Mechanisms of inhibitory effects of CoQ10 on UVB-induced wrinkle formation in vitro and in vivo.

Photodamaged skin exhibits wrinkles, pigmented spots, dryness and tumors. Solar UV radiation induces cyclobutane pyrimidine dimers (CPD) and further produces base oxidation by reactive oxygen species (ROS). ROS are thought to be a major factor to initiate the up-regulation of matrix metalloproteinases (MMPs) in keratinocytes and fibroblasts via activation of receptor proteins on the cell membrane of keratinocytes and fibroblasts, and to degrade fiber components in dermis, leading to wrinkle formation. Coenzyme Q10 (CoQ10) was reported to reduce ROS production and DNA damage triggered by UVA irradiation in human keratinocytes in vitro. Further, CoQ10 was shown to reduce UVA-induced MMPs in cultured human dermal fibroblasts. We speculated that UVB radiation-induced cytokine production in keratinocytes may be inhibited by CoQ10, resulting in the reduction of MMPs in fibroblasts leading to wrinkle reduction. Our in vitro studies showed that UVB-induced IL-6 production of normal human keratinocyte (NHKC) decreased in the presence of CoQ10. Furthermore, MMP-1 production of fibroblasts cultured with the medium containing CoQ10 collected from UVB-irradiated NHKC significantly decreased during 24 h culture. In the clinical trial study, we found that the use of 1% CoQ10 cream for five months reduced wrinkle score grade observed by a dermatologist. Taken together, our results indicate that CoQ10 may inhibit the production of IL-6 which stimulate fibroblasts in dermis by paracrine manner to up-regulate MMPs production, and contribute to protecting dermal fiber components from degradation, leading to rejuvenation of wrinkled skin.

The effect of sperm-oocyte incubation time on in vitro embryo development using sperm from a tetraparental chimeric bull.

The present study was designed as 5 x 4 factorial to investigate the effects of using sperm from 5 bulls, and varied sperm-oocyte incubation times (5, 10, 15 and 20 h) on the fertilization, cleavage rates and blastocyst formation on an in vitro bovine embryo production system. The bulls included a tetraparental Chimera, its sires (Japanese Black and Limousin), its maternal grand-sires (Japanese Brown and Holstein). The proportion of polyspermy, 2-pronuclei formation, fertilization, cleavage and development to blastocyst were affected (P < 0.001) by the duration of sperm-oocyte incubation, as well as by the interaction between bulls and their corresponding sperm-oocyte incubation time. Blastocyst rate observed after 5 h in oocytes inseminated with Chimera, Japanese Black and Limousin were higher (p < 0.05) than those observed at 20 h incubation. The proportion of blastocysts from oocytes inseminated with Japanese Black observed at 10 h of incubation did not differ from that of Chimera, but both were higher (p < 0.05) than those observed for the Limousin, Japanese Brown and Holstein sires. The present study showed that there was an effect by the duration of sperm-oocyte incubation on in vitro embryo development. The optimal time of sperm-oocyte incubation for the Chimera was similar to that of its sires (Japanese Black and Limousin) but differed from its maternal grand-sires (Japanese Brown and Holstein). The fertilization rates for the sperm from the Holstein bull increased up to 15 h suggesting that this might be the only bull that would benefit from a long incubation period for insemination.

Ultrasound-guided follicle aspiration and IVF in dairy cows treated with FSH after removal of the dominant follicle at different stages of the estrous cycle [corrected].

Recently, transvaginal ultrasound-guided follicle aspiration technology has been found to be of great value for in vitro fertilization (IVF) programs, even though the oocyte recovery rate and cleavage rate of transferrable embryos were low. In this study, we investigated the effect of the removal of the dominant follicle at different stages of the estrous cycle on the ovarian response of donor cows. Four experiments (EXPs) were devised. In EXP 1, 3 cows received 20 mg FSH on Day 1, ovulation occurred on Day 0, and on Day 3 follicles were aspirated. In EXP 2, the dominant follicle of the first wave was removed on Day 6 from 3 cows which received 20 mg FSH on Day 7 and on Day 9 follicles were aspirated. In EXP 3, 2 pregnant cows received 20 mg FSH on 70 d of pregnancy and 48 hr later follicles were aspirated a total of 5 times at 5-day intervals. In EXP 4, after ovulation on Day 0, 9 cows received 20 mg FSH on Days 8 to 14 of the estrous cycle and 48 hr after the last injection, follicles were aspirated once. The respective mean +/- SD numbers of aspirated follicles and recovered oocytes were higher (p < 0.01) in EXP 1 (13.4 +/- 1.7 and 8.7 +/- 2.3), EXP 2 (12.1 +/- 1.4 and 7.7 +/- 1.7) and EXP 3 (10.7 +/- 2.1 and 7.0 +/- 2.2) than in EXP 4 (5.8 +/- 2.3 and 3.1 +/- 1.6). The oocyte recovery rates were higher (p < 0.05) in EXP 1, EXP 2 and EXP 3 than in EXP 4. Similarly, the respective numbers of viable oocytes and cleavage rates were higher in EXP 1, EXP 2 and EXP 3 (6.0 +/- 1.3, 5.0 +/- 1.1 and 4.6 +/- 1.5 viable oocytes (p < 0.01); 66, 73 and 65% cleavage rates (p < 0.05)) than in EXP 4 (2.4 +/- 1.1; 46%). The numbers of morulae and blastocysts were higher (p < 0.05) in EXP 1, EXP 2 and EXP 3 than in EXP 4. In conclusion 1) removal of the dominant follicles from lactating and pregnant cows enabled viable oocytes to be recovered constantly and repeatedly by aspiration at different reproductive stages, and that viable blastocysts can be produced after IVF. 2) The presence or absence of a dominant follicle significantly affects the ovarian responses to FSH treatment. 3) This ultrasound-guided procedure proved to be an effective, repeatable and safe method for viable oocyte recovery from valuable pregnant donors.

Fertility of sperm from a tetraparental chimeric bull.

The purpose of the present study was to examine the ability of a tetraparental Chimera in producing IVF embryos. Cumulus oocytes complexes (COCs) were matured in vitro for 22 h. Frozen-thawed sperm of a Chimera (CH), as well as Japanese Black (JB), Limousin (L), Japanese Brown (JBr), Holstein (H) bulls were used for IVF. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h incubation was higher (P < 0.01) with the Chimera (CH) than with the Holstein and in Japanese Brown bulls, but did not differ from Japanese Black and Limousin bull sperm (79.0%, 71.2%, 72.5%, 57.8% and 57.0% for CH, JB, L, JBr and H sperm, respectively). Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera (O-CH) sperm were higher (P < 0.05) than with Japanese Brown (O-JBr) and (P < 0.01) than with Holstein (O-H) sperm, but did not differ from Japanese Black (O-JB) and Limousin (O-L) sperm (36/44, 81.8%; 28/35, 80.0%; 25/36, 69.4%; 19/43, 44.2% and 6/33, 18.2% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The cleavage rates of IVM oocytes inseminated with Chimera sperm were also higher (P < 0.001) than in Holstein, (P < 0.01) Japanese Brown and (P < 0.05) Limousin, but did not differ from Japanese Black sperm (181/239, 75.7%; 123/171, 71.9%; 108/186, 58.1%; 80/196, 40.8% and 30/186, 16.1% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The blastocyst rates of IVM oocytes inseminated with sperm were higher (P < 0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black (69/181, 38.1%; 48/123, 39.0%; 27/108, 25.0%; 7/30, 23.3% and 16/80, 17.8% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). Three findings suggested the sperm from this tetraparental Chimeric bull were able to be used for producing bovine IVF embryos.

Intracellular mechanism of ATP-induced depolarizing response in the bullfrog sympathetic ganglion cells.

Application of adenosine triphosphate (ATP) induced a depolarizing response in bullfrog sympathetic ganglion cells, which was associated with a decrease in membrane conductance. This depolarizing response was also produced by application of ADP, but not by adenosine, cyclic-AMP or cyclic-GMP. Suramin, an antagonist for the P2-purinoceptor, suppressed the response, while caffeine, an antagonist for the P1-purinoceptor, did not. Application of phospholipase C (PLC) inhibitors such as Li+ and 4-bromophenacyl bromide also suppressed the response. In addition, protein kinase C (PKC) inhibitors such as staurosporine and H-7 had a suppressant effect, while ryanodine, an inhibitor of Ca2 release, did not. These results suggest that ATP and ADP may stimulate P2-purinoceptor coupled with PLC, producing diacylglycerol, which activates PKC, resulting in the closing of K+ channel.

Dermatofibrosarcoma protuberans of the sole.

A 42-year-old Chinese man presented with a dome-shaped, red, tender, bony-hard tumor on his right sole. Histological examination revealed tumor nests which consisted mainly of fibroblast-like cells with large, spindle-shaped nuclei and histiocyte-like cells extending from the upper dermis to the subcutaneous tissue. The tumor cells showed both a storiform and a cartwheel pattern. The location of a dermatofibrosarcoma protuberans on the plantar aspect of the foot is exceptional.

A case of giant cell tumor of the tendon sheath developing on the palm.

A 22-year-old Japanese woman with a giant cell tumor of the tendon sheath on the palm is described. The location of the tumor is rare. Light microscopically, the tumor consisted of round or polygonal cells, giant cells, and foam cells. Electron microscopically, histiocyte-like cells and fibroblast-like cells were mainly observed, suggesting that the tumor may have originated from the synovial membrane.

Superovulation in the cow with a single intramuscular injection of FSH dissolved in polyvinylpyrrolidone.

It is desirable to reduce the number of treatments required to induce superovulation in cows. In this study we examined whether dissolving FSH in polyvinylpyrrolidone(PVP) would reduce the rate of absorption of FSH and allow it to be administered in a single dose for superovulation. In Experiment 1, 10 cows each received a single dose of FSH which contains 0.6% luteinizing hormone (FSH-R; 30 mg i.m.) dissolved in 30% PVP (10 ml) or in saline. In Experiment 2, a single injection of 30 mg FSH-R dissolved in 30% PVP was given to 25 cows, and 32 cows were injected twice daily in declining doses to receive a total of 28 mg FSH-R dissolved in saline. Prostaglandin F2alpha was given to all the cows 48 h after the first FSH treatment. Embryos were collected on Day 7 or 8 post insemination. In Experiment 1, the effect of FSH dissolved in PVP was compared with that dissolved in saline (number of recovered ova and embryos; 9.4+/-4.1 vs. 0). In Experiment 2, the rate of transferable embryos by single injection of FSH-R in PVP were significantly higher (P<0.05) than that of treatment of multiple injection groups. Progesterone concentration measured in serum collected 4 times from estrus (Day-0) to the day of embryo collection, indicated similar patterns in the 2 treatment groups. These findings suggest that PVP is a suitable solvent for prolonging the absorption of FSH given in a single injection thus providing a more practical approach of FSH administration.

[Effect of Y-25130, a selective 5-HT3 receptor antagonist, on the intestinal fluid secretion in rats].

We studied the effect of Y-25130 on the intestinal fluid secretion induced by 5-HT and cholera toxin in rats. 1) Net fluid secretion was increased dose-dependently by intramesenteric artery infusion of 5-HT (1-10 micrograms/min). 2) Y-25130 (0.01-1 mg/kg, i.v.) inhibited the net fluid secretion induced by 5-HT (3 micrograms/min). Granisetron and ondansetron also inhibited the net fluid secretion induced by 5-HT. 3) Methysergide did not inhibit the fluid secretion, and neither atropine nor tetrodotoxin inhibited it. 4) Cholera toxin (1-10 micrograms/2 ml into jejunal loops) caused profuse net fluid secretion. 5) Y-25130 (1 mg/kg, i.v.) inhibited the net fluid secretion induced by cholera toxin (3 micrograms/2 ml). Granisetron was inhibitory and ondansetron tended to inhibit the cholera toxin-induced secretion. These results suggest that 5-HT may increase the net fluid secretion through 5-HT3 receptors, and cholera toxin-induced fluid secretion may be at least partially mediated by 5-HT3 receptors. It is expected that Y-25130 may be useful for treating secretory diarrhea in humans.

Nuclear DNA content and distribution of Ki-67 positive cells in nodular fasciitis.

Nodular fasciitis (NF) is regarded as a disease of reactive growth of fibroblasts, although it frequently shows clinically rapid growth and sometimes has to be histologically discriminated from fibrosarcoma. In order to confirm the position of NF as a benign tumor, we studied 5 cases of NF by a quantitative analysis of the nuclear DNA content using flow cytometry in paraffin-embedded specimens. In 3 of the 5 cases, an immunohistochemical study using Ki-67 monoclonal antibody was also conducted in frozen sections. In the analysis of nuclear DNA content, DNA histograms showed a diploid pattern in all cases, and G1, S and G2 + M periods were calculated as 96.7%, 2.5% and 0.8% (on the average), respectively. Although the labeling index of Ki-67 antibody (positive cells/tumor cells; %) was as scattered as 1.3-7.8 (5.6 on the average), the mean value was higher than that in already reported dermatofibroma and dermatofibrosarcoma protuberans and lower than that in malignant fibrous histiocytoma. These results endorse the hypothesis that NF is a reactive benign disease and reflect the biological behavior of this disease, which may frequently show rapid growth.

Superovulation of Japanese black heifers treated with FSH-P and FSH-R.

The effects on superovulation in beef heifers of two kinds of porcine FSH preparations with different LH content, FSH-P and FSH-R, were compared. Forty-two heifers of the Japanese Black Breed received a total dose of 28AU of FSH-P containing 6.5% LH or FSH-R containing 0.58% LH intramuscularly, in a descending manner for four days. The mean numbers (+/- SD) of obtained ova/embryos and transferable embryos were 4.9 +/- 2.3, 1.5 +/- 0.9 and 11.2 +/- 2.4, 4.8 +/- 1.4, respectively. FSH-R treatment yielded a significantly greater number of ova/embryos and transferable embryos than FSH-P treatment (P < 0.05).

Comparative study of Ki-67 immunostaining and nuclear DNA content in histiofibrous tumors.

A comparative assessment was made of dermatofibroma, dermatofibrosarcoma protuberans, and malignant fibrous histiocytoma. The study was comprised of an immunohistochemical investigation using the Ki-67 monoclonal antibody and a quantitative analysis of cell nuclear DNA using flow cytometry. It was found that the index of Ki-67 monoclonal antibody labeling, the histogram pattern, and DNA index correlated with clinical malignancy, suggesting the usefulness of these indices for differential diagnosis and evaluation of prognosis.

Inhibition of jaw-opening reflex by stimulation of the central amygdaloid nucleus in the cat.

The effect of stimulation of the amygdaloid complex on the jaw-opening reflex (JOR) was studied in the cat anesthetized with pentobarbital sodium. The conditioning stimulation of the central amygdaloid nucleus (ACE), but not the other amygdaloid nuclei, markedly inhibited the JOR induced by the tooth pulp stimulation. Ipsilateral ACE conditioning stimulation with 300 microA produced inhibition which lasted approximately 500 ms from the cessation of the stimulation. Additionally, the microinjection of monosodium glutamate into ACE elicited inhibition of the JOR that lasted about 10 min. These findings suggest that the excitation of the cell bodies in ACE exerts inhibitory action on the trigeminal nociceptive reflex.

The projection pathway from the tooth pulp to the ipsilateral first somatosensory cortex (SI) in the cat.

About 40 per cent of tooth pulp-driven (TPD) neurones received afferent fibres from the ipsilateral tooth pulp. This ipsilateral afferent pathway was investigated in cats anaesthetized with nitrous oxide and halothane. The subcortical temperature of the brain was lowered to about 28 degrees C by perfusion of cold water within a thermode. Cooling the homotopic area contralateral to the recording site caused little change in the firing rate of short-latency TPD neurones upon ipsilateral pulp stimulation (n = 13). Microinjection (1-2 microliters) of 1 per cent lidocaine into the ipsilateral nucleus ventralis posteromedialis (VPM) caused significant diminution in the firing rate of short-latency TPD neurones (n = 11) to ipsilateral stimulation but not of long-latency TPD neurones (n = 8). About 35 per cent (13/35) of the TPD neurones distributed in the medial shell region of the VPM proper responded with short latency to ipsilateral pulp stimulation. These findings suggest that the ipsilateral input to short-latency TPD neurones in the oral area is carried via projection fibres from the ipsilateral VPM but not via commissural ones, and that the ipsilateral input to long-latency neurones is probably relayed in a site other than the ipsilateral VPM.