 has been shown effective in an animal model of disseminated candidiasis, even during severe granulocytopenia. Interferon- has been very effective as a modulator of resistance against a variety of fungal infectionsin vitro. The effect of interferon- against disseminated candidiasis has been demonstrated in a mouse model. Activation of neutrophils is the main mechanism by which interferon- enhances the elimination ofCandida, and consequently the agent is not effective in severly granulocytopenic animals. Data on the role of colony-stimulating factors against fungal pathogens are accumulating, and trials with these agents for hematologic patients with invasive fungal infections are now being performed.Abbreviations CGD chronic granulomatous disease - G-CSF granulocyte colony-stimulating factor - M-CSF monocyte colony-stimulating factor - GM-CSF granulocyte-monocyte colony-stimulating factor - IFN- interferon-gamma - IL interleukin - LAK lymphokine-activated killer - LPS lipopolysaccharide - MDP muramyl dipeptide - NK natural killer - PMN polymorphonuclear leukocytes - rh recombinant human - ROI reactive oxygen intermediates - TNF tumor necrosis factor     

In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Gal1-4GlcNAc1-3Gal1-4(Fuc1-3)Glc, lacto-N-neodifucohexaose Gal1-4(Fuc1-3)Glc-NAc1-3Gal1-4(Fuc1-3)Glc, and lacto-N-neodifucooctaose Gal1-4GlcNAc1-3Gal1-4(Fuc1-3)GlcNAc1-3Gal1-4(Fuc1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an -1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon.To prevent fucosylation at the glucosyl residue, lactulose Gal1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.     

Mechanisms of voltage transients during current clamp inNecturus gallbladder

Summary Microelectrode techniques were employed to study the mechanisms of the transepithelial voltage transients (V _ms ) observed during transmural current clamps in the isolatedNecturus gallbladder. The results indicate that: a) part of V _ms is due to a transepithelial resistance change (R _t ), and part to a tissue emf change. b) R _t is entirely caused by changes of the resistance of the paracellular pathway. At all current densities employed, the measured changes are probably due to changes in both fluid conductivity and width of the lateral intercellular spaces. At high currents, in addition to the effects on the lateral spaces, the resistance of other elements of the pathway (probably the limiting junction) drops, regardless of the direction of the current. c) The magnitude and polarity of the R _t -independent transepithelial and cell membrane potential transients indicate that the largest emf change takes place at the basolateral membrane (E _b ), with smaller changes at the luminal membrane (E _a ) and the paracellular (shunt) pathway (E _s ). It is shown that two-thirds of the transient are caused by E _s , and one-third by (E _b –E _a ). E _s can be explained by a diffusion potential generated by a current-dependent NaCl concentration gradient across the tissue. E _a and E _b are caused by [K] changes, mainly at the unstirred layer in contact with the basolateral membrane.     

Hydrogen ion dependence of carrier-mediated auxin uptake by suspension-cultured crown gall cells

1. The dependence on external hydrogen ion concentration of the carrier-mediated components of the uptakes of indol-3-yl acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4D) by suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch. is examined. The initiall rates of uptake of IAA and 2,4 D from 0.30 mol/l solutions exhibit pH optima of approximately pH 5.0 and pH 4.0 respectively.-2. The inherent difficulties, especially at low pH, of estimating the carrier-mediated uptake component in the presence of the large diffusive uptake of undissociated acid are discussed.-3. At pH 5.0, the uptakes of the two auxins are characterised by similar Michaelis Constants (0.65 and 0.80 mol/l) and Maximum Velocities (0.87 and 1.0 pmol/min/mg cells) for IAA and 2,4D respectively.-4. A dependence of carrier-mediated auxin uptake on cations other than hydrogen ions could not be demonstrated. It is suggested that uptake of auxin anions may occur by co-transport with hydrogen ions.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-γ

We investigated the influence of the combined use of sizofiran, a-1,3-glucan and a recombinant interferon- (rIFN-) upon biological activities of peritoneal macrophages (M). The number of peritoneal M and the production of cytokines (interleukin-1, interferon- and tumor necrosis factor) was increased by the combined treatment. Fully activated peritoneal M based on the increased number of elongated pseudopods were observed by electromicroscope. Sizofiran seems to assure a sufficient supply of M to kill tumor cells in the peritoneal cavity and co-administered rIFN- seems to directly stimulate the accumulated M in addition to its direct cytotoxicity against tumor cells. This combination therapy may be a step to the prevention of the recurrence of gynecological malignancies including ovarian cancer, after a negative second-look laparotomy.Abbreviations rIFN- recombinant interferon- - IL-1 interleukin-1 - TNF tumor necrosis factor - SLL second look laparotomy     

Correlations between in vivo resistance to Fusarium and in vitro response to fungal elicitors and toxic substances in carnation

Summary With the aim of ascertaining the existance of a correlation between in vivo resistance to Fusarium oxysporum f. sp. dianthi and in vitro response to fungal elicitors and toxic substances, phenylalanine ammonialyase and phytoalexin accumulation, on one hand, and resistance to culture filtrate, on the other, were assayed in in vitro cultures of three susceptible and four resistant Dianthus caryophyllus cultivars. Cultivars showing varying degrees of resistance in vivo either tolerated higher culture filtrate concentrations (Niki) or showed high PAL activity and phytoalexin production when treated with Fusarium elicitor (Duca), or responded positively to both treatments (Mei-Ling, Pulcino). No such responses were shown in tissue cultures of susceptible cultivars. The differential response to the fungal elicitor seemed to be highly specific as genetic differences between cultivars were not observed in tissue cultures treated with other biotic (Phytophthora infestans) and abiotic (HgCl₂) elicitors.Abbreviations FuCWC cell wall components from Fusarium oxysporum f. sp. dianthi race 2 - PhCWC cell wall components from Phytophthora infestans - PAL phenylalanine ammonia-lyase (EC 4.3.1.5)     

The pyridine-nucleotide cycle in tobacco

In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NADNMNnicotinamidenicotinic acidNaMNnicotinic acid adenine dinucleotideNAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.Abbreviations HPLC high-performance liquid chromatography - Na nicotinic acid - NaAD nicotinic acid adenine dinucleotide - NaMN nicotinic acid mononucleotide - NMN nicotinamide mononucleotide - PRPP 5-phosphoribosyl-1-pyrophosphate This contribution is dedicated to Professor Augustin Betz on the occasion of his 65^th birthday     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

Propionibacterium acnes induces acute TNFα-mediated apoptosis of hepatocytes followed by inflammatory T-cell-mediated granulomatous hepatitis in mice

The CD3+/TCR+ T-cell-mediated hepatic inflammation induced byPropionibacterium acnes could be divided into an acute and a chronic phase. The acute phase occurred within 72 h after injection and displayed hepatic apoptosis. Anti-TNF antibody inhibited both theP. acnes-induced hepatic apoptosis and lymphocyte infiltration seen in this phase, indicating the involvement of this cytokine. Thereafter, a chronic phase was manifested from days 7 to 14 after injection. It was characterized as granulomatous inflammation admixed with apoptosis of infiltrating lymphocytes and some hepatocytes. Immunohistochemical staining showed that the infiltrating lymphocytes displayed TNF, TNF type I receptor and a variety of cytokines including IL-1, IL-4, IL-6, IL-10, IFN or IL-12. Interestingly, in naive mice, the arteries in the liver constitutively expressed IFN. Its expression appeared to be substantially increased at 48 h, decreased at 72 h, and increased again on day 14 afterP. acnes injection. Furthermore, Fas or FasL was only detected on the lymphocytes within the granuloma. We conclude thatP. acnes can induce a TNF-mediated acute hepatic apoptosis which subsequently progress to a T-cell-mediated granulomatous hepatitis with increased expression of multiple cytokines and Fas/FasL.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.     

Discrimination of alfalfa weevil strains by allozyme analysis

Allozyme profiles of eastern weevils (Beltsville, Maryland; Washington Co., Illinois), western weevils (Logan and St. George, Utah), and Egyptian weevils (Yuma, Arizona; Westmorland, California) were compiled by acrylamide gel electrophoresis. Twenty-two gene loci from 12 enzymes (ACPH, ADH, AMY, AO, EST, GOT, G-6PDH, MDH, ME, SOD, TYR, XDH) were analyzed. Mean heterozygosity of these populations was 0.231, with an average proportion of polymorphic loci of 0.536. The mean genetic distance of all weevil populations was 0.033 and the fixation index was 0.024. Diagnostic loci were found which could distinguish western weevils from eastern and Egyptian weevils. The small genetic distance between the eastern and Egyptian weevils suggests that they may be the same strain and are certainly different from the western weevil strain. Based on this and other evidence, we conclude that all weevil strains in the United States are Hypera postica (Gyllenhal), and that the use of H. brunneipennis (Boheman) for the Egyptian alfalfa weevil of North America should be discontinued.

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Résumé Les allozymes d'Hypera de l'est des USA (Beltsville, Maryland; Washington Co., Illinois), de l'ouest des USA (Logan et St Georges, Utah) et égyptiens (Yuma, Arizona; Westmorland, Californie) ont été analysés par électrophorèse sur gel d'acrylamide. L'étude a porté sur 22 loci de 12 enzymes (ACPH, ADH, AMY, AO, EST, GOT, G-6PDH, MDH, ME, SOD, TYR, XDH). L'hétérozygotie moyenne de la population était 0.231, avec une moyenne de loci polymorphes de 0.536. La distance génétique moyenne de l'ensemble des populations était de 0.033 et l'indice de fixation de 0.024. Des loci caractéristiques ont été trouvés qui pourraient permettre de distinguer les Hypera occidentaux des orientaux et des égyptiens. La faible distance génétique entre les Hypera orientaux et égyptiens suggère qu'ils appartiennent à la même souche et sont certainement différents des occidentaux. A partir de cela et d'autres éléments, nous concluons que tous les Hypera des USA sont H. postica Gyllenhal et que l'utilisation d'H. brunneipennis Boheman pour désigner les Hypera égyptiens d'Amérique du Nord doit être abandonnée.

     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Transmission of primary trisomics in pearl millet

Summary Transmission rates of extra chromosomes found in the full set of trisomics of pearl millet (Pennisetum americanum) (2n = 14) were estimated by examining the progeny of selfed trisomics and the progeny of trisomics crossed to disomics. When the trisomics were selfed, dark green and tiny had the highest transmission rate (23.8% and 23.3%, respectively) and pseudonormal the lowest (13.8%). Other trisomics had an intermediate rate of transmission. When the trisomics were used as females in crosses with disomics, both dark green and tiny again had the highest transmission rate and pseudonormal the lowest. When the trisomics were used as males in crosses to disomics, no trisomic was transmitted to the progeny except for spindle, and this occurred with a very low frequency (2.0%). A variation in transmission rate was observed from plant to plant and season to season for the same trisomic type. A study of the transmission rate of the extra chromosomes indicated that the following factors were probably contributing to the lower rate of transmission: small- or light-weight seeds tended to have a higher proportion of trisomics than heavier seeds; lighter seeds had a lower percentage germination; a positive and significant correlation was noticed between trivalent frequency and transmission rate. Plants with reduced vigour produced a higher frequency of trisomics. Though trisomics involving longer extra chromosomes showed a high degree of pollen and ovule sterility, they were highly transmissible. This has resulted in a close relationship between gametic sterility and transmission rate of extra chromosome.     

Cell and endophyte structure of the nitrogen-fixing root nodules ofCeanothus integerrimus H.and A.

Summary The nitrogen fixing root nodules ofCeanothus integerrimus were very similar in appearance to other non-legume nodules. Each nodule was a cluster of small lobes. Each lobe in cross section had a central vascular cylinder and a hypertrophied cortex. The cortex contained very large infected cells, with large nuclei; among these infected cells were scattered small, normal-appearing cortex cells. The actinomycete endophyte consisted of wavy hyphae 0.4 m in diameter which terminated in pear-shaped vesicles 1.6 m×2 m. The vesicles were not septate. The function of the vesicles was unknown. The infected cells had apparently normal nuclei, chloroplasts and mitochondria and were probably alive, except at the base of the nodule where both infected cells and the endophyte they contained were dead.     

Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, 5-3-hydroxysteroid dehydrogenase (5-3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD). The activities of acid phosphatase, alkaline phosphatase and -glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular 5-3-HSD activity was higher in transgenic than in normal mice, while testicular 17-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of -glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.     

Restoration of the acute phase response after infection in cyclophosphamide-treated mice by granulocyte colony-stimulating factor

The therapeutic effect of granulocyte colony-stimulating factor (G-CSF) against intramuscular infection withPseudomonas aeruginosa in cyclophosphamide (CY)-treated mice was analyzed by measuring plasma levels of amyloid P-component (APC) and proinflammatory cytokine levels. CY (100mg/kg) treatment of mice significantly suppressed plasma concentrations of APC and tumor-necrosis factor- (TNF-) following infection withP. aeruginosa, in associated with enhanced susceptibility of the treated mice to this bacterium. A 4-day treatment of CY-treated mice with recombinant human G-CSF (rhG-CSF) increased resistance of CY-treated mice, together with the marked restoration of APC and TNF- productions. The capacity to produce interleukin 1- and TNF- of peritoneal macrophages and also that to produce IL-6 of spleen cells were significantly enhanced by thein vivo administration of rhG-CSF in CY-treated mice. These results indicate that G-CSF may increase the functions of monocytes/macrophages directly or indirectlyin vivo. Therefore, the therapeutic effect of rhG-CSF seems to consist of not only increases in the number and functions of neutrophills but also enhancement of monocyte/macrophage functions.Abbreviations rhG-CSF recombinant human granulocyte-colony stimulating factor - PMNs polymorphonuclear leukocytes - CY cyclophosphamide - HBSS Hanks' balanced salt solution - APC amyloid P-component - IEP immunoelectrophoresis - CFU colony-forming units - TNF- tumor-necrosis factor- - d IL interleukin     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work