Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.     

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis.

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.     

Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea

Fibroblast cells from two Japanese patients with Bloom's syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Induction of histidine decarboxylase in non-mast cells in the spleen of mice by injection of staphylococcal enterotoxin A

Injection of Staphylococcal enterotoxin A (SEA) into WBB6F1-W/WV mice genetically deficient in mast cells resulted in a 10-fold increase in the histidine decarboxylase [HDC, L-histidine carboxylase, EC 4.1.1.22] activity of their spleen. The nature of the spleen cells responsible for this increased HDC activity was studied. The HDC induction by SEA was abolished on day 1 after X-ray irradiation of the mice at 400 rad and restored by transplantation of bone marrow cells from normal WBB6F1-+/+ littermates into the X-ray irradiated WBB6F1-W/WV mice. Transplantation of cells from other organs of the normal mice, such as the thymus, mesenteric lymph node and spleen, did not restore the HDC increase significantly. Transplantation of cultured mast cells also did not restore the increase. Moreover, the high HDC activity of spleen cells induced by SEA was not affected by their treatment with anti-Thy-1,2 antibody and complement. Depletion of phagocytes from the spleen by treatment with carbonyl iron resulted in decrease in HDC activity. These results suggested that phagocytic cells derived from haemopoietic stem cells of the bone marrow were responsible for the increase in HDC activity induced by SEA.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

A low-temperature-sensitive intermediate state between S2 and S3 in photosynthetic water oxidation deduced by means of thermoluminescence measurements

The temperature dependence of S-state transitions in Photosystem II was measured by means of thermoluminescence using two different protocols for low-temperature flash excitation: protocol A, “last flash at low temperature”, and protocol B, “all flashes at low temperature”. Comparison of the temperature-dependence curves obtained by these two protocols revealed a marked difference particular for the three-flash experiments. The difference was attributed to the formation of a low-temperature sensitive precursor state between S₂ and S₃. The state is formed by two flash illumination given at −5 to −50°C, spontaneously transforms to normal S₃ on dark warming, and is not converted to S₀ by the 3rd flash. The precursor state was tentatively assigned to an S₃ in which H⁺ release is not completed.     

Substitution of an arginyl residue for the active site lysyl residue (Lys258) of aspartate aminotransferase

The active site lysyl residue (Lys258) of E. coli aspartate amino transferase was substituted for an arginyl residue by oligonucleotide-directed, site-specific mutagenesis. The mutant enzyme was obviously unable to form an aldimine bond with pyridoxal 5'-phosphate but firmly bound the coenzyme. The finding that the mutation did not lead to entire loss in the enzymic activity suggests that Lys258 may not be essential but auxiliary for enzymic catalysis. It is also conceived that the positive charge provided by Arg258 may contribute to the enzymic catalysis.