Hirosaki Medical Journal

Online ISSN: 0439-1721
Publications
Article
Amyloid molecules harboring pyroglutamate (pGlu) residue at the N-termini are considered to be important for the development of cerebral amyloidosis such as Alzheimer's disease and thought to be either spontaneously generated or being catalyzed by glutaminyl cyclase. Familial British dementia (FBD) is an autosomal dominant form of dementia neuropathologically characterized by parenchymal amyloid and preamyloid deposits, extensive cerebral amyloid angiopathy, and neurofibrillary tangles. FBD is caused by a stop to Arg mutation in the BRI2 gene, generating de novo created amyloid molecule ABri which accumulates in FBD brains but is not present in the normal population. Soluble ABri molecules present in the circulation of carriers of the BRI2 mutation are 34 amino acids long exclusively harboring Glu residue at the N-termini (ABri1-34E), whereas water- and formic acid-soluble ABri molecules extracted from FBD brains have abundant ABri species bearing pGlu residue (ABri1-34pE), suggesting that pyroglutamate formation occurs at the site of deposition. In order to further clarify the mechanism (s) of ABri deposition, we studied whether pyroglutamate formation indeed occurs outside the central nervous system taking advantage that FBD is also a systemic amyloidosis. Soluble and fibrillar ABri molecules extracted from systemic organs and analyzed biochemically using a combination of immunoprecipitation, mass spectrometry, and western blot analysis were oligomeric in size and contained a large proportion of ABri1-34pE. The data indicate that pyroglutamate formation at the N-termini of ABri molecules is an early step in the process of FBD amyloid deposition, and its formation is not restricted to the central nervous system.
 
Article
Cerebral amyloid angiopathy (CAA) is increasingly recognized as a major contributor of Alzheimer's disease (AD) pathogenesis. To date, vascular deposits and not parenchymal plaques appear more sensitive predictors of dementia. Amyloid deposition in and around cerebral blood vessels plays a central role in a series of response mechanisms that lead to changes in the integrity of the blood-brain barrier, extravasations of plasma proteins, edema formation, release of inflammatory mediators and matrix metalloproteases which, in turn, produce partial degradation of the basal lamina with the potential to develop hemorrhagic complications. The progressive buildup of amyloid deposits in and around blood vessels chronically limits blood supply and causes focal deprivation of oxygen, triggering a secondary cascade of metabolic events several of which involve the generation of nitrogen and oxygen free radicals with consequent oxidative stress and cell toxicity. Many aspects of CAA in early- and late-onset AD -the special preference of Aβ40 to deposit in the vessel walls, the favored vascular compromise associated with many Aβ genetic variants, the puzzling observation that some of these vasculotropic variants solely manifest with recurrent hemorrhagic episodes while others are mainly associated with dementia- await clarification. Non-Aβ cerebral amyloidoses reinforce the viewpoint that plaque burden is not indicative of dementia while highlighting the relevance of nonfibrillar lesions and vascular involvement in the disease pathogenesis. The lessons learned from the comparative study of Aβ and non-Aβ cerebral amyloidosis provide new avenues and alternative models to study the role of amyloid in the molecular basis of neurodegeneration.
 
Article
Cytokines including tumor necrosis factor alpha (TNF) play a role in sleep regulation in health and disease. Hypothalamic and cerebral cortical levels of TNF mRNA or TNF protein have diurnal variations with higher levels associated with greater sleep propensity. Sleep loss is associated with enhanced brain TNF. Central or systemic TNF injections enhance sleep. Inhibition of TNF using the soluble TNF receptor, or anti-TNF antibodies, or a TNF siRNA reduces spontaneous sleep. Mice lacking the TNF 55 kD receptor have less spontaneous sleep. Injection of TNF into sleep regulatory circuits, e.g. the hypothalamus, promotes sleep. In normal humans, plasma levels of TNF co-vary with EEG slow wave activity (SWA) and in multiple disease states plasma TNF increases in parallel with sleep propensity. Downstream mechanisms of TNF-enhanced sleep include nitric oxide, adenosine, prostaglandins and activation of nuclear factor kappa B. Neuronal use induces cortical neurons to express TNF and if applied directly to cortical columns TNF induces a functional sleep-like state within the column. TNF mechanistically has several synaptic functions. TNF-sleep data led to the idea that sleep is a fundamental property of neuronal/glial networks such as cortical columns and is dependent upon past activity within such assemblies. This view of brain organization of sleep has profound implications for sleep function that are briefly reviewed herein.
 
Article
TDP-43 proteinopathy (amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions) is a newly categorized group of neurodegenerative disorders characterized by abnormal accumulation and mislocalization of nuclear TDP-43 protein in the neuronal cytoplasm. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is non-enzymatically produced from PGD2, and plays roles in infl ammation and oxidative stress responses. Indeed, 15d-PGJ2 is up-regulated in the spinal motor neurons in ALS. In this study, biochemical and fluorescent staining analyses showed that 15d-PGJ2 modifi es expression, solubility, and subcellular localization of TDP-43. This alteration was at least partly related to a cyclopentenone ring structure containing an electrophilic carbon of 15d-PGJ2, because 15d-PGJ2 analogue, which lacks an cyclopentenone ring structure, had almost no eff ect on TDP-43 protein. Finally in vitro binding experiment indicated that 15d-PGJ2 is covalently bound to TDP-43 protein. These fi ndings suggest that a sustained high level of 15d-PGJ2 is involved in the pathogenesis of neurodegenerative disorders related to abnormal TDP-43 protein.
 
Article
Clumping factor A (ClfA) is an adhesin of Staphylococcus aureus, and the residues 40-559 of ClfA (ClfA40- 559) compose fi brinogen binding domain. It was reported that antibody to ClfA40-559 plays an important role in the protective eff ects. In this study, we investigated the role of IL-17 in the protective eff ects of an immunization with ClfA40-559 against S. aureus infection.  Mice immunized or non-immunized with ClfA40-559 were challenged with S. aureus. Bacterial numbers in the organs and survival rates were evaluated. RAW264.7, DC2.4 and spleen cells of naïve and immunized mice were stimulated with ClfA40-559. Cytokine production and mRNA expression in the spleens and cells was determined by ELISA and/or RT-PCR.   The survival rate was improved and the bacterial numbers in the organs were reduced in the mice immunized with ClfA40-559. ClfA40-559 induced IL-23p19 mRNA expression and IL-6 but not IL-12 production in DC2.4 and RAW264.7 cells. RORγt and IL-17A mRNA expression in the spleen cells of naïve mice was induced. In the spleen cells of immunized mice, ClfA40-559 induced the high production of IL-17 but not IFN-γ or IL-4. IL-17A, IL-6 and CXCL2 mRNA expression was also increased in the organs of immunized and challenged mice. These results suggest that an immunization with ClfA40-559 induces Th17, and that IL-17 contributes to host defense in early phase of S. aureus infection by promoting neutrophil recruitment.
 
Article
Nafamostat mesilate( FUT-175), a synthetic serine protease inhibitor, has been reported to have antitumour activities toward solid tumours. The objective of this study was to characterize the biological activities of FUT-175 in a malignant mesothelioma cell line. We used MSTO-211H, a biphasic-type human malignant pleural mesothelioma cell line. The effect on cell growth was evaluated by 3(- 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Secretion of urokinase-type plasminogen activator( u-PA) and plasminogen activator inhibitor-1( PAI-1) was analysed by enzyme-linked immunosorbent assay. The eff ects on relative mRNA expression levels were measured by reverse transcription polymerase chain reaction. The eff ect on cell invasiveness was evaluated by cell invasion assay. FUT-175 at 10-5 M signifi cantly inhibited cell growth and cell invasiveness. Cell growth reduced to 47.0 ± 2.1% compared with the control. The number of invasive cells also reduced to 16.0 ± 0.7 cells/hpf, while that of control cells was 41.4 ± 8.0 cells /hpf. U-PA and PAI-1 secreted from the cells were also reduced by FUT-175 in a dose-dependent manner. These results suggest that FUT-175 has the potential to act as a therapeutic agent against local growth and invasion, and functions by reducing PAI-1 and u-PA production of the human malignant mesothelioma(195 words)
 
Requirement of on-going protein synthesis for the TGF-/31 effect To examine whether the TGF-/31-elicited down-regulation of the BPAGI mRNA was dependent on on-going protein synthesis, similar inhibition studies were performed in the presence and absence of cycloheximide. Incubation of parallel cultures with cycloheximide (CHX) alone resulted in inhibition of BPAGI mRNA. However, the adition of TGF-/31 failed to down-regulate the BPAGI mRNA levels in the presence of CHX.  
Article
230-kD bullous pemphigoid antigen (BPAG1) is known as an autoantigen in bullous pemphigoid and is expressed exclusively in proliferating basal keratinocytes. TGF-β is a growth factor that has pleiotropic effects on a wide range of target cells and induces differentiation of basal keratinocytes. Therefore. TGF-β is postulated to inhibit BPAG1 expression. However. previous report conversely demonstrated an increase of BPAG1 expression by TGF-β. In this study. to understand regulatory role of TGF-β on BPAG1 functions. we examined the effect of TGF-β on BPAG1 gene expression using cultured keratinocytes. This study showed that BPAG1 mRNA expression was inhibited by TGF-β1 in concentration higher than 1.0 ng/ml. Furthermore. incubation of the cells with TGF-β1 in the presence of cycloheximide demonstrated that newly synthesized protein was required for BPAG1 regulation. To understand the detailed mechanisms of BPAGI modulation by TGF-β, we preformed transient transfection assay with a BPAGI promoter-CAT construct to know the detailed mechanisms of BPAG1 modulation by TGF-β. The results revealed that calcium and IFN-γ inhibited BPAG1 expression at transcriptional level, but TGF-β1 is not responsible for that transcriptional inhibition, suggesting that TGF-β may have differential molecular mechanism for down-regulation of BPAG1 gene expression from the events induced by IFN-γ.
 
Article
Coronary artery spasm plays an important role in the etiology of coronary spastic angina(CSA)and other acute coronary syndromes. Mice with a targeted disruption of the ATP-binding cassette transporter C9‒ABCC9 gene were developed as an animal model of CSA. Thus, the ABCC9 may be involved in the regulation of coronary artery vasomotility. The aim of this study was to investigate whether mutation in the coding region of the ABCC9 gene is detected in Japanese patients with CSA. The study included 9 Japanese patients with CSA (6 men and 3 women with a mean age of 51±13 years). Genomic DNA was extracted from the whole blood, and Mutation analysis of the coding region of ABCC9 was performed by direct sequencing. In one CSA patient, we found a single base substitution(G to A)at nucleotide position 126 in exon 21 of the coding region, which was heterozygous and did not cause amino acid substitution(T878T, silent mutation).In the remaining 8 patients, no base substitution was detected in the coding region of the ABCC9 gene. The results indicate that the mutation of the ABCC9 gene may not be involved in the genetic pathogenesis of CSA in humans.
 
Article
A 30-year-old woman underwent an emergency cesarean section because of fetal distress involved uterine rupture. As the placenta was firmly adhered to the uterine wall, following safe delivery, it took over one hour and more than 10000 ml blood loss until the placenta was removed. Additional blood was ordered, but there was delay in delivery. Bleeding was extremely beyond our estimation, and hardly controlled despite of transfusing 1400 ml of prepared blood. With stopping operation and continuing hyperoxic ventilation, we decided to start intraoperative blood salvage. Though hemoglobin, platelet, and base excess decreased to 2.7 g/dl, 1000/μl, and -11.2, hemodynamics could be maintained with use of a blood salvage system. Salvaged blood was very helpful to manage critical hemodilution until additional blood was reached. Overall estimated blood loss was 20190 ml and massive blood transfusion was needed. The patient emerged from anesthesia with no neurological complication. Rarely but actually cesarean section resulted in life-threatening hemorrhage. Blood salvage systems is safe and useful during cesarean section. Blood salvage should be usually considered one of the suitable choices in cesarean section patients when supply of homologous blood is limited.
 
Article
Electrophilic compounds, such as curcumin and neurite outgrowth-promoting prostaglandins (NEPPs), have been known to enhance neurite outgrowth in the presence of small amounts of nerve growth factor (NGF) in PC12 cells. However, the redox-sensitive molecular target for enhanced neurite outgrowth is largely unknown. NGF exerts its function by binding to the cell surface tyrosine kinase receptor, TrkA. Recently, Shibata et al. reported that PTP1B catalyses the dephosphorylation of TrkA. They also demonstrated that an electrophile from Japanese horseradish Wasabi, 6-methylsulfi nylhexyl isothiocyanate (6-HITC), inactivates PTP1B causing sustained phosphorylation and activation of TrkA and promoting neural diff erentiation of PC12 cells. Further, we recently discovered that carnosic acid (CA), a catechol-type electrophilic compound that is a major ingredient in the herb rosemary, strongly promotes neurite outgrowth of PC12h cells by activating the redox-sensitive transcription factor, Nrf2. Activation of Nrf2 by CA caused a marked induction of p62/ZIP, an important signaling scaff old protein for NGF signaling. Unexpectedly, NGF also activates Nrf2, which is essential for NGF-mediated neural diff erentiation. Thus, electrophiles may enhance neural diff erentiation by both TrkA-dependent and -independent mechanisms.
 
Article
To examine the effect of chronic hypertension on endothelium-derived hyperpolarizing factor( EDHF) responses, the hearts of Wistar-Kyoto rats( WKY) and spontaneously hypertensive rats( SHR) were isolated and perfused using Langendorff system with constant perfusion pressure. Bradykinin increased coronary fl ow( CF) dosedependently and this was not aff ected by NG-nitro-L-arginine methyl ester or indomethacin, indicating that bradykinin’s eff ect on CF was not mediated by nitric oxide or prostacyclin but by EDHF. Bradykinin-induced CF increase was smaller in SHR than in WKY. Tetrabutylammonium( a non-specific KCa channel blocker) abolished bradykinin-induced CF increase in both rats. 1-Ethyl-2-benzimedazolinone( 1-EBIO, an agonist of intermediate conductance KCa channel)-induced increase in CF was smaller in SHR than in WKY. 1,3-Dihydro-1-[2-hydroxy-5(- trifl uoromethyl) phenyl]-5(- trifl uoromethyl) -2H-benzimidazol-2-one( NS1619, an agonist of large conductance KCa channel)-induced increase in CF did not differ between SHR and WKY. In early stage of hypertension, there was no signifi cant diff erence between SHR and WKY in bradykinin- and 1-EBIO-induced increases in CF. In conclusion, EDHF response in coronary microcirculation is impaired in SHR due to dysfunction of intermediate-conductance calcium-activated potassium channels.
 
Article
Unmethylated CpG oligodeoxynucleotides (CpG) prevalent in bacteria and DNA viruses bind Toll-like receptor( TLR) 9 and directly stimulate DCs, thereby activating the innate and adaptive immune responses. CpG is potentially a powerful reagent for protective immunity against infection by a wide variety of pathogens, for cancer and allergy therapies, and for the development of prophylactic and therapeutic vaccines. Here we investigated the role of interleukin (IL)-15 in the activation of CpG-induced immune responses. We show that upon CpG-priming, both wild-type( WT) and NK cell-depleted WT mice produce interleukin( IL)-12 p70 and become resistant to a lethal dose of Listeria monocytogenes( LM), whereas IL-15-/- mice impair IL-12 p70 production and succumb to the infection. Notably, CpG-stimulated conventional dendritic cells (cDCs) are the major producer of both IL-15 and IL-12 p70, but cDCs do not produce IL-12 in the absence of plasmacytoid DCs( pDCs) in vivo. Importantly, cDC-derived IL-15 induces CD40 expression on cDCs, which interacts with CD40 ligand on pDCs, leading to CD40 cross-linking and IL-12 production. Collectively, these fi ndings show that IL-15-dependent cross-talk between cDCs and pDCs is essential for CpG-induced immune activation( recently published in Nat Immunol 2006;7:740-6)
 
Article
Action potentials are the fundamental signals for relaying information from one region to another in the nervous system. Action potentials are propagated along axons without decrease of their amplitudes and are conducted with constant velocity depending on axonal diameter and myelin. It is considered that the modulation of fi ring patterns of action potentials in the neural circuit infl uences the information processing in the brain. We investigated the modulatory eff ects of glial cells on the fi ring pattern and the axonal conduction of action potentials using rat hippocampal slice preparation. In our previous study, we focused on interneuron / perineuronal glial cell pairs in CA1 region and reported that perineuronal glial cells could be classifi ed into two groups, one group belong astrocytes (perineuronal astrocytes) and the other group oligodendrocytes (perineuronal oligodendrocytes), based on their membrane properties and immunohistochemical study. Direct depolarization of perineuronal astrocytes modulated the directly induced fi ring pattern of the interneuron, with initial facilitation and subsequent suppression. We also studied the oligodendrocytes in the alveus and examined their modulatory effects on the conduction of action potentials along axons of CA1 pyramidal cells. Direct repetitive depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that glial cells infl uence the firing pattern and axonal conduction of action potentials, and that their effects involve both facilitation and suppression.
 
Article
Objectives The purpose of this study was to test the hypothesis that adenosine-induced coronary microvascular dilation is blunted in the animals with diabetes mellitus( DM) through the impairment of KATP channel function.   Background Adenosine-induced coronary vasodilation is demonstrated to be mediated by activation of ATPsensitive potassium( KATP) channels and nitric oxide( NO).  Methods The hearts of Otsuka Long-Evans Tokushima fatty rats (OLETF, type 2 DM rats), and control Long- Evans Tokushima fatty rats( LETO) at the ages of 32 and 8 weeks were perfused using a Langendorff system with constant perfusion pressure (80 mmHg). Changes in coronary fl ow to adenosine, pinacidil and sodium nitroprusside (SNP) were examined before and after administration of glibenclamide( 10-7 M), or NG-nitro-L-arginine methyl ester (L-NAME, 10-4 M).   Results At the age of 32 weeks, adenosine- and pinacidil-induced increases in coronary fl ow were blunted in OLETF as compared with those in LETO (both p<0.05). Glibenclamide attenuated adenosine-induced increase in coronary fl ow in LETO (p<0.05), but not in OLETF. In contrast, L-NAME attenuated adenosine-induced increase in coronary flow in OLETF (p<0.05), but not in LETO. SNP-induced increases in coronary flow in LETO and OLETF were comparable and were not aff ected by glibenclamide. In 8-week-old OLETF and LETO, no diff erence was observed in adenosine-, pinacidil- and SNP-induced increases in coronary fl ow between OLETF and LETO.  Conclusions In this type 2 DM model, KATP channel function in coronary microcirculation is impaired. Adenosineinduced increase in coronary fl ow is mediated mainly by NO mechanism.
 
Article
We examined the therapeutic efficacy for the suicide gene introduction using two recombinant adenovirus vectors with HER2 promoter and Cre/loxP system in human gastric cancer cell lines, MKN-7 and MKN-28. HER2 protein level was more expressed in MKN-7 than in MKN-28. Next, we constructed a Cre recombinae expression vector in HER2-producing cell specifically, AxHER2NCre and AxCALNCD expressing cytosine deaminase (CD) gene under the control of the CAG promoter by the Cre switching system. Much higher CD messenger RNA (CDmRNA) and CD protein expression were induced in cells by the double infection method than by AxCALNCD only. Furthermore, CD mRNA and CD protein expression were induced higher in HER2-overexpressing cell line, MKN- 7 than in MKN-28. We examined the efficacy of cell growth inhibition using 5-fluorocytosine (5-FC) as anti-tumor prodrug. Inhibition effect was dose-dependent at each cell line and rate was more in MKN-7 in comparison with MKN-28. This system can be applied for HER2-overexpressing cancer specific gene therapy.
 
Article
Optimization of neuronal function and survival is an important goal in the treatment of cerebrovascular diseases in order to avoid or improve devastating long-term sequelae. Nerve growth factor (NGF) is essential for neuronal growth and survival in the central nervous system (CNS). Vascular endothelial growth factor (VEGF) is a potent mitogen specifi c for endothelial cells and a stimulator of neovascularization. VEGF also enhances vascular permeability, which may promote the development of brain edema during cerebral ischemia. These molecules aff ect the outcome of ischemia/reperfusion injury in the CNS. Edaravone, a brain-penetrant, free radical scavenger, is known to ameliorate postischemic neuronal dysfunction. Transcription factor Nrf2( nuclear factor-erythroid 2-related factor 2), a master regulator of antioxidant responses, plays an important role in the coordinated expressions of stress-inducible genes. Astrocytes express various genes involved in the regulation of neuronal functions, and the regulation of astrocyte gene expressions may be a potential therapeutic target in brain injury. This review aims to appraise the eff ects of radical scavenger edaravone and a natural Nrf2-inducer as neuroprotective agents in human astrocytes, particularly under an experimental model for hypoxia/reoxygenation.
 
Article
Candidate gene SNP study is a promising genetic approaches to complex common disorders. Arginine vasopressin (AVP), a peptide hormone released from the posterior pituitary, has been suggested to play important roles in the regulation of blood pressure, glycogenolysis and platelet aggregation through G protein coupled V1a receptor (V1aR). Thus, polymorphisms in the V1aR gene have been prospective as possible genetic markers for essential hypertension, type 2 diabetes mellitus and divergent platelet aggregation response to AVP. We identifi ed 4 novel single nucleotide polymorphisms( SNPs) in the promoter region of the V1aR gene and named according to the upstream locations such as, -6951G/A, -4112A/T, -3860T/C, and -242C/T. We investigated the association of 4 SNPs of the V1aR gene in 365 hypertensive and 255 healthy subjects, 186 T2DM patients and 188 non-diabetic control subjects (CS), and 33 young healthy volunteers living in the Aomori prefecture. Signifi cant association was identifi ed between SNP at -6951 and hypertension in nonobese individuals, at -6951 and type 2 diabetes mellitus. Positive association was also identifi ed between nonobese hypertension and haplotype H3. Signifi cant association was demonstrated between SNP at -6951 and glycemic status in young healthy subjects. However, there was no signifi cant association in the AVPinduced platelet aggregation with V1aR gene variants. The study suggests V1aR gene variants as increased risk for hypertension in nonobese and type 2 diabetes mellitus in the Aomori population; however, might not be useful as genetic markers for platelet aggregation heterogeneity.
 
Article
Chronic ultraviolet (UV) radiation results in photoaged skin characterized by deep wrinkle formation. Recent studies suggest that diminishment of anchoring fi brils( AF), stabilizing the association of the basement membrane to the underlying dermis, are involved in photoaged skin. Expression of COL7A1, encoding type VII collagen that is a major component of AF, is also decreased in photoaged skin. In this study, we have investigated COL7A1 transcription by UV and UV-related cytokines involved in photoaged skin. Nuclear run-on assay, luciferase assay and gel shift assay revealed that UV and UV-related cytokines( TNF-α, IL-1β) tissue specifi cally downregulated COL7A1 transcription in epidermal keratinocytes, whereas COL7A1 transcription was upregulated by each modulator in dermal fi broblasts. The responsive element of each modulator was located between nucleotide -524 and -22 of COL7A1 promoter and AP-1 and NF-κB families band to this region. Taken together, these data strongly suggest that the downregulation of COL7A1 transcription in epidermal keratinocytes, main cells expressing COL7A1 in the skin, and subsequent diminishment of AF are involved in photoaged skin. We also have discussed relation between these results and pathophysiology of wrinkle formation, characteristic feature of photoaged skin.
 
Article
The ability of murine allogeneic umbilical cord blood cells (UCBCs) to reconstitute the immune system was investigated. UCBCs obtained from fetuses of C57BL/6 (B6; H-2b) mice, which were transgenic for green fluorescent protein (GFP), were transplanted into RAG2 (-/-) BALB/c mice (H-2d). After transplantation, flow cytometric analysis revealed successful reconstitution of phenotypically mature GFP-positive immune cells of donor origin, including T cells, B cells, monocytes, and granulocytes in the peripheral blood of the recipient mice. Analysis of functional maturation of lymphocytes revealed that 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) -immunized UCBC-transplanted recipients produced both TNP-specific IgM and IgG antibodies. These results indicated that the recipient mice were capable of mounting antibody responses to T-dependent antigens; further, Ig class switching from IgM to IgG confi rmed that both B cells and CD4+ helper T cells derived from allogeneic UCBCs were immunologically competent. Furthermore, mice transplanted with allogeneic UCBCs accepted skin grafts from both B6 and BALB/c mice. However, these chimeric mice completely rejected skin grafts from third party C3H/ HeJ (H-2k) mice, indicating the presence of functional CD8+ killer T cells as well as CD4+ helper T cells. In terms of potential clinical application, our results indicate that allogeneic UCBC transplantation can enable recovery of the normal immune system in recipients.
 
Article
Although animal studies suggest that centrally active angiotensin-converting enzyme (ACE) inhibitors may protect against dementia beyond HTN control, the mechanism(s) underlying these improvements in cognitive function remains unclear. We measured the brain peptide levels in rat treated with captopril( 50 mg/kg) for 3 weeks by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF-MS). Two protein chip arrays were used for peptide profi ling: one with a strong anion- exchanger and the other with a weak cation-exchanger.   Comparing with control group, 15 mass peaks were considered specifi c to experimental animals, and 6 peaks were signifi cantly up-regulated and 5 down-regulated.
 
Article
By analyzing late onset Alzheimer’s disease (LOAD) in a genome wide association study of 3 American Caucasian series and evaluating the 25 SNPs with most significant allelic association in 4 additional series, we identifi ed a SNP (rs5984894) on Xq21.3 in PCDH11X that is strongly associated with LOAD in American Caucasians (total n=4,855; AD:2,391; control:2,464). Analysis of rs5984894 by logistic regression using sex as a covariate gave a global p value of 3.9 x 10-12 in the combined series. Odds ratios were 1.75( 95% CI 1.42-2.16) for female homozygotes (P=2.0x10-7) and 1.26 (95% CI 1.05-1.51) for female heterozygotes (P=0.01) compared to female non-carriers. For male hemizygotes (P=0.07) compared to male non-carriers the odds ratio was 1.18 (95% CI 0.99-1.41). The eff ect of this variant was dose dependent, as females homozygotes for the minor allele were at signifi cantly greater risk than heterozygous females and hemizygous males. We tested additional variants in PCDH11X for association with LOAD. One of these variants, rs2573905, showed association with LOAD similar to that for rs5984894, albeit with a slightly more significant p-value (5.4x10-13). This is not surprising since these two variants are in near perfect linkage disequilibrium (r2 = 0.98, D’ = 0.99), and the minor allele of these two SNPs occur on the same haplotype. However, rs2573905 is in a sequence that has been evolutionarily conserved between human and mice, with 70% sequence identity over 100bp, suggesting a possible functional role for this SNP. Joint analysis of APOE and rs2573905 genotypes showed that 75 women with LOAD (4.9%) were homozygous both for APOE e4 and for rs2573905 whereas only 2 unaff ected women (0.15%), both age 73, were double homozygotes. These fi nding suggest that the double homozygote may be fully penetrant in women over the age of 73 and that this combination may account for ~5% of the AD that occurs in women.
 
Article
Structural remodeling occurs in diverse heart diseases and affects their clinical courses. We reported that amiodarone suppresses both electrical and structural remodeling in a canine persistent atrial fi brillation model. As a mechanism for amiodarone’s effect on structural remodeling, we suggested its inhibitory effect on matrix metalloproteinase( MMP) activity. To elucidate it, we investigated the eff ect of amiodarone on MMP activity in a rat myocardial infarction model created by left coronary artery( LCA) ligation. Adult Sprague-Dawley rats were divided into sham-operated (Sham), sham-operated with amiodarone (Sham+AMD), LCA-ligated without amiodarone (MI) and LCA-ligated with amiodarone rats (MI+AMD). Amiodarone (20 mg/kg/day) was administered for 2 weeks before and for 4 weeks after operation. The hearts were excised at 4 weeks after operation. MMP-2 activity was measured by gelatin zymography. At 4 weeks after surgery, left ventricular fractional shortening was decreased in MI but not in MI+AMD rats. There was no diff erence in the infarct size between MI and MI+AMD rats (P=NS). As compared with Sham, MMP-2 activity was increased in MI (P<0.01), but not in MI+AMD (P=NS versus Sham; P<0.05 versus MI). MMP-2 activity was not increased in Sham+AMD( P=NS). Thus, amiodarone exerts an inhibitory eff ect on MMP activity. This may be related to the improvement left ventricular function in MI rats.
 
Article
Accumulating evidence supports that proinflammatory processes are implicated in the establishment of characteristic pathology in diabetic vascular and neurological complications. Altered cellular signaling during inflammatory processes is a new target for the treatment of diabetic complications and there appear to be some attempts to explore the eff ects of thiazolidinedione, a ligand of peroxisome proliferating activator receptor( PPAR)-γ for not only the treatment of diabetes itself but for the application to diabetic complications. We therefore explored the eff ects of PPAR-γ agonist, pioglitazone, on the experimental model of diabetic neuropathy. We found that pioglitazone signifi cantly improved nerve conduction velocities and depressed protein kinase C activity. This is a new area for the exploration of eff ective treatment of diabetic complications that pose a considerable socioeconomic burden in the current world.
 
Article
Cystatin C (CC), a cysteine protease inhibitor involved in protein degradation, is a marker of Bunina bodies in lower motor neurons in amyotrophic lateral sclerosis (ALS). TDP-43-immunoreactive inclusions are also histological hallmark of ALS. However, immunohistochemical localization of CC in ALS motor neurons with or without inclusions is uncertain. Recently, we demonstrated that the majority of anterior horn cells showed moderate to intense immunoreactivity for CC in controls and that CC immunoreactivity was severely decreased in anterior horn cells in ALS. The proportion of CC-immunolabeled anterior horn cells was reduced regardless of whether those neurons contained Bunina bodies or not. In contrast, the proportion of CC-immunolabeled anterior horn cells was signifi cantly reduced owing to the presence of TDP-43 inclusions. These fi ndings suggest that the formation of TDP-43 inclusions, but not of Bunina bodies, reduces the content of CC in spinal motor neurons and that perturbations in endogenous levels of CC in neurons may participate in neurodegenerative process in ALS.
 
Article
Methylone, an analog of 3,4-methylenedioxymethamphetamine(MDMA)is a narcotic drug which is forbidden due to its abusability. However, a detailed behavioral toxicity and rewarding effect of methylone has not yet been reported to this date. The aim of this study is to evaluate the toxicity and addictive effects of methylone. In order to detect the stimulant effect to central nerous system, a mouse behavioral toxicity test and a conditioned place preference(CPP)test were conducted by administering mehtylone, MDMA and methamphetamine(MAP).An immunohistochemical study was also performed to analyze ΔFosB, which is known to accumulate in the nucleus accumbens after chronic administration of the drugs of abuse. For determining the expression levels of ΔfosB mRNA in striatum, quantitative PCR analysis was also conducted by acute administration of methylone. Significant differences appeared in mice that were administered with 50 mg/kg and above of methylone. Methylone causes a similar qualitative behavioral effect as MAP, however not the same stereotyped behavior as MDMA. According to the result from CPP test, methylone of 2.5 mg/kg and above had shown a rewarding action. Chronic administration of methylone causes significant ΔFosB accumulation in the neurons of nucleus accumbens. Methylone and MDMA significantly induce ΔfosB mRNA in striatum. It is therefore suggested that methylone has psychoactive effects and can be considered as an addictive drug.
 
Article
The present study investigated the relationship between obesity and atherosclerosis according to age in the general female population. Six hundred fifty-five females were divided into 3 different age groups and the relationship between brachial-ankle pulse wave velocity (baPWV) and various obesity indices were investigated. In the youngest age group (20-39 years), BMI, percentage body fat and abdominal circumference were positively correlated with baPWV. In the middle-aged group (40-59 years), all four obesity indices (BMI, percentage body fat, abdominal circumference and the waist-to-hip ratio (WHR)) were positively correlated with baPWV. However, baPWV was found to be negatively correlated with BMI only in the oldest age group (60 years and over), though it was still positively correlated with WHR (but not abdominal circumference). The reason for this may be due to a gradual reduction of body weigh caused by some lifestyle-related diseases or aging. In this study, it was confirmed that abdominal obesity is a key indicator of arteriosclerosis, though the importance of BMI (general obesity) as an indicator may depend on the generation. Furthermore, our results suggested that abdominal circumference and WHR may have different significance as the indicators of arteriosclerosis.
 
The changes in plasma ET-l concentrations in patients with NTG by anthocyanins administration. Each color line represents the change in plasma ET-l concentration before and just after oral administration of anthocyanins in each patient with NTG. ET-l: endothelin-l; NTG: normal tension glaucoma
Effects of anthocyanins administration on the concentration of plasma ET-l (pg/mD
Scanning laser Doppler fiowmetry measurements of the neuroretinal rim of optic nerve head and peripapillary retina (a.uJ
Article
Purpose: To investigate the effects of anthocyanins in black currant on retinal blood flow circulation of patients with normal tension glaucoma (NTG). Methods: Thirty consecutive patients with NTG were included in this study. They were orally administrated anthocyanins extracted from black currant in tablet form once a day for a 6-month period. Systemic blood pressures. intraocular pressures (IOPs). concentrations of the plasma endothelin-1 (ET-l), blood flows at the neuroretinal rim of the optic nerve head and peripapillary retina. and visual field defects were measured before and just after the administration period. Results: Our study demonstrated that oral administration of anthocyanins tablets significantly increased the blood flows at both neuroretinal rim of the optic nerve head and peripapillary retina (p < 0.05). with no significant changes in mean blood pressures or IOPs. Furthermore. none of the subjects showed progression of their visual field defects. We also demonstrated that the oral administration of anthocyanins tablets significantly increased. and thus normalized the concentrations of plasma ET-1 (p < 0.05). Conclusion: These results suggest that anthocyanins orally administrated might be a safe and valuable choice for neuroprotective treatment of patients with NTG.
 
Article
Bacillus Calmette-Guerin( BCG) has been widely accepted as an eff ective treatment for CIS and superfi cial carcinoma of the bladder. However, it has considerable side eff ects and toxicity. We thought if eff ective bacterial cell wall elements as substitutes for live bacteria were identifi ed, useful treatment with lower toxicity while maintaining strong anti-tumor eff ects might be possible. For these reasons we generated a nano-particulated BCG complex, which does not contain live bacteria. Here, we present its direct in vitro anti-tumor eff ect on bladder cancer cell lines. Tokyo 172 BCG sub-strain was disrupted by French press with monitoring the particle distribution by the particle analyzer. After removing the not-disrupted bacteria by centrifuge at 6,800×g, supernatant was centrifuged at 18,000×g. Then the supernatant( Sup) and the precipitate( CW) were lyophilyzed to obtain nano-particulated BCG complex. Bladder cancer cell lines, J82 and KK47, were co-cultured with BCG, Sup, CW or Sup+CW( mix) for 5 days, then viable cell numbers were counted. In J82 cells, when separately added to the culture medium, both CW and Sup reduced cell number to about 70% of control cells. While they were mixed together, they reduced cell number equally compared with BCG; 59.2% (mix) vs 60.2%( BCG) in J82 cells, 67.3% vs 68.8% in KK47 cells. These preliminary in vitro experiments demonstrated the identical direct anti-tumor eff ect of nano-particulated BCG to that of live BCG. In vivo tumor assays are warranted for clinical application of nano-particulated BCG.
 
Article
S-1, an oral prodrug of 5-fluorouracil, has been employed in treatment of solid tumors in Japan. We report two patient cases whose international normalized ratio(INR)increased due to concomitant treatment with warfarin and S-1. In case 1, a 63-year-old man, who had taken 3.5 mg/day warfarin for dilated cardiomyopathy, had a subcutaneous hematoma in a left lower eyelid on the ninth course of combination chemotherapy with S-1 and docetaxel for metastatic gastric cancer. His INR increased to 3.68 despite careful monitoring of coagulation parameters. In case 2, a 68-year-old man, who received combination chemotherapy with S-1 and irinotecan for metastatic colorectal cancer, had to reduce the dose of warfarin twice during the same chemotherapy courses because of repeated prolongation of INR. We speculate that multiple mechanisms of unstable INR are involved in strengthening the anticoagulant effect of warfarin. Frequent monitoring of INR is required to prevent unexpected adverse events of coagulation abnormality induced by the interaction between S-1 and warfarin.
 
Article
D-Glucose is one of the most important energy sources for the survival of various organisms, from E. coli to mammals. For live-cell monitoring of glucose uptake at the single-cell level, a fl uorescent D-glucose derivative 2-[N- (7-nitrobenz-2-oxa-1,3-diazol-4- yl)amino]-2-deoxy-D-glucose [2-NBDG], which we developed, has been widely used in various research fi elds. For the last ten years, however, researchers have awaited an optical control substance for evaluating the extent of non-specifi c adsorption of 2-NBDG upon plasma membrane and/or the rate of unhealthy 2-NBDG uptake through partially( or transiently) damaged membrane.  Here we introduce a fluorescent L-glucose derivative, 2-[N(- 7-Nitrobenz-2-oxa- 1,3-diazol-4-yl)amino]- 2-deoxy-Lglucose [2-NBDLG]. L-Glucosamine is a key intermediate toward the synthesis of 2-NBDLG, but not commercially available. Although a few papers on the synthesis of L-glucosamine have been reported, a new synthetic method of L-glucosamine should be absolutely required in practical view of optical purity and preparative scale. We converted commercially available L-mannose into desired L-glucosamine by 10 steps in 14% of overall yield. The 1H-NMR data of synthetic L-glucosamine were completely identical with those of commercially available D-glucosamine. On the other hand, optical purity of L-glucosamine was confi rmed by comparison of specifi c rotation with that of D-glucosamine. L-Glucosamine thus obtained was coupled with NBD-halide to give 2-NBDLG. Use of transporterrecognizable (D-isomer) and unrecognizable (L-isomer) fluorescent analogues combined with real-time confocal microscopy, should provide valuable information on dynamism of glucose transport.
 
Article
Adipose tissue secretes various bioactive molecules (adipokines), and apelin is one kind of adipokines. Recently, it was shown that plasma apelin level is decreased in patients with chronic heart failure, and apelin might play an important role in the pathogenesis of cardiovascular disease. However, plasma apelin level in coronary artery disease( CAD) or other heart disease such as valvular heart disease( VHD) has not been elucidated. We enrolled 31 patients with CAD and 14 patients with VHD who underwent elective cardiac surgery. We also examined plasma apelin level in 20 healthy subjects (Control). Blood samples were obtained before the surgery. Paired samples of visceral and subcutaneous adipose tissues were harvested during surgery. Plasma apelin level was lower in both CAD and VHD than in Control. When compared between CAD and VHD, it was lower in CAD than in VHD, and was not aff ected by treatment with HMG-CoA reductase inhibitors (statins) which was shown to increase adiponectin level. Left ventricular ejection fraction( LVEF) was lower in CAD than in VHD. There was no correlation between plasma apelin level and LVEF. Gene expression of apelin in visceral adipose tissue was higher than that in subcutaneous adipose tissue, but it was similar between two groups. These suggest that plasma apelin level was decreased in patients with cardiac diseases, especially in those with CAD. Its role in the pathophysiology of CAD remains to be elucidated.
 
Article
Human infl uenza virus causes an annual epidemic infection. After infl uenza virus infection in the airway, infection sometimes spreads with severe neurologic complication and multiple organ failure, resulting in high mortality. In the process of viral spread from the lungs to other organs, signifi cant up-regulated trpsin was observed in various organs. Up-regulated trypsin eff ectively converted precursor of the viral envelope hemagglutinin( HA) into HA1 and HA2 subunits. The proteolytic activation of HA is a prerequisite for virus multiplication. Administration of the inhibitors of trypsin as new approaches for the treatment of infl uenza virus infection eff ectively suppressed viral multiplication. Almost vaccines for infl uenza virus infection are administered i.m. or s.c., which induce IgG-mediated protection in the systemic immune compartment, but this immunization off ers insuffi cient protection on the mucosal surface at the initial site of viral multiplication. To improve protective mucosal immunity, intranasal vaccination has been studied. The powerful mucosal adjuvants reported are toxin based, such as cholera toxin and Escherichia coli heat-labile toxin, but these enterotoxins cause severe side eff ects. We recently found natural mucosal adjuvant, pulmonary surfactant, in the lungs. Intranasal administration of infl uenza vaccine combined with Surfacten, a modifi ed pulmonary surfactant free of antigenic c-type lectins, induced high protective mucosal immunity in the airway and systemic immune responses in the blood, the effi cacy of both protective immunities by Surfacten being equivalent to those by cholera toxin. In this symposium, we reported the eff ects of Surfacten on mucosal and systemic immunities.
 
Article
Human parvovirus B19 (B19) is single stranded DNA virus, that causes erythema infectinosum in infant and/or acute onset polyarthritis in adult. We present the evidence showing the role of B19 on the etiopathogy of rheumatoid arthritis( RA).( 1) B19 DNA could be frequently amplifi ed in the samples from rheumatoid joints. The detection B19 RNA and B19 protein VP1 was specific for RA, and positive at T cells, B cells, macrophages and follicular dendritic cells in rheumatoid synovium. ( 2) B19 infection or transduction of B19 NS1gene caused TNFα, IL-6 and IL-8 production through activating AP1 and AP2 in macrophages or macrophage celll line U937. We also found Ku80 as a novel receptor for B19 on T cells, macrophages or erythroblasts. B19 used clathrin on the surface at their cell entry and caused enhanced actin polymerization, resulting in the migration of T cells. ( 3) B19-transgenic mice became susceptible to type II collagen-induced polyarthritis that is a model of RA. We also experienced 12 cases who developed RA after acute B19 infection. ( 4) Half of RA cases had a defective neutralizing ability to B19.
 
Article
Dietary vitamin A is an essential precursor of tissue retinol, which participates in a variety of biological processes including innate immunity. Functions of vitamin A mainly depend on retinoic acid( RA), principally all-trans- RA (atRA) and 9-cis-RA. We assessed whether atRA is benefi cial in host resistance against bacterial infections or not. Vitamin A-defi cient( VAD) mice were highly susceptible to infection with Listeria monocytogenes. Pre-treatment with atRA enhanced host resistance against L. monocytogenes infection in both VAD and VAS mice. Interferon( IFN) -γ production in atRA pre-treated VAS mice was not higher compared with the control VAD mice. The eff ect of atRA was independent of T cells and B cells. The bactericidal activity in macrophages obtained from atRA pre-treated VAS mice was almost the same level compared with the control VAS mice. Our results demonstrated that the treatment with atRA is benefi cial for host resistance against L. monocytogenes infection in the early phase and suggested a new therapeutic possibility of atRA in bacterial infections.
 
Article
α-Synucleinopathies comprise a group of neurodegenerative disorders that share α-synuclein (αS) accumulation in selected vulnerable neurons and glia, i.e. Parkinson’s disease (PD), dementia with Lewy bodies and multiple system atrophy( MSA). The histological hallmark of PD is neuronal αS aggregates called Lewy bodies( LBs). LB formation has been considered to be a marker for neuronal degeneration, because neuronal loss is found in the predilection sites for LBs. However, recent studies have suggested that oligomers and protofi brils of αS are cytotoxic, and that LBs may represent a cytoprotective mechanism in PD. The histological hallmark of MSA is αS aggregates in the oligodendrocytes referred to as glial cytoplasmic inclusions( GCIs). αS inclusions are also found in the neuronal somata, axons and nucleus. At present, two degenerative processes have been considered in this disease; one is due to the widespread occurrence of GCIs associated with oligodendroglia-myelin degeneration, and the other is due to the aggregation of αS in neurons in several brain regions. These two processes might synergistically cause neuronal depletion in MSA.
 
Article
Background: P38 mitogen-activated protein kinase(MAP kinase)plays on important role for progression of pathological cardiac hypertrophy. However, the role of p38 MAP kinase in cardiac hypertrophy induced by pressure overload remains unclear. We investigated the effect of chronic treatment with p38 MAP kinase inhibitor on the development of heart failure induced by transverse aortic constriction(TAC)in mice. Methods and Results: TAC increased left ventricular septal wall thickness(LVSWT)and cross-sectional area(CSA)of cardiomyocyte, and decreased LV fractional shortening(FS)compared with sham operation after 6 weeks. TAC also increased phosphorylation of p38 MAP kinase, whereas other hypertrophic signals were unchanged. In another experiment, TAC mice and sham operated mice were treated with subcutaneous injection of p38 MAP kinase inhibitor SB202190(5mg/kg/day)or placebo five times a week for six weeks. Treatment with p38 MAP kinase inhibitor attenuated the increase in LVSWT and CSA, and the decrease in FS in mice with TAC. Conclusions: Inhibition of p38 MAP kinase attenuated left ventricular hypertrophy and inhibited progression of systolic dysfunction in pressure overload-induced cardiac hypertrophy. These results suggest that inhibition of p38 MAP kinase has a protective effect for development of heart failure induced by pressure overload.
 
Article
To develop a Staphylococcus aureus vaccine, we constructed and expressed a non-toxic mutant staphylococcal enterotoxin C( mSEC) and investigated whether immunization with mSEC can protect against S. aureus infection. Mice were immunized with mSEC and challenged with viable S. aureus. Bacteria counts in the organs in mSEC-immunized mice were significantly lower and the survival rate was higher than those of the control group. mSEC vaccination induced the production of T-helper 2 type antibodies. The production of interleukin-10( IL-10) and IL-4 in vaccinated mice was signifi cantly higher compared with the control group, whereas the production of gamma interferon (IFN-γ. ) was signifi cantly decreased in the vaccinated mice. Furthermore, IFN-γ. and tumor necrosis factor-α・ production in vitro was signifi cantly inhibited by the sera from mSEC-vaccinated mice but not by those from control mice. These results suggest that vaccination with mSEC provides protection against S. aureus infection. SEC neutralizing antibodies and the IL-10 and IL-4 induction might play important role for the protection.
 
Article
Infection caused by methicillin-resistant Staphylococcus aureus (MRSA) has been the most commonly acquired types of nosocomial infections. It was reported that anterior nares are the major reservoir of S. aureus and the source of 80% of S. aureus bacteremia is endogenous. Considering these facts, elimination and reduction of nasal carriage are thought to be eff ective protection against systemic S. aureus infection and nosocomial infection. Toxic shock syndrome toxin 1( TSST-1) is one of superantigens secreted by S. aureus. Previously, it was reported that mutant form (H135A) of TSST-1( mTSST-1) was shown to be nontoxic, and subcutaneous vaccination with mTSST-1 could protect against systemic S. aureus infection in a mouse model. In this study, we investigated the protective eff ect of intranasal vaccination with mTSST-1 supplemented with non-toxic mutant( H44A) Escherichia coli heat labile toxin( mLT) as a mucosal adjuvant. The results demonstrated that intranasal immunization with mTSST-1 plus mLT could efficiently induce production of anti-TSST-1 antibodies in sera and also induce anti-TSST-1 IgA production in bronchoalveolar lavage fl uids( BALF) of vaccinated mice. In nasal-associated lymphoid tissues( NALT) of vaccinated mice, anti-TSST-1 IgA secreting cells were signifi cantly increased. To evaluate of the protective eff ect of this vaccine against systemic S. aureus infection, BALB/c mice were vaccinated with mTSST-1 plus mLT and challenged with clinical isolated S. aureus 834 intravenously. Bacterial numbers in spleen and liver, and cumulative mortality rate of vaccinated mice were lower than those of control mice. We further developed a mouse model of nasal S. aureus colonization. S. aureus bacterial numbers in nasal cavity of vaccinated mice were signifi cantly reduced compared with those of control mice. These results indicate that intranasal immunization with mTSST-1 plus mLT is able to induce systemic and mucous immune responses and of provide protection against systemic S. aureus infection and nasal colonization.
 
Article
To date, digital pathology is based on single focal plane images, and with its rapid development, image quality and information are becoming critical concern in the field. This paper presents a new multiple focal-plane based focus image fusion algorithm to enhance the image quality. First of all, we will introduce a new focus image fusion algorithm; next, we will demonstrate some experimental data and results on cytopathology slide images; and finally, we will verify the effectiveness of this algorithm using computer vision techniques and cytopathology diagnosis, and draw the conclusions.
 
Article
Background ̶Subarachnoid hemorrhage (SAH) remains a disease without any definitive treatment options. Research, to date, has concentrated on the pathophysiology of vasospasm. Recent evidence supports the concept of Early Brain Injury (EBI), a phenomena which may help to explain the complex pathophysiology seen in patients after a SAH. Summary ̶EBI aims to describe the pathophysiological events that occur in the brain within the fi rst seventy two hours after a SAH, before the onset of vasospasm. A number of pathways have been identifi ed which may play a role in the etiology of EBI. This review provides a brief synopsis of EBI and its implications for the future. Conclusions ̶EBI may represent a key event in the development of both vasospasm and Delayed Ischemic Neurological Deficit (DIND) after subarachnoid hemorrhage. Additional studies are required to determine the pathophysiology of EBI and to examine its role as a possible precursor to both vasospasm and DIND.
 
Poster
The mechanism regulating cerebral blood fl ow (CBF) during brain function (neurovascular coupling) has been widely investigated in animals under anesthetized conditions, though anesthesia is known to greatly aff ect neurovascular physiology. The present study aims to develop a novel model for neurovascular coupling studies in awake-behaving mice. Male C57BL/6J mice were initially anesthetized with isofl urane in preparation for attaching the study apparatus to the head. The animal was tethered to the study apparatus but allowed to move spontaneously on a fl oated ball. The animal behavior and regional CBF in the somatosensory barrel cortex were simultaneously measured with optical motion sensor and laser-Doppler fl owmetry (LDF), respectively. Anesthesia was discontinued during recovery, while whisker stimulation (frequency 10 Hz and duration 10 or 20 sec) was induced at either the contralateral or ipsilateral side of the LDF recording site. During the experiments, the animals showed no signs of struggling against the head restraint. The intensity of baseline CBF was higher while the animal was under 2% isofl urane aesthesia than it was after anesthesia was stopped. CBF response to stimulation was not observed under anesthesia. After the animal was recovered from anesthesia, an increase in CBF (34 ± 18%) was observed during contralateral stimulation but not during ipsilateral stimulation. The fl uctuation levels of CBF baseline during resting and walking conditions were ±2.7% and ±3.5%, respectively. We observed that these fl uctuations were not due to vibration noises caused by such as air-puff and animal motion in our experimental conditions.
 
Article
Massage therapy promotes psychosocial relaxation and reduce stress. In addition, this therapy has been reported to improve immune function. Although evaluation of psychosocial status has been performed with subjective psychological tests such as State-Trait Anxiety Inventory (STAI), subjective psychological tests are of limited value if the subjects fail to report reliably. Salivary biomarkers have been recently suggested as useful objective indicators for assessing psychosocial status. To determine whether salivary biomarkers are useful objective indices for assessing eff ects of back massage on psychological status in 25 young healthy female volunteers, we measured heart rate and salivary biomarkers( α-amylase, cortisol and chromogranin-A) and assessed STAI score before and after back massage. Back massage significantly reduced heart rate from 73.4±11.8 to 69.8±11.2 and STAI from 41.0±6.0 to 32.3±4.9. In contrast salivary chromogranin-A signifi cantly increased from 2.93±2.21 to 5.29±5.46 pmol/mg protein whilst salivary α-amylase and cortisol did not change. Therefore, salivary biomarkers tested may not indicate changes in psychological relaxation following back massage. Massage therapy has been reported to not only reduce psychosocial stress but also enhance immune functions in cancer patients. In the present study, massage therapy significantly increased chromogranin-A release. As several reports clearly show that chromogranin-A has antibacterial and antifungal activities, back massage may increase host defense with salivary chromogranin-A release against oral microbial invasion.
 
Article
Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors which are conserved from insects to humans. PGRPs can recognize bacteria and their cell wall components, peptidoglycans. Human PGRPs have bactericidal activities that can be accomplished by their amidase activities. Insect PGRPs consist of 16 subtypes and some have eff ector functions as the activation of Toll pathway (PGRP-SA), the activation of Imd pathway (PGRPLC) and induction of autophagosome formation (PGRP-LE), thus contribute to the elimination of bacteria. However, the role of mammalian PGRPs in bacterial infection remains unclear. In this study, we report the function of mouse PGRP-S, the homologue of PGRP-SA, in bacterial infection. The recombinant protein was produced in Escherichia coli overexpression system and used for specifi c antibody production. We investigated the role of PGRP-S in infection with Listeria monocytogenes, a facultative Gram-positive bacterium that can grow intracellularly. The administration of recombinant PGRP-S before L. monocytogenes infection decreased the number of bacteria in the organs of infected mice. When endogenous PGRP-S was neutralized by antibodies specifi c to PGRP-S, the bacterial number increased. The levels of proinfl ammatory cytokines that are essential in the protection against L. monocytogenes infection were lower when the specific antibody was administered prior to infection. Together with these, it is suggested that PGRP-S plays a role in the protection against L. monocytogenes infection.
 
Article
The Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from the Sprague-Dawley rat (SDR) and its inheritance is autosomal recessive. Our recent study has revealed that an 80-kb genomic DNA on 7q36 containing basic hair keratin genes, Kb21, Kb23, Kb26 and Krt2-25, is deleted in HHR. To characterize hair follicles in HHR, progression of hair cycle and expression profi les of basic hair keratins were immunohistochemically studied and compared with those of SDR. The HHR exhibited sparse hairs and their twisted hairs were shorter than SDR hairs. HHR hair follicles entered the catagen phase earlier than SDR and massive destruction of HHR hair follicles and infi ltration of infl ammatory cells occurred in the catagen phase. In HHR the hair medulla was enlarged and the inner root sheath was thinned while the hair cortex was formed where Kb25 was expressed. In SDR Kb25 was expressed in the hair matrix and medulla. Electron microscopy indicated loss of the cuticle in HHR. These results suggest that hypotrichosis of HHR is due to the deletion of hair keratin genes and expression of a keratin fusion gene. Thus, HHR seems to be a useful model to examine the role of hair keratins in the hair follicle formation.
 
Article
The circadian rhythms in mammals are regulated by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. Five clock-gene families, i.e. Clock, Bmal, Per, Cry and Dec, have been found to be involved in a transcription-translation feedback loop that generates the circadian rhythm at the intracellular level. In this study, we examined functional analysis of the Dec gene. DEC1 and DEC2 are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular diff erentiation, responses to hypoxia, and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was upregulated by hypoxia, however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is upregulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1 α (HIF-1α) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1α but not with ARNT1. The binding of HIF-1α to the hypoxia response element( HRE) in the VEGF promoter was decreased by DEC2 overexpression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.
 
Article
Congenital Hyperinsulinism in Infancy (HI) is a potentially-lethal condition of neonates and during early childhood. For many years the pathophysiology of this disorder was unknown. Recent advances in genetics, histopathology and molecule physiology have now revealed the causes of HI in a large cohort of patients. From defects in ion channel subunit genes to lesions in the control of pancreatic B-cell metabolism and anaplerosis, the causes of HI are both varied and numerous. However, in all cases they appear to share a common target protein - the ATPsensitive K-channel. The function of these channels is not only critical to the control of healthy normal insulin-secreting cell function, but “activating” defects in these channels lead to permanent neonatal diabetes and type 2 diabetes. HI can therefore arise through “channelopathies” of K-ATP channels: HI-KATP through gene defects in ABCC8 and KCNJ11 (Ch11.p15); or as a result of “metabolopathies” through defects in the genes encoding glucokinase HI-GK (GCK, Ch.7p15-p13), glutamate dehydrogenase HI-GDH (GLUD1, Ch.10q23.3) and Short-chain L-3-hydroxyacyl-CoA dehydrogenase HI-SCHAD( HADHSC, Ch.4q22-q26). Advances in the integration of genetic medicine and cell biology have provided key insights into the causes of HI, and this has been of key importance to the defi nition of pathogenesis. However, medical therapy for HI remains largely unchanged due to the availability of limited agents that are selective and specifi c for the termination of insulin release from β-cells. CHI can be a devastating disease, and in this review focuses upon the relationship between the basis of HI and current / future therapies, including stem cells.
 
Article
Responses to stimuli in cellar level are diverse and such hierarchical as secretion of stored factors, synthesis of lipid mediators and protein synthesis through genomic transcription. However, how can the cells respond in the case of necrosis? Recently a characteristic intranuclear protein, high-mobility group box 1 protein (HMGB1) is released from necrotic cells. The protein is an abundant nuclear protein with a dual function both inside and outside the cells. In physiological state, HMGB1 is present in the nucleus, and binds to DNA, playing a variety of crucial functions, including transcription and keeping the characteristic DNA architecture. However, the protein is released to extracellular space from most of necrotic cells, activated macrophages and dendritic cells. Out of the cells, HMGB1 acts as a signal of tissue damage and can promote infl ammation, immune responses, and results tissue regeneration. During sepsis and/or disseminated intravascular coagulation (DIC), however, massive accumulation of HMGB1 in the systemic circulation will cause multiple organ failure (MOF) and subsequent lethal outcome. Thus HMGB1 in the systemic circulation has been considered as a lethal mediator of sepsis, and a promising therapeutic target for sepsis. Recently we identified that thrombomodulin (TM), a natural anticoagulant glycoprotein expressed on the surface of endothelial cells, plays an important role in sequestering HMGB1. TM may prevent HMGB1 from reaching remote organs, thereby restricting the spectrum of HMGB1 action in the site of injury. Here we review recent progress made in defining the physiological and pathological roles of HMGB1 and therapeutic strategies aimed at blocking circulatory HMGB1.
 
Article
The localizations of nonheme-Fe( III) and Fe( II) were studied in the hypothalamo-neurohypophyseal system of the rat brain by light and electron microscopic nonheme iron-histochemistry. Fe (III)-deposit was heavily accumulated in the parvocellular part of the hypothalamic paraventricular nucleus( HPV), where numerous glias were stained. Fe( III)-deposit heavily fi lled the cytosol and lysosomes of microglia-, oligodendrocyte- and astrocyte-like cells, while neurons contained only a small number of Fe( III)-laden lysosomes. Fine processes of Fe( III)- laden microglia- and oligodendrocyte-like cells closely ensheathed the cell body and proximal dendrites of neurons. Fe (III)-laden astrocytelike cells tightly enclosed the capillary wall. The magnocellular part of the HPV and the supraoptic nucleus were less intensely stained than the parvocellular part of the HPV. The secretory axon terminals in the outer lamina of the median eminence( ME) and the posterior pituitary including the Herring bodies contained Fe( III)-laden small lysosomes among densely aggregated secretory granules. The secretory axon terminals in the ME were tightly enclosed by heavily Fe (III)-laden processes of tanycyte. The heavily Fe( III)-laden pituicytes and interstitial, pericapillary phagocytes closely approached the secretory axon terminals in the posterior pituitary. Fe (II) was largely localized in the lysosomes throughout the hypothalamo-neurohypophyseal system.
 
Article
Distinctive sex diff erences in reproductive behavior and physiology have been attributed not to diff erences in hormones gonad secretes in each sex but differences in particular brain structures. Interestingly enough, brain sex difference is determined mostly by gonadal hormones during ontogeny independent of genetic sex. In many laboratory mammals, brain sex can be manipulated at a particular stage of ontogeny called critical period for brain sex diff erentiation, by endocrine treatments. Thus, neonatal orchidectomy or administration of gonadal hormones to female pups reverses brain sex phenotype which culminates in sex-specifi c functions. The early, transient exposure to gonadal hormones or its absence leaves indelible marks on brain phenotype. This organizing action of gonadal hormones, combined with activational action of the hormones after puberty, accomplishes sex specific functions. Brain would undergo sex diff erentiation through neurogenesis, migration, or survival in addition to regulation of ion channels and other functional molecule. Two distinct structures, the sexually dimorphic nucleus of the preoptic area, which is larger in male rats than in males and the anteroventral periventricular nucleus, which furnish opposite characteristics, will provide good opportunity to understand the mechanism of brain sex diff erentiation.
 
Article
Neuronal activity mapping of cerebral functions using oxidative energetics has become an accepted functional magnetic resonance imaging (fMRI) technique, termed calibrated fMRI. It requires calculation of oxygen consumption (CMRO2) from blood oxygenation level dependent (BOLD) signal using multi-modal measurements of blood flow (CBF) and volume (CBV). This approach is based on a biophysical model which describes tissue oxygen extraction at steady-state, therefore it is unclear if this conventional steady-state BOLD model can be applied transiently for calculating dynamic CMRO2 changes. In particular, it is unknown whether calculation of CMRO2 from calibrated fMRI differs between brief and long stimuli. In this study linearity was experimentally demonstrated between BOLD-related components and neural activity. We used multi-modal fMRI (at 11.7T) and neuronal signal measurements of local fi eld potential (LFP) and multi-unit activity (MUA) in α-chloralose anesthetized rats during forepaw stimulation to show that respective transfer functions (of BOLD, CBV, CBF) generated by deconvolution with LFP( or MUA) are time invariant, for events in the millisecond to minute range. Since the transfer functions are time invariant for event-related and steady-state stimuli, it is possible to use calibrated fMRI in a dynamic manner. The multi-modal results allowed assignment of a significant component of the BOLD signal that can be ascribed to CMRO2 transients. Here we discuss the importance of minimizing residual signal, represented by the diff erence between modeled and raw signals, in convolution analysis using multi-modal fMRI and neural signals.
 
Top-cited authors
Mitsuru Nakazawa
  • Hirosaki University
Tamas Revesz
  • University College London
Blas Frangione
  • NYU Langone Medical Center
Yasushi Tomidokoro
  • University of Tsukuba
Agueda Rostagno
  • NYU Langone Medical Center