Effect of habitual exercise on urinary liver-type fatty acid-binding protein levels in middle-aged and older adults.

The purpose of this study was to examine the effect of habitual exercise on urinary liver-type fatty acid-binding protein (L-FABP), which can reflect the degree of various stresses on renal proximal tubule related to the progression of renal disease, in middle-aged and older adults. Cross-sectional and interventional approaches were used to comprehensively achieve this purpose. In the cross-sectional study, we investigated the relationship between physical activity levels and urinary L-FABP levels in 130 middle-aged and older adults. In the interventional study, subjects (n=31) were divided into two groups: exercise (n=19) and control group (n=12), whereby we examined the effects of 12-week aerobic exercise training on urinary L-FABP levels. The cross-sectional study showed that the urinary L-FABP levels were significantly lower in the higher physical activity group than in the lower physical activity group (P<.05). In the interventional study, 12-week aerobic exercise training significantly decreased urinary L-FABP levels (P<.01). Furthermore, the relative changes in urinary L-FABP levels were significantly correlated with the relative changes in physical activity levels and mean arterial pressure after intervention (r=-.374 and r=.530, respectively). Our results revealed that the urinary L-FABP levels were lower in the higher physical activity individuals, and aerobic exercise training decreased urinary L-FABP levels. These results suggest that habitual exercise appears to be associated with a decrease in the degree of several stresses on renal proximal tubule and to be beneficial for kidney health in middle-aged and older adults.

Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

Prevalent founder mutation c.736T>A of LIPH in autosomal recessive woolly hair of Japanese leads to variable severity of hypotrichosis in adulthood.

BACKGROUND: Mutations in LIPH are a cause of autosomal recessive woolly hair (ARWH). Homozygous c.736T>A (p.Cys246Ser), and compound heterozygous c.736T>A and c.742C>A (p.His248Asn) have been reported in 5 and 7 Japanese children with ARWH respectively. The severity of hypotrichosis is known to be able to change in the clinical course, and the mutation patterns of LIPH do not always correlate with the severity of hypotrichosis in ARWH caused by other mutation sites of LIPH. However, all 12 Japanese children previously reported to have ARWH have shown similar severity of hypotrichosis. OBJECTIVE: In this study, we investigated the clinical features and molecular basis of ARWH in patients including three adults (three adults and two children) from five non-related Japanese families. METHODS: Five families of Japanese origin that presented with woolly hair were studied. The phenotype was confirmed by clinical examination. Direct automated DNA sequencing of the LIPH gene was performed to identify the mutations in our probands. RESULTS: All patients had had woolly hair since birth. Homozygous c.736T>A mutations were found in four patients, including three adult cases, and compound heterozygous c.736T>A and c.742C>A mutations were found in one child patient. The two adults and two children had only sparse scalp hair, although one adult woman had mild hypotrichosis with long hairs. CONCLUSION: Some patients with homozygous c.736T>A can have a mild hypotrichosis phenotype with long hairs in adulthood.

Zinc biosorption by a zinc-resistant bacterium, Brevibacterium sp. strain HZM-1.

A zinc-resistant bacterium, Brevibacterium sp. strain HZM-1 which shows a high Zn2+ -adsorbing capacity, was isolated from the soil of an abandoned zinc mine. Kinetic analyses showed that Zn2+ binding to HZM-1 cells follows Langmuir isotherm kinetics with a maximum metal capacity of 0.64 mmol/g dry cells and an apparent metal dissociation constant of 0.34 mM. The observed metal-binding capacity was one of the highest values among those reported for known microbial Zn2+ biosorbents. The cells could also adsorb heavy metal ions such as Cu2+. HZM-1 cells could remove relatively low levels of the Zn2+ ion (0.1 mM), even in the presence of large excess amounts (total concentration, 10 mM) of alkali and alkali earth metal ions. Bound Zn2+ ions could be efficiently desorbed by treating the cells with 10 mM HCl or 10 mM EDTA, and the Zn2+ -adsorbing capacity of the cells was fully restored by treatment of the desorbed cells with 0.1 M NaOH. Thus, HZM-1 cells can serve as an excellent biosorbent for removal of Zn2+ from natural environments. The cells could grow in the presence of significant concentrations of ZnCl2 (at least up to 15 mM) and thus is potentially applicable to in situ bioremediation of Zn2+ -contaminated aqueous systems.

Synthesis, in vitro binding profile and biodistribution of a 125I-labeled N-benzyl pyrrolidinyl benzamide derivative: a potential radioligand for mapping dopamine D2 receptors.

cis-N-(1-Benzyl-2-methylpyrrolidine-3-yl)-5-iodo-2-methoxy-4-(methylamin o) benzamide (IYM), a YM-09151-2 analog iodinated at the 5-position of the benzoyl moiety, was synthesized and evaluated as a potential radiopharmaceutical for investigating brain dopamine D2 receptors by single photon emission computer tomography (SPECT). [125I]IYM was synthesized by a halogen exchange reaction and purified by high-performance liquid chromatography (HPLC). An in vitro competitive binding study with [3H]spiperone using rat striatal synaptosomal membranes revealed that IYM had higher affinity for dopamine D2 receptors than did YM-09151-2 or spiperone. In a saturation binding study using rat striatal synaptosomal membranes, IYM had a Kd of 0.04 nM. Biodistribution studies in mice disclosed that [125I]IYM exhibited high and specific striatal uptake, with the striatal/cerebellar uptake ratio being 14 at 120 min after injection. Furthermore, the striatal uptake of [125I]IYM was saturable, and [125I]IYM was displaced only by dopaminergic compounds. Ex vivo autoradiographic studies in rats further confirmed the high uptake and retention of this agent in the striatum and total blockade of its uptake by YM-09151-2. Thus, IYM showed specific binding to dopamine D2 receptors in the rodent striatum and therefore holds great potential for use in in vivo dopamine D2 receptor studies.

Porcine aorta smooth-muscle myosin contains three species made of different combinations of two 17-kDa essential light-chain isoforms.

Porcine aorta myosin was reacted with a bifunctional cross-linking reagent, N,N'-o-phenylenedimaleimide. The 17-kDa essential light chain (LC17) in each myosin head was intramolecularly cross-linked within a single myosin molecule. The 34-kDa cross-linked LC17 dimer was isolated and its peptide map, after lysylendopeptidase digestion, was obtained by reverse-phase HPLC. Based on the amino acid compositions of peptide fragments, the N-terminal Cys residues of LC17 subunits were assigned to be cross-linked to each other. To study the distribution of two LC17 isoforms, LC17nm and LC17gi [Hasegawa, Y., Ueda, Y., Watanabe, M. & Morita, F. (1992) J. Biochem. 111, 798-803], aorta myosin was reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). The LC17 dimer cross-linked with Nbs2 was resolved into three distinct bands on urea/PAGE using a 4% acrylamide gel. Densitometric analysis of the three band intensities showed that three pairs of LC17 isoforms in aorta myosin are present in the ratio of LC17nm-LC17nm/LC17nm-LC17gi/C17gi-LC17g i = 22:46:32. This ratio is consistent with the random combination of two LC17 isoforms with myosin heavy chains.

Myocardial accumulation of a dopamine D2 receptor-binding radioligand, 2'-iodospiperone.

125I-2'-iodospiperone (2'-ISP), which has a high and selective affinity for dopamine D2 receptors, produced a high myocardial accumulation of radioactivity in the early phase after intravenous injection into mice. A human scintigraphic study also showed that the myocardium was clearly visualized soon after intravenous injection of the tracer. Analysis of the myocardial homogenate obtained from mice showed that 125I-2'-ISP was metabolically stable and was taken up the myocardium in its intact form. Administration of spiperone significantly reduced the myocardial uptake of 125I-2'-ISP in mice. Treatment with haloperidol and (+) butaclamol, which have a high affinity for dopamine D2 receptors, also tended to reduce the myocardial uptake of radioactivity, while (-)-butaclamol, which has no affinity for dopamine D2 receptors, caused no change in uptake. These findings suggest that the myocardial accumulation of 2'-ISP occurred in association with dopamine D2 (DA2) receptors.

A sensitive serodiagnosis of hepatitis C virus (HCV) infection with two non-fused peptides: comparison of antibody responses detected with a newly developed assay and a commercial second-generation test.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of anti-HCV antibody. We assayed for antibodies against either oligopeptide (S29-1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false-positive results induced by non-specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non-fused S4 peptide was prepared by the recombinant DNA technique and site-specific proteolysis (by factor Xa). In 71 non-A, non-B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29-1/S4 ELISA as well as by a second-generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti-N14 (core) and C100-3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29-1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti-S29-1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of nonfused protein (S4) by recombinant DNA technique and a combination of S29-1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.

Regulatory mechanism by the phosphorylation of 20-kDa light chain of porcine aorta smooth muscle myosin.

Porcine aorta smooth muscle myosin was subjected to limited proteolysis by Staphylococcus aureus protease (V8-protease) at 30 mM KCl, under which condition the myosin is in the filamentous form. The heavy chain of the myosin molecule was mainly digested at the 68-160 kDa junction, which corresponds to the 50-20-kDa junction in the heavy chain of skeletal muscle myosin subfragment-1 (S-1). When the filamentous myosin formed a rigor complex in the presence of F-actin, this site was blocked, and the junction between S-1 and subfragment-2 (S-2) was in turn digested specifically. Both phosphorylated and unphosphorylated 20-kDa light chain (LC20) in the aorta myosin remained intact under these conditions. The actin-activated ATPase activity of phosphorylated myosin was not influenced by the cleavage of the S-1-S-2 junction. With unphosphorylated myosin, however, the actin-activated ATPase activity increased with the cleavage of the S-1-S-2 junction and reached the level of ATPase activity of phosphorylated myosin at the stage of complete cleavage. The increase of ATPase activity was found to be proportional to the loss of double-headed myosin. The overall data indicate that LC20 works to suppress the actin-activated ATPase activity, and the suppression is released by the phosphorylation of LC20. The presence of two heads in myosin is required to reveal such regulation by LC20.

Genetic background of high incidence of spontaneous tumors in BDX rats.

Genetic background of high incidence of spontaneous tumors in BDX rats was investigated with respect to susceptibility to physical or chemical agents and immune response. Percentages of colony forming ability of normal embryo fibroblasts from BDX rats after exposure to various doses of ultraviolet (UV) light, X-rays and Mitomycin C were almost identical to those of cells from control F344 rats. Furthermore, BDX rats showed similar level of rosette forming cells, blastogenic response, and plaque forming cell (PFC) response as compared with WKA rats used as a control, although BDX rats showed higher natural killer (NK) cell activity. These results indicate that high incidence of spontaneous tumors in BDX rats is not due to some defects in DNA repair or immune response. In 3 methylcholanthrene (MCA) carcinogenesis, however, BDX rats developed tumors very early; the mean latency periods for development of MCA-induced tumors were 135 days in BDX rats and 256 days in F344 rats. Measurement of the inducibility of specific aryl hydrocarbon hydroxylase (AHH) activity in liver microsomes revealed that BDX rats showed higher inducibility than F344 rats: the inducibilities were 23.7 in BDX rats and 5.7 in F344 rats. Higher inducibility of AHH in BDX rats seemed to be parallel to earlier development of tumors in MCA-carcinogenesis in the rats. Thus, it is most likely that high incidence of spontaneous tumors in BDX rats is due to high susceptibility to some chemical carcinogens which may be contained in foods or drinking water in a small dose.

Immunohistochemical localization of hexose 6-phosphate dehydrogenase in various organs of the rat.

Distribution of hexose 6-phosphate dehydrogenase in various organs of the rat has been studied by a peroxidase-labeled antibody method in order to find some clue to elucidating the, as yet unclear, function of this enzyme. As a result, the following cells were found to contain this enzyme in relative abundance: hepatic parenchymal cells, ovarian lutein and theca interna cells, testicular interstitial cells, striated ducts and serous tubular portions of the submandibular gland, plasma cells and the P3 segment of proximal convolutions, and collecting tubules of the cortex and inner medulla of the kidney. Although the role of this enzyme in salivary glands and in plasma cells is unclear at present, the results obtained with steroidogenic cells, liver cells, and renal tubules appear to suggest the possibility that this enzyme might be involved in drug and steroid metabolism.