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Biomedical subjects

R D Lane

Publications and source records attributed to R D Lane.

At least 55 records · Page 3Linked to original sources
PubMed1990

Successful fluoxetine treatment of pathologic jealousy.

A 39-year-old man suddenly became convinced that his wife had once been unfaithful to him, and soon thereafter he became depressed. Symptoms of depression responded to imipramine, but the jealousy did not. A trial of fluoxetine was attempted because of the obsessional nature of the jealousy. Fluoxetine 60 mg/day resulted in complete remission of the pathologic jealousy, consistent with the known efficacy of fluoxetine in obsessive compulsive disorder.

Adult↗
PubMed1989

The binding of large calpastatin to biologic membranes is mediated in part by interaction of an amino terminal region with acidic phospholipids.

Animal cells contain a non-lysosomal proteolytic system which degrades various protein substrates in the presence of calcium ion. The calcium-dependent proteinases (calpains) co-exist in cells with a specific protein inhibitor called calpastatin. Distribution of this inhibitor to different subcellular sites could be important in overall regulation of the calpains. Previously, myocardial calpastatin was shown to be present in preparations of sarcoplasmic reticulum and sarcolemma. In the present work, we show that purified bovine myocardial calpastatin binds to the acidic phospholipids, phosphatidylinositol and phosphatidylserine, but not to the neutral phospholipids, phosphatidylcholine and phosphatidylethanolamine. Large forms of calpastatin from canine myocardium and rabbit liver also were bound to phosphatidylinositol. Smaller forms of calpastatin present in the preparations did not bind to acidic phospholipids. Bovine large calpastatin was subjected to CNBr digestion, and a phospholipid-binding fragment representing approximately one-sixth of the intact protein mass was purified. Amino acid sequence analysis indicated that the phospholipid-binding fragment was derived from the amino terminus of the large calpastatin.

Amino Acid Sequence↗
PubMed1989

The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.

This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.

Animals↗
PubMed1989

Interhemispheric transfer deficit and alexithymia.

The hypothesis that alexithymia reflects a functional disconnection between the two cerebral hemispheres was examined in 25 male combat veterans with posttraumatic stress disorder (PTSD). Multiple regression analysis revealed that the efficiency of interhemispheric communication, evaluated with a tactile finger localization task, was a significant predictor of the degree of alexithymia, as measured by the Toronto Alexithymia Scale, independent of PTSD severity and IQ. The PTSD subjects without alexithymia did not differ on the finger localization task from 10 male control subjects without alexithymia. These data suggest that at least some forms of alexithymia may be mediated by an interhemispheric communication deficit.

Adult↗
PubMed1989

Differential effects of right unilateral and bilateral ECT on heart rate.

In a series of 11 melancholic patients who received both right unilateral and bilateral ECT during a single course of treatment, mean and minimum postictal heart rates were greater after bilateral ECT. Greater catecholamine release during bilateral ECT is the most likely explanation for these effects. These differences complement other evidence that bilateral ECT has a more pronounced physiological impact than unilateral ECT.

Catecholamines↗
PubMed1989

Subcellular localization of glycogen synthase with monoclonal antibodies.

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.

Amylases↗
PubMed1989

The non-lysosomal, calcium-dependent proteolytic system of mammalian cells.

The intracellular calcium-dependent proteases (calpains) and their endogenous protein inhibitor (calpastatin) are present in many different mammalian cells. There is emerging evidence for their importance in the turnover of membrane-associated proteins. Accordingly, it is important to understand how these proteinases and their inhibitor interact within cells, in particular at membranes. Bovine myocardial calpastatin appears to be associated in part with intracellular membranes, where it may effectively block the activity of calpain II on membrane-associated proteins. Immuno-electron microscopic studies suggest that canine myocardial calpain and calpastatin are associated with a number of membranous organelles. During canine myocardial autolysis, the amount of calpain at various organelles decreased, but the amount of calpastatin decreased to an even greater extent. Thus there may be a high calpain to calpastatin balance during heart ischemia at these sites. Calpain II aggregation may contribute to localization of the proteinase at sites of high calcium concentration within cells. A model is presented for interaction of calpain II and calpastatin at cellular membranes in the presence of calcium.

Amino Acid Sequence↗
PubMed1988

Myocardial calpain 2 is inhibited by monoclonal antibodies specific for the small, noncatalytic subunit.

Calpains (EC 3.4.22.17) are nonlysosomal intracellular proteinases which require calcium ion for activity. The calpains are heterodimers composed of a large catalytic subunit and a small subunit which may have a regulatory function during the catalytic cycle. However, whether calpains remain in the dimeric form or dissociate upon exposure to calcium is controversial. To resolve this issue, two monoclonal antibodies which specifically recognize the small calpain subunit were prepared using bovine calpain 2 heterodimer as the antigen. Both antibodies, designated P-1 and P-2, were capable of inhibiting bovine or canine calpain 2, and partially purified human erythrocyte calpain 1. However, neither could produce full inhibition. Further studies with P-1 and bovine calpain 2 indicated that the antibody decreased the calcium requirement for the proteinase. The Km for casein was increased and the Vmax was decreased. The addition of P-1 to the assay mixture several minutes after initiation of proteolytic activity resulted in a rapid inhibition. The P-1 antibody was also capable of decreasing the ability of the protein inhibitor of calpains (calpastatin) to inhibit bovine calpain 2. These studies indicate that the small subunit remains bound to the large subunit during catalysis and may influence its activity.

Animals↗
PubMed1988

An improved purification procedure for calpastatin, the inhibitor protein specific for the intracellular calcium-dependent proteinases, calpains.

The specific inhibitor protein (calpastatin) for the calcium-dependent intracellular proteinases (calpains) is an important regulator of these enzymes. In this communication we describe a one day procedure for purifying 3 to 5 mg of calpastatin from a kilogram of bovine myocardium. This represents a substantial improvement over previously described methods, and should facilitate future studies of calpastatin structure and function. A key, novel step in the purification was dye-matrix chromatography on an Affi-Gel Blue column. Contrary to previous indications, calpastatin purified by the new method did not contain significant amounts of carbohydrate. However, the presence of covalently bound phosphate in purified bovine myocardial calpastatin was confirmed and co-migration of phosphate and calpastatin activity was demonstrated on Bio-Gel A-1.5m chromatography. Thus, it is possible that calpastatin function is regulated by phosphorylation.

Animals↗
PubMed1988

The influence of stimulated peritoneal feeder cells and mitogens upon antibody secreting hybridomas.

Peritoneal exudate cells from mice injected with immunostimulatory agents were evaluated for their ability to promote hybridoma growth. Peritoneal cells from mice receiving peritoneal injections of either Freund's incomplete adjuvant or pristane, seven days prior to harvesting, produced the greatest number of antibody-producing hybridomas. Freund's incomplete adjuvant produced 16 fold more peritoneal cells than unstimulated mice, thus reducing the number of mice needed to supply feeder cells for the hybridoma cultures. In separate experiments a number of B-lymphocyte stimulating lectins and factors were tested for their ability to promote hybridoma growth. 2-mercaptoethanol (25 microM) routinely increased the number of antibody producing hybridomas by 5 to 15 fold. 2-mercaptoethanol had a varying ability to increase the numbers of hybridoma colonies. The cloning efficiency, rate of cell growth and antibody production of hybridoma cell lines, previously produced in the absence of 2-mercaptoethanol could also be increased when this reducing agent was added to the culture medium.

Animals↗
PubMed1988

An explant culture system for the study of atrial development.

An explant culture system for the study of chick atrial development was tested using media of varying composition. Right atria from chick embryos of 13 days incubation were excised, placed on stainless steel mesh grids and cultured for 16-21 days in supplemented media. The results indicated that DMEM supplemented with 15% fetal bovine serum maintained the highest percentage (57%) of contractile atria in culture. Even atria cultured in serum free DMEM gradually adapted and became completely contractile over a 15 day period. Gel electrophoresis was performed on NP-40 detergent extracts of contracting explants cultured for 1, 4, 6, 8, and 10 days, and from 13, 15, 16, 18, 20 day embryos and 1 day old chicks. The percent of total protein of five separate protein bands in the range of 17,000 to 60,000 Daltons were plotted against the days in vivo/in vitro. There was a dramatic change in the protein profile during the first day of culture (an increase in concentration in the 60 kDa protein and decreases in the 17, 31, 37 and 50 kDa proteins). The in vivo atria showed a relatively large increase in the concentration of several of the proteins (17, 37, 50 kDA), profiled just prior to hatching with subsequent fall after hatching. A comparison between the biochemical data and the morphology of the explants indicate that although the explants display characteristics of contractility and typical cardiocyte structure, they were operative at a different functional level than their in vivo counterparts. The loss of homeostatic regulation and other in vivo embryonic factors could play a role in this observed change. This culture system provides an alternative to the dissociation method, and will be a useful model to study factors that influence atrial growth and development.

Animals↗
PubMed1987

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity.

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.

Animals↗
PubMed1987

Isolated bovine myocardial sarcolemma and sarcoplasmic reticulum vesicles contain tightly bound calcium-dependent protease inhibitor.

Bovine myocardial sarcolemma and sarcoplasmic reticulum vesicle preparations contained calcium-dependent protease inhibitor protein. No inhibitor was detected in mitochondrial membranes. The membrane-bound inhibitor co-purified with the marker enzymes for sarcolemma and sarcoplasmic reticulum, Na+,K+-ATPase and Ca2+,K+-ATPase respectively, on isopycnic ultracentrifugation through linear sucrose density gradients. Sarcolemma and sarcoplasmic reticulum vesicles contained about 1 mg of inhibitor per g of membrane protein. However, about one-half of the inhibitor in sarcoplasmic reticulum vesicles was not tightly associated with the membrane. The membrane-bound inhibitor may function to modulate calcium-dependent proteolytic cleavage of sarcolemmal or sarcoplasmic reticulum-associated proteins.

Animals↗
PubMed1987

Sex differences in prolactin change during mourning.

Fourteen men and 12 women were interviewed eight weeks after conjugal bereavement to discuss the events prior to the spouse's death and the subsequent bereavement period. Prolactin (PRL) was measured at the beginning and end of the interview. Descriptions of the deceased spouse were obtained during the interview and rated for Developmental Level of Object Representation (DLOR), a measure of the cognitive complexity of the description. There were significant correlations between DLOR and PRL change for both men and women but the correlation for women was positive and the correlation for men was negative. These findings extend the literature on the psychological correlates of PRL change and suggest that the physiological changes associated with mourning are different for men and women.

Female↗
PubMed1987

Levels of emotional awareness: a cognitive-developmental theory and its application to psychopathology.

The authors present a cognitive-developmental theory of emotional awareness that creates a bridge between normal and abnormal emotional states. Their primary thesis is that emotional awareness is a type of cognitive processing which undergoes five levels of structural transformation along a cognitive-developmental sequence derived from an integration of the theories of Piaget and Werner. The five levels of structural transformation are awareness of bodily sensations, the body in action, individual feelings, blends of feelings, and blends of blends of feelings. The authors suggest applications of this model to current unresolved problems in psychiatric theory, research, and practice.

Affective Symptoms↗
PubMed1986

Computer-assisted determination of protein concentrations from dye-binding and bicinchoninic acid protein assays performed in microtiter plates.

The bicinchoninic acid protein assay was scaled down to operate in the wells of microtiter plates. The total volume of the assay was reduced to 210 microliters and required only 10 microliters of sample per assay. The assay can be rapidly performed and then read with a plate reader. The analysis of the data was performed with a microcomputer interfaced with the reader. A computer program was developed to analyze the data from the bicinchoninic acid assay as well as the dye-binding assay at either the high or low protein concentration ranges. Based upon the absorbance values of the standards, the program computed a linear regression formula which was then used to calculate the concentrations of the sample proteins. A series of assays performed using bovine serum albumin, alpha-chymotrypsin and gamma globulin demonstrated that the scaled down bicinchoninic acid assay produced linear absorption versus concentration data with average correlation coefficients between 0.9967 and 0.9940 at the high and low concentration ranges respectively. The protein-to-protein variation and proper selection of either protein assay based upon the presence of interfering materials was considered.

Adult↗
PubMed1986

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease.

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.

Animals↗