The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases

Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 microM outside calcium concentration and a maximal velocity of calcium efflux of 4.66 +/- 2.32 nmol X min-1 X mg-1. Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake.     

Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.     

Suppressed in vitro blastogenic responsiveness of rat spleen cells after continuous infusion of endotoxin by an implanted osmotic pump

Continuous infusion of a gram-negative bacterial endotoxin in relatively small doses into rats by means of an implanted osmotic pump was studied. The model system was designed to examine the effects of endotoxin on the blastogenic response of spleen cells to the endotoxin itself and to a nonspecific T-cell mitogen, concanavalin A (Con A). Rats were implanted with an osmotic pump which delivered saline for the first 42 hr to provide postsurgical recovery before the onset of endotoxin infusion. Previous studies had shown that during the first 1-4 days after administration of endotoxin marked alterations of metabolism and some changes in physiologic parameters such as blood pressure and in vitro myocardial performance occurred. In the present study the blastogenic responsiveness of spleen cells to endotoxin itself as well as to the nonspecific T-cell mitogen Con A was markedly decreased after several days of continuous administration of endotoxin. Control animals receiving only saline for the same period of time showed a similar depression of blastogenic responsiveness to the lipopolysaccharide (LPS), as well as to Con A, however, with a delay of 2-4 days before comparable levels of suppression became evident. These results indicate that marked alterations of immune competence as measured by blastogenesis of spleen cells to Escherichia coli LPS and to a mitogen such as Con A may occur after implantation of an osmotic pump, with or without continuous infusion of endotoxin. Further studies seem warranted to determine the role of the foreign body reaction to the osmotic pump as well as to the endotoxin administered by the pump.     

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

High colony-forming efficiency of primary human tumor cells cultured in the adhesive-tumor-cell culture system: improvements with medium and serum alterations

The colony-forming efficiency (CFE) of primary human tumor cells cultured in the adhesive-tumor-cell culture system (ATCCS) using Ham's F12 (F12) or Eagle's minimum essential medium, alpha modification (alphaMEM) and culture medium supplemented with either swine, equine or bovine sera were compared. AlphaMEM supplemented with equine serum provided the highest CFE of the combinations. The CFE increase due to the change from F12 to alphaMEM was approximately 5-fold, and the increase due to the change in serum from swine to equine was approximately 2-fold. Cytokeratin staining showed that this increase was not due to fibroblast growth. The high-average CFE with alphaMEM, approximately 3%, means that an inoculum of only 2 X 10(3) cells is needed to achieve formation of approximately 65 colonies in control cultures, thereby increasing the performance of this system when used in a chemosensitivity assay.     

Lipoxygenase and cyclooxygenase blockade by BW755C prevents endotoxin-induced hypotension but not changes in glucose metabolism

Arachidonic acid metabolites are mediators of various pathophysiologic events following endotoxin administration. However, their role in the endotoxin-induced increase in glucose metabolism has not been examined. Rats were administered either saline or BW755C (an inhibitor of both the cyclooxygenase and lipoxygenase pathways) 30 min prior to injection of E. coli endotoxin and whole body glucose kinetics assessed using a constant iv infusion of [6-3H] glucose. Treatment with BW755C prevented the endotoxin-induced hypotension and tachycardia. Endotoxin produced characteristic increases in the plasma glucose (23-70%) and lactate (2- to 9-fold) concentrations, as well as elevations in the rate of glucose appearance (34-63%) and metabolic clearance (40-92%). In contrast to the amelioration in hemodynamics, pretreatment with BW755C did not prevent these alterations in glucose metabolism normally seen after endotoxin. BW755C markedly reduced the endotoxin-induced increase in plasma catecholamine concentrations, but levels were still elevated 2- to 4-fold compared to control values. The results suggest that arachidonic acid metabolites mediate the early hypotensive response following endotoxin, but are not by themselves responsible for the elevated rates of glucose production and utilization.     

Molecular defect in factor IXBm Lake Elsinore. Substitution of Ala390 by Val in the catalytic domain

Earlier studies with factor IXBm Lake Elsinore (IXBmLE), a nonfunctional variant of factor IX, suggested that the defect in this protein may reside in the catalytic domain of the molecule (Usharani, P., Warn-Cramer, B. J., Kasper, C. K., and Bajaj, S. P. (1985) J. Clin. Invest. 75, 76-83). In this report, genomic DNA fragments from normal IX and IXBmLE alleles were cloned into phage lambda EMBL3 and the recombinant phage identified using normal IX cDNA and synthetic oligonucleotides. Exons VI, VII, and VIII of normal IX and IXBmLE gene were also amplified using a newly developed primer-directed polymerase chain reaction method. All eight exons and flanking regions of the normal IX and IXBmLE gene were sequenced by the dideoxy chain termination method. Comparison of the normal IX and IXBmLE sequences revealed a single base substitution (C----T) in the exon VIII of the BmLE variant, which results in the replacement of Ala390 by Val in the variant molecule. Although this mutation is in the catalytic domain of the molecule, purified factor IXaBmLE is indistinguishable from normal IXa in its activity toward a small synthetic substrate, L-tosylarginine methyl ester. These data, coupled with the previous data, identify a region (around residue 390) in the normal factor IXa which appears to play a major role in the extended macromolecular substrate binding site.     

Sorption von Cellulase an Weizenstroh und seine Komponenten

By comparing different activity data of the buffered cellulase solution before and after contact with the substrate the interaction between Penicillium janthinellum cellulase and wheat straw, resp. its components (holocellulose and isolated lignin) has been investigated. The loss of activity due to sorption or denaturation has been found to differ widely between the different activity data and between the various substrates. A remarkable loss of enzyme activity was observed after contact with isolated straw lignin. The differences in activity decrease between the cellulose and the lignin moiety were found to be largent with the cellobiase activity.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.