Biomedical subjects

Y Itoh

Publications and source records attributed to Y Itoh.

At least 55 records · Page 3Linked to original sources

PubMed2002

Changes of endothelin in streptozotocin-induced diabetic rats: effects of an angiotensin converting enzyme inhibitor, enalapril maleate.

Endothelin-1 (ET-1) concentrations are increased in patients with diabetes mellitus, particularly those with diabetic retinopathy, or essential hypertension. We hypothesized that ET-1 might participate in the development and progression of diabetic microangiopathy. In this study, the effects of the angiotensin converting enzyme (ACE) inhibitor, enalapril maleate, on diabetic angiopathy were examined in streptozotocin (STZ)-induced diabetic (STZ-DM) rats by monitoring variations in renal function and ET-1 concentrations in blood and organ tissues. Significant increases in kidney weight and in concentrations of urinary albumin, N-acetyl-fl-d-glucosamidase (NAG) and serum ET-1 were observed in the STZ-DM rats as compared with the non-diabetic rats, and the concentration of ET-1 in the kidneys tended to be increased. Microscopic and electron microscopic analyses showed increased mesangial cell proliferation, matrix expansion and enlarged mesangial area in the kidney of the diabetic rats. After administration of the ACE inhibitor, increased concentrations of urinary albumin and NAG in the STZ-DM rats were reduced to the control values with a slight improvement in the electron microscopic changes. These data suggest that ET-1 may be involved in the development and progression of diabetic nephropathy and may explain, in part, why diabetes is liable to complicate hypertension. ACE inhibitor may help to restore diabetic nephropathy in the STZ-induced diabetic rats.

Acetylglucosaminidase↗

PubMed2001

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Animals↗

PubMed2001

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Animals↗

PubMed2001

Involvement of interleukin-1beta/nitric oxide pathway in the postischemic impairment of long-term potentiation of the rat hippocampus.

To investigate whether postischemic cerebral dysfunction occurs via the interleukin-1 beta/nitric oxide (IL-1beta/NO) pathway, we examined the effects of an IL-1beta antagonist on long-term potentiation (LTP) impairment and excessive NO production in the rat hippocampus after 10-min global ischemia. Intracerebroventricilar administration of the IL-1beta antagonist attenuated NO production and rescued LTP impairment in the perforant path-dentate gyrus synapses, observed 1 day and 4 days after ischemic insult, respectively. There was an inverse relationship between LTP in the dentate gyrus synapses and hippocampal NO production. Centrally applied IL-1beta mimicked the consequences of transient ischemia in LTP formation and hippocampal NO production in non-ischemic rats. These findings indicate that the IL-1beta/NO pathway is involved in the hippocampal LTP impairment observed in the postischemic brain.

Animals↗

PubMed2001

Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion.

Activation of proMMP-2 by MT1-MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP-2 bound to MT1-MMP on the cell surface acts as a receptor for proMMP-2. Subsequently, adjacent TIMP-2-free MT1-MMP activates the proMMP-2 in the ternary complex. In this study, we demonstrate that MT1-MMP forms a homophilic complex through the hemopexin-like (PEX) domain that acts as a mechanism to keep MT1-MMP molecules close together to facilitate proMMP-2 activation. Deletion of the PEX domain in MT1-MMP, or swapping the domain with the one derived from MT4-MMP, abolished the ability to activate proMMP-2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1-MMP lacking the catalytic domain (MT1PEX-F) efficiently inhibited complex formation of the full-length enzymes and activation of pro MMP-2. Furthermore, expression of MT1PEX-F inhibited proMMP-2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1-MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue.

Animals↗

PubMed2001

Genomic DNA released by dying cells induces the maturation of APCs.

Mature APCs play a key role in the induction of Ag-specific immunity. This work examines whether genomic DNA released by dying cells provides a stimulus for APC maturation. Double-stranded but not single-stranded genomic DNA triggered APC to up-regulate expression of MHC class I/II and various costimulatory molecules. Functionally, dsDNA enhanced APC function in vitro and improved primary cellular and humoral immune responses in vivo. These effects were dependent on the length and concentration of the dsDNA but were independent of nucleotide sequence. The maturation of APC induced by dsDNA may promote host survival by improving immune surveillance at sites of tissue injury/infection.

Animals↗

PubMed2001

Retention and transit of intestinal mucoadhesive films in rat small intestine.

The retention and transit characteristics of intestinal mucoadhesive film systems have been studied after intraduodenal administration in rats. Small size four layered film preparations, 0.5x0.5 mm, were prepared, where the backing layer (45.1+/-2.9 microm thick) was made of a water-insoluble polymer, ethylcellulose (EC), the surface layer was made of enteric pH-sensitive polymers, Eudragit L100, S100 or HP-55 and the middle layer was made of cellulose membrane. The surface layer was attached to the middle layer with an adhesive layer composed of carboxyvinyl polymer (Hiviswako(R) 103). After administration of ten films to the duodenum, the rats were sacrificed hourly and the distribution of the films in the whole small intestine was directly observed after abdominal incision. The HP-55, Eudragit L100 and S100 film systems were found to adhere to the upper, middle and lower part of the small intestine after 1, 2 and 4 h, respectively, for 2-3 h. Direct inspection study suggests that intestinal mucoadhesive film system has functions of: (1) pH-dependent intestinal adhesion site specificity; (2) adhesion to the intestinal wall; and (3) retention in the small intestinal adhesion site for at least 2 h. Intestinal mucoadhesive film system has been suggested to be a targeting system for drugs to the gastrointestinal tract.

Adhesiveness↗

PubMed2001

IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10.

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8(+) DC preferentially induce Th1 responses, whereas CD8(-) DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8(-) than CD8(+) DC produced IL-12 after HKBA challenge, whereas only CD8(+) DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11c(low) cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8(+) and CD8(-) DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.

Adjuvants, Immunologic↗

PubMed2001

Brain glucose utilization in mice with a targeted mutation in the thyroid hormone alpha or beta receptor gene.

Brain glucose utilization is markedly depressed in adult rats made cretinous after birth. To ascertain which subtype of thyroid hormone (TH) receptors, TRalpha1 or TRbeta, is involved in the regulation of glucose utilization during brain development, we used the 2-[(14)C]deoxyglucose method in mice with a mutation in either their TRalpha or TRbeta gene. A C insertion produced a frameshift mutation in their carboxyl terminus. These mutants lacked TH binding and transactivation activities and exhibited potent dominant negative activity. Glucose utilization in the homozygous TRbetaPV mutant mice and their wild-type siblings was almost identical in 19 brain regions, whereas it was markedly reduced in all brain regions of the heterozygous TRalpha1PV mice. These suggest that the alpha1 receptor mediates the TH effects in brain. Inasmuch as local cerebral glucose utilization is closely related to local synaptic activity, we also examined which thyroid hormone receptor is involved in the expression of synaptotagmin-related gene 1 (Srg1), a TH-positively regulated gene involved in the formation and function of synapses [Thompson, C. C. (1996) J. Neurosci. 16, 7832-7840]. Northern analysis showed that Srg1 expression was markedly reduced in the cerebellum of TRalpha(PV/+) mice but not TRbeta(PV/PV) mice. These results show that the same receptor, TRalpha1, is involved in the regulation by TH of both glucose utilization and Srg1 expression.

Age Factors↗

PubMed2001

Reversal by NS-7, a neuroprotective compound, of the decrease in transcription factor CREB mRNA expression in rat brain after permanent middle cerebral artery occlusion.

The effect of a neuroprotective agent NS-7 on changes in mRNA expressions for cyclic AMP responsive element binding protein (CREB) and several neurotrophins was examined in the rat cerebral cortex after permanent middle cerebral artery occlusion (MCAO). Significant reduction in mRNA expressions for CREB was observed at 24h after MCAO. NS-7 (0.5mg/kg), when injected at 6h after MCAO, significantly reversed the decreased expression for CREB mRNA. In addition, the mRNA expression for basic fibroblast growth factor (bFGF) was also significantly enhanced by NS-7 in MCA-occluded but not in sham-operated rats. On the other hand, the mRNAs for interluekin-6 and inducible-type nitric oxide synthase were markedly induced in the cerebral cortex of MCA-occluded rats, which was not significantly reversed by NS-7. Therefore, it is suggested that the reversal of decrease in CREB mRNA and concomitant increase in mRNA expression for bFGF may contribute to the neuroprotective action of NS-7.

Animals↗

PubMed2001

Distribution of serum C-reactive protein and its association with atherosclerotic risk factors in a Japanese population : Jichi Medical School Cohort Study.

The distribution of serum C-reactive protein (CRP) levels and their association with age, sex, and atherosclerotic risk factors were studied in a large Japanese population between 1992 and 1995. The subjects consisted of 2,275 males and 3,832 females aged 30 years and over. CRP was measured by nephelometry. The distribution of CRP was highly skewed toward a lower level than that of previous studies and seemed to be a combination of two separate distribution curves. The increase in CRP with age was statistically significant, and males had higher CRP levels than did females. Males who were current smokers had higher CRP levels than did nonsmokers. Age, systolic blood pressure, diastolic blood pressure, triglycerides, fibrinogen, and body mass index were all positively associated with CRP in both sexes, while total cholesterol and blood glucose were positively related in females only. High density lipoprotein cholesterol was inversely related in both sexes. Multiple logistic regression analysis showed that sex, age, systolic pressure, high density lipoprotein cholesterol, triglycerides, fibrinogen, and body mass index were significant independent variables. In conclusion, the distribution of CRP among the Japanese was quite different from that among Westerners, although CRP levels correlated with other atherosclerotic risk factors, similar to those in Westerners.

Adult↗

PubMed2001

Effects of anesthesia on functional activation of cerebral blood flow and metabolism.

Functional brain mapping based on changes in local cerebral blood flow (lCBF) or glucose utilization (lCMR(glc)) induced by functional activation is generally carried out in animals under anesthesia, usually alpha-chloralose because of its lesser effects on cardiovascular, respiratory, and reflex functions. Results of studies on the role of nitric oxide (NO) in the mechanism of functional activation of lCBF have differed in unanesthetized and anesthetized animals. NO synthase inhibition markedly attenuates or eliminates the lCBF responses in anesthetized animals but not in unanesthetized animals. The present study examines in conscious rats and rats anesthetized with alpha-chloralose the effects of vibrissal stimulation on lCMR(glc) and lCBF in the whisker-to-barrel cortex pathway and on the effects of NO synthase inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME) on the magnitude of the responses. Anesthesia markedly reduced the lCBF and lCMR(glc) responses in the ventral posteromedial thalamic nucleus and barrel cortex but not in the spinal and principal trigeminal nuclei. L-NAME did not alter the lCBF responses in any of the structures of the pathway in the unanesthetized rats and also not in the trigeminal nuclei of the anesthetized rats. In the thalamus and sensory cortex of the anesthetized rats, where the lCBF responses to stimulation had already been drastically diminished by the anesthesia, L-NAME treatment resulted in loss of statistically significant activation of lCBF by vibrissal stimulation. These results indicate that NO does not mediate functional activation of lCBF under physiological conditions.

Anesthesia, General↗

PubMed2001

Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases.

Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.

Animals↗

PubMed2001

Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration.

Migratory cells including invasive tumor cells frequently express CD44, a major receptor for hyaluronan and membrane-type 1 matrix metalloproteinase (MT1-MMP) that degrades extracellular matrix at the pericellular region. In this study, we demonstrate that MT1-MMP acts as a processing enzyme for CD44H, releasing it into the medium as a soluble 70-kD fragment. Furthermore, this processing event stimulates cell motility; however, expression of either CD44H or MT1-MMP alone did not stimulate cell motility. Coexpression of MT1-MMP and mutant CD44H lacking the MT1-MMP-processing site did not result in shedding and did not promote cell migration, suggesting that the processing of CD44H by MT1-MMP is critical in the migratory stimulation. Moreover, expression of the mutant CD44H inhibited the cell migration promoted by CD44H and MT1-MMP in a dominant-negative manner. The pancreatic tumor cell line, MIA PaCa-2, was found to shed the 70-kD CD44H fragment in a MT1-MMP-dependent manner. Expression of the mutant CD44H in the cells as well as MMP inhibitor treatment effectively inhibited the migration, suggesting that MIA PaCa-2 cells indeed use the CD44H and MT1-MMP as migratory devices. These findings revealed a novel interaction of the two molecules that have each been implicated in tumor cell migration and invasion.

Amino Acid Sequence↗

PubMed2001

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at lambda >495 nm. Subsequent irradiation of the complex at lambda310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with T(alpha). For identification of the sites of crosslinks in T(alpha), the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T(alpha) species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T(alpha) derivatives and isolation of T(alpha) peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T(alpha) containing the amino acid sequences 310-313 and 342-345.

Amino Acid Sequence↗

PubMed2001

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent.

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at lambda > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the T(alpha) subunit. Crosslinking to T(alpha) was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in T(alpha) were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19-Arg-28 at the N-terminal region of T(alpha). The total results show that both the N and the C termini of T(alpha) are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Amino Acid Sequence↗

PubMed2001

Aichi Cancer Center 10-year experience with conservative breast treatment of early breast cancer: retrospective analysis regarding failure patterns and factors influencing local control.

PURPOSE: We analyzed the clinical results of conservative breast therapy in our institute to determine the risk factors influencing local and distant disease recurrence. METHODS AND MATERIALS: From 1989 to 1997, 301 breasts of 295 women with early breast cancer were treated with conservative surgery and adjuvant radiotherapy. There were 212 incidences of Stage I breast cancer, and 89 of Stage II. Patients were routinely treated with local resection, axillar dissection, and 46--50 Gy irradiation given in 23--25 fractions. Some also received a radiation boost to the tumor bed. RESULTS: The 5-/8-year overall survival, disease-free survival, and local control rates were 93.2/91.5%, 86.0/80.6%, and 95.1/92.5%, respectively. Using both univariate and multivariate analyses, tumor volume, estrogen receptor status, and age < 40 years were significant prognostic factors for disease-free survival. Both age < 40 years and surgical method had a strong effect on local control by uni- and multivariate analysis. Surgical margin status was a significant prognostic factor for local control at the univariate level (p < 0.0001), though it had only borderline significance at the multivariate level (p = 0.08). No patient experienced severe morbidity due to radiotherapy. CONCLUSION: The results obtained are comparable to previously reported data. Although the follow-up period was too short to draw definite conclusions about long-term outcomes, the outcome from conservative breast treatment was acceptable.

Adult↗

PubMed2001

Leptin receptor 5'untranslated regions in the rat: relative abundance, genomic organization and relation to putative response elements.

Hypothalamic sensitivity to leptin has been suggested to be important for regulation of body fat mass. Mice heterozygous for a mutation in the leptin receptor (leptin-R) have an increased body fat mass suggesting that the abundance of leptin-R may be an important determinator of leptin sensitivity. Leptin-R cDNAs from several species contain alternative 5'untranslated regions (5'UTRs), suggesting that several distinct regulatory regions may exist. To investigate possible mechanisms by which leptin-R expression may be regulated, we searched for possible alternative 5'UTRs of the leptin-R in the rat and determined their location in relation to putative response elements. Four leptin-R 5'UTRs (exons 1A-1D), which diverged 23 bp upstream of the start codon, were identified by 5'Rapid Amplification of cDNA Ends (5'RACE) and sequencing. Exons 1B and 1C were present in 31 and 61%, respectively, of all leptin-R transcripts in the hypothalamus as determined by a ribonuclease protection assay. Analysis of the 5' flanking genomic sequences revealed an imperfect estrogen response element (ERE), two Spl-sites, three CCAAT-boxes and one octamer. Exons 1A and 1D corresponded to a putative second gene, encoding the OB-Receptor Gene Related Protein (OB-RGRP), which is transcribed from a promoter shared with the leptin-R. DNA sequencing revealed that the rat OB-RGRP had 98 and 97% homology with the mouse and human sequence, respectively. We report here that transcription of the rat leptin-R gene may generate transcripts with four alternative 5'UTRs. The presence of a putative ERE, close to the most frequently used transcriptional start sites of the leptin-R gene in the hypothalamus, provides a possible mechanism by which estrogen may exert its effects on food intake.

5' Untranslated Regions↗