Molecular cloning and nucleotide sequence of murine 2,3-bisphosphoglycerate mutase cDNA

Cloning and sequencing of a murine cDNA with the entire coding region of 2,3-bisphosphoglycerate mutase is reported, as a prerequisite for further expression studies of this erythroid specific enzyme in Friend mouse erythroleukemia cells. A comparison between species of the deduced amino acid sequences of these proteins shows 20 substitutions between mouse and human and 21 between mouse and rabbit: none of these substitutions are in positions assumed to be in the active site. Amino acid alignment with the other related enzymes, the phosphoglycerate mutases, in combination with crystallographic data from yeast phosphoglycerate mutase, gives some insight into the structure/function correlation for this protein family. Amino acid residues which are most likely critical for either 2,3-bisphosphoglycerate mutase or phosphoglycerate mutase function are pointed out. Concerning the phylogenetic analysis, phosphoglycerate mutases B and M from mammalians appear to have diverged with the yeast enzyme from a common ancestor, before the emergence of the 2,3-bisphosphoglycerate mutases.     

Influence of somatostatine on plasma level of calcitonin in the calf and the swine]

In young calves receiving intraveinously a small dose of calcium to stimulate calcitonin release, intraveinous infusion of somatostatin did not significantly modify the jugular veinous plasma calcitonin levels measured by radioimmunoassay, using a porcine system which cross-reacts with bovine calcitonin. In piglets, intraveinous infusion of somatostatin also did not change the jugular veinous plasma calcitonin concentration.     

Limited proteolysis at the carboxy end modifies interactions between the subunits of Escherichia coli phosphofructokinase

The limited proteolysis of Escherichia coli phosphofructokinase by subtilisin involves the removal of a segment of 40-50 residues at the C-terminal end of each polypeptide chain [Le Bras, G., & Garel, J.R. (1985) J. Biol. Chem. 260, 13450-13453]. The time course of proteolysis has been followed by the appearance of shorter chains, the loss of allosteric inhibition by phosphoenolpyruvate, and the weakening of the tetrameric structure in the absence of fructose 6-phosphate. It is found that with only one shorter chain out of four the stability of the tetramer is altered so that it is no longer stable in the absence of fructose 6-phosphate. Also, the reduction in size of only two chains is sufficient to render the enzyme insensitive to allosteric effectors, albeit the protein still possesses the ability to bind such an effector (at least partially); the cleavage of all four chains is needed to lose all the effector binding ability. The C-terminal segment therefore plays an important role in subunit interactions as seen from the gradual changes in structural and functional properties which follow its removal from one, two, or four chains.     

1,25-Dihydroxycholecalciferol effects on plasma calcitonin levels and calcitonin mRNA in normal or partially vitamin D-depleted rats

The physiological effect of 1,25-(OH)2D3 on the regulation of calcitonin (CT) secretion was studied by measuring plasma CT levels and CT mRNAs extracted from thyroid glands of normal (D+) or partially vitamin D-depleted rats (D-). In both groups, acute 1,25-(OH)2D3 administration of 0.1 microgram/kg b.w. yielded an early drop in plasma calcium concentrations (around 0.6-1 mg/dl) with a maximum decrease 15 min after treatment. In spite of this hypocalcemia, a significant rise in plasma CT levels was observed within 5 min in D+ animals and within 30 min in D- animals after injection of the vitamin D metabolite. Nevertheless, the increased CT secretion was not associated with a marked and sustained rise in CT mRNA levels measured by dot-blot hybridization or CT mRNA activity evaluated by translation assay. By contrast to the observations made previously using supra-physiological doses of the vitamin D metabolites, no clear-cut effect on CT mRNA levels was found with lower doses. If we hypothesized that 1,25-(OH)2D3 plays a physiological role in CT secretion, our results suggest that this rapid control could be exerted at a post-translational level may be via an increase in the cytoplasmic ionized calcium concentration of C-cells.     

Fructose 6-phosphate prevents the proteolyzed derivative of Escherichia coli phosphofructokinase from dissociation and inactivation

N-terminal sequence analysis shows that the limited proteolysis of Escherichia coli phosphofructokinase results in the removal of the 40-50 C-terminal residues of each chain. When tetrameric, this proteolyzed derivative is still active albeit insensitive to allosteric effectors (Le Bras, G., and Garel, J.-R. (1982) Biochemistry 21, 6656-6660). In the absence of fructose 6-phosphate, the proteolyzed phosphofructokinase spontaneously loses its activity and dissociates into dimeric species. This inactivation/dissociation is slowed down by the binding of fructose 6-phosphate to only part of the sites; it is completely prevented by the saturation of all four fructose 6-phosphate sites. The other substrate ATP does not protect the proteolyzed phosphofructokinase against this inactivation/dissociation. This inactivation/dissociation is not due to denaturation and can be reversed in some conditions by the addition of fructose 6-phosphate. The active tetrameric structure of phosphofructokinase is stable when either the C-terminal segment is not removed or the fructose 6-phosphate sites are occupied.     

Phase II protocol for the evaluation of new treatments in patients with advanced gastric carcinoma: results of ECOG 5282

The study was a Phase II randomized study to evaluate the efficacy of new agents for the treatment of advanced gastric carcinoma. Patients were randomized to receive single agent chemotherapy with mitoxantrone, etoposide, aclacinomycin-A or spirogermanium. The patients were stratified by prior use of chemotherapy, prior doxorubicin use and ECOG performance status. Patients with a history of cardiac disease or prior doxorubicin exceeding a dose of 400 mg/m² were restrictively randomized to sopirogermanium or etoposide only. One hundred and fourteen patients were registered for the study. Among 98 evaluable patients there were only two partial responses (both in the etoposide arm), and one complete response in the mitoxantrone arm. The median survival on the study was 3.3 months. One hundred and six patients were analyzable for toxicity. There were four treatment-related deaths and four life-threatening toxicities. Because of low response rates and relatively high toxicities the studied compounds were not deemed worth further investigation for advanced gastric cancer.     

Plasma concentrations of immunoreactive calcitonin in rats during lactation

A mild hypocalcemia (0.5 mg/dl) is observed in rats after 14 days of lactation, but plasma and thyroidal calcitonin (CT) levels are both increased on day 7 of lactation. Plasma CT levels are higher (x2) in lactating females than those found in virgin females from day 7 to the end of lactation (21 days). In vitro, the CT secretion rate after calcium stimulation(3 mM) is not different between lactating and virgin females. The rapid removal of pups after parturition in control females induces a rebound in plasma calcium (+1.4 mg/dl) and plasma phosphate (+1.6 mg/dl) associated with elevated plasma CT values. Our results suggest, that the transient mild hypocalcemia of lactation is preceded by increased plasma CT levels; and that it is not the cause of elevated plasma parathyroid hormone levels already reported by us.