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Biomedical subjects

A Richmond

Publications and source records attributed to A Richmond.

At least 55 records · Page 3Linked to original sources
PubMed1995

Activation of protein kinase C enhances the phosphorylation of the type B interleukin-8 receptor and stimulates its degradation in non-hematopoietic cells.

We have previously characterized the stably transfected, clonally selected human placental cell line, 3ASubE P-3, which overexpresses the type B interleukin-8 receptor (IL-8RB) and responds to the chemokine melanoma growth stimulatory activity (MGSA) with enhanced phosphorylation of this receptor. In work described here, we demonstrate that the MGSA-enhanced phosphorylation of this receptor is mediated via a process involving pertussis toxin-sensitive G proteins. Furthermore, treatment of the 3ASubE P-3 cells with either 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (diC8), two different activators of protein kinase C (PKC), results in a concentration-dependent increase in the phosphorylation of the IL-8RB. Inhibition of PKC, by treatment with staurosporin (50 nM for 2 h), or down-regulation of PKC, by prolonged treatment with TPA (400 nM for 40 h) suppresses the TPA-enhanced receptor phosphorylation, but has no effect on the MGSA-enhanced receptor phosphorylation. These data suggest that the isoforms of PKC that are sensitive to these manipulations may not play a role in mediating the MGSA-enhanced phosphorylation of the IL-8RB. TPA treatment also results in a time-dependent decrease in 125I-MGSA binding to the 3ASubE P-3 cells. A 30-min treatment with 400 nM TPA results in approximately a 50% decrease in binding, whereas a 2-h treatment essentially eliminates specific binding of 125I-MGSA to these cells. The TPA-induced decrease in 125I-MGSA binding is accompanied by enhanced degradation of the IL-8RB, as indicated by Western blot analysis and pulse-chase experiments, suggesting a potential role for PKC as a negative regulator of the IL-8RB. MGSA treatment (50 nM for 2 h) also stimulates receptor degradation in the 3ASubE P-3 cells, indicating that this receptor is down-regulated in response to prolonged exposure to its ligand. In similar studies conducted on the promonocytic cell line, U937, MGSA treatment of the U937 cells resulted in receptor phosphorylation, whereas PKC activation failed to significantly modulate the phosphorylation state of the IL-8RB. Treatment of the U937 cells with MGSA, TPA, or diC8 resulted in a loss of receptor protein present in these cell types. These data imply that MGSA signaling through the IL-8RB is similar in both the non-hematopoietic and hematopoietic cell types, whereas activation of PKC by TPA or diC8 elicits different responses in these two distinct cell types.

Alkaloids↗
PubMed1995

Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.

The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.

Animals↗
PubMed1994

Melanoma growth stimulatory activity enhances the phosphorylation of the class II interleukin-8 receptor in non-hematopoietic cells.

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.

Base Sequence↗
PubMed1994

MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells.

We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift analyses indicate that IL-1 and TNF alpha induce NF-kappa B complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappa B complexes may result in the inability of the cytokines IL-1 and TNF alpha to activate transcription of the MGSA/GRO genes. IL-1 and TNF alpha posttranscriptionally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells, IL-1 increases the half-life of MGSA/GRO alpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNF alpha transcriptionally and posttranscriptionally regulate MGSA/GRO alpha, -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, the major effect of IL-1 and TNF alpha is on mRNA stability.

Base Sequence↗
PubMed1993

Localization of MGSA/GRO protein in cutaneous lesions.

Melanoma growth stimulatory activity (MGSA/GRO), a cytokine originally characterized as an autocrine growth factor for melanoma cells, is highly chemotactic for neutrophils and releases neutrophil elastase as well as other matrix-degrading enzymes. Previous work has demonstrated the presence of MGSA/GRO in melanocytic lesions and in the epidermal keratinocytes of non-lesional skin and psoriatic scale. Herein, MGSA/GRO localization was examined in a variety of human skin lesions exhibiting proliferative and/or differentiative disorders using immunohistochemical methods. Most lesions showed greater MGSA/GRO immunoreactivity in the more differentiated suprabasal keratinocytes of the stratum spinosum and stratum granulosum than in the stratum basalis, where the dividing basal cells are found. Hair follicles, sebaceous glands, and sweat glands were also frequently positive for MGSA/GRO. The highest level of immunoreactive MGSA/GRO in diseased epidermis was detected in verruca vulgaris, followed by psoriasis, keratoacanthoma, and squamous cell carcinoma. Detection of MGSA/GRO in basal cell carcinoma was variable, being present in the sclerosing variant and absent in the more common nodular variant. Melanocytic lesions stained less intensely for MGSA/GRO than keratinocytic lesions, where the levels of MGSA/GRO expression correlated with the inflammatory response and degree of keratinocyte differentiation.

Biopsy↗
PubMed1993

Expression of three forms of melanoma growth stimulating activity (MGSA)/gro in human retinal pigment epithelial cells.

PURPOSE: To characterize mRNA expression and protein production of the cytokine MGSA/gro in human retinal pigment epithelial (RPE) cells and to determine whether expression of MGSA/gro is modulated by serum and the cytokines interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), or transforming growth factor beta (TGF beta) mediators implicated in proliferative vitreoretinopathy (PVR). METHODS: Reverse-transcription polymerase chain reaction was used to determine the steady-state mRNA expression of three forms of MGSA/gro, alpha, beta, and gamma, by cultured human RPE cells in the presence or absence of recombinant IL-1 beta, TNF alpha, or TGF beta, or when serum-starved cells were re-fed with medium containing serum. Immunocytochemistry was used to characterize RPE cell-associated MGSA/gro protein, and immunoprecipitation of MGSA/gro from cell-conditioned medium was used to demonstrate MGSA/gro secretion. RESULTS: MGSA/gro mRNA was expressed minimally under basal conditions. Expression for all three forms of MGSA/gro mRNA was induced in a dose- and time-dependent manner after exposure to IL-1 beta, to a lesser extent after exposure to TNF alpha, but not after exposure to TGF beta. Serum induced MGSA/gro alpha and gamma transcripts, but not beta transcripts. Cell-associated MGSA/gro was identified on RPE cells grown in the absence of cytokines, but MGSA/gro was not secreted under these conditions. Exposure to IL-1 beta did not consistently cause increased cell-associated MGSA/gro; however, IL-1 beta induced secretion of MGSA/gro in a time-dependent manner. CONCLUSION: MGSA/gro is produced by human RPE in response to mediators implicated in PVR. Because MGSA/gro is a pleiotropic modulator of cell proliferation and inflammation, it may contribute to the intraocular wound healing response that characterizes PVR.

Blood Proteins↗
PubMed1992

The melanoma growth stimulatory activity receptor consists of two proteins. Ligand binding results in enhanced tyrosine phosphorylation.

The human melanoma growth-stimulatory activities (MGSA alpha, beta, gamma/GRO) are products of immediate early genes coding for cytokines that exhibit sequence similarity to platelet factor-4 and beta-thromboglobulin. MGSA/GRO alpha has been demonstrated to partially complete for binding to the approximately 58-kDa neutrophil receptor for another beta-thromboglobulin-related chemotactic protein, IL-8. We demonstrate that when [125I]MGSA/GRO alpha was cross-linked to receptors/binding proteins from human placenta, there were two major [125I]MGSA cross-linked bands of approximately 64,000 and approximately 84,000 Mr. Because [125I]MGSA exists primarily in monomer and dimer forms at the concentrations used here, it is not clear whether the receptor/binding proteins represented by the cross-linked bands are approximately 50,000 and approximately 70,000 or approximately 58,000 and approximately 78,000 Mr. Ligand binding to the receptor proteins is associated with enhanced tyrosine phosphorylation of a number of substrates, including proteins in the same Mr range as the MGSA/GRO receptor/binding proteins.

Binding, Competitive↗
PubMed1992

Modulation of inotropic therapy by venodilation in acute heart failure: a randomised comparison of four inotropic agents, alone and combined with isosorbide dinitrate.

The effects of four inotropic agents with differing ancillary properties [a cardiac glycoside (digoxin), a combined alpha- and beta-adrenergic agonist (dobutamine), a beta-adrenergic agonist (prenalterol), and a phosphodiesterase inhibitor (amrinone)] alone and with subsequent addition of isosorbide dinitrate were compared in 48 consecutive acute myocardial infarction patients with radiographic and haemodynamic (pulmonary artery occluded pressure greater than 18 mm Hg) left ventricular failure. All agents with the exception of dobutamine reduced the elevated left heart filling pressure; only digoxin and dobutamine augmented the cardiac stroke volume index. All drugs except digoxin reduced the SVRI; an arteriolar constrictor response was evident 60 min after digoxin and a tachycardia resulted after combined alpha- and beta- and beta-adrenergic stimulations (dobutamine and prenalterol, respectively). The addition of isosorbide dinitrate reversed the inotrope-induced elevations of systemic arterial pressure and resulted in additional reductions in left heart filling pressure. These data suggest that, in the absence of substantial venodilator properties in an inotropic compound, reduction in elevated left heart filling pressure is not achieved with inotropic therapy alone in acute left ventricular failure and combining a venodilator may be haemodynamically advantageous.

Adult↗
PubMed1992

Vasodilator therapy for acute heart failure: haemodynamic comparison of hydralazine/isosorbide, alpha-adrenoceptor blockade, and angiotensin-converting enzyme inhibition.

Haemodynamic comparison of three vasodilation regimens [intravenous (i.v.) hydralazine and isosorbide dinitrate (ISDN) combined, i.v. doxazosin, and i.v. enalaprilat] was undertaken in 36 patients with acute left ventricular (LV) failure due to recent myocardial infarction. Each regimen achieved similar reductions in pulmonary artery occluded pressure (PAOP, preload) and systemic arterial pressures (afterload), with increased cardiac and stroke volume (SV) indexes (p less than 0.01). Only the hydralazine and isosorbide combination induced resting tachycardia. Balanced vasodilatation after selective alpha-adrenoceptor blockade (doxazosin) and angiotensin-converting enzyme (ACE) inhibition (enalaprilat) without increase in heart rate (HR) suggests that these therapies may have definite haemodynamic advantages over the hydralazine/ISDN combination.

Adrenergic alpha-Antagonists↗
PubMed1991

Effects of MGSA/GRO alpha on melanocyte transformation.

In the work described here we demonstrate that the clonal cell line Mel-a-6, produced by transfection of mouse Melan-a cells with human MGSA, had an increased ability to form large colonies in soft agar and increased ability to form tumors when injected into nude mice as compared to cells transfected with the neomycin resistance gene alone. This effect appeared to be dependent on the levels of MGSA produced since another transfected clone, Mel-a-l, produced only a low level of MGSA transgene mRNA, formed only minimal large colonies in soft agar and had a tumorigenic rate equal to that of neomycin resistant controls. The histology of the Mel-a-6 tumors is compatible with features normally exhibited by melanoma tumors. The cells do not stain for melanin, and they are positive for the neural crest marker protein S-100 as well as the HMB 45 melanoma specific antigen. Immunohistochemical studies revealed expression of the human MGSA in tumor cells from tissues, excised from animals injected with cells from clone Mel-a-6. Whereas DNA ploidy analysis suggests that in vitro the Mel-a parent cell line, control Mel-a-neo, Mel-a-1 and Mel-a-6 cells show no evidence of aneuploidy, the nuclei isolated from the tumors from animals injected with Mel-a-6 do exhibit aneuploidy. These data suggest that over-expression of MGSA in immortalized melanocytes contributes to transformation.

Aneuploidy↗
PubMed1991

The pathogenic role of growth factors in melanoma.

Some early changes associated with atypical nevi, presumed to be progressing toward malignancy, include chromosomal abnormalities and altered production of growth factors, and/or growth factor receptors. Though normal epidermal melanocytes require a number of exogenous growth factors, nevi require fewer growth factors, and most metastatic melanomas are frequently capable of growing without an exogenous supply of growth factors. This is apparently caused by endogenous production of essential growth factors. Our laboratory focuses on melanoma growth stimulatory activity (MGSA), one of the endogenously produced growth factors, and the role it plays in tumor progression. MGSA is a member of the beta-thromboglobulin super family. These genes code for cytokines, which modulate the inflammatory response. The MGSA protein is highly chemotactic for neutrophils and competes with 125I-interleukin-8 for binding sites on neutrophil receptors. When normal immortalized mouse melanocytes are manipulated so that they overexpress the MGSA gene, the melanocytes form large colonies in soft agar and melanoma tumors in nude mice. These data suggest that the MGSA protein can potentially play a role in melanoma tumor progression.

Base Sequence↗
PubMed1990

Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes.

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.

Blotting, Northern↗
PubMed1989

Nitrate therapy for left ventricular failure complicating acute myocardial infarction: a haemodynamic comparison of intravenous, buccal, and transdermal delivery systems.

The haemodynamic effects of three different methods of nitrate administration [intravenous (i.v.) isosorbide dinitrate (ISDN) and buccal and transdermal nitroglycerin (NTG)] were evaluated in 36 patients with acute left ventricular failure due to a recent myocardial infarction. Similar reductions in pulmonary artery occluded pressure (p less than 0.01) followed all three regimens without change in heart rate and cardiac and stroke volume indices. Significant reductions in systemic arterial pressure and vascular resistance followed both ISDN and buccal NTG but not transdermal NTG. A disadvantage of the buccal NTG delivery was an abrupt and, on occasion, inappropriate reduction in blood pressure. The more gradual and controlled reduction of systemic blood pressure with substantial falls in pulmonary artery occluded pressure following ISDN infusion suggest that in the context of myocardial infarction this method of nitrate delivery may have safety advantages; however, when invasive haemodynamic monitoring facilities are not available, transdermal delivery may offer a practical alternative.

Administration, Buccal↗
PubMed1988

Chromosome and growth factor abnormalities in melanoma.

Cultures from metastatic melanomas of 15 patients had detailed melanoma growth stimulatory activity (MGSA) and cytogenetic analysis. The presence of melanoma cells was confirmed by microscopic identification of melanin, tyrosinase activity, and electron microscopy characterization of melanosomes. The MGSA is found in cytoplasmic granules after immunocytochemical stain. Three of the cultures did not produce MGSA and showed no distinctive cytogenetic differences. Breakpoints in derivative chromosomes were concentrated in region 1p1, and among all cultures chromosome 1 was the most frequently rearranged. It also has a low copy number of normal homologs. Chromosomes 18, X, and Y were never derivative, and chromosomes 2 and 4 were rarely so. Thus the cytogenetic data indicate that 4q13-21, the hybridization site for MGSA cDNA, is spared from gross change, although it could be under the influence of another site on chromosome 1 that is lost or rearranged. The ratio of abnormal to normal autosomes (mean per cell) in no culture exceeded 0.5, and for no autosome exceeded 0.8, suggesting a limit to the rearrangement tolerated for cell survival. If the Y is retained, the X:Y ratio varies around a normal figure of 1. The ratio of autosomes to sex chromosomes varies around a normal figure of 22. These data suggest stability of the X chromosome in cells undergoing multiple rearrangements of the autosomes.

Antibodies, Monoclonal↗