Resveratrol treatment increases mitochondrial biogenesis and improves viability of porcine germinal-vesicle stage vitrified-warmed oocytes.

Vitrification induces mitochondrial dysfunction in warmed oocytes, and degeneration and biogenesis of mitochondria are crucial for maintaining oocyte quality. The present study addresses a hypothesis that treatment of vitrified-warmed oocytes with resveratrol could improve the viability of oocytes by activating mitochondrial biosynthesis. Cumulus oocyte complexes (COCs) collected from gilt ovaries were vitrified, warmed, and cultured in a medium containing vehicle or 1 µM resveratrol. Resveratrol treatment improved survival, maturation, and mitochondrial membrane potential of vitrified-warmed oocytes, but did not improve the development into blastocysts after parthenogenetic activation. Resveratrol treatment increased mitochondrial synthesis, as determined by the expression levels of TOMM20 and mitochondrial DNA copy number, in vitrified-warmed oocytes, but not in non-vitrified oocytes. In addition, the effect of resveratrol treatment on mitochondrial synthesis was reduced by EX527, a SIRT1 inhibitor. Resveratrol treatment of vitrified-warmed oocytes increased the expression levels of genes involved in mitochondrial synthesis (TFAM, POLG, and PGC1α) and increased nuclear translocation of NRF2. Furthermore, vitrification induced mitophagy, as confirmed by a reduction in TOMM20 expression and decreased p62 aggregation, whereas resveratrol treatment did not affect these mitophagic features. In conclusion, vitrification induced mitochondrial clearance in oocytes, whereas activation of SIRT1 by resveratrol treatment facilitated the recovery of vitrified-warmed oocytes through the activation of mitochondrial synthesis.

Mitochondrial biogenesis and degradation are induced by CCCP treatment of porcine oocytes.

In this study, we investigated the mitochondrial quality control system in porcine oocytes during meiotic maturation. Cumulus cell oocyte complexes (COCs) collected from gilt ovaries were treated with 10  µM carbonyl cyanide-m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) for 2  h. The CCCP treatment was found to significantly reduce ATP content, increase the amount of phosphorylated AMP-activated protein kinase and elevate reactive oxygen species levels in oocytes. When the CCCP-treated COCs were cultured further for 44  h in maturation medium, the ATP levels were restored and the parthenogenetic developmental rate of oocytes to the blastocyst stage was comparable with that of untreated COCs. To examine the effects of CCCP treatment of oocytes on the kinetics of mitochondrial DNA copy number (Mt number), COCs treated with 0 or 10  µM CCCP were cultured for 44  h, after which the Mt number was determined by RT-PCR. CCCP treatment was found to increase the Mt number in the modified maturation medium in which mitochondrial degradation was inhibited by MG132, whereas CCCP treatment did not affect the Mt number in the maturation medium lacking MG132. The relative gene expression of TFAM was furthermore shown to be significantly higher in CCCP-treated oocytes than in untreated oocytes. Taken together, the finding presented here suggest that when the mitochondria are injured, mitochondrial biogenesis and degradation are induced, and that these processes may contribute to the recuperation of oocytes.

Escherichia coli lipopolysaccharide administration transiently suppresses luteal structure and function in diestrous cows.

The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1 ± 0.8 years old (mean ± s.e.m.), were given 10  ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 µg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm², P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P4) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P4 concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.

Expression of keratinocyte growth factor and its receptor in odontogenic keratocysts.

BACKGROUND: The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization. METHODS: The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR. RESULTS: KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1alpha increased the expression of KGF mRNA in the fibroblasts isolated from OKCs. CONCLUSION: KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1alpha.

Prostaglandin F 2 alpha stimulates endothelial nitric oxide synthase depending on the existence of bovine granulosa cells: analysis by co-culture system of endothelial cells, smooth muscle cells and granulosa cells.

Prostaglandin F(2 alpha) (PGF(2 alpha)) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF(2 alpha) receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF(2 alpha) and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage-dependent and the site-restricted effect of PGF(2 alpha) and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co-cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully-luteinized GC. PGF(2 alpha) stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix-cultures of EC and SMC with fully-luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF(2 alpha) at 1 h only when EC was cultured together with fully-luteinized GC but not with luteinizing GC. In all co-cultures, PGF(2 alpha) did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully-luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF(2 alpha).

Signaling pathways regulating IL-1alpha-induced COX-2 expression.

Interleukin-1alpha(IL-1alpha) stimulates the production of prostaglandin E(2) (PGE(2)) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1alphasignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1alphaincreased the expression of COX-2 mRNA and protein, and PGE(2) secretion in the fibroblasts. IL-1alphaincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC-which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-kappaB (NF-kappaB), respectively-attenuated the IL-1alpha-induced COX-2 mRNA expression and activated protein kinase C PGE(2) secretion. IL-1alpha(PKC), and PKC inhibitor staurosporine inhibited IL-1alpha-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1alpha-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1alphamay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-kappaB cascade.

Cloning and characterization of a GC-box binding protein, G10BP-1, responsible for repression of the rat fibronectin gene.

Fibronectin (FN) is an extracellular matrix protein that connects the extracellular matrix to intracellular cortical actin filaments through binding to its cell surface receptor, alpha5beta1, a member of the integrin superfamily. The expression level of FN is reduced in most tumor cells, facilitating their anchorage-independent growth by still unclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN gene, was isolated by using a yeast one-hybrid screen with the G10 stretch inserted upstream of the HIS3 and lacZ gene minimal promoters. G10BP-1 comprises 385 amino acids and contains two basic regions and a putative zipper structure. It has the same specificity of binding to three G-rich sequences in the FN promoter and the same size as the G10BP previously identified in adenovirus E1A- and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost undetectable in quiescent rat 3Y1 cells but increased steeply after growth stimulation by serum, reaching a maximum in late G1. Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G1-to-S progression. This down regulation was strictly dependent on the downstream GC box (GCd), and base substitutions within GCd abolished the sensitivity of the promoter to G10BP-1. In contrast, the level of Sp1, which competes with G10BP for binding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 determines the expression level of the FN gene. Preparation of glutathione S-transferase pulldowns of native proteins from the cell extracts containing exogenously or endogenously expressed G10BP-1, followed by Western blot analysis, showed that G10BP-1 forms homodimers through its basic-zipper structure.

A cooperative action of endothelin-1 with prostaglandin F(2alpha) on luteal function in the cow.

Prostaglandin F(2alpha) (PGF(2alpha)) is the primary luteolysin in the cow, and luteal endothelin-1 (ET-1) interacts with PGF(2alpha) during the process of luteolysis. In contrast, a developing corpus luteum (CL) is refractory to exogenous administration of PGF(2alpha). Thus, the present study was aimed to investigate the functional relationship between ET-1 and PGF(2alpha) in the mid-CL (PGF(2alpha)-sensitive) and early-CL (PGF(2alpha)-refractory). In the mid-CL model, cows (n = 6/treatment) were assigned to receive one of five types of treatments on day 10 of the estrous cycle: (1) an injection of saline; control, (2) a 500 microg of PGF(2alpha) analogue (sufficient dose to induce luteolytis); full-PG, (3) an intraluteal injection of 0.25 mg ET-1; ET-1, (4) a 125 micro g of PGF(2alpha) (insufficient dose to induce luteolytis); 1/4PG or (5) an intraluteal injection of 0.25 mg ET-1 after administration of a insufficient dose of PGF(2alpha) analogue; 1/4PG/ET. In the early-CL model, cows were assigned to receive one of two types of treatments on day 5 of the estrous cycle: (1) a sufficient dose of PGF(2alpha) analogue; PG (n = 5) or (2) an intraluteal injection ET-1 after a sufficient dose of PGF(2alpha); PG/ET (n = 7). In the mid-CL model, 1/4PG/ET resulted in a rapid reduction of progesterone (P) concentrations similar to that in full-PG from the next day. However, the levels of P in 1/4PG/ET (1.5-2.5 ng/ml) kept significantly higher than that in full-PG (< 0.5 ng/ml). ET-1 or 1/4PG did not decrease plasma P concentrations (4-6 ng/ml). The plasma ET-1 levels increased with the full-PG administration. In the early-CL model, both treatments had no effect on plasma P increase and ET-1 levels. The overall results indicate that the intraluteal ET-1 injection after administration of insufficient dose of PGF(2alpha) induces the depression of P secretion in vivo during the mid luteal phase in the cow, supporting the concept that ET-1 is one of a local mediator of functional luteolysis in the cow. The result further indicates that the early-CL is not only PG-refractory but also ET-1-refractory.

Isolation and characterization of different clones including myoepithelial-like variants from a clonal neoplastic epithelial duct cell line of human salivary gland origin.

A clonal neoplastic epithelial duct cell (HSGc) of human salivary gland origin has a fine structure similar to the intercalated duct cell and the capacity to express secretory component and lactoferrin. HSGc cells tend to form an occasional glandular arrangement in vitro and in vivo, and transplantation of cells into nude mice resulted in production of adenocarcinoma. By repeated single cell cloning, different types of clones could be isolated from HSGc. Cuboidal clones resemble the parent cell, but fail to form the glandular arrangement or express lactoferrin, suggesting a less differentiated type. Elongated clones have a fine structure similar to myoepithelial cells and carry myoepithelial markers such as S100 protein, actin, and myosin which are not detected in the HSGc and its cuboidal clones. These myoepithelial-like clones are able to express secretory component, lactoferrin, and lysozyme and to produce glycosaminoglycans, suggesting that they are a functionally active form of the neoplastic cell but different from the normal myoepithelial cell. Judging from their growth properties in vitro and in vivo, the myoepithelial-like clones are less malignant than HSGc or its cuboidal clones. Of four elongated clones, two did not produce tumors in athymic mice, while all of the cuboidal clones were tumorigenic. These findings suggest a possible conversion of the neoplastic duct cell to myoepithelial-like variants with low malignancy.

Extracellular matrix production and degradation by adenoid cystic carcinoma cells: participation of plasminogen activator and its inhibitor in matrix degradation.

Adenoid cystic carcinoma (AdCC) is characterized by low mitogenic activity, high invasiveness, and vigorous production and accumulation of extracellular matrix (ECM). As it does in vivo, a cell line (ACCS) derived from a human AdCC grows very slowly and displays potential for production of a large amount of ECM. ACCS cells also produce a significant amount of proteolytic enzymes, including urokinase-type plasminogen activator (uPA), and M(r) 72,000 and 92,000 gelatinases. These cells can degrade considerable amounts of ECM elaborated by normal mesenchymal cells, including rat muscle cells and human fibroblasts, mainly through a uPA-plasmin cascade. However, ECM elaborated by ACCS cells themselves is resistant to degradation by either the tumor cells or purified uPA in the presence of plasminogen, whereas the degradation rates of ACCS ECM and mesenchymal ECM by plasmin are comparable. Treatment of ECM with glycine (pH 2.7), which removes plasminogen activator inhibitor from the matrix, results in an increase in the rate of ACCS ECM degradation by uPA. Moreover, fibrin agarose reverse zymography, autoradiography, and immunoblotting showed a high level of plasminogen activator inhibitor type 1 in ACCS ECM. These findings suggest that the plasminogen activator inhibitor type 1 in ECM produced by AdCC cells may play a role in preventing matrix destruction by the tumor itself, and thus the ECM components of tumor origin are stably accumulated in the intercellular spaces and may support or promote the growth of AdCC cells, which have primitively low growth activity.

Biological characterization of pseudocyst-forming cell lines from human adenoid cystic carcinomas of minor salivary gland origin.

Two cell lines (ACCS and ACCY) were isolated from two individuals with adenoid cystic carcinoma (AdCC) using tissue culture techniques. Both cell lines have similar morphology, i.e., elongated and flattened cells with slender cytoplasmic processes. The two cell lines tend to form pseudocysts, which are a specific architectural feature of AdCC. Coexpression of cytokeratin and vimentin was found in the two cell lines, which occasionally also contained S-100 protein and lactoferrin or lysozyme immunoreactivity. Moreover, ACCS and ACCY displayed potential for the production of a large amount of extracellular matrix including basal lamina components such as fibronectin, laminin, and type IV collagen and glycosaminoglycans which are also part of the basal lamina. These findings suggest that the tumor cells, probably basal or myoepithelial like cells, are responsible for the formation of the peculiar stroma of AdCC consisting of a large amount of collagen-like fibers, basal lamina components, and mucopolysaccharides.

Growth inhibition and differentiation of human salivary adenocarcinoma cells by medium conditioned with normal human fibroblasts.

The present study demonstrates that normal human fibroblasts (WI-38) exert a profound influence on the growth and differentiation of HSGc-C5, a clonal neoplastic epithelial cell line of human salivary gland origin. Coculture of HSGc-C5 with WI-38 resulted in a slowing of growth and an increase in glycosaminoglycan synthesis by an indirect effect involving a diffusible factor(s). Conditioned medium (CM) from WI-38 grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum affected HSGc-C5 as follows. The CM suppressed growth of monolayer cells; inhibited DNA synthesis; suppressed growth (decrease in size of colonies) in semisolid agar; stimulated glycosaminoglycan synthesis, induced expression of functional markers of the salivary gland, such as the secretory component, lactoferrin, and lysozyme; inhibited expression of alkaline phosphatase; and induced morphological alteration into elongated cells. These findings strongly suggest that WI-38 CM contains a factor(s) which inhibits growth and induces differentiation of HSGc-C5. The CM was also active on other human cancer cells as a growth inhibitor, but not on normal human fibroblasts. Partial purification and characterization of the factor(s) suggests that it may be a novel protein carrying both tumor inhibiting and differentiation inducing activities.

Sp1 decoy transfected to carcinoma cells suppresses the expression of vascular endothelial growth factor, transforming growth factor beta1, and tissue factor and also cell growth and invasion activities.

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the angiogenic factors produced by tumor cells, vascular endothelial growth factor is considered to be the most potent and pathologically important. The synthesis of this growth factor has been shown to be modulated through Sp1 function following stimulation by tumor necrosis factor alpha (TNF-alpha). Oligodeoxynucleotides (ODNs) were synthesized with either the consensus sequence for Sp1 binding (Sp1 decoy ODNs) or a mutated form of this sequence (mt-Sp1 decoy ODNs). Using the hemagglutinating virus of Japan (HVJ)-liposome method, we transferred these ODNs into cultured cancer cells (A549 and U251 cells). The TNF-alpha-mediated expression of both VEGF and transforming growth factor beta1 and tissue factor (TF) by the cancer cells could be simultaneously suppressed to less than 30% by transfection of Sp1 decoy ODNs but not by mt-Sp1 decoy ODNs. In addition, in vitro invasiveness, synthesis of mRNA for urokinase-type plasminogen activator, and cell proliferation of both cell lines were also inhibited to 40% by the transfection of only Sp1 decoy ODNs. These results suggested that the Sp1 decoy strategy would be effective for regulating tumor growth by simultaneously reducing cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness.

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer.

The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.

Effects of positive pressure in odontogenic keratocysts.

Intracystic fluid pressure is thought to be involved in odontogenic cyst growth. In this study, we investigated the effects of positive pressure on the expression of interleukin-1alpha (IL-1alpha), matrix metalloproteinases (MMPs), and prostaglandin E2 (PGE2) in odontogenic keratocysts to determine whether this pressure stimulates inflammatory cytokine production and signaling of osteoclastogenic events. Positive pressure enhanced the expression of IL-1alpha mRNA and protein in odontogenic keratocyst epithelial cells, and increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and the epithelial cells. The pressure-induced secretions were inhibited by an interleukin-1 receptor antagonist. Recombinant human interleukin-1alpha (rhIL-1alpha) increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in the fibroblasts. Furthermore, in the fibroblasts, rhIL-1alpha enhanced the expression of macrophage colony-stimulating factor (M-CSF) mRNA, and rhIL-1alpha-induced PGE2 increased the expression of nuclear factor kappaB ligand (RANKL) mRNA. Thus, positive pressure may play a crucial role in odontogenic keratocyst growth via stimulating the expression of IL-1alpha in epithelial cells.

Blood flow: a key regulatory component of corpus luteum function in the cow.

Prostaglandin F2alpha (PGF2alpha) is the primary luteolysin in the cow. During the early luteal phase, the corpus luteum (CL) is resistant to the luteolytic effect of PGF2alpha. Once mature, the CL becomes responsive to PGF2alpha and undergoes luteal regression. These actions of PGF2alpha coincide with changes in luteal blood flow (BF): PGF2alpha has no effect on BF in the early CL, but acutely increases BF in the peripheral vasculature of the mature CL within 30 min of PGF2alpha injection. During spontaneous luteolysis, luteal BF increases on Days 17-18 of the estrous cycle, prior to any decrease in plasma progesterone (P). The increase in luteal BF is synchronous with an increase in plasma PGFM levels, suggesting that pulsatile release of PGF2alpha from uterus stimulates the increase in luteal BF. Serial biopsies of these CL showed that mRNA expression for endothelial nitric oxide synthase (eNOS) together with endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) increases on Days 17-18 when the luteal BF is elevated. On Day 19 when plasma P level firstly decreases, eNOS mRNA returns to the basal level whereas ET-1 and ACE mRNA remains elevated. Cyclooxygenase-2 (COX-2) mRNA expression increases on Day 19. In support of these data, an in vivo microdialysis study revealed that luteal ET-1 and angiotensin II (Ang II) secretion increases and precedes PGF2alpha secretion during spontaneous luteolysis. In conclusion, we show for the first time that an acute increase of BF occurs in the peripheral vasculature of the mature CL together with increases in eNOS expression and ET-1 and Ang II secretion in the CL during the early stages of luteolysis in the cow. We propose that the increase in luteal BF may be induced by NO from large arterioles surrounding the CL, and simultaneously uterine or exogenous PGF2alpha directly increases ET-1 and Ang II secretion from endothelial cells of microcapillary vessels within the CL, thereby suppressing P secretion by luteal cells. Taken together, our results indicate that an acute increase in luteal BF occurs as a first step of luteolysis in response to PGF2alpha. Therefore, local BF plays a key role to initiate luteal regression in the cow.

Severity of oral mucositis correlates with the response of oral cancer to preoperative radiochemotherapy.

Oral mucositis is a dose-limiting toxic effect of radiotherapy and chemotherapy on oral cancer. The purpose of the present study is to assess the relationship between tumor response and oral mucositis in preoperative radiochemotherapy for oral cancer retrospectively. Fifty-four cases of oral squamous cell carcinoma were treated with concurrent radiochemotherapy prior to surgery. When oral mucositis was evaluated with the WHO scale, severe oral mucositis (Grades 3 and 4) developed in 22 cases (41%). A more than 50% reduction in tumor size was clinically observed in 38 cases (70%). From histopathological analysis of the surgical specimens all tumor cells observed appeared to be non-viable in 16 cases (29%). The cases with Grade 1, Grade 2, Grade 3 and Grade 4 oral mucositis included 33%, 62%, 85% and 89% of clinical good-response cases and 0%, 24%, 31% and 55% of histopathological good-response cases, respectively. This retrospective study suggests that severe oral mucositis promises a good response of oral squamous cell carcinoma to radiochemotherapy.

Resveratrol improves the quality of pig oocytes derived from early antral follicles through sirtuin 1 activation.

During oocyte growth, the number of mitochondria drastically increases and mitochondrial function profoundly affects the oocyte competence. Resveratrol is a well-known activator of sirtuin 1 (SIRT1), which has a role in cellular energy homeostasis and mitochondrial biogenesis. The main aim of the present study was to examine the effect of supplementation of culture media with resveratrol on oocyte development and mitochondrial number and functions. Lipid contents and developmental ability of the oocytes grown in vitro were also examined. Oocyte-granulosa cell complexes were collected from early antral follicles of gilt ovaries and were cultured in medium containing 0 or 2 µM resveratrol for 14 days. Immunostaining revealed that resveratrol enhanced SIRT1 expression in oocytes. Antrum formation during the culture period and survivability of the granulosa cells surrounding the developed oocytes did not differ between the two concentrations of resveratrol. In addition, the ability of oocytes to complete meiotic maturation did not differ between the two concentrations of resveratrol, whereas the ability of oocytes to develop to the blastocyst stage was improved significantly by resveratrol (7.4% vs. 1.6%; P < 0.05). Resveratrol upregulated the ATP content in oocytes grown in vitro, and the addition of 2 µM of the SIRT1 inhibitor 6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX527) diminished this effect although EX527 alone had no effect on ATP content. The mitochondrial DNA copy number in oocytes determined by quantitative real-time polymerase chain reaction increased during in vitro oocyte development, but resveratrol did not affect the kinetics of the mitochondrial DNA copy number. We found that resveratrol also increased the expression level of phospho-5'-adenosine monophosphate-activated protein kinase in oocytes but decreased the lipid content in oocytes grown in vitro. These results suggest that resveratrol increased the ATP content in oocytes via energy homeostasis and improved the developmental ability of oocytes grown in vitro.

Establishment of bone morphogenetic protein 2 responsive chondrogenic cell line.

A clonal cell line named RMD-1 was established from the skeletal muscle of a 20-day fetal rat. RMD-1 represents a morphologically homogeneous population of undifferentiated mesenchymal cells, expressing alpha-smooth muscle actin and type I collagen, but no cartilage-associated genes. When cultured in agarose gel containing 100 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2; BMP-2), RMD-1 cells formed colonies and showed chondrocyte-like features as assessed by their ultrastructure, metachromatic staining with toluidine blue, and the production of large hydrodynamic-size proteoglycans. RMD-1 cells also differentiated into chondrocytes when the cells were plated at high density (over 2.5 x 10(5) cells/cm2) on type I collagen and incubated in medium containing 0.5% fetal bovine serum and 100 ng/ml of BMP-2. This chondrogenic differentiation was evidenced by a distinct morphological change into spherical cells, an increase in the levels of sulfated glycosaminoglycans, a decrease in type I collagen mRNA and the expression of cartilage-associated genes, including type II collagen, type IX collagen, aggrecan and alkaline phosphatase. In the presence of ascorbic acid and 10% serum, RMD-1 cells increased in size and expressed type X collagen as well as high alkaline phosphatase activity, then induced matrix mineralization. Thus, RMD-1 is a unique cell line that can differentiate from undifferentiated mesenchymal cells into hypertrophic chondrocytes.

Gelatinolytic activity of matrix metalloproteinase in tumor tissues correlates with the invasiveness of oral cancer.

We examined whether or not the gelatinolytic activity in tumor tissue was associated with the invasion and metastasis of oral squamous cell carcinoma (OSCC). Tissue homogenates were prepared from 57 biopsy specimens of OSCC. The gelatinolytic activities in the homogenates were measured by gelatin zymography and its densitometric analysis. The Immnunoblot findings revealed the major gelatinolytic activities to be due to matrix metalloproteinase (MMP)-2 and -9. The zymography-detected gelatinolytic activities of MMP-2 and MMP-9 in the tissue specimens significantly correlated with the degree of immunohistochemical staining detected in frozen sections of the same biopsy specimens. According to a histopathological analysis of the mode of invasion, highly invasive cases showed the increased gelatinolytic activities of MMP-2 as well as MMP-9 in the tissue specimens. Although no significant differences were observed in the gelatinase activities between the metastatic cases and the non-metastatic cases, the levels of tissue inhibitor of MMP (TIMP)-1 in the tumor tissue specimens were higher in the non-metastatic cases than in the metastatic cases. The cases with the high levels of MMPs and low levels of TIMP-1 thus seemed to have a high potential to metastasize. As a result, the zymographic measurement of the gelatinolytic activity in biopsy tissue specimens may therefore be useful in predicting the behavior and prognosis of OSCC.