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1.
Microbiology (Reading) ; 157(Pt 5): 1363-1374, 2011 May.
Article in English | MEDLINE | ID: mdl-21349975

ABSTRACT

HynSL from Alteromonas macleodii 'deep ecotype' (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD, hupH and hypCABDFE) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2, cyt and orf1) was cloned into an IPTG-inducible expression vector and transferred into an Escherichia coli mutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed in E. coli and was active, as determined by an in vitro hydrogen evolution assay. Subsequent genetic analysis revealed that orf2, cyt, orf1 and hupH are not essential for assembling an active hydrogenase. However, hupH and orf2 can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and its Thiocapsa roseopersicina homologue. When the structural genes for the T. roseopersicina hydrogenase, hynSL, were expressed along with known T. roseopersicina accessory genes (hynD, hupK, hypC1C2 and hypDEF), no active hydrogenase was produced. Further, co-expression of AltDE accessory genes hypA and hypB with the entire set of the T. roseopersicina genes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with the T. roseopersicina structural genes generated an active T. roseopersicina hydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts from T. roseopersicina and that the two hydrogenases share similar maturation mechanisms.


Subject(s)
Alteromonas/enzymology , Bacterial Proteins/genetics , Escherichia coli/genetics , Gene Expression , Hydrogenase/genetics , Thiocapsa roseopersicina/enzymology , Alteromonas/genetics , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Hydrogenase/metabolism , Thiocapsa roseopersicina/genetics
2.
Clin Exp Dermatol ; 29(3): 304-7, 2004 May.
Article in English | MEDLINE | ID: mdl-15115517

ABSTRACT

Epidermolysis bullosa (EB) pruriginosa is a subtype of dominant dystrophic EB (DDEB), characterized by severe pruritus and blistering localized to the extensor surface of the extremities. EB pruriginosa exhibits extensive clinical heterogeneity with variable expression and delayed age of onset. Mutations in the COL7A1 gene, especially in glycine residues within Gly-X-Y repeats, have been shown to cause this form of DDEB. Here, we report a novel COL7A1 mutation in a Taiwanese pedigree with EB pruriginosa. Using PCR and direct sequence analysis we have identified a G-->T transversion at nucleotide 7097 in exon 92 of COL7A1, converting a glycine residue to valine (G2366V). The mutation resides within a consecutive, uninterrupted stretch of 17 Gly-X-Y residues in the triple-helical domain of type VII collagen. Interestingly, an affected member of this family also displayed elevated IgE levels, previously reported in some patients with this disorder. Our finding further implicates COL7A1 mutation in the pathogenesis of EB pruriginosa and underscores the heterogeneous clinical symptoms of glycine mutations in DDEB.


Subject(s)
Collagen Type VII/genetics , Epidermolysis Bullosa Dystrophica/genetics , Mutation, Missense , Base Sequence , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Pedigree
3.
Adv Exp Med Biol ; 493: 81-7, 2001.
Article in English | MEDLINE | ID: mdl-11727785

ABSTRACT

Opioids such as morphine are potent analgesic and addictive compounds. Chronic morphine use also induces immunomodulatory and immunosuppressive effects, as especially evident in HIV-infected patients. Morphine acts on the immune cells primarily through its binding to mu-opioid receptors on the plasma membrane. However, morphine modulation of immune functions still exists in mu-opioid receptor knockout mice, suggesting that in addition to the mu opioid receptors, morphine may also act by mechanisms mediated by either delta or kappa opioid receptors. To determine whether morphine activates kappa opioid receptors (KOR), a quantitative competitive RT-PCR procedure was utilized to quantify the KOR gene expression of morphine-treated cells. A segment of KOR transcript spanning the second extracellular loop, which has the reported dynorphin specificity, and the seventh transmembrane domain of the receptor was amplified from the total RNA of morphine-treated CEM x174 lymphocytes, along with a competitor molecule. The competitor was constructed by deleting a 33-nucleotide fragment from KOR. The results of the competitive RT/PCR indicated that CEM x174 cells expressed KOR mRNA constitutively, in the order of femto-grams. Treatment of 10 microM of morphine resulted in the up-regulation of KOR gene expression 24 hr post-treatment. The observed morphine effect could be reversed by treating the cells with either naloxone (a KOR-partially selective antagonist) or nor-Binaltorphimine (a KOR-selective antagonist).


Subject(s)
Lymphocytes/drug effects , Lymphocytes/metabolism , Morphine/pharmacology , Naltrexone/analogs & derivatives , Receptors, Opioid, kappa/drug effects , Receptors, Opioid, kappa/genetics , Animals , Cell Line , Humans , Hybrid Cells , Immunosuppressive Agents/pharmacology , Mice , Naloxone/pharmacology , Naltrexone/pharmacology , Narcotic Antagonists/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Opioid, kappa/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/drug effects
4.
Int Immunopharmacol ; 1(9-10): 1733-42, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11562065

ABSTRACT

We have previously shown that classical brain-like kappa opioid receptors (KOR) are constitutively expressed in lymphocytic cells. including human CEM x174 T-B hybrid cells, Jurkat -T4 cells, human peripheral blood mononuclear cells (PBMC), human CD4+ cells and monkey PBMC (Biochem. Biophys. Res. Commun. 209 (1995) 1003). The present study further demonstrates that the KOR of lymphocytes are activated in the presence of extracellular morphine or U50,488H, a KOR selective agonist, and the activation causes an increase in the expression of KOR mRNA, as determined by a quantitative competitive Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) procedure. The observed agonist-induced KOR up-regulation was blocked by treating the cells with either naloxone (a KOR-partially selective antagonist) or nor-binaltorphimine (a KOR-selective antagonist). Up-regulation of lymphocytic KOR by morphine was also evidenced by flow cytometric analysis of phycoerythrin (PE) amplification of fluorescein isothiocyanate-conjugated arylacetamide labeling of the KOR. Although morphine binds primarily to mu-opioid receptors, together with the previously reported phenomenon that morphine modulation of immune functions also exists in mu-opioid receptor knockout mice, the present study confirms that opioids such as morphine may exert their effects through multiple opioid receptor types and that the effects of morphine or endogenous opioids on immune cells could not be simply adduced from the anticipated effects of a synthetic, selective opioid receptor ligand.


Subject(s)
Analgesics, Opioid/pharmacology , Lymphocytes/metabolism , Morphine/pharmacology , Naltrexone/analogs & derivatives , Receptors, Opioid, kappa/biosynthesis , Up-Regulation/drug effects , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Analgesics, Non-Narcotic/pharmacology , Cell Line , Fluorescent Antibody Technique, Indirect , Humans , Lymphocytes/drug effects , Morphine/antagonists & inhibitors , Naltrexone/pharmacology , Narcotic Antagonists/pharmacology , RNA, Messenger/biosynthesis , Receptors, Opioid, kappa/agonists , Reverse Transcriptase Polymerase Chain Reaction
5.
Biochem Biophys Res Commun ; 279(2): 621-8, 2000 Dec 20.
Article in English | MEDLINE | ID: mdl-11118335

ABSTRACT

Opioid receptors of subtypes delta, kappa, and mu similar to those found in brain cells have been identified in immune cells. The current study demonstrates by competitive polymerase chain reaction the treatment of human lymphocytic cells with morphine resulting in an increased amount of gene expression of mu opioid receptors. Antibodies against the MOR-1, the neuronal mu opioid receptor, were used in Western blot analysis of mu proteins and the results revealed a single band of approximately 50 kDa, the intensity of which was increased by morphine treatment. Similar results of mu opioid receptor activation were observed when monkey lymphocytes were treated with morphine. These studies suggest that in addition to causing an immune effect through communication with the neuroendocrine system, the psychoactive drug morphine may modulate immune functions by acting directly on the mu opioid receptors expressed on lymphocytes.


Subject(s)
Lymphocytes/physiology , Morphine/pharmacology , Receptors, Opioid, mu/genetics , Up-Regulation/drug effects , Animals , B-Lymphocytes , Cell Line , Cells, Cultured , Haplorhini , Humans , Hybrid Cells , Kinetics , Lymphocytes/drug effects , Naloxone/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes
6.
J Biol Chem ; 275(40): 31305-10, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-10887175

ABSTRACT

All HIV-1 strains studied to date use CCR5, CXCR4, or both receptors to enter cells. Simian immunodeficiency virus (SIV) infection of non-human primates has served as a useful model for understanding AIDS pathogenesis in humans. Research on several genetically divergent SIV isolates has revealed that SIV uses CCR5, and not CXCR4, for entry. CEM x174, a human lymphoid cell line, has been routinely used to cultivate and maintain various SIV strains. However, questions have arisen about how CEM x174, which reportedly was unable to express detectable amounts of CCR5 transcripts, efficiently supports the growth of SIV. In searching for an answer, we resorted to a sensitive competitive reverse transcriptase-polymerase chain reaction procedure in an attempt to detect as well as quantify the amount of CCR5 expression. Here we present our findings, which indicate that CEM x174 indeed expresses CCR5 and that the amount of CCR5 is increased in cells pretreated with morphine. These results correlate well with our previous observations that morphine treatment causes CEM x174 cells to be more susceptible to SIV infection. Similar morphine effect was not observed on CEM x174 cells infected with simian retroviruses, which do not depend on CCR5 for entry. These findings suggest a plausible mechanism whereby opiate drug users render themselves more susceptible to HIV infection, thereby explaining the vast prevalence of HIV infection among endemic drug use populations.


Subject(s)
Gene Expression/drug effects , Lymphocytes/drug effects , Lymphocytes/metabolism , Morphine/pharmacology , Narcotics/pharmacology , Receptors, CCR5/biosynthesis , Receptors, G-Protein-Coupled , Receptors, Virus , B-Lymphocytes/metabolism , Blotting, Western , Cell Line , Dose-Response Relationship, Drug , Flow Cytometry , HIV Infections/etiology , Humans , Kinetics , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Receptors, CXCR6 , Receptors, Chemokine , Receptors, Cytokine/metabolism , Receptors, Peptide/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Simian Acquired Immunodeficiency Syndrome/etiology , Simian Acquired Immunodeficiency Syndrome/metabolism , Substance-Related Disorders , T-Lymphocytes/metabolism , Time Factors
7.
Immunopharmacology ; 47(1): 53-62, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10708810

ABSTRACT

Opioid users having acquired human immunodeficiency syndrome (AIDS) are at a greater risk than non-users of contracting opportunistic infections. Opioid-administered and simian immunodeficiency virus (SIV)-infected rhesus monkeys have been an excellent model for studying AIDS and drug abuse in humans. In this study, chemotaxis of monkey leukocytes was evaluated using the chemokines interleukin-8 (IL-8) and regulated upon activation, normal T cell expressed (RANTES) as the chemoattractants, and the effects of various opioid agonists and antagonists on the efficiency of chemotaxis were examined. Opioids were either incubated with monkey leukocytes or added directly to chemokines, and the number of cells migrating toward IL-8 (for neutrophils) or RANTES (for monocytes) was scored. Inhibition of chemotaxis was seen with both assay conditions, and the inhibition was mediated by opioids binding to mu or kappa receptors. Binding to delta opiod receptors was rarely, if ever, observed. Although opioids themselves may act as weak chemoattractants for monkey leukocytes, addition of opioid agonists to chemokines would reduce the chemoattractant ability of the chemokines. Opioids did not cause the same inhibitory effect on the chemotactic migration of neutrophils when the complement component C5a or the chemotactic peptide N-formyl-MET-LEU-PHE (fMLP) was used as chemoattractant. These studies suggest that the presence of opioids during SIV infection immediately alters chemokine-mediated immune functions.


Subject(s)
Cell Movement/drug effects , Chemokines/pharmacology , Leukocytes/physiology , Narcotics/pharmacology , Animals , Chemokine CCL5/pharmacology , Chemotactic Factors/pharmacology , Chemotaxis, Leukocyte/drug effects , Complement C5a/pharmacology , Dose-Response Relationship, Drug , Interleukin-8/pharmacology , Leukocytes/drug effects , Macaca mulatta , Male , Monocytes/cytology , Monocytes/drug effects , Monocytes/physiology , Morphine/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Narcotic Antagonists/pharmacology , Neutrophils/cytology , Neutrophils/drug effects , Neutrophils/physiology , Time Factors
9.
J Biochem Mol Toxicol ; 14(1): 42-50, 2000.
Article in English | MEDLINE | ID: mdl-10561081

ABSTRACT

Heptachlor is an organochlorine insecticide used worldwide for the control of pests both agriculturally and domestically. Its lipophilic structure allows it to bioaccumulate and pass through the food chain, exposing those who come in contact with it to its tumor promoting and possible carcinogenic effects. As a mechanism of tumor promotion, we explored the possibility of heptachlor suppressing the apoptotic process in human CEM x 174 lymphocytes. In this article, we describe the effect of heptachlor on the activity of the apoptosis protease CPP32. We show that heptachlor by itself was able to stimulate CPP32 activity at relatively high concentrations. When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, a dual effect was observed. Low concentrations of heptachlor (5 microM-10 microM) suppressed doxorubicin-induced CPP32 activity, and high concentrations of heptachlor (80 microM-120 microM) augmented it. We also showed that heptachlor alone at relatively high concentrations induced apoptosis-associated changes in CEM x 174 cells including high molecular weight (HMW) DNA cleavage and chromatin condensation. From these results, it appears that heptachlor has tumor promoting-like effects at lower concentrations, and at higher concentrations induces apoptosis as a mechanism of cytotoxicity.


Subject(s)
Caspases/metabolism , Heptachlor/pharmacology , Insecticides/pharmacology , Apoptosis/drug effects , Caspase 3 , Cell Line , DNA/drug effects , Doxorubicin/pharmacology , Humans , Microscopy, Fluorescence
10.
In Vivo ; 13(2): 189-93, 1999.
Article in English | MEDLINE | ID: mdl-10363177

ABSTRACT

The extent of infection of monkey polymorphonuclear neutrophils (PMN) by simian immunodeficiency virus (SIV) has not yet been determined. Using the polymerase chain reaction (PCR) technique, we detected the presence of SIVmac239 DNA in rhesus macaque-derived PMN after 24 hrs of in vitro incubation of the cells with SIVmac239. Infection by SIVmac239 also down-regulated the expression of the bcl-2 apoptosis-blocking gene in the infected PMN. These SIVmac239-induced PMN intracellular alterations were correlated with an accelerated decrease in PMN viability over a period of 120 hrs compared to non-infected PMN. Evidence of chromatin condensation characteristic of programmed cell death (apoptosis) was also observed in SIVmac239-infected PMN. The results of this study provide a mechanism for the reduced chemotaxis/phagocytosis activities of PMN of SIVmac239-infected macaques and suggest that PMN is one of the target cells for SIVmac239 infection.


Subject(s)
DNA, Viral/analysis , Neutrophils/virology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Animals , Cell Survival , DNA Primers/chemistry , Disease Models, Animal , Genes, gag/genetics , Macaca mulatta , Microscopy, Electron , Neutrophils/ultrastructure , Polymerase Chain Reaction , Simian Acquired Immunodeficiency Syndrome/blood
11.
Proc Natl Acad Sci U S A ; 96(6): 2656-61, 1999 Mar 16.
Article in English | MEDLINE | ID: mdl-10077566

ABSTRACT

The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two distinct functional domains. The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs. The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain) binds to the Sc. pombe replication origin, ars1. The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc. pombe origins of replication, which differ significantly from those of Sa. cerevisiae.


Subject(s)
DNA Replication , DNA-Binding Proteins/genetics , Replication Origin/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , DNA, Fungal/genetics , Fungal Proteins/genetics , Humans , Molecular Sequence Data , Origin Recognition Complex , Repressor Proteins/genetics
12.
Toxicol Lett ; 104(1-2): 127-35, 1999 Jan 11.
Article in English | MEDLINE | ID: mdl-10048758

ABSTRACT

Organochlorine use over the past 50 years has resulted in the contamination of soil, water, plant and animal species. This contamination has created a long-lasting environmental problem, as the members of the organochlorine class of pesticides are resistant to degradation and have been labeled as persistent bioaccumulators. Studies have shown certain organochlorines to be tumor promoters, liver toxicants and to induce immune cell dysfunction in rats and mice. Our laboratory has shown that the organochlorines heptachlor and chlordane affect leukocytic gene expression and differentiation. In this study, experiments with CEM x 174 cells, a hybrid of human T and B cells, were performed to investigate the effects of the tumor promoter heptachlor and its congeners chlordane and toxaphene on retinoblastoma (Rb) gene expression. The results indicated that heptachlor, chlordane or toxaphene, in the range of 10-50 microM, were able to reduce Rb protein levels in a concentration-dependent manner. In the case of heptachlor, the reduction could be seen as early as 12 h and was time-dependent. Analysis of Rb mRNA levels revealed no detectable difference over the same concentration range. These results suggest that members of the organochlorine class are able to downregulate Rb expression at the post-transcriptional level, an effect similar to that on p53 tumor suppressor previously reported by our laboratory.


Subject(s)
Gene Expression Regulation , Genes, Retinoblastoma , Insecticides/toxicity , Lymphocytes/drug effects , Blotting, Western , Cells, Cultured , Chlordan/toxicity , Heptachlor/toxicity , Humans , Kinetics , Lymphocytes/ultrastructure , Protein Biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Toxaphene/toxicity , Transcription, Genetic
13.
In Vivo ; 13(6): 455-61, 1999.
Article in English | MEDLINE | ID: mdl-10757037

ABSTRACT

Heptachlor, a chlorinated hydrocarbon insecticide, has been considered an environmental contaminant with potential adverse health effects. Exposure to heptachlor may impair immune functions including the inhibition of leukocyte chemotaxis, which causes defects in host defense mechanisms. This study addresses the effects of heptachlor on the cell cycle progression of human lymphocytes. It has been found that addition of heptachlor to cultured lymphocytic cells prevents the cells from progression into the S phase of the cell cycle, with a concomitant accumulation of cells in G1 phase. An accompanying decrease (deactivation) in cyclin-dependent kinase cdk2 and dephosphorylation (activation) of cdc2 was observed. The altered cell cycle progression may trigger the cell's apoptotic potential, as indicated by the reduced amount of Bcl-2 synthesized inside heptachlor-treated cells. The interference of cell cycle progression by heptachlor was also seen with chlordane and toxaphene, two other chlorinated hydrocarbon insecticides.


Subject(s)
CDC2 Protein Kinase/biosynthesis , CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/biosynthesis , Heptachlor/pharmacology , Insecticides/pharmacology , Lymphocytes/drug effects , Lymphocytes/enzymology , Protein Serine-Threonine Kinases/biosynthesis , Apoptosis/immunology , Blotting, Western , CDC2 Protein Kinase/analysis , Cell Line , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/analysis , Flow Cytometry , G1 Phase/drug effects , Humans , Lymphocytes/cytology , Protein Serine-Threonine Kinases/analysis , S Phase/drug effects , Signal Transduction/drug effects , Toxaphene/pharmacology
14.
In Vivo ; 13(5): 389-96, 1999.
Article in English | MEDLINE | ID: mdl-10654191

ABSTRACT

It is recognized that chemotaxis and phagocytosis constitute the first line of defense in the immune system, and chemokines function mainly as chemoattractants for phagocytic cells, recruiting monocytes and neutrophils from the blood to sites of infection. In this study, chemotaxis of monkey leukocytes was evaluated using human chemokines IL-8 (interleukin-8), MIP-1 beta and RANTES as the chemoattractants, and the effects of micro-opioid receptor agonists, morphine, DAMGO, methadone and endomorphine, on the efficiency of chemotaxis were examined. It was found that human chemokines served well as chemoattractants for monkey leukocytes, and similar to the human system, chemokine-induced chemotaxis of monkey leukocytes was inhibited in the presence of micro-opioid receptor agonists. The inhibition could be reversed by naloxone, a specific micro-opioid receptor antagonist. These studies further support the value of the monkey model for drug abuse studies in humans, as well as suggest that opioids such as morphine may alter immune functions through micro-opioid receptors on leukocytes.


Subject(s)
Chemokines/antagonists & inhibitors , Chemokines/pharmacology , Chemotaxis, Leukocyte/drug effects , Monocytes/drug effects , Neutrophils/drug effects , Receptors, Opioid, mu/agonists , Analgesics, Opioid/pharmacology , Animals , Cell Migration Inhibition , Chemokine CCL4 , Chemokine CCL5/antagonists & inhibitors , Chemokine CCL5/pharmacology , Humans , Interleukin-8/antagonists & inhibitors , Interleukin-8/pharmacology , Macaca mulatta , Macrophage Inflammatory Proteins/antagonists & inhibitors , Macrophage Inflammatory Proteins/pharmacology , Male , Monocytes/cytology , Monocytes/immunology , Morphine/pharmacology , Neutrophils/cytology , Neutrophils/immunology , Receptors, Opioid, mu/antagonists & inhibitors
15.
In Vivo ; 12(5): 441-6, 1998.
Article in English | MEDLINE | ID: mdl-9827349

ABSTRACT

Chemokines, characterized as pro-inflammatory chemicals made by the immune system, consist of a family of low molecular weight proteins with potent in vitro chemotactic activity causing leukocyte accumulation in vivo. This study determines the effects of organochlorine pesticide exposure on the chemotactic functions of monkey neutrophils and monocytes, using a 48-well chemotaxis chamber. Chemokines IL-8 (interleukin-8) and RANTES were used as the chemoattractants to induce chemotaxis among these monkey leukocytes. Monkey neutrophils or monocytes were first treated with heptachlor, chlordane or toxaphene for 1 hour at 37 degrees C, and the number of cells migrating toward 200 ng/ml IL-8 (for neutrophils) or 100 ng/ml RANTES (for monocytes) were scored. Inhibition of chemotaxis was seen with all samples after treatment with heptachlor, chlordane and toxaphene at concentrations from as low as 10(-14) M to 10(-5) M. Among the three compounds studied, toxaphene was the least effective in preventing monocytes from migrating toward RANTES. The ability of these pesticides to inhibit chemotaxis did not correlate directly with their potential apoptotic effects on the monkey leukocytes. These studies suggest that exposure to organochlorine pesticides may alter leukocyte-related immune functions.


Subject(s)
Chemokines/pharmacology , Chemotaxis, Leukocyte/drug effects , Insecticides/pharmacology , Monocytes/drug effects , Neutrophils/drug effects , Animals , Apoptosis/drug effects , Cell Count , Chemokine CCL5/pharmacology , Chemotaxis, Leukocyte/physiology , Chlordan/pharmacology , DNA/analysis , Heptachlor/pharmacology , Interleukin-8/pharmacology , Macaca mulatta , Male , Monocytes/physiology , Neutrophils/physiology , Toxaphene/pharmacology
16.
Toxicology ; 128(1): 17-23, 1998 Jun 26.
Article in English | MEDLINE | ID: mdl-9704902

ABSTRACT

The organochlorine pesticide heptachlor constitutes a potential health hazard because of its persistence in nature, its reported contamination in food and milk, and its possible carcinogenic effects. As a tumor promoter, heptachlor induces human myeloblastic leukemia cells to differentiate, and also down-regulates the tumor suppressor gene p53 in human immune cells. In this study, the heptachlor signaling pathway in human lymphocytes was studied. Addition of heptachlor to human CEM x174 lymphocytic cells reduced the cellular levels of MAP kinase (MAPK, mitogen-activated protein kinase) cascade proteins, including ERK1 (a 44-kDa MAPK), ERK2 (a 42-kDa MAPK), a 85-kDa and a 54-kDa MAP kinase, MEK1 (a 45-kDa ERK kinase) and MEKK (a 78-kDa MEK kinase). However, heptachlor treatment caused a marked increase in the expression of the activated (Thr- and Tyr-dually phosphorylated) ERK1 and ERK2 in the cells. These studies indicate that mitogen-activated protein kinases are important intermediates in the signal transduction pathway of immune cells upon heptachlor exposure, and the observation of stimulation of activated MAP kinases without a simultaneous accumulation of basal enzymes may suggest the involvement of a negative feedback control mechanism in the pathway.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Heptachlor/toxicity , Insecticides/toxicity , Lymphocytes/enzymology , B-Lymphocytes/drug effects , B-Lymphocytes/enzymology , Blotting, Western , Cell Division/drug effects , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Humans , Hybrid Cells , Immunoblotting , Lymphocytes/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology
17.
Toxicol Lett ; 94(1): 29-36, 1998 Jan 16.
Article in English | MEDLINE | ID: mdl-9544696

ABSTRACT

Previous studies have shown that heptachlor, a chlorinated hydrocarbon insecticide, is a liver tumor promoter in rats and mice and induces tumor promoting-like alterations in human myeloblastic leukemia cells. The nature of tumor promotion is multifaceted and has recently been shown to include suppression of programmed cell death (apoptosis) as a mechanism by which a tumor promoter can prolong cell viability. The ability of tumor promoters to suppress apoptosis prompted us to address the question of whether heptachlor is capable of effecting the expression of genes involved in lymphocyte apoptosis, in particular, the p53 tumor suppressor gene. Experiments with a CEM x 174 cell line, a hybrid of human T and B cells, revealed that heptachlor downregulated p53 gene expression at the post-transcriptional level without changing levels of mRNA in the cells. The heptachlor-induced reduction in the basal levels of expression of this gene was both in a concentration and time-dependent manner.


Subject(s)
Apoptosis/drug effects , Genes, p53/drug effects , Heptachlor/toxicity , Insecticides/toxicity , Lymphocytes/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Down-Regulation , Gene Expression , Genes, p53/genetics , Humans , In Vitro Techniques
18.
J Biol Chem ; 272(43): 26815-7, 1997 Oct 24.
Article in English | MEDLINE | ID: mdl-9341110

ABSTRACT

Morphine sulfate causes immunomodulatory and immunosuppressive effects in human. In this study, the signaling pathway involved in these morphine effects was studied. Addition of morphine sulfate to human CEMx174 lymphocytic cells resulted in increased expression of mitogen-activated protein kinase cascade proteins. Morphine enhanced the cellular levels of ERK1 (44 kDa), ERK2 (42 kDa), a 54-kDa ERK, MEK1 (45 kDa), and MEKK (78 kDa). A time-dependent increase in the activated (Thr and Tyr dually phosphorylated) state of ERK1 and ERK2 was also observed. Naloxone, a morphine antagonist, reversed the observed morphine effects, implicating a micro opioid receptor-mediated process. These findings suggest that mitogen-activated protein kinases are important intermediates in signal transduction pathways initiated by morphine receptors in immune cells.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Lymphocytes/physiology , MAP Kinase Kinase Kinase 1 , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Morphine/pharmacology , Signal Transduction/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Cell Line , Enzyme Induction , Humans , Lymphocytes/drug effects , Lymphocytes/enzymology , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism
19.
Science ; 275(5305): 1468-71, 1997 Mar 07.
Article in English | MEDLINE | ID: mdl-9045610

ABSTRACT

The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.


Subject(s)
Protein Biosynthesis , RNA Nucleotidyltransferases/metabolism , RNA, Fungal/genetics , RNA, Messenger/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Animals , Cytoplasm/metabolism , DEAD-box RNA Helicases , Eukaryotic Initiation Factor-4A , Genes, Fungal , Mice , Mutation , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Phenotype , RNA Helicases , RNA Nucleotidyltransferases/genetics , RNA Splicing , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/enzymology
20.
Addict Biol ; 2(4): 421-30, 1997 Oct.
Article in English | MEDLINE | ID: mdl-26735947

ABSTRACT

Rhesus monkeys (Macaca mulatta) and the simian immunodeficiency virus (SIV) were used as an animal model system to evaluate longitudinally the effects of opioid dependence on the development of AIDS. Results have shown that in addition to weakening the host's T cell and PMN functions, chronic opioid treatment of SIVmac239-infected animals caused (a) an increased virus replication rate, (b) an increased rate of viral mutation, (c) a nascent humoral (antibody) response against mutated autologous strains of virus, (d) an alteration of CD8 + cell-mediated immunity toward the mutant virus, (e) an increased tolerance of AZT among the infectious virus and (f) a shorter life span for the infected animals. These results suggest that chronic administration of opioids alters general fundamental aspects of the AIDS viremia.

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