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Biomedical subjects

M Iwata

Publications and source records attributed to M Iwata.

At least 19 recordsLinked to original sources
PubMed1995

Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.

The goal of this study was to determine whether sphingosine and ceramide, second messengers derived from sphingolipid breakdown, alter kidney proximal tubular cell viability and their adaptive responses to further damage. Adult human kidney proximal tubular (HK-2) cells were cultured for 0-20 hr in the presence or absence of sphingosine, sphingosine metabolites (sphingosine 1-phosphate, dimethylsphingosine), or C2, C8, or C16 ceramide. Acute cell injury was assessed by vital dye exclusion and tetrazolium dye transport. Their subsequent impact on superimposed ATP depletion/Ca2+ ionophore-induced damage was also assessed. Sphingosine (> or = 10 microM), sphingosine 1-phosphate, dimethylsphingosine, and selected ceramides (C2 and C8, but not C16) each induced rapid, dose-dependent cytotoxicity. This occurred in the absence of DNA laddering or morphologic changes of apoptosis, suggesting a necrotic form of cell death. Prolonged exposure (20 hr) to subtoxic sphingosine doses (< or = 7.5 microM) induced substantial cytoresistance to superimposed ATP depletion/Ca2+ ionophore-mediated damage. Conversely, neither short-term sphingosine treatment (< or = 8.5 hr) nor 20-hr exposures to any of the above sphingosine/ceramide derivatives/metabolites or various free fatty acids reproduced this effect. Sphingosine-induced cytoresistance was dissociated from the extent of cytosolic Ca2+ loading (indo-1 fluorescence), indicating a direct increase in cell resistance to attack. We conclude that sphingosine can exert dual effects on proximal renal tubular viability: in high concentrations it induces cell necrosis, whereas in low doses it initiates a cytoresistant state. These results could be reproduced in human foreskin fibroblasts, suggesting broad-based relevance to the area of acute cell injury and repair.

Acute Kidney Injury↗
PubMed1995

AG dinucleotide insertion in a patient with chronic granulomatous disease lacking cytosolic 67-kD protein.

The 67-kD cytosolic protein (p67-phox) is an essential component of the superoxide-generating system in phagocytes, and its defect is known to cause chronic granulomatous disease (CGD). We sequenced p67-phox cDNA from one of seven patients found in Japan and his parents. In the patient's cDNA, homozygous AG dinucleotide insertion at position 399 (or 401) was found together with three other homozygous substitutions in a coding region (A-542 to G, T-895 to C and A-983 to G) compared with the sequence reported for HL-60 cells. In cDNA from his parents, the AG insertion was found to be heterozygous. In contrast, the other three substitutions were found homozygously in his father's specimen and the latter two in his mother's specimen. The substitution of A-542 to G was heterozygous in his mother's cDNA. The AG insertion would induce a frame shift and bring about a stop codon at the position of 433. However, the other three differences would give insignificant changes for the protein function, if any, because the substitutions of A-542 to G and A-983 to G result in the conservative amino acid transitions, ie, Lys-180 to Arg and Lys-327 to Arg, and that of T-895 to C no amino acid change. Neutrophils from the patients completely lacked superoxide generating activity whereas those from his parents generated substantial amounts of superoxide anion upon stimulation. Thus, it is concluded that the AG dinucleotide insertion is responsible for the disease in this patient.

Adult↗
PubMed1995

Calcineurin activation protects T cells from glucocorticoid-induced apoptosis.

In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway.

Animals↗
PubMed1995

Cell-mediated catabolism of aggrecan. Evidence that cleavage at the "aggrecanase" site (Glu373-Ala374) is a primary event in proteolysis of the interglobular domain.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

Aggrecans↗
PubMed1995

Structural organization of the gene for CD40 ligand: molecular analysis for diagnosis of X-linked hyper-IgM syndrome.

CD40 ligand (CD40L) on activated T cells plays a crucial role for Ig heavy-chain class switching and the mutation of the gene for this ligand in the X-chromosome causes immunodeficiency with hyper-IgM (X-HIM). We isolated and characterized the human genomic clone for CD40 L to obtain information about the transcriptional regulatory regions of the gene and to develop a molecular diagnostic method for X-HIM patients. The genomic DNA isolated was over 12 kb long containing 5 exons that cover full sequence of mRNA for the ligand. DNA motif analysis based on transcription factor database revealed the presence of a GATA 1 box at around -265 bp. The typical TATA box, CAT site or GC rich region was not found in the 5' flanking region. However, two possible TATA like sequences were found at around -27 and -136 bp. Using the oligonucleotide primers corresponding to the introns, we performed a PCR-SSCP analysis of each exon from a patient with X-HIM syndrome and detected abnormality in exon 5. When sequenced, dinucleotide deletion in this exon was found in the patient and in one X allele of his mother as the only different sequence from that of the control gene. This procedure is simple and could be used for diagnosis of the X-HIM syndrome.

Adolescent↗
PubMed1995

Synaptic loss in the proximal axon of anterior horn neurons in motor neuron disease.

This report deals with an ultrastructural investigation of the synapses of the proximal axons of normal-appearing anterior horn neurons of 7 patients with amyotrophic lateral sclerosis (ALS) and 4 patients with motor neuron disease who had no upper motor neuron and corticospinal tract involvement (lower motor neuron disease, LMND). Specimens from 12 age-matched individuals who died of non-neurological diseases served as controls. Proximal axons directly emanating from the normal-appearing neurons were examined: 42 axons were from ALS patients, 43 from LMND patients and 87 from controls. Our results show that the number of synapses on axon hillocks, as well as the lengths of the synaptic contact and of the active zone were reduced in both groups of patients (P < 0.0001), but no significant differences were seen between patients and controls with respect to the synaptic parameters of initial axon segments. There was no overall difference between ALS and LMND patients. These findings suggest that the electrophysiological functions pertaining to integration of electrical inputs into the axon and information transduction on the axon may be greatly impaired in the early stages of motor neuron diseases, and that the observed synaptic alterations may be pathological events, likely to be due to anterior horn neuron degeneration.

Aged↗
PubMed1995

Biological characteristics of neuroblastoma with spontaneous tumor reduction: a case report.

The authors examined the biological characteristics of a neuroblastoma with spontaneous tumor reduction. A 6-month-old boy with a pelvic neuroblastoma underwent surgical extirpation of the tumor 1 month after diagnosis. The size of the tumor reduced spontaneously while he was awaiting operation. The low proliferative activity of the tumor cells and the presence of apoptosis in the tumor tissue were shown by an immunohistochemical method using anti-PCNA antibody and a DNA fragmentation analysis, respectively. These results suggest that the spontaneous tumor reduction seen in this patient may well be caused by the overwhelming apoptosis of tumor cells.

Apoptosis↗
PubMed1995

Cross-linking of the TCR-CD3 complex with CD4, CD8 or LFA-1 induces an anti-apoptotic signal in thymocytes: the signal is canceled by FK506.

The immunosuppressant FK506 did not block differentiation of double-negative thymocytes into double-positive (DP) cells, but interfered with differentiation of DP cells into mature single-positive cells in a fetal thymus organ culture system, suggesting that FK506 inhibits positive selection. The drug also reduced the number of DP cells recovered after the culture. As positive selection depends on the inhibition of thymocyte apoptosis at its DP stage by signaling through the TCR-CD3 complex and some of the accessory molecules, including CD4, CD8 and LFA-1, we studied the possibility that FK506 enhanced apoptosis by itself or canceled the inhibition of apoptosis. The results indicated that FK506 was hardly toxic or hardly affected anti-CD3-induced DNA fragmentation in isolated thymocytes in vitro. On the other hand, upon cross-linking TCR-CD3 together with CD4, CD8 or LFA-1, FK506 markedly enhanced DNA fragmentation and cytolysis. The drug, however, hardly or only slightly enhanced these responses upon cross-linking TCR-CD3 together with CD2, CD28, Thy-1 or H-2Kd. Cross-linking of TCR-CD3 together with CD4, CD8 or LFA-1 markedly inhibited glucocorticoid-induced death and the inhibition was canceled by FK506. Furthermore, cross-linking of TCR-CD3 together with LFA-1 potentially induces both an apoptosis-inducing signal and an FK506-sensitive anti-apoptotic signal, and that the latter signal may be related to an essential signal for positive selection.

Animals↗
PubMed1995

Evidence for a short form of alpha 1(IV) as a major polypeptide in bovine lens capsule.

The extracts from bovine lens capsule with acetic acid contained, after reduction, three major collagenous polypeptides with M(r) = 180k, 175k, and 160k, which were specifically immuno-stained with anti-type IV collagen polyclonal antibody. The biochemical properties of 180k and 160k polypeptides were akin and were distinct from that of 175k polypeptide [J. Biochem. (1993) 114,358-362]. In the present study, evidence that the 160k and 180k polypeptides from bovine lens capsule both originated from alpha 1(IV) was obtained on the basis of reactivity with a monoclonal antibody that recognizes alpha 1(IV) chain at the collagenous sequence contained in [KGEPGLPGRGFPGFP]. The epitope-bearing sequence was identified from the following three experiments. Pepsin-solubilized polypeptides from human placenta were purified by affinity chromatography on the antibody-coupled column and sequenced. The restriction map of the clones positively reactive with the monoclonal antibody from human placenta cDNA library was superimposed on that of human alpha 1(IV) cDNA at a specific region. Synthetic peptides corresponding to the sequence were assayed for inhibitory activity against the reaction between epitope-bearing pepsin fragments and the antibody. The 180k and 160k polypeptides showed similar intensities in protein staining as well as in immuno-staining with the monoclonal antibody. In contrast, the 175k polypeptide did not react with the monoclonal antibody, indicating that it is a genetically distinct type IV collagen chain, presumably alpha 2(IV) from its abundance. The 160k, a major type IV collagen polypeptide, is a short form of alpha 1(IV) present as a tissue form in bovine lens capsule.

Amino Acid Sequence↗
PubMed1995

Comprehensive treatment of advanced neuroblastoma involving autologous bone marrow transplant.

Encouraging results are reported with high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation in the treatment of advanced neuroblastoma. However, relapse remains a significant problem. We used high-dose chemotherapy, surgery, intraoperative radiation and an autologous bone marrow transplant treated in vitro to remove tumor cells followed by 13-cis-retinoic acid to treat 36 children with advanced neuroblastoma. This comprehensive treatment appears to improve the survival rate of patients with advanced neuroblastoma, including those with N-myc amplification and bony involvement. The disease-free survival rate was 66% (95% confidence interval, 49-84%) at 3 years. All patients who received 13-cis-retinoic acid developed cheilitis, but no bone marrow depression occurred in these patients. Five patients developed hemolytic uremic syndrome (HUS) post-transplant. This may have been related to the procedure used for total body irradiation. Patients who had their kidneys shielded during this procedure did not develop this syndrome. Patients who received local irradiation at the primary site showed no evidence of relapse in this region, indicating that such therapy may help to prevent a relapse. These data suggest a high rate of 3 year disease-free survival with this treatment strategy. The nonrandomized nature of the study and use of multiple modalities precludes analysis of the specific contribution of each.

Adrenal Gland Neoplasms↗
PubMed1995

Evaluation of new medium with chromogenic substrates for members of the family Enterobacteriaceae in urine samples.

A new medium containing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt (Glu agar) for Escherichia coli and a new medium containing 5-bromo-3-indolyl-beta-D-galactoside (Gal agar) for beta-galactosidase-positive members of the family Enterobacteriaceae were compared with MacConkey agar in a diagnostic trial with 3,562 urine specimens. The isolation rates of E. coli and beta-galactosidase-positive Enterobacteriaceae were increased 8.4 and 19.5%, respectively. The sensitivities and specificities of Glu agar and Gal agar were 98.5 and 100% and 99.2 and 99.5%, respectively.

Bacterial Typing Techniques↗
PubMed1995

Protein synthesis inhibition induces cytoresistance in cultured human proximal tubular (HK-2) cells.

After sublethal injury, proximal tubular cells acquire resistance to further attack. This study evaluated whether this could be a possible consequence of decreased protein synthesis, a potential correlate of cell damage. To this end, cultured human proximal tubular cells (HK-2) were subjected to 0-24 h of protein synthesis inhibition (> 98%), either by adding protein synthesis inhibitors [cycloheximide (CH) or verrucarin A] or by inducing sublethal ATP depletion (antimycin A + 2-deoxyglucose). After 24 h of these treatments, significant resistance to Ca2+ ionophore/ATP depletion-induced attack was noted (assessed by vital dye exclusion, compared with normal cells). That < or = 6 h of protein synthesis inhibition caused no cytoresistance implied the importance of evolving protein depletion rather than nonspecific drug effects or protein synthesis inhibition per se. CH plus ATP depletion did not induce additive benefits, suggesting a common mechanism. Cytoresistance was dissociated from the extent of free Ca2+ loading and ATP depletion but was associated with a decrease in membrane deacylation. CH removal promptly restored protein synthesis and cytoresistance was lost; conversely, ATP recovery did not restore protein synthesis and cytoresistance persisted. The emergence of cytoresistance correlated with the disappearance/dephosphorylation of an unidentified 130-kDa tyrosine-phosphorylated protein/protein complex (denoted pp-130). The functional significance of this change was suggested by the fact that tyrosine phosphatase inhibition with orthovanadate maintained pp-130 expression and prevented the cytoresistant state. We conclude that protein synthesis inhibition in HK-2 cells can induce a cytoresistant state. Suppression in phospholipase activity and altered tyrosine phosphorylation events may have functional significance in this regard.

Adenosine Triphosphate↗
PubMed1995

Signaling through CD40 rescues IgE but not IgG or IgA secretion in X-linked immunodeficiency with hyper-IgM.

The ligand for CD40 (CD40L) is a membrane protein on activated T cells that induces B cell proliferation and differentiation. Several mutations of the CD40L gene were reported responsible for defective class switching of B cells in an X-linked immunodeficiency with hyper IgM (X-HIM). We studied four affected males from three families and found three independent mutations including new mutations of CD40L gene. In every X-HIM patient tested, however, anti-CD40 plus IL-10 did not induce class switching from IgM to IgG or IgA, even in the presence of Staphylococcus aureus Cowan I strain (SAC). CD4+ T cell clones, expressing CD40L on their surface, also did not rescue IgG or IgA induction by X-HIM peripheral blood B cells in vitro. But signaling through CD40 induced both B cell proliferation and IgE secretion when IL-4 was added to the culture. Taken together, these results show that in vitro signaling through CD40 rescues IgE but not IgG or IgA secretion by peripheral blood X-HIM B cells and suggest that in vivo CD40 and CD40L interaction might be necessary for IgG and IgA differentiation in X-HIM.

Amino Acid Sequence↗
PubMed1995

[A case of localized fibrous mesothelioma arising from visceral pleura of the right lower lobe].

A 56-year-old male who had been followed for chronic hepatitis had cough and hemosputum. Chest X-ray and CT films showed an abnormal mass shadow in the right thorax. On suspicion of intrathoracic tumor, resection was performed. The tumor, which was on the diaphragm without invasion, was pedunculated and arose from the bottom surface of visceral pleura of the right lower lobe. From the microscopic findings and immunohistochemical staining, the tumor was diagnosed as localized fibrous mesothelioma. Some localized fibrous mesothelioma exhibit as malignant tumor. Therefore, complete resection and intensive follow-up should be done.

Humans↗
PubMed1995

[The clinical usefulness of botulinum toxin type A for spasmodic torticollis and facial spasm].

We studied the clinical effectiveness of botulinum toxin type A for spasmodic torticollis and that for facial spasm by multicenter, non-blinded study. The freeze-dried crystalline botulinum toxin type A (AGN 191622; Allergan Inc., Irvine, CA) was injected into the hyperactive muscles and the clinical course was followed for 22 weeks. Repeated injections were done, if necessary, with an interval of 4 weeks. The toxin was highly effective in both disorders. In spasmodic torticollis, clinical severity improved in 38 (63.3%) and the global improvement was seen in 39 (65.0%) out of 60 patients. Subjective improvement was seen in 56 (93.3%). In facial spasm, 52 (92.9%) out of 56 patients improved after the treatment. Unfavorable reactions, mainly consisting of neck muscle weakness and dysphagia in torticollis and facial weakness in facial spasm, were mostly due to the excessive action of the toxin. They were usually mild and transient. No patients discontinued the trial because of side effects. Botulinum toxin injection is a very useful and safe method for the symptomatic treatment of spasmodic torticollis and facial spasm.

Adolescent↗
PubMed1995

Subcutaneous pheomycotic cyst of the finger caused by Exophiala jeanselmei: association with a wooden splinter.

A 58-year-old man with mild glucose intolerance noted a subcutaneous cystic nodule on the ventral aspect of his ring finger. He had experienced trauma to the finger while gardening five weeks before the lesion occurred. Exophiala jeanselmei was isolated and identified on culturing of aspirate from the cystic nodule. The nodule was then removed surgically, and a wooden splinter was found close to the subcutaneous fibrous cyst, suggesting that the causal organism had entered the subcutaneous tissue with the wooden splinter.

Cysts↗