Application of 15N-nuclear magnetic resonance (N.M.R.) spectroscopy for the study of nitrogen metabolism of a parasite: transamination in Angiostrongylus cantonensis eggs

In an attempt to study nitrogen metabolism in a parasite, we applied 15N-nuclear magnetic resonance (NMR) technology with a stable isotope, a 15N-labeled compound, for the study of the transamination system in A. cantonensis eggs, and demonstrated that 15N-aspartic acid can serve as an amino group donor for both the 2-oxoglutaric-glutamic acid and the pyruvic acid-alanine transamination systems in the eggs.     

In vitro aggregation of platelets induced by alpha-toxin (phospholipase C) of Clostridium perfringens.

Highly purified alpha-toxin (phospholipase C) of Clostridium perfringens prepared by affinity chromatography on agarose-linked egg-yolk lipoprotein induced the in vitro aggregation of platelets of an irreversible type. The aggregation started after a time lag, the length of which depended on the concentration of the toxin; the reciprocal of the time lag was found to be directly proportional to the toxin concentration. Using this assay method, we demonstrated that the platelet-aggregating activity of alpha-toxin reached minimum at around 70 C but heating at higher temperatures inactivated it to a lesser extent; the same anomaly in heat inactivation was observed with phospholipase C activity possessed by the toxin. By subjecting purified alpha-toxin to isoelectric focusing, four molecular forms were isolated, all of which were associated with both the platelet-aggregating and phospholipase C activities. From all these results we concluded that the entity responsible for the platelet-aggregating activity is identical with alpha-toxin (phospholipase C).     

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

Normal Japanese individuals harbor polymorphisms in the p14 ARF /INK4 locus promoters and/or other gene introns. — Variation in nucleotide sequences in each individual

In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced promoters in the p14 ^ARF /INK4 (p16 ^INK4a and p15 ^INK4b ) locus genes and introns in murine double minute 2, vitamin D receptor, and presenilin 1 genes. We found nucleotide alterations in the promoters, including substitutions at positions ?2610 and ?1536 and deletions at positions ?4489, ?4488 to ?4483, ?2241 to ?2240, and ?2221 to ?2218 in p14 ^ARF and substitutions at positions ?1643 and ?1270 in p15 ^INK4b , and we found that each individual harbored polymorphisms in the locus promoters and/or introns. Some polymorphic nucleotides were included in the same set of associatively altered nucleotides. Reporter gene analysis by using luciferase revealed that altered nucleotides, including those containing the set, changed luciferase gene activity in some cell lines. The results of this study show that multi-nucleotide sequences are altered in each normal Japanese individual.     

Comparison of enzymatic properties between hPADI2 and hPADI4

In the sera of rheumatoid arthritis (RA) patients, autoantibodies directed to citrullinated proteins are found with high specificity for RA. Peptidylarginine deiminases (PADIs) are enzymes responsible for protein citrullination. Among many isoforms of PADIs, only PADI4 has been identified as an RA-susceptibility gene. To understand the mechanisms of the initiation and progression of RA, we compared the properties of two PADIs, human PADI2 and human PADI4, which are present in the synovial tissues of RA patients. We confirmed their precise distribution in the RA synovium and compared the stability, Ca2+ dependency, optimal pH range, and substrate specificity. Small but significant differences were found in the above-mentioned properties between hPADI2 and hPADI4. Using LC/MS/MS analysis, we identified the sequences in human fibrinogen indicating that hPADI2 and hPADI4 citrullinate in different manners. Our results indicate that hPADI2 and hPADI4 have different roles under physiological and pathological conditions. Further studies are needed for the better understanding of the role of hPADIs in the initiation and progression of RA.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.