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Biomedical subjects

R C Newton

Publications and source records attributed to R C Newton.

At least 19 recordsLinked to original sources
PubMed1995

Modification of tumor necrosis factor-alpha (TNF-alpha) production by the Na(+)-dependent HCO3- cotransport in lipopolysaccharide-activated human monocytes.

Tumor necrosis factor-alpha (TNF-alpha) is produced and secreted from monocytes in response to activation with lipopolysaccharide (LPS). The role of Na+ and HCO3- in the production of TNF-alpha by monocytes was investigated; it was observed that replacement of Na+ in the culture medium with sucrose or choline chloride inhibited TNF-alpha production completely. The addition of Na+ to Na(+)-free culture medium restored TNF-alpha production with an EC50 value of 35 mmol/l. The amiloride analog 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ antiporter, inhibited TNF-alpha production with an EC50 of 3.3 microM. Without HCO3- in the culture medium TNF-alpha production was inhibited by 92%. Total protein synthesis was inhibited by 85% in the absence of Na+ but did not change in the absence of bicarbonate in the culture medium. Intracellular pH (pHi) which increased from 6.90 in control monocyte to 7.40 in response to activation with LPS was abrogated to pHi of 6.95 in the absence of Na+ but did not change in the absence of HCO3- in the culture medium. In the presence of 100 microM phloretin or DIDS the pHi of activated monocyte was reduced to control value, TNF-alpha production was inhibited completely and total protein synthesis was inhibited by 61%. These data suggest that (1) TNF-alpha production, as other proteins, is dependent on the pHi of monocytes,and (2) TNF-alpha production, in contrast to total protein, is modulated by Na(+)-dependent HCO3-.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Regulation of monocyte integrin expression by beta-family chemokines.

In the present study we investigated the ability of three monocyte chemokines (MCP-1, MIP-1 alpha, and RANTES) to modulate monocyte adhesion molecules in an attempt to evaluate their potential to induce tissue infiltration of macrophages in vivo. All three chemokines tested induced increased expression of the alpha-chains of two members of beta 2 family of integrins, CD11b and CD11c, and their common beta-chain (CD18). They had no effect on CD11a expression. Enhancement of CD11b and CD11c was dose dependent and followed a distinct time course with peak levels at 4 h. Levels declined to reach basal levels by 24 h. In contrast, IL-1 induced enhancement remained high after 24 h of stimulation. However, the increases caused by chemokines were not mediated by IL-1 as indicated by lack of inhibition by the IL-1R antagonist. Studies on the mechanism of integrin up-regulation showed that mobilization of cytosolic free calcium is an important signaling event in this response and that up-regulation is associated with mobilization from intracellular pools mediated by microtubules. Enhanced CD11b and CD11c expression by chemokines was also found to result in enhancement of monocyte binding to endothelial cells. Further studies indicated that monocyte binding to endothelial cells follows similar dose-response kinetics as the up-regulation of integrins and can be partially blocked by Abs to CD11b and CD11c. These results suggest that modulation of the integrin expression by chemokines may facilitate the tissue trafficking of monocytes during inflammation.

CD18 Antigens↗
PubMed1994

Comparison of biological responses of human monocytes and THP-1 cells to chemokines of the intercrine-beta family.

The biological responses of human monocytes and cells of the monomyelocytic THP-1 cell line to stimulation with members of the beta chemokine family are described in this report. All three chemokines tested, MCP-1, MIP-1 alpha, and RANTES, elicited mobilization of intracellular free calcium in monocytes and THP-1 cells. The magnitude of response was highest with MCP-1 stimulation. MCP-1 desensitized monocyte responses to MIP-1 alpha and RANTES, but no such desensitization was observed in THP-1 cells. MIP-1 alpha or RANTES did not desensitize either monocytes or THP-1 cells to MCP-1 stimulation. All three chemokines elicited a potent chemotactic response in monocytes that was comparable in magnitude to that of f-Met-Leu-Phe. MIP-1 alpha and RANTES required a fivefold higher dose than MCP-1 to elicit a peak response. On the contrary, THP-1 cells showed no significant chemotactic response. Studies of the desensitization of the monocyte chemotactic response indicated that all three chemokines are capable of causing complete homologous desensitization. Heterologous desensitization was observed only when monocytes were treated with MCP-1 followed by MIP-1 alpha or RANTES. Studies of actin polymerization and cell polarization responses of monocytes indicated that these two responses attained peak magnitude after 10 min of stimulation with any of the chemokines. Dose-response kinetics were similar to those of the chemotactic response. THP-1 cells again failed to show either of these two responses. Finally, the activation potential of the chemokines was measured by their ability to induce respiratory burst. A tenfold higher concentration than that causing peak chemotactic response was required to elicit respiratory burst and no heterologous desensitization was noticed. Respiratory burst could be induced in THP-1 cells with a direct protein kinase C activator but not with any of the chemokines. These results indicate that, of the three examples tested, MCP-1 is the most potent member of the beta chemokine family in the biological responses examined. Although a calcium response was elicited in THP-1 cells with chemokines, a lack of subsequent responses indicates some missing links in the downstream signal transduction pathways.

Actins↗
PubMed1994

Selective non-peptide ligands for an accommodating peptide receptor. Imidazobenzodiazepines as potent cholecystokinin type B receptor antagonists.

A series of imidazobenzodiazepines, non-peptide antagonists of the peptide hormone cholecystokinin (CCK), are described. Derived by chemical modification of the benzodiazepine ring system embedded within the CCK-B antagonist L-365,260, these compounds display CCK-B/CCK-A selectivity and some analogs have receptor binding affinities in the subnanomolar range. This group of novel imidazobenzodiazepines, among which N-[(2S,4R)-methyl-6-phenyl-2,4-dihydro-1H-imidazo[1,2- alpha][1,4]benzodiazepin-4-yl]-N'-[3-methylphenyl]-urea (12) is the principal compound, expands the structural diversity of the collection of non-peptide CCK-B antagonists and will be useful in further delineating the function of CCK in the central nervous system.

Animals↗
PubMed1993

Species differences in [125I]interleukin-1 binding in brain, endocrine and immune tissues.

There were dramatic species differences in the level of [125I]recombinant human interleukin-1 alpha ([125I]hIL-1 alpha) binding with high levels of binding present in mouse and rabbit tissues, while no specific binding was present in rat and guinea pig tissues. Utilizing [125I]hIL-1 alpha, moderate to high levels of specific binding were observed in EL-4 6.1 cells (representative of Type I IL-1 receptors) and in mouse hippocampus, spleen and testis; however, no specific [125I]hIL-1 alpha binding was present in Raji cells (representative of Type II IL-1 receptors) and in rat tissues. On the other hand, utilizing [125I]hIL-1 beta, high specific IL-1 binding was present in EL-4 6.1 and Raji cells and moderate binding was evident in mouse tissues, whereas specific [125I]hIL-1 beta binding to rat tissues was not detectable. Moreover, no IL-1 binding in rat tissues was observed using [125I]hIL-1 receptor antagonist, [125I]mouse IL-1 beta or the homologous radioligands [125I]rat IL-1 beta or [125I]rat IL-1 receptor antagonist. These data demonstrate that under optimal conditions for labeling Type I or Type II IL-1 receptors, no specific binding is observed in rat tissues suggesting the presence of novel IL-1 receptor(s) in rat tissues.

Animals↗
PubMed1993

Role of glucose in interleukin-1 beta production by lipopolysaccharide-activated human monocytes.

When monocytes are activated with endotoxin (lipopolysaccharide [LPS]), they make and release several mediators, including interleukin-1 beta (IL-1 beta). This study was undertaken to investigate the role of glucose in IL-1 beta production by these cells. IL-1 beta was produced in a dose-dependent manner to glucose concentration in the culture medium. The uptake of (3H)2-deoxyglucose in monocytes was stimulated by LPS 1,554% after 10 minutes, 6,095% after 2 hours, then gradually declined after 4 hours of incubation. The inhibition of the uptake of (3H)2-deoxyglucose by either 10 microM cytochalasin B or phloretin, added at the time of monocyte activation, was accompanied by significant reduction in ATP/ADP ratio and the inhibition of the production of IL-1 beta by activated monocytes. The synthesis of total protein did not change in monocytes activated in the absence of glucose in the culture medium, nor in the presence of either 10 microM cytochalasin B or phloretin. The export of IL-1 beta from LPS-activated monocytes was not inhibited by either 10 microM cytochalasin B or phloretin, nor in the absence of glucose in the culture medium. These data suggest that 1) glucose is required for LPS-induced IL-1 beta production by monocytes; 2) glucose is the major source of ATP for IL-1 beta production; 3) glucose transporter (GLUT 1) does not control the export of IL-1 beta.

Biological Transport↗
PubMed1993

Escherichia coli: derived murine interleukin-1 beta with N-terminus partially N alpha-acetylated.

Two forms of murine recombinant interleukin-1 beta (mrIL-1 beta) from Escherichia coli were purified by ion exchange column chromatography; each exhibited equivalent biological activity in the murine thymocyte proliferation assay. It was determined by mass spectrometry of tryptic peptides that both retained the initiating methionine but one form was N alpha-acetylated at the N-terminus.

Acetylation↗
PubMed1992

Activation and distribution of inflammatory cells in the mouse uterus during the preimplantation period.

Northern blot analysis of mouse uterine RNA showed that IL-1 (alpha and beta), and TNF-alpha mRNA were abundant on day (D) 1 of pregnancy, reduced on D2, and remained basal throughout the remainder of the preimplantation period (D3 and D4). Elevated IL-1 beta and TNF-alpha mRNA levels on D1 were accompanied by increased levels of immunoreactive protein in uterine cytosol preparations as determined by ELISA. In situ hybridization detected IL-1 beta mRNA in cells located in the endometrial stroma and concentrated in subepithelial regions on D1. Immunocytochemical localization of IL-1 beta and TNF-alpha identified cells scattered throughout the endometrial stroma, but more concentrated in the subepithelial region on D1. On D3 and D4, cytokine-immunopositive cells decreased in number and became located predominantly at the endometrial-myometrial junction. Histochemical localization of peroxidase as a marker predominantly for eosinophils showed an abundance of these cells in the D1 uterus. The distribution of peroxidase-positive cells in the uterus followed the same temporal and spatial changes as cytokine-immunopositive cells during the preimplantation period. These data document the occurrence of an inflammatory response in the uterus on D1 of pregnancy, and demonstrate that as the preimplantation period progresses the distribution of inflammatory cells changes from the subepithelial region of the endometrial stroma to the periphery of the uterus at the endometrial-myometrial junction. Mechanisms regulating the uterine inflammatory response on D1 were investigated. Cytokine mRNA levels were not significantly elevated during the estrous cycle or after treatment of adult ovariectomized mice with estradiol-17 beta. In contrast, mating with vasectomized males resulted in an inflammatory response on D1 of pseudopregnancy similar to that on D1 of normal pregnancy, whereas mechanical stimulation of the uterine cervix failed to elicit such a response. These results strongly suggest a role for some factor(s) in the ejaculate, other than spermatozoa, in the initiation of a uterine inflammatory response after mating, but an effect of the act of mating cannot be excluded.

Animals↗
PubMed1992

Strongyloides stercoralis hyperinfection in a carrier of HTLV-I virus with evidence of selective immunosuppression.

A patient with near fatal Strongyloides hyperinfection syndrome is briefly described. Investigation for possible risk factors for this parasitic infection disclosed that he was a carrier of human T-cell leukemia virus type I (HTLV-I), but without evidence of disease due to this retrovirus. Over the next few years, the patient's serum antibody levels of IgG to S. stercoralis larvae declined and became undetectable despite continued infection with the parasite. Repeated courses of appropriate treatment cleared the parasitic infection only temporarily. The patient was also found to have undetectable total serum IgE and a negative immediate hypersensitivity skin test to S. stercoralis antigens. Five of six other patients with HTLV-I-associated disease and with or without strongyloidiasis were also found to have very low total serum IgE levels. It is postulated that HTLV-I infection in certain individuals may selectively impair immune responses that are critical in controlling strongyloidiasis.

Antibodies, Helminth↗
PubMed1992

Type I interleukin-1 receptors in the mouse brain-endocrine-immune axis labelled with [125I]recombinant human interleukin-1 receptor antagonist.

Iodine-125-labelled recombinant human interleukin-1 (IL-1) receptor antagonist ([125I]IL-1ra) was utilized to further determine the characteristics of IL-1 receptors in the brain-endocrine-immune axis. The binding of [125I]IL-1ra in homogenates of mouse hippocampus, spleen and testis was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity (KD, 20-30 pM). In competition studies, IL-1ra, recombinant human IL-1 alpha, IL-1 beta and a weak IL-1 beta analog inhibited [125I]IL-1ra binding to mouse tissues in parallel with their biological activities. In autoradiographic studies, [125I]IL-1ra and [125I]IL-1 alpha binding showed comparable distribution patterns with highest densities of binding sites present in the dentate gyrus of the hippocampus, choroid plexus, anterior pituitary, marginal zones and red pulp regions of the spleen, epididymis and interstitial area of the testis. The binding characteristics and distribution of [125I]IL-1ra are comparable to those of previously characterized Type I IL-1 receptors. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immune responses to physiological and pharmacological stimuli.

Animals↗
PubMed1992

Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini.

A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.

Amino Acid Sequence↗
PubMed1992

Effects of intracellular ions on interleukin-1 beta production by lipopolysaccharide-activated human monocytes.

Following the observation that interleukin 1 beta (IL-1 beta) production in lipopolysaccharide (LPS)activated monocytes increases in concert with a rise in intracellular pH (pHi), the role of ion transport in IL-1 beta production was investigated. The amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)-H+ antiporter, inhibited extracellular IL-1 beta. The replacement of Na+ in the culture medium with sucrose or choline chloride also prevented monocyte activation. The sodium ionophore monensin, in doses from 100 pM to 1 microM, potentiated LPS-stimulated extracellular IL-1 beta when compared with LPS alone. In the absence of LPS activation, monensin by itself at 10 nM stimulated IL-1 beta production to 63%. EIPA at 10 microM inhibited the Na+ influx, the rise in pHi, and intra- and extracellular IL-1 beta production in activated monocytes; this inhibition was reversed by 10 nM monensin. In the absence of bicarbonate, or in the presence of 10 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, the pHi of activated monocytes and the total protein synthesis did not change, but the production of IL-1 beta was inhibited. The data suggest that the stimulated influx of Na+ via the Na(+)-H+ antiporter regulates both pHi and IL-1 beta production in LPS-activated monocytes. The requirement for bicarbonate indicates an additional mechanism(s), separate from the modulation of pHi and intracellular Na+.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Modulation of synovial fibroblast plasminogen activator and plasminogen activator inhibitor production by protein kinase C.

Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Internalization and nuclear localization of interleukin 1 are not sufficient for function.

The human fibroblast interleukin 1 (IL-1) receptor is a glycosylated transmembrane protein with a cytoplasmic domain of 213 amino acids. We have constructed a series of deletion mutants of the cytoplasmic region of the IL 1 receptor and have used these mutants to examine its role in ligand binding, internalization, signal transduction, and nuclear localization of IL-1. Mutant receptors lacking most of the cytoplasmic domain are expressed at the cell surface and can bind, internalize, and localize IL-1 at the nucleus, but they do not allow IL-1-mediated induction of interleukin 2 and SV40 promoters. We have localized a critical region for signal transduction to a 50-amino acid segment of the cytoplasmic domain of the receptor. These studies demonstrate that IL-1 internalization and nuclear localization are not sufficient to trigger IL-1 activation of gene expression in T-cells.

Autoradiography↗
PubMed1990

Identification of soluble interleukin-1 binding protein in cell-free supernatants. Evidence for soluble interleukin-1 receptor.

This study describes the identification and characterization of a soluble interleukin-1 (IL-1) binding protein in the conditioned media from Raji human B-lymphoma cells. The soluble IL-1 binding material was isolated by IL-1 affinity chromatography, and treatment with trypsin decreased its ability to bind to IL-1 demonstrating its protein nature. The soluble IL-1 binding protein was specific for IL-1 and was able to discriminate between Il-1 alpha and IL-1 beta in a manner analogous to the membrane-bound Raji IL-1 receptor. The specificity of the IL-1 binding protein was further established in two ways. 1) Cell-free supernatants from Raji "receptor-negative" cells did not contain any IL-1 binding protein, thus ruling out nonspecific interactions between IL-1 and a serum or other protein present in the conditioned medium; and 2) the soluble binding protein inhibited IL-1 binding to Raji cells in a dose-dependent manner. Scatchard analysis of IL-1 beta binding showed the dissociation constant (KD) to be 5.1 nM for the soluble IL-1 binding protein compared with 0.8 nM for the membrane-bound IL-1 receptor. Gel chromatography of the soluble binding protein yielded a major peak of IL-1 binding activity with a molecular mass of 35-45 kDa. The characteristics of the soluble IL-1 binding protein described above are consistent with those of the extracellular binding domain of the membrane-bound Raji IL-1 receptor.

Cell Line↗
PubMed1990

A clinical trial of topical terbinafine (a new allylamine antifungal) in the treatment of tinea pedis.

Twenty-three patients were enrolled in a randomized, double-blind trial of terbinafine 1% cream compared with placebo vehicle in the treatment of tinea pedis. Of the 20 patients who were evaluated for efficacy, 10 received terbinafine and 10 received placebo. Except for the terbinafine-treated patients being an average of 11 years older than the patients receiving placebo and the median duration of disease being 6 weeks longer in the placebo group, the two groups were demographically and clinically similar. Results of mycologic tests and clinical findings showed terbinafine to be significantly more effective than placebo in the treatment of tinea pedis. Significantly more terbinafine-treated patients than placebo-treated patients showed conversion to negative culture and microscopy at end of therapy and a significant reduction in scored signs and symptoms. Overall efficacy at follow-up (combined mycologic and clinical findings) was also significantly greater in the terbinafine group (78%) than in the placebo group (zero) (p less than 0.001). Unexplained elevation of liver function test results was noted in three placebo-treated patients and in one terbinafine-treated patient, but these changes were not considered clinically relevant or drug related.

Administration, Cutaneous↗
PubMed1990

Human N-terminal analogs of interleukin 1 beta demonstrate altered binding and function in hematopoiesis.

This study describes the structure-function relationship of interleukin 1 beta (IL-1 beta) using two amino-terminal muteins of human IL-1 beta. One mutein, clone 18, which substitutes a threonine and methionine for the alanine and proline at positions 1 and 2 of the N-terminus of fully processed and active IL-1 beta, demonstrated similar activity to that of native IL-1 beta in inducing granulocyte-macrophage colony-stimulating activity (GM-CSA) from cultured fibroblasts. Clone 18 also demonstrated similar binding to IL-1 beta receptors on fibroblasts when using a competitive binding assay. The second mutein was GLU-4, which in addition to substituting alanine and proline by threonine and methionine also substituted glutamine for arginine at position 4 of the processed IL-1 beta molecule. GLU-4 required a 3-log increase in concentration to obtain the same GM-CSA release from fibroblasts and to produce the same amount of competitive binding inhibition as clone 18 and native IL-1 beta. In addition, preincubation of bone marrow cells with clone 18 and native IL-1 beta demonstrated a greater ability to protect early hematopoietic progenitors from the lethal effects of 4-hydroperoxycyclophosphamide when compared to similar concentrations of GLU-4. A greater number of large granulocyte-macrophage, erythroid, and mixed colonies as well as blast cell colonies were observed when bone marrow cells were preincubated for 20 h with clone 18 or native IL-1 beta as compared to preincubation with GLU-4 or medium alone. Therefore, arginine at position 4 of the processed IL-1 beta molecule was shown to be a key residue in the function of IL-1 beta as a hematopoietic regulator. These results also suggest that minor changes in the N-terminal sequence of IL-1 beta result in decreased interaction with its receptor and a subsequent reduction in biological activity.

Binding, Competitive↗