Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.     

Lactic acid concentration as an indicator of acceptability in refrigerated or freeze-thawed ground beef.

Lactic acid concentrations increased in refrigerated and freeze-thawed anaerobically stored ground beef. Bacterial counts were higher in refrigerated samples, but the ratios of gram-positive bacteria in refrigerated and freeze-thawed samples were the same. No differences in appearance or odor between refrigerated and freeze-thawed samples were noted after 2 days of aerobic storage. Initial lactic acid concentration can be used to predict the shelf life of frozen beef.     

A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Collagen fibrillogenesis in vitro: an investigation of the thermal memory effect and of the early events occurring during fibril assembly using dynamic light scattering

Dynamic light scattering has been used to characterize a variety of lathyritic rat skin collagen solutions. The technique was used to monitor the onset of fibril assembly in vitro and to investigate the thermal memory effect. Although the incorporation of thermal memory was demonstrated by reheating the sample and subsequently observing a shortened turbidimetric lag phase, no significant differences between naive solutions and ones exhibiting thermal memory could be detected using photon correlation spectroscopy. This suggests that subtle changes in the state of the collagen molecules rather than extensive changes in the degree of aggregation are responsible for the thermal memory effect. During fibrillogenesis, no large-scale changes in the distribution of monomers or aggregates occur until near the end of the lag phase.