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Biomedical subjects

A Sheldon

Publications and source records attributed to A Sheldon.

At least 19 recordsLinked to original sources
PubMed1995

Three-dimensional energy-minimized model of human type II "Smith" collagen microfibril.

A procedure is described for constructing a three-dimensional model of fibril-forming human type II collagen based on the "Smith" microfibril model. This model is a complex of five individual collagen triple-helical molecules, and is based on known structural parameters for collagen. Both experimental and theoretical data were used as constraints to guide the modeling. The resulting fibril model for type II collagen is in agreement with both physical and chemical characteristics produced by experimental staining patterns of type II fibrils. Some advantages of the type II model are that the stereochemistry of all the sidechain groups is accounted for, and specific atomic interactions can now be studied. This model is useful for: development of therapeutics for collagen related diseases; development of synthetic collagen tissues; design of chemical reagents (i.e., tanning agents) to treat collagen-related products; and study of the structural and functional aspects of type II collagen. Described is the procedure by which the Smith microfibril of type II collagen was developed using molecular modeling tools, validation of the model by comparison to electron-microscopic images of fibril staining patterns, and some applications of this microfibril model.

Amino Acid Sequence↗
PubMed1995

Effects of stromelysin activity on proteoglycan degradation of canine articular cartilage explants.

We investigated whether stromelysin activity in the medium of canine articular cartilage explants is associated with proteoglycan degradation in these explants. Cartilage explants were treated with recombinant human interleukin 1 alpha (rh-IL-1 alpha), lipopolysaccharide, or canine monocyte-conditioned medium. Proteoglycan synthesis and degradation were measured. Metalloproteinase activity (inhibitable by tissue inhibitor of metalloproteinase 2) in the culture medium was measured by use of fluorimetry with a quenched fluorescent substrate. Western blots of the medium were probed with polyclonal antibodies to human stromelysin, collagenase, and gelatinase. Neither metalloproteinase activity nor proteoglycan degradation were inducible in canine cartilage explants treated with rh-IL-1 alpha. However, proteoglycan synthesis was significantly (P < 0.05) decreased by concentrations of 10 and 100 ng of rh-IL-1 alpha/ml. Metalloproteinase activity in the medium accompanied proteoglycan degradation of cartilage treated with lipopolysaccharide and monocyte-conditioned medium. The metalloproteinase released into the medium was identified as prostromelysin by results of western blotting.

Animals↗
PubMed1995

Interleukin-4, an inhibitor of cartilage breakdown in bovine articular cartilage explants.

OBJECTIVE: To determine the ability of interleukin-4 (IL-4) to inhibit the degradation of proteoglycan in bovine articular cartilage explants stimulated by human interleukin-1 (IL-1 alpha), tumor necrosis factor (TNF-alpha), a combination of TNF-alpha and IL-1 alpha, and lipopolysaccharide (LPS). METHODS: 35SO4 radiolabelled bovine radiocarpal cartilage explants were treated with IL-1 alpha, TNF-alpha, TNF-alpha plus IL-1 alpha, or LPS, plus various concentrations of IL-4 for 72 h. Proteoglycan released to the media was analyzed by scintillation counting and composite gel electrophoresis. Media samples were also analyzed by Western immunoblotting for metalloproteinases and TIMP. RESULTS: IL-4 significantly reduced the cartilage proteoglycan degradation induced by IL-1 alpha, TNF-alpha, TNF-alpha plus IL-1 alpha, or LPS (50% inhibitory concentration, IC50 for IL-4 ranged from about 15 to 50 ng/ml). Western blotting showed that media stromelysin levels were increased by IL-1 alpha, TNF-alpha, and LPS, but that IL-4 had no observable effect. Composite gel electrophoresis demonstrated quantitative and qualitative differences in proteoglycan degradation after IL-4 treatment. CONCLUSION: IL-4 has a potent inhibitory effect on cartilage degradation after stimulation with IL-1 alpha, TNF-alpha, TNF-alpha plus IL-1 alpha, or LPS. These results suggest that IL-4 should be investigated further for therapeutic value as a chondroprotective agent for the treatment of arthritis.

Animals↗
PubMed1993

Coexpression of IL-2 receptor p55 and p75 by circulating blood lymphocytes.

By using high-sensitivity fluorescence and flow cytometry, it is possible to show that 30-40% of lymphocytes from PBL express the p55 chain of the IL-2 receptor, whereas the p75 chain is expressed at low concentrations on most lymphocytes without in vitro activation. The availability of a second fluorochrome capable of high sensitivity allows simultaneous analysis of p55 and p75, albeit with some sacrifice in sensitivity. Two-color analysis shows that a small proportion of cells (1-6%) coexpress measurable concentrations of both chains of the IL-2 receptor, and three-color studies show that these cells are predominantly CD4-positive T cells and express the CD45R0 isoform of the leucocyte-common antigen, i.e., have the phenotype of activated helper T cells. These cells may be a useful indicator of immune activation.

Antibodies, Monoclonal↗
PubMed1993

Structure-function correlations of calcium binding and calcium channel activities based on 3-dimensional models of human annexins I, II, III, V and VII.

The annexins are a family of calcium-dependent phospholipid-binding proteins which share a high degree of primary sequence similarity. Using a model of the crystal structure of annexin V as a template, 3-dimensional models of human annexins I, II, III and VII were constructed by homology modeling (J. Greer, J. Mol. Biol. 153, 1027-1042, 1981; J.M. Chen, G. Lee, R.B. Murphy, R.P. Carty, P.W. Brant-Rauf, E. Friedman and M.R. Pincus, J. Biomolec. Str. Dyn. 6, 859-87, 1989) for the 316 amino acid portions corresponding to the annexin V structure published by Huber et al. (J. Mol. Biol. 223, 683-704, 1992). These methods were used to study structure-function correlations for calcium ion binding and calcium channel activity. Published experimental data are specifically shown to be consistent with the annexin models. Possible intramolecular disulfide bridges were identified in annexin I (between Cys297 and Cys316) and in annexins II and VII (between Cys115 and Cys243). Each of the annexin models have 3 postulated calcium binding sites, usually via a Gly-Xxx-Gly-Thr loop with an acidic Glu or Asp residue 42 positions C-terminal to the first Gly. Despite a nonconserved binding site sequence, annexins I and II are able to coordinate calcium in domain 3 since the residue in the second loop position is directed toward the solvent away from the binding pocket. This finding also suggests a mechanism for a conformational change upon binding calcium. Highly conserved Arg and acidic sidechains stabilize the channel pore structure; annexin channels probably exist in a closed state normally. Arg271 may be involved in channel opening upon activation: basic residue 254 can stabilize Glu112, which allows Arg271 to interact with residue 95 instead of Glu112. Residue 267, found on the convex surface at the pore opening, may also be important in modifying channel activity.

Amino Acid Sequence↗
PubMed1993

Anxious attachment as a determinant of adult psychopathology.

Traditionally, an excess of interpersonal dependency has been used to characterize disturbed interpersonal relationships in adults. This approach stands in sharp contrast to that of attachment theorists who conceptualize attachment as functionally distinct from dependency. Attachment theory focuses on anxious attachment in the definition and dynamics of disturbed adult interpersonal relationships. The purpose of this study was to examine the relevance of anxious attachment to the differentiation of psychiatric outpatients from nonpatients. Empirically, three scales (feared loss of the attachment figure, proximity seeking, and separation protest) measure the components of anxious attachment as defined by Bowlby. The scales were administered to two research samples. Sample 1 was composed of 136 respondents to a survey within the Calgary community. Sample 2 consisted of 110 psychiatric outpatients drawn from the psychotherapy clinic of the Calgary General Hospital. The results indicate that the three components of anxious attachment offer a clinically relevant system for differentiating between psychiatric outpatients and nonpatients. Of these three components, feared loss has the predominant effect. The implications of this finding for the delineation of disturbed interpersonal relationships in adults is discussed.

Adult↗
PubMed1993

cAMP levels in fast- and slow-twitch skeletal muscle after an acute bout of aerobic exercise.

The present study examined whether exercise duration was associated with elevated and/or sustained elevations of postexercise adenosine 3',5'-cyclic monophosphate (cAMP) by measuring cAMP levels in skeletal muscle for up to 4 h after acute exercise bouts of durations that are known to either produce (60 min) or not produce (10 min) mitochondrial proliferation after chronic training. Treadmill-acclimatized, but untrained, rats were run at 22 m/min for 0 (control), 10, or 60 min and were killed at various postexercise (0, 0.5, 1, 2, and 4 h) time points. Fast-twitch white and red (quadriceps) and slow-twitch (soleus) muscles were quickly excised, frozen in liquid nitrogen, and assayed for cAMP with a commercial kit. Unexpectedly, cAMP contents in all three muscles were similar to control (nonexercise) at most (21 of 30) time points after a single 10- or 60-min run. Values at 9 of 30 time points were significantly different from control (P < 0.05); i.e., 3 time points were significantly higher than control and 6 were significantly less than control. These data suggest that the cAMP concentration of untrained skeletal muscle after a single bout of endurance-type exercise is not, by itself, associated with exercise duration.

Animals↗
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↗