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.     

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.     

Enzymatic evidence for involvement of the methylmalonyl-CoA pathway in propionate oxidation by Syntrophobacter wolinii

Enzyme measurements were carried out with crude cell-free extracts of the propionate oxidizing coculture of Syntrophobacter wolinii and Desulfovibrio G11. Using cell-free extracts of a pure culture of Desulfovibrio G11 as a blank, most of the enzymes involved in the methylmalonyl-CoA pathway for propionate oxidation, including a propionyl-CoA: oxaloacetate transcarboxylase, were demonstrated in S. wolinii.     

A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

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.