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Biomedical subjects

P E Clark

Publications and source records attributed to P E Clark.

8 recordsLinked to original sources
PubMed1992

Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage.

The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.

Animals↗
PubMed1989

Increasing gene expression in yeast by fusion to ubiquitin.

Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene. An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein. The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP). The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo. Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast. The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli. This technology should greatly enhance the utility of yeast for heterologous protein production.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Small proteoglycan synthesis by skin fibroblasts cultured from elderly donors and patients with defined defects in types I and III collagen metabolism.

The relative synthesis of two different types of small proteoglycans with potentially distinct roles in tissue function (PGI and PGII) was investigated in human skin fibroblast cultures initiated from donors of increasing age (fetal to 92 y) and from patients with defined defects in type I and type III collagen metabolism. Because these two small proteoglycans are not distinguished by the usual methods of ion-exchange and sieve chromatography, we have separated them using gel electrophoresis and confirmed this by specific immunoprecipitation. Small proteoglycans of the PGII type were the predominant species found in the medium of all cultures from normal donors, regardless of age. Most of the mutant cell lines showed a profile of small proteoglycan synthesis like that of the normal cells (i.e., predominantly PGII) although an increased ratio of PGI/PGII was seen for two cell strains from patients with Ehlers-Danlos syndrome type IV characterized by intracellular accumulation of type III procollagen. We conclude that mutations affecting collagen primary structure and secretion appear to have little effect on the cells' synthesis and secretion of small proteoglycans. These findings fail to support an hypothesis suggesting that the metabolism of normal cellular synthetic products (proteoglycans) is altered by abnormal cellular processing of a defective product (collagen).

Adult↗
PubMed1986

The modulating effects of lipids on purified rat liver Golgi galactosyltransferase.

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.

Animals↗
PubMed1986

Regulation of the yeast metallothionein gene.

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.

Copper↗