Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Acid hydrolase activity of cultured bovine oligodendrocytes

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8–11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.     

Molecular design of novel metal-binding oligomeric human metallothioneins

 Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apo-metallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245–255 nm. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These novel oligomeric hMTs may be useful in bioremediation for heavy metals. Received: 18 October 1999 / Received last revision: 21 January 2000 / Accepted: 13 February 2000     

Uphill energy transfer in a chlorophyll d-dominating oxygenic photosynthetic prokaryote, Acaryochloris marina

The steady-state fluorescence properties and uphill energy transfer were analyzed on intact cells of a chlorophyll (Chl) d-dominating photosynthetic prokaryote, Acaryochloris marina. Observed spectra revealed clear differences, depending on the cell pigments that had been sensitized; using these properties, it was possible to assign fluorescence components to specific Chl pigments. At 22 degrees C, the main emission at 724 nm came from photosystem (PS) II Chl d, which was also the source of one additional band at 704 nm. Chl a emissions were observed at 681 nm and 671 nm. This emission pattern essentially matched that observed at -196 degrees C, as the main emission of Chl d was located at 735 nm, and three minor bands were observed at 704 nm, 683 nm, and 667 nm, originating from Chl d, Chl a, and Chl a, respectively. These three minor bands, however, had not been sensitized by carotenoids, suggesting specific localization in PS II. At 22 degrees C, excitation of the red edge of the absorption band (which, at 736 nm, was 20 nm longer than the absorption maximum), resulted in fluorescence bands of Chl d at 724 nm and of Chl a at 682 nm, directly demonstrating an uphill energy transfer in this alga. This transfer is a critical factor for in vivo activity, due to an inversion of energy levels between antenna Chl d and the primary electron donor of Chl a in PS II.     

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.     

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.     

Clinical effects of elastic bandage on neurogenic orthostatic hypotension

Neurogenic orthostatic hypotension (OH) often causes troublesome symptoms such as dizziness, syncope and falling, interfering active daily life or various therapies in rehabilitation. Nonpharmacologic measures for treating patients with OH include wearing elastic leotard, head-up tilting at night, etc. Elastic garment or antigravity suits is certainly effective, but it may be uncomfortable and not practical. Although elastic bandage (EB) bound on the lower limbs has been thought to be useful, there is few clinical report about its beneficial evidence. We investigated short-term clinical effects of commercially available EB on OH, and estimated the mechanism of its effectiveness by measuring some blood pressure-related humoral variables in neurodegenerative patients with OH.     

Coumarins as novel 17β-hydroxysteroid dehydrogenase type 3 inhibitors for potential treatment of prostate cancer

The synthesis and SAR studies of 3- and 4-substituted 7-hydroxycoumarins as novel 17β-HSD3 inhibitors are discussed. The most potent compounds from this series exhibited low nanomolar inhibitory activity with acceptable selectivity versus other 17β-HSD isoenzymes and nuclear receptors.