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T Kosaka

Publications and source records attributed to T Kosaka.

At least 55 records · Page 3Linked to original sources
PubMed2000

Proline- and alanine-rich Ste20-related kinase associates with F-actin and translocates from the cytosol to cytoskeleton upon cellular stresses.

Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeleton in vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 degrees C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock.

3T3 Cells↗
PubMed2000

Identification and C-terminal characterization of proteins from two-dimensional polyacrylamide gels by a combination of isotopic labeling and nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry.

We propose a novel method for the identification and C-terminal characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were digested in a gel in a buffer solution containing 50% 18O-labeled water, and mixtures of 18O/16O-labeled peptides were analyzed by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). This method was evaluated using horse skeletal muscle myoglobin as the model protein in SDS gel. The high resolution of FT-ICR MS minimized the overlapping of peptide peaks and facilitated identification of the C-terminal peptide, which was done by observing the undisrupted isotope peak pattern. As well, with its low ppm-level high mass accuracy, it can rapidly and reliably identify the in-gel-separated protein and determine its C-terminal by peptide mass fingerprinting alone. Therefore, this method should be applicable to routine and high-throughput proteome studies. Here, the method was applied to the analysis of rat liver proteins separated by 2D-PAGE. The C-termini of eight proteins were successfully identified out of 10 randomly picked Coomassie brilliant blue-stained spots. The feasibility and limitations of this approach are reported in this paper.

Amino Acid Sequence↗
PubMed2000

Gap junctions linking the dendritic network of GABAergic interneurons in the hippocampus.

The network of GABAergic interneurons connected by chemical synapses is a candidate for the generator of synchronized oscillations in the hippocampus. We present evidence that parvalbumin (PV)-containing GABAergic neurons in the rat hippocampal CA1 region, known to form a network by mutual synaptic contacts, also form another network connected by dendrodendritic gap junctions. Distal dendrites of PV neurons run parallel to the alveus (hippocampal white matter) and establish multiple contacts with one another at the border between the stratum oriens and the alveus. In electron microscopic serial section analysis, gap junctions could be identified clearly at 24% of these contact sites. A dendrodendritic chemical synapse and a mixed synapse also were found between PV-immunoreactive dendrites. Three-dimensional reconstruction of the dendritic arborization revealed that both PV neurons of the well known vertical type (presumptive basket cells and axoaxonic cells) and those of another horizontal type constitute the dendritic network at the light microscopic level. The extent of dendritic fields of single PV neurons in the lateral direction was 538 +/- 201 micrometer (n = 5) in the vertical type and 838 +/- 159 micrometer (n = 6) in the horizontal type. Our previous and present observations indicate that PV-containing GABAergic neurons in the hippocampus form the dual networks connected by chemical and electrical synapses located at axosomatic and dendrodendritic contact sites, respectively. Gap junctions linking the dendritic network may mediate coherent synaptic inputs to distant interneurons and thereby facilitate the synchronization of oscillatory activities generated in the interneuron network.

Animals↗
PubMed2000

Activation of nuclear factor kappaB at the onset of ossification of the spinal ligaments.

We examined the correlation between the activation of nuclear factor kappaB (NFkappaB), stimulated by environmental factors involving cytokines and growth factors in ligament cells, and the onset of ossification of the spinal ligaments (OSL) or diffuse idiopathic skeletal hyperostosis (DISH). Aseptic samples were taken carefully from non-ossified sites during surgery (75 patients). We carried out preliminary hematoxylin and eosin and toluidine blue staining, using five portions of each specimen, and excluded samples containing chondrocytic, osteoblastic, or inflammatory cells (n = 25). We used specimens from the remaining 50 patients (35 men and 15 women, ranging in age from 45-81 years); average age, 59.5 years (18 nuchal ligament specimens, and 32 yellow ligament specimens). OSL or DISH had occurred in 25 patients, 20 patients were in the non-OSL group (8 with cervical spondylotic myelopathy, and 12 with lumbar canal stenosis), and the remaining 5 samples were collected from patients with injury. For culture study, we used portions of the 14 largest samples from the above 50 patients. We extracted nuclear proteins and cytoplasmic proteins from non-ossified spinal ligaments in 50 patients and detected p65RelA/NFkappaB by Western blotting. Tumor necrosis factor-alpha (TNF alpha), interleukin 1beta (IL-1beta), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1) in cytoplasm were quantified by enzyme-linked immunosorbent assays (ELISA). Cultured cells from the 14 samples were then stimulated with 10, 100, 250, or 500 ng/ml of recombinant human (rh)PDGF-B or TGFbeta1. A control experiment was performed without rhPDGF-BB or TGFbeta1 stimulation. Alkaline phosphatase (ALP) activity was standardized by the DNA content of the cells. The number of NFkappaB-positive samples was significantly higher in patients with OSL or DISH than in non-OSL patients. This tendency was obvious in the case of OSL or DISH with non-insulin-dependent diabetes mellitus (NIDDM). In OSL and in DISH patients, significantly greater amounts of PDGF-BB and TGFbeta1 were seen in ligament cells than in non-OSL patients (P < 0.05). There was a positive correlation between the detection of p65RelA/NFkappaB band and the content of PDGF-BB and TGFbeta1 in ligament cells (P < 0.05). ALP activity tended to be higher in cells in the OSL group not receiving any other treatment. Our results indicate the possibility that NFkappaB, stimulated by environmental factors involving PDGF-BB and TGFbeta1 in ligament cells, influences the osteoblastic differentiation of undifferentiated mesenchymal cells.

Actins↗
PubMed2000

Spectrophotometric assay for serum platelet-activating factor acetylhydrolase activity.

We developed a spectrophotometric assay for serum platelet-activating factor acetylhydrolase (PAF-AH, EC 3.1.1.47.) activity using a platelet-activating factor (PAF) analogue with a 4-nitrophenyl group as substrate. PAF-AH hydrolyzes the sn-2 position of the substrate ¿1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine, producing p-nitrophenyl succinate. This liberation was spectrophotometrically monitored and the activity determined from the change in absorption. The assay does not require radioisotopes and is applicable to an automatic analyzer. Utilizing this assay with an automatic analyzer, it is possible to measure the activities of thousands of samples in a few hours with excellent precision (CV 0.5%, n=30) and high correlation (r=0.979, n=100) with the results of a conventional radioisotopic assay. The assay should be particularly useful for clinical diagnostics.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2000

The dual network of GABAergic interneurons linked by both chemical and electrical synapses: a possible infrastructure of the cerebral cortex.

To know the structural feature of individual nerve cells and of the network they form is essentially important for understanding how the brain works. We have recently shown that a certain subpopulation of hippocampal GABAergic neurons that contain a calcium-binding protein parvalbumin form the dual network connected by both chemical synapses and gap junctions. The mutual chemical synaptic contacts are formed between their axon terminals and somata whereas gap junctions are located between their dendrites. In this article, we demonstrate that the dual network of parvalbumin-containing GABAergic interneurons is not restricted to the hippocampus but found also in the neocortex and, therefore, appears to be a fundamental structure of the cerebral cortex, possibly having some relevance to the synchronized activities observed broadly in various cortical areas.

Animals↗
PubMed2000

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb--IV. Intraglomerular synapses of tyrosine hydroxylase-immunoreactive neurons.

Synapses of intraglomerular processes of tyrosine hydroxylase-immunoreactive neurons in the rat main olfactory bulb were examined by electron microscopic immunocytochemistry. Prominent characteristics of intraglomerular synapses of tyrosine hydroxylase-immunoreactive elements were that the vast majority (about 80%) of their synaptic inputs were asymmetrical synapses from olfactory nerve terminals and, though far smaller in proportion, one half of the remaining were asymmetrical synapses from mitral/tufted cell dendrites and the other half were symmetrical synapses from gamma-aminobutyric acid-like immunoreactive elements. So far, we have observed no typical reciprocal synapses between tyrosine hydroxylase-immunoreactive processes and mitral/tufted dendrites; however, we have often identified serial synapses; that is, asymmetrical synapses from olfactory nerve terminals or mitral/tufted cell dendrites to tyrosine hydroxylase-immunoreactive processes, and then symmetrical synapses from the latter to different mitral/tufted cell dendrites. These synaptic connections of tyrosine hydroxylase-immunoreactive neurons were very different from those of Calbindin-D(28k)-immunoreactive neurons, which received no synaptic contact directly from olfactory nerve terminals but formed reciprocal synapses with mitral/tufted cells as we analysed previously.Thus, our present and previous electron microscopic studies combined with confocal laser scanning light microscopy clearly indicated for the first time the heterogeneity of periglomerular neurons, not only in their chemical and morphological features, but also in their synaptic organization in the olfactory glomerulus.

Animals↗
PubMed2000

Therapeutic immunization against Helicobacter pylori infection in the absence of antibodies.

Helicobacter pylori is an important human pathogen. Prophylactic immunization with bacterial antigen plus an adjuvant protects mice against challenge with live H. pylori. Surprisingly, it was found that immunizations of mice already infected with Helicobacter also influenced bacterial colonization. This concept of therapeutic immunization is a novel phenomenon. Because H. pylori lives in the lumen of the stomach, it was initially hypothesized that the protective mechanism would involve induction of secretory IgA. However, work with knockout mice has demonstrated that prophylactic immunization is equally effective in mice deficient in IgA and even in microMT mice lacking B lymphocytes. Currently nothing is known about therapeutic vaccination and the effect of immunizing a host with an ongoing ineffective immune response. To address this, we infected B-cell deficient, microMT mice with H. pylori and therapeutically immunized them four times in 3 weeks with bacterial sonicate and cholera toxin adjuvant. These immunizations significantly reduced colonization by H. pylori. The antibody- negative status of the microMT mice was confirmed by ELISA. Thus, therapeutic immunization stimulates an immune response, which reduces H. pylori infection via a mechanism that is antibody independent. How this is achieved remains to be determined, but may well involve a novel immune mechanism.

Adjuvants, Immunologic↗
PubMed2000

Comparison of long-term therapeutic effect of an ACE inhibitor, temocapril, with that of a diuretic on microalbuminuria in non-diabetic essential hypertension.

Many investigators have reported that angiotensin-converting enzyme (ACE) inhibitors have antiproteinuric effects and retard the progression of renal impairment in diabetic patients. On the other hand, those effects of ACE inhibitors have not been well established in patients with essential hypertension. This study was conducted to prospectively evaluate whether an ACE inhibitor, temocapril, could modify the urinary microalbumin excretion rate (UAE) in hypertensive outpatients who had no signs of renal impairment. To compare the long-term effect of temocapril with that of a diuretic on UAE, hypertensive patients treated with a diuretic (trichlormethiazide) were enrolled in a prospective study if they had normal serum creatinine levels and no overt proteinuria during a 3-month screening period. A urinary microalbumin-to-urinary-creatinine ratio (mg albumin/mmol Cr) was used as an estimate of UAE. Patients visited the hospital monthly to determine blood pressure (BP) and UAE. After baseline observation during the treatment with the diuretic, the subjects were randomly divided into two groups. In group A, the diuretic was switched to temocapril, 2 to 4 mg once daily for 12 months. In group B, the subjects continued to receive the diuretic for an additional 12 months. Seventy-six outpatients (41 men and 35 women; mean age, 59.0+/-1.4 years) with essential hypertension entered the study. The effects of temocapril on BP appeared to be clinically similar to those of the trichlormethiazide, but the use of temocapril significantly decreased UAE. In group A (n=37), UAE decreased significantly (p<0.01) from the baseline value of 4.19+/-0.37 mg albumin/mmol Cr to 2.47+/-0.29 and 2.68+/-0.28 mg albumin/mmol Cr at the 6th and 12th month of temocapril therapy, respectively. In contrast, in group B (n=39) UAE was unchanged (baseline, 4.16+/-0.63 mg albumin/mmol Cr; 6 months, 4.92+/-0.72; 12 months, 4.71+/-0.74). These results indicate that long-term therapy with temocapril may be superior in reducing UAE than is diuretic therapy in patients with essential hypertension who had no signs of renal impairment.

Albuminuria↗
PubMed2000

Active-oxygen involvement in canine NK-mediated cytotoxicity.

We examined the relationship between natural killer (NK) cell-mediated cytotoxicity, the produced active-oxygen and cytotoxic factor (CF) release in co-culturing canine NK cells with tumor cells (CL-1 target cells). In co-culturing, the adding of n-propyl gallate (active-oxide scavenger) removed the produced active-oxygen, which inhibited NK cell-mediated cytotoxicity and the CF release. Moreover, adding of this agent inhibited the tyrosine phosphorylation of NK intracellular protein which observed in co-culturing. Therefore, the active-oxygen produced from canine NK cells are thought to relate the signal transduction in NK-mediated cytotoxicity.

Animals↗
PubMed1999

Diversity of the calretinin immunoreactivity in the dentate gyrus of gerbils, hamsters, guinea pigs, and laboratory shrews.

We have recently reported that calretinin (CR) immunoreactivity in the mouse dentate gyrus (DG) is prominently different from that in the rat and monkey dentate gyrus. The CR-immunoreactive (IR) neuronal components characteristic of mouse DG were (1) mossy cells in the ventral hilus, (2) punctate elements in the inner molecular layer, (3) Cajal-Retzius cells in the molecular layer, and (4) immature granule cells at the basal part of the granule cell layer, which were also IR for highly polysialylated neural cell adhesion molecule. In this study, we examine the CR-IR elements in the DG of the gerbil, hamster, guinea pig, and laboratory shrew, and compare them with those of the rat, monkey, and mouse, reported previously. We show that mossy cells are distributed throughout the dorsoventral axis in all these animals, but mossy cells in the ventral hilus of the hamster, gerbil, and laboratory shrew are CR-IR, resembling those of the mouse, whereas mossy cells of the guinea pig are CR negative, like those of the rat. The inner molecular layer, the target zone of mossy cells, was observed to contain CR-IR punctae in the hamster, gerbil, and laboratory shrew, which corresponds to the CR immunoreactivity of the mossy cells. In addition, we observed CR-IR presumed Cajal-Retzius cells in all animals examined. On the other hand, CR-IR immature granule cells were encountered in the laboratory shrew, but not in other animals. The present study reveals prominent species differences in the CR-IR elements of the DG.

Animals↗
PubMed1999

Quantitative analysis of neuronal nitric oxide synthase-immunoreactive neurons in the mouse hippocampus with optical disector.

A detailed quantitative analysis of immunocytochemically identified nonprincipal neurons containing neuronal nitric oxide synthase (nNOS) was performed on the mouse hippocampus, with particular reference to the dorsoventral gradient. The present study applied two variations of a stereologic technique, the optical disector--one that used confocal laser-scanning microscope optical sections to examine colocalization of nNOS and glutamic acid decarboxylase 67 (GAD67), and the other that used conventional thick sections to examine numerical densities (NDs) and cell sizes of nNOS-immunoreactive (IR) neurons. Colocalization analysis indicated that practically all nNOS-IR neurons (97.6%) were GAD67-IR, whereas a part of the GAD67-IR neurons (about 30%) were nNOS-IR in the whole hippocampus at both dorsal and ventral levels. The percentages of GAD67-IR neurons containing nNOS were higher in the dentate gyrus (DG, about 50%), and lower in the Ammon's horn (about 20%). Laminar analysis revealed that the majority of GAD67-IR neurons contained nNOS in the stratum lacunosum-moleculare of the CA3 region (about 60%) and in the molecular layer of the DG (about 80%). The NDs of nNOS-IR neurons in the whole hippocampus showed a dorsoventral gradient, which increased from dorsal (1.6 x 10(3)/mm3) to ventral (2.2 x 10(3)/mm3) levels. The NDs were relatively higher in the principal cell layers, where about 40% of nNOS-IR neurons were situated both in the Ammon's horn and DG. The mean cell sizes of nNOS-IR neurons showed no remarkable laminar differences or dorsoventral gradient in the Ammon's horn, but they were extensively larger in the hilus of the DG than in other layers. These results indicate that nNOS-IR neurons in the mouse hippocampus represent a subpopulation of gamma-aminobutyric acid (GABA)ergic neurons and suggest that the laminar distributions of nNOS-IR neurons related to possible functional heterogeneity of GABAergic neurons in each hippocampal layer.

Animals↗
PubMed1999

Polyunsaturated fatty acid anilides as inhibitors of acyl-coA: cholesterol acyltransferase (ACAT).

A series of polyunsaturated fatty acid anilides were synthesized and evaluated as ACAT inhibitors. Compound 24 had potent inhibitory activity against microsomal ACAT derived from U937, HepG2 and Caco-2 cell lines. Therefore, it might be expected to act as an antiarteriosclerotic and hypocholesterolemic agent. Interestingly, the ACAT inhibitory potency of 24 varied significantly depending on the source of the enzyme.

Adrenal Glands↗
PubMed1999

Differential effects of Bcl-2 overexpression on hippocampal CA1 neurons and dentate granule cells following hypoxic ischemia in adult mice.

In contrast to its known anti-apoptotic activity in sympathetic neurons, immortal neuronal cell lines, and primary cultured immature neurons of the central nervous system (CNS), the role of Bcl-2 in CNS neurons in the adult brain is poorly understood. In the present study, we examined effects of overexpression of Bcl-2 on selective neuronal death of the hippocampal CA1 neurons and the dentate granule cells induced by hypoxic ischemia in adult transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase (NSE-hbcl-2). At the light microscopic level, numbers of TUNEL-positive cells with pyknotic nuclei were observed in the CA1 subfield of NSE-hbcl-2 transgenic mice, as well as that of wild-type mice, after hypoxic ischemic insult, although the onset of neuronal death was apparently delayed in NSE-hbcl-2 transgenic mice. The electron microscopic studies showed that morphological changes of the degenerating CA1 neurons from both groups were clearly distinct from ordinary apoptosis. In contrast, a significant amount of degenerating dentate granule cells from wild-type but not from transgenic mice had typical apoptotic nuclei by the treatment. The activation of caspase-3 was detected in the dentate granule cells but not that of the CA1 neurons. These results indicate that the overexpression of Bcl-2 effectively suppressed dentate granule cell apoptosis but only delayed cell death of the CA1 neurons induced by hypoxic ischemia, suggesting the occurrence of a non-apoptotic, caspase-3-independent mechanism for neuronal death in the CA1 subfield.

Animals↗
PubMed1999

Distribution of a specific calcium-binding protein of the S100 protein family, S100A6 (calcyclin), in subpopulations of neurons and glial cells of the adult rat nervous system.

S100A6 (calcyclin) is a member of the large S100 Ca2+-binding protein family, considered to activate several processes along the calcium signal transduction pathway including the regulation of cell growth, proliferation, secretion, and exocytosis. In the present study, the distribution of S100A6 in the rat nervous system was examined by immunohistochemistry with a goat antiserum against recombinant human S100A6, which recognizes the rat S100A6 homologue. The main S100A6-immunoreactive elements were 1) neuronal somata and dendrites in some specific regions of the limbic system (e.g., the basolateral amygdaloid nucleus, ventral tip of the CA1-subicular border region, entorhinal cortex, and parasubiculum), most of which were identified as a subpopulation of pyramidal cells; 2) olfactory receptor cells and olfactory nerve fibers and terminals in the olfactory bulb; 3) some tracts of the hindbrain and spinal cord (e.g., the spinal trigeminal tract, solitary tract, dorsal root fibers, and the tract of Lissauer) and their terminals (e.g., the principal sensory trigeminal nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, marginal zone, substantia gelatinosa, and proper sensory nucleus of the dorsal horn), as well as some sensory neurons of their origins in the dorsal root and trigeminal ganglia; 4) a subpopulation of astrocytes in the white matter (e.g., the corpus callosum, cingulum, external capsule, internal capsule, and fimbria of the hippocampus) and around the ventricles; 5) some ependymal cells, especially around the central canal; and 6) Schwann cells. These results will improve our understanding of the diverse function of Ca2+-binding proteins in the CNS.

Animals↗
PubMed1999

Distinctive neuronal organization of the olfactory bulb of the laboratory shrew.

The organization of the main olfactory bulb of an insectivore, the laboratory shrew (Suncus murinus), was studied morphologically. We found particular small spherical regions, nidi, at the border between the glomerular and external plexiform layer (EPL), which were intensely GAD positive, 30-60 microm in diameter, and where no olfactory nerves were seen. Around the nidus small calbindin D28k-positive GABAergic neurons, perinidal cells, were clustered. Furthermore, a distinctive type of newly discovered neurons, which we named tasseled cells, located at the middle of the EPL extended dendrites to the nidus, where their small tuft-like complicated branches made synapses with perinidal cells. The present study showed that the basic components of the olfactory bulb are not necessarily constant in all mammals.

Animals↗
PubMed1999

Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis.

Previously we showed that large multipolar cells immunoreactive for calretinin and subunits 2 and 3 of amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptors (GluR2/3) clustered in the ventral hilus of the mouse dentate gyrus and revealed that they were mossy cells. Although such large calretinin immunoreactive cells were not seen in the dorsal hilus, our Golgi study revealed the presence of mossy cells in the dorsal hilus. As we observed large intensely GluR2/3 immunoreactive cells in the dorsal hilus, we suggested that these calretinin negative but intensely GluR2/3 positive large cells in the dorsal hilus were also mossy cells. In the present study we confirmed this identification with several methods. The extracellular tracer labeling studies revealed that all of 47 mossy cells identified morphologically were intensely GluR2/3 positive but calretinin negative, whereas none of 22 non-mossy hilar neurons were intensely GluR2/3 positive. Electron microscopically most of intensely GluR2/3 positive somata and dendritic processes showed the characteristic ultrastructural features of mossy cells. Furthermore, the fimbria-fornix-hippocampal commissure transection procedures induced the calretinin expression in some of these dorsal GluR2/3 immunoreactive cells. On the basis of these observations, we concluded that the vast majority of intensely GluR2/3 immunoreactive large cells in the mouse dorsal hilus were mossy cells. Then we evaluated the presumed difference in the distribution of mossy cells along the dorsoventral axis by the disector. The numerical density of mossy cells was about 1.4 times larger at the ventral level than at the dorsal level, indicating that the dorsoventral difference in the distribution of mossy cells in the mouse hilus was far smaller than that previously speculated.

Animals↗