Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.

We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue.     

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.     

Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3',4,4',5-pentachlorobiphenyl

Coplanar polychlorinated biphenyls included in dioxin-like compounds are bio-accumulated and adversely affect wildlife and human health. Although many researchers have studied the metabolism of PCBs, there have been few reports of the in vitro metabolism of 3,3',4,4',5-pentachlorobiphenyl (PCB126), despite the fact that it has the highest toxicity among PCB congeners. Cytochrome P450 (CYP) 1A1 proteins can metabolize some dioxins and PCBs by hydroxylation, but the activities of human and rat CYP1A1 proteins are very different. The mechanism remains unclear. From our results, rat CYP1A1 metabolized PCB126 into 4-OH-3,3',4',5-tetrachlorobiphenyl and 4-OH-3,3',4',5,5'-pentachlorobiphenyl, but human CYP1A1 did not metabolize. Homology models of the two CYP proteins, and docking studies, showed that differences in the amino acid residues forming their substrate-binding cavities led to differences in the size and shape of the cavities; only the cavity of rat CYP1A1 allowed PCB126 close enough to the haem to be metabolized. Comparison of the amino acid residues of other mammalian CYP1A1 proteins suggested that rats have a unique metabolism of xenobiotics. Our results suggest that it is necessary to be careful in human extrapolation of toxicity data estimated by using the rat as an experimental animal, especially in the case of compounds metabolized by CYP1A1.     

Direct repeats surrounding the ribosomal RNA genes of Physarum polycephalum

Sequence homology was found between the external transcribed spacer and the terminal non-transcribed spacer of Physarum polycephalum rDNA. The homologous sequences were located 2kb upstream from the 19s rRNA gene and 0.3kb downstream from 26S rRNA gene, respectively, and were arranged in a direct repeat manner. Sequence analyses showed that the direct repeats consisted of two parts: one was sequences of about 130bp which showed over 90% sequence homology with each other. The other consisted mainly of many tandem repeats of a 50 to 52bp unit. The direct repeat-rRNA genes-direct repeat unit was found to be flanked by short direct repetitious sequences. Based on these findings, the significance of the direct repeat is discussed in terms of evolution of rDNA.     

Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO2-fumigated leaves of spinach

Chlorophyll a and carotenoids of spinach began to be destroyed2 to 3 hr after fumigation with 2 ppm SO₂ under light, whereaschlorophyll b was undamaged during 8 hr of exposure to SO₂.Pheophytin a was not affected by the fumigation. When disks excised from leaves fumigated with SO₂ at 2 ppm for2 hr were illuminated, chlorophyll a and carotenoids were brokendown, while they were not destroyed in darkness. The destructionof these pigments was suppressed under nitrogen. Chlorophylla destruction was inhibited by l,2-dihydroxybenzene-3,5-disulfonate(tiron), hydro-quinone and ascorbate, but not by l,4-diazabicyclo-[2,2,2]-octane(DABCO), methio-nine, histidine, benzoate and formate. Chlorophylla destruction was inhibited by phenazine methosulfate but stimulatedby methyl viologen. Addition of superoxide dismutase (SOD) tothe homogenate of SO₂-fumigated leaves inhibited the chlorophylla destruction. The activity of endogenous SOD was reduced to40% by 2-hr fumigation before the loss of chlorophyll was observed.These results suggest that chlorophyll a destruction by SO₂was due to superoxide radicals (O₂^–). Moreover, malondialdehyde (MDA), a product of lipid peroxidation,was formed in SO₂-fumigated leaves. MDA formation was inhibitedby tiron, hydroquinone and DABCO but not by benzoate and formate.MDA formation was increased by D₂O. These results suggest thatlipid peroxidation in SO₂-fumigated leaves was due to singletoxygen ¹O₂ produced from O_2^–. (Received May 15, 1980; )     

Expression of c-myc oncogene during differentiation of human burst-forming unit, erythroid (BFU-E)

We investigated the serial expression of c-myc oncogene during differentiation of normal human burst-forming unit, erythroid (BFU-E), in vitro, together with the replating efficiency and the expression of transferrin receptors. We found that the c-myc oncogene was expressed in the early progeny of BFU-E and that this expression decreased during erythroid differentiation. This change of the c-myc expression was well correlated to changes in the replating efficiency and the expression of transferrin receptors. These observations indicate that the c-myc oncogene probably has a physiological role in normal human hematopoiesis as well as leukemic hematopoiesis.     

Free Fatty acids regulate two galactosyltransferases in chloroplast envelope membranes isolated from spinach leaves

Effects of MgCl₂ and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl₂ with a saturated concentration of more than 5 millimolar. Free α-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl₂, without affecting its maximum activity at higher MgCl₂ concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl₂ were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl₂, CaCl₂ and a high concentration of KCl in place of MgCl₂. UDGT activity was hyperbolically enhanced by MgCl₂ with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl₂-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl₂ concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves.