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Biomedical subjects

Y Ueda

Publications and source records attributed to Y Ueda.

At least 379 records · Page 21Linked to original sources

SEM study on microvascular changes following implantation of bone morphogenetic protein combined with hydroxyapatite into experimental bone defects.

BMP-HAP complex was implanted into a bone defect in the femur of Wistar-strain rats, and animals were allowed to heal for one to 8 weeks prior to sacrifice. Similar bone defects without BMP-HAP complex served as controls. Osseous healing and microvascular changes, as revealed by plastic microcorrosion castings, were subsequently examined under a scanning electron microscope. One week after implantation, sproutings and congregate sinusoidal capillary plexuses and primary bone trabeculae (woven bone) were observed around and between the BMP-HAP complexes. Control specimens revealed a fine and immature sinusoidal capillary plexus arising from sproutings and elongations of pre-existing blood vessels, but bone formation was not observed. At two weeks, newly-formed trabeculae were observed around and on the surface of the HAPs, and a network of thick, newly-formed vessels was observed in intervening space. At three weeks, networks of newly-formed vessels were observed on the surface of the HAPs, and surrounding newly-formed trabeculae had become thickened. At four weeks, bone defects were filled, and HAP was completely embedded in new bone. At eight weeks, the HAP was fused with new bone, and the boundary between the HAP and new bone was unclear. In comparison with comparable surgically created control bone defects without implanted BMP-HAP complex, BMP apparently stimulates new vascularization. Further, implanted BMP-HAP apparently stimulates undifferentiated mesenchymal cells to differentiate into angioblasts and osteoblasts, or vice versa (dedifferentiation).

Animals↗

[A case of acquired stuttering resulting from striatocapsular infarction].

Acquired stuttering resulting from a striatocapsular infarction is reported. A 54-year-old man was admitted to our hospital because of acute onset of stuttering and right facial palsy. The patient spoke very slowly. Speech was characterized by repetitions and prolongations. We considered that there was no aphasia, because comprehensive faculty was normal and neither paraphasia nor word-finding difficulty was recognized. Brain CTs and MRI revealed a striatocapsular infarction extending from the putamen to the caudate nucleus in the left hemisphere. Previously, there were reports of stuttering derived from parkinsonism and lesions in the supplementary motor area and thalamus. Therefore, we proposed that the stuttering arose from a collapse of basal ganglia circuits that connect the cerebral cortex and basal ganglia, including the extrapyramidal tract system.

Cerebral Infarction↗

Enhancement by baclofen of the Gs-coupled receptor-mediated cAMP production in Xenopus oocytes expressing rat brain cortex poly (A)+ RNA: a role of G-protein beta gamma subunits.

We investigated the mechanism by which GABA-B receptors enhance the Gs-coupled receptor-mediated cAMP production in Xenopus oocytes expressing poly (A)+ RNA derived from rat brain cortex. We expressed the cystic fibrosis transmembrane conductance regulator gene (CFTR) as a reporter for cAMP changes in oocytes. The GABA-B agonist (-)baclofen enhanced the adrenergic beta 2 agonist isoproterenol- or vasoactive intestinal peptide (VIP)-induced CFTR currents, whereas (-)baclofen alone did not cause any currents. The (-)baclofen-enhanced currents were inhibited by the GABA-B antagonist 2-OH saclofen. The enhancement by (-)baclofen was further augmented by coexpressing adenylyl cyclase (AC) type II, an isotype activated by G beta gamma and G alpha s, but not by coexpressing AC type III, an isotype insensitive to G beta gamma. Moreover, pretreatment of the oocytes with pertussis toxin (PTX) abolished the enhanced effect of (-)baclofen. These results indicate that upon GABA-B activation, the G beta gamma released from PTX-sensitive G-proteins activates the AC type II (or IV), and this process requires the G alpha s activation by Gs-coupled receptors.

Adrenergic beta-Agonists↗

Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells.

To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)-dependent murine cell line, Ba/F3, the cells showed IL-3-independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Delta(5-195)GCRER, whose product lacks the extracellular G-CSF-binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Delta(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Animals↗

Role of protein kinase C-alpha in activation of ecto-5'-nucleotidase in the preconditioned canine myocardium.

We have reported that activation of protein kinase C (PKC) increases ecto-5'-nucleotidase activity, which may contribute to the infarct size-limiting effect of ischemic preconditioning. Since we have reported that Ca(2+)- and phospholipid-sensitive PKC is activated due to ischemic preconditioning, we further tested 1) whether PKC-alpha or -beta is translocated to the cellular membrane of the preconditioned canine myocardium, and 2) whether activation of PKC contributes to the increase in ecto-5'-nucleotidase activity via phosphorylation-dependent mechanisms. Four times of 5 minutes coronary occlusion separated by 5 minutes of reperfusion (ischemic preconditioning) translocated PKC-alpha to the cellular membrane in the canine hearts, although PKC-beta, -delta, -epsilon, and -zeta were not translocated. The activity of Ca(2+)- and phospholipid-sensitive PKC increased, which was attenuated by the removal of either Ca2+ or phosphatidylserine. Ecto-5'-nucleotidase was also activated in the preconditioned myocardium compared with control. Inhibition of PKC due to GF109203X blunted the activation of myocardial ecto-5'-nucleotidase. Okadaic acid (an inhibitor of phosphatase) enhanced the increases in ecto-5'-nucleotidase activity due to preconditioning, and this enhancement was blunted by GF109203X. We conclude that ischemic preconditioning activates PKC-alpha, and thus ecto-5'-nucleotidase.

Adenosine Triphosphatases↗

Local spread of metastatic vertebral tumors. A histologic study.

STUDY DESIGN: Nineteen solitary metastatic vertebral tumors obtained by total en bloc spondylectomy were analyzed histologically. OBJECTIVES: To determine the mechanisms of local spread of vertebral tumors and identity barrier tissues to tumor progression in the vertebra. SUMMARY OF BACKGROUND DATA: Histologic studies of specimens resected at amputation or en bloc excision have determined the pattern of local spread of bone and soft tissue tumors in the extremities and identified barrier tissues to tumor spread. However, a similar assessment of vertebral malignancies is difficult. The development of total en bloc spondylectomy for primary vertebral malignancy and solitary has allowed the collection of tissue samples sufficient to analyze the pattern of local spread of tumors, using the concept of compartment and barrier. METHODS: Histologic sections of all vertebral elements were prepared from specimens collected after en bloc spondylectomy. Serial 5-mm sections in the sagittal plane were also prepared and examined. RESULTS: Analysis of tumor location showed that metastatic tumors reached the vertebral column by invading the bone marrow of the dorsal region of the vertebral body. The anterior longitudinal ligament, posterior longitudinal ligament, periosteum abutting the spinal canal, ligamentum flavum, periosteum of the lamina and spinous process, interspinous ligament, supraspinous ligament, cartilaginous endplate and the anulus fibrosus served as barriers to tumor progression. The posterior longitudinal ligament was the weakest barrier tissue and was gradually destroyed by the tumor at the point of perforating vessels. Even after destruction of the barrier tissue, tumor cells were covered with a thin, fibrous reactive membrane. Two pathways allowed tumor spread to the adjacent vertebrae: 1) from the edge of the vertebral body to the adjacent vertebral body beneath the longitudinal ligament, and 2) through the paravertebral muscles to the neighboring lamina. CONCLUSIONS: Each vertebra can be represented by a compartment surrounded by several barriers. The most common path for tumor spread is through the posterior longitudinal ligament to the epidural space.

Aged↗

Serial lectin affinity chromatography with concavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures of prostatic acid phosphatase in human prostate carcinoma.

Differences between human prostate carcinoma (PCA, five cases) and benign prostatic hyperplasia (BPH, five cases) in asparagine-linked (Asn) sugar-chain structure of prostatic acid phosphatase (PAP) were investigated using lectin affinity chromatography with concanavalin A (Con A) and wheat germ agglutinin (WGA). PAP activities were significantly decreased in PCA-derived PAP, while no significant differences between the two PAP preparations were observed in the enzymatic properties (Michaelis-Menten value, optimal pH, thermal stability, and inhibition study). In these PAP preparations, all activities were found only in the fractions which bound strongly to the Con A column and were undetectable in the Con A unbound fractions and in the fractions which bound weakly to the Con A column. The relative amounts of PAP which bound strongly to the Con A column but passed through the WGA column, were significantly greater in BPH-derived PAP than in PCA-derived PAP. In contrast, the relative amounts of PAP which bound strongly to the Con A column and bound to the WGA column, were significantly greater in PCA-derived PAP than in BPH-derived PAP. The findings suggest that Asn-linked sugar-chain structures are altered during oncogenesis in human prostate and also suggest that studies of qualitative differences of sugar-chain structures of PAP might lead to a useful diagnostic tool for PCA.

Acid Phosphatase↗

Tat protein induces self-perpetuating permissivity for productive HIV-1 infection.

We report that human immunodeficiency virus type 1 (HIV-1) has evolved a self-perpetuating mechanism to actively generate cells permissive for productive and cytopathic infection. Only activated T cells can be productively infected, which leads to their rapid depletion (2 x 10(9)/day in an infected individual). Establishment of productive HIV-1 infection therefore requires continual activations from the large pool of quiescent T cells. Tat protein, which is secreted by infected cells, activated uninfected quiescent T cells in vitro and in vivo. These Tat-activated uninfected cells became highly permissive for productive HIV-1 infection. Activation of primary T cells by Tat protein involved integrin receptors and was associated with activation of mitogen-activated protein kinases, including ERK1 and JNK kinase. Accordingly, these primary T cells progressed from G0 to the late G1 phase of the cell cycle.

Calcium-Calmodulin-Dependent Protein Kinases↗

The Ig heavy chain gene is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization.

Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing gamma constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11;14)(q13.3; q32.33) was detected in 5 patients, t(8;14)(q24.1;q32.33) in 2, t(14;18)(q32.33;q21.3) in 2, and t(7;14)(q32.1;q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7;14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.

Adult↗

Detection of 14q32.33 translocation and t(11;14) in interphase nuclei of chronic B-cell leukemia/lymphomas by in situ hybridization.

Abnormalities of chromosome 14 involving band q32.33 are among the most commonly observed cytogenetic alterations in B-cell malignancies. To assess the incidence and pathogenetic implications of 14q32.33 translocation in chronic B-cell leukemia/lymphomas, we performed fluorescence in situ hybridization (FISH) analysis with variable region (V(H)) and gamma constant region (Cgamma) gene probes in 37 patients with these disorders. Chromosome 14q32.33 translocation was detected in 2 of 18 patients with chronic lymphocytic leukemia (CLL), 1 of 2 with CLL of mixed cell types (CLL/PL), 1 of 2 with pro-lymphocytic leukemia (PLL), 5 of 6 with leukemic mantle-cell lymphoma (MCL), 2 of 7 with splenic B-cell leukemia/lymphoma of possible marginal zone origin (SBLL) and 2 with leukemic follicular lymphoma (FL). To further characterize 14q32.33 translocations in these patients, we developed a new procedure using double-color FISH with PRAD1, BCL2, V(H) and Cgamma gene probes. Chromosome t(11;14) was detected in 1 patient with CLL/PL, 1 with PLL and 5 with MCL. Chromosome t(14;18) was detected in 2 patients with FL. In a PLL patient with t(11;14), the cosmid CPP29 containing the PRAD1 gene and its 5'-flanking region split and co-localized with both Cgamma and V(H) gene probes, thus spanning the breakpoint. In CLL and SBLL patients, donor chromosomes were other than chromosomes 2, 11, 18 and 19, suggesting the involvement of a novel oncogene(s) in the pathogenesis of these diseases. Interphase FISH rapidly detected 14q32.33 translocation, t(11;14) and t(14;18) in B-cell malignancies with low mitotic activity at the single-cell level, facilitating the correlation of the molecular features of these translocations with clinical characteristics.

Adolescent↗

The mGluR6 5' upstream transgene sequence directs a cell-specific and developmentally regulated expression in retinal rod and ON-type cone bipolar cells.

We generated transgenic mice, using 9.5 kilobase pairs of the 5' upstream sequence from the mouse metabotropic glutamate receptor subtype 6 (mGluR6) gene fused to the beta-galactosidase (lacZ) reporter gene, and investigated the promoter function of the cell-specific and developmentally regulated expression of mGluR6. Most of the independent transgenic lines commonly showed the lacZ expression in the defined cell layers of the retina, and four transgenic lines were characterized in detail for cell-specific lacZ expression patterns by X-gal staining and lacZ immunostaining. The lacZ-expressing retinal cells were classified into two cell types. One cell type was identified as rod bipolar cells on the basis of colocalization of protein kinase C (PKC) immunoreactivity and morphological criteria. The other cell type was PKC-immunonegative and resided at the cell layers corresponding precisely to ON-type cone bipolar cells. The latter bipolar cells were found to exist as a large cell population comparable to rod bipolar cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and PKC antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of mGluR6 during retinal development. This study demonstrates that the mGluR6 5' upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of mGluR6 in ON-type bipolar cells and supports the view that mGluR6 is responsible for ON responses in both the rod and cone systems.

Animals↗

Relationship between BDNF- and trk-expressing neurones in rat dorsal root ganglion: an analysis by in situ hybridization.

Brain-derived neurotrophic factor (BDNF) is synthesized in sensory neurones and suggested to operate on these same neurones by an autocrine mechanism, but it is unclear whether these neurones express the functional receptor (TrkB) for BDNF. We therefore examined the co-localization of BDNF and neurotrophin receptor mRNAs in adult rat dorsal root ganglion (DRG) neurones by in situ hybridization histochemistry. BDNF mRNA signals were detected in about 40-50% of DRG neurones (L4-5). Almost all the trkA mRNA-expressing neurones (95%) were positive for BDNF mRNA, while no trkB mRNA- and few trkC mRNA-expressing neurones displayed BDNF mRNA signals. These findings suggest that BDNF is mainly synthesized in the sensory neurones that are responsive to nerve growth factor but not to BDNF or neurotrophin-3. It is unlikely that BDNF is involved in an autocrine loop in sensory ganglia.

Animals↗

Excitatory effects of algesic compounds on neuronal processes in murine dorsal root ganglion cell culture.

The effects of algesic compounds on the distal portion of the processes of cultured dorsal root ganglion cells (C-fiber) of mouse were studied by patch-clamp whole-cell recording at the cell soma (cell body). The processes of the cell were isolated from the cell body with a separator. Bradykinin (BK, 10 microM), prostaglandin E2 (PGE2, 20 microM), and capsaicin (CAP, 2 microM) were applied to the processes of a cell on the third day after seeding, each of which evoked action potentials in the cell body. No desensitization was seen by the repeated application of BK to the processes. No action potentials in the cell body were observed when BK (10 microM) was applied concomitantly with tetrodotoxin (6 microM). These results suggest that the stimuli of algesic compounds to the neuronal processes of the cultured dorsal root ganglion cells are useful for studying the neuronal mechanism involved in pain.

Action Potentials↗

Mitral valve repair in a patient with severe porcelain aorta.

We repaired the mitral valve in a patient with severe porcelain aorta. Significant mitral regurgitation developed in a 66-year-old woman with heavy calcification throughout the whole aorta. At operation, cardiopulmonary bypass was properly established by combined axillary and femoral arterial cannulations for sufficient systemic flow. Likewise, the combination of a superior mitral approach and profound hypothermic fibrillatory arrest in conjunction with low-flow cardiopulmonary bypass allowed us to repair the mitral valve successfully.

Aged↗

Expression of vasoactive intestinal polypeptide receptor mRNA and secretory regulation by vasoactive intestinal polypeptide in rat submandibular and sublingual salivary glands.

Vasoactive intestinal polypeptide (VIP)-receptor mRNA was strongly expressed in the acinar cells in the submandibular gland but not in the sublingual gland. VIP-containing nerve fibres were richly distributed around acini in the submandibular gland but were rare around acini of the sublingual gland. In the submandibular gland, the chorda was stimulated at various frequencies (1-40 Hz) together with an infusion of (N-Ac-Tyr1, D-Phe2)-GRF(1-29)-NH2 (109 M), VIP antagonist, which reduced salivary flow from the submandibular gland only at high-frequency stimulation (> 20 Hz), and more markedly reduced the salivary protein concentration. When the chorda was continuously stimulated the antagonist reduced the salivary flow only during the initial 5 min. Exogenous VIP 10(-12) - 10(-8) M) infusion at the same time as chorda stimulation caused no increase in salivary flow, but the salivary protein concentration was increased in a dose-dependent manner. In the sublingual gland, neither VIP nor the VIP antagonist affected chorda-evoked salivary flow and protein concentration. Thus, endogenous VIP may play a part in the regulation of both fluid and protein secretion, especially of protein, evoked by chorda stimulation at high frequency in the submandibular gland. These phenomena occurred only in the initial phase of secretion. In the sublingual gland, it seems likely that VIP plays no part in the regulatory mechanism, at least with regard to salivary fluid secretion in the acinar cells.

Animals↗

Ca2+/Mg(2+)-dependent endonuclease in marrow CD34 positive and erythroid cells in myelodysplasia.

Endonucleases capable of producing internucleosomal DNA cleavage are one of the key enzymes in apoptosis. We examined endonuclease activities contained in nuclei of CD34+ and erythroid cells in the bone marrow (BM) from 12 patients with the myelodysplastic syndromes. The levels of Mg(2+)-dependent and acidic endonucleases showed little changes as compared with those from normal BM. By contrast, the level of Ca2+/Mg(2+)-dependent endonuclease was appreciably higher in MDS erythroid cells than normal counterparts, although the activity varied markedly in CD34+ and erythroid cells. Our results suggested that Ca2+/Mg(2+)-dependent endonuclease is related to ineffective erythropoiesis in MDS.

Adult↗

Interphase detection of BCL6/IgH fusion gene in non-Hodgkin lymphoma by fluorescence in situ hybridization.

We characterized a t(3;14)(q27;q32) translocation in nine patients with B-cell, non-Hodgkin lymphoma (B-NHL) by fluorescence in situ hybridization (FISH). Fluorescence in situ hybridization with immunoglobulin heavy chain (IgH) and BCL6 gene probes detected t(3;14) rapidly and accurately, including complex t(3;14) in three patients; one with t(3;12;8;14)(q27;p13;q24.1;q32) and two with t(3;?;14)(q27;?;q32). Among these nine patients, seven escaped from cytogenetic detection by our G-banding analysis. Double-color FISH with IgH (Y6) and BCL6 (cosB5-1) showed fusion of BCL6 and IgH genes on der(3)t(3;14) in all nine patients, suggesting that der(3) may play a critical role in the development of lymphoma carrying complex as well as standard t(3;14) translocations. BCL6/IgH fusion gene was also demonstrated in interphase nuclei at a frequency of 23% to 91.5% over the cut-off value in control studies (9.0 +/- 2.76%). The breakpoints assessed by FISH with two cosmid clones containing BCL6 probes, cosB5-1 and cosB5-2, were within the cluster region in seven patients including one with complex type, but were not evaluated in two patients with t(3;?;14), because of the loss of partner chromosome. Using double-color FISH with these two BCL6-specific probes, none of an additional 32 patients in whom mitotic spreads were available showed 3q27 translocations. Fluorescence in situ hybridization with IgH and BCL6 gene probes is a rapid and sensitive method to detect t(3;14) in routine cytogenetic studies.

Aged↗