Voluntary running exercise attenuates the progression of endothelial dysfunction and arterial calcification in ovariectomized rats.

AIM: Loss of oestrogen synthesis capacity after menopause contributes to increases in arterial stiffness and calcification. Exercise training improves arterial stiffness and calcification. However, the mechanism of exercise training-induced improvement of arterial stiffness and calcification remains unclear. METHOD: We examined the mechanism by using aortas of sham-operated rats (sham control; SC), ovariectomized rats (OVX control; OC), OVX plus treatment with vitamin D(3) plus nicotine (VDN) rats (OV sedentary; OVSe), which is an animal model of endothelial dysfunction and arterial calcification, and voluntary running wheel exercise for 8 weeks plus OVX plus VDN rats (OV exercise; OVEx). RESULTS: The arterial tissue calcium and endothelin-1 (ET-1: a vasoconstrictor peptide and a potent regulator of arterial calcification) levels were significantly higher in OVSe rats compared with the SC and OC rats, whereas these levels in the OVEx rats were significantly lower than in the OVSe rats. Additionally, arterial expression of endothelial nitric oxide synthase (eNOS), which is an enzyme that produces nitric oxide (NO: a vasodilator substance), was reduced in OVSe rats. However, exercise training prevented the decrease in eNOS expression. Moreover, there was a significant positive correlation between arterial calcium level and arterial ET-1 level. CONCLUSION: These findings suggest that exercise training-induced improvement of ET-1 and NO prevents the impairment of endothelial function after menopause in females, and this improvement may result in less arterial calcification.

Interference of Long-Distance Movement of Grapevine berry inner necrosis virus in Transgenic Plants Expressing a Defective Movement Protein of Apple chlorotic leaf spot virus.

ABSTRACT Transgenic Nicotiana occidentalis plants expressing a movement protein (P50) and partially functional deletion mutants (DeltaA and DeltaC) of the Apple chlorotic leaf spot virus (ACLSV) showed resistance to Grapevine berry inner necrosis virus (GINV). The resistance is highly effective and GINV was below the level of detection in both inoculated and uninoculated upper leaves. In contrast, GINV accumulated in inoculated and uninoculated leaves of nontransgenic (NT) plants and transgenic plants expressing a dysfunctional mutant (DeltaG). On the other hand, in some plants of a transgenic plant line expressing a deletion mutant (DeltaA', deletion of the C-terminal 42 amino acids), GINV could spread in inoculated leaves, but not move into uninoculated leaves. In a tissue blot hybridization analysis of DeltaA'-plants inoculated with GINV, virus could be detected in leaf blade, midribs, and petiole of inoculated leaves, but neither in stems immediately above inoculated leaves nor in any tissues of uninoculated leaves. Immunohistochemical analysis of GINV-inoculated leaves of DeltaA'-plants showed that GINV could invade into phloem parenchyma cells through bundle sheath of minor veins, suggesting that the long-distance transport of GINV might be inhibited between the phloem cells and sieve element (and/or within sieve element) rather than bundle sheath-phloem interfaces. Immunogold electron microscopy using an anti-P50 antiserum showed that P50 accumulated on the parietal layer of sieve elements and on sieve plates. The results suggested that resistance in P50-transgenic plants to GINV is due to the interference of both long-distance and cell-to-cell movement of the virus.

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs.

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.

Assessment of proliferative potentials of canine osteosarcomas and chondrosarcomas by MIB-1 immunohistochemistry and bromodeoxyuridine incorporation.

The proliferative potential of 17 canine osteosarcomas (OSs) (13 osteoblastic, two anaplastic, one fibroblastic and one chondroblastic), 18 chondrosarcomas (CSs) (13 mesenchymal and five ordinary), three osteomas, and one chondroma was evaluated immunohistochemically by labelling Ki-67 antigen with MIB-1 antibody, and incorporated bromodeoxyuridine (BrdU) with anti-BrdU antibody. The location of BrdU-positive cells in OSs and CSs was similar to that of MIB-1 positive cells, and the mean value of the BrdU labelling index (BrdU LI) and the MIB-1 positive index (MIB-1 PI) in each case were significantly correlated (rs = 0.942, P < 0.05 with Spearman rank correlation coefficient; r = 0.779 P < 0.05 with linear regression analysis). The mean MIB-1 PI of OSs was 29.5%, which was approximately 2.5 times that of CSs, and the highest MIB-1 PI was 34.8% +/- 1.8 S.E.M. in areas without osteoid. In CS cases, the survival rate after 24 months was significantly higher than in OS cases. The high MIB-1 PI therefore supports the view that OSs are clinically more aggressive than CSs in dogs. On the other hand, the highest MIB-1 PI values of mesenchymal CS components occurred in transitional areas, which were composed of poorly differentiated cells embedded in a myxomatous matrix between the chondroidal and mesenchymal regions. The MIB-1 PI was 21.3% +/- 3.0 S.E.M. P < 0.001 in transitional areas. Proliferative markers may be useful in diagnosis and prognosis.

Effects of combustion temperature on PCDD/Fs formation in laboratory-scale fluidized-bed incineration.

Combustion experiments in a laboratory-scale fluidized-bed reactor were performed to elucidate the effects of combustion temperature on PCDD/Fs formation during incineration of model wastes with poly(vinyl chloride) or sodium chloride as a chlorine source and copper chloride as a catalyst. Each temperature of primary and secondary combustion zones in the reactor was set independently to 700, 800, and 900 degrees C using external electric heaters. The PCDD/Fs concentration is reduced as the temperature of the secondary combustion zone increases. It is effective to keep the temperature of the secondary combustion zone high enough to reduce their release during the waste incineration. On the other hand, as the temperature of the primary combustion zone rises, the PCDD/Fs concentration also increases. Lower temperature of the primary combustion zone results in less PCDD/Fs concentration in these experimental conditions. This result is probably related to the devolatilization rate of the solid waste in the primary combustion zone. The temperature decrease slows the devolatilization rate and promotes mixing of oxygen and volatile matters from the solid waste. This contributes to completing combustion reactions, resulting in reducing the PCDD/Fs concentration.

Synthesis and pharmacological activity of 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4- pyrrolidinyl]benzamide (TKS159) and its optical isomers.

Of 4-amino-5-chloro-2-methoxy-N-(1-ethyl-2-hydroxymethyl-4- pyrrolidinyl)benzamide, four optical isomers, (2S,4S)-1 (TKS159), (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27, were prepared from optically active 4-amino-1-ethyl-2-hydroxymethylpyrrolidine di-p-toluenesulfonate [(2S,4S)-14, (2S,4R)-17, (2R,4S)-20 and (2R,4R)-23, respectively]. The requisites, (2S,4S)-14, (2S,4R)-17, (2R,4S)-20 and (2R,4R)-23, were prepared from a commercially available trans-4-hydroxy-L-proline. The absolute configurations of (2S,4S)-1 (TKS159), (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27 were spectroscopically determined. Of the benzamide derivatives, four optical isomers, (2S,4S)-1, (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27, showed a relatively potent affinity for 5-hydroxytryptamine 4 (5-HT4) receptors in a radioligand binding assay ([3H]GR113808). The activities of 25-27 were less effective than that of 1 for the gastric emptying of a phenol red semisolid meal in rats. All this suggests that the most potent of the isomers was 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2- hydroxymethyl-4-pyrrolidinyl]benzamide (1).

[Potassium Sucrose Octa Sulfate Reference Standard (Control 961) of National Institute of Health Sciences].

The raw material of potassium sucrose octa sulfate was examined for the preparation of the "Potassium Sucrose Octa Sulfate Reference Standard (Control 961)". Analytical data obtained were as follows: infrared spectrum, the same as that of the Potassium Sucrose Octa Sulfate Reference Standard (Control 901); high-performance liquid chromatography, one impurity was detected; water content, 8.1%; assay of sucrose octa sulfate, 99.6%. Based on the above results, the raw material was authorized to be the Potassium Sucrose Octa Sulfate Reference Standard of the National Institute of Health Sciences.

[Thrombin Reference Standard (Control 961) of National Institute of Health Sciences].

The "Thrombin Reference Standard (Control 961)" of National Institute of Health Sciences was prepared. The precision of filling into ampoule was about 1% as C.V. The content of a-thrombin was about 87%. The thrombin potency of the standard material was assayed against the Thrombin Reference Standard (Control 8710) according to the method of JP XIII and the potency was 1033 +/- 59 unit/ampoule. From the results, the potency of the proposed material for Thrombin Reference Standard was defined as 1,030 units per ampoule.

[Kallidinogenase Reference Standard (Control 971) of National Institute of Health Sciences].

The "Kallidinogenase Reference Standard (Control 971)" of National Institute of Health Sciences was prepared. The kallidinogenase potency of the standard material was assayed against the 2nd Kallidinogenase Reference Standard (Control 854) by the enzyme assay method using H-D-valyl-L-leucyl-L-arginine-p-nitro-anilide as the substrate. The potency of the Kallidinogenase Reference Standard material thus obtained was defined as 119 unit per ampoule.

[Retinol Acetate Reference Standard (Control 971) and Retinol Palmitate Reference Standard (Control 971) of National Institute of Health Sciences].

The "Retinol Acetate Reference Standard (Control 971)" and "Retinol Palmitate Reference Standard (Control 971)" of National Institute of Health Sciences using the assay of vitamin A ester were prepared. The proposed materials were evaluated in collaboration with four laboratories. Analytical data obtained were as follows. 1) The purities of retinol acetate and retinol palmitate measured by HPLC were 99.9 +/- 0.06% and 94.5 +/- 0.06%, respectively. 2) ultraviolet spectrum of retinol acetate and retinol palmitate showed the lambda max at 326-327 nm. 3) The difference in relative extinction of retinol acetate and retinol palmitate at 300 nm, 310 nm, 320 nm, 330 nm, 340 nm and 350 nm are within the range provided in JPXIII. 4) The contents of retinol acetate and retinol palmitate were 52,000 IU/g and 52,200 IU/g, respectively. Based on the above results, these proposed materials were authorized to be the Reference Standards of the National Institute of Health Sciences.

[Cholecalciferol Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material for cholecalciferol was examined for preparation of the "Cholecalciferol Reference Standard (Control 971)". Analytical data obtained were as follows: melting point, 83.8 degrees C; UV and infrared spectra, the same as those for JP Cholecalciferol Reference Standard (Control 945), respectively; specific absorbance at 265 nm, E1 cm 1% = 485; optical rotation, [alpha]D20 = +107.4 degrees; thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurity was detected; assay, 98.9% by HPLC. Based on the above results, the raw material was authorized as the Cholecalciferol Reference Standard (Control 971) of National Institute of Health Science.

[Ergocalciferol Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material for ergocalciferol was examined for preparation of the "Ergocalciferol Reference Standard (Control 971)". Analytical data obtained were as follows: melting point, 116.7 degrees C; UV and infrared spectra, the same as those for JP Cholecalciferol Reference Standard; specific absorbance, E1 cm 1% = 461(265 nm); optical rotation, [alpha]D20 = +102.5 degrees; thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurity was detected; assay, 102.4% by HPLC. Based on the above results, the raw material was authorized as the Ergocalciferol Reference Standard (Control 971) of National Institute of Health Sciences.

[Tocopherol Acetate Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material of tocopherol acetate was examined for the preparation of the "Tocopherol Acetate Reference Standard (Control 971)". Analytical data obtained were as follows: infrared spectrum, the same as that of the Tocopherol Acetate Reference Standard (Control 941); specific absorbance, E1 cm 1% (284 nm) = 43.0; thin-layer chromatography, no impurities were detected until 50.0 micrograms of the loaded raw material; high-performance liquid chromatography (HPLC), one impurity was detected and the amount was estimated to be about 0.68%; assay by HPLC, 100.2%. Based on the above results, the raw material was authorized as the Tocopherol Acetate Reference Standard (Control 971) of National Institute of Health Sciences.

[Thiamine Hydrochloride Solution Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material of thiamine hydrochloride solution was examined for the preparation of the "Thiamine Hydrochloride Solution Reference Standard (Control 971)". Analytical data obtained were as follows: assay by HPLC, 100.8%; spectrophotometric assay, 99.8%. Based on the above results, the raw material was authorized to be the Thiamine Hydrochloride Solution Reference Standard of the National Institute of Health Sciences.

[Prednisolone Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material of prednisolone was examined for the preparation of the "Prednisolone Reference Standard (Control 971)". Analytical data obtained were as follows: melting point, 238.4 degrees C (decomposition); infrared spectrum, the same as that of the Prednisolone Reference Standard (Control 911); UV spectrum, lambda max = 243 nm; specific absorbance, E1 cm 1% (243 nm) = 414.8; loss on drying, 0.06%; thin-layer chromatography, one impurity was detected; high-performance liquid chromatography (HPLC), 4 to 5 impurities were detected and the total amount was estimated to be about 0.51%; assay by HPLC, 100.6%. Based on the above results, the raw material was authorized as the Prednisolone Reference Standard (Control 971) of National Institute of Health Sciences.

[Application of thermogravimetry to loss on drying test and water-content determination of drugs].

Thermogravimetry, one of the techniques of thermal analysis, was applied to the quality control of drug raw materials as a "Loss on Drying" or for "Water Content Determination". Twenty two kinds of drugs were selected for the comparison of the applicability of thermogravimetry with that of Loss on Drying Test and/or Water Content Determination by the Karl-Fisher method. In all kinds of drugs, it was ascertained that the results with thermogravimetry agreed well with those obtained by Loss on Drying test and/or Karl-Fisher method. In conclusion, thermogravimetry can be used as a substitute for the Loss on Drying test in cases where drug possess a water bound strongly. Further, thermogravimetry can be utilized for some drugs to which the Karl-Fisher method cannot be applied due to their insolubility in Karl-Fisher reagents.

[Epinephrine Bitartrate Reference Standard (Control 951) of National Institute of Health Sciences].

The raw material for epinephrine bitartrate was tested for preparation of the "Epinephrine Bitartrate Reference Standard (Control 951)" of National Institute of Health Sciences. Analytical data obtained were as follows: melting point, 148.6 degrees C (decomposition); UV spectrum, lambda max = 279 nm; IR spectrum, the same as that of JP Epinephrine Bitartrate Reference Standard (Control 792); optical rotation, [alpha]20D = -52.8 degrees; thin-layer chromatography, one impurity was detected; high-performance liquid chromatography, no impurity was detected; loss on drying, 0.01%; assay, 99.6% by potentiometric titration, 100.3% by spectrophotometry. Based on the above results, the raw material was authorized as the Epinephrine Bitartrate Reference Standard (Control 951) of National Institute of Health Sciences (Japanese Pharmacopoeia).

[Riboflavin Reference Standard (Control 951) of National Institute of Health Sciences].

The raw material for riboflavin was tested for preparation of the "Riboflavin Reference Standard (Control 951)" of National Institute of Health Sciences. Analytical data obtained were as follows: melting point, 284.6 degrees C (decomposition): specific absorbance, E1cm1% = 857 (267 nm), 277 (373 nm), 326 (445 nm); IR spectrum, the same as that of JP Riboflavin Reference Standard (Control 921); optical rotation, [alpha]20D = -135.6 degrees; thin-layer chromatography, three impurities were detected; high-performance liquid chromatography, a small amount of 10 impurities were detected: loss on drying, 0.10%; assay, 100.4% by spectrophotometry. Based on the above results, the raw material was authorized as the Riboflavin Reference Standard (Control 951) of National Institute of Health Sciences (Japanese Pharmacopoeia).

[Estradiol Reference Standard (Control 961) of National Institute of Health Sciences].

The raw material for estradiol was examined for preparation of the "Estradiol Reference Standard (Control 961)" of National Institute of Health Sciences. Analytical data obtained were as follows: melting point, 179.1 degrees C; UV spectrum, lambda max = 281 nm; IR spectrum, the same as that of the Estradiol Reference Standard of National Institute of Health Sciences (Control 931); optical rotation, [alpha]20D = +79.5 degrees; thin-layer chromatography, one impurity was detected; high-performance liquid chromatography (HPLC), a trace amount of three impurities were detected; loss on drying, 3.17%; assay, 99.4% by UV spectrophotometry and 99.0% by HPLC. Based on the above results, the raw material was authorized as the Estradiol Reference Standard (Control 961) of National Institute of Health Sciences.

[dl-Camphor Reference Standard (Control 961) of National Institute of Health Sciences].

The raw material of dl-camphor was examined for the preparation of the "dl-Camphor Reference Standard (Control 961)" of National Institute of Health Sciences. Analytical data obtained are as follows: UV spectrum, lambda max = 290 nm; IR spectrum, the same as that of the present JP Camphor Reference Standard (Control 953); melting point, 179.1 degrees C; purity test by gas-chromatography (GC), three kinds of impurities were detected; assay by GC, 99.8%. Based on the above results, the candidate raw material was authorized as the dl-Camphor Reference standard (Control 961) National Institute of Health Sciences (Japanese Pharmacopoeia).