Improved efficiency of asymmetric hydrolysis of 3-substituted glutaric acid diamides with an engineered amidase.

AIMS: To improve the efficiency of asymmetric hydrolysis of 3-(4-chlorophenyl) glutaric acid diamide (CGD) using a recombinant Comamonas sp. KNK3-7 amidase (CoAM) produced in Escherichia coli. METHODS AND RESULTS: The CoAM gene was cloned, sequenced and found to comprise 1512 bp, encoding a polypeptide of 54 054 Da. CoAM-transformed E. coli were able to perform R-selective hydrolysis of CGD; however, complete conversion of 166·2 mmol l(-1) CGD in 28 h could not be obtained. We attempted to optimize the reactivity of CoAM by mutating single amino acids in the substrate-binding domain. Notably, the methionine-substituted L146M mutant enzyme showed increased reactivity, completing the conversion of 166·2 mmol l(-1) CGD in just 4 h. The Km value for L146M was lower than that of CoAM. CONCLUSIONS: We succeeded in creating the L146M mutant of CoAM with increased substrate affinity and found that this was the best mutant for the hydrolysis of CGD. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the efficiency of hydrolysis of 3-substituted glutaric acid diamides is useful to improve the synthesis of optically active 3-substituted gamma-aminobutyric acid. This is the first report of efficient hydrolysis of CGD using amidase mutant-producing E. coli cells.

MDR1 RNA levels in human renal cell carcinomas: correlation with grade and prediction of reversal of doxorubicin resistance by quinidine in tumor explants.

We examined the distribution of RNA levels expressed by the multidrug-resistance gene (MDR1, also known as PGY1) in 42 renal cell carcinoma (RCC) samples (38 primary and four metastatic lesions). The median MDR1 RNA level for the 38 primary lesions, expressed relative to the level for KB-3-1 cells, was approximately one-half of the level in multidrug-resistant KB-8-5 cells. Elevated MDR1 RNA levels were also observed in three of the four metastatic lesions. The mean MDR1 RNA level was higher in well-differentiated RCCs than in those that were poorly differentiated, suggesting that the increased expression of the MDR1 gene in RCCs originates from the increased expression in renal proximal tubule cells. To clarify the association of the MDR1 protein product P-glycoprotein with natural resistance to doxorubicin (ADR) in RCCs, we evaluated the effects of quinidine on in vitro sensitivity to ADR in 16 RCC samples, using a [3H]thymidine incorporation assay. The enhancing effect of quinidine (7.5 micrograms/mL) on sensitivity to ADR was statistically significant only in the group with high MDR1 RNA levels. Similar enhancement by quinidine of sensitivity to ADR was also observed in the established RCC cell lines in which MDR1 RNA levels were high. These results suggest that P-glycoprotein is active in the natural resistance of RCCs to ADR.

Enhanced hepatocyte growth factor level in human prostate cancer treated with endocrine therapy.

Hepatocyte growth factor (HGF) has been revealed to have various functions such as regeneration, cancer invasion and tumor suppression in normal and cancer cells of different organs. To compare HGF expression in non-malignant prostate tissues and prostate cancers pretreated with or without neoadjuvant endocrine therapy (pretreated PC and non-treated PC), the amounts of HGF protein and mRNA were quantitated by Western blot analysis and reverse transcription-competitive polymerase chain reaction. The biologically active HGF (baHGF), which was converted by an HGF activator protein from secretory type proHGF, showed significantly higher expression in pretreated PC than in non-treated PC. The amounts of baHGF in pretreated PC were similar to those in non-malignant prostate tissues. There was no difference in the expression level of full-length HGF mRNA encoding secretory type HGF among the three groups. No variance of tissue content of short variant HGF mRNA encoding a competitive HGF antagonist among the three groups indicated that short variant HGF protein in prostate cancer seemed not to function similarly to a competitive HGF antagonist in benign prostate tissues. These results suggest that the tissue level of baHGF in non-treated and pretreated PC depends on activation of proHGF supplied from distal organs rather than de novo HGF mRNA synthesis in prostate glands. Increased baHGF in pretreated PC is expected to relate to tumor suppression in vivo.

Expression of c-MET/HGF receptor mRNA and protein in human non-malignant and malignant prostate tissues.

We previously reported the expression of hepatocyte growth factor (HGF), a ligand for c-MET, at the level of mRNA and protein in human prostate tissues. The present study was carried out to evaluate the relationship between c-MET expression and cancer cell proliferation or effect of cancer therapy. The expression both in mRNA and protein levels of c-MET proto-oncogene was determined semi-quantitatively by reverse transcription-competitive polymerase chain reaction (RT-competitive PCR) and Western blot analysis for prostate tissues from 32 Japanese male subjects. In addition, tissue localization of c-MET translation product was examined by immunohistochemistry for corresponding specimens. Although there was significantly higher c-MET protein expression in malignant (prostate cancer treated with/without neoadjuvant endocrine therapy) than in non-malignant prostate tissues (normal prostate and benign prostate hyperplasia; BPH), unexpectedly, c-MET mRNA showed high expression in the non-malignant group. Thus, there was no parallelism between mRNA and protein expressions of c-MET. Endocrine therapy did not alter c-MET mRNA and protein expressions in human prostate cancer. Immunohistochemical localization and expression of c-MET protein was found to be intense in cancer cells and weak in epithelia of normal and hyperplastic prostates. Unconcerted expression of mRNA and protein of c-MET, the reason of which is uninterpretable, is supposed to be one of characteristics of human prostate cancer.

Obturator hernia: the usefulness of computed tomography in diagnosis.

BACKGROUND: Delay in diagnosis and treatment of the rare obturator hernia often leads to high resection and mortality rates. Reviews of the clinical courses and the reliability of computed tomography (CT) in early diagnosis are reported. METHODS: Seventeen patients with obturator hernias were treated between April 1983 and December 1994. Except for one man, all were emaciated women who had undergone an average of 4.7 live deliveries. The mean age was 79.9 years. RESULTS: All patients had small bowel obstruction symptoms. Howship-Romberg sign was present in 11 patients. Twelve cases were correctly diagnosed before operation. After the introduction of CT in the diagnosis of suspected hernia cases, preoperative diagnosis was made on 9 of the 10 patients (90%) and CT was performed on 8 patients with 100% accuracy. CONCLUSIONS: Definite and early diagnosis is possible with the awareness of the clinical courses of this hernia and by performing CT on suspected cases.

Estrogen potentiates N-methyl-D-aspartate receptor subunit R2B mRNA expression during the late prepubertal period in female rats.

To study interactions between estrogen and excitatory amino acid, changes in the mRNA level of the N-methyl-D-aspartate R2B subunit (NR2B) in the hypothalamus and hippocampus were measured following estrogen treatment in prepubertal female rats. Three hours after estrogen injection, the hypothalamic and hippocampal tissues were subjected to a competitive RT-PCR assay. Estrogen significantly increased the mRNA levels of the hypothalamic NR2B in day 30 females, whereas no increase was seen in day 15 females. No such change was detected in the hippocampus. These results suggest that gene expression of hypothalamic NR2B is regulated by estrogen in peripubertal females. Differential potentiation of NR2B mRNA expression by estrogen between early and late prepubertal females suggests the existence of some neural maturational mechanism, which may be correlated with the onset of puberty.

Pharmacological characterization of [3H]-JTH-601, a novel alpha1-adrenoceptor antagonist: binding to recombinant human alpha1-adrenoceptors and human prostates.

Several alpha1-adrenoceptor (AR) selective antagonists are now widely used to improve lower urinary tract symptoms in benign prostatic hyperplasia patients. However, these drugs often result in orthostatic hypotension, because of their poor uroselectivity; the blockade of alpha1-AR not only in prostate but also in vasculature. Here we have investigated uroselectivity of JTH-601, a newly developed antagonist, in radioligand binding experiment using recombinant human alpha1-AR subtypes and human prostate. In saturation experiments, [3H]-JTH-601 showed subtype selectivity: high affinity to alpha1a-AR (pKd; 9.88+/-0.09), lower affinity to alpha1b-AR (pKd; 8.96+/-0.17) and no specific binding at concentrations up to 3000 pM to alpha1d-AR. In competition experiments, JTH-601 and its metabolic compound (JTH-601-G1) also showed alpha1a-AR selectivity, exhibiting approximately 5 times higher affinity for alpha1a-AR than for alpha1b-AR, 10 to 20 times higher affinity than for alpha1d-AR, respectively. [3H]-JTH-601 also bound to human prostate membranes in monophasic manner with high affinity constant (pKd; 9.89+/-0.12, Bmax=123.6+/-16 fmol/mg protein). JTH-601 is a unique alpha1-AR antagonist that shows high affinity and selectivity for human recombinant alpha1a- and human prostate. This new compound is useful for understanding alpha1-AR pharmacology and may have a therapeutic value.

Distribution and metabolism of F6-1,25(OH)2 vitamin D3 and 1,25(OH)2 vitamin D3 in the bones of rats dosed with tritium-labeled compounds.

26,26,26,27,27,27-Hexafluo-1,25(OH)2 vitamin D3, the hexafluorinated analog of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biologic activity in the kidneys and small intestine appears to be related to F6-1,25(OH)2 vitamin D3 metabolism to ST-232, 26,26,26,27,27,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3, a bioactive 23S-hydroxylated form that is resistant to further metabolism. Since F6-1,25(OH)2 vitamin D3 is considered to prevent osteoporotic decrease in bone mass by suppressing bone turnover, we here compared the distribution and metabolism of [1 beta-3H]F6-1,25(OH)2 vitamin D3 and [1 beta-3H]1,25(OH)2 vitamin D3 in bones of rats by autoradiography and radio-HPLC. In the dosed groups, radioactivity was detected locally in the metaphysis, the modeling site in bones. As compared with the [1 beta-3H]1,25(OH)2 vitamin D3 case, [1 beta-3H]F6-1,25(OH)2 vitamin D3 was significantly retained in this site, and moreover, it mainly persisted as unchanged compound and ST-232. These findings indicate that the reason for the higher potency of F6-1,25(OH)2 vitamin D3 than 1,25(OH)2 vitamin D3 in bones are linked with increased distribution and reduced metabolism.

FDG PET for evaluating the change of glucose metabolism in prostate cancer after androgen ablation.

In the clinical study of prostate cancer, the effect of androgen ablation on glucose metabolism in cancer tissue has not been elucidated. The purpose of this study was to investigate the change in glucose utilization due to endocrine therapy for prostate adenocarcinoma. Ten patients with histologically proven prostate cancer were prospectively investigated with (18)F-fluorodeoxyglucose and positron emission tomography (FDG PET) prior to and after the initiation of endocrine therapy. FDG uptake was calculated to measure glucose utilization in cancer tissue. The change in FDG accumulation was compared with changes in serum prostate specific antigen (PSA) level and prostate size. FDG accumulation in the prostate decreased in all patients 1-5 months after the initiation of hormone therapy. The serum PSA level and prostate size measured on computerized tomography (CT) also decreased in these periods. A decrease in FDG accumulation was also demonstrated in metastatic sites. In this study, there appeared to be a decrease in FDG uptake in prostate cancer after endocrine therapy not only in primary prostate cancer lesions but also at metastatic sites, suggesting that the glucose utilization by tumours was suppressed by androgen ablation.

Tissue distribution and pharmacological potential of SM-16896, a novel oestrogen-bisphosphonate hybrid compound.

Postmenopausal osteoporosis is caused mainly by a deficiency of oestrogen with rapid bone loss. To target oestrogen to the bone effectively, we have synthesized and evaluated the effects of a novel hybrid compound of oestrogen and bisphosphonate, SM-16896. The tissue distribution pattern and pharmacological potential are reported. Although the affinity for calf uterine oestrogen receptor was very low (IC50: 73.3 microM; 1/25000 of that of 17beta-oestradiol (2.84 nM)), SM-16896 showed oestrogenic activity. SM-16896 (1 microM) induced a 4.5-fold transcriptional activity in rat osteosarcoma UMR-106 cells compared with vehicle-treated control, when we used the expression vector for human oestrogen receptor and a CAT reporter plasmid containing an oestrogen-responsive element. The distribution of SM-16896 after a subcutaneous administration to 7-week-old female rats was examined by radioluminography using 3H-labelled SM-16896. At 30 min after the administration, significant radioactivity was detected in the bone. At 24 h after administration, a high level of radioactivity was detected in the bone, but in the uterus it was only at a background level. Daily subcutaneous administration of 0.5 mgkg(-1) SM-16896 for 12 weeks (five times per week) to 13-week-old ovariectomized rats suppressed the ovariectomized-induced reduction in bone mineral density. A bone mineral density ratio of 120% was maintained compared with sham-operated rats, whereas a relatively low suppression of uterine weight was observed (about 50% loss compared with sham-operated rats). In the same experiment, the implantation of a 17beta-oestradiol time-release pellet (0.25 mg/pellet/90 days) almost completely suppressed the reduction of both the bone mineral density and uterine tissue weight. It is likely that the effect of SM-16896 on bone was due to its oestrogenic activity, since 1.0 mgkg(-1) SM-18108, the bisphosphonate moiety of this compound, had no effect on bone in 7-week-old ovariectomized rats. The results suggest that SM-16896, a bisphosphonate-conjugated oestrogen, showed a preference profile in the uterus and bone due to its characteristic distribution pattern compared with the natural oestrogen analogue 17beta-oestradiol. Thus, bisphosphonate-conjugated oestrogens have the potential to improve patient compliance in oestrogen therapy by minimizing adverse effects and reducing the frequency of medication.

Metabolism of 4-[1-(2-fluoro-4-biphenylyl)ethyl]-2-methylaminothiazole (SM-8849) in rats.

Metabolism of 4-[1-(2-fluoro-4-biphenylyl)ethyl]-2-methylaminothiazole (SM-8849), a novel immunomodulatory agent, in rats was investigated. By co-chromatography with authentic samples, desmethylated (SM-8800) p-hydroxylated (SL-5512) and desmethylated-p-hydroxylated SM-8849 (SL-5515) were detected in the bile. Thermospray mass spectrometry (TSP-MS) analysis of five metabolites isolated from the bile revealed molecular ions of both conjugates (glucuronides and a sulfate) and their aglycones. Aglycone structures were determined by comparison of their product spectra with those of authentic standards. Further analyses of conjugation sites were carried out by 1H-NMR including differential NOE. As a result, the sulfate of SL-5515 (5515-S), the N-glucuronides of SL-5512 and SM-8849 (5512-NG and 8849-NG, respectively), the glucuronide of SL-5512 (5512-G) and the O-glucuronide of 4-hydroxy-3-methoxy-SM-8849 (CatOMe-OG) were identified. In addition, N-methylthiouras was identified in urine by LC/MS/MS.

[A case of renal leiomyosarcoma].

A case of renal leiomyosarcoma is presented. A 65-year-old man was admitted with asymptomatic gross hematuria. On excretory pyelography, the left kidney was not visualized. Computed tomography showed a left intrarenal mass. The patient underwent left radical nephrectomy. The histological diagnosis was leiomyosarcoma probably originating from the left renal pelvis.

[A clinical study of urolithiasis in Shizuoka City Hospital--stone analysis and 24-hour urine calcium and uric acid levels].

Stone analysis was performed for 216 urinary calculi which were obtained from 205 patients in our hospital from January, 1980 to June, 1984. The results revealed 161 calcium stones, 21 uric acid stones, 19 struvite stones, 2 cystine stones and 13 others. Sixty one of the 205 patients (male 44, female 17) and 19 controls (male 11, female 8) were investigated for 24-hour urine calcium and uric acid. Forty seven of the patients had calcium stones, 7 of the patients had uric acid stones and 7 of the patients had struvite stones. The mean 24-hour urine calcium level was 146.8 +/- 76.5 mg/day for the male controls and 139.1 +/- 69.9 mg/day for the female controls. The mean 24-hour urine uric acid level was 528.1 +/- 132.6 mg/day for the male controls and 362.0 +/- 135.2 mg/day for the female controls. The mean 24-hour urine calcium level for the male calcium-stone group was 214.6 +/- 96.8 mg/day, and it was significantly higher than that for the male controls (p less than 0.05). The 24-hour urine analysis revealed abnormalities in 17 urolithiasis patients (27.9%) including hypercalciuria in 11 patients (18.0%) and hyperuricosuria in 9 patients (14.1%).

[Preoperative management of pheochromocytoma with prazosin: report of three cases].

We used prazosin (alpha-blocker) for preoperative management of three patients with pheochromocytoma. All patients had episodic hypertension with headache and palpitation. The first dose of prazosin caused blood pressure to drop in one of three patients, but no significant hypotension was observed. By the daily dose of 6-8 mg prazosin with or without propranolol, subjective symptoms of the patients were improved and the frequency of the hypertensive attack was decreased Operations were carried out without significant complications, but phentramine and nitroprusside were required because of rapid rise of blood pressure during induction of anesthesia and surgical manipulation.

[Assessment of the response of human bladder cancer cell lines to radiation using colony formation assay and [3H] thymidine incorporation assay].

The effect of radiation on the human bladder cancer cell lines, KK47 and J82, was studied using colony formation assay and [3H] thymidine incorporation assay. The response to radiation was dose-related for both assays, but the curve for the colony formation assay was steeper than that for [3H] thymidine incorporation assay. However, the steepness of the curve for [3H] thymidine incorporation assay increased to be approximately the same as that for the colony formation assay as the incubation period increased. [3H] Thymidine incorporation assay can provide a rapid assessment of radiation sensitivity comparable to that obtained by the colony formation assay to select a radiosensitive bladder cancer patient for definitive or adjunctive radiotherapy.

[A case of renal cell carcinoma with erythrocytosis].

A case of renal cell carcinoma with erythrocytosis is presented. A 51-year-old man was referred to us for evaluation of left renal mass. Laboratory data revealed marked erythrocytosis and elevated serum erythropoietin level. These data were normalized after removal of the left kidney. Pathological diagnosis was adenocarcinoma of clear cell type.

[Evaluation of prostate cancer using FDG-PET].

The clinical usefulness of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) was examined in 54 patients with prostate cancer. FDG accumulation was positive in 38 of 54 the prostates (70%), 3 of 8 the lymph node metastases (38%) and 10 of 16 the bone metastases (63%), which suggested that FDG-PET is not superior to other conventional imaging methods as a tool for tumor detection. On the other hand, a quantitative value for FDG uptake in the prostate, expressed as a standardized uptake value (SUV), significantly correlated to the histological grade, clinical stage and serum PSA of the patients. A decrease of SUV was observed in all the patients who responded to endocrine treatment, and the patients with high pre-treatment SUV were shown to be at the risk for disease progression after initial treatment. The present results indicated that FDG-PET could provide us with useful prognostic information for the patients with prostate cancer by evaluating the malignant potential of the tumor.

[Treatment of localized prostate cancer: radical prostatectomy versus radiation therapy].

Between 1982 and 1990, 57 patients with prostate cancer (clinical stage T2-3) underwent pelvic lymphadenectomy at Fukui Medical School or Toyooka Public Hospital. The patients were subsequently treated either by radical prostatectomy (39 cases) or external radiation therapy (18 cases). The patients were between 56 and 85 years old (Mean 73). The outcome of the 47 patients without lymph node metastasis (prostatectomy 33, radiation 14) was examined. The 5-year disease-specific survival rate was 96% for the patients treated by prostatectomy and 93% for those treated by radiation. The 5-year progression-free survival rate for the prostatectomy group and radiation group was 94% and 41%, respectively (P = 0.004). The outcome of the 10 patients with lymph node metastasis (prostatectomy 6, radiation 4) was not satisfactory because of the high progression rate in the two groups (5-year progression-free survival, 22% in prostatectomy and 25% in radiation group). In the patients with no metastasized lymph nodes, we should choose radical prostatectomy, if our goal is to cure the patients.

[In vitro chemosensitivity test by human tumor colony forming assay].

In vitro chemosensitivity of urological cancers was assessed by a human tumor colony forming assay (HTCA) and a 3H-thymidine incorporation assay. Primary tumor cells from 160 of 253 (63%) urological cancers showed adequate colony growth (greater than 30 colonies per well), and there was a 57% true positive and 100% true negative rate for predicting clinical response of anticancer agents. On the other hand, cells from 37 of 45 (82%) urologic cancers incorporated a sufficient amount of 3H-thymidine (greater than 300 cpm). However, when the positive control drug (chromomycin A3 100 micrograms/ml) was used for the assay quality control, the successful assay rate of the HTCA (38%) was lower than that of the 3H-thymidine incorporation assay (75%), while there was a significant correlation in drug sensitivities between the two assays. Thus, the 3H-thymidine incorporation assay seemed to be more useful than the HTCA for evaluating the chemosensitivity of urologic cancers.

[20 cases of hypercalcemia associated with urogenital malignancies].

Twenty cases (3.3%) of hypercalcemia of more than 11.0 mg/dl associated with urogenital malignancy were observed in 610 inpatients during the past 5 years and 6 months (Jan. 1978-June 1983). Incidences were 10 (16.1%) out of 62 cases of renal cell carcinoma, 6 (1.9%) out of 321 cases of bladder cancer, 3 (6.7%) out of 45 cases of renal pelvic and ureteral cancer, and one (1.1%) out of 95 cases of prostatic cancer. As treatment, surgery (radical nephrectomy) and anti-cancer chemotherapy were effective in 3 cases (2 renal cell carcinomas and one renal pelvic cancer). Conservative therapy with hydration combined with either indomethacin, steroid or eel calcitonin was effective in 11 cases, and s-Ca level was decreased by 3.7 mg/dl on the average. Eighteen patients were in the terminal stage of malignancy when hypercalcemia was observed, and died 5 days to 9 months (mean; 2 months) after the onset of hypercalcemia.