[Possible pathogenic role of IL-16 on SLE: with reference to HIV-1 infection].
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CD4 Antigens↗
Biomedical subjects
S Lee
Publications and source records attributed to S Lee.
Promoter region of the Drosophila melanogaster norpA gene.
We have sequenced the 5'-flanking region of the Drosophila melanogaster norpA gene and characterized its promoter. The potential promoter region, which was deduced from the determination of the transcription start point (tsp), lacks a distinct TATA box sequence. Deletion analysis of the promoter region suggests that the minimal promoter necessary for efficient expression of the gene is located between -138 (PstI) and +278 relative to the tsp. Within this minimal promoter region, at least two downstream regulatory elements responsible for the stimulation of gene expression seem to exist in the DNA fragments between +44 and +121 and between +214 and +278. Among these, the DNA fragment between +44 and +121 affects promoter activity more dramatically (about 6-7 fold). This DNA fragment contains the consensus promoter element previously reported to be important for photoreceptor cell-specific expression, and this promoter element seems to be working in the norpA gene expression.
Animals↗
Specific gas chromatographic analysis of diethylcarbamazine in human plasma using solid-phase extraction.
Diethylcarbamazine (DEC, 1-diethylcarbamyl-4-methylpiperazine) is an antiparasitic piperazine derivative used in the treatment of lymphatic filariasis. DEC-N-oxide is a major metabolite in humans which has antifilarial activity. Gas chromatographic analysis of DEC in plasma can be complicated by the presence of the metabolite, since the thermally unstable DEC-N-oxide is converted to a material which coelutes with DEC under the conditions of the analysis. We now report a method to separate DEC-N-oxide from DEC in plasma using solid-phase extraction with subsequent gas chromatographic analysis using a nitrogen specific detector. 1-Diethylcarbamyl-4-ethylpiperazine (E-DEC) was the internal standard. The standard curve of DEC is linear in the range of 10 to 200 ng/ml. The limit of detection is 4 ng/ml. Reproducibility at 10, 100 and 200 ng/ml concentration points of the standard curve gives coefficients of variation of 6.1%, 7.8% and 1.6%, respectively. Recovery following solid-phase extraction is 99.3% for DEC and 94.8% for the internal standard. This sensitive and specific analytical method is suitable for pharmacokinetic studies of DEC.
Calorimetry, Differential Scanning↗
Structure-activity relationship of 3-substituted N-(pyridinylacetyl)-4- (8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene )- piperidine inhibitors of farnesyl-protein transferase: design and synthesis of in vivo active antitumor compounds.
Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.
3T3 Cells↗
Alcohol increases the expression of type 1, but not type 2 alpha corticotropin-releasing factor (CRF) receptor messenger ribonucleic acid in the rat hypothalamus.
We investigated the ability of a moderately intoxicating dose of alcohol (3 g/kg, injected i.p. 3 h earlier) to up-regulate the genetic expression of CRF receptor type 1 (CRF-R1) and 2 (CRF-R2alpha) in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus as well as in the amygdala. The mRNA encoding CRF-R1 was not constitutively expressed in the PVN or the SON but was present in the amygdala. Alcohol selectively up-regulated CRF-R1 transcripts in the PVN. Basal levels of CRF-R2alpha transcripts were present in the limbic system and the ventromedial hypothalamic nucleus but were not altered by alcohol. We then determined whether the up-regulation of hypothalamic CRF-R1 mRNA levels was functionally connected to CRF-dependent pathways. We first showed that the i.c.v. injection of CRF significantly (P < 0.01) increased CRF-R1 but not CRF-R2alpha mRNA levels. We then injected the CRF antagonist, astressin, i.c.v. 30 min prior to alcohol, at a dose previously shown to completely block many CRF-dependent events in the brain, and found that it did not significantly interfere with-alcohol-induced gene expression of PVN CRF-R1. These results indicate that acute alcohol treatment selectively activates CRF-R1 in the endocrine hypothalamus and that this response does not appear to depend on the stimulation of CRF receptors. In contrast, no up-regulation of CRF-R1 or CRF-R2alpha gene expression was observed in extrahypothalamic regions thought to participate in the behavioral influence of alcohol.
Animals↗
An initial, three-day-long treatment with alcohol induces a long-lasting phenomenon of selective tolerance in the activity of the rat hypothalamic-pituitary-adrenal axis.
We determined whether an initial alcohol challenge induced long-lasting changes in the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Adult male rats received intragastric injections of the vehicle or a moderately intoxicating dose of alcohol (3.0 gm/kg) daily for 3 d. When animals were acutely challenged with alcohol 3-12 d later, their ACTH and corticosterone responses were significantly blunted, compared with that of vehicle-pretreated rats. In contrast, exposure to mild electric foot shocks induced a pattern of ACTH secretion that was comparable in animals administered alcohol or the vehicle previously, indicating a lack of cross-tolerance. No significant differences were observed in pituitary responsiveness to corticotropin-releasing factor or vasopressin in rats pretreated with the vehicle or alcohol. The influence of the initial drug treatment was not mimicked by exposure to foot shocks, nor was it prevented by administering a potent corticotropin-releasing factor antagonist to block the elevations in plasma ACTH and corticosterone induced by this initial treatment. Finally, we found that rats injected initially with the vehicle and challenged subsequently with alcohol exhibited the expected increased neuronal activation (measured by the upregulation of steady-state mRNA and protein levels of immediate early genes) in the paraventricular nucleus of their hypothalamus. In contrast, this response was markedly decreased in animals exposed previously to the drug. To our knowledge, this is the first report that exposure to a stress (i.e., alcohol), although not immediately altering the response of the HPA axis to this particular signal, induces a selective tolerance that is both slow to develop and long-lasting. The primary mechanism mediating the ability of an initial drug treatment to decrease subsequent responses of the HPA axis to a second drug challenge seems to be the inability of hypothalamic neurons to respond adequately to this second challenge.
Adrenocorticotropic Hormone↗
Crystals of Rous sarcoma virus capsid protein show a helical arrangement of protein subunits.
Crystals of Rous sarcoma virus (RSV) capsid protein diffract X rays to 3.5 A resolution and belong to the monoclinic space group C2 with unit cell parameters a = 374.4 A, b = 128.1 A, c = 200.2 A, and beta = 121.8 degrees. One asymmetric unit of the crystal may contain between 28 and 35 molecules, based on reasonable crystal density assumptions. A self-rotation function and Patterson synthesis suggest that RSV capsid protein crystallizes as a helical array. The determinants of the viral particle morphology are not encoded in the capsid alone. The assembly of a helical array in the crystal reflects the absence of any conformational switching. However, it is expected that the subunit interactions seen in the crystal will be preferred and will relate to those found in the immature or mature virion.
Amino Acid Sequence↗
Characterization of citrate synthase purified from Drosophila melanogaster.
Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrate was purified from Drosophila melanogaster. Some physicochemical as well as enzymatical properties were investigated. The optimum pH and temperature were pH 8.0-9.0 and 45 degrees C, respectively. The molecular weight of the enzyme was determined as 81,000 Da by gel filtration and the purified active enzyme consisted of two identical subunits which had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purified enzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K(m)) of the enzyme for acetyl-CoA and oxaloacetate were 6.7 microM and 3.1 microM, respectively. Kinetic studies showed that citrate synthase follows the concerted mechanism which forms a ternary complex. Propionyl-CoA, ATP, and intermediates of the TCA cycle, succinyl-CoA and alpha-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparison, we found similarities at the N-terminal region. However, in the Ouchterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.
Animals↗
Nucleotide sequence and genetic complementation analysis of lep from Azotobacter vinelandii.
The lep of Azotobacter vinelandii is an 852-base-pair open reading frame (ORF) which encodes a protein of 284 amino acid residues. The translated protein shares 75% homology with leader peptidase I isolated from Pseudomonas fluorescens and 37% homology with leader peptidase I isolated from Escherichia coli. Five highly conserved regions found in the family of leader peptidase I proteins are conserved in A. vinelandii Lep. The putative membrane topology of the protein seems similar to that of E. coli leader peptidase I based on the hydrophobicity analysis of the predicted amino acid sequence. Southern blotting analysis of the A. vinelandii chromosome by probing with lep specific DNA revealed that lep is present as a single copy per the chromosome. A multicopy plasmid carrying A. vinelandii lep could complement a temperature sensitive lep mutant of E. coli strain IT41, suggesting that we have identified the functional copy of lep present on A. vinelandii genome.
Amino Acid Sequence↗
Implications of a possible clustering of highest-energy cosmic rays.
Recently, a possible clustering of a subset of observed ultra-high energy cosmic rays above approximately 40 EeV (4 x 10(19) eV) in pairs near the supergalactic plane was reported. We show that a confirmation of this effect would provide information on the origin and nature of these events and, in case of charged primaries, imply interesting constraints on the extragalactic magnetic field. Possible implications for the most common models of ultra-high energy cosmic ray production in the literature are discussed.
Journal Article↗
Allelotype analysis in the evolution of chronic myelocytic leukemia.
To elucidate the genetic events that may play important roles in the progression of chronic myelocytic leukemia (CML), we performed allelotype analysis in 30 patients with CML as the disease transformed to accelerated phase or blast crisis (21 myeloid and 9 lymphoid cases). DNAs were extracted from slides of bone marrow smears or from freshly isolated bone marrow mononuclear cells. The DNAs from the same individuals in both chronic phase and either blast crisis or accelerated phase were analyzed at 82 microsatellite markers, which mapped to each of the autosomal arms except the short arms of the acrocentric chromosomes. Loss of heterozygosity (LOH) on at least one locus was observed in 21 of the 30 cases (70%) as the disease progressed. Frequent allelic loss of > or = 20% of the informative cases was observed on chromosome arms 1p (35%), 7p (21%), 19p (20%), and 20q (29%). Allelic losses were also analyzed according to phenotypes. LOH of > or = 20% was detected on 1p (29%), 18p (20%), and 20q (27%) in myeloid blast crisis, and on 1p (50%), 4p (25%), 7p (43%), 9p (29%), 18q (25%), 19p (43%), and 20q (33%) in lymphoid blast crisis. Serial cytogenetic information was available for most of our cases with LOH on these arms, and only one case had loss of both chromosomes 9 and 20. Fractional allelic loss, calculated for each sample as the total number of chromosomal arms lost/total number of arms with information, showed a median value of 0.06 and a mean of 0.098 (range 0 to 0.60). These results suggest that tumor suppressor genes especially on 1p, 7p, 19p, and 20q probably have an important role in the progression to blast crisis of CML.
Adult↗
Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes.
Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.
Amino Acid Sequence↗
Chemopreventive effects of S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine against benzo[a]pyrene.
The putative antimutagenic/anticarcinogenic organosulfur compound, S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC), has been demonstrated to inhibit the metabolic activation and the genotoxicity of N-nitrosodiethylamine. We have investigated the chemopreventive activity of AC-DDTC against benzo[a]pyrene (B[a]P) in the Salmonella typhimurium bacterial mutation assay, in the chromosome aberration assay using Chinese hamster lung fibroblast (CHL), and in the mouse micronucleus assay in bone marrow cells. In the bacterial mutation assay, AC-DDTC produced a concentration dependent decrease in the number of mutant colonies induced by B[a]P. The chromosome damaging responses of B[a]P in CHL cells were abolished by the treatment of AC-DDTC, approximately to the level of the control. In the in vivo mouse bone marrow micronucleus test, pretreatment of AC-DDTC 1 h prior to B[a]P reduced the frequency of micronucleated polychromatic erythrocytes. The inhibitory effects were statistically significant and dose-dependent. Our results demonstrate that AC-DDTC, one of the mixed disulfide model compounds of disulfiram, prevents the mutagenic effects of B[a]P.
Acetylcysteine↗
Substituted N-phenylisothioureas: potent inhibitors of human nitric oxide synthase with neuronal isoform selectivity.
S-Ethyl N-phenylisothiourea (4) has been found to be a potent inhibitor of both the human constitutive and inducible isoforms of nitric oxide synthase. A series of substituted N-phenylisothiourea analogues was synthesized to investigate the structure-activity relationship of this class of inhibitor. Each analogue was evaluated for human isoform selectivity. One analogue, S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (39), exhibited 115-fold and 29-fold selectivity for the neuronal isoform versus the inducible and endothelial derived constitutive isoforms, respectively. Studies have shown the substituted N-phenylisothiourea 39 binds competitively with L-arginine.
Animals↗
Simultaneous determination of imipramine, desipramine and their 2- and 10-hydroxylated metabolites in human plasma and urine by high-performance liquid chromatography.
A simultaneous assay for imipramine, desipramine and their 2- and 10-hydroxy-metabolites using high-performance liquid chromatography (HPLC) is described. The drugs and internal standard, pericyazine, were extracted from plasma or urine at pH 9.6 with diethyl ether and back-extracted into 0.1 M orthophosphoric acid. The recovery of the compounds ranged from 78.6% for imipramine to 94.3% for 2-hydroxydesipramine. The extracts were analysed by reversed-phase HPLC with electrochemical detection using a mobile phase of 30% acetonitrile in 0.1 M K2HPO4 at pH 6.0 delivered at 2 ml/min. All compounds were resolved in a run time of 15 min with lower limits of quantification of 1.5 ng/ml for hydroxy-metabolites and 3 ng/ml for imipramine and desipramine. The intra- and inter-day coefficients of variation at 50 ng/ml were 5.2% and 6.8%, respectively (n=8).
Adult↗
Inhibition of HIV infection of H9 cells by chlorpromazine derivatives.
The binding between the HIV surface protein, gp120, and the CD4 coreceptor is known to be initiated by electrostatic interactions. Because of the ability of chlorpromazine to interact with proteins by charge transfer, we tested several derivatives for their ability to block binding of HIV to CD4+ cells. We have shown that 7,8-dioxo-chlorpromazine blocks binding of fluorescein isothiocyanate-labeled anti-Leu3a and rgp120 to peripheral human blood T4 cells and blocks syncytia formation between gp120- and CD4-expressing cells. We also found that 7,8-dioxo-chlorpromazine blocks HIV infectivity of H9 cells and acts synergistically with zidovudine.
Anti-HIV Agents↗
Inhibition of tumor necrosis factor-alpha action within the CNS markedly reduces the plasma adrenocorticotropin response to peripheral local inflammation in rats.
The present study tested the hypothesis that the cytokine tumor necrosis factor-alpha (TNF-alpha) is an important CNS mediator of the hypothalamo-pituitary-adrenal (HPA) axis response to local inflammation in the rat. Recombinant murine TNF-alpha administered directly into the cerebroventricles of normal rats produced a dose-dependent increase in plasma adrenocorticotropin (ACTH) concentration. Local inflammation induced by the intramuscular injection of turpentine (50 microl/100 gm body weight) also produced an increase in plasma ACTH, peaking at 160-200 pg/ml at 7.5 hr after injection (compared with 10-30 pg/ml in controls). Intracerebroventricular pretreatment with either 5 microl of anti-TNF-alpha antiserum or 1-50 microg of soluble TNF receptor construct (rhTNFR:Fc) reduced the peak of the ACTH response to local inflammation by 62-72%. In contrast, intravenous treatment with the same doses of anti-TNF-alpha or rhTNFR:Fc had no significant effect on the ACTH response to local inflammation. Although these data indicated an action of TNF-alpha specifically within the brain, no increase in brain TNF-alpha protein (measured by bioassay) or mRNA (assessed using either in situ hybridization histochemical or semi-quantitative RT-PCR procedures) was demonstrable during the onset or peak of HPA activation produced by local inflammation. Furthermore, increased passage of TNF-alpha from blood to brain seems unlikely, because inflammation did not affect plasma TNF-alpha biological activity. Collectively these data demonstrate that TNF-alpha action within the brain is critical to the elaboration of the HPA axis response to local inflammation in the rat, but they indicate that increases in cerebral TNF-alpha synthesis are not a necessary accompaniment.
Adrenocorticotropic Hormone↗
Glycinergic inhibition contributes to the generation of rostral scratch motor patterns in the turtle spinal cord.
Cutaneous stimulation within the rostral scratch receptive field in a low spinal-immobilized turtle elicits a fictive rostral scratch reflex characterized by robust rhythmic motor output from ipsilateral hindlimb muscle nerves and weaker, alternating motor discharge in contralateral nerves. Simultaneous bilateral stimulation elicits bilateral rostral scratch motor patterns in which activity on the right and left sides alternates. We investigated the role of glycinergic inhibition in the generation and coordination of fictive rostral scratch motor patterns. Glycine (2 or 5 mM) and strychnine (5-50 microM), a glycine antagonist, were superfused over the anterior spinal hindlimb enlargement while fictive rostral scratch motor output was recorded bilaterally from hindlimb muscle nerves in the form of electroneurograms (ENGs). Although glycine reduced rostral scratch burst frequencies, strychnine tended to increase burst frequency. Strychnine also changed the shape of hip flexor ENG bursts, resulting in more abrupt burst onsets, indicating an earlier recruitment of motor neurons with large ENG spikes. During bilateral stimulation, strychnine increased the variability of interlimb phase values (left vs right hip flexor bursts) but did not abolish right-left alternation. These results indicate that glycinergic neurons in or near the anterior hindlimb enlargement contribute to the overall timing of the rostral scratch rhythm and to the recruitment timing of individual hip flexor motor neurons within each scratch burst. Our data also indicate that glycinergic mechanisms contribute to, but are not critically important for, maintaining an alternating interlimb coordination during bilateral scratch motor patterns.
Animals↗