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A Sheldon

Publications and source records attributed to A Sheldon.

At least 19 recordsLinked to original sources
PubMed1992

Detection of cytokine receptors by high-sensitivity immunofluorescence/flow cytometry.

Cytokines have profound effects on cells, and act through receptors which need only be at low concentrations (around 100 copies per cell) to transmit activation signals. The detection of such low concentrations is possible using monoclonal antibodies and fluorescence/flow cytometry, but only by using specialized techniques. The best results so far have been obtained using biotinylated second antibody followed by phycoerythrin-streptavidin, and batches of these reagents have to be carefully selected. Analysis of the fluorescence is best done using 546 nm excitation from a mercury arc lamp, but 512 nm excitation from an argon-ion laser can also be used. With appropriate alignment, instruments with 488 nm fixed-wavelength lasers can give sensitivity almost as good as the 546 nm system. Working at high sensitivity, background levels also increase, particularly for B lymphocytes. Background staining can be reduced to acceptable levels by blocking the two major mechanisms for non-specific binding. Applications of these methods to the detection of cytokine receptors on normal and malignant cells are reviewed.

Animals↗
PubMed1991

Annexin VI is associated with calcium-sequestering organelles.

Annexin VI is a member of a Ca(2+)-dependent, phospholipid-binding protein family. Although functions for this annexin have been proposed from in vitro studies, most remain controversial. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) demonstrated that annexin VI modified, in a Ca(2+)-dependent manner, the gating behavior of the sarcoplasmic reticulum Ca(2+)-release channel, reconstituted into artificial bilayers, by increasing both the open probability and the mean open time. This effect was specific to the trans chamber, which represents the luminal side of the sarcoplasmic reticulum. In agreement with those findings, we show herein that annexin VI produced no effect on Ca(2+)-uptake or -release by intact heavy sarcoplasmic reticulum vesicles (analogous to the cis chamber). We also used monospecific antibodies to evaluate the subcellular localization of annexin VI by immunofluorescent microscopy. Studies in rat skeletal muscle suggest that annexin VI is present surrounding individual myofibrils. Double immunolocalization studies with cultured muscle cells (chick myotubes) using anti-annexin VI and anti-SR Ca(2+)-ATPase antibodies demonstrated superimposable staining patterns. In non-muscle tissue (normal rat kidney (NRK) cells), a punctate, perinuclear anti-annexin VI staining pattern was observed. Collectively, these data suggest that annexin VI may play a regulatory role in the Ca(2+)-release/uptake cycle in the sarcoplasmic reticulum as well as in non-muscle organelles, a key process in stimulus-response systems.

Animals↗
PubMed1991

Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI.

Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.

Amino Acid Sequence↗
PubMed1990

Purification of the microsomal Ca2(+)-ATPase from rat liver.

The Ca2(+)-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficoll-sucrose treatment, column chromatography with agarose-hexane adenosine 5'-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca2(+)-ATPase was stable for at least two weeks when stored at -70 degrees C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca2(+)-ATPase. Further characterization of the ER Ca2(+)-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca2(+)-ATPase cross-reacted with the purified Ca2(+)-ATPase from rat liver ER membranes.

Animals↗
PubMed1989

Attachment theory and brief psychotherapy: applying current research to clinical interventions.

Recent advances in attachment research provide a framework for defining the content and process of brief psychotherapy with adults. Attachment theory emphasizes a number of issues crucial to therapeutic work. Specifically, attachment relationships are enduring components of a individual's pattern of interpersonal behaviours. Functionally, attachment relationships address security needs. Insecure attachment arises from a representational model based on feared loss of the attachment figure, which predisposes the individual to have little confidence in the attachment figure's availability, responsiveness, and permanence. Behavioural responses to insecure attachment can lead to specific patterns of interpersonal relationships which, in turn, strengthen the representational model. Thus, a relatively stable, self-reinforcing system evolves and results in a consistent inability to experience security within attachment relationships. In this article, the authors describe the current framework for understanding adult attachment relationships and present clinical vignettes illustrating the saliency of attachment theory to common clinical presentations. The goal of the clinical intervention is defined as increasing the "permeability" of the individual's working model of attachment through affective and cognitive re-assessment of attachment experiences and expectations. Finally, the utility of this approach in evaluating the outcome and effectiveness of psychotherapy is highlighted.

Adult↗
PubMed1988

Calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobe.

We have studied the calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobes: one, squid calmodulin (SCaM), similar to bovine brain calmodulin (BCaM), the other, squid calcium-binding protein (SCaBP), distinct (Head, J.F., Spielberg, S., and Kaminer, B. (1983) Biochem J. 209, 797-802). Equilibrium dialysis measurements on the squid proteins (and BCaM) were made at 100 mM KCl in the presence and absence of 3 mM Mg2+, and at 400 mM KCl in the presence of 3 mM Mg2+, which more closely resembles the conditions in the squid. SCaM, SCaBP, and BCaM each bind a maximum of 4 Ca2+ ions/molecule of protein under the ionic conditions tested. SCaBP has a higher affinity than SCaM or BCaM for Ca2+ at 100 mM KCl in the absence of Mg2+. However, in the presence of Mg2+, half-maximal binding to SCaBP occurs at a similar pCa value to that observed with calmodulin. Increasing the KCl concentration reduces the affinity of all three proteins for Ca2+. UV absorption measurements showed that the binding of 4 Ca2+ ions/molecule is necessary to complete spectral changes in SCaBP, compared to two for the calmodulins. While Ca2+ causes perturbations in aromatic chromophores in SCaM and SCaBP, Mg2+ causes a significant perturbation only in SCaBP. These Mg2+-induced changes differ qualitatively from those induced by Ca2+.

Animals↗
PubMed1987

Diversity in smooth muscle thin filament composition.

Two classes of smooth muscle thin filament can be identified and separated based on their interaction with antibodies specific either to filamin or to caldesmon. One type is composed of actin, tropomyosin and filamin and the other of actin, tropomyosin and caldesmon.

Actins↗
PubMed1987

An approach to the delineation of adult attachment. Scale development and reliability.

The authors present the results of a project to develop scales to measure the features of adult attachment using a construct-oriented approach. Adult attachment is defined in terms of eight features: proximity seeking, secure base effect, separation protest, feared loss of the attachment figure, reciprocity, availability, responsiveness of the attachment figure, and use of the attachment figure. Each feature was defined and a large item pool to tap all dimensions was derived from item nominations by clinicians, researchers, and lay subjects. The resultant scales were administered to a small sample for preliminary empirical testing. Analyses of internal reliability for each scale resulted in the elimination of items that detracted from reliability. After editing, all scales had satisfactory internal consistency. A discriminant-functions analysis provided suggestive evidence that this method of defining and measuring adult attachment would have relevance for identifying psychopathology.

Adult↗
PubMed1986

The place of attachment in the life events model of stress and illness.

Recent evidence indicates that perceived adequacy of support has substantial predictive power for the subsequent onset of minor psychiatric symptoms. Such findings suggest that social support is not purely a property of the environment, but is also a function of personality. The problem, however, with a re-emphasis on personality variables is that any one of scores of dimensions can be chosen for study. This paper argues that precision may be gained by conceptualizing personality variables in terms of basic behavioral systems that have been extensively investigated in ethological and developmental studies. One such basic system is attachment. The present paper discusses the relevance of attachment theory for understanding susceptibility to stress and individual differences in the use of social support during times of stress.

Adaptation, Psychological↗
PubMed1973

Friendly models.

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Game Theory↗