Study of insulin response to oral glucose load after acute and chronic glycemic control in type 2 diabetic subjects

To investigate whether correction of fasting hyperglycemia per se improves the insulin secretion in type 2 diabetic subjects, plasma insulin response to 75 g oral glucose load has been studied after acute and chronic normalization of fasting plasma glucose levels in 7 overt type 2 diabetic subjects. For the acute normalization of elevated fasting plasma glucose levels, an artificial endocrine pancreas was employed. Although fasting plasma glucose concentrations were normalized before the oral glucose challenge, insulin response to oral glucose was not improved compared to those without normalization of fasting plasma glucose levels. After 1-3 month control of hyperglycemia, the insulin response to glucose in the subjects was significantly improved compared to those without treatments. Results indicate that chronic metabolic control is essential for the improvement of insulin response to glucose in type 2 diabetic subjects, and also suggest that the impaired insulin secretion in type 2 diabetes is not due to hyperglycemia per se, but due to the metabolic derangements which lead to chronic hyperglycemia.     

Effects of monoenergetic X-rays with resonance energy of bromine K-absorption edge on bromouracil-labelled E. coli cells

In order to examine enhanced killing that might be induced by Auger cascades in the incorporated atoms in cells, bromouracil(BrU)-labelled E. coli cells were irradiated with monoenergetic X-rays at 13.49 and 12.40keV, just above and below the K-absorption edge of bromine. In both cases BrU-labelled cells were more sensitive for killing than were normal cells. However, when the degree of BrU-sensitization was compared between the two energies of X-rays, the enhanced killing at 13.49 keV was only small, 2 +/- 8 per cent based on the D0 value in saline. By the addition of DMSO, which is believed to suppress radical-mediated effects, killing of BrU-labelled cells was enhanced at 13.49 keV by 8 +/- 4 per cent as compared with 12.40 keV, based on D0. These results have been examined in terms of absorbed energy in BrU-labelled cells and in terms of the number of induced Auger events.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.     

The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus, a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.