Immunoprecipitation studies of monoclonal antibodies submitted to the Second International Swine CD Workshop.
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Animals↗
Biomedical subjects
M Shimizu
Publications and source records attributed to M Shimizu.
A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia. Mutations in brief no. 117. Online.
Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of all steroids including glucocorticoids, mineralocorticoids and sexsteriods. It has recently been reported that mutations in the steriodogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel framshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene.
Adrenal Hyperplasia, Congenital↗
Evidence for a putative telomerase repressor gene in the 3p14.2-p21.1 region.
Telomeres, which are the repeated sequences located on both ends of chromosomes in eukaryotes, are known to shorten with each cell division, and their eventual loss is thought to result in cellular senescence. Unlike normal somatic cells, most tumor cells show activation of telomerase, a ribonucleoprotein enzyme that stably maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres. The KC12 cell line derived from a renal cell carcinoma in a patient with von Hippel-Lindau disease showed telomerase activity and loss of heterozygosity on the short arm of chromosome 3. Introduction of a normal human chromosome 3 into KC12 cells by microcell fusion induced cellular senescence, accompanied by suppression of telomerase activity and shortening of telomere length. Microcell hybrids that escaped from cellular senescence maintained telomere length and telomerase activity similar to those of the parental KC12 cells. We previously showed a similar suppression of telomerase activity by introduction of chromosome 3 into another renal cell carcinoma cell line, RCC23. The putative telomerase repressor gene was mapped to chromosome region 3p14.2-p21.1 by deletion mapping of KC12 + chromosome 3 revertants that escaped from cellular senescence and by transfer of subchromosomal fragments of chromosome 3 into RCC23 cells.
Carcinoma, Renal Cell↗
A repressor function for telomerase activity in telomerase-negative immortal cells.
Human telomerase, a ribonucleoprotein that adds TTAGGG repeats onto telomeres and compensates for their shortening, is repressed in most normal human somatic cells. Human somatic cells are considered to have a limited proliferation capacity because of the telomere shortening. Although immortalization of somatic cells is often associated with telomerase reactivation, there are some immortal cells in which telomerase activity is undetectable. In these cells, telomeres may be maintained by an unknown mechanism other than telomerase reactivation. To examine the genetic regulation of telomerase activity, we constructed hybrids between immortal cells with (HepG2) and without (KMST6) telomerase activity. These two cell lines had relatively short and long telomeres, respectively. The hybrid cells continued to proliferate without detectable telomerase activity even after 100 population doublings. Telomerase-positive subpopulations occasionally appeared after serial passages. Southern blot analysis revealed that the hybrids had long terminal restriction fragments similar to that of KMST6, regardless of telomerase activity, and fluorescence in situ hybridization with a telomeric probe showed high-intensity hybridization signals on telomeres, indicating relatively long telomeric repeats. These results suggest that the telomerase-negative immortal cells contain a gene or genes functioning as a telomerase repressor and maintain telomere length by a dominant mechanism other than telomerase reactivation.
Cells, Cultured↗
Novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia.
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Adrenal Hyperplasia, Congenital↗
Urinary levels of chemokines (MCAF/MCP-1, IL-8) reflect distinct disease activities and phases of human IgA nephropathy.
Leukocytes have been implicated to be involved in the pathogenesis of IgA nephropathy (IgAN). To clarify the precise molecular mechanism of recruitment and activation of leukocytes in the subgroups of IgAN, latent, acute, and chronic types, we studied monocyte chemotactic and activating factor (MCAF/MCP-1) and interleukin (IL)-8 in urines and renal expression of these cytokines. Urinary MCAF levels were significantly higher in chronic type, and were correlated with pathological progressive factors such as mesangial proliferation and interstitial cellular infiltration associated with CD68-positive macrophage. On the other hand, urinary IL-8 elevated only in acute type and were correlated with glomerular endocapillary proliferation and the degree of hematuria. In immunohistochemical study, IL-8 was mainly observed in glomeruli, otherwise MCAF in vascular endothelial cells, tubular epithelial cells, and infiltrated mononuclear cells in the interstitial lesions. These observations demonstrated that MCAF and IL-8 were differentially expressed in kidneys with IgAN, and their subtypes, and suggest that chemokines may be involved in the pathogenesis of IgAN at distinct phases or pathological lesions, possibly through the recruitment and activation of a distinct type of leukocyte.
Adolescent↗
Age-related change in cadmium-induced hepatotoxicity in Wistar rats: role of Kupffer cells and neutrophils.
The hepatotoxicity of cadmium was studied in 1-, 2-, and 6-month-old male Wistar rats. Liver damage, indicated by the increase in serum alanine aminotransferase activity 24 h after sc administration of 3 and 6 mg/kg cadmium, was observed only in 6-month-old rats. Dose-dependent increases in the cadmium content of the liver were similar for all three age groups. Basal and induced metallothionein contents were higher in livers of 1-month-old rats than in those of 2- and 6-month-old rats. In contrast, the basal glutathione content of the liver was higher in 6-month-old rats than in 1- and 2-month-old rats, and glutathione content increased slightly in all three age groups after cadmium administration. Thus, the higher susceptibility to cadmium-induced hepatotoxicity in 6-month-old rats seemed not to be explained by differences in cadmium uptake or by the metallothionein and glutathione contents of the liver. Inactivation of Kupffer cells with gadolinium chloride or depletion of neutrophils with cyclophosphamide relieved cadmium hepatotoxicity only in 6-month-old rats. In addition, 6-month-old rats were more susceptible than 2-month-old rats to lipopolysaccharide-induced hepatotoxicity. The results suggest that age-associated changes in Kupffer cell function and infiltration of neutrophils are important determinants of cadmium-induced hepatotoxicity in rats.
Age Factors↗
Effect of glutathione depletion and metallothionein gene expression on arsenic-induced cytotoxicity and c-myc expression in vitro.
Arsenic exposure is clearly linked to human cancer. In rodent cells, arsenic has been reported to induce aberrant gene expression, including activation of the proto-oncogene c-myc. Abnormal or altered expression of such oncogenes can be involved in the acquisition of a malignant phenotype. Although its mechanism of action is unclear, arsenic is known to exert at least some of its toxic effects through interaction with sulfhydryl groups, and the non-protein sulfhydryl glutathione (GSH) appears to play an important role in detoxication of arsenic. Similarly, metallothionein (MT), a metal-binding protein with high sulfhydryl content, often functions in defense against metal-induced or oxidative cellular injury. Therefore, we examined the relationship among GSH, MT gene expression, and arsenic-induced toxicity or c-myc expression in cultured rat myoblast (L6) cells. In initial toxicity studies, arsenic was used in both the trivalent (arsenite) and pentavalent (arsenate) forms. The role of GSH was studied by pretreating cells with L-buthionine sulfoximine (BSO), which induces a marked depletion of GSH. In vitro exposure of L6 cells to BSO (1 to 25 microM) resulted in dose-dependent decreases in GSH. GSH depletion sensitized cells to both arsenite and arsenate. Zinc pretreatment, at levels which highly activated MT expression, had no effect on arsenite-induced cytotoxicity. Arsenite (1 microM) alone modestly increased c-myc expression from 1 to 4 h after treatment (maximum of 2.0-fold over control). After GSH depletion cells responded to arsenite exposure with much larger increases in c-myc transcription (3.2-fold over control). Zinc pretreatment had no reductive effect on arsenite-induced c-myc expression despite markedly activating the MT gene. Thus, it appears that the cellular levels of GSH, but not MT gene expression, play an important role in resistance to arsenic toxicity and aberrant gene activation. Moreover, depletion of GSH enhances arsenic-induced proto-oncogene activation, which might contribute to subsequent transformation.
Animals↗
Comparative effects of repeated administration of cadmium on kidney, spleen, thymus, and bone marrow in 2-, 4-, and 8-month-old male Wistar rats.
Male Wistar rats at 2, 4, and 8 months were given s.c. injections of CdCl2 at doses of 0.5, 1, and 2 mg Cd/kg, 3 days/week, for 4 weeks. Dose-related adverse effects observed at the end of the injections were as follows: decrease in body weight gain, increases in liver, kidney, and spleen weights, decrease in red blood cell counts, and increase in white blood cell counts accountable by an increased percentage of neutrophils in peripheral white blood cells. Essentially, all of these changes were age-related, i.e., the extents of the effects in each age group were in the order of 8- > 4- > 2-month-old rats when compared at the same dose level of Cd. The sensitive indicators of histological changes were increase in hematopoietic cells in bone marrow and fibrous tissue proliferation in the thymus > fibrous tissue proliferation and hyperplasia of lymph follicle in the spleen > renal tubular degeneration. These histological changes became to be marked at lower doses with aging. Dose-dependent increases in total Cd concentrations in the liver, kidney, and spleen were slightly higher with aging, while metallothionein (MT) contents in these organs were induced exactly in the same pattern as Cd concentrations in each organ in various age groups. This study revealed that the adverse effects after repeated administration of Cd on the kidney, spleen, thymus, and bone marrow worsened with increasing animal age and that this phenomenon could not be explained simply by differences in Cd disposition or MT induction in the organs of the different age groups.
Age Factors↗
System B0,(+)-mediated regulation of lysine transport in Caco-2 human intestinal cells.
We investigated whether lysine transport would be subject to adaptive regulation in Caco-2 human intestinal cells. The activity of Lys transport in Caco-2 cells decreased with increasing incubation time with 10 mM Lys. Among the two systems involved in Lys transport, the system b0,+ component was greatly decreased by incubating cells with 10 mM Lys, whereas the system y+ component did not change. These results suggest that system b0,+ mainly contributes to the adaptive regulation of Lys transport in Caco-2 cells.
Amino Acid Transport Systems↗
Diffusion-weighted echo-planar MRI of lacunar infarcts.
We studied 35 patients with lacunar infarcts, using diffusion-weighted echo-planar imaging (DW-EPI) at 1.5 T. The relative apparent diffusion coefficient ratio (ADCR) of each lesion was calculated and lesion conspicuity on DW-EPI was compared to that on images acquired with fast fluid-attenuated inversion recovery and T2-weighted fast spin-echo sequences. Acute small infarcts (within 3 days) were identified with DW-EPI as an area of decreased ADCR (range 0.33-0.87; mean 0.67) and high signal, subacute small infarcts (4-30 days) as a high-signal or isointense areas of decreased or nearly normal ADCR (0.54-0.98; 0.73), and chronic small infarcts (> 30 days) as low- or high-signal areas of nearly normal or increased ADCR (0.97-1.92; 1.32). In three patients, small infarcts of the brain stem in the hyperacute phase (within 6 h) were seen only with DW-EPI. In five patients, fresh small infarcts adjacent to multiple old infarcts could be distinguished only with DW-EPI.
Aged↗
Antitumor activity exhibited by Fas ligand (CD95L) overexpressed on lymphoid cells against Fas+ tumor cells.
Lymph node (LN) cells of Fas-mutant mice lpr/lpr (lpr) and lpr(cg)/lpr(cg) (lpr(cg)) express an increased level of Fas ligand (FasL) (CD95L). We examined the antitumor potential of cell-bound FasL on these LN cells against Fas+ tumor cells. Fas+ F6b and Fas- N1d cells were produced from murine hepatoma MH134 (Fas-) by gene transfection. lpr and lpr(cg) LN cells inhibited growth of F6b but not N1d cells in vitro. Neither gld/gld lpr/lpr (gld/lpr) LN cells, which lack both FasL and Fas, nor wild-type LN cells showed growth-inhibitory activities against F6b and N1d cells. The effector cells and molecule were CD4-CD8- T cells and FasL, respectively. The tumor neutralization test and adoptive transfer demonstrated that lpr and lpr(cg), but not gld/lpr, LN cells retarded the growth of F6b cells. Although anti-Fas antibody and FasL cause severe liver failure, wild-type mice injected with lpr LN cells appeared clinically normal. Adoptive transfer of lpr LN cells to F6b-bearing mice exerted the same antitumor activity in wild-type and gld/lpr recipient mice, indicating the applicability of cell-bound FasL for Fas-mediated target therapy of cancer. These results suggest that antitumor activity was dependent on the Fas-FasL system and that lymphoid cells overexpressing FasL can be powerful antitumor effector cells against Fas+ tumor cells.
Animals↗
Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs.
Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.
Amino Acid Sequence↗
Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma.
We found that lysophospholipase D (LPLD) in rat plasma prefers unsaturated to saturated lysophosphatidylcholines as substrates, generating a biologically active lipid, lysophosphatidic acid, but it does not hydrolyze diacyl-phospholipids. In this study, this LPLD required a metal ion for activity, Co2+ being the most effective, followed in order by Zn2+, Mn2+, and Ni2+. This metal-ion-stimulated LPLD with unique substrate specificity, which has not been described previously, was susceptible to thiol-blocking reagents and serine esterase inhibitors, but not to a histidine-modifying reagent. Consistent with results using thiol-modifying agents, short-chain fatty aldehydes, secondary products of lipid peroxidation, were found to inhibit LPLD. Addition of dibutylhydroxytoluene or butylhydroxyanisole to the plasma increased the activity of this enzyme, probably in a manner independent of its antioxidant activity, since another antioxidant, propyl gallate, was rather inhibitory. These results suggest that rat plasma contains an active LPLD that differs in some properties from other members of the known phospholipase D family detected in animal tissues and body fluids.
1-Acylglycerophosphocholine O-Acyltransferase↗
Primary low-grade, B-cell, mucosa-associated lymphoid tissue lymphoma of the colorectum: clinical and colonoscopic features in six cases.
BACKGROUND: Unlike gastric lymphoma, the endoscopic features of low-grade, mucosa-associated lymphoid tissue (MALT) lymphoma of the colorectum have not been defined. METHODS: We analyzed colonoscopy, endoscopic ultrasonography, and histologic findings in six patients with MALT lymphoma restricted to the colorectum. Histologic confirmation of the proliferation of centrocyte-like cells with lymphoepithelial lesions formed the criteria for the selection of patients. RESULTS: Based on endoscopic findings, the patients were divided into cases of solitary (4 patients) or multifocal (2 patients) involvement. Endoscopic findings in cases of solitary involvement were uniform in that the lesions were either smooth or nodular protrusions, demonstrated by a hypoechoic mass under endoscopic ultrasonography. Multifocal involvement was endoscopically recognized as protrusions, dilated vasculature, or small ulcers. CONCLUSIONS: Protruding lesions are the main pattern of involvement in MALT lymphoma of the colorectum, although variable and multifocal involvement is also possible in this type of lymphoma.
Aged↗
Elongated non-neoplastic colonic polyp: endoscopic features.
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Aged↗
The effect of nitric oxide synthase inhibitor on reperfusion injury of the brain under hypothermic circulatory arrest.
OBJECTIVE: The objective of this study was to investigate the protective effects of nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride, on reperfusion injury of the brain under hypothermic circulatory arrest. METHODS: After cardiopulmonary bypass was established using 12 piglets each weighing about 30 kg, the animals were cooled to a brain temperature of 20 degrees C and circulatory arrest was performed for 90 minutes followed by reperfusion for 120 minutes. The level of nitric oxide within the brain was measured with a needle electrode inserted into the brain. In the treatment group, N(G)-nitro-L-arginine methyl ester hydrochloride was administered with an intravenous injection of 1.5 mg/kg at the onset of the reperfusion followed by a 60-minute continuous venous infusion of 1.5 mg/kg/hr. RESULTS: In the control group, nitric oxide levels within the brain increased not during ischemia but during reperfusion, and the level after 120 minutes of reperfusion increased significantly compared with that of before circulatory arrest. But in the treatment group, N(G)-nitro-L-arginine methyl ester hydrochloride administered at the onset of reperfusion inhibited nitric oxide production during reperfusion. A significant difference was observed between the groups regarding the nitric oxide level after 120 minutes of reperfusion. Regarding cerebral blood flow, excess lactate, and cerebral tissue water content, no significant difference was observed between the groups. However, recovery of somatosensory evoked potential after 120 minutes of reperfusion was detected in all six animals in the treatment group, but none in the control group (p = 0.001). CONCLUSION: These data suggest that N(G)-nitro-L-arginine methyl ester hydrochloride protects the brain against reperfusion injury under hypothermic circulatory arrest.
Animals↗
Oncocytic lipoadenoma of the submandibular gland.
We report a case of oncocytic lipoadenoma of the submandibular gland, previously unrecognized benign tumor of the salivary gland. The patient was a 66-year-old Japanese woman with a left submandibular mass, measuring 11 x 7.5 x 5 cm. Microscopically, the mass was completely surrounded by a thin fibrous connective tissue capsule, and was composed of an intimate admixture of mature fat cells and oncocytes. There have been no English reports of such a neoplasm in the salivary gland with the exception of one recent case report of lipoadenoma of the parotid gland without oncocytic features. We propose the diagnostic term "oncocytic lipoadenoma" for this benign tumor.
Adenoma↗