Polar and Neutral Lipid Changes in Spinach Leaves with Ozone Fumigation: Triacyiglycerol Synthesis from Polar Lipids

When mature spinach (Spinacia oleracea L. cv. New Asia) plantswere continuously fumigated with 0.5 ppm (v/v) ozone, the totalfatty acids in the leaf lipids remained unchanged in both contentand composition during the initial 8 h, but thereafter they,especially hexadecatrienoic acid (16:3) and -linolenic acid(18:3), began to decrease with marked accumulation of malondialdehyde,indicating fatty acid peroxidation. During the first 6 h, fattyacids in polar lipids decreased to 68% of their initial level,and most were recovered in the neutral lipid fraction. Amongthe polar lipids, monogalactosyl diacylglycerol (MGDG), digalactosyldiacyiglycerol (DGDG) and phosphatidylcholine (PC) decreasedwhereas sulfoquinovosyl diacylglycerol, phosphatidyl glycerol(PG), phosphatidylethanolamine and phosphatidylinositol werestable during 6 h of ozone fumigation. Phosphatidic acid (PA)increased 3.4 times during the same period. Among the neutrallipids, triacyiglycerol (TG) and 1,2-diacylglycerol (1,2-DG)increased markedly with ozone fumigation. The constituent fatty acids of 16:3, oleic acid (18:1), linoleicacid (18:2) and 18:3 increased markedly in TG and 1,2-DG within6 h of ozone fumigation, whereas 3-trans-hexadecenoic acid (16:1),18:1, 18:2 and 18:3 increased in PA. Since 16:3 is specificto MGDG and DGDG, and 18:1 and 18:2 to PC among the glycerolipidsmainly reduced by ozone, the results suggest that the acyl moietiesof the galactolipids were converted to TG via 1,2-DG, and thoseof PC to TG through PA and 1,2-DG. The appearance of 16:1 inPA indicates that a small amount of PG, specifically acylatedwith 16:1, was degraded to PA by ozone fumigation. (Received August 21, 1984; Accepted November 16, 1984)     

The 717Val----Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups

Alzheimer's disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.     

Sequence analysis of a KpnI family member near the 3' end of human beta-globin gene.

We determined the complete nucleotide sequence (6125 bp) of a full-length member of human KpnI family, designated T beta G41, which is located about 3 kb downstream from the beta-globin gene. Comparison of the sequence with the KpnI family sequence compiled by Singer revealed that a new 131 bp sequence is present in the T beta G41. Hybridization analyses showed that a few thousand of human KpnI family members are carrying this additional sequence. Computer search of DNA databases for T beta G41-homologous sequence showed that some T beta G41-homologous sequences were closely associated with pseudogenes. The T beta G41 sequence also showed significant sequence homology with ChBlym-1, a transferrin-like transforming gene of chicken. Furthermore, an amino acid sequence deduced from the T beta G41 nucleotide sequence revealed a relatively-high homology to those of human transferrin and lactotransferrin.     

Whole-cell-dependent biosynthesis of sulfo-conjugate using human sulfotransferase expressing budding yeast

Cytosolic sulfotransferases (SULTs), one of the predominant phase II drug metabolizing enzymes (DME), play important roles in metabolism of xeno- and endobiotics to generate their sulfo-conjugates. These sulfo-conjugates often have biological activities but are difficult to study, because even though only small amounts are required to evaluate their efficacy and safety, chemical or biological synthesis of sulfo-conjugatesis is often challenging. Previously, we constructed a DME expression system for cytochrome P450 and UGT, using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell-dependent manner. In this study, we developed a yeast expression system for human SULTs, including SULT1A1, 1A3, 1B1, 1C4, 1E1, and 2A1, in Saccharomyces cerevisiae and examined its sulfo-conjugate productivity. The recombinant yeast cells expressing each of the SULTs successfully produced several hundred milligram per liter of xeno- or endobioticsulfo-conjugates within 6 h. This whole-cell-dependent biosynthesis enabled us to produce sulfo-conjugates without the use of 3’-phosphoadenosine-5’-phosphosulfate, an expensive cofactor. Additionally, the production of regiospecific sulfo-conjugates of several polyphenols was possible with this method, making this novel yeast expression system a powerful tool for uncovering the metabolic pathways and biological actions of sulfo-conjugates.

     

Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis

The disease model of familial amyloidotic polyneuropathy—7.2-hMet30 mice—manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21–24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.     

Molecular evolution of group II phospholipases A2

The nucleotide sequences of 13 cDNAs encoding group II phospholipases A₂ (PLA₂ ^S), which are from viperidae snake venoms and from mammalian sources, were aligned and analyzed by phylogenetic trees constructed using various components of the sequences. The evolutionary trees derived from the combined sequences of the untranslated (5 and 3) region and the signal peptide region of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. These observations indicated that the mature protein-coding region has evolved through a process differently from the untranslated and signal peptide regions. The trees built from the nucleotide differences at each of three positions of codons in the mature protein-coding region suggested that snakevenom-gland PLA₂ genes have evolved via a process different from mammalian PLA₂ genes. The occurrence of accelerated evolution has been recently discovered in Trimeresurus flavoviridis venom-gland group II PLA₂ isozyme genes (Nakashima et al. 1993, Proc Natl Acad Sci USA 90:5964–5968), so the present phylogenetic analysis together with the estimation of nucleotide divergence of cDNAs provides further evidence that snakevenom-group II PLA₂ isozyme genes have evolved by accelerated evolution to gain diverse physiological activities. Correspondence to: M. Ohno     

Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24.

Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43-48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1alpha,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.     

Inactivation of the ATP-dependent DNase of Escherichia coli After Infection with Double-Stranded DNA Phages

The ATP-dependent DNase activity of Escherichia coli disappeared or was markedly reduced after infection with double-stranded DNA phages, T2, T3, T4, T5, T6, T7, lambda, phi80, and P1, but not with the single-stranded DNA phage f1, or the RNA phage Qbeta. This DNase activity was not reduced when chloramphenicol was added prior to phage infection.     

Expression of rat alpha 2-macroglobulin gene during pregnancy

Rat alpha 2-macroglobulin (alpha 2M) is a typical acute phase protein, the concentration of which in serum increases more than 100-fold after inflammation. It is also known that the protein increases during pregnant (and neonatal) stages. Using a specific cDNA probe, expression of the alpha 2M gene during pregnancy was studied at the mRNA level. During inflammation, the liver is almost the only organ producing alpha 2M, but during pregnancy the placenta and uterus were found to be major organs producing a large amount (70-80% of that of inflamed liver) of alpha 2M mRNA at days 12-15. The yolk sac, maternal liver and fetal (or neonatal) liver also produced a small but significant amount (5-20% of that of inflamed liver) of the mRNA. Southern blotting analysis showed that only one copy of the alpha 2M gene was present in a haploid rat genome. These results indicated that a single alpha 2M gene has the ability to respond to two completely-different physiological states.     

Two types of coagulogen mRNAs found in horseshoe crab (Tachypleus tridentatus) hemocytes: molecular cloning and nucleotide sequences

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.