Search PubMed⌕ Search

Biomedical subjects

R E Lewis

Publications and source records attributed to R E Lewis.

At least 91 records · Page 5Linked to original sources
PubMed1990

Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C.

The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1990

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase.

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.

Amino Acids↗
PubMed1989

Multifunctional glycoprotein receptors for insulin and the insulin-like growth factors.

Insulin and the insulin-like growth factors (IGF) I and II are structurally related peptides that elicit a large number of similar biological effects in target cells. Three well-characterized receptor complexes bind one or more of these peptides with high affinity. Two of these receptors, denoted as type I, are ligand-activated tyrosine kinases with similar heterotetrameric alpha 2 beta 2 subunit structures which bind insulin or IGF-I, respectively, with highest affinity. Ligand-stimulated tyrosine autophosphorylation of these receptors further activates their intrinsic tyrosine kinase activities both in vitro and in intact cells. Rapid signal transduction follows such receptor autophosphorylation and tyrosine kinase activation, leading to increased serine phosphorylation of many cellular proteins and decreased serine phosphorylation of several others. Experiments in our laboratory have identified three distinct insulin-activated serine kinase activities in cell-free extracts that appear to account for the insulin-stimulated serine phosphorylation of the insulin receptor itself, ATP citrate lyase, and acetyl CoA carboxylase, respectively. A third receptor in this group binds IGF-I and II, lacks kinase activity and is denoted as type II IGF receptor. Amino acid sequences of this receptor deduced from isolated rat cDNA clones show a high degree of homology with those of the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor. We demonstrated that these receptors are indeed identical. The IGF-II/Man-6-P receptor rapidly recycles between the cell surface membrane and intracellular membrane compartments, providing for the rapid uptake of both IGF-II and mannose 6-phosphate-linked lysosomal enzymes. Insulin action markedly increases the proportion of receptors in the plasma membrane and the uptake of bound ligands. We also observe that large amounts of the extracellular domain of the IGF-II/Man-6-P receptor are released into the serum of fetal, neonatal and adult rats. The biological role of this receptor in IGF-II function is yet to be determined.

Animals↗
PubMed1989

Acute microvascular effects of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine: comparisons with leukotriene B4.

Acute effects of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) on microvessels of the hamster cheek pouch were studied using intravital and electron microscopy. FMLP (1.0 microM) applied topically to the pouch microvasculature produced transient vasoconstriction of second- and third-order arterioles, which was accompanied by modest narrowing of pericytic venules, leukocyte adherence, diapedesis, and macromolecular extravasation. Contrary to observations of leukotriene B4 (LTB4)-exposed venules in the same animal model, electron micrographs of fMLP-treated venules supported junctional, vesicular, and potential transcellular modes of transendothelial protein transport. Endothelial junctions were not characterized by large gaps, but failure of endothelial processes to effectively bridge diapedesing cells and reestablish junctional contact resulted in substantial deposition of subendothelial reaction product. Venous microvessels 18-21 micron in diameter were particularly susceptible to the deleterious effects of fMLP and revealed the highest percentage of total junctions or endothelial cells labeled by reaction product. Implied barrier dysfunction was more prevalent in fMLP-treated vessels exhibiting adherence and/or diapedesis; however, the presence of barrier defects was not correlated with leukocyte margination and/or emigration (Marg/Emig). The absence of simple correlation, without persuasive evidence for direct vasopermeability effects, recognizes that some Marg/Emig events may not have been present at the time of fixation and suggests the potential for neutrophil-mediated endothelial dysfunction, governed by multiple determinants, is often unrealized.

Animals↗
PubMed1989

Intercellular adhesion molecule expression in the evolving human cutaneous delayed hypersensitivity reaction.

Intercellular adhesion molecule-1 (ICAM-1), putatively expressed by antigen-presenting or target skin cells, is a ligand for the lymphocyte function-associated antigen (LFA-1) present on circulating lymphocytes. Immunohistochemistry of normal adult human skin using monoclonal antiserum to ICAM-1 demonstrated focal reactivity restricted to endothelium lining the dermal microvasculature. Delayed hypersensitivity responses elicited with dinitrochlorobenzene in the skin of the same subject were evaluated sequentially over a 96 h period using immunohistochemical and ultrastructural techniques. The first alteration observed consisted of mast cell degranulation within perivenular foci in the superficial dermis at 4 h after antigen challenge. Sparse superficial perivascular T-cell infiltrates were present by 24 h. Progressive staining for ICAM-1 was observed in microvascular endothelium and in dermal dendritic cells between 24 and 48 h. ICAM-1 expression was documented focally within the lower epidermis at 48 h and diffusely within the lower and upper epidermal layers at 96 h. ICAM-1 expression by keratinocytes was consistently associated with T-cell migration into the epidermis, whereas migration was never observed in the absence of ICAM-1 reactivity. Immunoelectron microscopy confirmed ICAM-1 to be exclusively present on endothelial cells, dermal dendritic cells, mononuclear cells, and keratinocytes, and permitted characterization of the patterns of membrane reactivity. ICAM-1 expression by epidermal cells appears to be closely linked to the progressive migration of T cells from the dermis into the epidermis that characterizes cutaneous delayed hypersensitivity.

CD4-Positive T-Lymphocytes↗
PubMed1989

Anti-class II antibody production prolongs renal allograft survival.

It is widely accepted that transfusions are beneficial to the outcome of renal allotransplantation. Whereas some investigators suggested that transfusions may induce both specific and nonspecific suppression of the cell-mediated immune response, others disagree. To lend clarity to this discrepancy, we collected 40 serum samples before and after blood transfusion therapy of first-time cadaveric renal allograft recipients and evaluated each for T cell and B cell cytotoxic antibodies using an Amos modified complement-dependent microlymphocytotoxicity assay. When greater than 10% of the panel cells reacted with a grade 4 or better, the panel was considered significant, and when a lymphocyte specificity was lysed by antibody-rich serum greater than 50% of the time, the antibody was considered specific. Control T and B cell PRA assays employed sera from 27 normal nontransfused volunteers of similar age and sex. Survival distributions of differences in the PRA before and after blood transfusions and posttransfusion PRA levels were compared using the Gehan generalized Wilcoxon test. Other factors which influence allograft survival such as HLA-A, -B and -DR matches, number of blood transfusions, immunosuppressive therapy, age, sex, parity, previous positive crossmatch, circulating cytotoxic antibodies matching the graft, prior dialysis, length of time on the waiting list, lapse of time between transfusion and transplantation and the underlying primary diagnosis were also considered using the Gehan generalized Wilcoxon test or the chi 2 approximation. Transfusion-related B cell cytotoxic antibodies, HLA-DR monospecific or multispecific antibodies and HLA-A, -B matching extended graft survival in a significant manner. Sex influenced the production of B and T cell transfusion-related cytotoxic antibodies with females producing greater quantities of antibodies than males. Parity and the production of monospecific or multispecific antibody were associated with an increase in transfusion-related B cell cytotoxic antibody. A difference in sex was not linked to the production of monospecific or multispecific HLA-DR antibodies. The majority of males failed to respond to multiple blood transfusions with the production of B cell cytotoxic antibodies although more than half were successfully grafted. All females and males who responded with the production of B cell cytotoxic antibodies monospecific or multispecific, with the exception of 1 female, demonstrated an allograft survival of greater than 1 year. In conclusion, differences between pre- and post-transfusion B cell PRAs and monospecific or multispecific HLA-DR antibodies identified in patient sera following transfusions were good predictors of renal allograft survival in both males and females.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Transfusion↗
PubMed1989

Contrasting quantitative alterations in CD4+ and CD8+ lymphocytes in HIV-infected African Americans compared with the Caucasian population.

Enumeration of circulating T lymphocytes is crucial in the investigation of AIDS and related conditions. The single best measure of disease progression and prognosis is the absolute number of helper/inducer T lymphocytes in the peripheral blood. Although the phenotypic identification of a particular subset reflects no direct information on the function of the population, the information provided by the analysis furnishes new insight regarding racial differences in the immune deficiency associated with AIDS. The severity of the HIV illness in the African American population, as reflected by a decrease in the absolute number of circulating CD4+ lymphocytes, was marked compared to the Caucasian population with AIDS. Consequently, the CD4/CD8 ratio was lower in the African American HIV+ population. A higher level of activated mononuclear lymphocytes and NK cells in this population may indicate active disease. The incidence of life-threatening opportunistic infections such as PCP was greater in the adult/adolescent African Americans compared to Caucasians. In contrast, PGL was found more frequently in the Caucasian participants. Although the rate of HIV infection in the adult/adolescent African American population was not different from population estimates for the area under study, the incidence in the pediatric African American population was twice the population estimates for the race. This increased rate occurred in the parent-at-risk as well as in the hemophiliac group.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Diapedesis and the permeability of venous microvessels to protein macromolecules: the impact of leukotriene B4 (LTB4).

The cellular basis of the reported neutrophil-dependent increase in microvascular permeability evoked by leukotriene B4 (LTB4) was probed using protein intravascular fluorescent and electron dense tracers in conjunction with intravital, light and electron microscopy. LTB4 (1 microM) applied topically to the hamster cheek pouch promoted neutrophil (PMN) adherence to endothelial cells (ECs) and resulted in limited, diffuse leakage of fluorescein-labeled albumin from pericytic venules. ECs to which PMNs adhered displayed many vesicles and a prominent cytoskeletal reorganization of microfilaments. During emigration, microfilament-rich luminal extensions of ECs effectively resealed the endothelial barrier prior to or in conjunction with PMN penetration of the basal lamina. Intimate associations between PMNs and ECs, coupled with efficient bridging of the lumen by EC processes, argued against significant transendothelial escape of macromolecules concomitant with PMN egress. Studies employing horseradish peroxidase as an intravascular tracer suggested minimal enhancement of junctional permeability in small venules and refuted a role for vesicular transport. The primary apparent barrier defect was a selective alteration in the membrane integrity of a proportion of ECs.

Animals↗
PubMed1988

Lymphocyte subset T4/T8 ratio in systemic lupus erythematosus: correlation with disease activity, laboratory abnormalities and treatment.

Lymphocyte subsets were studied in forty-nine patients with SLE using monoclonal antibodies and flow cytometry. A decrease in T4+ reactive cells (helper/inducer) was the most frequent observation. Decreased T4/T8 ratios were seen in patients with increasing clinically active disease, patients with positive anti-DS-DNA, positive anti-RNP antibodies and patients with low CH50 activity. However, low T4/T8 ratios were seen in patients with negative anti-Sm. Low T4/T8 ratios were also observed in patients taking prednisone at more than 10 mg/day and in patients treated with immunosuppressive drugs.

Adolescent↗