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.

Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride.

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.

Urachal anomalies: ultrasonography and management.

BACKGROUND: Fifty-six children with anomalies of the urachus remnant identified by ultrasound scan have been encountered in the authors' hospital over the last 4 years. METHODS: Twenty of these 56 cases were symptomatic urachal remnants, whereas the urachus remnants were seen incidentally by ultrasound scanning in the other 36 patients. Symptomatic cases were treated with antibiotics or observation. Then, symptomatic cases were divided into 2 groups. One group, surgical group, was treated with surgical resection of the urachal remnant. The other group, observation group, was followed up without its surgical resection. Forty-four patients, 11 cases of symptomatic urachal remnant and 33 asymptomatic cases, were followed up, excluding patients who had surgical treatment and who were lost to follow-up. RESULTS: Thirty patients underwent periodical ultrasonographic examination during follow-up. In 9 cases, including 2 symptomatic cases, urachal remnants have disappeared during the follow-up period spontaneously. No symptom had developed during follow-up from asymptomatic cases. CONCLUSIONS: The patients with asymptomatic urachal remnants do not require follow-up, and urachal remnants, especially those under 1 year of age, do not require surgical resection unless the patient has multiple episodes.

Long-term disposition of a novel lipophilic platinum complex SM-11355 in dog after intrahepatic arterial administration: highly sensitive detection of platinum and radioactivity.

1. The disposition of SM-11355, an anticancer platinum complex for hepatocellular carcinoma, was investigated in dog by measuring platinum (Pt) and radioactivity levels following intrahepatic arterial administration of (14)C-SM-11355 suspended in Lipiodol, an oily lymphographic agent. Plasma and excretion profiles were monitored in six animals, with tissue distribution studied after 1 day, 4 and 13 weeks (n = 2/time point). 2. SM-11355 was released very slowly into the systemic circulation from Lipiodol, resulting in very low levels of Pt compounds in plasma, urine, faeces and organs. Plasma levels of Pt and radioactivity declined with apparent half-lives of 5-7 weeks. Excretion continued even at 3 months after the administration with proportions excreted for Pt and radioactivity up to 30-60% in urine and 8-10% in faeces. 3. The Pt and radioactivity in the liver accounted for 80-100% of the dose at 1 day and for 20-50% at 13 weeks after the administration, predominately as intact SM-11355. The concentrations were highest in the left lobe of the liver, the administration site, but levels in the remainder of the liver were also markedly higher than those in plasma and other tissues. 4. The results strongly support the concept that SM-11355 targets the liver with highly selectivity and sustained release of Pt compounds.

Non-invasive measurement of coronary flow reserve in children with Kawasaki disease.

OBJECTIVE: To investigate whether transthoracic Doppler echocardiography (TTE) can reliably measure the coronary flow reserve in the left anterior descending coronary artery in children with Kawasaki disease. DESIGN: Coronary flow velocity in the distal left anterior descending coronary artery was measured by TTE and was compared with that obtained by intracoronary Doppler guide wire. The ratio of maximum hyperaemia (intravenous administration of adenosine triphosphate, 160 microg/kg/min) to baseline peak (mean) diastolic coronary flow velocity in the distal artery was used as an estimate of coronary flow reserve. SETTING: University hospital. PATIENTS: 10 patients with significant left anterior descending coronary stenosis (> 70% diameter stenosis) (group A) in the proximal or middle portion of the artery and 14 patients (group B) without significant stenosis, all with Kawasaki disease documented by previous coronary angiography. RESULTS: The reduced hyperaemic coronary flow velocity in group A compared with group B resulted in a markedly lower coronary flow reserve, derived from both peak diastolic velocity and mean diastolic velocity by either technique of investigation. Multivariate analysis identified the best predictor of left anterior descending coronary artery stenosis to be a coronary flow reserve of < or = 2.2, derived from mean diastolic flow velocity measured using TTE (sensitivity 90%, specificity 100%, accuracy 96%). A good correlation was found between diastolic velocity derived values for coronary flow reserve measured using both TTE and Doppler guide wire (r = 0.92, p = 0.0001). CONCLUSIONS: Coronary flow reserve in the distal left anterior descending coronary artery can be accurately measured using TTE without any intravascular instrumentation in children with Kawasaki disease.

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.

Pelvic venoablation with ethanol for the treatment of erectile dysfunction due to veno-occlusive dysfunction.

OBJECTIVES: To perform pelvic venoablation with ethanol injection into the deep dorsal vein for the treatment of 10 patients with venogenic erectile dysfunction. This procedure was easily performed without any selective embolization technique. The efficacy and safety of this technique are discussed. METHODS: A total of 10 patients with veno-occlusive dysfunction, severe enough to make vaginal insertion impossible, underwent pelvic venoablation with ethanol. The mean patient age was 67.1 years. Under spinal anesthesia, after the venous leaks were identified by cavernosography, a 20-gauge flexible needle was inserted into the deep dorsal vein. The pelvic venogram obtained with deep dorsal venography was included in what was revealed by the venogram obtained with cavernosography. A mixture of absolute ethanol and contrast medium (4:1) was used as a sclerosing agent. Under fluoroscopic control, the sclerosing agents were injected into the deep dorsal vein through a flexible needle. Success was defined as the ability to achieve vaginal insertion without the aid of any drugs, vasoactive injections, penile prosthesis, or vacuum device. RESULTS: The follow-up ranged from 25 to 37 months (mean 32.3). At the short-term follow-up visit (less than 6 months), 7 patients (70%) reported erections sufficient for vaginal insertion; at the long-term follow-up visit, 5 men (50%) reported sustained, sufficient potency and 5 (50%) reported persistent erectile dysfunction. No serious complications occurred. CONCLUSIONS: Our pelvic venoablation technique using ethanol was effective, minimally invasive, and cost-effective.

Prognostic value of nuclear area index in combination with the World Health Organization grading system for patients with renal cell carcinoma.

OBJECTIVES: To assess the diagnostic accuracy of the World Health Organization (WHO) grading system for renal cell carcinoma (RCC) in terms of nuclear size evaluation. Furthermore, the prognostic usefulness of the nuclear area index (NAI), a new nuclear morphometric parameter expressed as the mean nuclear area (MNA) ratio of cancer to normal tubular cells, is investigated. METHODS: Measurement of the nuclear areas of cancer and normal tubular cells was performed on the histologic slides from the 76 patients with RCC, and the distribution of MNA and NAI was compared among the WHO grades. The clinical usefulness of MNA, NAI, grade, and TNM categories for the prediction of the progression-free and cause-specific survival of the patients was examined. RESULTS: MNA for cancer cells and NAI significantly increased according to the grade. NAI was 1.0 or less in 9 of the 10 patients with G1 tumors and more than 1.0 in 12 of the 13 patients with G3 tumors, whereas the NAI ranged widely from 0.53 to 2.0 in 53 patients with G2 tumors. By multivariate analysis, including grade and TNM categories, NAI and MNA were independent variables for survival in all the patients as well as for cancer progression in localized disease. CONCLUSIONS: WHO G2 RCCs are actually composed of tumors with varying nuclear size, and the prognosis of the patients with G2 tumors varied as well. NAI could provide improved prognostic information for the patients with RCC, especially in G2 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.

Preoperative diagnosis of ileal lipoma by endoscopic ultrasonography probe.

The occurrence of tumor in the small intestine is relatively rare. It has been demonstrated that lipoma of the ileum is a cause of intussusception. We report a 59-year-old man admitted to our hospital for lower abdominal pain. Diagnosis of intussusception was made by abdominal x-ray and ultrasonography. Enema contrast studies revealed ileocolic intussusception. Colonoscopy revealed a tumor with an submucosal tumor (SMT)-like head and coil-spring appearance in the ascending colon. Endoscopic ultrasonography (EUS) revealed a hyperechoic submucosal lesion with features compatible with lipoma. Subsequently, this was confirmed histopathologically after resection. To our knowledge, this is the first report of preoperative diagnosis of ileal lipoma by EUS.

Local thrombus formation in the site of intravenous injection of chlorpromazine: possible colloid-osmotic lysis of the local endothelial cells.

Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artificial membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakage. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenously dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.

Analysis of the mechanism of discrepant nuclear morphometric results comparing preoperative biopsy and prostatectomy specimens.

OBJECTIVES: To explore the mechanism for the differing nuclear morphometric results between needle biopsy and surgical specimens of the prostate. METHODS: In experiment 1, a comparison of mean nuclear area (MNA), volume-weighted mean nuclear volume (MNV), and form factor (FF) for prostatic epithelial cells was performed between preoperative needle biopsy and prostatectomy specimens from 5 patients with benign prostatic hyperplasia (BPH). In experiment 2, a scheduled, sequential ex vivo needle sampling from the enucleated prostates (at 0, 2, 6, and 24 hours after surgical resection) was also performed for 7 patients with BPH. The prostatectomy specimens were left unfixed for 2 hours until the second needle sampling was done. Nuclear morphometric parameters were measured on the needle-sampled as well as on the prostatectomy specimens. RESULTS: MNA, MNV, and FF of BPH cells measured on preoperative biopsy specimens were smaller than those of surgical specimens in all 5 of the cases. The results of nuclear morphometry on the materials obtained by ex vivo needle sampling of prostates before and during fixation revealed that the MNA, MNV, and FF for BPH cells of 0-hour specimens were significantly smaller than those for needle samples at 2, 6, and 24 hours after surgical resection as well as those for prostatectomy specimens. CONCLUSIONS: The present study provided further evidence that the ischemic damage caused by delayed fixation could result in a substantial change of the nuclear morphology of prostate cells. An immediate start, as well as a rapid completion, of the fixation procedure seems critical for an accurate nuclear morphometry of prostatectomy specimens.

Prognostic value of nuclear morphometry on needle biopsy from patients with prostate cancer: is volume-weighted mean nuclear volume superior to other morphometric parameters?

OBJECTIVES: To compare the prognostic value of stereologically estimated volume-weighted mean nuclear volume (MNV) with other nuclear morphometric parameters using pretreatment needle-biopsy specimens of prostate cancer. METHODS: The MNV, mean nuclear area, form factor, and coefficients of variation for nuclear area (VNA) and form factor were measured on pretreatment needle biopsy specimens from 66 patients with prostate cancer (clinical Stage B, n = 9; Stage C, n = 14; and Stage D, n = 43), all of whom underwent androgen deprivation therapy. The prognostic value of those morphometric parameters, as well as Gleason score and clinical stage, was examined in terms of cause-specific patient survival using univariate and multivariate analysis (Cox proportional hazard model). RESULTS: Univariate analysis of the nuclear morphometric parameters revealed that MNV, mean nuclear area, VNA, coefficient of variation for form factor, and clinical stage were significant prognostic factors for cause-specific patient survival. However, when the patients with Stage D disease were selectively analyzed for survival, only the VNA was a significant prognostic parameter. Furthermore, the multivariate analysis, including the morphometric parameters, clinical stage, and Gleason score revealed that only VNA and clinical stage were independent variables. CONCLUSIONS: The present comparative study could not demonstrate any prognostic superiority of MNV over other nuclear morphometric parameters in patients with prostate cancer.

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.

[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.

Ratio of gamma-seminoprotein to prostate-specific antigen for the detection of prostate cancer: its discrimination power could be influenced by the assay methods of PSA and/or gamma-seminoprotein.

BACKGROUND: The ratio of gamma-seminoprotein (gamma-Sm) and prostate-specific antigen (PSA) has been regarded as being superior over PSA alone as a discriminator between prostate cancer and benign prostatic diseases. In previous studies, PSA and gamma-Sm were measured by the Eiken kit and the old-version or revised Chugai kit, respectively. We compared the power of gamma-Sm ratio with that of PSA alone when using Markit-M PSA assay and the revised Chugai gamma-Sm assay. METHODS: Fifty-three patients with prostate cancer having no metastasis and 116 with benign prostatic diseases were enrolled in this study. Prostate-specific antigen was measured by Markit-M kit and gamma-Sm was measured by the revised Chugai kit. The discrimination power of gamma-Sm ratio and PSA alone was evaluated with receiver operating characteristic (ROC) curves. Comparisons between prostate cancer and benign diseases were performed with Mann Whitney U-test and Fisher's exact test. RESULTS: The optimal cut-off value was set at 3.1 ng/mL for PSA and 0.935 for gamma-Sm ratio. Sensitivity, specificity and positive predictive value of PSA alone were 81.1, 81.0 and 66.2%, respectively, while those of gamma-Sm ratio were 73.6, 90.5 and 78.0%, respectively. There was no statistical significance in each value between PSA and gamma-Sm ratio. Areas under the ROC curves of PSA and gamma-Sm ratio were 0.881 and 0.866, respectively (P>0.05). CONCLUSION: Contrary to the previous reports, gamma-Sm ratio and PSA were not different in the discrimination between prostate cancer and benign prostatic diseases, which suggested that the discrimination power of gamma-Sm ratio, and presumably that of the free PSA to total PSA ratio as well, could be considerably influenced by the assay kits for serum PSA and/or gamma-Sm (free PSA) used. Therefore, the clinical significance of gamma-Sm ratio should be evaluated for each PSA assay kit.

Comparison of nuclear morphometric results between needle biopsy and surgical specimens from patients with prostate cancer.

OBJECTIVES: To compare nuclear morphometric values and Gleason scores between biopsy and radical prostatectomy specimens in patients with clinically localized prostate cancer. METHODS: The mean nuclear area (MNA), volume-weighted mean nuclear volume (MNV), and form factor (FF) were measured on the 18-gauge needle biopsy and radical prostatectomy specimens of 25 patients with clinically localized prostate cancer. The correlation between biopsy and surgical specimens was investigated for MNA, MNV, FF, and Gleason scores. RESULTS: The average values for the MNA, MNV, and FF of the biopsy specimens (36.2 microm2, 366 microm3, and 0.86, respectively) were significantly smaller than those of the prostatectomy specimens (51.4 microm2, 646 microm3, and 0.91) by Student's paired t test. The Pearson correlation of morphometric parameters between the biopsy and surgical specimens was significant only for FF. A comparison of histologic grading between the biopsy and surgical specimens revealed identical Gleason scores in 32% and identical grades (on a three-grade system) in 68% of all the cases. CONCLUSIONS: Discrepant nuclear morphometric results were observed between biopsy and surgical specimens of localized prostate cancer. The reason for such differing results is unclear but may be caused by artifacts associated with tissue sampling and processing. It is recommended that data obtained by biopsy should be considered separately from that obtained from surgical specimens.

Immunohistochemical expression of p53 and bcl-2 proteins is not associated with sarcomatoid change in renal cell carcinoma.

An immunohistochemical study was conducted to examine the expression of p53 and bcl-2 proteins in RCC (renal cell carcinoma) with sarcomatoid change in order to determine whether abnormalities in those proteins are associated with an enhanced malignant potential of RCC. Paraffin-embedded tissues from 11 patients with RCC, in which sarcomatoid change was prominent, were stained using anti-p53, bcl-2 and Ki-67 antibodies. Immunoreactivities for these antibodies were compared between the sarcomatoid components and corresponding basic histologic (clear or papillary) components in individual cases. Measurement of the mean nuclear areas of each component was also performed using an image analyzer system. There was no substantial increase in immunoreactivity for p53 or bcl-2 proteins in sarcomatoid components as compared with basic components. In contrast, the percentage of Ki-67-positive cells and the mean nuclear area were significantly larger in sarcomatous components than in basic components. The expression of p53 and bcl-2 proteins was not likely to play a major role in the sarcomatoid change of RCC.

In vivo and in vitro pharmacokinetics and metabolism studies of 26,26,26,27,27,27-F6-1,25(OH)2 vitamin D3 (Falecalcitriol) in rat: induction of vitamin D3-24-hydroxylase (CYP24) responsible for 23S-hydroxylation in target tissues and the drop in serum levels.

1. 26,26,26,27,27,27-F6,-1,25(OH)2 vitamin D3, Falecalcitriol, the hexafluorinated analogue 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 biological activity appears related to F6-1,25(OH)2 vitamin D3 metabolism to F6-1,23S,25(OH)3 vitamin D3, a bioactive 23S-hydroxylated form which is resistant to further metabolism. 2. In the present in vivo studies, the repeated oral administration of [3H]F6-1,25(OH)2 vitamin D3 to rat resulted in a significant reduction of the radioactivity and the F6-1,25(OH)2 vitamin D3 concentrations in serum, especially at the 2 h maximum point after each dosing. Additionally, F6-1,23S,25(OH)3 vitamin D3 in the serum and small intestine was increased by the prior administration of F6-1,25(OH)2 vitamin D3. 3. Further in vitro investigation showed [3H]F6-1,25(OH)2 vitamin D3 to be metabolized to F6-1,23S,25(OH)3 vitamin D3 by kidney and small intestine homogenates of rat, the reaction being increased by the prior administration of F6-1,25(OH)2 vitamin D3. Moreover, this latter treatment was associated with a marked increase of CYP24 mRNA in the small intestine within 4 h after dosing. 4. The results indicate that in vivo metabolism of F6-1,25(OH)2 vitamin D3 to F6-1,23S,25(OH)3 vitamin D3 is catalysed by CYP24, the enzyme being induced by prior substrate exposure.