Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture

Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X₂Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X₂Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X₂Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X₂Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X₂Y, 20% type I trimer, 13% type III and 1% type II.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

The myeloproliferative sarcoma virus causes transformation or erythroid progenitor cells in vitro

The myeloproliferative sarcoma virus induces spleen focus formation in vivo and transforms fibroblasts in vitro. We showed in this study that in vitro infection of spleen or bone marrow cells from susceptible mice with the myeloproliferative sarcoma virus leads to the formation of erythroid bursts. Under optimal conditions erythroid bursts formed in the absence of added erythropoietin, but the addition of as little as 0.05 U of erythropoietin per ml to infected cultures resulted in a significant increase in numbers of erythroid bursts and the proportion of hemoglobinized cells. A comparison of the kinetics of burst formation and the size of the induced bursts with those induced with Friend virus suggested that either sarcoma virus such as the myeloproliferative sarcoma virus or the target cells for the two viruses were not the same. Density characterization and heat lability studies indicated that the increased erythroid proliferation in vitro was a virus-induced event, but the possibility that the induced erythroid burst formation is mediated via interaction with a nonerythroid target cell and subsequent release of a soluble factor cannot be ruled out.     

Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.     

Immobilization of luciferase from the firefly Luciola mingrelica — Catalytic properties and stability of the immobilized enzyme

Firefly (Luciola mingrelica) luciferase [Photinus luciferin 4-monooxygenase (ATP-hydrolysing); Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing), EC 1.13.12.7] has been immobilized on albumin and polyacrylamide gel, on AH-, CH- and CNBr-Sepharose 4B as well as on Ultragel, Ultradex and cellophane film activated by cyanogen bromide. Only immobilization on cyanogen bromide-activated polysaccharide carriers resulted in highly active immobilized luciferase. Kinetic properties of immobilized luciferase hardly differed from those of the soluble enzyme. The inactivation rate constants of soluble and immobilized luciferase were measured at pH 5.5–9.0 and 25°C as well as at pH 7.8 and 20–40°C. The ΔH^≠ and ΔS^≠ values for inactivation of soluble and immobilized luciferases were obtained. A 1000-fold stabilization effect was noted for the luciferase immobilized on CNBr-Sepharose 4B at pH 7.5 and 25°C. A stabilization mechanism for the immobilized luciferase is discussed.     

Correction to: Integrative transcriptomics reveals genotypic impact on sugar beet storability

Plant Molecular Biology - In the above mentioned publication, part of Fig. 6B was distorted (extra diagonal lines appeared). The original article has been corrected and the proper version...