Availability of immunocytochemistry using cocktail antibody targeting p63/cytokeratin14 for the differential diagnosis of fibroadenoma and ductal carcinoma in situ in fine needle aspiration cytology of the breast.

OBJECTIVE: The differential diagnosis of fibroadenoma (FA) and ductal carcinoma in situ (DCIS) has been problematic in fine needle aspiration biopsy (FNAC) because it has been difficult to differentiate between the "large epithelial clusters" associated with FA and those associated with DCIS. The purpose of this study was to prospectively validate the usefulness of immunocytochemical staining using cocktail antibody targeting p63/CK14 in the differential diagnosis of FA and DCIS. MATERIALS AND METHODS: Twenty patients diagnosed as having an uncertain malignant potential (indeterminate) for breast cancer on the basis of a FNAC finding were selected randomly: ten patients with FA and ten with DCIS. The cover glass on a specimen stained with the Papanicolaou stain on a glass slide was peeled off, and the specimen was restained by immunocytochemical staining of cocktail antibody targeting p63 and CK14. RESULTS: Six of the twenty patients were CK14-immunopositive: FA, 6; DCIS, 0. The remaining patients were CK14-immunonegative: FA, 4; DCIS, 10. The number of CK14-immunopositive DCIS patients was significantly different from that of FA patients (P=.0054). Eight out of the twenty patients were p63-immunopositive: FA, 8; DCIS, 0. The remaining patients were p63-immunonegative: FA, 2; DCIS, 10. The number of p63-immunopositive DCIS patients was significantly different from that of FA patients (P=.0004). CONCLUSIONS: Immunocytochemical staining using cocktail antibody targeting p63/CK14 was useful for the differential diagnosis of FA and DCIS in FNAC of the breast.

Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi.

Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, post-embryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.

Preoperative diagnosis of thyroid papillary and anaplastic carcinomas by real-time quantitative reverse transcription-polymerase chain reaction of oncofetal fibronectin messenger RNA.

The restricted expression of oncofetal fibronectin (onfFN) mRNA in thyroid papillary and anaplastic carcinomas was recently reported. In this study, we measured the copy number of onfFN mRNA in RNAs extracted from fine needle aspiration biopsies by real-time quantitative reverse transcription-PCR using thyroglobulin mRNA as an internal control. By measuring the onfFN:thyroglobulin mRNA ratio, preoperative aspirates from 31 papillary carcinomas and an anaplastic carcinoma can be distinguished from those from 5 adenomatous goiters, 5 follicular adenomas, and 4 follicular carcinomas. Thus, quantification of onfFN by real-time quantitative reverse transcription-PCR may be useful for the preoperative diagnosis of papillary and anaplastic carcinomas.

In vitro ubiquitination of cyclin D1 by ROC1-CUL1 and ROC1-CUL3.

Overexpression of cyclin D1 has been implicated in a variety of tumors, such as breast cancers, gastrointestinal cancers and lymphomas. Both gene amplification and protein degradation mediated by ubiquitin (Ub)-dependent proteolysis regulate the abundance of cyclin D1. Here we report that ROC1 interacted with all three D type cyclins in vivo but did not bind to other cyclins tested. The ROC1-CUL1 and ROC1-CUL3, but not ROC1-CUL2, -CUL3 and -CUL4, immunocomplexes promoted polyubiquitination of bacterially purified cyclin D1 in vitro. RING finger mutations of ROC1 eliminated the Ub ligase activity toward cyclin D1. In all cases the ubiquitination of cyclin D1 was accompanied by autoubiquitination of the cullins. The results suggest the involvement of ROC1-cullin ligases in cyclin D1 ubiquitination and a potential mechanism whereby the cullin subunit is ubiquitinated itself while ubiquitinating a substrate.

Insulin resistance in Werner's syndrome.

Insulin resistance in Werner's syndrome (WS) was studied using the glucose clamp technique, and compared with physiologically aged and young subjects. Fasting immuno-reactive insulin (IRI) was increased in patients with Werner's syndrome compared with aged and young subjects. Metabolic clearance rate (MCR) of glucose was decreased in the aged and WS. A rightward shift of the dose-response curves of insulin and MCR of glucose was observed in the aged and WS with a more pronounced shift in the latter. MCR of insulin was also decreased in WS. [125I]insulin binding to erythrocytes was similar in the three groups. These results suggest that insulin resistance associated with WS is due to a post-binding defect manifested by a rightward shift of the dose-response curve of insulin-induced glucose disposal and a decrease in insulin clearance rate.

Evidence that portal vein decompression improves survival of canine quarter orthotopic liver transplantation.

The minimum graft volume still remains unclear in reduced-size liver transplantation (RLT). This study reports the improved survival of canine RLT using a quarter graft with the aid of a portahepatic vein shunt (PHVS). In beagles, the donor liver was reduced to the right lateral and caudate lobes (quarter graft) with or without provision of PHVS, and transplanted orthotopically in the recipient. The PHVS was established by an end-to-end anastomosis of the portal vein branch and the hepatic vein in the resected left lateral lobe. Liver chemistries including arterial blood ketone body ratio (AKBR) were serially measured during and after surgery. All seven animals with PHVS survived more than 3 days (mean +/- SD; 5.3 +/- 1.7 days), whereas all six without PHVS died within 3 days (1.8 +/- 0.8 days, P < 0.01). Portal vein pressures immediately after recirculation in animals with and without PHVS were 8.5 +/- 1.2 mmHg and 16.9 +/- 3.1 mmHg, respectively (P < 0.01). Regardless of the presence or absence of PHVS, AKBR dropped to a level lower than 0.7 during the anhepatic period and returned promptly to above 1.0 as early as 30 min after recirculation. Thereafter, the AKBR values in animals with PHVS remained higher than 1.0, whereas those in animals without PHVS showed a progressive decrease, showing a statistically significant difference between the two groups after 12 hr (P < 0.05). Graft function, as assessed by AKBR, was well correlated with survival and other liver chemistries. These results indicate that, in an extreme RLT, portal hypertension is a risk factor predisposing to graft failure, most likely by increasing microvascular injury after recirculation.

Regulation of glucose transporter synthesis in cultured human skin fibroblasts.

The present study was designed to see the effects of glucose on glucose transporter expression and glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in approximately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.

Chymotrypsin inhibition induced by side chain-side chain intramolecular CH/pi interaction in D-Thr-L-Phe benzylamide.

The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (K1 = 4.5 x 10(-6) M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH2)n-C6H5 (n = 0-4) were tested, inhibitory potency peaked at n = 1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH3)-C6H5, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (about 5-fold, K1 = 9.1 x 10(-7) M). In conformational analysis by 400 mHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield shifts of D-Thr-beta OH (shifts in ppm, 0.09-0.17), D-Thr-beta CH (0.23-0.32), and D-Thr-gamma CH3 (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-gamma CH3 and Phe-phenyl groups were evidenced by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/pi interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1 site, while the hydrophobic core with CH/pi interaction binds at the S2 or S1' site.

Repression of nitrogenase by ethanol in nitrogen-deprived cultures of Rhodovulum sulfidophilum.

Light-dependent H2 evolution did not occur in nitrogen-deprived cultures of Rhodovulum sulfidophilum in the presence of ethanol. When ethanol was added to cells which had been grown with ammonia, derepression of the nitrogen fixation genes (nifHD) was inhibited at an ethanol concentration of 1 mM. On the other hand, when cells had nitrogenase-catalyzed proton-reducing activity prior to ethanol addition, reduction of the nifHD transcript level did not occur after the addition. In cells grown with ammonia, concomitant addition of an auxiliary oxidant such as dimethylsulfoxide or sodium bicarbonate resulted in derepression of nitrogenase activity in the presence of ethanol. These results suggest that the electron-accepting process is necessary for derepression of nif genes in cultures which use ethanol as the electron donor.

Pharmacokinetics of adriamycin and cisplatin for anhepatic chemotherapy during liver transplantation.

We investigated the pharmacokinetics of cytotoxic anticancer agents administered under anhepatic conditions. Beagle dogs underwent either a sham operation consisting of laparotomy only (control group, n = 11) or a laparotomy and total hepatectomy under venovenous bypass (anhepatic group, n = 12). Each dog received a bolus intravenous injection of either Adriamycin (1 mg/kg) or cisplatin (1 mg/kg). The plasma and urine concentrations of each drug were measured at intervals for up to 2 h after drug injection. The dogs given Adriamycin were then sacrificed to determine tissue drug concentrations in the liver (controls only), spleen, kidney, heart, lung, skeletal muscle and small intestine. The control and anhepatic groups showed similar Adriamycin profiles during the initial 5 min after drug injection. However, subsequently, the plasma Adriamycin concentrations remained persistently higher in the anhepatic dogs than in the controls, yielding a two-fold elevation of the mean area under the concentration-time curve in the anhepatic group (P < 0.01 vs controls). The renal clearance values did not significantly differ between the two groups. The tissue Adriamycin concentrations in all measured organs, excluding the liver, were higher in the anhepatic group than in the controls. In a second set of experiments with cisplatin, the plasma platinum concentrations did not significantly differ between the two groups throughout the time course. However, the renal clearance of platinum in the anhepatic dogs showed a fourfold increase compared with that in the controls (P < 0.01). These pharmacokinetic data suggest that Adriamycin carries the risk of increased systemic toxicities, while cisplatin may be associated with increased renal toxicity when administered during the anhepatic period of liver transplantation.

Screening for primary hyperparathyroidism (PHPT) in clinic patients: differential diagnosis between PHPT and malignancy-associated hypercalcemia by routine blood tests.

Screening for primary hyperparathyroidism (PHPT) by measurement of the serum calcium concentration detects one patient per 500-1000 individuals in Western countries, and one patient per 2500-5000 subjects in Japan. Among clinic patients, however, the presence of many false-positive cases due to malignancy-associated hypercalcemia (MAH) reduces the benefit of such screening. We evaluated a new method of screening for PHPT based on the results of routine blood tests using the hospital information system (HIS) at our hospital. This new method could distinguish PHPT from MAH. This study included 25179 blood samples in which the serum calcium (Ca), albumin (Alb), chloride (Cl) and inorganic phosphate (IP) concentrations had been measured between March, 1994 and February, 1995 at Osaka University Medical Hospital. The HIS was programmed to pick blood samples that satisfied Formula 1 [Ca(mEq/ml) > 0.3 x Alb(g/dl) + 4.1] and Formula 2 ([Cl(mEq/ml)-84] x [10 x Alb-15]/[IP(mg/dl)/3.1] > 400). Of data from 25179 blood samples collected, those from 54 patients satisfied both Formulae 1 and 2. The patients from which these samples were derived from were subject to further analysis: medical records were studied and the intact-parathyroid hormone concentration was measured if necessary. Of these 54 cases, 19 patients (35.2%) were subsequently diagnosed with PHPT, including two, who were newly diagnosed with PHPT by this screening procedure. Although 35 (64.8%) of 54 patients were false-positive, many of them were treated with blood purification therapies in the Department of Pediatrics or the Intensive Care Unit (ICU). On the other hand, there were four false-positive cases (7.4%) caused by MAH. False-negative case in this study was only one patient (5%), whose diagnosis was normocalcemic PHPT. When omitting samples from pediatric patients and those in ICU, this screening procedure for PHPT has the advantage of being able to differentiate this diagnosis from MAH.

Single catheter technique of hepatic venous isolation and extracorporeal charcoal hemoperfusion for malignant liver tumors.

BACKGROUND: A single catheter technique of hepatic venous isolation and charcoal hemoperfusion (HVI-CHP) using a 4-lumen/2-balloon (4L-2B) catheter was developed to perform high-dose intra-arterial chemotherapy of the liver. Herein we report the technique, safety, and pharmacokinetics of this system in comparison with the original double-balloon technique. PATIENTS AND METHODS: Sixteen patients with malignant liver tumors were treated by hepatic arterial infusion (HAI) with adriamycin at a dose of 100 mg/m2 under HVI-CHP. Seven patients underwent HVI-CHP by the double-balloon technique (group A), in which filtered hepatic effluent and the rest of the inferior vena caval blood were separately drawn and returned to the left axillary vein. The other nine patients were treated by the single catheter technique (group B). In group B, hepatic effluent was isolated by balloon inflations and directed to filters through fenestrations of one major lumen of a 4L-2B catheter. The filtered blood was returned straight to the right atrium through the other major lumen of the catheter. RESULTS: All patients in group A had a smooth stepwise induction of HVI-CHP, whereas one of nine patients in group B developed severe hypotension requiring interruption of HVI. The hepatic venous flow rate in group B during HVI-CHP was significantly higher than that in group A (P < 0.05). Systemic adriamycin exposure, as assessed by the area under the time concentration curve in systemic serum, was significantly higher in group A compared to that in group B (P < 0.01). CONCLUSIONS: The single catheter technique is hemodynamically tolerable and feasible in the majority of patients with malignant liver tumors. In view of systemic drug exposure, the single catheter technique is superior to the original double-balloon technique.

Effect of bovine small intestine thioredoxin on aldose reductase activity.

Under oxidative stress mediated by H(2)O(2), significant activation of purified aldose reductase from bovine small intestine was observed in the presence of purified thioredoxin from bovine small intestine.

Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens.

Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens.

Broad spectrum and mode of action of an antibiotic produced by Scytonema sp. TISTR 8208 in a seaweed-type bioreactor.

A photobioreactor was constructed using anchored polyurethane foam strips (1 x 1 x 40 cm) fixed onto a stainless-steel ring to prevent flotation, as a biomass support material (BSM). This type of reactor was named a seaweed-type bioreactor. A filamentous cyanobacterium, Scytonema sp. TISTR 8208, which produces a novel cyclic dodecapeptide antibiotic, was immobilized in seaweed-type photobioreactor and cultivated with air containing 5% CO2 sparged at a gas flow rate of 250 mL/min under illumination at a light intensity of 200 mmol photon m-2 s-1. The antibiotic produced in the seaweed-type photobioreactor was purified by HPLC and examined regarding its spectrum and mode of action. The antibiotic effectively inhibited the growth of Gram-positive bacteria, pathogenic yeasts, and filamentous fungi, but it had only a weak effect on Gram-negative bacteria. Scanning electron micrograph analysis showed that the most characteristic change was swelling of the cells after exposure to the antibiotic. The antibiotic seems to alter the conformation of the microbial cell membrane, thereby changing its permeability, leading to osmotic shock.

Improvement of substrate conversion to molecular hydrogen by three-stage cultivation of a photosynthetic bacterium, Rhodovulum sulfidophilum.

In photosynthetic bacteria, after transition to light-anaerobic and nitrogen-deficient conditions, hydrogen evolution starts with expression of nitrogenase activity. Until the expression of enough activity, Rhodovulum sulfidophilum consumed substrates and converted them to poly(3-hydroxybutyrate) (PHB), resulting in a decrease in the proportion of substrate converted into hydrogen gas. To prevent conversion to PHB during the period when nitrogenase activity is derepressed, the authors employed a cultivation method consisting of three stages: cell growth, nitrogenase derepression, and hydrogen production. Cells cultivated by this method exhibited no lag time before the commencement of hydrogen evolution and gave an improved yield of hydrogen from the algal fermentative products.

Ocular hypotensive effects of monoamine oxidase-A inhibitors in rabbit.

The effects of monoamine oxidase-A (MAO-A) inhibitors with epinephrine on intraocular pressure in the pigmented rabbit were studied. MAO-A inhibitors were used topically with or without various concentrations of epinephrine. For the measurement of intraocular pressure, applanation pneumatonography was used and tissue MAO activities were determined by radiometric assay. After topical administration with clorgyline, MAO-A activities in the bulbar conjunctiva and the iris-ciliary body were remarkably inhibited, whereas MAO-B inhibition was minimal. Maximal reduction of intraocular pressure with 0.05% epinephrine was 3.2 mmHg. Single administration of clorgyline, amiflamine, moclobemide or CGP 11305-A caused decreases in the intraocular pressure of 2.0, 2.5, 1.8 and 2.4 mmHg, respectively. In the coadministration experiments with epinephrine, the ocular hypotensive effects of epinephrine were potentiated with clorgyline, amiflamine, moclobemide and CGP 11305-A (6.6, 4.8, 5.6 and 5.8 mmHg). On the contrary, they were not influenced by the MAO-B inhibitor deprenyl. These results indicated that MAO-A inhibitors potentiated the ocular hypotensive effects of epinephrine, and that the coadministration of a reversible MAO-A inhibitor with epinephrine might be useful for patients with glaucoma.

[Idea and practice with the systematization of clinical laboratory in the Central Laboratory, Osaka University Hospital].

On 1 September 1993, we left our old hospital and moved to our brand new establishment, and at that time we adopted the order-entry and reporting system. In this paper we report on our new laboratory computer system that has been developed to manage a lot of information and to analyze rapidly many test tubes (4000 samples per day) and to elevate the service for our patients. We developed the automated clinical laboratory system and this new system was named as the Clinical Laboratory Supervised System (CLASSY). We used the NEC system 3500 Model 10, NEC N5200 Model 03 sx and NEC PC9821 Ae as a laboratory host computer, an interface unit and a terminal for routine work, respectively. CLASSY covers the automated analysis not only for clinical chemistry, but also for hematology, urinalysis and microbiology. As the ordering and reporting system is applied to the hospital information system, order information for clinical test is transferred to our laboratory host computer when the bar-code label is printed out from the automatic bar-code labeller. Then it is transferred from the laboratory host computer to some subsystems or automatically to an analyzer through the interface units or modems.

[Informed consent for the use of the remaining portion of laboratory specimens for education, research, and quality control of laboratory tests].

The remaining portion of a laboratory specimen is usually used for education, research, and quality control of laboratory tests in hospitals, but informed consent has not been obtained because of the high volume of patients who undergo laboratory tests. However, patients must be informed in some manner. Therefore, we decided to inform patients that any remaining specimen would be used for various purposes by placing such a notice on walls in the central clinical laboratory and hospital lobby. We then obtained a signature on a dissent document, instead of a consent document, from any patient who dissented from such use. This indirect process for obtaining informed consent was approved by the ethics committee of Osaka University Medical School. The number of dissent documents sent in to the director was 54 of about 400,000 patients who underwent laboratory tests over the last 3 years, and there was no complaint against this "informed consent process".

[The effect of percutaneous hepatic venous isolation and charcoal hemoperfusion for high-dose chemotherapy for hepatoma].

We herein report the efficacy of percutaneous high-dose chemotherapy under hepatic venous isolation and charcoal hemoperfusion (HVI.CHP) in the treatment of hepatoma patients. This study included 23 patients with bilobar multiple intrahepatic metastases and 1 patient with high risk for recurrence after hepatectomy. All patients received adriamycin at doses ranging from 60-150 mg/m2 through the hepatic artery. Sixteen patients had HVI.CHP by the double-balloon technique, while a recent 8 patients had the single catheter technique using a 4L.2B catheter; 4 of these 8 patients had repeated treatment. Except for two early patients with hepatic arterial thrombosis and necrotizing pancreatitis, there was no lethal complication, and quality of life after treatment was remarkably improved in patients treated by the single catheter technique. Among 22 evaluable patients, 3 had CR and 11 had PR, yielding a response rate of 63%. Mean survival duration was prolonged to 13 months in responders, against only 5 months in nonresponders. In conclusion, HVI.CHP was highly effective for advanced hepatoma patients and the single catheter technique facilitated a repeated high-dose intraarterial chemotherapy, which may offer a possibility of complete remission even in highly advanced cases.