The Journal of biological chemistry
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Osteogenesis imperfecta (OI) type IB is a rare subset of the mildest form of OI, clinically characterized by moderate bone fragility, blue sclera, and dentinogenesis imperfecta. Cultured skin fibroblasts from two unrelated individuals (OI-197 and OI-165) with the typical features of OI type IB produced shortened alpha2(I) chains. Reverse transcription-polymerase chain reaction of the alpha2(I)-cDNA revealed deletions in the triple helical domain of 5 exons (exons 7-11) in OI-197, and 8 exons (exons 10-17) in OI-165. ⋯ In contrast, bone extracts from OI-197 showed the presence of the mutant collagen. This incorporation of the abnormal collagen into the mature matrix was also demonstrated in long term cultures of the patient's osteoblasts. The deposition of the mutant collagen by bone osteoblasts but not by skin fibroblasts demonstrates a tissue specificity in the incorporation of mutant collagen into the matrix which may explain the primary involvement of bone and not skin in these patients.
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Adozelesin is a member of a family of extraordinarily cytotoxic DNA damaging agents that bind to the DNA minor groove in a sequence-specific manner and form covalent adducts with adenines. Previous studies employing purified enzymes and adozelesin-modified template DNAs suggested that adozelesin-DNA adducts inhibit DNA replication at the level of nascent DNA chain elongation. In this study, neutral/neutral two-dimensional agarose gel electrophoresis was employed to analyze simian virus 40 (SV40) DNA replication intermediates recovered from adozelesin-treated SV40 virus-infected cells. ⋯ We conclude that the disappearance of SV40 replication intermediates induced by adozelesin treatment was a consequence of maturation of these intermediates in the absence of new initiation events. Adozelesin inhibition of nascent chain elongation is first observed at concentrations above those needed to block initiation. Adozelesin treatment inhibits SV40 DNA replication at concentrations that produce adducts on just a small fraction of the intracellular population of SV40 DNA molecules.
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The in vivo studies of transcriptional regulation by glucose, in general, have yielded ambiguous interpretations due to the closed loop relationship between insulin and glucose. Insulin cannot be held as a constant since elevated glucose levels will elicit a corresponding rise in insulin and current animal models of insulinopenia are associated with a plethora of counter-regulatory hormone responses. One potential solution to increase intracellular glucose flux without a further increase in insulin was achieved by transgenic overexpression of the insulin-sensitive glucose transporter, GLUT4, in specific skeletal muscles (previously described in Tsao, T.-S., Burcelin, R., Katz, E. B., Huang, L., and Charron, M. J. (1996) Diabetes 45, 28-36). Using these MLC-GLUT4 transgenic mice as a model, we investigated the effects of increased glucose flux on hexokinase II (HK II) gene expression in skeletal muscle. Under conditions where blood glucose levels were normal and insulin levels decreased by 36%, HK II mRNA level was reduced in non-GLUT4-overexpressing tissues (i.e. heart and adipose tissue) of 2-4-month-old male MLC-GLUT4 transgenic mice. This reduction in HK II mRNA was prevented in skeletal muscle, where overexpression of GLUT4 caused a 2.5-fold increase in basal and insulin-stimulated glucose uptake. The levels of HK II mRNA in heart, muscle, and adipose tissue are paralleled by HK II enzymatic activity. ⋯ 1) due to relative mild insulinopenia, HK II expression is decreased in non-GLUT4-overexpressing tissues of MLC-GLUT4 mice compared to age/sex-matched controls, and 2) GLUT4-mediated increase in cellular glucose flux can prevent the decrease in HK II expression (in GLUT4-overexpressing tissues) as a result of relative mild insulinopenia. Indeed, during the process of aging, the return of circulating insulin levels of MLC-GLUT4 mice to normal levels is associated with the normalization of HK II expression in all tissues of MLC-GLUT4 and age/sex-matched control mice. We propose that: 1) glucose flux has an amplifying effect on the ability of insulin to stimulate skeletal muscle HK II gene expression and 2) insulin-dependent glucose flux may be a potential mechanism by which HK II gene expression is regulated by sensitivity to insulin.
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Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. ⋯ Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.
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In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP). Addition of 5 microM D-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G0/G1 phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. ⋯ Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.