Isolation of cDNA and genomic DNA clones encoding type II collagen.

A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs.     

Solubilization of bovine heart-value collagen.

Complete solubilization of bovine heart-valve gollagen was obtained by using sequential pepsin digestion and extraction with dithiothreitol under non-denaturing conditions. Use of the reducing agent was the crucial step resulting in solubilization. These findings suggest that disulphide-bonded proteins may be in part responsible for the insolubility of bovine heart-valve collagen.     

Identification of collagen alpha1(I) trimer in embryonic chick tendons and calvaria

Collagen with a molecular composition [α₁(I)]₃ has been identified in acetic acid extracts from lathyritic chick embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1-6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F1 hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1a) mice were low responders. The V(H) recombinant strains BAB.14 and CB-8KN that possess the Ig-1b allotype of C57BL, but have some of the V(H) genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

The size of collagen fibrils that stimulate platelet aggregation in human plasma.

Suspensions of collagen fibrils of different size were prepared from solutions of radioactive tropocollagen type I by either differential centrifugation or differential incubation at elevated temperature. The fractions were compared with respect to their ability to stimulate human blood platelet aggregation in plasma, their binding to human platelets, and their morphology, as seen in the electron microscope. Although small particles with a sedimentation coefficient as low as 4.5 S bound to platelets, aggregation was not observed in the presence of collagen multimers and protofibrils without visible cross-bands in stained specimens. The onset of platelet-aggregating activity before the appearance of turbidity in collagen solutions incubated at elevated temperature is due to the formation of a few banded fibrils; this early onset and the fibrils do not appear in collagen solutions that have been ulctracentrifuged before incubation.     

Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase.

1. Adipocytes isolated from rats 6--9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.     

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F_2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF_2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF_2α