In Vitro and In Vivo Detection of Drug-induced Apoptosis Using Annexin V-conjugated Ultrasmall Superparamagnetic Iron Oxide (USPIO): A Pilot Study.

PURPOSE: To investigate the binding potential of newly developed Annexin V-conjugated ultrasmall superparamagnetic iron oxide (V-USPIO) for detection of drug-induced apoptosis in vitro and in vivo. METHODS: Apoptotic cells induced by camptothecin were incubated with or without Annexin V-USPIO at a concentration of 0.089 mmol Fe/L in vitro. T2 values of the two cell suspensions were measured by 0.47T nuclear magnetic resonance (NMR) spectrometer. Tumor-bearing mice were subjected to 1.5T MR scanner at 2 h after intraperitoneal injection of etoposide and cyclophosphamide. Following the pre-contrast T1- and T2-weighted imaging (0 h), the post-contrast scan was performed at 2, 4, 6 and 24 h after intravenous injection of Annexin V-USPIO (100 µmol Fe/kg). As a control, MRI was also obtained at 4 h after injection of USPIO without Annexin V. The ratio of tumor signal intensity (SI) on post-MRI for that on pre-MRI (Post/Pre-SI ratio) was calculated. After scanning, tumors were resected for pathological analysis to evaluate the distribution of iron and apoptotic cells. RESULTS: The suspension of apoptotic cells incubated with Annexin V-USPIO showed shorter T2 value than that without it. On T1-weighted imaging post/pre-SI ratio at 4 h after injection of Annexin V-USPIO showed 1.46, while after injection of USPIO without Annexin V was 1.17. The similar distribution of iron and apoptotic cells was observed in concordance with high signal intensity area on post-T1-weighted imaging. CONCLUSION: A newly developed Annexin V-USPIO could have the potential for detection of drug-induced apoptosis.

Correlation between port-to-target distance and procedural difficulty in single-incision laparoscopic cholecystectomy: An observational study.

INTRODUCTION: Single-incision laparoscopic cholecystectomy (SILC) is more challenging than conventional (multiport) laparoscopic cholecystectomy (CLC) because of the increased likelihood of instrument collision and the limited surgical workspace. In SILC, procedural difficulties may increase when the port-to-target distance is long. We aimed to assess the correlation between port-to-target distance and procedural difficulty. METHODS: Thirty-six consecutive patients who underwent SILC at our hospital were included in this study. The umbilicus-to-Calot's triangle distance (UCD) was measured intraoperatively. The correlations between the UCD and operative time were analyzed, and for comparison, CLC cases during the same period (n = 28) were similarly analyzed. Moreover, UCD was estimated from preoperative CT (UCD-CT), and the usefulness of UCD-CT was assessed during SILC and CLC. RESULTS: Thirty-four patients successfully underwent SILC. There were positive correlations between the UCD and pneumoperitoneum time. Multivariate linear regression analysis, including BMI and height, which were previously reported to have a correlation with longer operative time in SILC, showed that UCD is an independent predictive factor for prolonged operative duration. However, BMI and height were not independent predictive factors. UCD and UCD-CT had a very strong positive correlation; therefore, UCD was estimated from CT. UCD-CT had a strong positive correlation with operative time in SILC, but not in CLC. CONCLUSIONS: A longer UCD is an important predictive factor for difficult cases of SILC, but this finding is not applicable in CLC. The usefulness of UCD is specific to SILC.

Voltammetric study of the boric acid-salicylaldehyde-H-acid ternary system and its application to the voltammetric determination of boron.

The ternary system of boric acid, salicylaldehyde (SA) and H-acid (HA) was voltammetrically studied from kinetic and equilibrium points of view. The effect of the SA substituents was also studied by using two analogs, 5-fluorosalicylaldehyde (F-SA) and 5-methylsalicylaldehyde (Me-SA). The three cathodic peaks of Azomethine H (AzH), Azomethine H-boric acid complex (AzB), and free SA were observed in the solution containing boric acid, SA and HA. The peak potentials of AzH and SA were shifted to negative potentials with increasing pH, while the peak potential of AzB was pH-independent. This difference indicates that a proton participates in the charge-transfer steps of the AzH and SA reductions, but not in that of the AzB reduction. The formation constants for the AzB complexation were similar among all the examined analogs. In the kinetic study, the reaction rate was higher in an acidic condition for the AzH formation, but in a neutral condition for the AzB formation. The rate constants for the AzB complexes were in the order of F-SA > SA ≈ Me-SA, indicating that the fluoro group accelerates the F-AzB complexation. The AzB complexation mechanism is considered to consist of more than three steps, i.e., the pre-equilibrium of the salicylaldehyde-boric acid complex (SA-B) formation, the nucleophilic attack of HA on SA-B, and the remaining some steps to form AzB. Based on these results, the voltammetric determination method of boron using F-SA was optimized, which allowed the boron concentration to be determined within only 5 min with a 0.03 mg B dm(-3) detection limit.

AlCl3-mediated aldol cyclocondensation of 1,6- and 1,7-diones to cyclopentene and cyclohexene derivatives.

Exactly 1/3 mol of AlCl3 is sufficient to cyclize 1 mol of 1,ω-dibenzoylbutane (or pentane) to a cyclopentenone (or hexenone) derivative in high yield at room temperature in 40 min to several hours. This condensation is driven by removing elements of water as HCl and Al(OH)3, and the product enones are exclusively unconjugated, unlike the base-catalyzed condensations providing thermodynamically more stable conjugated enones.

Evaluation of magnetic nanoparticle samples made from biocompatible ferucarbotran by time-correlation magnetic particle imaging reconstruction method.

BACKGROUND: Molecular imaging using magnetic nanoparticles (MNPs)-magnetic particle imaging (MPI)-has attracted interest for the early diagnosis of cancer and cardiovascular disease. However, because a steep local magnetic field distribution is required to obtain a defined image, sophisticated hardware is required. Therefore, it is desirable to realize excellent image quality even with low-performance hardware. In this study, the spatial resolution of MPI was evaluated using an image reconstruction method based on the correlation information of the magnetization signal in a time domain and by applying MNP samples made from biocompatible ferucarbotran that have adjusted particle diameters. METHODS: The magnetization characteristics and particle diameters of four types of MNP samples made from ferucarbotran were evaluated. A numerical analysis based on our proposed method that calculates the image intensity from correlation information between the magnetization signal generated from MNPs and the system function was attempted, and the obtained image quality was compared with that using the prototype in terms of image resolution and image artifacts. RESULTS: MNP samples obtained by adjusting ferucarbotran showed superior properties to conventional ferucarbotran samples, and numerical analysis showed that the same image quality could be obtained using a gradient magnetic field generator with 0.6 times the performance. However, because image blurring was included theoretically by the proposed method, an algorithm will be required to improve performance. CONCLUSIONS: MNP samples obtained by adjusting ferucarbotran showed magnetizing properties superior to conventional ferucarbotran samples, and by using such samples, comparable image quality (spatial resolution) could be obtained with a lower gradient magnetic field intensity.

Local skin flap reconstruction for abdominal wound dehiscence after abdominal surgery with a stoma: report of two cases.

Abdominal wound dehiscence is a serious complication of laparotomy, and fascial dehiscence in a patient with a stoma is especially difficult to manage. We describe how we performed local skin flap reconstruction for abdominal wound dehiscence in two patients with stomas. One patient underwent sigmoidectomy with a colostomy for peritonitis caused by perforated diverticulitis of the sigmoid colon. Postoperative fascial dehiscence was repaired by rhomboid flap reconstruction. The other patient underwent total gastrectomy, cholecystectomy, and splenectomy. An ileostomy was performed for digestive tract perforation, which was complicated by abdominal dehiscence with necrosis of the fascia. This was repaired by rotation flap reconstruction. The abdominal walls in both patients were repaired successfully without tension.

Analysis of alteration of p75NTR processing and signalling by PS2 mutation and gamma-secretase inhibition.

The presenilins (PSs) were identified as causative genes in cases of early-onset familial Alzheimer's disease (AD) and current evidence indicates that PSs are part of the gamma-secretase complex responsible for proteolytic processing of type I membrane proteins. p75NTR, a common neurotrophin receptor, was shown to be subject to gamma-secretase processing. However, it is not clear if the p75NTR downstream signal is altered in response to gamma-secretase cleavage, and further there is a possibility that AD-related PS mutations may affect this cleavage, resulting in pathogenic alterations in signal transduction. In this study, we confirmed that p75NTR downstream signalling is altered by PS2 mutation or gamma-secretase inhibition in SHSY-5Y cells. The activity of the small GTPase RhoA is strongly affected by these treatments. This study demonstrates that gamma-secretase and PS2 play an important role in regulating neurotrophin signal transduction and either mutation of PS2 or inhibition of gamma-secretase disturbs this function.

[Evaluation of nickel allergy in mice].

This study was performed to establish a convenient mouse model for the evaluation of nickel allergy. For sensitization, 0.2 g of nickel sulfate in petrolatum was applied 4 times every other day to the shaved dorsal skin. Seven days after the first application of the nickel sulfate salt sample, the antigen-specific metal allergy reaction was estimated based on the swelling response of the footpad injected with 20 microliters of nickel sulfate salt in saline. Percutaneously applied nickel powder in petrolatum, as well as the original nickel salt sample, induced a significant nickel allergy reaction. Therefore, it is suggested that this system is applicable for the evaluation of antimetal allergic substances.

Anti-inflammatory and anti-allergic activities of hydroxylamine and related compounds.

The anti-inflammatory activities of several novel oximes and O-acyl oximes that we synthesized have been reported based on carrageenan-induced rat foot-pad swelling assay and histamine-induced rat vascular permeability assay. A cyclooxygenase (COX)-1 inhibitory effect has also been reported for 4'-piperidinoacetophenone and 4'-morpholinoacetophenone oximes and their O-acyl derivatives. To further search for more effective non-steroidal anti-inflammatory or anti-allergic drugs, 1-hydroxylamino-1-(4'-piperidinophenyl) ethane (P-HA) and 1-hydroxylamino-1-(4'-morpholinophenyl) ethane (M-HA) were synthesized from the corresponding oximes with sodium cyanoborohydride, and N,O-diacetyl hydroxylamines (P-HA-Ac and M-HA-Ac) were prepared from these hydroxylamines using acetyl chloride. These hydroxylamines and N,O-diacetyl hydroxylamines clearly exhibited inhibitory effects on mouse carrageenan-induced foot-pad swelling induced by oral administration (150, 37.5 mg/kg). An oral dose of P-HA-Ac (150 mg/kg) significantly inhibited the mouse anaphylactic reaction to ovalbumin measured by the abdominal wall (AW) method. Percutaneous administration of P-HA and M-HA significantly inhibited 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity reaction (type IV) in mice at a dose of 0.5 and 0.1 mg/ear, respectively. All tested hydroxylamines and N,O-diacetyl hydroxylamines clearly inhibited both COX-1 and COX-2 enzyme activities with IC(50) values of 1.9-28.7 and 1.6-2.9 micro M against COX-1 and COX-2, respectively. Hydroxylamines (P-HA and M-HA) also showed a 5-lipoxygenase inhibitory effect.

Assignment of UVB-responsive cis-element and protoplastization-(dilution-) and elicitor-responsive ones in the promoter region of a carrot phenylalanine ammonia-lyase gene (gDcPAL1).

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al. [1997] Photochem. Photobiol. 66, 464-470), L4 is UVB responsive, and L1 is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated g-DcPAL1 promoter-luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.

Simultaneous Determination of Aldicarb, Ethiofencarb, Methiocarb and Their Oxidized Metabolites in Grains, Fruits and Vegetables by High Performance Liquid Chromatography.

Three carbamate pesticides, aldicarb (A0), ethiofencarb (E0), methiocarb (M0) and six of their oxidized metabolites, sulphoxides (A1, E1, M1) and sulphones (A2, E2, M2) were simultaneously determined. Five grams of a sample were homogenized with acetone, and then treated with dichloromethane-hexane mixture (1:1) and sodium chloride (NaCl), homogenized and centrifuged. The organic layer was removed and the aqueous residue was reextracted a second time with dichloromethane - hexane mixture. The combined organic extracts were evaporated in vacuo. The residue was dissolved in dichloromethane and charged on a Sep-Pak® aminopropyl cartridge. Carbamates were eluted from the cartridge with 1% methanol in dichloromethane. The eluate was evaporated to dryness and 0.5 ml of methanol and 1.5 ml of 0.001 N-hydrochloric acid (HCl) solution were added. Individual carbamates were separated by gradient elution high performance liquid chromatography (HPLC) using octyldecylsaline (ODS) column. Derivatization of separated carbamates to fluorescent derivatives was achieved in-line. Recovery of pesticides and their oxidized metabolites from rice, apple, cabbage and other foods ranged from 60 to 103% following fortification at 20 ppb. Detection limits were 1 ppb for A1, A2, E1, E2, 2 ppb for A0, E0, M1 and 4 ppb for M0 and M2 (S/N>3).

Simultaneous Determination of Aldicarb, Ethiofencarb, Methiocarb and Their Oxidized Metabolites in Grains, Fruits and Vegetables by High Performance Liquid Chromatography.

Three carbamate pesticides, aldicarb (A0), ethiofencarb (E0), methiocarb (M0) and six of their oxidized metabolites, sulphoxides (A1, E1, M1) and sulphones (A2, E2, M2) were simultaneously determined. Five grams of a sample were homogenized with acetone, and then treated with dichloromethane-hexane mixture (1:1) and sodium chloride (NaCl), homogenized and centrifuged. The organic layer was removed and the aqueous residue was re-extracted a second time with dichloromethane - hexane mixture. The combined organic extracts were evaporated in vacuo. The residue was dissolved in dichloromethane and charged on a Sep-Pak® aminopropyl cartridge. Carbamates were eluted from the cartridge with 1% methanol in dichloromethane. The eluate was evaporated to dryness and 0.5 ml of methanol and 1.5 ml of 0.001 N-hydrochloric acid (HCl) solution were added. Individual carbamates were separated by gradient elution high performance liquid chromatography (HPLC) using octyldecylsaline (ODS) column. Derivatization of separated carbamates to fluorescent derivatives was achieved in-line. Recovery of pesticides and their oxidized metabolites from rice, apple, cabbage and other foods ranged from 60 to 103% following fortification at 20 ppb. Detection limits were 1 ppb for A1, A2, E1, E2, 2 ppb for A0, E0, M1 and 4 ppb for M0 and M2 (S/N>3).

Simultaneous Determination of Vamidothion and Its Oxidation Metabolites in Potatoes and Apples by Gas Chromatography.

A method for simultaneous determination of vamidothion (V0) and its oxidation metabolites vamidothion sulphoxide (V1) and vamidothion sulphone (V2) in potatoes and apples has been developed. Fifty grams of a sample was homogenized and extracted with acetone followed by evaporation to remove the acetone. To the residual aqueous solution, a 10% sodium chloride solution was added, and the coextractives were eliminated by washing the aqueous solution with 10% ethyl acetate in hexane. Then, V0, V1, and V2 were extracted from the aqueous solution using dichloromethane. The organic layer was evaporated to dryness and filled up to 2 ml with ethyl acetate. Two microliters of the extract were injected into gas chromatograph-mass spectrometer set for selected ion monitoring. The column used was a CBP-1 capillary column (0.2 mm inside diameter × 15 m, 0.25 µm film thickness). Gas chromatographic conditions were investigated in detail and only nonpolar capillary columns gave satisfactory results. The retention time of undecomposed V1 has been reported for the first time. The recoveries for the fortified potatoes and apples were 93-109% for V0, 62-108% for V1, and 64-89% for V2, when they were fortified at levels of 0.01-5.0 ppm. Detection limits were 0.01, 0.2, and 0.05 ppm for V0, V1, and V2, respectively.

Determination of Ethyl Carbamate in Various Fermented Foods by Selected Ion Monitoring.

A simple and sensitive method for the determination of ethyl carbamate (urethane) in various fermented foods has been developed. Twenty g of sample was homogenized and extracted with 150 ml of acetone. Twenty ml of water was added to the extract which was then evaporated to remove organic solvent. The residual aqueous solution was extracted three times with 100 ml of dichloromethane. The organic layer was concentrated by rotary evaporator and was charged on an acid-celite column. After washing the column with 100 ml of pentane and 100 ml of pentane-dichloromethane mixture (80:20), ethyl carbamate was eluted with 60 ml of dichloromethane. The eluant was concentrated to 1 ml using a rotary evaporator, and ethyl carbamate was determined by gas chromatography-mass spectrometry in the selected ion monitoring mode. For liquid samples, extraction with acetone was not necessary. The column purification step was also unnecessary for alcoholic beverages. The detection limit for this procedure was 0.5 ppb/sample, and the recovery was 70 to 105% for miso, moromi, natto, soy sauce, sake, yogurt, and bread when they were fortified with 5 to 50 ppb of ethyl carbamate. Ethyl carbamate levels in various fermented foods including yogurt, bread, mirin, or sakekasu were determined by this method. Relatively high levels of ethyl carbamate were detected in sake and soy sauce.

Determination and Identification of Daminozide in Fresh Fruits Purchased in Japan.

The concentration of residual daminozide (trade name of formulation: Alar or B-Nine) in cherries, grapes, peaches, and apples purchased in Japan was investigated using the colorimetric method. Daminozide hydrolyzes in boiling strong alkaline solution to release unsymmetrical dimethylhydrazine which is distilled and reacted with trisodium pentacyanoamine ferroate to form a specific red color at pH 4.5. This color is measured spectrophotometrically. The levels of daminozide detected were 0.07-1.39 ppm in cherries (11 samples), 0-0.36 ppm in grapes (10 samples), 0-0.42 ppm in peaches (9 samples), and 0-0.88 ppm in apples (10 samples). Furthermore, daminozide in each sample was identified as methyl daminozide by the gas chromatography/mass spectrometry (GC/MS) method.

Simultaneous Determination of Ten Kinds of Organochlorine Insecticides in Beef by Gas Chromatography.

A simple and rapid method for simultaneous determination of 10 kinds of organochlorine insecticides (ß-BHC, heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin, p,p'-DDT, p,p'-DDE, p,p'-DDD, t-chlordane) in beef was established. The pesticides were extracted from samples with acetone hexane (3:1) mixture, and purified by Florisil column chromatography, then determined by use of a gas chromatograph equipped with an electron capture detector (ECD-GLC). Recoveries of the 10 pesticides added to beef at 0.02 ppm and 0.1 ppm were within the range of 63.9 - 89.0% and 76.8 - 90.5% by the proposed method, respectively. One hundred and twenty six samples of Australian beef received at the Osaka and Kobe quarantine stations from August to December in 1987 were analyzed, and only one sample was over the limit of dieldrin (+aldrin) referring to Japanese temporary tolerance.

Determination of Ethylene Dibromide in Tropical Fresh Fruits Using Dean-Stark Apparatus and ECD-Gas Chromatography.

The concentration of residual EDB (ethylene dibromide) in tropical fresh fruits imported to Japan were investigated using the Dean-Stark apparatus and ECD-gas chromatography (ECD-GC) i.e. EDB was distilled into a small amount of n-hexane using a Dean-Stark apparatus and determined by ECD-GC. Recovery of EDB added to papaya pulp at the concentration of 0.1 ppm was more than 98.1% by the proposed method. The relationship between storage conditions and concentration of residual EDB was investigated for mango pulps; it was clarified that EDB content in mango pulps decreased with the storage time at room temperature (25°C) and refrigerated (7°C), but the content did not decrease for 6 d at -20°C. We surveyed the concentration of residual EDB in pulps, peels and seeds of the fresh fruits (grapefruits, papayas, mangos and litchis) imported to Japan in 1987 separately. Extremely high concentrations of EDB were found in the seeds of grapefruits. A similar trend was also observed in other fruits.

Time Courses for Viable Counts, pH and Total Volatile Basic Nitrogen in Squid Homogenates ( Ommastraphes bartrami ) Inoculated with Spoilage Yeast or Bacteria.

The spoilage patterns of Candida lipolytica , Pseudomonas fluorescens and Staphylococcus epidermidis in sterile squid homogenates were compared. The three species were inoculated singly or as a mixture, incubated for up to 7 d, and analyzed for viable counts, pH values and the amounts of total volatile basic nitrogen (TVB-N) produced. Incubation temperatures were 5, 15 and 25°C. When each species was separately inoculated, C. lipolytica showed slower growth than that of the two bacterial species, but TVB-N production in the homogenates increased to level that indicated spoilage. The TVB-N exceeded the concentration corresponding to the threshold value of spoilage on d 4 at 15°C or on d 2 at 25°C. The TVB-N values in the C. lipolytica inoculated homogenates reached 112 and 215 mg per 100 g of homogenate on d 7 at 15 and 25°C, respectively. On these occasions the respective pH values were 7.9 and 8.1, and the homogenates inoculated with the yeast showed spoilage emitting an ammoniacal odor. In general, TVB-N production by C. lipolytica was slightly less than that by P. fluorescens , but much more than that by S. epidermidis . The TVB-N curves obtained from the mixed cultures containing the three species were very similar to those obtained from the single cultures of P. fluorescens . Viable counts of the individual strains in the mixed cultures showed that the yeast strain grew independently without being suppressed by the growth of the two bacterial strains, and even 3 weeks after incubation the survival counts of the yeast strain were higher than those of the bacterial strain at 15 and 25°C, respectively.

Determination of Rosin Ester Gum Emulsifiers in Fruit Juices by Gas Chromatography.

A method for detection and determination of rosin ester gum in fruit juice was established as follows. Rosin ester as a component of ester gum was extracted with benzene from the sample, and saponified with N/2 KOH-ethanol solution. The rosin acids were extracted with diethyl ether in acidic condition, derivatized with TMS reagent and determined by gas chromatography (GC). Seven kinds of ester gum standard were analyzed by the proposed method, and it was found that contents of dihydroabietic acid and abietic acid in the ester gums ranged between 33.8-75.1% and 0-36.7%, respectively, but the total contents of them were 66.7-75.1% (average, 70.9 ± 3.0%). Dihydroabietic acid and abietic acid derived from ester gum in 6 kinds of imported fruit juice were identified by GC-mass spectra and quantitated by GC. The contents of ester gum in samples estimated from the two peaks were 15.2-33.9 ppm. Recoveries of ester gum added to sample at 50 and 500 ppm were more than 92.7%, and the detection limit of ester gum was 0.5 µg (2 ppm in sample) by the proposed method.

Rapid Determination of Mono-, Di- and Tri-isopropyl Citrate in Foods by Gas Chromatography.

A simple and rapid method for determining mono-, di- and tri-isopropyl citrates in foods was developed. Isopropyl citrates in butter and milk powder were extracted with ethyl acetate under acid condition and in edible oil with hexane. The ethyl acetate containing isopropyl citrates was evaporated and the residue was taken up in hexane. The isopropyl citrates in hexane were extracted into acetonitrile. After evaporation of the solvent, isopropyl citrates were methylated with diazomethane, and they were determined by gas chromatography. Recoveries of mono-, di- and tri-isopropyl citrates from edible oil, butter and milk powder by this method were more than 92.9%, 95.7% and 94.8%, respectively. The detection limits of isopropyl citrates were 1 µg/g of sample.