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Biomedical subjects

M Kinoshita

Publications and source records attributed to M Kinoshita.

At least 109 records · Page 6Linked to original sources
PubMed2000

Neural responses in the retinotopic representation of the blind spot in the macaque V1 to stimuli for perceptual filling-in.

When visual stimuli that cover the entire blind spot are presented monocularly, the color and brightness of the surrounding field are seen within the blind spot, although it receives no retinal input. Important questions about such perceptual filling-in are whether neurons in the visual system representing visual field locations within the blind spot are activated when filling-in occurs and, if so, what the properties of these neurons are. To address these questions, we recorded the activities of single neurons in the primary visual cortex (V1) of the awake monkey. We first identified the area of V1 representing the region of the blind spot and then assessed neural responses to stationary visual stimuli of various size. We found that there are neurons in layer 4 and deeper laminae, particularly layer 6, that respond to large stimuli covering the blind spot which induces perceptual filling-in. Most of these neurons had very large binocular receptive fields that extended outside the blind spot. These neurons also preferred relatively large stimuli and exhibited color selectivity. These results indicate that when a large uniform surface is presented on the blind spot, neurons at the V1 region representing the blind spot transmit signals essential for filling-in that inform of the presence of a large surface as well as the absence of smaller stimuli at the blind spot.

Action Potentials↗
PubMed2000

Differential localization of septins in the mouse brain.

We have carried out a comparative immunohistochemical study on four members of the septin family, CDCrel-1, Septin6, CDC10, and H5, which are abundantly expressed in the adult mouse brain. We found that each septin showed overlapping but distinct distribution at the levels of light and electron microscopy. CDCrel-1 was abundant in inhibitory presynaptic terminals and associated with GABAergic vesicles in the thalamus, globus pallidus, and cerebellar nuclei. Septin6 was associated with synaptic vesicles in various brain regions, including glomeruli of the olfactory bulb. CDC10 was diffusely expressed in the brain and was localized beneath presynaptic membrane and astroglial processes. H5 was localized in the astroglial processes in some specific brain regions. The differential expression and subcellular localization of these septins indicates that a given neuron or glial cell expresses a specific set of septin monomers and that the resulting septin complexes with distinct compositions may play distinct roles in the brain.

Animals↗
PubMed2000

A novel protein, RTN-XS, interacts with both Bcl-XL and Bcl-2 on endoplasmic reticulum and reduces their anti-apoptotic activity.

Bcl-2 and Bcl-XL serve as critical inhibitors of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. We identified two members of the reticulon (RTN) family as Bcl-XL binding proteins, i.e., NSP-C (RTN1-C) and a new family member, RTN-XS, both of which did not belong to the Bcl-2 family and were predominantly localized on the endoplasmic reticulum (ER). RTN-XS interacted with both Bcl-XL and Bcl-2, increased the localization of Bcl-XL and Bcl-2 on the ER, and reduced the anti-apoptotic activity of Bcl-XL and Bcl-2. On the other hand, NSP-C interacted only with Bcl-XL, affected the localization of Bcl-XL, and reduced Bcl-XL activity, but had no effect on Bcl-2. These results suggest that RTN family proteins can modulate the anti-apoptotic activity of Bcl-XL and Bcl-2 by binding with them and can change their localization to the ER.

Amino Acid Sequence↗
PubMed2000

Ionotropic glutamate receptors expressed in human retinoblastoma Y79 cells.

Mammalian retinal photoreceptors and pinealocytes have common characteristic that they secrete melatonin and L-glutamate as chemical transmitters. Although pinealocytes express glutamate receptors and receive glutamate signals, whether or not photoreceptors express glutamate receptors is unknown. Here, we investigated the expression of the glutamate receptors in cultured Y79 clonal human retinoblastoma cells, as model systems of photoreceptors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that GluR1, GluR5, GluR7, EAA2, NR1, NR2A and NR2D mRNAs were present in the cultured cells. Northern analysis confirmed the presence of GluR7, EAA2, NR1, NR2A and NR2D mRNAs, while other mRNAs were under the detection limit. Addition of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate increases intracellular (Ca(2+)) in Fura-2 loaded cells, which is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. N-methyl-D-aspartate also increases intracellular (Ca(2+)). These results demonstrated the presence of functional ionotropic receptors in Y79 cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

High levels of plasma brain natriuretic peptide and interleukin-6 after optimized treatment for heart failure are independent risk factors for morbidity and mortality in patients with congestive heart failure.

OBJECTIVES: The aim of this study was to evaluate whether repetitive measurements of plasma levels of neurohumoral factors and cytokines before and after additional treatment are useful for predicting mortality in patients with congestive heart failure (CHF). BACKGROUND: Neurohumoral and immune activation play an important role in the pathophysiology of CHF. However, the effects of serial changes in these factors on the prognostic value remain unknown. METHODS: We measured plasma levels of neurohumoral factors and cytokines and left ventricular ejection fraction (LVEF) before and three months after optimized treatment for CHF in 102 consecutive patients with severe CHF (New York Heart Association class III to IV) on admission to our hospital. Physicians who were blind to the plasma neurohumoral factors until study completion treated patients using standard drugs. Patients were monitored for a mean follow-up period of 807 days. RESULTS: Plasma levels of neurohumoral factors, cytokines and LVEF were significantly improved three months after optimized treatment. Cardiac death occurred in 26 patients. Among 19 variables including LVEF, only a high level of brain natriuretic peptide (BNP) and interleukin-6 (IL-6) at three months after optimized treatment showed significant independent relationships by Cox proportional hazard analysis with a high mortality for patients with CHF. CONCLUSIONS: These findings indicate that high plasma BNP and IL-6 levels three months after optimized treatment are independent risk factors for mortality in patients with CHF, suggesting that sustained high plasma levels of BNP and IL-6 after additional standard treatment were independent risk factors for mortality in patients with CHF despite improvements in LVEF and symptoms.

Aged↗
PubMed2000

Transcardiac extraction of circulating endothelin-1 across the failing heart.

To determine the transcardiac gradient of plasma endothelin-1 (ET-1) in patients with congestive heart failure (CHF), we measured plasma levels of ET-1 in both the aortic root and the coronary sinus in 14 normal subjects and 79 consecutive patients with CHF. In normal subjects, plasma ET-1 was significantly higher in the coronary sinus than in the aortic root; these findings were also shown in patients with mild CHF, suggesting that there was ET-1 spillover across the heart. In contrast, plasma ET-1 was significantly lower in the coronary sinus than in the aortic root in patients with severe CHF, suggesting there was ET-1 extraction across the heart in patients with severe CHF. The transcardiac gradient of plasma ET-1 was correlated with the left ventricular end-diastolic volume index (r = 0.501, p <0.0001) and plasma level of procollagen type III amino terminal peptide in the coronary sinus (r = 0.54, p = 0.0008), a marker of myocardial fibrosis. Stepwise multivariate analysis showed that the transcardiac gradient of plasma ET-1 was an independent and significant relation with the left ventricular end-diastolic volume index in patients with CHF (r = 0.665, p <0.0001). These findings suggest that elevated circulating ET-1 is extracted across the failing heart with a significant correlation between the transcardiac gradient of plasma ET-1 and the left ventricular end-diastolic volume index, suggesting that ET receptors are upregulated in the failing ventricle and that the elevated circulating ET-1 might stimulate the process of left ventricular remodeling in patients with severe CHF.

Angiotensin II↗
PubMed2000

Crocus sativus lectin recognizes Man3GlcNAc in the N-glycan core structure.

Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Manalpha(1-3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Manalpha(1-6)Man branch. Most interestingly, the beta-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn in N-glycans and the l-Fuc linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications.

Animals↗
PubMed2000

Introduction of a foreign gene into medakafish using the particle gun method.

We developed a procedure to introduce a foreign gene into fertilized eggs of medakafish (Oryzias latipes) using the particle gun method, which is one of the easiest and most reliable techniques for gene transfer. A plasmid construct with the green fluorescence protein (GFP) gene driven by the madakafish beta-actin gene promoter was successfully introduced into eggs, and the expression of GFP was observed in 20% of the primary transfectant (chimera) fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant fish. The new application described here should enable us to investigate gene expression using the fish model on a routine basis without high technical sophistication. J. Exp. Zool. 287:285-293, 2000.

Animals↗
PubMed2000

Fluorometric determination of mucin-type glycoproteins by the galactose oxidase-peroxidase method.

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.

Animals↗
PubMed2000

Molecular cloning and characterization of antigens expressed on rat tumor vascular endothelial cells.

We have previously prepared monoclonal antibodies (MAbs) against tumor vasculature using cultured rat tumor endothelial cells (TECs) isolated from solid KMT-17 tumors and identified 40 and 80 kDa antigens recognized by TES-17 and TES-23 MAbs, respectively. To clarify the nature of antigens on tumor vasculature, molecular cloning was conducted by screening the rat TEC cDNA library. Two antigens were detected: a 40 kDa OTS-8 antigen, which had been defined as a differentiation marker for osteoblastic lineage, and an 80 kDa CD44H antigen. Northern blots showed that OTS-8 mRNA was expressed exclusively in the lung, in addition to TECs, while CD44H mRNA was detected in the lung, intestine, spleen, thymus and peripheral blood cells, in addition to TECs. Immuno-histochemistry of KMT-17 tumors revealed that OTS-8 and CD44 were expressed on sprouting TECs. In addition, TES-23 MAb stained TECs of tubular vessels as well as sprouting TECs, but anti-rat CD44 MAbs stained only sprouting TECs. PCR showed that CD44 cDNA with a splice in exon 6 (CD44ex6) was present in rat TECs at low levels. Our results indicate that OTS-8 and CD44 are expressed on rat sprouting TECs and that TES-23 MAb recognizes CD44H on sprouting TECs as well as an unknown epitope on TECs of tubular vessels that could not be recognized by anti-rat CD44 MAbs.

Animals↗
PubMed2000

Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase.

The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. We purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in vitro by PKG, but relatively poorly by the related cAMP-dependent protein kinase and not by protein kinase C. Two splice variants of G-septin (alpha and beta) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C-terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [alpha-(32)P]GTP confirmed that G-septin is a GTP-binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons.

Alternative Splicing↗
PubMed2000

T-588, a novel neuroprotective agent, delays progression of neuromuscular dysfunction in wobbler mouse motoneuron disease.

R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588) enhances acetylcholine release from the frontal cortex and hippocampus in rats, and can ameliorate cognitive dysfunction in various amnesia models of rodents. T-588 protects rat cerebellar granule cells from glutamate neurotoxicity in culture. This agent also inhibits facilitation in the crayfish neuromuscular junction and mammalian cerebellum. Clinical trials of T-588 are underway in patients with Alzheimer's disease. We attempted to determine whether T-588 treatment ameliorates neuromuscular dysfunction in the wobbler mouse, an animal model of motoneuron disease (MND). After the initial diagnosis of MND at the age of 3-4 weeks, wobbler mice were orally administered T-588 (3, 10, 30 mg/kg) or vehicle daily for 4 weeks in a blinded fashion. We compared symptomatic, pathological and biochemical changes among the groups. In comparison with vehicle, T-588 administration potentiated grip strength, attenuated forelimb contracture and increased the weight of the biceps muscles. T-588-treated mice had retarded denervation muscle atrophy and elevated activities of choline acetyltransferase (ChAT) or lactate dehydrogenase in the biceps muscles. T-588 treatment also enhanced ChAT activities and promoted formation of cyclic adenosine monophosphate in the cervical cord. Pharmacokinetic study also showed that T-588 was transported efficiently into the cerebrum and spinal cord following oral administration. Thus, T-588 treatment delayed the progression of wobbler murine MND. Our findings suggest that this agent has therapeutic potential in human motor neuropathy or MND.

Administration, Oral↗