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Biomedical subjects

M R Clark

Publications and source records attributed to M R Clark.

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

Excess of red cell membrane proteins in hereditary high-phosphatidylcholine hemolytic anemia.

Previous descriptions of hereditary high-phosphatidylcholine hemolytic anemia (HPCHA) have highlighted the association of increased erythrocyte membrane phosphatidylcholine with abnormal membrane cation permeability. We studied the function and composition of erythrocyte membranes from three individuals with HPCHA to characterize further the membrane abnormalities in this disorder. Despite significant macrocytosis, HPCHA red cells were dehydrated and showed an increased surface area to volume ratio compared to normal red cells. The passive efflux of K+ from HPCHA erythrocytes was increased fourfold at 37 degrees C. Total membrane phospholipid was increased 7-42%, largely due to excess phosphatidylcholine, which made up 35.8-37.2% of total phospholipid. Membrane cholesterol:phospholipid ratios were in the normal range. It appears that the excess phosphatidylcholine was not acquired during circulation, since plasma lipids were normal and all subpopulations of density-separated HPCHA erythrocytes were similarly abnormal. The ratio of total protein to phospholipid in white ghosts was increased, indicating that membrane protein was increased to an even greater extent than membrane lipids. No abnormal membrane proteins were identified by Coomassie or periodic acid Schiff (PAS) staining. Quantitation of the major membrane proteins indicated that the total protein excess in HPCHA membranes was due to a proportional increase in all major proteins. We conclude that HPCHA erythrocytes have excess membrane proteins and hypothesize that the changes in lipid composition and cation permeability are secondary to underlying protein abnormalities, which remain to be defined.

Adult↗
PubMed1990

Immunohistochemical localization of prostaglandin H synthase in the embryo and uterus of the mouse from ovulation through implantation.

Prostaglandins (PGs) in the embryo and endometrium are involved in processes that are important for implantation. Although the presence of PGs (PGE2, PGF2 alpha, PGI2) in decidualized endometrium has been widely reported, less is known about the capacity of the pre-implantation embryo to synthesize PGs. Prostaglandin H (PGH) synthase is necessary for the production of PGs. Using an immunohistochemical method, PGH synthase was localized in the mouse embryo and uterus from superovulation through embryo implantation. No PGH synthase was detected in oocytes at the time of ovulation or in single-cell embryos 1 day post-fertilization (PF). Circular areas of immunostaining became evident in the cytoplasm of blastomeres at the morula stage (day 3 PF). After implantation (day 5 PF), a low level of PGH synthase reactivity was observed in embryonic cells; no PGH synthase was detected in the embryo by day 7 PF. The endometrial glands exhibited maximal immunostaining by day 3 PF, and after implantation, PGH synthase appeared in decidual cells along the border of placentation. Low levels of PGH synthase reactivity were detected in myometrial cells during the period after superovulation through day 7 PF. This is the first demonstration of PGH synthase in the mouse embryo prior to apposition with glandular endometrial epithelium, supporting the hypothesis that the embryo has the potential to produce PGs that may mediate autocrine and/or paracrine responses at the time of nidation.

Animals↗
PubMed1990

Immunohistochemical localization of prostaglandin H synthase in the female reproductive tract and endometriosis.

Prostaglandin H synthase, also known as cyclooxygenase, is an essential enzyme in prostaglandin synthesis. Using a monoclonal antibody and immunohistochemical techniques, we studied the localization of this enzyme in the female reproductive tract and in endometriosis. In the uterus, prostaglandin H synthase was primarily localized to endometrial epithelium with surface epithelium containing more immunoreactive prostaglandin H synthase than glandular epithelium. Fallopian tube secretory epithelial cells also contained prostaglandin H synthase while ciliated epithelial cells did not. Cervical epithelium and myometrial cells did not contain prostaglandin H synthase. A higher percentage of glands in endometriosis and adenomyosis, as compared with eutopic endometrium, contained prostaglandin H synthase suggesting that these tissues are also capable of prostaglandin synthesis. These findings may have important physiologic significance in the mechanisms of embryo implantation, in menstruation, and in the pathophysiologic features of endometriosis.

Adult↗
PubMed1990

Transient hyperprolactinemia during cycle stimulation and its influence on oocyte retrieval and fertilization rates.

Prolactin (PRL) has been shown to have inhibitory effect on follicle-stimulating hormone induced aromatase activity and estrogen biosynthesis in human granulosa cells cultured in vitro. To investigate the validity of the hypothesis that transient hyperprolactinemia during controlled ovarian hyperstimulation might influence follicular steroidogenesis and oocyte maturation, we measured serum PRL, estradiol, and progesterone before aspiration of oocytes in women undergoing ovarian stimulation (n = 108) in in vitro fertilization-embryo transfer. No correlation was detected between PRL and total number oocytes, number mature oocytes, fertilization rate, cleavage rate, and pregnancy rate. Transient elevation of PRL was a common finding in patients (57%) but was not associated with a poor clinical outcome.

Clomiphene↗
PubMed1990

Liquid chromatographic studies on the aqueous solution conformation of substituted benzamides related to remoxipride.

A series of disubstituted benzamides related to the sterically hindered 2,6-dimethoxybenzamide drugs, such as remoxipride, are prepared. The structure-retention relationships for these compounds are studied on hydrocarbon stationary phases in various hydroorganic mobile phases. The 2,6-dimethoxybenzamide displays a very low capacity factor under these reversed-phase conditions, suggesting that steric crowding prevents the formation of an amide-methoxy N-H...O intramolecular hydrogen bond. The corresponding 2-hydroxy-6-methoxybenzamide shows a dramatic increase in affinity for the hydrocarbon stationary phase, which is characteristic of strong intramolecular hydrogen bonding in these compounds. These results suggest that the aromatic ring--carbonyl aqueous solution conformation is almost 90 degrees in amides like remoxipride and changes to coplanarity upon demethylation of one methoxy group.

Antipsychotic Agents↗
PubMed1990

Localization of prostaglandin endoperoxide synthase in the human corpus luteum.

Prostaglandins have been implicated in both maintenance and luteolysis of the primate corpus luteum. Central to the production of prostaglandins is the enzyme prostaglandin endoperoxide synthase (PGHS). In the present study, we identified the cell types which contain PGHS in 44 human corpora lutea, using immunoperoxidase staining techniques. Intense granular staining was present in the cytoplasm of granulosa lutein cells of tissues obtained from the mid-luteal phase. Theca lutein cells demonstrated a diffuse cytoplasmic staining which was less intense than that observed in granulosa lutein cells. Staining appeared less intense in tissues from the early or late phase. Ovarian stromal cells demonstrated little or no PGHS immunoreactivity. PGHS staining in the corpus luteum of pregnancy was similar in intensity and cell distribution to that of mid-luteal corpus luteum. In summary, human corpus luteum contains immunoreactive PGHS which localized mainly to well-differentiated granulosa lutein cells.

Adolescent↗
PubMed1990

Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle.

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase was studied throughout the rat estrous cycle. Animals were necropsied at 1300 h on each day of the 4-day cycle, and an additional group was necropsied at 2300 h on proestrus. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. At all stages of the cycle, intense immunostaining was observed in newly formed corpora lutea. Luteal cells were immunoreactive, but the connective tissue centrum was unstained. Interstitial tissue contained heavily labeled cells, whereas the germinal epithelium exhibited faint staining. During estrus, metestrus, and diestrus, thecal cells from preantral and antral follicles contained PGH synthase immunoreactivity, but granulosa cells were unstained. Faint staining of mural granulosa cells was observed first in 78% of preovulatory follicles (less than 400-microns diameter) in ovaries collected on the afternoon of proestrus. After the luteinizing hormone surge, 95% of the preovulatory follicles exhibited PGH synthase staining. The percentage of immunoreactive granulosa cells in these preovulatory follicles increased 4-fold in ovaries collected at 2300 h on proestrus. The presence of ovarian PGH synthase throughout the rat estrous cycle and the changes in cellular localization may reflect the potential role of PGs in follicular and luteal function.

Animals↗
PubMed1990

An abnormality of the gene that encodes neutrophil Fc receptor III in a patient with systemic lupus erythematosus.

In the course of examining the structure and function of Fc receptors on peripheral blood cells of patients with systemic lupus erythematosus, we identified a patient whose neutrophils did not react with either monoclonal or polyclonal antibodies to Fc receptor III. However, neutrophils from the patient were comparable to neutrophils from healthy controls with respect to their expression of Fc receptor II, complement receptor 1, complement receptor 3, and the phosphatidylinositol-linked, complement regulatory protein, decay-accelerating factor. The abnormality of expression of Fc receptor III was limited to the patient's neutrophils (her natural killer cells reacted normally with anti-Fc receptor III antibodies), and was associated with abnormal recognition and binding of IgG-coated erythrocytes. Analysis of genomic DNA revealed evidence that failure of the patient's neutrophils to express Fc receptor III was most likely due to an abnormality of the gene that encodes this receptor.

Adult↗
PubMed1990

Humanisation of monoclonal antibodies for therapy.

Monoclonal antibodies are playing an increasing role in many experimental therapies. A major limitation of their use is that they are recognised by the patient as being of foreign origin and an antiglobulin response is provoked. Recombinant DNA technology offers the ability to convert these rodent antibodies into a more human form. Different degrees of humanisation can be achieved ranging from chimeric antibodies with a combination of human constant regions with rodent variable regions to fully reshaped antibodies where the variable regions are also humanised. Encouraging preliminary results have been obtained and it seems likely that these will dictate the use of humanisation as a required procedure in the therapeutic use of monoclonal antibodies.

Animals↗
PubMed1990

Purging in auto- and allografts: monoclonal antibodies which use human complement and other natural effector mechanisms.

CAMPATH-1M is a rat IgM monoclonal antibody which binds to an antigen on all human lymphocytes and monocytes, but which is not present on marrow stem cells (Hale et al., 1983). Lymphocytes can be efficiently killed in bone marrow buffy coat preparations using the antibody and donor human serum as a means to avoid graft versus host disease (GVHD) (Waldmann et al., 1984). An analysis of 520 matched sibling bone marrow transplants (BMT) for leukaemia demonstrates that T-cell depletion using CAMPATH-1M markedly reduces the incidence and severity of GVHD, but there is an increased risk of graft rejection. In the case of CGL in chronic phase, there is also an associated extra risk of relapse, particularly in patients where engraftment may have been compromised (Hale et al., 1988a). A rat IgG2b antibody of the same specificity as CAMPATH-1 (CAMPATH-1G) was developed which is able to both fix human complement and opsonize lymphocytes in vivo (Dyer et al., 1989). Initial studies for the prophylaxis of bone marrow rejection in 55 mismatched and matched unrelated donor (MUD) BMTs suggest that CAMPATH-1G treatment of the recipient may reduce, but not eliminate, marrow graft rejection. The broad CAMPATH-1 specificity means that it is also ideal for purging a range of lymphoid malignancies prior to autologous BMT, or even for direct serotherapy of leukaemic patients. However, there may be limitations of monoclonal antibody purging using complement or other natural effector mechanisms either in vivo or in vitro; in particular, antigenic modulation and an antiglobulin response. Phase 1 studies of "in vivo purging" with a monovalent CD3 antibody (Clark et al., 1989), and also with a genetically engineered humanized IgG1 (CAMPATH-1H) (Hale et al., 1988b) suggest that these limitations can be overcome.

Antibodies, Monoclonal↗
PubMed1989

Nonsteroidal antiestrogen inhibition of protein kinase C in human corpus luteum and placenta.

These studies were undertaken to determine whether nonsteroidal antiestrogens would inhibit the calcium/lipid-dependent protein kinase (protein kinase C) activity in hormonally-responsive human reproductive tissues. Cytosol was prepared from human corpus luteum and term placenta. Protein kinase C activity was examined with various antiestrogens, estrogens, and catecholestrogens. The nonsteroidal antiestrogens tamoxifen, clomiphene and Z-4-hydroxytamoxifen inhibited protein kinase C in cytosol from human corpora lutea and placentae in a concentration-dependent manner. The IC50 values were 35-45 microM for tamoxifen, 58-66 microM for clomiphene, and 88 microM for hydroxytamoxifen. Protein kinase C purified 600-fold from human placenta was also inhibited by tamoxifen. The estrogens, estradiol and diethylstilbestrol (DES), and the catecholestrogens, 2-hydroxyestradiol and 4-hydroxyestradiol, had no effect on protein kinase C activity, nor were they able to prevent the inhibition of protein kinase C by the antiestrogens. Inhibition of the enzyme by the antiestrogens was competitive with phosphatidylserine and 1,2-diolein. In addition, tamoxifen inhibited enzyme activity stimulated by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). The data suggest that the action of these antiestrogens on protein kinase C was a direct inhibition of the enzyme. Furthermore, the site of interaction showed markedly different structural specificity from that of the estrogen receptor.

Corpus Luteum↗
PubMed1989

Molecular basis for a polymorphism involving Fc receptor II on human monocytes.

IgG Fc receptor II (Fc gamma RII) on human monocytes is polymorphic with respect to its appearance on gels after isoelectric focusing and with respect to its ability to mediate T lymphocyte proliferation induced by murine anti-CD3 mAb of the IgG1 isotype (i.e., its ability to bind murine IgG1). To determine the molecular basis for this polymorphism, we isolated total cellular RNA from PBMC of responders and nonresponders (defined by Leu-4-induced [3H] thymidine incorporation) and synthesized corresponding cDNA. Sequences encoding the extracellular domain of Fc gamma RII were then amplified using the Taq polymerase chain reaction. Amplified DNA fragments were cloned into pUC vectors, and sequenced. Analysis of clones from two nonresponders revealed a single base change (G for A) at position 519, which would result in the substitution of a histidine for an arginine at residue 133 in the mature Fc gamma RII protein. These findings suggest that the polymorphism involving human monocyte Fc gamma RII results from allelic variation of a single gene.

Adult↗
PubMed1989

Recording sound from human skeletal muscle: technical and physiological aspects.

In order to examine technical factors that influence muscle sound recording, single twitches of muscle were utilized since their waveforms were likely to be reproducible. We observed that satisfactory recordings could be made with either Archer air interface, or Hewlett-Packard direct contact sensor, microphones. Firm contact and stability between the microphone and the skin surface were particularly important. Frequencies below 20 Hz, the lower limit of the human auditory range, must be recorded, since they account for at least 90% of the power of the muscle sound wave. The chief frequencies were below 4 Hz. The sound wave produced by a maximal twitch of human thenar muscle induced by median nerve stimulation at the wrist is maximal in amplitude over the center and recedes to near zero at the margins of the muscle. It is preceded by the muscle compound action potential and is followed by the force curve, recorded with a strain gauge attached to the thumb. The sound resembles force in total time course, and it increases with increasing strengths of nerve stimulation. However, it differs in its latency, phase relationships, and response to nerve stimulation at different frequencies. Some of the features of muscle sound suggest it relates to both the active contractile and the parallel elastic components of muscle during a twitch contraction, but not the series elastic component.

Adult↗
PubMed1989

Suppression of LH-stimulated prostaglandin and progesterone accumulation in rat granulosa cells by isoquinolinesulfonamide protein kinase inhibitors.

Rat granulosa cells were incubated with isoquinolinesulfonamide inhibitors of protein kinases A and C and/or LH, dibutyryl cAMP (dbcAMP), tetradecanoylphorbol acetate (TPA), cholera toxin, or forskolin for 5 h. H7 (25 microM) was observed to inhibit LH, cholera toxin or dbcAMP stimulation of prostaglandin (PGE), and progesterone accumulation. H7 produced inhibition when added as little as 2 min before and as long as 1 h after LH. HA1004 was ineffective against LH or cholera toxin stimulation of PGE or progesterone at up to 100 microM. H9 blocked some LH and forskolin responses at 25 microM, but required a 50 microM concentration to minimally affect TPA stimulation. Cytotoxicity was not observed at the concentrations and times of isoquinolinesulfonamides tested. H7 and H9, therefore, suppress LH stimulation of granulosa cell functions in a dose- and time-dependent manner consistent with inhibition of protein kinases A and/or C, and consonant with a requirement for such kinases in LH action.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Immunohistochemical localization of prostaglandin H synthase in the epididymis and vas deferens of the mouse.

Prostaglandins (PGE2, PGF2 alpha) in the excurrent ducts of the male reproductive tract appear to be both modulators of ductal contractility for transport of spermatozoa and factors involved in the regulation of sperm maturation. To identify the tissue sites for the production of prostaglandins (PGs) in the excurrent ductal system, we have employed an immunohistochemical technique to localize prostaglandin H (PGH) synthase in the epididymis and vas deferens of the mouse. A mouse monoclonal antibody to PGH synthase was used and was shown to be specific for the mouse enzyme by Western blot analysis. In sexually mature mice, PGH synthase was primarily localized to the epithelium of the epididymis and vas deferens. Within the epididymal epithelium, immunoactivity appeared in all cell types of the initial segment, in a subpopulation of cells with predominantly apically oriented nuclei in the caput and corpus, and in low levels in the cauda. PGH synthase reactivity was the most intense in the epithelial cells of the vas deferens. PGH synthase was not detected in smooth muscle cells, spermatozoa, or luminal fluid. This study suggests that the epithelium of the excurrent ductal system of the mouse is the major site for PG production. The regionalization of PGH synthase to cells in the epididymis thought to be involved in the absorption of luminal fluid suggests that PGs may play a role in fluid and ion transport.

Animals↗
PubMed1989

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua.

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.

Blotting, Western↗
PubMed1989

Mean corpuscular hemoglobin concentration and cell deformability.

A prominent characteristic of sickle cells is the presence of a subpopulation of severely dehydrated cells. Apart from its effect on the rate and extent of Hb S polymerization at low oxygen tension, dehydration has adverse effects on deformability of oxygenated sickle cells. In general, the degree of deformability defect induced by cellular dehydration measured in vitro depends on the method used to measure deformability. Methods in which the extent or rate of cellular deformation are measured are especially sensitive to intracellular Hb concentration, whereas filtration methods are more sensitive to cell size. Studies of naturally occurring cellular dehydration in red cell disorders other than sickle cell disease have indicated that red cells can tolerate dehydration and a substantial increase in intracellular viscosity much more readily than a loss of deformability that results from loss of surface area or increase in cell volume. Further, the major clinical problems in sickle cell disease, involving occlusion of small vessels, do not correlate with the proportion of poorly deformable, dehydrated cells. Thus, the direct rheologic effects of cellular dehydration in sickle cell disease are probably much less important than the effects of MCHC on the kinetics of Hb S polymerization.

Erythrocyte Deformability↗