The phosphate groups of the high mobility group like protein P1 strengthens its affinity for DNA.

PCA soluble proteins isolated from rat liver and proliferating HeLa interphase cells were subjected to chromatography on columns containing immobilized s.s and d.s. DNA. P1 from rat liver was eluted from s.s. and d.s. DNA between 0.20 and 0.45 M NaCl, while dephosphorylated P1 was not retained by s.s. and d.s. DNA columns at 0.25 M, suggesting that phosphate groups enhance the affinity of P1 for DNA. P1 from proliferating HeLa interphase cells exhibit increased affinity for d.s. as well as s.s. DNA when compared to rat liver P1. The higher extent of phosphorylation in proliferating cells supports the finding that phosphate enhances rather than reduces the affinity of P1 for DNA.     

Metabolic conversion of the biologically active phospholipid, lysophosphatidic acid, in fibroblasts.

Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

The ageing pineal gland and its physiological consequences.

Melatonin, the chief hormone of the pineal gland, is produced and secreted into the blood in a circadian manner with maximal production always occurring during the dark phase of the light:dark cycle. Whereas the 24h rhythm of melatonin production is very robust in young animals including humans, the cycle deteriorates during ageing. The rhythm of melatonin can be substantially preserved during ageing by restricting the food intake of experimental animals; this same treatment increases the life span of the animals. The exogenous administration of melatonin to non-food restricted animals also reportedly increases their survival. Moreover, melatonin has been shown to have immunoenhancing effects and oncostatic properties. The implication of these studies is that melatonin may have both direct and indirect beneficial effects in delaying ageing processes or it may retard the development of processes (e.g., immunodeficiency and tumor growth) which contribute to a reduced life span.     

Failure of thapsigargin to alter ion transport in human sweat gland epithelia while intracellular Ca2+ concentration is raised.

Cai in cultured human sweat gland epithelial monolayers was measured using Fura-2 fluorescence. Thapsigargin (Tg) caused a sustained increase in Cai, the rate of rise being slower but the magnitude greater than with the agonists lysylbradykinin and ATP. Tg caused an irreversible change such that even after it was removed Cai was dependent on the ambient calcium concentration, consistent with the hypothesis that Ca2+ entry is controlled by the state of the intracellular stores. Calcium entry after Tg was not modified by nimodipine, omega-conotoxin, or BAY K8644 but could be blocked by low concentrations (0.5 mM) of La3+. High concentrations of La3+ (2 mM) caused an increase in the response to Tg, suggesting that membrane ATPase exerts a major Cai lowering effect. Intracellular Ca2+ ion chelation with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid significantly blunted the response to Tg. Finally, Mn2+ entry rate into epithelial cells was doubled by Tg. In spite of the evidence that Tg raises Cai to values greater and for longer than calcium requiring agonists only the latter affected transepithelial transport processes. It is shown that Tg neither affects transepithelial sodium transport nor chloride conductance, both of which increase in response to lysylbradykinin or ATP. It is concluded that spatio-temporal patterns of Cai increase after Tg and other agonists are different.     

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.