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Biomedical subjects

M R Sherman

Publications and source records attributed to M R Sherman.

At least 19 recordsLinked to original sources
PubMed1995

Pharmacokinetics and tolerability of ventricularly administered superoxide dismutase in monkeys and preliminary clinical observations in familial ALS.

The discovery of mutations in the gene for Cu/Zn superoxide dismutase (SOD) in some cases of familial amyotrophic lateral sclerosis (FALS) provides a rationale for enzyme replacement therapy. The inability of SOD to cross the blood-brain barrier motivated this study of the safety, tolerability and pharmacokinetics of bovine SOD (bSOD) administered into the CSF of rhesus monkeys and one late-stage, SOD-deficient FALS patient. Kinetic analyses in the patient indicated that intracerebroventricular (i.c.v.) administration, but not lumbar administration, delivered bSOD to the entire CSF pathway. Daily bolus i.c.v. injections (32 mg/day) and continuous i.c.v. infusion (30 mg/day) were well tolerated by the patient. During the period of daily bolus injections, the patient's performance on manual muscle tests was nearly stable, in contrast with the rapid decline before and after that period. These results justify further investigation of bSOD therapy in SOD-deficient FALS patients.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Local immunity in lung-associated lymph nodes in a murine model of pulmonary histoplasmosis.

Local immunity against acute pulmonary histoplasmosis was studied in the lung-associated lymph nodes of normal nonimmune mice infected intratracheally with live Histoplasma capsulatum yeasts. The phenotypes and distribution of cells in lung-associated lymph nodes and spleens were determined by flow cytometry. In addition, the immune responsiveness of these cells was evaluated by in vitro blastogenesis. Anti-H. capsulatum antibodies in serum and H. capsulatum antigen in tissue were measured by immunoassays. Cellular immune responses were greater in the lymph nodes than in the spleens. In lymph nodes 7 days after infection, a marked increase in the number of B lymphocytes caused the percentage to rise to 43%, compared with 26% in controls, and it remained elevated throughout the course of infection. A CD3+ cell that did not express CD4 or CD8 increased in number until it constituted 21% of lymph node cells, compared with 5% in controls, by day 14. The numbers of CD4+ and CD8+ T lymphocytes were modestly increased from days 7 to 35, but their percentages dropped because of the greater numbers of B lymphocytes and CD3+4-8- cells. Macrophages consistently constituted 2 to 3% of lymph node cells during the study. In spleens 7 days after infection, the percentage of macrophages in infected mice rose to 21%, compared with 9% in controls, but the total spleen cell number did not increase until day 14, when all cell subsets were nearly double in number. The in vitro blastogenic response of lymph node cells to H. capsulatum peaked at day 7, but spleen cell response was minimal during the course of infection. Histoplasma-specific serum immunoglobulin G antibodies reached peak levels by day 21 and remained high to the end of the study. In contrast, levels of antigen-specific immunoglobulin M antibodies were very low. These data suggest that antigen-specific immune responses occur in lung-associated lymph nodes and that this draining lymph node response may be an important component in host defense against Histoplasma lung infection.

Acute Disease↗
PubMed1992

Incidence of HIV infection in monocyte subpopulations characterized by CD4 and HLA-DR surface density.

OBJECTIVE: The purpose of this study was to determine any correlation between the expression of CD4 antigen on the surface of monocytes, and the frequency with which these cells are infected with HIV. DESIGN: CD4 surface expression on monocytes is significantly less than that expressed on CD4+ lymphocytes. Nevertheless, all monocytes express the HIV CD4 receptor and infected individuals have a significant decrease in the number of monocytes that express a higher density of CD4 surface fluorescence. METHODS: Three-color flow cytometric analysis was used to characterize monocyte-enriched peripheral blood mononuclear cells (PBMC) in terms of surface expression of CD4, CD14 (macrophage antigen), and class II major histocompatibility antigen (HLA-DR). HLA-DR+ monocytes from HIV-positive individuals were sorted into two subpopulations based on either 'bright' or 'dim' CD4 surface expression. A polymerase chain reaction (PCR) assay was used to detect the presence of proviral HIV sequences within the DNA from 10(5) cells from each sorted population. RESULTS: Post-sort analysis revealed that the dim CD4+ monocyte subset expressed dim HLA-DR surface antigen, while the bright CD4+ monocyte subset contained both bright and dim HLA-DR+ cells. PCR results showed that four out of eight dim CD4+ monocyte subsets contained proviral HIV DNA, compared with one out of eight bright CD4+ monocyte subsets.

Adult↗
PubMed1987

Pathogenesis of vertebral artery occlusion following cervical spine manipulation.

Sporadic cases of vertebral artery occlusion following neck manipulation have been reported since 1947. The pathogenesis of occlusion, however, has remained largely speculative. Autopsy examination of the intracranial and extracranial portions of the vertebral arteries in the case presented here clearly demonstrates that intramural hematoma arising within granulation tissue in the tunica media was the acute, occlusive event. Furthermore, prior neck manipulation may have caused asymptomatic development of granulation tissue within the tunica media, thereby predisposing the patient to acute intramural hematoma during subsequent neck manipulation.

Adult↗
PubMed1986

"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Triamcinolone acetonide 21-oic acid methyl ester: a potent local antiinflammatory steroid without detectable systemic effects.

There have been many previous attempts to prepare glucocorticoid analogs that would have high antiinflammatory activity at the site of application but minimal systemic side effects. In principle, esters of cortoic acids could fulfill these criteria, if they had sufficient affinity for the glucocorticoid receptors but were rapidly hydrolyzed to the inactive acids in the circulation. With this rationale, we have synthesized esters of the 21-oic acid of triamcinolone acetonide (TA, 9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1, 4-diene-3,20-dione 16,17-acetonide), a potent synthetic glucocorticoid, in both tritiated and unlabeled forms. The synthesis involves 1) oxidation to the 21-dehydro compound with methanolic cupric acetate, 2) further oxidation to the acid with methylene blue in the presence of KCN at pH 6.5, 3) esterification with diazomethane in the presence of methanol or ethanol, to produce the methyl ester of TA (TAme) or ethyl ester, respectively, and 4) purification of the products by TLC and HPLC. The molecular weights and structures of the esters were established by mass spectrometry and nuclear magnetic resonance. The binding of [3H]TAme to steroid receptors or serum steroid-binding proteins and the in vitro hydrolysis of the ester were evaluated simultaneously, by chromatography on Sephadex LH-20 columns in aqueous buffer. [3H]TAme is bound with high affinity by receptors from human leukemic cells and rat liver. The pattern of competition for this binding is characteristic of glucocorticoid receptors: TA approximately equal to TAme greater than R5020 (a synthetic progestin) approximately equal to aldosterone greater than 5 alpha-dihydrotestosterone. [3H]TAme is not bound detectably by serum steroid-binding proteins and is rapidly hydrolyzed during incubation with serum at 37 C. The acidic product has a very low affinity for the glucocorticoid receptor. Complexes of [3H]TAme with human and rat receptors have sedimentation coefficients of 9-10S in hypotonic buffer containing 20 mM Na2MoO4 and approximately 4S in hypertonic, molybdate-free buffer. These values of s20,w are similar to those of the oligomeric and monomeric forms, respectively, of the same receptors labeled with [3H]TA, and of mammalian steroid receptors, in general. The antiinflammatory activity of TAme in rats is comparable to that of prednisolone, but the ester is devoid of the side effects associated with prednisolone treatment (suppression of thymic weight and of serum corticosterone concentration). These bioassay data and the high affinity of the ester for human glucocorticoid receptors suggest that TAme may eventually be useful clinically, as a loc

Animals↗
PubMed1984

Multiple forms and fragments of cytosolic glucocorticoid receptors from human leukemic cells and normal lymphocytes.

Therapy with glucocorticoids is generally more effective in acute lymphoblastic leukemia than in other types of human leukemia. Previous studies, however, have not revealed any consistent relationship between clinical responsiveness and the cellular or cytosolic concentration of glucocorticoid-binding sites. The objectives of this study were to determine whether there are intrinsic structural differences among the glucocorticoid receptors in various types of leukemic cells and normal lymphocytes and to investigate the role of endogenous peptidases in receptor degradation. Cytosols were prepared from fresh or rapidly frozen leukocytes from 6 healthy adults and 35 high-risk leukemia patients (median white blood cell count, 150,000 cells/microliter; median age, 13 years). Receptors were labeled with [3H]triamcinolone acetonide and quantitated by charcoal-dextran treatment or Sephadex LH-20 chromatography. Mean and median cytosolic receptor concentrations in 12 acute lymphoblastic leukemia specimens lacking the standard B-cell or T-cell markers ("null cells") were approximately 4-fold higher than in 23 other leukemic cell specimens. No other consistent differences in receptor content were observed. Agarose filtration and ultracentrifugation in hypotonic buffers containing 20 mM Na2MoO4 revealed complexes of similar size and shape in all clinical specimens tested and two established leukemic cell lines. They had Stokes radii (Rs) of 8.1 +/- 0.5 (S.D.) nm (n = 50), sedimentation coefficients of 9.5 +/- 0.3S (n = 40), molecular weights of approximately 330,000, and axial ratios (a/b) of approximately 12. In hypertonic, molybdate-free buffer, these oligomeric complexes were dissociated into subunits with Rs of 5.9 +/- 0.3 nm (n = 12) and a/b of 11 to 12, as observed previously for other receptors. Fragmentation of the oligomer and the subunit was evident in some cytosols. High activities of peptidases of various specificities were detected in leukemic cell cytosols, as in other cytosols, by fluorometric assays with derivatives of 7-amino-4-methylcoumarin. Receptor cleavage by these and other endogenous enzymes may account for previous observations of "abnormal" receptors in cytosols from some leukemic specimens. We conclude that intrinsic structural defects in the receptors are unlikely explanations for the unresponsiveness of some types of leukemia to steroid therapy.

Adult↗
PubMed1983

Structure, dissociation, and proteolysis of mammalian steroid receptors. Multiplicity of glucocorticoid receptor forms and proteolytic enzymes in rat liver and kidney cytosols.

The forms of steroid receptors detected in mammalian tissue cytosols vary from a globular fragment, the mero-receptor, with a molecular weight (Mr) of approximately 23,000 to highly asymmetric molybdate-stabilized complexes (Mr approximately 330,000). Our objectives were to investigate the relationships among various receptor forms and mechanisms of stabilization by Na2MoO4, to characterize endogenous proteolytic enzymes, and to evaluate the effects of freezing the tissues on receptor structure and protease activities in the resultant cytosols. Glucocorticoid receptors and proteases were analyzed in cytosols from fresh and frozen rat liver and kidney. Enzymes were assayed fluorometrically at approximately 24 degrees C, with peptidyl tyrosine, lysine, and phenylalanine derivatives of 7-amino-4-methylcoumarin. Rates of cleavage of the tyrosine-containing substrate by crude and partially purified liver cytosol enzymes were markedly suppressed by Na2MoO4. While receptor cleavage by these enzymes has not been demonstrated, these results illustrate the direct inhibition of proteolysis by molybdate. Rates of cleavage of the lysine-containing substrate were 7- to 40-fold higher in cytosols from frozen liver and fresh or frozen kidney, in which the Stokes radius (Rs) of the largest receptor form was 70-74 A, than in fresh liver cytosol, in which Rs was 84 +/- 2 A (n = 20). Filtration of the 84 A complex (Mr approximately 330,000) in hypertonic buffer without Na2MoO4 revealed a 7:3 mixture of forms with Rs of 50-60 A (Mr approximately 90,000) and Rs of 30-40 A (Mr approximately 50,000). The latter are derived from the 50-60 A forms by proteolysis and/or dissociation. We conclude that the 84 A (untransformed) receptor is an oligomer, probably a tetramer, containing the 50-60 A subunits and that purification of the intact structure will require removal or inactivation of contaminating proteases.

Animals↗
PubMed1981

Human breast tumor estrogen receptor: effects of molybdate and electrophoretic analyses.

The largest and smallest discrete forms of the estrogen receptor in human breast tumor cytosol were characterized by competitive steroid binding, ultracentrifugation, gel filtration, and electrophoresis in polyacrylamide gels of several concentrations. Incubation of cytosol with [3H]estradiol and centrifugation in glycerol gradients containing 20 mM Na2MoO4 and 0 or 150 mM KCl revealed a 9-10S form of the receptor. It resembles the molybdate-stabilized complexes in cytosols of other human and rodent, malignant and healthy tissues, and the complex detected in breast tumor cytosol containing leupeptin, a bacterial protease inhibitor. Preservation of receptor integrity during purification and discrimination from serum steroid-binding components are facilitated by inclusion of molybdate in all buffers. Possible mechanisms of action of molybdate include the inhibition of ribonuclease action on RNA-associated receptor forms and protection against specific proteolytic cleavage by stabilization of a phosphate group on the vulnerable residue or a neighboring one. During fractionation of tumor cytosol in the absence of molybdate, the receptor is converted to a mixture of fragments. The smallest that retains the bound steroid, the mero-receptor, resembles the products of endogenous and exogenous protease action on receptors for all classes of steroids in a wide range of tissues. The similarities between both the largest and the smallest known forms of the breast tumor estrogen receptor and corresponding forms of other receptors support the notion of the common architecture of steroid receptors in normal and malignant tissues of diverse origins.

Binding, Competitive↗
PubMed1980

Estrogen receptor cleavage and plasminogen activation by enzymes in human breast tumor cytosol.

Estrogen receptors in cytoplasmic extracts of breast tumors from more than 40 patients were separately analyzed by gel filtration and/or ultracentrifugation under diverse conditions. Resultant patterns are presented for specimens from 11 women with infiltrating duct carcinoma and are representative of results obtained in all samples of sufficient size and receptor content (approximately 40 fmol/mg cytosol protein) for accurate determination of hydrodynamic parameters. Estradiol-binding components of intracellular origin were distinguished from the serum contaminant, sex hormone-binding globulin by their high affinity for diethylstilbestrol and negligible affinity for 5 alpha-dihydrotestosterone. The predominant molecular forms of the receptors, but not the steroid specificity, varied dramatically with experimental factors, including the duration of the fractionation procedure, ionic strength, and the presence of protease inhibitors, particularly the bacterial tripeptides N-acetyl- and N-propionyl-L-leucyl-L-leucyl-DL-arginine aldehydes (leupeptin). At least three discrete forms of the intracellular receptors were detected. The smallest labeled complex, the mero-receptor, with a sedimentation coefficient of about 3S and a Stokes radius of about 24 A, was formed during prolonged analysis of control cytosol in hypotonic or hypertonic buffers. Complexes with an intermediate sedimentation coefficient (approximately 5S) and Stokes radius (approximately 34A) were detected when control cytosol was analyzed rapidly in hypotonic buffer or when cytosol containing 50 nM leupeptin was analyzed in hypertonic buffer. The largest receptor form (10.5S, 71A) was predominant in cytosol prepared with 50 mM leupeptin and analyzed in hypotonic buffer. In this small series of patients, there was no obvious correlation between the molecular form of the receptors and the clinical status or eventual responsiveness to endocrine therapy. Preliminary studies of endogenous proteolytic enzymes in breast tumor cytosol that may be involved in mero-receptor formation included assays of plasminogen activators (EC 3.4.21.-) by fibrinolytic and spectrofluorometric techniques. The detection of high concentrations of plasminogen activators in several tumor cytosols and the inhibition of this activity by leupeptin, which stabilizes the large receptor forms in this and other systems, are consistent with a possible role of these enzymes in receptor cleavage.

Aged↗
PubMed1979

Glucocorticoid receptor cleavage by leupeptin-sensitive enzymes in rat kidney cytosol.

The proteolytic origin of small fragments of both the glucocorticoid and mineralocorticoid receptors in rat kidney cytosol was inferred from the effects of leupeptin, a bacterial tripeptide that inhibits many proteases [Sherman, M.R. et al., (1978). Federation Proc. 37:167--173]. In the present study, the smallest fragment of the glucocorticoid receptor containing the steroid-binding site, the mero-receptor, was characterized with respect to the Stokes radius (RS = 23 +/- 3 A) and the isoelectric point (pI = 5.9 at 4 degrees). Chromatography of cytosol labeled with [3H]triamcinolone acetonide on Sephadex LH-20 (Pharmacia) in aqueous buffer resolved the steroid-receptor complex from the unmodified free steroid and from steroid metabolites and contaminants. This technique facilitated analyses of the leupeptin-stabilized receptor form by isoelectric focusing (pU = 5.9 at 4 degrees) and centrifugation in glycerol gradients (s20,w = 9--11 S in 50mM KCl). When this large complex in fresh cytosol was analyzed on Agarose (Bio-Rad) at a high flow rate, it had RS congruent to 60 A in 50 mM KCl and RS congruent to 30 A in 400 mM KCl. These analytical studies with leupeptin indicate the need for inexpensive, irreversible inhibitors of proteolytic enzymes for the purification of intact receptors, holo-receptors, from kidney and other tissues. Specific proteases can then be applied to dissect the holo-receptor into the globular mero-receptor, proximal to the steroid-binding site, and the asymmetric region(s), distal segment(s), that may be involved in the nuclear interactions.

Animals↗