Molecular genetic analysis of 30 families with Joubert syndrome.

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the 'molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer.

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.

Mutation analysis of transforming growth factor beta type II receptor, Smad2, Smad3 and Smad4 in esophageal squamous cell carcinoma.

Mutations of the transforming growth factor beta type II receptor (TGFbetaRII) gene and Smad family genes have been observed in several human cancers. However, there has been no report on mutation analysis of the entire coding regions of these genes in esophageal cancer, and the role of these genes in the development of esophageal cancer remains unknown. We performed polymerase chain reaction-single strand conformation polymorphism analysis of TGFbetaRII, Smad2, Smad3 and Smad4 genes and microsatellite assay in 20 esophageal squamous cell carcinomas (ESCC). We detected polymorphisms at exon 2 of Smad3 and intron 6 of Smad4. No mutation was found in TGFbetaRII, Smad2, Smad3 and Smad4 genes. These results suggest that mutation of TGFbetaRII, Smad2, Smad3 and Smad4 genes is a rare event in ESCC.

Mutation of the transforming growth factor-beta type II receptor gene is a rare event in human sporadic gastric carcinomas.

Mutations of the transforming growth factor-beta type II receptor (TGF-beta RII) gene have been detected in several human cancers. However, mutation analysis of coding sequences of TGF-beta RII in gastric carcinomas has not yet been fully elucidated. We performed PCR-SSCP analysis and direct DNA sequencing of the entire coding region of TGF- RII in 38 human sporadic gastric cancers and 8 gastric cancer cell lines. Mutations of the TGF-beta RII were detected in two tumors and three cell lines. Two tumors had one base deletion in the polyadenine tract in exon 3, the cystein-rich extracellular domain. Three cell lines had a silent mutation in the kinase domain located in exon 4. Polymorphisms were detected in introns 2 and 3. An a/g polymorphism was observed at the seventh base in intron 2 and an a/t polymorphism was observed at the fourth to last base in intron 3. There were no mutations in exons 1, 2, 5, 6 and 7. These results indicate that the polyadenine tract in the TGF-beta RII is a mutational hot spot in human gastric cancer. However, these results also suggest that mutations of the gene are rare events in human sporadic gastric cancer.

Molecular analysis of the h-warts/LATS1 gene in human breast cancer.

Loss of heterozygosity (LOH) on chromosome 6q is often observed in breast cancer, suggesting the existence of a putative tumor suppressor. Recently, a human homolog of the Drosophila warts tumor suppressor gene, h-warts/LATS1, was identified and mapped at chromosome 6q24-25.1. Mutation analysis of the h-warts/LATS1 was performed using 25 breast cancer tissues by RT-PCR SSCP analysis. Although LOH of the h-warts/LATS1 was found in one patient, no mutations were found. Two polymorphisms were found, but neither of them caused amino acid substitutions. Further investigations are necessary to elucidate the role of the h-warts/LATS1 gene in the carcinogenesis of breast cancer.

Mutation analysis of the Smad3 gene in human ovarian cancers.

The Smad3 gene is a member of the Smad family, vertebrate homologues of Drosophila Mad, and its gene product is a cytoplasmic element in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad3 gene mutations in 36 human ovarian cancers, and found that 15 cases (41. 7%) had a polymorphism at codon 103. Because this mutation was not accompanied by amino acid replacement, the present results show that the mutations in the Smad3 gene are unlikely to be involved in human ovarian cancers.

Effect of brefeldin A and lysosomotropic reagents on intracellular trafficking of epidermal growth factor and transferrin in Madin-Darby canine kidney epithelial cells.

To regulate intracellular sorting of epidermal growth factor (EGF) or transferrin (Tf), the effect of brefeldin A (BFA) or lysosomotropic reagents was investigated. To examine the effect of them on the net transcellular transport of 125I-EGF or 125I-Tf, their transcytosis was investigated in the presence or absence of reagents. For the investigation of their fate after internalization, radiolabeled ligands were internalized at 37 degreesC, followed by extensive washing and subsequent incubation at 37 degreesC in the ligand-free medium (pulse-chase study). BFA enhanced transcytosis of 125I-Tf, but had no effect on 125I-EGF. Kinetic analysis in the pulse-chase study showed that BFA does not affect cell-surface binding or intracellular sorting of EGF, while it only increases the transcytosis rate constant of Tf. From the lysosomotropic reagents study, both ammonium chloride and monensin suppressed transcytosis and recycling as well as the degradation of EGF, while both chloroquine and bafilomycin A selectively suppressed the degradation process with only a minimal effect on transcytosis, resulting in an increase in the amount transcytosed. It is concluded the that enhancement effect of BFA on transcytosis depends upon the type of receptor targeted. Lysosomotropic reagents can be divided into two types as far as the specificity of the effect on the net amount of EGF transcytosed in Madin-Darby canine kidney (MDCK) cells is concerned.

Inhibitory effects of combined administration of uft and krestin on intraperitoneal metastases in mice.

In this study we investigated the effect of combined administration of UFT and Krestin (PSK) on the growth of intraperitoneal metastasis and temporal changes in the immune-endocrine system under this immunochemotherapy. At 7 days, Plasma ACTH levels of the PSK group were significantly lower than that of the control group (p<0.01). At 14 days, the tumor weight in the combined and the PSK alone group was significantly decreased, compared to the control group (p<0.05). Serum IAP levels of the UFT group were significantly lower than that of the control group (p<0.05). Serum cortisol levels of the combined group were lower than that of the other groups.

Influence of surgical insults for colorectal cancers on neuroendocrine and immune parameters.

In surgery for colorectal cancer, a tendency of immune suppression was noted as seen in surgery for other types of gastro-intestinal cancer. This tendency was also found in the biological response, and it was related to the operative procedures and insults for rectal cancer. The degree of immunosuppression was milder in patients who underwent procedures that preserve the natural sphincter than in those who underwent colostomy. It is important for surgeons to avoid postoperative immunosuppression in order to prevent recurrence and improve prognosis. Given similar degrees of operative insult and stage of cancer, an operative procedure which causes as little immunosuppression as possible should be selected.

Genetic variations of the midkine (MK) gene in human sporadic colorectal and gastric cancers.

Midkine (MK), a retinoic acid responsible protein, is regulated during development and may play an important role in tumorigenesis. A search for genetic variations of the MK gene, located on chromosome 11q11.2 in humans, has not yet been conducted in cancers. To examine the entire coding region, as well as 4 regions of the promoter covering all functional motifs, 8 sets of intron-based and promoter region primers were designed. Using these primers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of genomic DNA samples from 60 sporadic colorectal and 37 sporadic gastric cancer patients was carried out. This analysis, followed by DNA sequencing, revealed a heterozygous g/t polymorphism at the 62nd base on intron 3 in five colorectal tumors (8.3%) and one gastric tumor (2.7%). In the promoter region, a heterozygous CTT deletion, creating a (CTTTT)2 repeat, in one colorectal cancer sample (1.67%) and a heterozygous 2-bp deletion in the G7 tract in another colorectal cancer patient were detected. A/G and A/A alleles were also detected at nt. -1741 in 36 (97.3%) and one (2.7%) gastric cancer samples, respectively. The A/G alleles were observed in all colorectal cancer patients (100%). All variations observed in the promoter region showed polymorphism. These results suggest that in sporadic colorectal and gastric cancers some gene alterations are present in the MK promoter region, but alterations in the coding region are rare.

Efficacy of immunochemotherapy with Ftorafur and Krestin in rats.

The effectiveness of combined administration of Ftorafur (FT) and Krestin (PSK) on experimentally-induced liver cancer has not been established. This study was undertaken to elucidate the effect of combined administration of these drugs on tumor growth and temporal changes in the immuno-endocrine system under this immunochemotherapy. Male inbred WKA/H strain rats were used. The drugs used were FT and PSK, each dissolved in water and fed orally. The drugs were administered separately but concomitantly in standardized cycles to the tumor-bearing animals. KDH-8 ascitic liver cancer cells were subcutaneously transplanted into WKA rats. The tumor growth inhibition rate of FT and PSK was then determined. Twenty-one days after subcutaneous transplantation, tumor growth in the combined administration transplantation, tumor growth in the combined administration group was significantly inhibited, compared to the control group (p < 0.001). At fourteen days, plasma ACTH levels of the FT + PSK combined group were significantly lower than those of the control group (p < 0.001).

Immunosuppressive acidic protein (IAP) level in serum and peritoneal washings, and its implication in determining multidisciplinary treatments.

In this study we examined the efficacy of the measurement of IAP (serum ancl peritoneal washings) during the treatment of colorectal cancers, and determined the possible benefits of using the IAP measurements from both serum and peritoneal washings. One hundred and six patients (peritoneal washings: 58, peritoneal fluid: 67) were investigated. Serum IAP levels were significantly higher in patients who underwent complicated surgical procedures which involved greater time and blood loss compared to those patients who underwent less involved surgical procedures with little loss of blood and time. In peritoneal washings, up to 66. 7% of cases with peritoneal dissemination showed elevated levels of IAP in the peritoneal cavity, while 37% of cases with no dissemination at surgery macroscopically had positive IAP levels (8 mg/g protein). The results suggest the value of IAP in determining the need for combined immunochemotherapy for patients in an immunosuppressive environment.

Oral absorption of cephalosporins is quantitatively predicted from in vitro uptake into intestinal brush border membrane vesicles.

In order to establish a method to predict oral absorption of drugs, which are absorbed by the oligopeptide transporter (PepT1), fraction absorbed (F) of cephalosporin antibiotics was predicted from in vitro uptake into rat intestinal brush border membrane vesicles (BBMV). Using in vitro uptake data, F values of cephalosporins in humans were predicted using the equation derived from the complete radial mixing (CRM) model, which was proposed by Amidon et al. (Amidon et al., J. Pharm. Sci. 69 (1980) 1369). In the present study, uptake into BBMV was measured at 25 and 4 degrees C in the presence of an H+ -gradient, and the uptake clearance (CLuptake) was calculated. Clearance for the uptake mediated by PepT1 (DeltaCLuptake) was then calculated as CLuptake at 25 degrees C minus that at 4 degrees C. When DeltaCLuptake and F values were analyzed according to the present equation, fairly good correlation between DeltaCLuptake and F was observed. It was further demonstrated that the present method may be able to quantitatively predict F values of cephalosporins by using several cephalosporins as standards.

Inhibitory effects of combined administration of 5-FU and Krestin on liver cancer KDH-8 in WKA/H rats.

The effectiveness of the combined administration of 5-fluorouracil (5-FU) and Krestin (PSK) on experimentally induced liver cancer has not been established. This study was undertaken to elucidate the effect of the combined administration of these drugs on tumor growth and metastasis. Male inbred WKA/H strain rats were used. The drugs used were 5-FU and PSK, each dissolved in water and fed orally. The drugs were administered separately or concurrently in standardized cycles to the tumor-bearing animals. KDH-8 ascitic liver cancer cells were subcutaneously transplanted into WKA rats. The tumor growth inhibition rates of 5-FU and PSK were then determined. Eighteen days after subcutaneous transplantation, tumor growth in the combined administration group was significantly inhibited, compared to the control group and the single treatment groups (p < .05). In addition, a liver metastatic model was prepared by transplanting KDH-8 cells into the spleen. Then the metastatic inhibitory effects of 5-FU and PSK were analyzed. At 14 days, the mean number of liver metastatic nodules was approximately 63 in the control group. However, the combined-medicated group showed a much lower number of nodules (40), indicating that metastasis was significantly inhibited (p < .05).

Invasive pulmonary aspergillosis in a puerperant with drug-induced agranulocytosis.

Invasive pulmonary aspergillosis (IPA) is an acute infection of Aspergillus species to the lungs. It generally occurs in immunocompromised hosts, especially with neutropenia. We report a 30-year-old puerperant, who developed IPA from agranulocytosis. She had been treated for threatened labor with ritodrine and cefepime, one of which induced agranulocytosis. After vaginal delivery of twins, pneumonia emerged in the right lower lobe. She was diagnosed to have IPA according to the halo sign on computed tomography (CT) and positive circulating antibody against Aspergillus, and was treated successfully with oral itraconazole followed by surgical resection. It is important to note that IPA might arise in otherwise immunocompetent hosts when neutropenia is long-standing.

[Fundamental study on T-1982 (cefbuperazone) in obstetrics and gynecology].

Following results were obtained from intravenous administration of T-1982 (cefbuperazone) 1 g by measuring its concentrations in uterine arterial serum, cubital venous serum, oviduct, ovary and several sites in uterine tissue. Endometrium showed the highest concentration among various uterine tissues by any administration (bolus injection, dripping infusion for 1 or 2 hours). Transfer concentrations about 1 hour after the end of 1 hour drip infusion proved to be almost the same as 2 hours drip infusion. In the field of obstetrics and gynecology, it was considered that T-1982 has good efficacy in infections especially caused by E. coli, Klebsiella and Proteus.

[Absorption and excretion of cefotaxime and its levels in uterine arterial blood, female internal genital organ tissue and pelvic cavity fluid (author's transl)].

Following results were obtained from intravenous single administration of cefotaxime 2 g by measuring its levels in uterine arterial blood, elbow venous blood, ovary, oviduct, several sites in uterine tissue, and in the fluid excreted in the dead space formed by radical hysterectomy. (1) Uterine artery and elbow vein blood levels revealed marked increase immediately after administration followed by gradual reduction at slow rate. (2) Peak levels in female internal genital organ tissue were achieved 12 approximately 19 minutes after cefotaxime administration, and ranged between 5 to 8 mcg/g independently from measurement site. In cervix uteri, portio vaginalis high concentration of 4.9 approximately 5.7 mcg/g was achieved within ten minutes after drug administration. (3) In the fluid excreted in the dead space formed by radical hysterectomy cefotaxime levels were maintained around 15 mcg/ml level throughout 5 approximately 6 hours following its administration, and gradually decreased at slow rate later.

Measurement and compartmental modeling of the effect of CYP3A5 gene variation on systemic and intrarenal tacrolimus disposition.

We evaluated the hypothesis that cytochrome P450 3A5 (CYP3A5) expression can affect intrarenal tacrolimus accumulation. Tacrolimus was administered orally to 24 healthy volunteers who were selected on the basis of their CYP3A5 genotype. As compared with CYP3A5 nonexpressors, expressors had a 1.6-fold higher oral tacrolimus clearance and 2.0- to 2.7-fold higher metabolite/parent area under the curve (AUC) ratios for 31-desmethyl tacrolimus (31-DMT), 12-hydroxy tacrolimus, and 13-desmethyl tacrolimus (13-DMT). In addition, the apparent urinary tacrolimus clearance was 36% lower in CYP3A5 expressors as compared with nonexpressors. To explore the mechanism behind this observation, we developed a semiphysiological model of renal tacrolimus disposition and predicted that tacrolimus exposure in the renal epithelium of CYP3A5 expressors is 53% of that for CYP3A5 nonexpressors, when normalized to blood AUC. These data suggest that, at steady state, intrarenal accumulation of tacrolimus and its primary metabolites will depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to interpatient differences in the risk of tacrolimus-induced nephrotoxicity.

Fucosyltransferase of the peritoneum contributed to the adhesion of cancer cells to the mesothelium.

Adhesion molecules associating with peritoneal dissemination were investigated using human gastric (MKN45 and MKN74) and colon (KM12C and KM12SM) cancer cells and the mouse peritoneum. Adhesion of cancer cells to the peritoneum was determined by a recently reported novel ex vivo method. MKN45 cells established from poorly differentiated adenocarcinoma with less glycosylated sugar chains on their cell surface showed higher adhesion activities to the peritoneum ex vivo and produced large amount of metastases in the abdominal cavity of nude mice, whereas MKN74 cells from differentiated adenocarcinoma with more glycosylated sugar chains showed slightly low adhesion activity. KM12SM cells with highly metastatic potential to liver showed fairly low adhesion activity to the peritoneum compared with KM12C cells. The mouse peritoneum was found to contain alpha 1 --> 2, alpha 1 --> 3, and alpha 1 --> 4 fucosyltransferases, and adhesion of cancer cells was observed to the cellulose ester membrane, on which partially purified alpha-fucosyltransferases from mouse peritoneum were immobilized. The adhesion of cancer cells to fucosyltransferase-immobilized membrane was specifically inhibited by the addition of oligosaccharides and glycoproteins, which could serve as substrates for alpha-fucosyltransferases. These results indicate the contribution of alpha-fucosyltransferases to the adhesion of disseminated cancer cells to the peritoneum and support the possibility of antiadhesion therapy of peritoneal dissemination by treatment with substrates for alpha-fucosyltransferases.

Kinetic analysis of transcytosis of epidermal growth factor in Madin-Darby canine kidney epithelial cells.

PURPOSE: The aim of this study is to clarify the intracellular fate and a rate limiting step in transcytosis of epidermal growth factor (EGF) in Madin-Darby Canine Kidney (MDCK) epithelial cells. METHODS: The kinetics of transcytosis of 125I-EGF was investigated. To examine the fate of EGF molecules bound to its receptor on the cell surface. 125I-EGF was prebound to the basal surface at 4 degrees C, followed by extensive washing and subsequent incubation at 37 degrees C in EGF-free medium. RESULTS: Saturable transport of 125I-EGF through the cell monolayer could only be observed from the basal to apical side. Most (approximately 90%) of the EGF molecules bound to the surface receptor are internalized with a half-life of 1-3 min, followed by intracellular degradation with a half-life of 20-50 min. The exocytosis of internalized EGF into the apical medium is much slower with a half-life of 130-250 min. Even when 125I-EGF was incubated with MDCK cells at 37 degrees C and washed with acid to remove cell-surface 125I-EGF, intact 125I-EGF appeared in the basal medium with a half life of 160-170 min. CONCLUSIONS: The exocytosis of internalized EGF into the apical medium is a rate limiting step in EGF transcytosis. At least a small amount of internalized EGF is recycled.