Frequency distribution of axon diameters in the left aortic nerve of the rat raised in space--Neurolab Program.

Introduction: To study development of the aortic baroreflex mechanisms under conditions of microgravity, we carried out the various experiments in the neonate rats aged 25 days raised in microgravity for 16 days (flight: FLT group) in Neurolab program (STS-90, space shuttle Columbia, launch date: Apr 17, '98. Some results of the experiments were already reported. The results of histological examination of the aortic nerve which is the afferent of the aortic baroreflex showed that the number of unmyelinated fibers in FLT was significantly less in than those in two control groups and there was no difference between FLT and each control group in the analysis of myelin. In the present paper, the frequency distribution of axon diameters of the left aortic nerves in FLT was compared to that in two ground control groups to examine the growth of the aortic nerve fibers in space. Methods: After breeding Sprague-Dawley rats for 16 days in the shuttle in space and in the animal center in the Kennedy space center, a total of 43 deeply anesthetized rats were perfused with 1% parahormaldehyde and 1% glutaraldehyde or 4% parahormaldehyde solution buffered at pH7.4 with 0.12 M phosphate solution. Concerning the control groups, one group was the asynchronous ground control (AGC) group in which the rats were housed in the same cages as those on the shuttle, and the other was the vivarium ground control (VIV) group in which the rats were housed in commercial cages. The cervical region of the left aortic nerve which is a branch of the vagus was cut off and stored in the same fixative as that used for perfusion, and postfixed in the solution of 1% OsO4, for 2 hours within 24 hours after the perfusion. The fixed specimens were embedded in epoxy resin blocks by the usual method for electron microscopy following dehydration. Electron microscopic montages of transverse sections of these nerve trunks were made from the five left aortic nerves in each group. The magnification of the montages was approximately 13400 times. The long and short axes (a and b) of the nerve fibers and the myelin thickness (T) were measured with a caliper and the axon diameters (R were calculated by following formula: R2=[(a-2T)2+(b-2T)2]/2.     

Critical Amino Acid Variants in HLA-DRB1 and -DQB1 Allotypes in the Development of Classical Type 1 Diabetes and Latent Autoimmune Diabetes in Adults in the Japanese Population

The effects of amino acid variants encoded by the human leukocyte antigen (HLA) class II on the development of classical type 1 diabetes (T1D) and latent autoimmune diabetes in adults (LADA) have not been fully elucidated. We retrospectively investigated the HLA-DRB1 and -DQB1 genes of 72 patients with classical T1D and 102 patients with LADA in the Japanese population and compared the frequencies of HLA-DRB1 and -DQB1 alleles between these patients and the Japanese populations previously reported by another institution. We also performed a blind association analysis with all amino acid positions in classical T1D and LADA, and compared the associations of HLA-DRB1 and -DQB1 amino acid positions in classical T1D and LADA. The frequency of DRß-Phe-13 was significantly higher and those of DRß-Arg-13 and DQß-Gly-70 were significantly lower in patients with classical T1D and LADA than in controls. The frequencies of DRß-His-13 and DQß-Glu-70 were significantly higher in classical T1D patients than in controls. The frequency of DRß-Ser-13 was significantly lower and that of DQß-Arg-70 was significantly higher in LADA patients than in controls. HLA-DRß1 position 13 and HLA-DQß1 position 70 could be critical amino acid positions in the development of classical T1D and LADA.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

Molecular cloning and functional characterization of mouse Nxf family gene products

Tap, a member of the evolutionarily conserved nuclear RNA export factor (NXF) family of proteins, has been implicated in the nuclear export of bulk poly(A)+ RNAs. cDNAs encoding the mouse NXF proteins (Tap, NXF7, NXF2, and NXF3) were prepared and the gene products were characterized in terms of their genomic organization, expression patterns, and biochemical properties. Mouse Tap was found to be ubiquitously expressed, whereas tissue- and developmental stage specific expression of mouse Nxf2, Nxf3, and Nxf7 was observed. Although mouse Tap and NXF2 bound to the phenylalanine-glycine repeat sequences of nucleoporins, NXF7 and NXF3 did not. GFP-tagged mouse Tap and NXF2 were localized predominantly in the nucleus. In contrast, GFP-tagged NXF7 and NXF3 were localized exclusively in the cytoplasm. As shown for the human counterpart, disruption of the leucine-rich nuclear export signal or leptomycin B treatment abolishes the cytoplasmic localization of mouse NXF3. p15/NXT1, an essential cofactor for human Tap in the export of mRNAs, was able to bind to mouse Tap, NXF2, and NXF3, but NXF7 did not form a stable heterodimeric complex. Transient transfection experiments indicated that only mouse Tap and NXF2 enhance the nuclear export of an otherwise inefficiently exported mRNA substrate. The orthologous relationship between human and mouse Nxf genes is discussed on the basis of these data.     

Phosphorylation of RanGAP1 stabilizes its interaction with Ran and RanBP1

Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 (358S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358S phosphorylation of RanGAP1 affects the Ran system.     

Revisiting the importance of model fitting for model-based fMRI: It does matter in computational psychiatry

Computational modeling has been applied for data analysis in psychology, neuroscience, and psychiatry. One of its important uses is to infer the latent variables underlying behavior by which researchers can evaluate corresponding neural, physiological, or behavioral measures. This feature is especially crucial for computational psychiatry, in which altered computational processes underlying mental disorders are of interest. For instance, several studies employing model-based fMRI—a method for identifying brain regions correlated with latent variables—have shown that patients with mental disorders (e.g., depression) exhibit diminished neural responses to reward prediction errors (RPEs), which are the differences between experienced and predicted rewards. Such model-based analysis has the drawback that the parameter estimates and inference of latent variables are not necessarily correct—rather, they usually contain some errors. A previous study theoretically and empirically showed that the error in model-fitting does not necessarily cause a serious error in model-based fMRI. However, the study did not deal with certain situations relevant to psychiatry, such as group comparisons between patients and healthy controls. We developed a theoretical framework to explore such situations. We demonstrate that the parameter-misspecification can critically affect the results of group comparison. We demonstrate that even if the RPE response in patients is completely intact, a spurious difference to healthy controls is observable. Such a situation occurs when the ground-truth learning rate differs between groups but a common learning rate is used, as per previous studies. Furthermore, even if the parameters are appropriately fitted to individual participants, spurious group differences in RPE responses are observable when the model lacks a component that differs between groups. These results highlight the importance of appropriate model-fitting and the need for caution when interpreting the results of model-based fMRI.     

Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts

DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl₂, NiCl₂, ZnCl₂ and MnCl₂. In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.     

Comparative Studies of the Thermodynamic Stabilities Between Sheared A:G and Watson-Crick A:U(T) Base Pairs in RNA and DNA

Abstract

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)₂ and d(GGACGAGTCC)₂, both of which form two consecutive ‘sheared’ A:G base pairs at the center [Katahira et al. (1993) Nucleic Acids Res. 21, 5418–5424; Katahira et al., (1994) Nucleic Acids Res. 22, 2752–27591. The parameters were determined also for r(GGACUAGUCC)₂ and d(GGACTAGTCC)₂, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes. The characteristic features in CD spectra of RNA and DNA containing the sheared A:G base pairs are also reported.

     

Nishiyama Reservoir: Lead Sources,Inventory, and the Influence of the Nagasaki Atomic Bomb

Sediment core samples were obtained from Nishiyama Reservoir, 3 km east of the hypocenter of the Nagasaki atomic bomb, to evaluate both sources and inventories of lead delivery to the sediments. Using microwave digestions and ICP-MS analytical techniques, elevated concentrations of isotopically unique lead were identified in the 1945 sediment horizon. These results illustrate that the Nagasaki bomb attack can be linked with unique lead loading to the sediments in the region of the hypocenter. There is a possibility that the roll-up of in-situ lead accounted for a majority of the increase in lead flux delivered to reservoir sediments during the atomic bomb attack.     

Hypoxia-induced phenotypic transition from highly invasive to less invasive tumors in glioma stem-like cells: Significance of CD44 and osteopontin as therapeutic targets in glioblastoma

The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive glioma stem-like cells (GSCs) in tumors. Upon GBM recurrence, GSCs with highly invasive and highly migratory activities must assume a less-motile state and proliferate to regenerate tumor mass. Elucidating the molecular mechanism underlying this transition from a highly invasive phenotype to a less-invasive, proliferative tumor could facilitate the identification of effective molecular targets for treating GBM. Here, we demonstrate that severe hypoxia (1% O₂) upregulates CD44 expression via activation of hypoxia-inducible factor (HIF-1α), inducing GSCs to assume a highly invasive tumor. In contrast, moderate hypoxia (5% O₂) upregulates osteopontin expression via activation of HIF-2α. The upregulated osteopontin inhibits CD44-promoted GSC migration and invasion and stimulates GSC proliferation, inducing GSCs to assume a less-invasive, highly proliferative tumor. These data indicate that the GSC phenotype is determined by interaction between CD44 and osteopontin. The expression of both CD44 and osteopontin is regulated by differential hypoxia levels. We found that CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo. Mouse brain tumors generated from CD44-knockdown GSCs exhibited diminished invasiveness, and the mice survived significantly longer than control mice. In contrast, siRNA-mediated silencing of the osteopontin gene decreased GSC proliferation. These results suggest that interaction between CD44 and osteopontin plays a key role in tumor progression in GBM; inhibition of both CD44 and osteopontin may represent an effective therapeutic approach for suppressing tumor progression, thus resulting in a better prognosis for patients with GBM.