Biomedical subjects
Publications and source records attributed to M Ikeda.
[reaction: see text] A cerium(IV) double decker porphyrin (1) bearing four 4-methoxyphenyl groups was synthesized. Compound 1 shows a positive, homotoropic allosteric effect in metal recognition of Ag(+) ion, and the peripheral pi clefts of 1 act as effective binding sites for Ag(+) ion.
Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.
In myelinated fibers of the sural nerves in Charcot-Marie-Tooth disease 1A (CMT 1A), with peripheral myelin protein (PMP) 22 gene duplication, it has been controversial whether axonal attenuation occurs or the myelin sheath thickens. Therefore, the relationship between the transverse axonal area and the number of myelin lamellae was morphometrically studied in myelinated fibers of the sural nerves in CMT 1A with PMP22 gene duplication to re-evaluate such relationships as are revealed in hereditary motor sensory neuropathy, type 1 without genetic diagnosis. In electron microscopic studies both the axonal circumference and natural log (ln) axonal area were significantly (P < 0.01) smaller in the CMT 1A group (n = 8) than in the control group (n = 9). Myelin lamellae numbers, however, were similar in both groups. In a regression analysis relating ln axonal areas to the numbers of myelin lamellae, ln axonal area in the CMT 1A group was significantly (P < 0.05) smaller than in the control group in myelinated fibers with the numbers of myelin lamellae equal to or over 40. Therefore, it was concluded that the axonal area was smaller in large-diameter myelinated fibers in the CMT 1A group than in the control group. We speculated on the existence of axonal attenuation due to the impaired effect of Schwann cells on myelinated axons in CMT 1A with PMP22 gene duplication.
Despite the lower site 1 affinity of the 20-kDa human growth hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22-kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K- and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH.
To assess the molecular epidemiology of HIV-1 in Republic of Congo (Congo), we investigated 29 HIV-1s obtained from 82 Congolese AIDS and ARC patients in 1996 and 1997. Part of the env region including the V3 loop was phylogenetically analyzed. The genotypes observed were varied: of 29 specimens, 12 (41 %) were subtype A, 1 (3%) was subtype D, 6 (21%) were subtype G, 6 (21%) were subtype H, 2 (7%) were subtype J, and 2 (7%) could not be classified as any known subtypes (U, unclassified). The heterogeneous profile of HIV-1 infection was different from the profiles of neighboring Central African countries. These data show that subtypes G and H as well as subtype A were circulating with high prevalence. The fact that new genetic subtypes (J and U) are circulating indicates a need for a greater surveillance for these subtypes both in Congo as well as in other parts of the world.
Ca(2+)-activated K(+) channels (K(Ca)) regulate a wide variety of cellular functions by coupling intracellular Ca(2+) concentration to membrane potential. There are three major groups of K(Ca) classified by their unit conductances: large (BK), intermediate (IK), and small (SK) conductance of channels. BK channel is gated by combined influences of Ca(2+) and voltage, while IK and SK channels are gated solely by Ca(2+). Volatile anesthetics inhibit BK channel activity by interfering with the Ca(2+) gating mechanism. However, the effects of anesthetics on IK and SK channels are unknown. Using cloned IK and SK channels, hIK1 and hSK1-3, respectively, we found that the currents of hIK1 were inhibited rapidly and reversibly by volatile anesthetics, whereas those of SK channels were not affected. The IC(50) values of the volatile anesthetics, halothane, sevoflurane, enflurane, and isoflurane for hIK1 inhibition were 0.69, 0.42, 1.01 and 1.03 mM, respectively, and were in the clinically used concentration range. In contrast to BK channel, halothane inhibition of hIK1 currents was independent of Ca(2+) concentration, suggesting that Ca(2+) gating mechanism is not involved. These results demonstrate that volatile anesthetics, such as halothane, enflurane, isoflurane, and sevoflurane, affect BK, IK, and SK channels in distinct ways.
We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting that expressed hGHR is functionally active. Comparative analysis using bGH and hGH revealed that 70% of lipolysis stimulation by 1-10 ng/ml hGH could be attributed to hGHR-mediated response. Analyses on inhibition and phosphorylation of signaling molecules suggested that GH-induced lipolysis stimulation is dependent on gene expression and not mediated through PKA-, PKC-, PLA-, PLC-, nor MAPK-pathway but possibly through JAK-STATs pathway. Duration of STAT5 activation by hGH continued up to 48 h. We also revealed that 22 K hGH isoform, 20K hGH which has been reported as a weaker agonist for GH-induced lipolysis stimulation, possesses equipotent activity and shows stronger action in the presence of hGHBP as compared to 22 K hGH. Taken together we conclude that the hGH-induced lipolysis was not mediated through MAP-, PKA-, PKC-, nor PLA-pathway but might be mediated through STAT pathway and that 20K hGH might show higher lipolytic activity than 22 K hGH in adipose tissue that produces a large amount of GHBP.
Information is still scarce for many Asian countries on general population exposure to two potentially toxic heavy metals of lead (Pb) and cadmium (Cd). The present review on publications from this study group is intended to provide an overview of the non-occupational exposure to Pb and Cd among general populations in east and south-east Asia. During the period of 1991-1998, surveys were conducted in four cities in south-east Asia, five cities in mainland China, and two cities each in Japan and Korea. Peripheral blood, morning spot urine and 24-h food duplicate samples were collected from 20-50 non-smoking adult women in each survey site. The samples were digested by heating in the presence of mineral acids, and then analyzed by inductively-coupled plasma mass spectrometry for the metals in blood (Pb-B and Cd-B), in urine (Pb-U and Cd-U), and in food duplicate (Pb-F and Cd-F). Measures were subjected to statistical analysis with an assumption of log-normal distribution. Pb-B, Pb-U and Pb-F levels varied substantially among the 11 urban sites; GM distributed in ranges of 32-65 microg/l for Pb-B, 2.1-7.5 microg/g cr (creatinine) for Pb-U, and 7-32 microg/day for Pb-F. The same was also the case for Cd exposure parameters, the distribution ranges being 0.5-1.8 microg/l for Cd-B, 1.2-3.1 microg/g cr for Cd-U, and 5-32 microg/day for Cd-F. It appeared that the exposure to Pb and Cd was highest in Kuala Lumpur, and Tokyo + Kyoto, respectively, and lowest in Tokyo + Kyoto and in Manila. Additional surveys in Japan showed that Pb exposure was even lower in rural areas than in Tokyo + Kyoto. When compared with the values reported in the literature, Pb exposure levels among Asian populations appear to be similar to the levels in Europe and in the United States, whereas Cd exposure seems to be higher in Asia than in Europe. The contribution of the dietary route in Pb absorption was variable and was inversely related to the extent of air pollution, whereas Cd uptake was almost exclusively via the dietary route with little contribution of the respiratory route.
To clarify the role of glial fibrillary acidic protein (GFAP)-expressed glial cells in the circadian clock, we examined GFAP expression in the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) under various lighting conditions in mice. We demonstrated that GFAP expression did not show daily change in the SCN under a light-dark cycle; however, long-term housing under constant lighting conditions led to dramatic changes in GFAP expression, i.e., a decrease in the SCN and an increase in the IGL. Furthermore, mice that had a targeted deletion in the GFAP gene (GFAP mutant mice) showed longer and more arrhythmic circadian activity rhythms in constant lighting conditions than wild-type mice, while GFAP mutant mice exhibited stable circadian rhythms both in a light-dark cycle and constant darkness, and showed normal entrainment to environmental light stimuli. These results suggest that the GFAP-expressed astroglial cells in the SCN and the IGL may have some role in circadian oscillation under constant lighting conditions.
Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells.
Cholinergic regulation of the suprachiasmatic nucleus (SCN) has been extensively studied although the intracellular signaling mechanisms are not well understood. We examined immunostaining for phospholipase C-beta (PLC-beta) families that couple to muscarinic acetylcholine receptors (mAChR) and demonstrated the expression of PLC-beta 1 and beta 4 in the mouse SCN. Ca2+ imaging analysis indicated that the MI-mAChR antagonist, pirenzepine blocked carbachol-induced Ca2+ elevation in the SCN and the response was equivalent between the wild type and the PLC-beta 4-knockout mice. In addition, the knockout mice displayed locomotor and temperature rhythms coupling to 24 h light/dark cycles. Therefore, it was proposed that PLC-beta 1 but not PLC-beta 4 was involved in the mAChR-mediated Ca2+ signaling in the SCN.
Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. LMP2A functions to downregulate B-cell signal transduction and viral reactivation from latency in EBV-immortalized B cells in vitro, and acts to provide B cells with both a survival and developmental signal in vivo. Identification of proteins associated with LMP2A is important for elucidation of the mechanism that LMP2A employs to regulate B-cell signal transduction and EBV latency. LMP2A is constitutively tyrosine phosphorylated and is associated with protein tyrosine kinases such as Lyn and Syk when specific LMP2A tyrosines are phosphorylated. The amino-terminal domain of LMP2A includes multiple proline-rich regions, which may provide binding sites for proteins containing SH3 or WW domains. In this study, we demonstrate that four cellular proteins bind specifically to two PPPPY (PY) motifs present within the LMP2A amino-terminal domain. Protein microsequence analysis determined that three of these proteins were AIP4, WWP2/AIP2, and Nedd4. All of these proteins are members of the Nedd4-like ubiquitin-protein ligases family and have conserved domains including the C2, WW, and ubiquitin-protein ligase domain. The mutation of both PY motifs completely abolished binding activity of these proteins to LMP2A and the interaction of AIP4 and WWP2 with LMP2A was confirmed in cell lines expressing LMP2A, WWP2, and AIP4. Furthermore, a reduction in the level of Lyn and the rapid turnover of LMP2A and Lyn were observed in LMP2A-expressing cells. These findings suggest that LMP2A recruits Nedd4-like ubiquitin-protein ligases and B-cell signal transduction molecules, resulting in the degradation of LMP2A and Lyn by a ubiquitin-dependent mechanism. This provides a new means by which LMP2A may modulate B-cell signal transduction.
In Japan, the proportion of hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) is 40%, whereas more than 90% are infected with hepatitis C virus (HCV). To evaluate the immunological status of hemophiliacs infected with HIV-1, we investigated the pattern of cytokine production in peripheral blood mononuclear cells (PBMCs) of HIV-1-seropositive and -seronegative hemophiliacs, HIV-1-seropositive non-hemophiliacs, and healthy individuals. The production of IL-18 and IL-1beta from PBMCs stimulated with Staphylococcus aureus Cowan strain 1 (SAC) in the HIV-1-seropositive hemophiliacs was significantly decreased in comparison with the other groups. On the other hand, IL-12 production in both HIV-1-seropositive groups was significantly lower than in HIV-1-seronegative groups. TNF-alpha and IL-6 production was similar among the four groups. In contrast, plasma levels of TGF-beta1 were increased in HIV-1-seropositive hemophiliacs, HIV-1-seropositive nonhemophiliacs, and HIV-1-seronegative hemophiliacs, with the highest levels being in HIV-1-seropositive hemophiliacs, suggesting that coinfection with HIV-1 and HCV increases the level of plasma TGF-beta in HIV-1-seropositive hemophiliacs. Treatment of PBMCs from healthy individuals with TGF-beta1 inhibited IL-18 and IL-1beta production without affecting IL-6, IL-10, or TNF-alpha production. Suppression of the expression of caspase 1 mRNA, which is known to be an IL-1beta-converting enzyme and which also cleaves the precursor of IL-18, was observed in the SAC-stimulated PBMCs from healthy individuals after treatment with TGF-beta1 and in the SAC-stimulated PBMCs from HIV-1-seropositive hemophiliacs, suggesting that the decreased production of IL-18 and IL-1beta in HIV-1-seropositive hemophiliacs may be related to the downregulation of caspase 1 mRNA induced by high levels of TGF-beta1 in plasma.
The objectives of the present study are: (a) to clarify the current levels of environmental exposure to lead (Pb) and cadmium (Cd) in Shaanxi Province in China in comparison with levels in other parts of mainland China; (b) to examine if there is any urban-rural difference in Pb and Cd exposure; and (c) to quantify the role of cereals as the dietary source of environmental exposure to these metals. For this purpose, triplet surveys on lead and cadmium exposure were conducted in the provincial capital of Xian and two farming villages A and B in Shaanxi Province, China in 1997. The grand geometric mean for lead (Pb) intake via foods (Pb-F), Pb in blood (Pb-B) and Pb in urine as corrected for creatinine concentration (Pb-Ucr) were 30 micrograms/day, 33 micrograms/l and 5 micrograms/g creatinine, respectively, with significant differences among the survey sites, e.g. Pb-B being higher in Xian (43 micrograms/l) than in the two villages (38 and 22 micrograms/l). The counterpart values for cadmium (Cd) intake via foods (Cd-F), Cd in blood (Cd-B) and Cd in urine (Cd-Ucr) were 6.1 micrograms/day, 0.46 microgram/l and 2.8 micrograms/g creatinine, respectively, with no substantial inter-survey site difference. Thus, it was possible to conclude that, from comparison with the values reported in 1990s literature, the exposure of Shaanxi people to Pb and Cd is no higher than, and even possibly lower than, the levels reported for people in other parts of mainland China. The exposure to Cd was almost exclusively from foods, whereas the exposure to air-borne Pb was large enough in Xian to explain higher Pb-B and Pb-Ucr than the level in Village B despite lower Pb-F in Xian than in Village B. Cereals (wheat, rice, maize and foxtail millet) contributed 26 and 84% of dietary Pb and Cd intake, respectively.
Mammalian secretory phospholipase A(2)s (sPLA(2)s) form a family of structurally related enzymes that are involved in a variety of physiological and pathological processes via the release of arachidonic acid from membrane phospholipids or the binding to specific membrane receptors. Here, we report the cloning and characterization of a novel sPLA(2) that is the sixth isoform of the sPLA(2) family found in humans. The novel human mature sPLA(2) consists of 123 amino acids (M(r) = 14,000) and is most similar to group IIA sPLA(2) (sPLA(2)-IIA) with respect to the number and positions of cysteine residues as well as overall identity (51%). Therefore, this novel sPLA(2) should be categorized into group II and called group IIE (sPLA(2)-IIE) following the recently identified group IID sPLA(2) (sPLA(2)-IID). The enzymatic properties of recombinant human sPLA(2)-IIE were almost identical to those of sPLA(2)-IIA and IID in terms of Ca(2+) requirement, optimal pH, substrate specificity, as well as high susceptibility to the sPLA(2) inhibitor indoxam. Along with the biochemical properties of proteins, genetic and evolutional similarities were also observed among these three types of group II sPLA(2)s as to the chromosomal location of the human gene (1p36) and the exon/intron organization. The expression of sPLA(2)-IIE transcripts in humans was restricted to the brain, heart, lung, and placenta in contrast to broad expression profiles for sPLA(2)-IIA and -IID. In sPLA(2)-IIA-deficient mice, the expression of sPLA(2)-IIE was markedly enhanced in the lung and small intestine upon endotoxin challenge, which contrasted with the reduced expression of sPLA(2)-IID mRNA. In situ hybridization analysis revealed elevation of sPLA(2)-IIE mRNA at alveolar macrophage-like cells in the lung of endotoxin-treated mice. These findings suggest a distinct functional role of novel sPLA(2)-IIE in the progression of inflammatory processes.
Recent studies suggest that melatonin 1b (Mel1b) receptor, as well as melatonin 1a (Mel1a) receptor, is involved in the modulation of circadian rhythms in mammals. Mutational analysis was performed in the entire coding region of the human Mel1b receptor gene using genomic DNA from sleep disorder subjects. We have identified two missense mutations, G24E and L66F. However, neither is likely to be associated with sleep disorders in our study population. One of the subjects with non-24-h sleep-wake syndrome carries missense mutations in both the Mel1a and Mel1b receptor genes.
The objectives of the present study are to examine if there exists a quantitative relationship between lead in urine (Pb-U) and that in blood (Pb-B), and also between cadmium in urine (Cd-U) and that in blood (Cd-B) among the general populations who are environmentally (and not occupationally) exposed to these elements at various levels. For this purpose, peripheral blood and morning spot urine samples were collected in 1991-1998 from approximately 50 non-smoking adult women each in four cities in south-east Asia and five cities in mainland China, and two cities each in Japan and Korea. The samples were wet-ashed and then analyzed by inductively-coupled plasma mass spectrometry for Pb-B, Cd-B, Pb-U and Cd-U. Measured values were subjected to analysis to detect possible correlation between the pairs of parameters. A significant correlation between Pb-B and Pb-U was observed when the intensity of Pb exposure (as expressed by Pb-B) was relatively high so that the correlation was significant in all cases studied when Pb-B level was, e.g. 50 microg/l or above. It was also observed that the correlation between Cd-B and Cd-U was significant when Cd-B was, e.g. > 1 microg/l. Thus, it is possible to deduce that, in environmental health, Pb-B and Cd-U levels can be estimated on a group basis from Pb-U and Cd-B, respectively, when Pb and Cd exposure levels are relatively high, e.g. with Pb-B and Cd-B of > 50 microg/l and > 1 microg/l.
Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.