Biomedical subjects
Publications and source records attributed to K Iijima.
We synthesized a series of thienopyrimidine derivatives and examined their cytotoxic effects on several cell lines. One of the derivatives, NSL-1406, was shown to exert potent cytotoxic effects on leukemia cell line including P388 cells and J774 cells. It was also inhibitory on mouse osteoclasts and suppressed the in vitro bone resorption by osteoclasts at nanomolar concentrations.
We prepared a potent and relatively selective human chymase inhibitor 9 (-), based on the study of SAR of a symmetrical anhydride-type serine protease inhibitor 1. Kinetic studies suggested that 9 (-) reacts with the Ser residue at the active site of the enzyme, forming a stable acyl enzyme complex. We also showed the importance of the tri-substituted beta-amino acid structure for the potent anti-enzymatic activity.
We introduce a new potent inhibitor, N-[2, 2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine alpha-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (kinact) and enzyme-inhibitor dissociation constant (Ki) against alpha-chymotrypsin were 0.0028 s-1 and 0.0045 microM, respectively (kinact/Ki = 630 000 M-1 s-1). These kinetic parameters indicate that this inhibitor is one of the most powerful alpha-chymotrypsin inactivators ever reported. On the basis of structure-activity relationship (SAR) and structure-stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl group, beta-substituent at the beta-amino acid residue, and ester structure, we suggest that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the beta-position and 4-cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.
Prolonged tracheotomy and endotracheal intubation often induce symptoms of airway obstruction and delay decannulation and extubation. Bronchoscopic examination of patients undergoing these treatments usually shows the presence of exuberant (pseudopapillary or nodular) granulation tissue occupying the airway lumen. An immunohistochemical analysis was undertaken of vascular endothelial growth factor (VEGF) expression in exuberant tracheal granulation tissue (n=17) obtained from children treated with prolonged tracheotomy or endotracheal intubation. Increased levels of VEGF protein and mRNA were expressed mainly by tracheal epithelial cells that migrated to cover the granulation tissue and partly by pericapillary macrophages in this tissue, whereas normal tracheal epithelium did not express VEGF. The VEGF expression level correlated significantly with the severity of the exuberant granulation tissue response (p=0.0018). As VEGF induces angiogenesis and vascular permeability, characteristics of granulation tissue, and plays a pivotal role in granulation tissue development, enhanced VEGF expression may be involved in the development of exuberant tracheal granulation tissue.
The microwave spectrum of (E)-benzaldehyde oxime, C(6)H(5)-CH&dbond;NOH and C(6)H(5)-CH&dbond;NOD, has been observed in the frequency range from 26.5 to 40.0 GHz. The spectrum of the ground vibrational state was assigned and fitted to the Watson's A-reduced Hamiltonian to obtain these rotational and centrifugal distortion constants: A = 5183.13(29) MHz, B = 895.367(3) MHz, C = 763.819(3) MHz, Delta(J) = 0.019(3) kHz, and Delta(JK) = 0.204(7) kHz for the normal species, and A = 5158.4(23) MHz, B = 869.44(2) MHz, C = 744.34(2) MHz, Delta(J) = 0.023(2) kHz, and Delta(JK) = 0.193(7) kHz for the deuterated species. The values of the DeltaI (=I(c) - I(a) - I(b)) obtained for the normal and deuterated species were -0.295(6) and -0.28(5) a.m.u. Å(2), respectively. The molecular conformation of this molecule was a planar one in which the values of the dihedral angles, CCNO and CNOH, were almost 180 degrees and 180 degrees, respectively. Copyright 1999 Academic Press.
Intrapericardial organized hematoma, which compresses cardiac chambers late after open heart surgery, is extremely rare. We report a case of intrapericardial organized hematoma in a 63-year-old man who underwent aortic valve replacement 8 years prior, which may have aggravated rheumatic mitral valve regurgitation compressing the mitral valve anulus. Under extracardiopulmonary bypass and cardioplegic heart arrest, we removed the hematoma and replaced the mitral valve with a 27 mm St. Jude Medical valve. There were no bleeding points on the heart and pericardium at operation and no history of blunt chest trauma. The etiology of the hematoma is uncertain.
We describe a prospective study of 2-year moderate-dose cyclosporin (CS) treatment in 13 children with steroid-dependent minimal change nephrotic syndrome (MCNS). CS treatment was commenced at 100-150 mg/m2 per day after remission was attained with prednisolone therapy, was adjusted to a target trough level of 100 ng/ml, and was administered for 2 years. The number of relapses during CS treatment significantly decreased compared with before CS treatment, all patients were able to discontinue prednisolone therapy, and steroid toxicity was reduced; 54% of patients remained in remission during CS treatment. Renal biopsies performed before CS treatment all showed MCNS without tubulointerstitial lesions. Creatinine clearance and urinary beta 2-microglobulin levels during CS treatment were normal in all patients, but renal biopsies performed after CS treatment revealed chronic CS nephrotoxicity in 7 patients. Clinical data, including CS dose and CS trough blood levels, were not significantly different between patients with and without nephrotoxicity. In conclusion, 2-year moderate-dose CS treatment in children with steroid-dependent MCNS is effective in preventing relapse and decreasing steroid toxicity. This treatment can, however, result in a high incidence of chronic nephrotoxicity. Renal function is not a reliable indicator of chronic CS nephrotoxicity. Renal biopsy is therefore necessary to monitor chronic CS nephrotoxicity.
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X-linked Alport's syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen alpha5 chain (alpha5[IV]). Polymerase chain reaction-single-str and conformation polymorphism (PCR-SSCP) on genomic DNA has previously been used to screen for mutations in the COL4A5 gene, but this method was relatively insensitive, with mutations detected in less than 50% of patients. Here, we report a systematic analysis of the entire coding region of the COL4A5 gene, using nested reverse-transcription-polymerase chain reaction (RT-PCR) and the direct sequence method using leukocytes. This study examines twenty-two unrelated Japanese patients with X-linked Alport's syndrome showing abnormal expression of alpha5(IV) in the glomerular or epidermal basement membranes. Mutations that were predicted to be pathogenic were identified in 12 of the 13 male patients (92%) and five of the nine female patients (56%). Six patients had missense mutations, four had out-of-frame deletion mutations, three had nonsense mutations, and three had mutations causing exon loss of the transcript. The current study shows that nested RT-PCR and the direct sequence method using leukocytes are highly sensitive and offer a useful approach for systematic gene analysis in patients with X-linked Alport's syndrome.
Platelet-activating factor (PAF) is a potent mediator of inflammatory injury in renal diseases. PAF is degraded to inactive products by PAF acetylhydrolase. Recently, a point mutation (G to T transversion) of the PAF acetylhydrolase gene was observed at position 994, and this mutation was found to contribute to the variability in plasma PAF levels, with undetectable plasma PAF acetylhydrolase activity occurring in homozygous patients (TT genotype) and reduced levels of activity in heterozygous patients (GT genotype). Therefore, we investigated the effect of the PAF acetylhydrolase gene mutation on the pathogenesis and progression of immunoglobulin A (IgA) nephropathy. Genomic DNA was obtained from 89 children with IgA nephropathy and 100 controls. We identified the PAF acetylhydrolase gene mutation (G994T) by polymerase chain reaction. There was no significant difference in genotypic frequency between patients and controls. However, urinary protein excretion at the time of biopsy was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. The percentage of glomeruli with mesangial cell proliferation was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. These results indicate the PAF acetylhydrolase gene mutation may influence the degree of proteinuria and the extent of mesangial proliferation in the early stage of childhood IgA nephropathy.
We previously reported a novel monoclonal antibody KOR-SA3544 which predominantly reacted with a surface antigen (sSA3544) expressed on Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL). In the present study, we demonstrate that the antibody specifically recognized nonspecific cross-reacting antigen (NCA)-50/90 (CD66c), one of the carcinoembryonic antigen (CEA)-related glycoproteins encoded by a member of the CEA gene family. In addition, we show that the SA3544 antigen (NCA-50/90) was invariably expressed in cytoplasm of all of the human leukemic cell lines examined (sSA3544-positive B-lymphoid two, sSA3544-negative T or B-lymphoid and non-lymphoid 24) regardless of the presence or absence of surface expression of this antigen. Immunoelectromicroscopic examination revealed that the cytoplasmic antigen was mainly present in granules in sSA3544-positive leukemia cells, whereas it was diffusely present in cytosol in sSA3544-negative leukemia cells. Thus, among members of the CEA family, NCA-50/90 was first demonstrated to be expressed not only on the surface of some leukemia cells, but also in cytoplasm of various types of leukemia cells.
The p16 gene encoding a specific inhibitor of cyclin-dependent kinases 4 and 6 has been reported to be inactivated at a variety of rates in malignant tumors. We studied frequency and mechanism of inactivation of the p16 gene in various types of childhood acute lymphoblastic leukemia (ALL) using 36 leukemic cell lines established from children (B precursor-ALL, 28; B-ALL/Burkitt's lymphoma, 3; and T-ALL, 5). On Southern blot, homozygous deletions or hemizygous deletions with rearrangement were detected in 14 cell lines. The expression of p16 protein was not observed on Western blot in 18 of 22 cell lines with intact p16 gene, but induced in 16 cell lines after treatment with the demethylating agent, indicating the silencing of the p16 gene by hypermethylation. Of note, the p16 gene was inactivated by hypermethylation of the 5' CpG island in nine of nine cell lines with 11q23 translocation, but was restored with the treatment of the demethylating agent. Partial methylation of the p16 gene was also demonstrated in three of eight primary leukemia samples with this translocation, suggesting that the p16 gene inactivation by hypermethylation might play a role in the leukemogenesis and disease progression of ALL with 11q23 translocation.
BACKGROUND: The influence of the degree of cell differentiation and of physical stimulation on gastric mucosal wound healing is not completely understood. METHODS: A gastric mucosal cell line, GSM06, derived from the gastric mucosal cells of transgenic mice harbouring the simian virus 40 large T antigen, was cultured at 33 degrees C to make a confluent cell sheet. Artificial wounds of constant size were created. Wound healing was monitored at different temperatures (33, 37 and 39 degrees C), which altered the degree of differentiation. Cell proliferation was detected by bromodeoxyuridine staining. Mechanical strain was applied to adherent GSM06 cells after wounding in order to increase their length by an average of 5 or 10% at 5 cycles/min for 60 h. Repair of the wound was monitored every 12 h. RESULTS: Differentiated gastric epithelial cells showed a higher speed of migration. The number of proliferating cells around the wound was greatest at 33 degrees C and barely detectable at 39 degrees C. Under conditions of mechanical strain, the migration speed of differentiated cells (at 39 degrees C) slowed in a strain strength-dependent manner. CONCLUSIONS: It is suggested that cell differentiation status and physical stimulants might play a role in gastric wound healing in vivo by modifying cellular migration.
Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.
Benzo[a]pyrene (B[a]P), a tobacco-derived carcinogen, induces lung tumors in rodents through its carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE). Tumorigenesis is inhibited by dietary myo-inositol in the post-initiation phase. However, little is known about how B[a]PDE and myo-inositol affect normal human lung cells. We addressed this question using untransformed human small airway epithelial (SAE) cells. SAE cell viability decreased <50% in parallel to an increase of apoptotic cells (>20%) 2 days after the cells were treated for 1 h with B[a]PDE (>100 nM). In contrast, the cell number and viability were not altered in A549 human lung cancer cells by B[a]PDE treatment up to 10 microM with <5% apoptotic cells and <10 U/l LDH in the medium. SAE cells retain the features of basal cells in serum-free, low Ca2+ (4 nM) medium up to 4-5 passages, but in serum-supplemented or serum-free, high Ca2+ (1 mM) cultures, they differentiate into non-ciliated epithelial cells expressing Clara cell secretory protein (CCSP). A non-toxic, physiologically relevant dose of B[a]PDE (1 nM) partially inhibited serum and Ca2+-induced SAE cell differentiation. This effect was abolished by wortmannin, a phosphatidylinositol-3 kinase (PI-3K) inhibitor, and PD98059, a mitogen activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor, but not by SB202190, a p38 MAPK inhibitor, or melittin, a protein kinase C inhibitor. Myo-inositol (10-100 microM) did not alter growth or differentiation of untreated SAE or A549 cells, but reversed the inhibitory effect of B[a]PDE on serum and Ca2+-induced SAE cell differentiation when supplemented to the culture after B[a]PDE treatment. This myo-inositol action was not altered by PD98059, wortmannin or melittin, but was partially suppressed by SB202190. Collectively, these results indicate that B[a]PDE inhibits serum-induced SAE cell differentiation, possibly involving activating signals through a PI-3K/MEK1 mediated MAPK pathway, whereas myo-inositol protects SAE cells against this inhibitory effect of B[a]PDE perhaps through both PI-3K/MEK1 and p38 MAPK pathways.
PURPOSE: This study investigated the area size of the flow of a gold braze alloy on commercially pure titanium and Ti-6Al-4V alloy plates, and elemental composition at the interface was determined. In the second part of this study, the tensile strengths of titanium plates brazed using a gold alloy were investigated. MATERIALS AND METHODS: Chips of Type IV gold alloy and silver braze alloys were melted onto commercially pure titanium and Ti-6Al-4V surfaces in a dental infrared radiation unit. Flow area of each braze alloy was measured using a digital image analyzer. Tensile specimens (n = 5) were also prepared by infrared brazing using the braze alloys. Five specimens for each combination of the two titanium plates and the two braze alloys were subjected to tensile loading using a Universal testing machine. Electronprobe microanalysis of x-rays at cross-section of the brazed joints to determine elemental composition across the interface, as well as scanning electron microscopic observation at the fracture surfaces, were also conducted. RESULTS: The braze alloys flowed well and spread over the Ti and Ti-6Al-4V plates. Braze alloy type significantly influenced flow, and the gold alloy flowed less on the titanium materials. The mean tensile strengths of Ti and Ti-6Al-4V plates brazed using the gold braze alloy were 219 MPa and 417 MPa, respectively. The fracture surfaces of Ti-6Al-4V specimens with the gold braze alloy exhibited typical ductile behavior. Ti with the same braze alloy showed brittle surfaces. A greater concentration of Cu was found at the Ti with gold braze interface. CONCLUSION: The flow and the tensile strength of the gold alloy coating on titanium surface by means of an infrared brazing is adequate for dental use.
OBJECTIVE: Although it is widely accepted that Helicobacter pylori (H. pylori) infection is an important cause of atrophic gastritis, few studies have examined the relationship between H. pylori-induced atrophic gastritis and the occurrence of reflux esophagitis. The present study was aimed to examine the relationship between H. pylori infection, atrophic gastritis, and reflux esophagitis in Japan. METHODS: A total of 175 patients with reflux esophagitis were compared with sex- and age-matched 175 control subjects. Diagnosis of H. pylori infection was made by gastric mucosal biopsy, rapid urease test, and serum IgG antibodies. Severity of atrophic gastritis was assessed by histology and serum pepsinogen I/II ratio. RESULTS: H. pylori infection was found in 59 (33.7%) patients with reflux esophagitis, whereas it was found in 126 (72.0%) control subjects. The grade of atrophic gastritis was significantly lower in the former than in the latter. Among the H. pylori-positive patients, atrophic gastritis was milder in the patients with reflux esophagitis than in the patients without it. CONCLUSIONS: These findings suggest that most cases of reflux esophagitis in Japan occur in the absence of H. pylori infection and atrophic gastritis, and it may also tend to occur in patients with milder gastritis even in the presence of H. pylori infection. Therefore, H. pylori infection may be an inhibitory factor of reflux esophagitis through inducing atrophic gastritis and concomitant hypoacidity.
Aging is associated with skewed type 2 (T2) T cell responses that may be modulated by herbal medicines. A group of Japanese herbal medicines, so-called "Hozai," have been used to improve the physical condition of the elderly. One representative "Hozai," Juzen-Taiho-To (JTX) appears to have beneficial effects on cancer patients. In this study we hypothesized that JTX modulated skewed T2 responses in the elderly. T1 and T2 responses against ovalbumin (OVA) were examined in old BALB/c mice fed JTX (0.2% w/w). We measured anti-OVA IgG1, IgG2a, and IgG2b antibody (Ab) levels after the primary and secondary OVA challenges; T1 and T2 responses augment IgG2a/IgG2b Ab and IgG1/IgE Ab production, respectively. We also assessed production of T1 and T2 cytokines (IFN-gamma and IL-5, respectively), and co-stimulatory molecule expression by regional draining lymph node cells. JTX-fed mice had higher IgG2b Ab and IFN-gamma production than controls along with lower IgG1 Ab. JTX did not alter IL-5 production or co-stimulatory molecule expression. Hoelen, an herbal component, induced similar changes. Our results indicate that JTX and Hoelen modulate T cell responses against OVA toward more balanced T1/T2 responses in old BALB/c mice. Such effects of JTX may help prevent the development of diseases associated with immunodisregulation in the elderly.