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M R Clark

Publications and source records attributed to M R Clark.

At least 127 records · Page 7Linked to original sources
PubMed1987

Ovarian prostaglandin synthase: immunohistochemical localization in the rat.

Ovarian prostaglandin synthase is stimulated by the luteinizing hormone surge resulting in the preovulatory increase in prostaglandins. In the present study, the ovarian cellular localization of prostaglandin synthase was identified by immunohistochemistry. Ovaries were collected from 27-day-old rats at the time of stimulation with pregnant mare serum gonadotropin (8 IU) (zero hours), 48 hours later, or 8 hours after administration of human chorionic gonadotropin (5 IU). At zero hours prostaglandin synthase immunostaining was present in the theca of larger follicles and interstitial regions. The number and intensity of immunostained cells in the theca increased between zero and 48 hours. The granulosa cell layer adjacent to the basement membrane in large antral follicles exhibited immunostaining. A similar staining pattern was observed 8 hours after human chorionic gonadotropin. These observations indicate that in the rat, the theca, interstitium, and granulosa contain prostaglandin synthase immunoreactivity and may contribute prostaglandins during follicular development and ovulation.

Animals↗
PubMed1987

Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1----3Gal epitope in primates.

Anti-Gal is a natural antibody, which constitutes as much as 1% of circulating IgG in humans and displays a distinct specificity for the structure Gal alpha 1----3Gal. This glycosidic structure has been found on various tissues of many nonprimate mammals. A comparative study of the occurrence of anti-Gal versus the expression of the Gal alpha 1----3Gal epitope was performed in primates, and a distinct evolutionary pattern was observed. Whereas anti-Gal was found to be present in Old World monkeys and apes in titers comparable to those in humans, its corresponding antigenic epitope is abundantly expressed on erythrocytes of New World monkeys. Immunostaining with anti-Gal of glycolipids from New World monkey erythrocytes indicated that the molecules to which anti-Gal binds are similar to those found in rabbit and bovine erythrocytes. These findings indicate that there is an evolutionary reciprocity between New World and Old World primates in the production of the Gal alpha 1----3Gal structure and the antibody that recognizes it. The expression of the Gal alpha 1----3Gal epitope was evolutionarily conserved in New World monkeys, but it was suppressed in ancestral lineages of Old World primates. The suppression of this epitope was accompanied by the production of anti-Gal. The observed in vivo binding of anti-Gal to human normal senescent and some pathologic erythrocytes implies that the Gal alpha 1----3Gal epitope is present in man in a cryptic form.

Animals↗
PubMed1987

T-cell killing of target cells induced by hybrid antibodies: comparison of two bispecific monoclonal antibodies.

Two different bispecific hybrid antibodies were generated by cell fusion of pairs of existing hybrid-myeloma cell lines. Both hybrid antibodies had similar specificity for human CD3 and mouse Thy-1 but differed in the isotypes of the immunoglobulin heavy chains. Hybrid HA2b/2b was a hybrid between a rat IgG2b (CD3) and a rat IgG2b anti-Thy-1, whereas HA2b/2c was a hybrid between the same rat IgG2b (CD3) and a rat IgG2c anti-Thy-1. Both hybrid antibodies were found to be very potent in inducing the killing of Thy-1-positive targets by human T-cell blasts, with the hetero-hybrid HA2b/2c showing a higher titer. T-cell blasts generated from resting peripheral blood mononuclear cells by a novel mitogenic antibody, YTH361, were exploited as effector cells. In addition to the CD3-dependent killing, the rat IgG2b anti-Thy-1 antibody and the hybrid antibody HA2b/2b but not the rat IgG2c anti-Thy-1 or the hybrid antibody HA2b/2c were also able to elicit antibody-dependent cell-mediated cytotoxicity (ADCC). This ADCC was inhibited by an anti-FcRlow (CD16) monoclonal antibody, which suggests that these effectors were K-cells. Toxicity toward the T-cell blast effector population was also observed, but in this instance the hetero-hybrid HA2b/2c had a lower cytotoxic titer. In conclusion, mixed isotype hybrid antibodies may have some advantages for eliciting T-cell-mediated killing of tumor cell targets by exhibiting a better therapeutic ratio of target cell to effector cell cytotoxicity.

Animals↗
PubMed1987

Ektacytometric measurement of sickle cell deformability as a continuous function of oxygen tension.

In an effort to study the rheologic effects of small amounts of hemoglobin S (HbS) polymer in sickle red cells, we have used the ektacytometer, a laser diffraction couette viscometer, to measure sickle cell deformability as a function of oxygen tension. Sickle cell populations of defined intracellular hemoglobin concentration (MCHC) were isolated using Stractan density gradients and were resuspended in buffered polyvinylpyrrolidone solutions for deformability measurements. Using a gas-porous, hollow fiber gas exchange system to establish a linear gradient in oxygen tension, deformability was measured over a pO2 range of 76 to 0 mm Hg. Parallel spectroscopic determinations of oxygen saturation permitted determination of cell deformability as a function of oxygen saturation for each discrete MCHC population. From these measurements the level of oxygen saturation at which a loss in cell deformability was first detected could be defined. Then, using the data of Noguchi and Schecter, the amount of polymerized HbS in the cells at that defined level of oxygen saturation was estimated. The results of this analysis suggested that the quantity of polymer that caused a detectable loss in cell deformability increased with increasing MCHC. In addition, for MCHC above 30 g/dL, this represented a substantial fraction of the total HbS in the cell.

Anemia, Sickle Cell↗
PubMed1986

Tolerance to rat monoclonal antibodies. Implications for serotherapy.

The antiglobulin response is a major complication of mAb therapy. It has been suggested that, in clinical practice, this might be avoided by using human or chimeric mAbs, or by prior induction of tolerance to the therapeutic mAb. In this study, we show that it is possible to induce tolerance in mice to the constant regions of rat IgG2b mAbs by both classical deaggregation methods and by anti-L3T4 mAb therapy. Mice tolerant to IgG2b constant region determinants failed to make an antiglobulin response when immunized with a number of mAbs of the same isotype that had no binding specificity for mouse cells, but produced vigorous antiidiotypic responses to cell-binding mAbs. Binding of antibodies to hemopoietic cells rends their idiotypic determinants major immunogens even in the presence of tolerance to constant region epitopes. These findings suggest that the use of human or chimeric mAbs will not be sufficient to eliminate the antiglobulin response, and that additional methods need to be investigated.

Animals↗
PubMed1986

Acute digitalis poisoning: the role of intravenous magnesium sulfate.

Acute digitalis poisoning is a complex emergency with a reported mortality rate of 3% to 25%. In severe overdose, the sodium, potassium-adenosine triphosphatase system is severely inhibited, leading to cardiac dysrhythmias and an elevation of the serum potassium. Magnesium, a cofactor regulating this ion transport system, can successfully treat acute digitalis-induced rhythm disturbances and restore the transmembrane potassium gradient. This paper discusses the cellular mechanism involved in digitalis toxicity and reviews the literature concerning the use of magnesium in acute cardiac glycoside poisoning.

Acute Disease↗
PubMed1986

Mechanisms of action of gonadotropin releasing hormone on rat granulosa cells.

To elucidate the mechanisms of stimulatory actions of GnRH on rat granulosa cells (GC), we have compared the actions of a GnRH agonist with those of a tumor-promoting phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA) and Ca+2 ionophore, A23187. GC were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG. Following prelabeling with 3[H]arachidonic acid (AA), the cells were incubated with the test substances for 10 min and AA release determined. A GnRH agonist, [D-Ala6, des-Gly-NH2(10)] GnRH ethylamide (GnRHa; 10 ng/ml) increased AA release 175% compared to the control value. AA release in the presence of GnRHa was larger than that due to 1 microM A23187 or 40 nM TPA alone. A23187 or TPA increased GnRHa-stimulated AA release further. GC were incubated with the test substances for longer time periods, i.e., up to 5 h. GnRHa caused a 4-fold increase in prostaglandin (PG) synthase activity at 5 h. GnRHa increased PGE accumulation to the same extent as TPA, but only increased PG synthase activity about half as much. In combination with TPA, GnRHa had no influence on TPA-stimulated PG synthase activity, but increased PGE accumulation to levels comparable to those with A23187 plus TPA. GnRHa caused a 2.5 fold increase in progesterone (P) accumulation, which was the same as TPA. P accumulation in the presence of GnRHa was affected by neither A23187 nor TPA. These data indicate that the combination of TPA and A23187 can substitute for GnRH action on PGE and P accumulation in rat GC.

Animals↗
PubMed1986

The mechanisms by which phorbol ester inhibits LH stimulation of progesterone production in rat granulosa cells.

Tumor-promoting phorbol esters are believed to affect cell functions by activating a Ca+2-and lipid-dependent protein kinase (protein kinase C). 12-0-Tetradecanoyl phorbol-13-acetate (TPA) inhibits LH stimulation of progesterone (P) accumulation in rat granulosa cells. To determine the mechanisms by which TPA inhibits LH stimulation of P accumulation, TPA regulation of various ovarian steroidogenic enzymes was investigated. Cells were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h. TPA decreased the P responses to LH, cholera toxin, and (Bu)2cAMP by 20%, 24%, and 28%, respectively. One locus of inhibition of LH action, therefore, was after cAMP. TPA decreased LH-stimulated 3-beta-hydroxysteroid dehydrogenase activity. Furthermore, TPA stimulated 20-alpha-hydroxysteroid dehydrogenase activity. The combination of TPA and A23187 inhibited LH-stimulated P accumulation to the same extent as TPA alone. These data suggest that TPA-induced decreases in LH-stimulated P production result from both the inhibition of P biosynthesis and the stimulation of P breakdown.

20-Hydroxysteroid Dehydrogenases↗
PubMed1986

Excessive binding of natural anti-alpha-galactosyl immunoglobin G to sickle erythrocytes may contribute to extravascular cell destruction.

A large proportion of sickle erythrocytes is removed from the circulation by the macrophages of the reticuloendothelial system. In view of the proposed role for natural antibodies in the destruction of normal senescent erythrocytes, we looked for a possible similarity in the antibodies that bind in situ to senescent and sickle cells. Bound IgG molecules were detected by a highly sensitive rosetting antiglobulin test, using K562 myeloid cells. After separation on Stractan density gradients, the 0.6% most dense (senescent) normal cells and the most dense 40% sickle cells displayed membrane-bound IgG as reflected by the high proportion of rosettes formed. No antibody was found on low-density cells of either type. The bound antibodies were readily eluted from both sickle and normal senescent cells by carbohydrates containing alpha-galactosyl residues. These antibodies appear identical to the recently discovered human natural anti-alpha-galactosyl IgG (anti-Gal), an IgG antibody present in high titers in normal sera. Moreover, affinity-purified anti-Gal interacted specifically with sickle and normal cells depleted of the autologous antibodies. A similar pattern of binding to the various erythrocyte subpopulations was observed when the radiolabeled lectin with anti-alpha-galactosyl specificity, Bandeiraea simplicifolia, was used. In vitro phagocytosis of normal and sickle erythrocyte subpopulations correlated with the presence of anti-Gal on these cells. The in situ binding of anti-Gal to a large proportion of sickle erythrocytes may reflect an accelerated physiologic aging process by which immune recognition of prematurely exposed alpha-galactosyl-bearing antigenic sites contributes to shortened cell survival.

Anemia, Sickle Cell↗
PubMed1986

Effects of a leukotriene antagonist on the early hemodynamic manifestations of group B streptococcal sepsis in piglets.

In order to evaluate the influence of leukotrienes on group B streptococcal (GBS) sepsis we studied the effect of a leukotriene antagonist, FPL 57231, on the early hemodynamic changes occurring secondary to an infusion of live GBS. Paralyzed, mechanically ventilated piglets received a continuous intravenous infusion of bacteria (5 X 10(8) org/kg/min) while systemic arterial pressure and pulmonary artery pressure were measured continuously. Cardiac output was measured by thermodilution; and plasma samples for determination of thromboxane B2 and 6-keto-PGF1 alpha were taken at preset intervals. In addition to GBS, treatment animals received a continuous infusion of FPL 57231 starting 15 min after the bacterial infusion was begun. Study animals as a whole responded to bacteria within 15 min with a marked elevation in pulmonary artery pressure from 13.6 +/- 4 to 44.6 +/- 6 mm Hg (p less than 0.001), and a decline in PaO2 (79 +/- 9 to 44 +/- 5 mm Hg) (p less than 0.001) and a cardiac output (0.27 +/- 0.07 to 0.15 +/- 0.06 liter/min/kg) (p less than 0.0001). In animals treated with FPL 57231 these changes were reversed or significantly attenuated by 60 min. In the control group pH deteriorated significantly to 7.17 +/- 0.1 compared to baseline values (p less than 0.01) by 60 min, while treatment group animals maintained a pH of 7.3 +/- 0.23. Thromboxane B2 and 6-keto PGF1 alpha were similar in both groups and did not change during the study period. In addition, survival was significantly longer in treatment (191 +/- 44 min) compared to control animals (100 +/- 32 min) (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1986

The preovulatory increase in ovarian collagenase activity in the rat is independent of prostaglandin production.

In the present study, we have examined the role of gonadotropins and prostaglandins in the preovulatory increase of ovarian collagenase activity in the rat. Whole ovaries of immature PMSG-primed rats (20 IU) were removed before and 8 h after the rats were treated with human (h) CG, Nembutal, and/or indomethacin. The ovaries were homogenized in a solution containing Triton X-100 (0.25%) and centrifuged. Collagenase was extracted by resuspending the pellets in buffer containing 100 mM CaCl2, heating to 60 C for 6 min, and centrifuging. The supernatants were treated with dithiothreitol (2 mM) and iodoacetamide (5 mM) to inactivate collagenase inhibitors. Collagenase activity was measured as the percent digestion of 3H-type I collagen/100 microliters aliquot of ovarian sample. At zero time (52 h after PMSG), ovarian collagenase activity was 4.2 +/- 1.2% digestion (mean +/- SEM, n = 3). In ovaries collected 8 h after the endogenous LH surge or 8 h after the administration of 10 IU hCG at time zero, collagenase activity rose to 19.6 +/- 2.1 (n = 6) and 22.5 +/- 1.7% digestion (n = 11), respectively. Indomethacin (1.5 mg/100 g BW) administered 30 min after hCG, produced no change in collagenase activity (24.8 +/- 2.5% digestion, n = 7) although the expected increase in ovarian prostaglandin E after hCG treatment was blocked. When the endogenous LH surge was blocked with Nembutal (3 mg/100 g BW), collagenase activity in 8-h ovaries was 6.8 +/- 1.1% digestion (n = 10). The Nembutal block of the preovulatory collagenase increase was overcome by administration of hCG (8-h ovarian enzyme activity = 22.7 +/- 3.2% digestion, n = 8). These observations demonstrate that hCG stimulates ovarian collagenase activity and that this stimulation is not dependent on prostaglandin synthesis.

Animals↗
PubMed1986

Medical applications of monoclonal antibodies.

Since the first publication of the production of monoclonal antibodies in 1975 there has been enormous interest in the possible medical applications. Monoclonal antibodies are now widely used in all areas of biological and medical research as well as in clinical diagnostic tests and in therapy. This review concentrates on the clinical use of antibodies in therapy particularly with regard to the properties of the antibodies which seem most relevant to their usefulness. In-vitro tests using human effector systems and in-vivo animal models have demonstrated the importance of the antibody isotype and valency for antigen as well as the specificity of binding. With the appropriate choice of antibody, used either alone, or conjugated to radioisotopes, drugs or toxins, it is possible to specifically deplete subpopulations of cells both in-vitro and in-vivo. Such antibodies can be used for tumour therapy, and for immunosuppression in organ transplantation and in autoimmune disorders.

Animals↗
PubMed1986

Association between morphologic distortion of sickle cells and deoxygenation-induced cation permeability increase.

We hypothesized that the deoxygenation-induced increase in cation permeability of sickle cells was related to mechanical distention of the membrane by growing HbS polymer within the cell. To test this hypothesis, we determined the effect of deoxygenation on cation fluxes in sickle cells under conditions that restricted or permitted extensive growth of polymer, producing different degrees of membrane distention. Manipulation of suspending medium osmolality for density-isolated high and low mean cell hemoglobin concentration (MCHC) cells was used to regulate the extensional growth of polymer bundles and hence membrane distortion. For initially low MCHC cells, the deoxygenation-induced increase in both Na and K fluxes was markedly suppressed when the MCHC was increased by increasing the osmolality. This suppression corresponded to the inhibition of extensive morphologic cellular distortion. For initially high MCHC, ISC-rich cells, deoxygenation had minimal effect on K permeability. However, reduction of MCHC by a decrease in osmolality produced a concomitant increase in cation permeability and cellular distortion. These observations support the idea that the sickling-associated increase in membrane permeability is related to mechanical stress imposed on the membrane by bundles of HbS polymer.

Anemia, Sickle Cell↗
PubMed1985

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes.

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.

Acyltransferases↗
PubMed1985

AUR memorial Award. Induced alignment of flowing sickle erythrocytes in a magnetic field. A preliminary report.

Deoxygenated sickle erythrocytes in static suspension align perpendicular to a magnetic field. To assess the importance of this observation to MRI of sickle-cell disease, an in vitro flow apparatus was devised and the orientation of sickle erythrocytes flowing through a 0.38 T magnetic field was investigated. We showed a significant perpendicular alignment of fully deoxygenated sickle erythrocytes flowing at 3 to 4 mm/minute (P less than .001). These results suggest that deoxygenated erythrocytes in a sickle-cell patient could orient perpendicular to a magnetic field, and therefore that MRI of such patients could possibly result in worsening of vaso-occlusive complications. Further studies are needed to assess the possible hazards of MRI of sickle-cell disease, especially at high field strengths.

Anemia, Sickle Cell↗
PubMed1985

Phorbol ester regulation of rat granulosa cell prostaglandin and progesterone accumulation.

Tumor-promoting phorbol esters are believed to affect cell functions by activating a Ca+2- and lipid-dependent protein kinase (protein kinase C). Since such protein kinases may be involved in ovarian granulosa cell metabolism, the effects of phorbol esters on prostaglandin (PG) and progesterone (P) accumulation were investigated. Cells were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h. A tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), at a concentration of 25 ng/ml, caused 4-fold increases in PGE and 6-keto-PGF1 alpha accumulation at 5 h. LH (10 ng/ml) caused 7- and 4-fold increases in PGE and 6-keto-PGF1 alpha accumulation, respectively. When tested in combination, the increases in PGE and 6-keto PGF1 alpha due to TPA and LH were additive. Like the effect of LH, the TPA stimulation of PG synthesis occurred after a delay of 2-3 h. By 5 h of incubation, cells exposed to TPA exhibited increased PG synthase activity in whole homogenates. TPA caused a smaller (2-fold) increase in P accumulation than was observed with LH (10-fold). When tested in combination, however, TPA decreased the P response to LH by approximately 25%. These effects of TPA on basal and LH-stimulated PG and P accumulation were very similar to the actions of GnRH. We, therefore, investigated the effect of exposure to the combination of GnRH and TPA. A GnRH agonist, [D-Ala6,des-Gly-NH2(10)] GnRH ethylamide (GnRHa; 10 ng/ml) caused a 4-fold increase in PGE accumulation. The effect of TPA on PGE accumulation was also additive to that of GnRHa. TPA, on the other hand, did not affect the 2.5-fold P response to GnRHa. Neither stimulation or inhibition of PGE or P accumulation was observed in the presence of a nontumor-promoting phorbol ester. Furthermore, TPA did not affect basal or LH-stimulated cAMP accumulation or basal or LH-stimulated protein kinase A activity. These data indicate that protein kinase C activation can influence granulosa cell PG and P accumulation.

Animals↗