Characterization of an essential histidine residue in thermophilic malate dehydrogenase

Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate. The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme. The enzyme was protected from inactivation by NADH. The enzyme was also inactivated by dye-sensitized photooxidation. Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme. Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C. From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation. The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

A microassay for the rapid and selective binding of cells from solid tumors to mouse macrophages

Summary A microassay was developed to study the rapid binding characteristics of murine macrophages activated by gamma interferon and muramyl dipeptide to adherent neoplastic or nonneoplastic target cells. The binding of tumor cells to both activated and nonactivated macrophages was time- and temperature-dependent, and independent of tumor cell type. Activated macrophages bound more tumor cells than nonactivated macrophages. The initial binding of macrophages to target cells did not necessarily lead to lysis. First, primed macrophages bound tumor cells but did not lyse them, and second, nonactivated macrophages bound nontumorigenic cells without subsequent lysis. The rapid binding assay described here could prove useful in investigating the recognition mechanism(s) between macrophages and tumor cells derived from solid primary and metastatic cancers.     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Heme O biosynthesis in Escherichia coli: the cyoE gene in the cytochrome bo operon encodes a protoheme IX farnesyltransferase.

The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme O present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme O. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel enzyme, protoheme IX farnesyltransferase, essential for heme O biosynthesis.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Pinus MUTOI (Pinaceae), a new species of permineralized seed cone from the upper Cretaceous of Hokkaido,Japan

Pinus mutoi is described as a new species on the basis of a permineralized seed cone from the Upper Cretaceous of Hokkaido, Japan. The cone is at least 20 cm long and up to 6 cm in diameter, consisting of a cone axis and numerous cone-scale complexes that are arranged helically around the axis. Two winged seeds are borne on the adaxial surface of each ovuliferous scale. Each complex receives a single trace from the vascular cylinder of the cone axis. In the scale base, all the resin canals occur abaxially to the vascular strand. The spatulate bract of the fossil is unique to the specimen among the cones of both living and fossil Pinus. The central umbo, broad sclerotic cortex of cone axis, and absence of serotinous features of the fossil cone suggest affinity with the subsection Sylvestres of the section Pinus, subgenus Pinus. This is the first record of permineralized preserved Pinus cone from the Cretaceous of Eastern Eurasia.     

Neutron activation analysis of biological samples at the Radiochemistry Division of IPEN-CNEN/SP

Biological Trace Element Research - Neutron activation analysis is a very useful method for determination of a great number of elements in biological samples. At the Radiochemistry Division of the...