Molecular genetic analysis of the Prader-Willi syndrome by using fluorescent multiplex PCR of the dinucleotide repeats on chromosome 15q11-q13.
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Biomedical subjects
Publications and source records attributed to T Yuki.
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1. Recombinant human serum albumin (rHSA) (1 g/kg) significantly decreased the weight of ascites in rats with puromycin aminonucleoside-induced nephropathy. 2. Furosemide (1-30 mg/kg) did not significantly reduce the weight of ascites in this model. 3. A combination of rHSA (1 g/kg) with furosemide (5 mg/kg) significantly decreased the weight of ascites in this model compared with furosemide alone. 4. In consideration of these results, rHSA can be a substitute for human serum albumin products prepared from human plasma in therapy for ascites or edema in furosemide-resistant nephrotic syndrome.
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Using 2 in vitro systems in which normal human salivary gland cells can be immortalized by transfection with SV40 DNA (NS-SV-DC), and in which a non-metastasizing human salivary gland adenocarcinoma cell clone (HSGc) can be converted to biologically aggressive and metastasizing cancer cells, we analyzed the relationship between the expression of basic fibroblast growth factor (bFGF) and malignant phenotype in human salivary gland cell clones. By enzyme immunoassay, intracellular contents of bFGF were equally detected in all the cell clones, while bFGF contained in serum-free conditioned medium of each cell clone was different: HSGc and metastasizing cell clones, respectively, released 2- and 10-fold more bFGF into media than did NS-SV-DC. Immunoblot analysis also revealed a similar result as that detected by enzyme immunoassay. By Northern blot analysis, bFGF mRNA expression was consistently detected in all the cell clones examined. We then quantitated bFGF contents in sera of tumor-bearing mice by enzyme immunoassay. Although sera from nude mice bearing an HSGc tumor showed significantly higher amounts of bFGF than those of control mice, bFGF concentrations in sera gradually decreased after removal of the tumor. For metastasizing cell clones, serum concentrations of bFGF were 2.2- to 2.6-fold higher than those of HSGc. In addition, bFGF amounts in sera after removal of the tumors were significantly higher in mice bearing metastasizing cell clone tumors compared with those of HSGc tumor-bearing mice. Our results, therefore, indicate that increase in bFGF release occurs along with the progression of human salivary gland cancer cells and that serum bFGF may be useful for evaluation of tumor presence.
OBJECTIVE: The purpose of the study is to clarify the manifestation of the ciliary body after retinal photocoagulation for diabetic retinopathy. DESIGN: Ultrasound biomicroscopy (UBM) was applied to obtain the tomographic features of the anterior ocular segment. PARTICIPANTS: Twenty-one eyes of 17 consecutive patients with diabetic retinopathy were included in this study. INTERVENTION: These eyes were treated by panretinal photocoagulation divided in four sessions. In the first session, the nasal quadrant of the fundus was treated with argon or dye (590 nm) laser totaling 208 to 331 spots per eye. MAIN OUTCOME MEASURES: The eyes were examined by UBM before, immediately after, 3, and 7 days after the first session of photocoagulation. RESULTS: Three days after photocoagulation, ciliochoroidal detachment was observed in 19 eyes (90%). The detachment was present along the whole circumference. The ciliary detachment was most prominent in the pars plana in 19 eyes and extended to the pars plicata in 9 eyes. It spontaneously had disappeared 7 days after photocoagulation. CONCLUSIONS: Ciliochoroidal detachment was a very frequent feature after sector photocoagulation in diabetic eyes. Although this complication was asymptomatic and transient so far, this potential hazard merits due attention in the planning of panretinal photocoagulation in diabetic eyes.
Mesenteric lymph node (MLN) and spleen lymphocytes of Sprague-Dawley rats were cultured with 1 mM unsaturated fatty acids (UFAs) with or without 100 microM alpha-tocopherol (Toc), and the immunoglobulin content and thiobarbituric acid (TBA) value of the culture media were measured to clarify the relationship between lipid peroxidation and the IgE level in the culture medium. The increase in the IgE content and TBA value induced by UFAs was alleviated in the presence of Toc in both lymphocytes, and was correlated well with their oxidation rates in most cases. Gamma-linolenic acid enhanced the IgE level much more than would be expected from its oxidation rate in both lymphocytes, and linoleic acid showed similarly high activity only in splenocytes. These results suggest that lipid peroxidation is partly responsible for the enhancement of IgE level induced by UFAs.
To confirm the relationship between the layer structure of the common bile duct by a microscanner (MS) and its histological features, we performed a study using the pinning method with needles and catgut. The locations of 67 needles inserted at random depths in 29 slices of the resected common bile duct from 18 patients were confirmed both by a MS and a microscope. The wall of the common bile duct was delineated as a two- (42 points) or three-layer structure (25 points); "low and high" or "high, low, and high echoic layers" from the mucosal side. A fibrotic layer (ss 1) was often (56/67 points, 16/18 patients) seen in the subserosa (ss) containing nerves and vessels larger than 100 mu in diameter. Among the 51 needle echoes demonstrated in the inner hypoechoic layer, 2 were located in the mucosa (m), 12 in the fm, 19 in the af, 17 in the ss 1, and 1 in the fatty layer of the ss (ss 2). Five of the 6 needles in the outer hyperechoic layer were in the ss 2, and 1 was in the pancreatic parenchyma (pa). Four of the 8 needles at the border between the inner hypoechoic layer and the outer hyperechoic layer histologically corresponded to fm, af, ss 1, and pa, respectively, and the other four corresponded to ss 2. From these results, we conclude that the inner hypoechoic layer contains not only m, fm, and af, but also ss 1. Therefore, this should be kept in mind for the preoperative assessment of the depth of bile duct carcinoma by MS.
In this study, we have examined the expression of integrin subunits in normal and malignant human salivary gland cell clones as well as its regulation by transforming growth factor-beta 1 (TGF-beta 1). By the analysis using immunofluorescence staining, an SV40 immortalized normal human salivary gland duct cell clone (NS-SVDC) with no tumorigenic ability by s.c. implantation into nude mice was identified to express the integrin beta 1, alpha 2, alpha 3 and alpha 6 subunits on the cell surface, while the expression of these subunits, except for beta 1 subunit, was reduced or completely diminished in a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic but not metastatic potential in nude mice and metastatic cell clones derived after in vitro exposure of HSGc to N-methyl-N-nitrosourea. In addition, immunoblot analysis also exhibited the same results as those obtained with immunofluorescence staining. The alpha 1 subunit was not demonstrable in any of the cell clones by both techniques. TGF-beta 1 augmented the expression of the beta 1 subunit in NS-SV-DC, while HSGc and metastatic cell clones demonstrated no changes in the expression of the beta 1 subunit in response to TGF-beta 1. These findings, therefore, suggest that there is an inverse relationship between the malignancy and the expression mode of integrin subunits, especially alpha 2 subunit, in human salivary gland cell clones with varying degrees of malignant potential, and that TGF-beta 1 is a positive regulatory factor in the expression of the beta 1 subunit in normal but not malignant cell clones.
To understand the molecular mechanisms whereby normal human salivary gland cells become malignant and escape growth-inhibitory control by transforming growth factor (TGF)-betaI, we examined the effect of TGF-betaI on the proliferation and expression of TGF-beta receptors in cells and the expression of TGF-beta type II receptor (TbetaR-II) mRNA. An SV40-immortalized normal human salivary gland duct cell clone (NS-SV-DC) with no tumorigenic ability, originally obtained via s.c. implantation into nude mice, was partially resistant to the growth-inhibitory effect of TGF-betaI, while a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic, but not metastatic, potential in nude mice was more resistant to the growth-suppressive effect of TGF-betaI than NS-SV-DC. Metastatic cell clones derived from carcinogen-treated HSGc were completely refractory to the anti-proliferative effect of TGF-betaI. Affinity cross-linking revealed that NS-SV-DC possesses the types I, II (TbetaR-II) and III receptors. However, HSGc and metastatic cell clones lacked expression of detectable levels of the TbetaR-II protein. Moreover, we evaluated TbetaR-II mRNA expression in these cell clones by Northern blot analysis and observed that, although NS-SV-DC expressed a large amount of TbetaR-II mRNA, a small amount of TbetaR-II mRNA was detectable in HSGc. In contrast, no significant bands were detected in metastatic cell clones. Our results, therefore, suggest that one of the possible mechanisms of escape from autocrine or paracrine growth inhibition by TGF-betaI during human salivary gland carcinogenesis involves reduced expression or lack of TbetaR-II.
To understand the specific cell type responsible for the synthesis of basement membrane components of the salivary gland, the effects of transforming growth factor (TGF)-beta 1 on morphological change, cellular proliferation and collagen synthesis were examined in these immortalized duct and myoepithelial cell clones, and the expression forms of their TGF-beta receptors analysed. When TGF-beta 1 was added to the cell clones in vitro, it induced a morphological alteration, with flattening in myoepithelial but not in duct cells. Although the growth of Mv1Lu mink lung epithelial cells was almost completely inhibited by TGF-beta 1, the duct and myoepithelial cells were partially resistant to such inhibition. By immunoblot analysis of immunoprecipitates, p53 was found bound to the simian virus-40 large T antigen, suggesting a functional loss of p53 in regulation of cell-cycle arrest. In the cloned myoepithelial cells but not the duct cells, TGF-beta 1 stimulated the production of type IV collagen. To attempt to understand the distinct responsiveness of cell clones to TGF-beta 1, the expression forms of TGF-beta receptors were examined by affinity cross-linking. Although the intensities of the cross-linked bands of the TGF-beta type II and type III receptors, particularly the type II, were weaker in the duct than the myoepithelial cell clones, the expression of the type II receptor mRNA was consistently detected in both clones. Accordingly, the reduction of TGF-beta 1 binding may have occurred at the post-transcriptional level. These findings imply that the cloned myoepithelial cells but not the cloned duct cells produce type IV collagen in response to TGF-beta 1 through the receptor-mediated signal transduction pathway, which is presumably disrupted in the cloned duct cells.
Proteolytic enzyme activity has been shown to be important for cyst formation. In this study, we constructed a cyst-like structure in vivo and analyzed molecular mechanisms involved in the development of the lesion. When SV40-immortalized duct cells of normal human salivary gland (NS-SV-DC) were treated with TGF-beta 1 at a concentration of 1 ng/ml or 5 ng/ml followed by co-inoculation with Matrigel into the backs of nude mice, they formed large cysts containing fluid when 5 ng/ml of TGF-beta 1 was used. Analysis of the fluid demonstrated high MMP activity. Immunohistochemical staining exhibited strong reactivity with anti-MMP-2 antibody in TGF-beta 1 (5 ng/ml)-treated NS-SV-DC. Northern blot analysis indicated that the expression of TGF-beta 1 and MMP-2 mRNAs in cells was greatly enhanced by treatment with 5 ng/ml TGF-beta 1. These findings suggest that the in vivo cyst formation by TGF-beta 1-treated cells is associated with continuous induction of MMP-2 activity.
The detectability of superficial-type carcinoma of the gallbladder by ultrasonography (US) and endoscopic ultrasonography (EUS) were investigated. Eight patients with such carcinoma of the gallbladder were reviewed. They had undergone both US and EUS previous to surgery. The tumor was detected in 3 patients by US, 2 patients with IIa + IIb-type carcinoma and 1 patient with IIa-type carcinoma. Those lesions were visualized as localized thickening of the gallbladder wall or as broad-based tumor. In 2 patients, lesions were not initially detected by US, but were shown by US after the examination by EUS. The tumor was detected by EUS also in 1 case of IIb + IIa-type carcinoma besides those 3 patients mentioned above. All four of these lesions were visualized as broad-based tumor by EUS. It was difficult to detect pure IIb-type or small IIa-type carcinoma even by EUS. In patients with concomitant acute cholecystitis or gallbladder stone, it was difficult to evaluate the abnormal findings of the gallbladder wall.
To compare intraluminal ultrasonographic (ILUS) findings with histological findings of rectal carcinoid tumors, 35 patients with rectal carcinoid tumors were reviewed. The results were as follows: 1) The rectal wall was visualized as a seven- or nine-layer structure by means of ILUS in 81% of the patients. 2) The possibility that the thin hyperechoic third layer above the tumor on ILUS corresponds to the muscularis mucosae and fibrointerstitium above the tumor histologically. 3) In cases with relatively high internal echoes, the amount of fibrointerstitium exceeded that of tumor cells histologically. 4) In cases with nonuniform internal echo patterns, tumor cells were separated by thick fibrointerstitium forming nodular nests.
We examined the content of type IV collagenases and plasminogen activators (PAs) in luminal fluid of four extravasation mucoceles by means of gelatin or casein zymography. Immunohistochemical staining for type IV collagenases and PAs was performed to identify the possible source of these enzymes. The luminal fluid examined by gelatin zymography showed a high level of type IV collagenases compared with saliva from Wharton's duct. PAs were detected in only one of the cases by casein zymography, and none was detected in the saliva. By immunohistochemical staining, type IV collagenases and PAs were detected in duct and myoepithelial cells of the adjacent salivary gland and in macrophages and fibroblasts of the cyst wall. The results suggest that proteolytic enzymes are involved in the pathogenesis of mucoceles.
We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.
Between April 1992 and May 1993, 13 patients (age, 66-79) with localized prostate cancer underwent laparoscopic pelvic lymphadenectomy at Asahi General Hospital. Clinical stage comprised A2 for 3 patients, B1 for 1 and C for 9. Lymphadenectomy covered inner half of external iliac nodes and obturator nodes, from pubic bone to proximal end of umbilical ligament. Operating time ranged from 70 minutes to 133 minutes with median of 102 minutes. The number of total lymph nodes dissected ranged from 3 to 17 nodes with median of 7. Lymph nodal involvement was detected in one patient. Two patients needed laparotomy due to bleeding; from abdominal wall caused at insertion of trocar in one, and oozing of blood for 10 hours after procedure in the other. Other serious complications were not observed. In conclusion, laparoscopic pelvic lymphadenectomy was a good staging procedure for localized prostate cancer.
The ftsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene. It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain. The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala. Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively. Possible implications of these observations are discussed.
A 46-year-old male without any subjective complaints was diagnosed by radiography as having one polypoid lesion at the distal esophagus. Endoscopy indicated a semipedunculated polyp about 1 cm in size, the specimens of which included atypical cells with a high N/C ratio in the submucosa. Squamous cell carcinoma of the esophagus was suspected. Cytological studies of PAS, CEA and keratin staining to atypical cells were all negative, with Lugol solution staining of the tumor. After polypectomy the lesion was diagnosed as a squamous cell papilloma of the esophagus; and the atypical cells were determined to be immature fibroblasts and/or endothelium.