Depigmentation efficacy of galacturonic acid through tyrosinase regulation in B16 murine melanoma cells and a three-dimensional human skin equivalent.

Sugar is a well-known cosmetic ingredient for moisturizing skin with minimal side-effects. Several reports have demonstrated an antimelanogenic effect of sugar in melanocytes. We evaluated the whitening efficacy of galacturonic acid (GA), the main component of pectin, as an anti-melanogenic agent. GA significantly suppressed melanin synthesis and secretion in a concentration-dependent manner in α-melanocyte stimulating hormone-treated B16 melanoma cells, and inhibited tyrosinase activity and expression at a dose of 10 mmol/L. In a three-dimensional human skin equivalent (MelanoDerm), GA clearly brightened tissue colour. Haematoxylin and eosin and Fontana-Masson (F&M) staining of tissue sections revealed decreased melanin production without skin tissue collapse in the presence of GA. Interestingly, GA dramatically suppressed gene expression of the melanogenic proteins tyrosinase, tyrosinase-related protein (TYRP)-1 and microphthalmia-associated transcription factor, but not TYRP-2. The results support the utility of GA as an effective candidate antimelanogenic agent.

A novel antagonist to the inhibitors of apoptosis (IAPs) potentiates cell death in EGFR-overexpressing non-small-cell lung cancer cells.

In the effort to develop an efficient chemotherapy drug for the treatment of non-small-cell lung cancer (NSCLC), we analyzed the anti-tumorigenic effects of a novel small molecule targeting the inhibitor of apoptosis (IAPs), HM90822B, on NSCLC cells. HM90822B efficiently decreased IAP expression, especially that of XIAP and survivin, in several NSCLC cells. Interestingly, cells overexpressing epidermal growth factor receptor (EGFR) due to the mutations were more sensitive to HM90822B, undergoing cell cycle arrest and apoptosis when treated. In xenograft experiments, inoculated EGFR-overexpressing NSCLC cells showed tumor regression when treated with the inhibitor, demonstrating the chemotherapeutic potential of this agent. Mechanistically, decreased levels of EGFR, Akt and phospho-MAPKs were observed in inhibitor-treated PC-9 cells on phosphorylation array and western blotting analysis, indicating that the reagent inhibited cell growth by preventing critical cell survival signaling pathways. In addition, gene-specific knockdown studies against XIAP and/or EGFR further uncovered the involvement of Akt and MAPK pathways in HM90822B-mediated downregulation of NSCLC cell growth. Together, these results support that HM90822B is a promising candidate to be developed as lung tumor chemotherapeutics by targeting oncogenic activities of IAP together with inhibiting cell survival signaling pathways.

Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine.

This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.

Genitourinary rhabdomyosarcoma with systemic metastasis in a young dog.

A 2-year-old intact female Golden Retriever presented due to rapidly progressing depression, ascites, dysuria, abdominal pain, and severe vaginal bleeding. At necropsy, the retroperitoneal space was expanded by multiple coalescing neoplastic nodules and the uterine wall was thickened with poorly defined neoplastic infiltrates. The urinary bladder was markedly thickened due to botryoid nodules exhibiting exophytic growth into the lumen. Metastases to lung, liver, kidney, and abdominal and thoracic lymph nodes were also noted. Microscopically, the genital tract and retroperitoneal masses were consistent with the alveolar subtype of rhabdomysarcoma, while the urinary bladder mass had characteristics of the embryonal subtype. Immunohistochemically, the neoplastic cells in all these tissue sites were intensely positive for desmin, sacromeric actin, and vimentin, while they were uniformly negative for cytokeratin and smooth muscle actin. Phosphotungstic acid hematoxylin stain revealed cross-striations in the cytoplasm of scattered neoplastic cells. Based on the gross findings, histopathology, and immunohistochemistry, genitourinary rhabdomyosarcoma with multisystemic metastases was made.

The study on photoreflectance characteristic of semi-insulating GaAs surface region by its exposure to 6 MeV electron beam.

Photoreflectance measurements were performed to investigate the optical properties in the electron beam irradiation semi-insulating GaAs(e-beam irradiation GaAs) and semi-insulating GaAs(SI-GaAs). A considerable increase of the PR amplitudes has been registered after the e-beam irradiation in comparison with the GaAs. It is that result of a higher electron scattering on the lattice defects created by the e-beam.

Subcellular redistribution of protein kinase C isozymes is associated with rat liver cirrhotic changes induced by carbon tetrachloride or thioacetamide.

BACKGROUND AND AIMS: Protein kinase C (PKC) plays a key role in the alteration of signal transduction in the liver, which may contribute to the development of liver cirrhosis. The aim of the present study was to examine the subcellular redistribution of PKC isozymes in rat liver cirrhosis, which is induced by two different cirrhotic chemical agents, carbon tetrachloride (CCl4) and thioacetamide (TAA). METHODS AND RESULTS: Thioacetamide and CCl4 were administered to rats for 8 and 30 weeks, respectively before rats were killed and autopsies performed at 9, 20 and 30 weeks later. The TAA induced a fibrotic pattern in the liver that differed from that produced by CCl4, notably in the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear-cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed that severe cirrhotic changes were present 9 weeks after the commencement of CCl4 treatment and 30 weeks after TAA treatment. DISCUSSION: When the subcellular redistribution of PKC isozymes (PKCalpha, -beta1, -delta, and -epsilon) was examined, all the PKC isozymes in CCl4-treated rats were found to be translocated to the membrane fraction, which may mean PKC activation, and then downregulated by proteolytic degradation after 9 weeks of treatment, which coincided with peak cirrhotic changes. All rats treated with CCl4 recovered to the control level after 20 weeks of treatment. In the case of TAA-treated rats, PKC isozymes were translocated to the particulate fraction of the liver after 9 weeks of treatment and this persisted in most of the rats for the duration of the experiment. CONCLUSIONS: From these results, it would appear that PKC translocation preceded morphologic changes, and that an altered subcellular distribution of the PKC isozyme may be associated with the response to liver damage and carcinogenesis.

Radiation augments a sequential program of differentiation in PKC inhibitor- pretreated mouse epidermal cells.

The aim of this study was to determine whether gamma-rays affect differentiation in mouse epidermal cells. After a pre-treatment with the PKC inhibitor staurosporin (STS) or 1-(5-isoquinolinesulfomyl)-2-methylpiperazine (H7), gamma-rays were irradiated with or without an elevation of 0.12 mM Ca2+ and expressions of differentiation markers and each PKC isozyme were examined in normal primary and v-rasHa transformed mouse keratinocytes. Gamma-rays induced the expressions of differentiation markers of keratin 1 and 10 (K1 and 10), filaggrin, loricrin and SPR-1 in normal keratinocytes when the Ca2+ concentration was increased, and these phenomena were augmented in H7 pretreated cells. Similar results were obtained in STS pretreated cells; in this case, gamma-rays enhanced the expressions of the differentiation markers even without an elevated Ca2+ concentration. In v-rasHa transformed cells, gamma-rays induced the expression of differentiation markers not only at 0.05 mM Ca2+, but in 0.12 mM Ca(2+)-shifted cells, and in H7 pretreated cells, these phenomena were augmented. The translocation of PKC alpha to the particulate fraction was seen in H7 pretreated normal keratinocytes. Radiation also induced PKC alpha expression in STS pretreated cells, independent of Ca(2+)-shift, as well as altered expressions of PKC delta and -eta, while expressions of PKC alpha, -delta, -epsilon, and -eta were enhanced in v-rasHa transformed cells. In conclusion, gamma-rays augmented the expressions of both spinous and granular differentiation markers in normal and v-rasHa transformed keratinocytes and this effect was augmented when PKC inhibitors were used, which may be mediated by the cellular redistribution of PKC isozymes.

Retrospective analysis of 305 consecutive cases of endometrial ablation and partial endomyometrial resection.

STUDY OBJECTIVE: To assess the efficacy and safety of operative resectoscopy, partial endomyometrial resection, and endometrial ablation in the evaluation and treatment of abnormal uterine bleeding. DESIGN: Retrospective analysis of 305 consecutive cases of endometrial ablation and partial endomyometrial resection. SETTING: Midwestern urban obstetric and gynecology group practice and teaching hospitals. PATIENTS: Three hundred five women (age 30-72 yrs) with abnormal uterine bleeding. Interventions. Partial endomyometrial resection and endometrial ablation. MEASUREMENTS AND MAIN RESULTS: Of the 301 patients who completed surgery and follow-up, 283 (97%) reported improvements in amenorrhea (55%), hypomenorrhea (41%), and eumenorrhea (1%). Ten (3%) failed to report improvement. In 24 (7.9%) women, hysterectomy was performed for various reasons after endometrial ablation, including recurrent bleeding in 4. Four uterine perforations occurred, infection was suspected in one patient, and loss of Laminaria occurred in another; all patients, however, were observed appropriately and discharged the same day of surgery. CONCLUSIONS: Partial endomyometrial resection and endometrial ablation is a safe and effective treatment of abnormal uterine bleeding, and may be an alternative to hysterectomy in selected patients.

Postablation-tubal sterilization syndrome.

Operative resectoscopy and endometrial ablation are often performed to treat abnormal uterine bleeding, but little is known about the potential late complications of these procedures. We reviewed the records of 305 women who underwent endometrial ablation at a midwestern obstetrics and gynecology group practice and teaching hospital between July 1990 and October 1995. For 71 women, tubal ligation, salpingectomy, or tubal sterilization was performed at the time of ablation. Of these, six (8.4%) developed intense cyclic pain 5 to 40 months after surgery. Four subsequently underwent exploratory laparotomy and hysterectomy, and two others underwent laparoscopic tubal resection and destruction. Gross pathologic findings revealed hematosalpinx, and microscopic examination showed endometriosis, acute and chronic inflammation of the fallopian tubes, and acute and chronic myometritis. We believe these characteristic clinical and pathologic findings are consistent with postablation-tubal sterilization syndrome, a distinct clinical entity arising as a late complication of endometrial ablation in patients with a history of tubal ligations and/or obstruction.

Current concepts of cell-cycle regulation and its relationship to oocyte maturation, fertilization and embryo development.

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.

Effect of calcium ion on the maturation of cumulus-enclosed pig follicular oocytes isolated from medium-sized graafian follicles.

Porcine follicular oocytes from medium-sized follicles (3-5 mm in diameter) were cultured in modified Hank's balanced salts solution (MHBS) to which pyruvate, lactate, and glucose were added as an energy source. Bovine serum albumin (0.4%) was added as a protein source and the oocytes were cultured for 42 h at 37 degrees C in 5% CO2 in air. In this medium porcine oocytes underwent 80-90% nuclear maturation after 42 h. Oocytes were cultured in MHBS with various amounts of CaCl2 as well as in the presence of verapamil, a Ca2+ channel blocker, and the divalent cationophore A23187. It was found that the lowest concentration of Ca2+ required for oocyte maturation was around 0.0265-0.053 mM. Such a requirement for Ca2+ in the culture medium extended through metaphase II. If Ca2+ was omitted during the final 4 h of culture, the metaphase II chromosomes appeared extremely condensed or degenerated. Verapamil at a level of 0.2 mM inhibited germinal vesicle breakdown or resulted in degeneration, whereas lower concentrations did not affect oocyte maturation. In the presence of 0.02 mM verapamil, the maturation of cumulus-enclosed oocytes was not affected, whereas at the same dose of verapamil the maturation of denuded oocytes was inhibited. Less than 3.8 X 10(-7) M divalent cationophore did not inhibit oocyte maturation. Maturation was inhibited by 3.8 X 10(-7) and 3.8 X 10(-6) M divalent cationophore. In conclusion, maintenance of oocytes in a nondegenerated state also requires the constant presence of Ca2+ in the culture medium.

Actions of hormones and other factors upon oocyte maturation.

Oocyte maturation is controlled by a combination of hormonal and local follicular factors. Osmolarity, pH, and perhaps Ca2+ concentration of the surrounding medium are also important. Follicular fluid contains a low molecular weight OMI which acts to keep the oocyte from maturing. Luteinizing hormone added to cultured cumulus enclosed porcine oocytes can reverse the inhibitory action of OMI. The level of OMI in the follicular fluid appears to decrease as the follicle matures. Addition of FSH and prolactin to cultured granulosa cells stimulates OMI secretin whereas addition of testosterone or dihydrotesterone brings about a decrease in OMI secretion. Elevated LH in vivo may bring about oocyte maturation before ovulation by (a) an antagonist action on OMI; (b) stimulating the synthesis of testosterone by theca cells and thus inhibiting the synthesis of OMI by granulosa cells; and (c) action on the granulosa cells to promote luteinization which may also cause a decrease in OMI synthesis. The hastened oocyte maturation associated with follicular atresia could be due to a decline in OMI due to granulosa cell death and/or elevated follicular androgens.

Porcine granulosa and cumulus cell properties. LH/hCG receptors, ability to secrete progesterone and ability to respond to LH.

In order to compare the properties of isolated cumulus and granulosa cells, granulosa cells and cumulus cells surrounding oocytes were harvested from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) porcine antral follicles and the number of LH/hCG receptors was measured by the binding of [125I]hCG. The ability of the cells to secrete progesterone in culture was examined in the presence and absence of hCG and LH. In 3 separate experiments of 1-h incubations at 37 degrees C using cells harvested from medium-sized follicles, granulosa cells bound 10--15-fold more iodinated hCG than an equivalent number of cumulus cells. During a 2-day culture period, cumulus cells secreted less progesterone than granulosa cells from medium- and large-sized antral follicles (p less than 0.01). The potential of both cumulus and granulosa cells to secrete progesterone in culture increased as the follicle progressed from small to large size. Also, the ability of the oocyte to mature in culture increased with antral follicle size. Concurrently the ability of cumulus-oocyte complexes to form monolayers in culture decreased as the follicle matured. Cumulus and granulosa cells harvested from small- and medium-sized follicles responded similarly to LH and hCG with a stimulation in progesterone secretion after 2-6 days in culture.

Maturation of rabbit follicular oocytes in a defined medium of varied osmolality.

Rabbit oocytes from follicles greater than or equal to 1 mm in diameter were cultured for 18 h in a defined medium with osmolality adjusted in 20 mosmol increments from 230 to 350 mosmol by altering only the NaCl concentration. Adjustment, based upon determination of the osmolality of the medium, was necessary because a difference existed between calculated and achieved osmolality in this complex solution. The proportions of oocytes which matured to meiosis II with polar body formation were 64, 68, 64 and 65% in media of 250, 270, 290 and 310 mosmol, respectively.

Effects of hormones on the maturation of rabbit oocytes recovered from follicles of various sizes.

Progesterone stimulated oocytes to develop more rapidly in culture. The time-dependent effect was more pronounced on large preovulatory Graafian follicles than on small- and medium-sized follicles. Treatment with LH had no effect.