Biomedical subjects

K Tsujikawa

Publications and source records attributed to K Tsujikawa.

At least 19 recordsLinked to original sources

PubMed2005

A study of the metabolism of methamphetamine and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in isolated rat hepatocytes.

The metabolism of methamphetamine (MA) and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in freshly isolated rat hepatocytes was investigated, and compared with in vivo results. A suspended hepatocyte culture, established from male Wistar rats using a collagenase perfusion technique, was incubated in the presence of MA or 2C-B. After enzymatic hydrolysis of the conjugated forms, the metabolites were extracted by liquid-liquid partition and analyzed by gas chromatography/mass spectrometry (GC/MS). Amphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine were detected in the culture fluids of the rat hepatocytes inoculated with MA. The alcohol derivative, carboxylic acid derivative, 2-O-desmethyl-2C-B, 2-O-desmethyl-N-acetyl-2C-B and 5-O-desmethyl-N-acetyl-2C-B were detected in the case of 2C-B. The major metabolite of MA in rat hepatocytes was p-hydroxymethamphetamine. This is in good agreement with the urinary excretion profile for rats that were fed MA. 2-O-Desmethyl-2C-B and the carboxylic acid derivative were the major recovered metabolites of 2C-B in the rat hepatocyte culture, a slight deviation from the in vivo findings, in which 5-O-desmethyl-N-acetyl-2C-B was found to be the main component. Metabolites with a hydroxy group were largely present in their conjugated forms in the culture fluids, except for 2-O-desmethyl-2C-B. Taking these results into consideration, a primary hepatocyte culture system has the potential to provide a quick and handy method for estimating the in vivo metabolic fate of abused drugs.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗

PubMed2003

Overexpression of leucocyte common antigen (LAR) P-subunit in thyroid carcinomas.

Protein tyrosine phosphatase (PTPase) dephosphorylation and protein tyrosine kinase (PTKs) phosphorylation of key signal transduction proteins may be regulated by extracellular signals, making PTPases important in the regulation of cell proliferation. Leucocyte common antigen (LAR), a receptor-like PTPase, consists of E-subunit, containing the cell adhesion molecule-like receptor region, and P-subunit specific for a short segment of the extracellular region, the transmembrane peptide, and two cytoplasmic PTPase domains. We produced a monoclonal antibody against the LAR P-subunit for immunohistochemical screening of LAR expression in normal and tumourous tissues. Gliomas and gastric, colorectal, lung, breast and prostate cancers showed weak and relatively infrequent expression. Intense and diffuse expression, however, was detected in 95% (227 out of 239) of thyroid carcinomas, but only 12% (22 out of 128) of adenomas and no cases of benign thyroid disease were immunopositive. In contrast to broad staining in carcinomas, LAR expression in thyroid adenomas was often found in small focal or locally invasive areas. Western blot analysis similarly detected LAR P-subunit protein in thyroid carcinomas, but not in normal tissues. We believe this to be the first demonstration of LAR overexpression in thyroid carcinoma and may help to elucidate the role of PTPases in the development of malignancy.

Antigens, CD↗

PubMed2002

Molecular cloning and characterization of mouse calcitonin gene-related peptide receptor.

The calcitonin gene-related peptide (CGRP) plays important roles as a neurotransmitter/neuromodulator in the central nervous system, and as a potent vasodilator when secreted from peripheral, perivascular nerves through its specific receptors. In this study, we cloned mouse cDNA counterparts of the human CGRP receptor composed of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) and examined the signal transduction mechanism through the CGRP receptor. Mouse CRLR (mCRLR) is a 462-amino acid G protein-coupled heptahelical receptor, and mouse RAMP1 (mRAMP1) is a 148-amino acid single membrane-spanning protein with a short cytoplasmic portion. Specific binding of (125)I-CGRP was detected only when both mCRLR and mRAMP1 cDNAs were cotransfected to COS-7 cells, and the Kd value of the receptor was 2.2 x 10(-10) M. CGRP induced a marked elevation of the intracellular cAMP levels in COS-7 cells cotransfected with mCRLR and mRAMP1. CGRP signaling through the mCRLR/mRAMP1 receptor complex was found to increase the promoter activities of cyclic AMP responsive element and serum responsive element in the co-transfected HeLa cells. These results indicate that mCRLR and mRAMP1 constitute a functional mouse CGRP receptor for the transduction of CGRP signaling by PKA and extracellular signal-regulated kinase signal transduction pathways.

Amino Acid Sequence↗

PubMed2001

Indocyanine green and fluorescein hyperfluorescence at the site of occlusion in branch retinal vein occlusion.

BACKGROUND: In patients with branch retinal vein occlusion (BRVO), we investigated the presence of indocyanine green (ICG) and fluorescein hyperfluorescence at the site of occlusion. We also assessed the association of this feature with the clinical outcome of these patients. METHODS: Both indocyanine green (ICG) videoangiography and fluorescein angiography (FAG) were performed in 21 eyes with BRVO of less than 1 month duration. Deterioration of the disease was defined clinically as an increase in retinal hemorrhages or retinal edema. Capillary nonperfusion was quantified with computer image analysis from the FAG pictures. RESULTS: ICG videoangiography showed focal hyperfluorescence along the venous wall at the site of the affected A-V crossing in 9 of the 21 eyes, and FAG showed this feature in 10 eyes. The ICG hyperfluorescence was more prominently and focally detected than the hyperfluorescence on FAG, which was sometimes diffusely seen throughout the whole occluded area. Eight of the nine eyes showing ICG hyperfluorescence had clinical deterioration with an increase in retinal hemorrhage or edema. This deterioration occurred more frequently in eyes with hyperfluorescence and/or late leakage than in ones without these features. The mean nonperfused area was significantly larger in eyes with hyperfluorescence than in eyes without these features. CONCLUSION: The ICG hyperfluorescence at the site of A-V crossing is associated with disease deterioration in patients with fresh BRVO. The ICG hyperfluorescence was more easily detectable than the hyperfluorescence on FAG, although the difference in sensitivity between the two methods is not great.

Adult↗

PubMed2001

Two patterns of opacity in corneal dystrophy caused by the homozygous BIG-H3 R124H mutation.

PURPOSE: To investigate the opacity pattern in corneas with an Arg124His (R124H) homozygous mutation of the BIG-H3 gene. METHODS: Slit-lamp examination was performed on eight patients with corneal dystrophy resulting from a genetically confirmed BIG-H3 R124H homozygous mutation. The birthplace of each patient also was determined. RESULTS: Slit-lamp examination disclosed two types of opacity patterns in corneas with the BIG-H3 R124H homozygous mutation. Type I (n = 4) is a spot-like opacity present in the anterior stroma in which the lesions are confluent. Type I is the same pattern that previous reports have shown to be caused by the BIG-H3 R124H homozygous mutation. The type II corneal opacity pattern (n = 4) is a reticular opacity in the anterior stroma with round translucent spaces. Type II opacity has not been reported previously in association with any corneal dystrophy. The patients with the type I opacity do not share a common birthplace; however, interestingly, the patients with the type II opacity traced their origin to Tottori prefecture in western Japan. CONCLUSION: The BIG-H3 homozygous R124H mutation induces the development of two distinct patterns of corneal opacity, the recognition of which can establish an accurate diagnosis of corneal dystrophy caused by the homozygous BIG-H3 R124H mutation independent of genetic analysis. In addition, genetic factors or circumstantial influences other than the gene responsible for the corneal dystrophy may influence the pattern of corneal opacity.

Adult↗

PubMed2001

Distinct functions of the two protein tyrosine phosphatase domains of LAR (leukocyte common antigen-related) on tyrosine dephosphorylation of insulin receptor.

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.

Animals↗

PubMed2001

[A giant testicular tumor detected with dyspnea due to lung metastases: a case report].

A 19-year-old male presented with dyspnea. Clinical examination revealed the left infant-head-sized testicular tumor, multiple lung metastases and retroperitoneal bulky lymph node metastasis with marked elevation of serum lactic dehydrogenase (LDH) and alpha-fetoprotein. Left radical orchiectomy followed by the chemotherapy with etoposide and cisplatin (EP) for 4 cycles was performed. The tumor weighed 1,700 g, and was pathologically diagnosed as mixed germ cell tumor consisting of embryonal carcinoma and yolk sac tumor. After the treatment, the tumor markers were normalized with partial response (PR) of lung metastases and complete response (CR) of retroperitoneal lymph node metastasis. Thereafter, biopsy of lung metastases through video-assisted thoracoscopic surgery (VATS) was performed, and pathologically no viable cells were detected. Five months after the treatment, he was seized with convulsion due to brain metastasis with hemorrhage. Therefore, a surgical resection of brain metastasis and 2nd line chemotherapy with etoposide, ifosfamide and cisplatin (VIP) chemotherapy for 3 cycles was performed. The patient has been free of recurrence for 21 months after the 2nd line chemotherapy.

Adult↗

PubMed2001

[Usefulness of 99mTc-methoxy-isobutyl-isonitrile scintigraphy for preoperative localization of adenoma in primary hyperparathyroidism].

We evaluated the usefulness of 99mTc-methoxy-isobutyl-isonitrile (MIBI) dual phase scintigraphy for detecting hyperfunctioning parathyroid adenoma. We retrospectively reviewed 18 hyperparathyroid patients who received MIBI prior to neck exploration and compared the radiological findings of MIBI with ultrasonography (US) and magnetic resonance imaging (MRI). Fifteen patients were studied with MRI, and 17 patients were examined with US. All patients were found to have a solitary parathyroid adenoma histopathologically. MIBI correctly revealed the location of 17 adenomas among 18 confirmed tumors. In our series, there was one false-positive case that was found to have thyroid adenoma. The diagnostic sensitivity of MIBI MRI and US was 94.4%, 80% and 52.5%, respectively. The positive predictive value (PPV) was 94.4% for MIBI, 81.8% for MRI and 92.3% for US. We conclude that MIBI is useful and accurate for the preoperative localization of adenoma in primary hyperparathyroidism.

Adenoma↗

PubMed2000

Molecular cloning and characterization of a novel human STE20-like kinase, hSLK.

We have cloned a human counterpart to a guinea pig STE20-like kinase cDNA, designated human SLK (hSLK), from a human lung carcinomatous cell line A549 cDNA library. hSLK cDNA encodes a novel 1204 amino acid serine/threonine kinase for which the kinase domain located at the N-terminus shares considerable homology to that of the STE20-like kinase family. The C-terminal domain of hSLK includes both the coiled-coil structure and four Pro/Glu/Ser/Thr-rich (PEST) sequences, but not the GTPase-binding domain (GBD) that is characteristic of the p21-activated kinase (PAK) family, polyproline consensus binding sites, or the Leu-rich domain seen in the group I germinal center kinases (GCKs). Northern blot analysis indicated that hSLK was ubiquitously expressed. hSLK overexpressed in COS-7 cells phosphorylates itself as well as myelin basic protein used as a substrate. On the other hand, hSLK cannot activate any of the three well-characterized mitogen-activated protein kinase MAPK (ERK, JNK/SAPK and p38) pathways. Moreover, hSLK kinase activity is not upregulated by constitutive active forms of GTPases (RasV12, RacV12 and Cdc42V12). These structural and functional properties indicate that hSLK should be considered to be a new member of group II GCKs.

Amino Acid Sequence↗

PubMed2000

Detection of CD45iota mRNA in murine Th1 but not Th2 clones.

CD45, a prototype of the receptor-like protein tyrosine phosphatase (PTPase) family, is one of the essential molecules in signal transduction through T cell receptors. Because at least 8 types of CD45 isoforms can potentially be produced by alternative mRNA splicing of exons 4, 5, and 6, the analyses at the transcription and protein levels of CD45 during the development and differentiation of T cells have been performed using RT-PCR and isoform-specific monoclonal antibodies, respectively. We report here that the ninth and smallest isoform of CD45, designated as CD45iota (CD45t), which is alternatively spiced from exons 4, 5, and 6 as well as exon 7, is present in the fetal thymus and splenic T cells of mice, and in murine Th1 clones, but not in Th2 clones. The expression of full-length CD45t mRNA as the functional CD45 PTPase was confirmed by RT-PCR analysis. Furthermore, the expression vector of CD45t was constructed, and its expression was detected in combination with anti-pan CD45 mAb and our newly established anti-LAR/CD45 PTPase domain mAb. These results suggested that CD45t might be an important isoform of CD45 for differentiation signaling of Th cells, and might be used as a marker to distinguish between Th1 and Th2 cells.

Alternative Splicing↗

PubMed2000

Ste20-like kinase (SLK), a regulatory kinase for polo-like kinase (Plk) during the G2/M transition in somatic cells.

BACKGROUND: Activation of the cyclin-dependent kinase cdc2-cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine-14 and tyrosine-15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo-like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo-like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown. RESULTS: We show here that human Ste20-like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation-dependent enhancement of SLK activity. CONCLUSIONS: We propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.

3T3 Cells↗

PubMed2000

Interaction of merosin (laminin 2) with very late activation antigen-6 is necessary for the survival of CD4+ CD8+ immature thymocytes.

The laminin alpha2-chain is a component of merosin, a member of the laminin family molecules, which is mainly expressed in the basement membranes of striated muscle. It is known that laminin alpha2 gene (lama2) null mutant mice (dy3k/dy3k) exhibit congenital muscular dystrophy (CMD). Because the laminin alpha2-chain is also expressed in the thymus, the role of merosin in the thymus was examined. In association with the onset of muscular dystrophy, CD4+ CD8+ double-positive (DP) thymocytes disappear by apoptotic cell death, while CD4+ CD8- or CD4- CD8+ thymocytes remain. In order to study the mechanisms leading to the selective death of DP cells in the absence of merosin, the role of the interaction between very late activation antigen-6 (VLA-6), a candidate merosin ligand in the thymus, and merosin was examined. The in vitro survival of thymocytes from normal mice was maintained by the addition of either anti-VLA-6 monoclonal antibodies (mAbs) or merosin. Furthermore, when the normal thymocytes were cultured on thymic epithelial cell lines, viable DP cell recoveries on wild-type epithelial cells were better than on cells from null mutant mice. The results suggest that DP cells are more sensitive to an uncharacterized apoptotic death signal, and that survival is supported by the interaction between VLA-6 and merosin.

Animals↗

PubMed2000

Rapid detection of M1S1 mutations by the protein truncation test.

PURPOSE: To determine a method of rapid detection of M1S1 gene mutations in patients with gelatinous droplike corneal dystrophy. METHODS: Forty-one patients from 35 families with gelatinous drop-like corneal dystrophy were studied. The entire coding region of the M1S1 gene was screened using the protein truncation test (PYT), with a polymerase chain reaction fragment amplified from genomic DNA serving as a template of in vitro translation. RESULTS: Homozygous or compound heterozygous mutations were detected in all patients by a single reaction of the PTT. This result matched those obtained using the polymerase chain reaction-restriction fragment length polymorphism and direct sequence analyses. The Q118X mutation was present in 63 of the 70 alleles, accounting for 90% of the disease-associated chromosomes in Japanese patients. CONCLUSIONS: The PTT is useful for detecting mutations in the M1S1 gene. This technique showed that the Q118X mutation is a founder mutation in Japanese patients with gelatinous droplike corneal dystrophy, and it reflects the linkage disequilibrium reported previously.

Antigens, Neoplasm↗

PubMed1999

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development.

Green fluorescent protein (GFP) transgenic (GFP+) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP+ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP+ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3epsilon stimulation between GFP+ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP+ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP+ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP+ mice to the analysis of lymphocyte development. When bone marrow cells from GFP+ mice were transplanted. T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP+ and CD4+8+ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP+ mice is a powerful tool for visualizing lymphocyte development.

Animals↗

PubMed1999

Roles of integrins and CD44 on the adhesion and migration of fetal liver cells to the fetal thymus.

Adhesion and migration of mouse fetal liver (FL) cells to the thymus were investigated using cells from green fluorescent protein transgenic (GFP+) mice. FL cells from GFP+ embryos at 12 gestational days (E12) of mice were incubated with 2'-deoxyguanosine-treated fetal thymus lobe (from E14) by thymic repopulation (hanging drop) culture methods. GFP+ cells were observed in the thymus lobe at the end of the repopulation culture period. A large part of the infiltrated cells expressed CD44 until day 2 of culture on a permeable membrane, then lost the expression. CD25 expression was observed from day 1 to day 4. Around day 8, GFP+ cells became both CD4+ and CD8+. The results support the early observation of the sequential expression of CD44, CD25, and CD4/8 during the early stages of thymocyte development. When anti-CD44 mAb was added at the beginning of the repopulation culture period, GFP+ FL cells adhered to the surface of the thymus lobe but did not migrate into the thymus. Pretreatment of the thymus with hyaluronidase or hyaluronate produced results similar to the results of anti-CD44 treatment. On the other hand, the addition of anti-integrin alpha4 mAb inhibited adhesion to the thymus, and almost no GFP+ cells were seen on the surface of the thymus lobe. The data suggest that integrin alpha4 and CD44 play different roles, i.e., integrin alpha4 is required for the adhesion of FL cells to the thymus lobe and CD44 is required for the migration of the cells into the thymus.

Animals↗

PubMed1999

Protease-sensitive urinary pheromones induce region-specific Fos-expression in rat accessory olfactory bulb.

Vomeronasal organs of female Wistar rats were exposed with sprayed urine preparations of male Wistar rats prior to sacrifice. Exposure to crude urine and ultrafiltrated urine preparation (<5000 Da) induced significant Fos expression, which is correlated with cellular activity, in the mitral/tufted cell layer of the accessory olfactory bulb (AOB), while exposure to the remaining substances after ultrafiltration (>5000 Da) and control salt solution did not. Exposure to urine preparation treated with papain induced expression of Fos-immunoreactive cells in the rostral region of the AOB, but did not induce such expression in the caudal region. Exposure to urine preparation treated with pronase induced urine-specific Fos immunoreactivity neither in the rostral nor in the caudal region. These results suggest that at least two different peptides carrying pheromonal activities are contained in male Wistar rat urine.

Animals↗

PubMed1999

Vitrectomy for rhegmatogenous or tractional retinal detachment with familial exudative vitreoretinopathy.

OBJECTIVE: To examine the anatomic features and surgical indications of familial exudative vitreoretinopathy (FEVR) complicated with rhegmatogenous or tractional retinal detachment. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Twenty-eight eyes of 25 patients who had either clinically suspected or fully diagnosed FEVR. Of these, 25 had rhegmatogenous retinal detachment, 2 had tractional retinal detachment, and 1 had tractional retinal detachment plus vitreous hemorrhage. INTERVENTIONS: The authors carefully observed the vitreoretinal interface during surgery, studied the clinical and anatomic features of FEVR, and then evaluated the surgical results. RESULTS: The vitreoretinal adhesions were so strong in the peripheral avascular area that iatrogenic retinal breaks easily occurred in 22 of 28 eyes. In all cases, the bimanual technique with vitreous scissors and forceps was required to dissect the posterior vitreous membrane from the retinal surface. The retina was reattached in 24 of 28 cases (85.7%), and visual acuity improved in 20 eyes (71.4%). CONCLUSION: Dissection of the vitreous in the peripheral avascular area is very difficult in FEVR, and those patients for whom this procedure was not successfully performed may have a poorer prognosis.

Adolescent↗

PubMed1999

Simultaneous bilateral renal pelvic tumors.

BACKGROUND: A 72-year-old man was admitted with gross hematuria. Investigations revealed bilateral renal pelvic tumors. METHODS/RESULTS: Via a midline incision, left nephroureterectomy with bladder cuff resection was performed for the large left-sided tumor. The right small solitary right-sided tumor was endoscopically resected simultaneously. Histologically, both tumors were grade 2 transitional cell carcinomas without muscular invasion. CONCLUSION: There has been no evidence of recurrence or metastasis 30 months postoperatively.

Aged↗