Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP.

Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase.

The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R. J., and Reimann, E.M. (1990) J. Biol. Chem. 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase. We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007). The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit. Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells. Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose. Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene. Southern blots indicate the presence of a single copy of GLC7 in S. cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability. The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.     

The effect of morphologic variety on the biostatics of lumbosacral transition

Variants shown by the orientation of the cover-plate and of the superior articular facets of the sacrum have a considerable influence on the lumbosacral biostatistics. This can be illustrated by parallelograms of forces. If we assume a certain force stressing upon the sacrum, we are able to calculate the distribution of stress upon the cover-plate and the articular facets by help of the size and the orientation of these weight bearing faces.     

ATP utilizing reactions of human erythrocyte membranes and the influence of modulator proteins

The ATP production of human erythrocytes in the steady state (approximately 2 mmoles . 1 cells-1 . h-1, 37 degrees C, pHi 7.2) is maintained by glycolysis and the ATP consumption is essentially limited to the cell membrane. About 25% of the ATP consumption is used for ion transport ATPases. The bulk of the ATP consuming processes in intact erythrocytes remains poorly understood. "Isotonic" erythrocyte membranes prepared under approximate intracellular conditions after freeze-thaw hemolysis have high (Ca2+, Mg2+)-ATPase activities (80% of the total membrane ATPase activity). There is a great discrepancy between the high capacity of the (Ca2+, Mg2+)-ATPase in isotonic membranes and the actual activity in the intact cell. The (Ca2+, Mg2+)-ATPase of isotonic membranes has a "high" Ca2+-affinity (Ka less than 0.5 microM) and a "low" Mg-ATP affinity (Km approximately 760 microM). This state of (Ca2+, Mg2+)-ATPase is caused by the association of calmodulin and 30000 Dalton polypeptides (ATP affinity modulator protein). Hypotonic washings of isotonic membranes result in a loss of the 30 kD polypeptides. EGTA (0.5 mM) extracts derived from isotonic membranes contain the 30 kD modulator protein and restore the properties of the (Ca2+, Mg2+)-ATPase of hypotonic membrane preparations to the isotonic characteristics. The Mg-ATP affinity modulator protein is assumed to form a complex with calmodulin and (Ca2+, Mg2+)-ATPase.     

Early molecular events in the interaction of enveloped viruses with cells

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells.The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell.Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to a label transfer from virus to cell membranes or vice versa.While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.     

The influence of implant number and abutment design on the biomechanical behaviour of bone for an implant-supported fixed prosthesis: a finite element study in the upper anterior region

It is always recommended to use more implants for supporting a prosthesis in the immediate loading condition than in the classical two-stage treatment procedure. By means of the finite element (FE) method, the influence of the number of implants used in immediately loaded fixed partial prosthesis (FPP) on the load distribution was investigated, considering the abutment geometry. Two 3D FE models were studied employing four implants to support a FPP in the premaxilla. One model was designed with straight abutments and the other with 20°-angled abutments. The results concerning implant displacements, stresses and strains were compared with those of two implant-supported FPPs, obtained in a previous study. A noticeable reduction in the determined biomechanical bone loading was observed with the use of more implants in supporting an immediately loaded prosthesis. This study confirms that the use of additional numbers of implants in an immediately loaded prosthesis is highly recommended.     

Fungizide: Risiken der resistenzentwicklung und suche nach neuen targets

Trotz intensiver Bemühungen im Bereich des Pflanzenschutzes sind weltweit erhebliche Verluste im Bereich pflanzlicher Erntegüter zu verzeichnen. Rund ein Drittel der Verluste werden mikrobiellen Schaderregern, also Pilzen, Bakterien und Viren, zugeschrieben. Besonders schwierig ist der Tatsache zu begegnen, dass Pilze gegenüber spezifisch wirkenden Fungiziden Insensitivitäten bzw. Resistenzen entwickeln. Aus diesem Grunde muss über geeignete Strategien beim Fungizideinsatz, beim ‘Disease management’ und bei der Suche nach neuen, hochwirksamen Fungiziden nachgedacht werden. Der Erfolg bei der Suche nach neuen Fungiziden wird stark davon abhängen, ob neue Zielmoleküle für potentielle Fungizide entdeckt werden können. Dabei können moderne molekulargenetische Techniken von großem Nutzen sein.     

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.