Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein

An inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction. Five cDNA clones were isolated from the human placental lambda gt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe. The longest insert consists of 1,566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail. The deduced amino acid sequence was corroborated by chemical analyses of the protein. The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, and endonexin-related proteins. The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.     

Synthesis of Collagen-Like Proteins in Embryonic Organs of the Sea Urchin, Hemicentrotus pulcherrimus

In sea urchin embryos exposed to ¹⁴C-proline at 20°C for 3 hr at the gastrula, prism or pluteus stage, ¹⁴C-radioactivity was found in hot acid-extractable proteins, in which more than 4% of the radioactivity was detectable in hydroxyproline residues. In these embryos, ¹⁴C-radioactivity in collagen-like proteins was found in the archenteron, spicule and embryo-wall cells. The rate of synthesis of collagen-like proteins was highest in the archenteron in the mid-gastrula stage, in the embryo-wall cells in the prism stage and in the spicule in the pluteus stage. The rate of synthesis decreased in the archenteron and increased in embryo-wall cells in the period between the mid- and late-gastrula stages, when the rate of synthesis in the spicule was quite low. Thereafter, the rate decreased slightly in the embryo-wall cells, was maintained in archenteron and increased markedly in the spicule. The rates of synthesis of collagen-like proteins are high in these embryonic organs at stages at which development and growth respectively, occur in embryos. Therefore, synthesis of collagen-like proteins probably supports morphogenesis in these embryonic organs.     

Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s

At least two GTP-binding proteins (G proteins) with Mr values of about 20,000 were extracted from bovine aortic smooth muscle membranes by sodium cholate. The most abundant G protein (22K G) was purified to near homogeneity by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite and Mono Q HR5/5. 22K G showed kinetic and physical properties very similar to those of smg p21, a G protein recently isolated from bovine brain and human platelet membranes, having the same effector domain as ras p21s. Moreover, 22K G was recognized specifically by the anti-smg p21 antibody. These results indicate that the major G protein in bovine aortic smooth muscle membranes is smg p21.     

Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF-7 cell line and its TNF-resistant variant

Tumor necrosis factor (TNF)-resistant variant of human mammary cancer MCF-7 cell line was isolated by stepwise selection. The final TNF-resistant variant Tnf-1000 showed more than 100-fold higher resistance than the parental MCF-7 cell. Saturation kinetics for 125I-TNF binding showed that TNF-1000 cells had similar TNF receptor numbers as MCF-7 cells, but of a lower affinity. Induction of superoxide dismutase (SOD) was compared between MCF-7 and Tnf-1000 cells treated with TNF: SOD scavenges potentially toxic superoxide radicals. TNF induced more mitochondrial manganese SOD (SODm) in MCF-7 than in Tnf-1000 whereas there appeared to be no significant induction of cytosolic copper/zinc SOD (SODc) by TNF in both MCF-7 and Tnf-1000 cell lines. Acquirement of TNF-resistance in MCF-7 cells might be correlated with expression of SODm.     

The effect of cytokines on the ploidy of megakaryocytes

The effects of recombinant cytokines on the ploidy of human megakaryocytes derived from megakaryocyte progenitors were studied using serum-free agar cultures. Nonadherent and T cell-depleted marrow cells were cultured for 14 days. Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet IIb/IIIa. The ploidy of individual megakaryocytes in colonies was determined by microfluorometry with DAPI (4',6-diamidino-2-phenylindole) staining. Recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) supported megakaryocyte colony formation in a dose-dependent manner. However, both rhIL-3 and rhGM-CSF had no definite ability to increase the ploidy values. Recombinant human erythropoietin (rhEpo) or recombinant human macrophage colony-stimulating factor (rhM-CSF) by itself did not stimulate the growth of megakaryocyte progenitors. rhEpo or rhM-CSF, however, stimulated increases in the number, size and ploidy values of megakaryocyte colonies in the presence of rhIL-3 or rhGM-CSF. Recombinant human interleukin 6 (rhIL-6) showed no capacity to generate or enhance megakaryocyte colony formation when added to the culture alone or in combination with rhIL-3. rhIL-6, however, increased the ploidy values in colonies when added with rhIL-3. These results show that rhEpo, rhM-CSF and rhIL-6 affect endomitosis and that two factors are required for megakaryocyte development.     

Structure of retinular cells in a Drosophila melanogaster visual mutant,rdgA, at early stages of degeneration

Summary A Drosophila visual mutant rdgA has photoreceptive cells which degenerate gradually after eclosion. Fine structure of the retinular cells of rdgA ^KS60 and rdgA ^K014 was studied during early stages of degeneration to determine the initial morphological defects. The retinular cells of these two alleles showed the following structural abnormality within 1 day after eclosion: (1) rhabdomeres were small and irregular in shape; (2) cisternae of the rough endoplasmic reticulum were more numerous than those in normal retinular cells; (3) submicrovillar cisternae were absent; and (4) lysosomes were fewer than normal. Three-dimensional reconstruction of serial sections of the ommatidia showed that the degeneration of mutant rhabdomeres proceeds more rapidly in regions remote from the nuclei. These results suggest that the process of turnover of rhabdomeric microvilli is abnormal in rdgA. We also confirmed an increase of lysosomes and destruction of cellular organelles, as reported by previous investigators at more advanced stages of degeneration.     

Induction of tumor growth inhibitory factor (TGIF) in human mononuclear cells by OK-432, a streptococcal preparation

Summary A tumor growth inhibitory factor (TGIF) was induced in the culture supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and a streptococcal preparation, OK-432, in vitro. The activity generated in the supernatant increased in a time-dependent fashion and first appeared 6 h after the initiation of culture, reaching its maximum around 48 h. The TGIF was cytostatic against seven of ten human tumor targets, but not against three murine tumor targets. Tumor cell growth was inhibited by a transient contact, i.e., 1 h, with TGIF. The TGIF was produced by lymphocytes but not by monocytes, because the activity was usually enhanced by elimination of plastic-adherent cells from the original PBMC fraction. The TGIF was relatively stable against heating at 56° C for 30 min, but the activity was totally destroyed after heating at 70° C for 5 min. The molecular weight of TGIF was estimated to be about 43×10³ daltons by gel filtration. No interferon (IFN) activity was detected in the TGIF-positive fractions obtained by gel filtration, and the TGIF-positive fractions did not inhibit the growth of tumor necrosis factor (TNF)-sensitive mouse L929 cells. The TGIF activity was not significantly affected in neutralizing tests using specific antibodies against human IFN and TNF. The OK-432 was administered i.p. for management of cancer patients with malignant ascites. Ascites-derived mononuclear cells (ASMC) were obtained before and 3 to 5 days after OK-432 injection. The ASMC obtained after the injection produced TGIF in vitro in the absence of OK-432; the preinjection ASMC showed no such production. A positive correlation was found between TGIF-producing activity by ASMC and the effect of OK-432 injection on ascites volume. These results indicate that TGIF is induced in mononuclear cells by OK-432 not only in vitro but also in vivo and plays an important role in inhibition of tumor growth in cancer patients.     

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos

Summary Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5–12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinet regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in paralled with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria. The relationship between, on the one hand, changes in the enzymatic patterns with a bearing on the energy-yielding metabolism and, on the other hand, cellular differentiation during major organogenesis in rat embryos is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Effect of diabetes mellitus induced by streptozotocin on renal Superoxide dismutases in the rat

Two forms of superoxide dismutase, CuZn-SOD and MnSOD, have been investigated in the kidneys of streptozotocin-induced diabetic rats using both radio-immunoassay and immunoenzyme staining. The rats were killed 2, 8 and 12 weeks after the induction of diabetes mellitus and the kidneys excised. Two weeks after the induction of diabetes, the kidneys were hypertrophied because of the proliferation of renal tubular epithelium. However, the total CuZnSOD content of the kidneys did not increase and, because of the epithelial proliferation, the CuZnSOD concentration in each proximal tubular cell was decreased. Armanni-Ebstein lesions were found in the distal tubules 8 and 12 weeks after the induction of diabetes. The cells in these lesions were intensely stained for CuZnSOD, suggesting an adaptive response to the enhanced oxidative stress. The MnSOD staining in the thick ascending limbs of Henle's loops was enhanced in the diabetic kidneys, while that in the cortical tubules was unaltered. MnSOD was assumed to increase in response to hypermetabolism associated with the proliferation of renal tubules. This was most marked in the cells which were rich in mitochondria, again suggesting an adaptive response to enhanced oxidative stress induced by diabetes mellitus. The glomeruli of both the diabetic and control groups were not stained for SODs, and no significant microscopic change was found even 12 weeks after the induction of diabetes mellitus.