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Biomedical subjects

S A Morris

Publications and source records attributed to S A Morris.

At least 91 records · Page 5Linked to original sources
PubMed1988

Interactions of spectrin in hereditary elliptocytes containing truncated spectrin beta-chains.

An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up approximately 30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of beta-type, using monoclonal antibodies. Because a univalent terminal spectrin alpha-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (alpha beta') would readily enter into an association with normal (alpha beta) dimers to give alpha 2 beta beta' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by alpha beta' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of alpha beta' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the beta-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of alpha beta' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of alpha beta and alpha beta' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30 degrees C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.

Elliptocytosis, Hereditary↗
PubMed1988

Rapid purification of clathrin-coated vesicles by free-flow electrophoresis.

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.

Animals↗
PubMed1987

The influence of EDTA on adenylate cyclase activity in membranes from rat and mouse myocardium.

Inclusion of EDTA in the homogenization buffer of both mouse and rat myocardium profoundly alters the properties of the adenylate cyclase complex. EDTA leads to an increase in the Vmax for adenylate cyclase activity due to all of the following agents: isoproterenol, Gpp[NH]p, forskolin and Mg2+. For forskolin and Mg2+, the EDTA-associated increase in Vmax is not accompanied by a change in sensitivity to activation. However, EDTA is associated with enhanced sensitivity to activation by isoproterenol and increased sensitivity to the effect of Mg2+ on isoproterenol-dependent adenylate cyclase activity. A result of greater isoproterenol-dependent adenylate cyclase activity, due to the presence of EDTA, is an attenuated synergistic contribution of Gpp[NH]p. Changes in stimulatable adenylate cyclase activity as a result of EDTA occurs in concert with effects of cholera toxin upon ADPribosylation of the guanine nucleotide regulatory protein, Ns. Substantial auto-ADP-ribosylation occurs in a cholera toxin-sensitive 42 kDa band in membranes prepared in the presence of EDTA. In addition, cofactor and substrate requirements in the cholera toxin-dependent ADP-ribosylation reaction depend on the method of membrane preparation. The results suggest that the integrity of the adenylate cyclase complex depends in part on the attention given to proteolysis that may be activated during the course of homogenization.

Adenosine Diphosphate Ribose↗
PubMed1987

Infection of L6E9 myoblasts with Trypanosoma cruzi alters adenylate cyclase activity and guanine nucleotide binding proteins.

We studied the consequences of infection of L6E9 myoblasts with T. cruzi on the adenylate cyclase complex to test the hypothesis that infection alters the functional properties of the guanine nucleotide regulatory proteins, Ns and Ni. Stimulating activities of adenylate cyclase due to isoproterenol, isoproterenol plus Gpp(NH)p, or forskolin (activities mediated by Ns) are not altered by infection. However, inhibitory activities mediated by Ni [Gpp(NH)p, acetylcholine, and adenosine inhibition of forskolin-dependent adenylate cyclase activity] are compromised by infection. The reduction in adenosine's inhibition of forskolin-dependent adenylate cyclase activity is seen throughout the effective concentration range of adenosine. Pertussis toxin does not change basal or stimulated adenylate cyclase activity in infected cells compared with normal uninfected cells, nor does it alter the inhibiting action of adenosine. To evaluate the coupling proteins (Ns and Ni) involved in the stimulation and inhibition of adenylate cyclase more directly, cholera- and pertussis-toxin-dependent ADP ribosylation studies were performed. The incorporation of [32P]ADP ribose in the presence (specific) or absence (nonspecific) of the toxins was markedly decreased in membranes prepared from infected cells. However, in membranes prepared from infected or uninfected cells previously treated with pertussis toxin, there was a significant reduction in specific pertussis-toxin dependent ADP ribosylation. The infection-associated diminution in toxin-dependent ADP ribosylation complements the impaired inhibition of adenylate cyclase data. Collectively, the data further substantiate an infection-associated alteration in the adenylate cyclase complex, probably at the level of the guanine nucleotide binding proteins.

Acetylcholine↗
PubMed1987

Specificity of sperm-binding Wolffian duct proteins in the rooster and their persistence on spermatozoa in the female host glands.

Unlike those of mammals, chicken spermatozoa can develop their fertilizing ability before they leave the testis to pass into the Wolffian duct; moreover, chicken spermatozoa do not require a period of capacitation in the female tract. A question arises, therefore, as to the significance of secretory proteins shown to bind to the surface of chicken spermatozoa as they pass into and through the Wolffian duct. Using anti-Wolffian duct fluid IgG as a probe visualized by immunoperoxidase staining, the present investigation confirms that testicular spermatozoa of quail and turkey as well as chicken do not have any surface determinants in common with those present in Wolffian duct secretions. By contrast, those in the lower portion of the vas deferens display a strong reaction with anti-Wolffian fluid IgG over their entire surface, and immunoprecipitation studies suggest that this reflects the binding of four Wolffian duct proteins. Since a reaction to the antichicken fluid IgG is shown also by mature quail and turkey, but not duck and pigeon spermatozoa, the Wolffian components that coat spermatozoa in birds appear to have a specificity confined to the same order, in this case the Galliformes. Following vaginal or intramagnal insemination, spermatozoa present 48 hours later in the uterovaginal host glands and infundibulum glands, respectively, still reacted strongly. This finding that Wolffian duct components persist on the surface of spermatozoa in the female tract is consistent with the possibility that they have some role in sperm storage or survival in female birds.

Animals↗
PubMed1987

Stomatocytosis, abnormal platelets and pseudo-homozygous hypercholesterolaemia.

A 13-yr-old girl with congenital haemolytic anaemia associated with pseudo-homozygous hypercholesterolaemia is described. The erythrocyte morphology showed 50-80% stomatocytes, but no abnormality of membrane lipid or protein composition or of cation transport was detected. The platelets were reduced in number, abnormally large and showed reduced adhesion. Successful treatment of the hypercholesterolaemia did not influence the stomatocytosis.

Adolescent↗
PubMed1986

Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts.

Alterations in receptor-independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3, 6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low-and high-affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low- and high-affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p-dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 microM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera- and pertussis toxin-dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.

Adenosine Diphosphate Ribose↗
PubMed1986

Prenatal diagnosis of hereditary red cell membrane defect.

The red cells of a severely anaemic 2-year-old child of a white British family showed high haemolytic fragility with poikilocytosis. The cells showed markedly impaired thermal stability. The mother was phenotypically normal, but the father's red cells showed mild elliptocytosis. The spectrin from the latter, extracted at low temperature, was 30% dimeric (cf. 5-10% in normals). Tryptic digests of the spectrin from both father and daughter showed a reduction in the fragment of 80,000 molecular weight, derived from the terminus of the alpha-chain, and the elevation of a fragment of molecular weight 46,000, as well as one of 53,000. These characteristics and the autosomal recessive inheritance lead to a diagnosis of type II hereditary pyropoikilocytosis, so far reported only in two black American families (Lawler et al, 1983). The spectrin from the father was examined with respect to thermal conformational stability, and was found to be normal. The spectrin from the cells of the daughter gave evidence of the presence of oxidative (disulphide) cross-links, as well as of extensive noncovalent aggregation. Blood was obtained from the umbilical cord vein of the 19-week fetus of the pregnant mother: 250 microliters of blood was used for preparation of red cell membranes for SDS-gel electrophoresis and for extraction of spectrin. Analysis of the spectrin by gel electrophoresis in the native state revealed that the proportion of dimer was within the normal range, and the fetus therefore did not possess the hereditary pyropoikilocytosis phenotype. It is suggested that the procedures described could be generally applied to the prenatal identification of phenotypes associated with severe haemolytic anaemias.

Anemia, Hemolytic, Congenital↗
PubMed1985

The regulation of adenylate cyclase activity in turkey erythrocyte membranes by forskolin.

The mechanisms by which forskolin stimulates adenylate cyclase activity in turkey erythrocyte membranes and is influenced by manganese and Gpp(NH)p were studied. Forskolin-dependent adenylate cyclase activity in particulate turkey erythrocyte membranes is enhanced following preincubation of membranes with isoproterenol and GMP (cleared membranes). In contrast, solubilization of turkey erythrocyte membranes, previously cleared, renders them relatively refractory to forskolin but not to Gpp(NH)p. Whereas adenylate cyclase activity due to the simultaneous presence of forskolin and Mn2+ in particulate turkey erythrocyte membranes is additive, their copresence becomes synergistic after solubilization. The apparent Kact for forskolin activation of adenylate cyclase is not influenced by clearance or by the presence of Mn2+ in particulate turkey erythrocyte membranes. Following solubilization, the Vmax for forskolin-dependent adenylate cyclase activation determined in the presence of Mn2+ is also independent of clearance. Forskolin activation of turkey erythrocyte adenylate cyclase appears to be influenced at sites in addition to the catalytic unit.

Adenylyl Cyclases↗
PubMed1984

Alteration of the pattern of beta-adrenergic desensitization in cultured L6E9 muscle cells infected with Trypanosoma cruzi.

L6E9 myoblasts infected with Trypanosoma cruzi undergo desensitization to beta-adrenergic catecholamines in a manner distinct from uninfected control myoblasts. Following incubation of intact cells with isoproterenol for 2 h, homogenates prepared from differentiated, high density uninfected L6E9 cells retain isoproterenol-dependent adenylate cyclase activity. In addition, previous exposure to isoproterenol is accompanied by a decrease in the number of beta-adrenergic receptors. Homogenates of high density L6E9 cells infected with T. cruzi retain their adenylate cyclase responsivity to isoproterenol but demonstrate a marked decrease in beta-adrenergic receptors. Following desensitization infected cell homogenates lose their responsiveness to isoproterenol and demonstrate a more marked decrease in beta-receptors. There does not appear to be any effect of T. cruzi infection on affinity of beta-adrenergic agonists for the beta-receptor or on changes in agonist affinity associated with desensitization. Infection of low density undifferentiated cells results in no apparent change in adenylate cyclase activity or in beta-receptors. Their behavior in the setting of desensitization--decreased whole cell cyclic AMP, decreased adenylate cyclase, unchanged beta-receptors--is also not affected by infection. The pattern of desensitization to beta-adrenergic agonists in high density infected cells shares several properties with the pattern of desensitization in low density uninfected cells, suggesting that infection may be associated with part of the more primitive cellular response pattern.

Adenylyl Cyclases↗
PubMed1983

Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site.

The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.

Adenylyl Cyclases↗
PubMed1983

The use of [125I]iodocyanopindolol as a specific probe for beta-adrenergic receptors in differentiating cultured rat skeletal muscle.

In order to examine more precisely the role of beta-adrenergic receptors in the process of differentiation we used the new radioligand iodocyanopindolol ([125I]ICYP), which we found to be a very useful probe to identify beta receptors. Binding characteristics conformed to those expected for a physiologically relevant beta receptor. L6E9 cells grown in horse serum, which allows differentiation, exhibit increased beta receptor density in intact cells as a function of age. In contrast, cells grown in fetal calf serum, which does not allow differentiation, exhibit constant beta receptor density. In broken cells, however, both differentiating and non-differentiating cells show an increase in beta receptors. These results suggest that the process of differentiation is associated with an unmasking of beta receptors which are increasing but cryptic in undifferentiated cells.

Adenylyl Cyclases↗
PubMed1982

Conditions associated with the appearance of guanine nucleotide-dependent adenylate cyclase activity in turkey erythrocyte membranes.

The mechanisms involved in the activation of adenylate cyclase in turkey erythrocyte membranes may include the release of inhibitory nucleotides from the guanine nucleotide regulatory protein (N) and the subsequent formation of an active GTP-N complex. We have studied the conditions required for the appearance of guanine nucleotide-dependent adenylate cyclase activity in turkey erythrocyte membranes in an effort to understand further the mechanisms involved in its activation. Turkey erythrocyte membranes, whose adenylate cyclase enzyme is usually poorly responsive to the direct actions of GTP or its analogue, Gpp(NH)p, became markedly responsive after previous exposure to isoproterenol in a sucrose-containing buffer. Without sucrose, isoproterenol alone did not lead to Gpp(NH)p responsiveness. Maximum expression of Gpp(NH)p-dependent adenylate cyclase activity required prior incubation with both GMP and isoproterenol. The requirement for sucrose when isoproterenol was used without GMP was lost when GMP was present. With or without GMP, isoproterenol induced a half-maximum effect after 5 min at 37 degrees. The development and maintenance of Gpp(NH)p-dependent adenylate cyclase activity was mediated, in part, by beta-adrenergic receptors. Following a 10-min incubation period with isoproterenol and GMP, the addition of the beta-adrenergic inhibitor, propranolol, for 4 additional min completely reversed the stimulation produced by isoproterenol and GMP. In addition, high-affinity agonist binding to beta-receptors was necessary, but not sufficient, for the development of Gpp(NH)p responsiveness.

Adenylyl Cyclases↗
PubMed1978

In vivo smooth muscle function in normal and obstructed rabbit vas deferens.

We characterized smooth muscle function in the rabbit vas deferens in vivo by means of a new strainometer. Intraluminal pressure was measured and smooth muscle tension in the wall was calculated. Our studies revealed a low baseline pressure, no spontaneous contractions, and a constant wall tension. Studies of the effect of vasectomy on in vivo smooth muscle function showed increased intraluminal pressure, diameter, and baseline wall tension, and development of spontaneous contractions with increased wall tension, intraluminal pressure, and diameter with each contraction.

Animals↗
PubMed1978

Response of the rabbit vas deferens in vivo to vasoactive drugs.

We evaluated the smooth muscle response to the systemic administration of various vasoactive drugs and hormones in vivo in the rabbit vas deferens. Norepinephrine, epinephrine, and urecholine caused contractions of the vas deferens that caused it to shorten and thicken. Isuprel inhibited contractions that were initiated by obstruction of the vas deferens. Catecholamine depletion from guanethidine caused dilation of the vas deferens, increased intraluminal pressures spontaneous contractions, and a spermpacked lumen. The vas deferens in the immature rabbit showed normal response to norepinephrine and obstruction. Estrogen administration prevented the normal dilation and increased peristalsis observed with obstruction.

Animals↗
PubMed1977

Renal plasmacytoma.

Plasmacytoma, a localized extramedullary mass of plasma cells, is one manifestation of multiple myeloma. Renal plasmacytomas are rare lesions with neovascularity on arteriogram. This may cause confusion with the much more common renal cell carcinoma unless the clinician is aware of this entity. A case is presented, the literature reviewed, and problems in management discussed.

Adenocarcinoma↗