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.     

Influence of nitrate concentration at the root surface on yield and nitrate uptake of kohlrabi (Brassica oleracea gongyloides L.) and spinach (Spinacia oleracea L.)

The objective of this study was to determine if plant roots have to take up nitrate at their maximum rate for achieving maximum yield. This was investigated in a flowing-solution system which kept nutrient concentrations at constant levels. Nitrate concentrations were maintained in the range 20 to 1000 μM. Maximum uptake rate for both species was obtained at 100 μM. Concentrations below 100 μM resulted in decreases in uptake rate per cm root (inflow) for both spinach and kohlrabi by 1/3 and 2/3, respectively. However, only with kohlrabi this caused a reduction in N uptake and yield. Thus indicating that this crop has to take up nitrate at the maximum inflow. Spinach, however, compensated for lower inflows by enhancing its root absorbing surface with more and longer roots hairs. Both species increased their root length by 1/3 at low nitrate concentrations.     

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.     

Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans

To simulate production-scale conditions of gluconic acid fermentation by Gluconobacter oxydans, different experimental setups are presented in this study. From the determination of the time constants of a production-scale reactor, it can be concluded that mixing and oxygen transfer are the rate-limiting mechanisms. This results in oxygen concentration gradients which were simulated in a one-compartment reactor in which the oxygen concentration was fluctuated by a fluctuated gassing with air and nitrogen. It could be concluded that only very long periods of absence of oxygen (ca. 180 s) results in lower specific oxygen uptake rates by Gluconobacter oxydans. From scale-down studies carried out in a two-compartment system to simulate a production-scale reactor more accurately, it could be concluded that not only the residence time in the aerated part of the system is important, but the liquid flow in between the different parts of the reactor is also an essential parameter. It could also be concluded that the microorganisms are not influenced negatively by the fluctuated oxygen concentrations with respect to their maximal oxidation capacity. The two-compartment system can also be used for optimization experiments in which the "aerated" compartment was gassed with pure oxygen. From these experiments it was concluded that also a short residence of the cells at high oxygen concentrations diminished the growth and product formation rates. These experiments show the necessity of the scale-down experiments if optimization is carried out. The two-compartment system presented in this study is a very attractive tool for reliable scale-down experiments.     

Relative developmental success of interspecific Lepomis hybrids as an estimate of gene regulatory divergence between species

The developmental success of interspecific Lepomis hybrids is used as an index of gene regulatory divergence between the green sunfish, L. cyanellus, and each of three other parental species, longear sunfish, L. megalotis, warmouth, L. gulosus, and bluegill, L. macrochirus. This gene regulatory divergence is compared to the degree of structural gene divergence among these four species (genetic distance [Nei, '78], D, ranged from 0.206 to 0.586). The developmental success of the hybrid embryos at the level of morphogenesis was higher than expected from the genetic distance between the parental species. The rates of morphogenesis of the hybrid embryos were the same as that for the green sunfish embryos. The percentage of embryos that hatched was relatively high in all crosses. However, two of the hybrid crosses resulted in enhanced percentages of hatched embryos. Slight increases in the extent of morphological abnormalities were observed in hybrids from crosses between more distantly related parental species. The schedules and levels of enzyme locus expression of the hybrids, assessed spectrophotometrically and electrophoretically for nine enzyme systems (encoded in a total of 14 loci), were different from each other and from those of the green sunfish embryos. Alterations in the time of first enzyme appearance and in the time of first increase in enzyme activity in the developing hybrid embryos were not correlated with genetic distance between parental species. However, the extents of alteration of enzyme activities over the entire period of hybrid embryogenesis were correlated with the genetic distance. We attribute the morphological and molecular anomalies observed in the hybrids to gene regulatory incompatibilities between species. Although the exact number of mutational differences and their relative developmental impacts are not known, some inferences can be drawn about the degree of divergence in gene regulation between species. It appears that an uncoupling of the rates of structural and regulatory gene evolution can occur between species of some taxa, an observation that has implications for the roles of gene regulatory differences in organismic evolution.