Effects of ABCB1 3435C>T genotype on serum levels of cortisol and aldosterone in women with normal menstrual cycles.

ABCB1, also known as MDR1/P-glycoprotein, can transport cortisol and aldosterone. We examined the effects of ABCB1 polymorphisms on serum levels of cortisol and aldosterone among different phases of the normal menstrual cycle in 51 non-pregnant healthy Japanese female volunteers (22 +/- 1 years old). The menstrual cycle was divided into three phases: premenstrual phase (14 days preceding the onset of menstruation, N = 22; menstrual phase, N = 11, and postmenstrual phase, N = 18). ABCB1 -129T>C, 1236C>T, 2677G>A/T, and 3435C>T genotypes were determined. Serum levels of cortisol, aldosterone, estradiol, progesterone, and testosterone were measured. The serum levels of estradiol in the pre- and post-menstrual phases and of progesterone in the premenstrual phase were significantly increased when compared to their serum levels in the menstrual phase (P < 0.005). In the postmenstrual phase, the mean serum cortisol level in subjects with the 3435CT and 3435TT genotype was 7.6 +/- 3.4 microg/dL (mean +/- SD, N = 7), which was significantly lower than in women with the 3435CC genotype (9.9 +/- 1.8 microg/dL, N = 11) (P = 0.037). The opposite effect was observed in the serum aldosterone level during the postmenstrual phase (97.2 +/- 23.4 and 141.2 +/- 48.5 pg/mL for 3435CC and 3435CT + 3435TT, respectively; P = 0.041). These findings suggest that ABCB1 3435C>T genotype can influence serum levels of cortisol and aldosterone during the postmenstrual phase of the normal menstrual cycle.

MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas.

BACKGROUND: Many patients with invasive ductal carcinoma of the pancreas (IDC) have a poor outcome. MUC4 expression has been implicated as a marker for diagnosis and progression of IDC, but there are no studies of the relation between MUC4 expression and patient prognosis in IDC. AIMS: To investigate the prognostic significance of MUC4 expression in IDC. METHODS: The expression profiles of MUC4, ErbB2, p27, and MUC1 were investigated in IDC tissues from 135 patients by means of immunohistochemistry. RESULTS: MUC4 was expressed in 43 of the 135 patients with IDC (31.9%). The survival of 21 patients with high MUC4 expression (>20% of neoplastic cells stained) was significantly worse than that of the 114 patients with low MUC4 expression (<20% of neoplastic cells stained) (p = 0.0043). Univariate analysis showed that high MUC4 expression (p = 0.0061), large primary tumour status (>T2) (p = 0.0436), distant metastasis (p = 0.0383), lymphatic invasion (p = 0.0243), and surgical margins (p = 0.0333) were significant risk factors affecting the outcome of patients with IDC. Backward stepwise multivariate analysis showed that MUC4 expression (p = 0.0121), lymph node metastasis (p = 0.0245), and lymphatic invasion (p = 0.0239) were significant independent risk factors. ErbB2, p27, and MUC1 were not independent risk factors. CONCLUSIONS: This study shows that MUC4 expression in IDC is a new independent factor for poor prognosis and predicts the outcome of patients with IDC.

Characterization and identification of genes essential for dimethyl sulfide utilization in Pseudomonas putida strain DS1.

Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO(2)) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon 5 (Tn 5) mutagenesis had a Tn 5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441.

Comamonas testosteroni metabolizes testosterone as the sole carbon source via a meta-cleavage reaction. A meta-cleavage enzyme gene, tesB, was cloned from C. testosteroni TA441. The deduced N-terminal amino acid sequence of tesB matched that of the purified meta-cleavage enzyme which is induced in TA441 during growth on testosterone as the sole carbon source. The tesB-disrupted mutant did not show growth on testosterone, suggesting that tesB is necessary for TA441 to grow on testosterone. Downstream from tesB, three putative ORFs which encode products also necessary for growth of TA441 on testosterone were identified. The usual substrate of TesB is probably 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione. Although this compound was not identified in the gene disrupted mutants, accumulation of upstream metabolites of testosterone degradation, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione, was shown by TLC analysis.

MDR1 genotype-related pharmacokinetics of digoxin after single oral administration in healthy Japanese subjects.

PURPOSE: To evaluate the MDR1 genotype frequency in the Japanese population and to study the relationship between the MDR1 genotype and the pharmacokinetics of digoxin after single oral administration in healthy subjects. METHODS: The MDR1 genotype at exon 26 was determined in 114 healthy volunteers by polymerase chain reaction-restriction fragment length polymorphism. The serum concentration-time profile of digoxin was examined after single oral administration at a dose of 0.25 mg. RESULTS: It was found that 35.1 % (40/114) of subjects were homozygous for the wild-type allele (C/C). 52.6% (60/114) were compound heterozygotes with a mutant T-allele (C3435T) (C/T), and 12.3% (14/114) were homozygous for the mutant allele (T/T). There was no effect of gender or age on the distribution. The serum concentration of digoxin after a single oral administration increased rapidly, attaining a steady state in all subjects; however, it was lower in the subjects harboring the T-allele. AUC0-4 h values (+/-SD) were 4.11 +/- 0.57, 3.20 +/- 0.49. and 3.27 +/- 0.58 ng h/ml, respectively, with a significant difference between C/C and C/T or T/T. CONCLUSIONS: The serum concentration of digoxin after single oral administration was lower in the subjects harboring a mutant allele (C3435T) at exon 26 of the MDR1 gene.

Molecular and pharmacokinetic properties of 222 commercially available oral drugs in humans.

This study was performed to determine the exclusion criteria that differentiate poorly absorbed drugs from good drug candidates, and to accelerate drug development by exclusion of unnecessary assessment. The molecular and pharmacokinetic properties of 222 commercially available oral drugs were tabulated and their correlations were analyzed. The exclusion criteria obtained were 1) a molecular weight of more than 500, and 2) a ClogP value of more than 5. Exceptions to molecular weight criteria were compounds with a sugar moiety, high atomic weight, and large cyclic structure. It was also suggested that being a substrate for MDRI (P-glycoprotein) does not always result in poor bioavailability, and that drug development by chemical modification of a seed or lead compound with quantitative structure activity relationship analysis can result in lower bioavailability, higher bound fraction and lower urinary excretion, which would hamper later development processes and might result in considerable drug-drug interaction. The criteria should be adjusted according to the pharmacological profiles of the agents in question and depending on the estimated profit, but ignoring these criteria may result in a significant waste of time and money during drug development.

Time-dependent expression of intestinal phenotype in signet ring cell carcinomas of the human stomach.

Signet ring cell carcinomas of the stomach are thought to arise from the proper gastric mucosa without intestinal metaplasia. It was recently reported that intestinal phenotypes appear along with tumor progression. In this study, we performed several experiments to reconsider the significance of this intestinalization in the growth of signet ring cell carcinoma. We applied mucin histochemistry with monoclonal antibodies MUC2 (Ccp58) and M1 (45M1), and paradoxical concanavalin A staining for class III mucin [PCS(III)] reaction to 29 intramucosal and 25 deeply invasive carcinomas of this type and correlated the phenotypic expression with the size of the mucosal spread and the depth of tumor invasion. It was found that the larger the size of the mucosal lesion, the more frequently the intestinal phenotypes were demonstrated. There was no significant increase in the expression of the intestinal phenotype as the tumor invaded the deeper part of the mucosa or as the intestinal metaplasia increased in the background mucosa. The intestinal expression appeared to be suppressed in the earlier phase of deep invasion. In the mucosal part of the tumor, the intestinal phenotype was often expressed regionally and incompletely, coexisting with gastric phenotypes at the cellular and the tissue levels. These findings indicate that the expression of the intestinal phenotype is a time-dependent and unstable phenomenon probably based on the accumulation of genetic changes and plays a neutral role in progression of signet ring cell carcinomas.

Expression of MUC1 and MUC2 mucins and relationship with cell proliferative activity in human colorectal neoplasia.

Our previous studies on MUC1 and MUC2 mucin expression in various human neoplasms have found that MUC1 expression is related with a poor outcome whereas MUC2 expression is related with a favorable outcome. In the present study, we examined the alteration of MUC1 and MUC2 antigens on malignant transformation of colorectal mucosa, and also its relationship with cell proliferative activity (Ki-67 labeling index) of neoplastic epithelial cells in 200 adenomas and 58 carcinomas. In the 200 adenomas, we analyzed a total of 400 adenomatous lesions (mild dysplasia, 200 lesions; moderate dysplasia, 153 lesions; severe dysplasia, 47 lesions). MUC1 was expressed in carcinomas (24%) and adenomas with severe dysplasia (4%), but was not expressed in adenomas with mild or moderate dysplasia. MUC2 was expressed in a significantly greater number of adenomas with mild dysplasia (72%) than in adenomas with moderate dysplasia (45%) or severe dysplasia (47%), as well as in the carcinomas (38%; P < 0.0001). The Ki-67 labeling index was significantly lower in the MUC2-positive cases than in the MUC2-negative cases in the adenomas with mild dysplasia (13.6 vs 24.2%; P < 0.0001) or moderate dysplasia (25.7 vs 44.4%; P < 0.0001), and in the carcinomas (32.5 vs 48.4%; P < 0.05). In conclusion, the data from our study indicate that increased MUC1 expression and reduced MUC2 expression may be related to malignant transformation of colorectal neoplasia. We also demonstrated that decreased MUC2 expression, which is correlated with increased Ki-67 labeling, may play an important role in the progression of colorectal adenomatous change.

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S.

A CH3OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH3OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants ( > ca. 700 mg glucose equivalent/l) were > 11-22 times higher than those produced by the wild-type strain (

Gene expression of gastric type mucin (MUC5AC) in pancreatic tumors: its relationship with the biological behavior of the tumor.

Previously it has been found that the MUC2 gene for intestinal type secretory mucin is highly expressed in intraductal papillary mucinous tumors (IPMT), which are characterized by non-invasive growth and a favorable outcome. In contrast, MUC2 mRNA is rarely expressed in invasive ductal carcinomas (IDC), which have poor outcomes. The gastric type secretory mucin, MUC5AC, is strongly expressed in the surface mucous cells of gastric mucosa. As both MUC2 and MUC5AC mucins share the characteristics of forming highly viscous gels, it is expected that not only MUC2 mucin expression but also MUC5AC mucin expression may be associated with a favorable prognosis in patients with pancreatic tumors. MUC5AC mucin gene expression was examined in 24 cases of IPMT and 38 cases of IDC by in situ hybridization using a digoxigenin-labeled oligonucleotide. The results were compared with MUC2 mucin gene expression. Neither MUC5AC mRNA nor MUC2 mRNA was detected in normal pancreatic tissues. MUC5AC mRNA was expressed in 20 of 24 cases of IPMT (83%) and in five of 38 cases of IDC (13%). In contrast, MUC2 mRNA was expressed in 14 of 24 cases of IPMT (58%) and in none of the 38 cases of IDC (0%). The expression rates of MUC5AC mRNA and MUC2 mRNA in IPMT were significantly higher than those in IDC (P< 0.001, respectively). Intraductal papillary mucinous tumors are characterized by three histological types: (i) villous dark cell type; (ii) papillary clear cell type; and (iii) compact cell type. The villous dark cell type generally expressed both MUC5AC+ and MUC2+ genes. Alternatively, the papillary clear cell type and the compact cell type usually showed MUC5AC+ and MUC2- expression. Patients with MUC5AC mRNA expression had a significantly better survival prognosis than those with no MUC5AC mRNA expression (P< 0.005). In conclusion, MUC5AC gene expression occurs in a majority of IPMT cases, even in those with no MUC2 production. MUC5AC expression can be

Polypeptide Requirement of Multicomponent Monooxygenase DsoABCDEF for Dimethyl Sulfide Oxidizing Activity.

In our previous study, the multicomponent monooxygenase DsoABCDEF in Acinetobacter sp. strain 20B was cloned based on its ability to oxidize dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO) in E. coli cells, which had high sequence similarity with phenol hydroxylase MopKLMNOP in Acinetobacter calcoaceticus NCIB8250, DmpKLMNOP in Pseudomonas sp. CF600 and some other multicomponent monooxygenases. In this study, DsoB, C, D, E, and F were found to be needed for DMS-oxidizing activity in polypeptide requirement experiments, while DsoA was not necessary for it. It was also found that complementation of the deletion mutants lacking DsoC or F with the corresponding Dmp polypeptides supported the DMS-oxidizing activity, while complementation of the deletion mutants lacking any of the oxygenase subunits (DsoB, D, or E) with the corresponding Dmp polypeptides reduced or nullified the activity.

Cloning and characterization of genes encoding an enzyme which oxidizes dimethyl sulfide in Acinetobacter sp. strain 20B.

Acinetobacter sp. strain 20B was isolated based on the ability to utilize dimethyl sulfide as the sole sulfur source. Since strain 20B oxidized indole as well as dimethyl sulfide, indigo production by recombinant Escherichia coli clones carrying Acinetobacter DNA was used as a selection for cloning genes encoding dimethyl sulfide oxidation genes. The gene encoding an indole-oxidizing enzyme was also found to oxidize dimethyl sulfide. The dimethyl sulfide-oxidizing enzyme genes consisted of six open reading flames designated dsoABCDEF. The deduced amino acid sequences of dsoABCDEF were homologous with those of the multicomponent phenol hydroxylases. DsoABCDEF oxidized dimethyl sulfide to dimethyl sulfoxide, and dimethyl sulfoxide to dimethyl sulfone.

[Successful chronic daily administration of oral etoposide for a case of adult T cell leukemia].

A 54-year-old woman with leucocytosis and skin lesion was hospitalized and diagnosed as chronic type adult T cell leukemia (ATL) in August 1989. Since her ATL cell count and LDH level increased after hospitalization, oral administration of etoposide was started at a dose of 100 mg/day for seven days. The oral administration of etoposide induced another chronic state of ATL. After 10 months without medication, she was readmitted because of an acute ATL crisis. After daily administration of etoposide at a dose of 50 mg/day, the white blood cell count and serum LDH level decreased to the normal range, and abnormal lymphocytes of peripheral blood disappeared. The low-dose daily administration of etoposide at a dose of 25 approximately 50 mg/day could be maintained over six months. No severe side effects except for alopecia and mild myelosuppression were noted during the treatment. Chronic daily administration of oral etoposide is one candidate for the treatment of ATL in an outpatient clinic.

Percutaneous absorption of elcatonin and hypocalcemic effect in rat.

The percutaneous absorption of elcatonin (EC), a hypocalcemic peptide, was investigated. A transdermal dosage form of EC was produced using a gel base, absorption enhancer and protease inhibitor, and applied to rats for 24 h. The combination of bile salt such as taurocholate and glycocholate, and n-octyl-beta-D-glucoside or n-octyl-beta-D-thioglucoside (OTG) exerted the potent enhancing effect on the absorption of EC, and a potent hypocalcemic effect was shown for 24 h or longer. The least level of plasma calcium was obtained 6 h or longer after application, suggesting the relatively rapid absorption of EC. The apparent bioavailability of EC in system 5 was 4.6%, this value being noteworthy in the percutaneous absorption of peptides. When the enhancing effect of taurocholate and OTG was separately measured, both agents acted additively on the absorption of EC. An EC ointment maintained the hypocalcemin effect after storage for 15 d at 40 degrees C. The transdermal dosage form has the potential to be an efficient drug delivery system for Paget's disease and osteoporosis.

Inhibition of paraquat accumulation in rabbit kidney cortex slices by ascorbic acid.

Effects of ascorbic acid and Fe2+ on the accumulation of paraquat into rabbit kidney cortical slices were studied. Ascorbic acid showed dose-dependent inhibition of paraquat accumulation. Fe2+ had a more powerful stimulatory effect on the lipid peroxidation of cortical slices than ascorbic acid, but it did not reduce paraquat accumulation. These results suggest that ascorbic acid is able to inhibit the accumulation of paraquat independent of its peroxidative action, and that ascorbic acid may serve as an antidote for the toxicity of paraquat.