Biomedical subjects
M Iizuka
Publications and source records attributed to M Iizuka.
Coreceptor function of mutant human CD4 molecules without affinity to gp120 of human immunodeficiency virus.
Despite extensive mutational studies on the human CD4 molecule and its affinity to human immunodeficiency virus (HIV) envelope glycoprotein gp120, coreceptor functions of such mutant molecules have only been examined by indirect measurement of their affinity to class II major histocompatibility complex (MHC) molecules. In this report, coreceptor functions of mutant human CD4 molecules, which have no or reduced affinity to an HIV envelope protein, gp120, were assessed in a murine T cell receptor/class II MHC recognition system. The substitution of human C" beta strand with the murine homologous segment resulted in the loss of the coreceptor function as well as in the complete loss of gp120 binding capacity, corroborating the consensus that Phe-43 in C" beta strand plays crucial roles in both situations. However, simultaneous replacement of the C'-C" loop along with the C" beta strand by homologous murine segments rescued the coreceptor function, whereas gp120 binding capacity remained negative. Further analysis indicated that insertion of lysine between Gly-41 and Ser-42 can partially compensate for the coreceptor function lost by the Phe-43 --> Val mutation. Although the coreceptor function of these mutant CD4 molecules in a human T cell recognition system is yet to be determined, these observations necessitate a re-evaluation of the role played by Phe-43 in coreceptor function. Examination of the sensitivities of the mutant CD4 molecules expressed on HeLa cells to infection by a T cell-tropic HIV-1 strain indicated that only those mutants that had completely lost gp120 binding capacity were resistant to the infection. All mutants having whole C" substitution, irrespective of additional substitutions or their coreceptor functions, were resistant to the infection.
Characterization of the anticonvulsant and neuroprotectant BIIR 561 CL in vitro: effects on native and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.
BIIR 561 CL is a novel blocker of AMPA receptors and voltage-dependent sodium channels. In this study we further describe the effects of BIIR 561 CL on AMPA receptor-mediated membrane currents in rodent neurons, as well as in cells expressing recombinant human GluR1/2 receptors in more detail. BIIR 561 CL suppressed responses to kainate in neuronal cultures from rat cortex with an IC50 of 9.8 microM. Similar effects were observed using acutely dissociated neurons from the CA1 region of rat hippocampus (IC50 = 9.5 microM). Inhibition of kainate responses by BIIR 561 CL was prevented by preapplication of GYKI 53655, suggesting that both non-competitive inhibitors bind to a common site of the receptor. The effect of 10 microM BIIR 561 CL on kainate-induced currents was dependent on extracellular pH, with more pronounced block (84.1%) under acidic conditions (pHextern=6.4), compared to only 30.1% at a pHextern of 8.4. Thus, it can be hypothesized that BIIR 561 CL inhibits AMPA receptors in ischaemic brain regions more effectively than in healthy tissue. BIIR 561 CL inhibited responses to 1 mM glutamate in cells expressing recombinant human GluR1/2 receptors with similar potency, as compared to kainate responses in rat neurons (IC50=17.3 microM). The reference compound NBQX had an IC50 of 25.2 nM. None of the two compounds affected the glutamate-induced receptor desensitization at any tested concentration. The block by BIIR 561 CL was not use-dependent and had fast on- and off-kinetics (tauon=6.8 s; tauoff=1.3 s in hGluR1/2 receptors with 30 microM BIIR 561 CL). Thus, BIIR 561 CL can be anticipated to have a promising profile for the treatment of neurological disorders like brain ischaemia and head trauma.
Genetic polymorphisms in the cell growth regulated gene, SC1 telomeric of the HLA-C gene and lack of association of psoriasis vulgaris.
Psoriasis vulgaris is associated with the HLA-Cw6 and Cw7 antigens. We have previously narrowed down the critical region most likely to contain the psoriasis vulgaris gene to 111 kb spanning 89 kb to 200 kb telomeric of the HLA-C locus by microsatellite mapping. This segment includes three known genes (POU5F1, SC1 and S) and four new expressed genes. Among them, SC1 (TCF19) is the cell growth regulated gene possibly with trans-activator activity. Since psoriasis vulgaris is a common skin disorder characterized by hyperproliferation of epidermal cells, it is tempting to speculate that the SCI gene is one of the strong candidate genes responsible for the development of psoriasis vulgaris. Here, we investigated genetic polymorphisms in the SC1 gene by direct DNA sequencing and polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) techniques. Three single nucleotide polymorphisms in exon 2, two of which are accompanied by amino-acid substitutions, were identified. Further, one 4-bp deletion polymorphism was detected around the acceptor site of the lariat-shaped structure necessary for RNA splicing in intron 2. No significant difference in the dimorphic or haplotypic distribution at these four polymorphic sites was observed between the patients with psoriasis vulgaris and healthy controls. This suggests that the susceptible gene for psoriasis vulgaris is not the SC1 gene itself, although a unique homozygous haplotype was identified in the patients.
Human T-cell receptor BV6 gene polymorphism in relation to expression level and CD4/CD8 skewness.
Using 50 samples of umbilical cord blood lymphocytes from Japanese donors, we analysed two human T-cell receptor beta variable (TCRBV) genes, BV6S4 and BV6S5, for their polymorphism, usage frequencies and CD4/CD8 skewness. They showed contrasting CD4/CD8 skewness, BV6S4 to CD8+ T cells and BV6S5 to CD4+ T cells. Genotyping of the BV6S4 alleles (A1, A2 and A3) revealed two of the six possible BV6S4 genotypes, A1/A2 and A2/A2. Among the two BV6S4 genotypes, no significant difference was detected in usage frequency or CD4/CD8 skewness. On the other hand, genotyping of the BV6S5 alleles (A1 and A2) revealed all three possible BV6S5 genotypes, A1/A1, A1/A2 and A2/A2, and the gene usage frequency was high, in the order A1/A1 > A1/A2 > A2/A2. These results indicate that the amino acid substitutions in BV6S5 (S36R and G70E) are in some way associated with the expression level of this gene. In the analysis of CD4/CD8 skewness, the three BV6S5 genotypes had similar skewness, indicating that A1 alleles are expressed more frequently than A2 alleles in both CD4+ and CD8+ T-cell populations. Although BV6S5 exhibits marked skewness to CD4+ T cells, our results indicate that the higher expression of A1 alleles is not associated with the increased ratio of CD4+ T cells.
The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells.
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) -type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1-GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+-permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2-lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip-mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor-mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.
Presence of bacterial 16S ribosomal RNA gene segments in human intestinal lymph follicles.
BACKGROUND: There is currently no information regarding microbial agents inside the intestinal lymph follicles. METHODS: Biopsy or resected specimens, mostly from macroscopically normal areas, were sectioned with a cryostat. DNA was extracted from microdissected samples, exclusively from the lymph follicle. Amplification of DNA was performed using universal primers designed from conserved regions of bacterial 16S ribosomal RNA (rRNA). Several clones with inserts of around 400 base pairs were subjected to DNA sequence analysis followed by a database homology search. RESULTS: Bacterial 16S rRNA gene segments were detected in the lymph follicle in 2 of 14 (14%) non-inflammatory bowel disease (IBD) cases, 4 of 14 (28%) Crohn disease cases, and in 2 of 5 (40%) ulcerative colitis cases. Nineteen 16S rRNA gene segments were recognized in the eight positive cases. Five segments showed 100% identity to known bacterial 16S rRNAs, namely staphylococcus species, Streptococcus sanguis, and Paracoccus marcusii. However, the other 14 segments showed below 100% identity, indicating either the presence of unknown bacteria or of bacteria without known DNA data. No single identified or unidentified bacterium, characteristic of IBD, including Mycobacterium paratuberculosis and Listeria monocytogenes, was detected. CONCLUSIONS: The present study confirms the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles and paves the way for new investigations into the microbiology of the lymph follicle. Whether or not bacteria inside the lymph follicle is a primary stimulus in IBD has yet to be clarified.
Dynamic contrast enhanced CT measurement of blood flow during interstitial laser photocoagulation: comparison with an Arrhenius damage model.
One effect of heating during interstitial laser photocoagulation (ILP) is to directly destroy the tumour vasculature resulting in a loss of viable blood supply. Therefore, blood flow measured during and after treatment can be a useful indicator of tissue thermal damage. In this study, the effect of ILP treatment on rabbit thigh tumours was investigated by measuring blood flow changes using dynamic contrast enhanced computed tomography (CT). The CT measured changes in blood flow of treated tumour tissue were fitted to an Arrhenius model assuming first order rate kinetics. Our results show that changes in blood flow of tumour tissue distant from surrounding normal tissue are well described by an Arrhenius model. By contrast, the temperature profile of tumour tissue adjacent to normal tissue must be modified to account for heat dissipation by the latter. Finally, the Arrhenius parameters derived in the study are similar to those derived by heating tumour tissue to a lower temperature (<47 degrees C) than the current study. In conclusion, CT can be used to monitor blood flow changes during ILP and these measurements are related to the thermal damage predicted by the Arrhenius model.
Immunohistochemical analysis of the distribution of measles related antigen in the intestinal mucosa in inflammatory bowel disease.
BACKGROUND: Measles virus is implicated in the aetiology of Crohn's disease. This measles hypothesis is mainly supported by immunohistochemical findings that the measles related antigen is present in the intestine of patients with Crohn's disease. Recently we isolated this antigen from the intestine of a patient with Crohn's disease using a molecular cloning technique and produced the monoclonal antibody against it (designated 4F12). AIM: To discover whether the measles related antigen is uniquely present in Crohn's disease. SUBJECTS/METHODS: Colonic mucosa samples from 20 patients with Crohn's disease, 20 with ulcerative colitis, 11 with non-inflammatory bowel disease (IBD) colitis, and nine controls were immunohistochemically stained with the anti-measles monoclonal antibody 4F12. The numbers of positive cells, the ratio of positive cells to nucleated cells, and the staining intensity of the positive cells were compared. Furthermore, the distribution of the measles antigen in other human organs was examined. RESULTS: Both the number of positive cells and the ratio of positive cells to nucleated cells were significantly increased in Crohn's disease, ulcerative colitis, and non-IBD colitis compared with controls (p<0.05) but were similar among the three disease groups. The staining intensity of the positive cells was also similar among the three disease groups. Small numbers of positive cells were observed in the oesophagus, stomach, duodenum, jejunum, and lung. CONCLUSIONS: The presence of the measles related antigen in the colonic mucosa was not unique to Crohn's disease. These results, together with the observation that such a measles related antigen was derived from host protein, do not support the hypothesis that measles virus causes Crohn's disease.
Fe(III)-IDA induces proliferation and sustains functions of various types of cultured cells as a substitute for transferrin.
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[Comparison of whole-body MR imaging and bone scintigraphy in the detection of bone metastases from breast cancer].
The aim of this study was to investigate the usefulness of whole body MR imaging (WB-MRI) in the detection of bone metastases from breast cancer and to compare the results with those from bone scintigraphy. In 21 patients with suspected bone metastasis from breast cancer, both bone scintigraphy and WB-MRI were performed. With WB-MRI, coronal images were obtained using a body coil in an FOV of 48 cm, and sequences of fast short TI inversion recovery (STIR) and gadolinium-enhanced fast spoiled GRASS (SPGR) were used in three parts: from the head to the thorax, the abdomen to the pelvis, and the lower extremities. Of the total 105 metastatic bone lesions, 65 (61.9%) were detected by bone scintigraphy, 98 (93.3%) by fast STIR, and 74 (70.5%) by fast SPGR. Thus, the detection of bone metastases by WB-MRI was excellent. However, detectability in the ribs was lower for WB-MRI than for bone scintigraphy. Contrast-enhanced MRI was useful in the differentiation of osteosclerotic lesions, in which high signal intensity is rare, pleural effusion, which has high signal intensity on STIR, and bone metastatic lesions. In conclusion, WB-MRI showed high reliability in the detection of bone metastatic lesions from breast cancer.
Lymph-node staining with activated carbon CH40: a new method for axillary lymph-node dissection in breast cancer.
OBJECTIVE: To demonstrate the usefulness of activated carbon particles (CH40) as a vital staining dye for visualizing lymphatic vessels and lymph nodes in breast cancer. DESIGN: A retrospective evaluation. SETTING: Department of Surgery in Sendai National Hospital, Japan, a 716-bed teaching hospital. METHODS: To identify as many lymph nodes as possible in the axillary fat, by which we might decrease the possibility of the presence of undetected metastatic nodes, an emulsion of activated carbon particles (CH40) was injected into the centre of the mammary gland, close to the tumour site, 3 days before radical surgery. MAIN OUTCOME MEASURE: The number of lymph nodes found by the traditional method and by the CH40-injection method were recorded. RESULTS: After injection, the CH40 was readily adsorbed into regional lymphatics and streamed along with the lymph flow to blacken regional lymph nodes. The CH40-guided method increased the mean number of nodes per case found in the axilla from 8.4, by the traditional method, to 14.0 nodes per case. CONCLUSIONS: The use of the CH40 technique has two technical advantages; one is that it allows surgeons to locate the blackened lymph nodes at the time of surgery and the other is that it allows pathologists to look for the nodes in fatty tissue. Lymph-node dissection with the aid of activated carbon particles is inexpensive, easy to perform and enables the smallest lymph nodes to be easily recognized. CH40 is the technique of choice for the detection of axillary lymph nodes in cases where the number of lymph nodes detected by the traditional method is too small for accurate surgery. In conclusion, the present study demonstrates that CH40 could be an appropriate tool for more accurate staging of breast cancer axillary specimens.
Stable expression of human homomeric and heteromeric AMPA receptor subunits in HEK293 cells.
Human homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors (GluRs) were stably expressed in HEK293 cells with cDNAs encoding the flip splice variant of GluR1, GluR2, GluR3, GluR4 subunit, and the GluR1/GluR2, GluR3/GluR2, and GluR4/GluR2 combination. The lethal combination of GluR2 and GluR4 subunits was found in high expression levels of both receptors. The AMPA-evoked current voltage relationships demonstrated the functional channel properties, such as a double rectification in GluR1, GluR3, and GluR4 receptors, and a linear relation in receptors assembled from GluR2 alone and coexpression of GluR2 with the other subunits. All the transfectants exhibited higher selectivity for AMPA than glutamate in dose-dependent current responses. [3H]AMPA binding revealed that the homomeric and heteromeric receptors displayed a single binding site in Scatchard analysis, with dissociation constant (Kd) values in the range of 14.5-49.3 nM. The Bmax values were in the range of 0.57-7.66 pmol/mg protein. The ligand displacement potency for [3H]AMPA binding was CNQX > glutamate > NS257 in all of the transfectants. These results suggest that stable transformants expressing human homomeric and heteromeric AMPA receptors will be useful tools to define selectivity and potential site of action for AMPA receptor modulators.
[Case suspected to be tuberculous myelitis with pulmonary tuberculosis].
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Intercostal expiratory activity in an in vitro brainstem-spinal cord-rib preparation from the neonatal rat.
1. We examined whether expiratory activity can be observed when central chemoreceptors are activated by a decrement in the extracellular pH in an isolated brainstem-spinal cord-rib preparation from 0- to 3-day-old rats. Expiratory activity was defined as the burst activity that occurs in an internal intercostal muscle (IIM) during the silent period between the periodic inspiratory bursts in the C4 ventral root (which contains phrenic motor axons). 2. During perfusion with modified Krebs solution (26 mM HCO3-, 5 % CO2, pH 7.4), there was no consistent activity in IIM, though rhythmic inspiratory motor activity always appeared in the C4 ventral root. 3. When the pH of the perfusate was lowered from about 7.4 to 7.1 by reducing [HCO3-] from 26 to 10 mM, the frequency of the C4 inspiratory rhythm increased, and rhythmic activity appeared in IIM. In most cases, the rhythmic burst in IIM started just after the cessation of the C4 inspiratory burst and coincided with movement of the ribs in a caudal direction. This intercostal expiratory burst was limited to the first half of the expiratory phase. 4. The coordinated reciprocal motor activity between the C4 ventral root and IIM changed to a largely overlapping pattern when strychnine (5-10 microM), a glycine receptor antagonist, was added to the perfusate. 5. These results suggest (i) that the neuronal mechanisms responsible for expiratory motor activity are preserved in this in vitro preparation and (ii) that the glycinergic inhibitory system plays an important role in the coordination between inspiratory and expiratory motor activity during respiration.
Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein.
The origin recognition complex (ORC) is an initiator protein for DNA replication, but also effects transcriptional silencing in Saccharomyces cerevisiae and heterochromatin function in Drosophila. It is not known, however, whether any of these functions of ORC is conserved in mammals. We report the identification of a novel protein, HBO1 (histone acetyltransferase binding to ORC), that interacts with human ORC1 protein, the largest subunit of ORC. HBO1 exists as part of a multisubunit complex that possesses histone H3 and H4 acetyltransferase activities. A fraction of the relatively abundant HBO1 protein associates with ORC1 in human cell extracts. HBO1 is a member of the MYST domain family that includes S. cerevisiae Sas2p, a protein involved in control of transcriptional silencing that also has been genetically linked to ORC function. Thus the interaction between ORC and a MYST domain acetyltransferase is widely conserved. We suggest roles for ORC-mediated acetylation of chromatin in control of both DNA replication and gene expression.
L-selectin and its ligands mediate infiltration of mononuclear cells into kidney interstitium after ureteric obstruction.
It was previously reported that the L-selectin ligands detected by a rat L-selectin and human IgG chimeric molecule (rLEC-IgG) are expressed in the distal tubules of the kidney, where no leukocyte traffic is seen under physiological conditions. In the present study, the role of L-selectin ligands in leukocyte infiltration into the kidney interstitium was investigated using a rat ureteric obstruction model. After ligation of the ureter, ligands for L-selectin rapidly disappeared from tubular epithelial cells and were relocated to the interstitium and peritubular capillary walls, where infiltration of monocytes and CD8(+) T cells subsequently occurred. Mononuclear cell infiltration was significantly inhibited by intravenous injection of a neutralizing monoclonal antibody (MAb) against L-selectin, indicating the possible involvement of an L-selectin-mediated pathway. Interestingly, immunohistochemical studies with a MAb against sulphatide showed that the distribution of sulphatide, known to be one of the candidates of L-selectin ligand, was almost indistinguishable from the staining pattern of rLEC-IgG in both normal and ureteric obstructed kidneys, suggesting that sulphatide and/or related molecule(s) relocated to the renal interstitium were recognized by leukocyte L-selectin, leading to interstitial leukocyte infiltration. In line with this notion, intravenous injection of sulphatide markedly inhibited leukocyte infiltration, suggesting that L-selectin-sulphatide interaction may play a pivotal role in interstitial leukocyte infiltration in the kidney following ureteric obstruction.
In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats.
This study describes the in vitro digestibility and fermentability of high molecular weight (ca. 2,000,000) levan and its effect on the metabolism of lipids in growing rats fed cholesterol-free diets. Levan was synthesized from sucrose using bacterial levansucrase immobilized on a honeycomb-shaped ceramic support. Although body weight gain, weight of visceral organs, morphologic changes in the digestive tract, and the serum triacylglycerol and glucose concentrations were not affected by feeding levan diets for 4 weeks, a significant hypocholesterolemic effect was observed. Serum cholesterol level was decreased to 83% or 59% by feeding a 1% or 5% levan diet, respectively. The hypocholesterolemic effect was accompanied by a significant increase in fecal excretion of sterols and lipids. High molecular weight levan, though not hydrolyzed by the salivary amylases, was hydrolyzed by artificial gastric juice and was changed to a low molecular weight (ca. 4,000) levan with a small amount of fructose, but did not produce any fructooligosaccharides. Low molecular weight (ca. 6,000) levan was not hydrolyzed by either pancreatic juice or small intestinal enzymes. This suggests that, in vivo, low molecular weight levan derived from the high molecular weight material is not further digested and reaches the colon intact. The fermentation of low molecular weight levan (ca. 6,000) by several strains of bifidobacteria was not observed. These results showed that the hypocholesterolemic effect of levan may result from the prevention of intestinal sterol absorption, and not from the action of the fermentation products of levan.