Therapeutic potential of dedifferentiated fat cells in a rat model of osteoarthritis of the knee.

Introduction: Mature adipocyte-derived dedifferentiated fat cells (DFATs) represent a subtype of multipotent cells that exhibit comparable phenotypic and functional characteristics to adipose-derived stem cells (ASCs). In this study, we assessed the chondroprotective properties of intra-articularly administrated DFATs in a rat model of osteoarthritis (OA). We also investigated in vitro the expression of anti-inflammatory and chondroprotective genes in DFATs prepared from the infrapatellar fat pad (IFP) and subcutaneous adipose-tissue (SC) of human origin. Methods: In the cell transplantation experiment, rats were assigned to the DFAT and Control group (n = 10 in each group) and underwent anterior cruciate ligament transection (ACLT) accompanied by medial meniscus resection (MMx) to induce OA. One week later, they received intra-articular injections of 1 × 106 DFATs (DFAT group) or PBS (control group) four times, with a weekly administration frequency. Macroscopic and microscopic evaluations were conducted five weeks post-surgery. In the in vitro experiments. DFATs derived from the IFP (IFP-DFATs) and SC (SC-DFATs) were prepared from donor-matched tissue samples (n = 3). The gene expression of PTGS2, TNFAIP6, PRG4, BMP2, and BMP6 under TNF-α or IFN-γ stimulation in these cells was evaluated using RT-PCR. Furthermore, the effect of co-culturing synovial fibroblasts with DFATs on the gene expression of ADAMTS4 and IL-6 were evaluated. Results: Intra-articular injections of DFATs significantly inhibited cartilage degeneration in the rat OA model induced by ACLT and MMx. RT-PCR analysis revealed that both IFP-DFATs and SC-DFATs upregulated the expression of genes involved in immune regulation, anti-inflammation, and cartilage protection such as PTGS2, TNFAIP6, and BMP2, under stimulation by inflammatory cytokines. Co-culture with DFATs suppressed the expression of ADAMTS4 and IL6 in synovial fibroblasts. Conclusions: The intra-articular injection of DFATs resulted in chondroprotective effects in the rat OA model. Both SC-DFATs and IFP-DFATs induced the expression of anti-inflammatory and chondroprotective genes in vitro. These results indicate that DFATs appear to possess therapeutic potential in inhibiting cartilage degradation and could serve as a promising cellular resource for OA treatment.

Transplantation of dedifferentiated fat cell-derived micromass pellets contributed to cartilage repair in the rat osteochondral defect model.

BACKGROUND: Mature adipocyte-derived dedifferentiated fat (DFAT) cells possesses the ability to proliferate effectively and the potential to differentiate into multiple linages of mesenchymal tissue; similar to adipose-derived stem cells (ASCs). The purpose of this study is to examine the effects of DFAT cell transplantation on cartilage repair in a rat model of osteochondral defects. METHODS: Full-thickness osteochondral defects were created in the knees of Sprague-Dawley rats bilaterally. Cartilage-like micromass pellets were prepared from green fluorescent protein (GFP)-labeled rat DFAT cells and subsequently transplanted into the affected right knee of these rats. Defects in the left knee were used as a control. Macroscopic and microscopic changes of treated and control defects were evaluated up to 12 weeks post-treatment with DFAT cells. To observe the transplanted cells, sectioned femurs were immunostained for GFP and type II collagen. RESULTS: DFAT cells formed micromass pellets expressing characteristics of immature cartilage in vitro. In the DFAT cell-transplanted limbs, the defects were completely filled with white micromass pellets as early as 2 weeks post-treatment. These limbs became smooth at 4 weeks. Conversely, the defects in the control limbs were still not repaired by 4 weeks. Macroscopic ICRS scores at 2 and 4 weeks were significantly higher in the DFAT cells-transplanted limbs compared to those of the control limbs. The modified O'Driscol histological scores for the DFAT cell-transplanted limbs were significantly higher than those of the control limbs at corresponding time points. GFP-positive DAFT cells were detected in the transplanted area at 2 weeks but hardly visible at 12 weeks post-operation. CONCLUSIONS: Transplantation of DFAT cell-derived micromass pellets contribute to cartilage repair in a rat osteochondral defect model. DFAT cell transplantation may be a viable therapeutic strategy for the repair of osteochondral injuries.

Chattonella (Raphidophyceae) bloom spatio-temporal variations in Tachibana Bay and the southern area of Ariake Sea, Japan: Interregional displacement patterns with Skeletonema (Bacillariophyceae).

In 2010, a massive bloom of the raphidophycean flagellate Chattonella occurred in the Ariake Sea and Tachibana Bay. Bloom dynamics and hydrographical conditions were examined by field survey. The development and decline of the bloom occurred three times in Tachibana Bay. First and third bloom developments synchronized with precipitation, and the second bloom developed in synchronization with a salinity decrease which occurred in relation to an increase of river discharge from the Chikugo River which takes several days to flow from the Ariake Sea. These results imply that the bloom was transported with the low salinity water from the Ariake Sea to Tachibana Bay. During blooms along the northern coast of Shimabara Peninsula, the predominant phytoplankton species changed from Chattonella to Skeletonema. Low salinity water intrusion induced an interregional difference of the Chattonella and Skeletonema bloom spatially-differentiated by the salinity in the Ariake Sea and Tachibana Bay.

Variability of factors driving spatial and temporal dispersion in river plume and Chattonella antiqua bloom in the Yatsushiro Sea, Japan.

The dynamics of river plume in relation to harmful blooms of the raphidophycean flagellate, Chattonella antiqua in summer 2008-2010 in the Yatsushiro Sea, Japan were studied using a hydrodynamic model and monitoring data. In the southern area, the bloom formed in the waters stratified by a halocline caused by the southward expansion of riverine water from the Kuma River after the bloom initially forming in the northern area. The timing of the southward riverine water advection can be explained by the balance between the wind stress term and the pressure gradient term calculated from the horizontal density difference between the northern and southern areas. The wind stress and pressure gradient terms were evaluated using the sea surface temperature, salinity, wind speed and direction at two stations. Real time monitoring or continuous observations in these areas will enable nowcasts of bloom expansion when a bloom develops in the northern area.

Production of fertile offspring from oocytes grown in vitro by nuclear transfer in cattle.

Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360,000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence.

In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 µM of milrinone, the enucleation rate was significantly improved by 100 µM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 µM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 µM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.

Optimization of oxygen concentration for growing bovine oocytes in vitro: constant low and high oxygen concentrations compromise the yield of fully grown oocytes.

The oxygen environment in cell culture has a significant impact on the health and performance of cells. Here, we compared the effects of reduced (5%) and ambient (20%) oxygen concentrations on bovine oocyte-granulosa cell complexes, each containing a growing oocyte 90-102 µm in diameter, cultured for 14 days. Both oxygen concentrations showed some advantages and disadvantages; in 5% oxygen, the survival rate of oocytes was significantly higher than in 20% oxygen, but the resulting oocytes were significantly smaller, which was a serious disadvantage. During the first 4 days of culture, the growth and viability of oocytes were satisfactory using 5% oxygen. This observation led us to examine the effect of changing the oxygen concentration from 5% to 20% on Day 4 in order to minimize the expected disadvantages of constant 5% and 20% oxygen. The largest population of fully grown oocytes was obtained from cultures in which the oxygen concentration was changed in this way, which also led to higher oocyte viability than in constant 20% oxygen. A similar tendency was found in the frequency of oocytes becoming blastocysts after in vitro fertilization. Surviving oocytes eventually became located within an enlarged dome-like structure, and although the 5% oxygen environment may have been appropriate for oocyte growth in the early stages, 20% oxygen may have been necessary for the growth of oocytes in the dome-like structure. These results indicate an effective way of modulating oxygen concentration according to the growth of oocyte-granulosa cell complexes in vitro.

Growth of bovine oocyte-granulosa cell complexes cultured individually in microdrops of various sizes.

In mammalian embryo culture, the embryo:medium volume ratio can substantially affect embryo developmental performance. In the present study, we tested the possibility of improving the growth of bovine oocytes by reducing the medium volume, from a typical volume used in mouse follicle culture to a minimum possible level. A total of 282 complexes, each containing a growing oocyte 87-100 mum in diameter, were individually placed in microdrops of 2, 5, 10 or 20 microl and cultured for 13 days in a modified TCM-199 supplemented with 4% polyvinylpyrrolidone (molecular weight: 360 kDa). Oocyte diameter was measured every other day to trace the growth of each oocyte. Half the medium was replaced every other day or every day, and comparison revealed that daily replacement was more favorable for culture of these microdrops. The highest survival rate, 95%, occurred in the 20-microl microdrops, where most oocytes continued to grow throughout the culture period. In comparison, in the 5- and 10-microl microdrops, more oocytes died, and growth slowed towards the end of culture. In the 2-microl microdrops, which had the highest death rate, growth virtually ceased after 9 days. The surviving oocytes were usually accompanied by a characteristic dome-like structure of the granulosa cell mass, except in the 2-microl microdrops. In conclusion, the 20-microl microdrops allowed oocyte growth at an acceptable level, and any further reduction of the volume only had a negative impact on oocytes.

Large duodenal somatostatinoma in the third portion associated with severe glucose intolerance.

A 51-year-old man was admitted with hyperglycemia and a duodenal tumor. Although his glycemic control was poor, basal C-peptide levels were not suppressed. Further examination revealed a mass measuring 7.8 cm in diameter in the third portion of the duodenum. Duodenectomy revealed a slow-growing sessile tumor located near Treitz's ligament. The immunohistochemical profile of sections of the specimen revealed the presence of somatostatin. The patient's serum somatostatin was elevated to 300 pg/ml preoperatively, but was reduced to 10 pg/ml postoperatively. Glycemic control also normalized after the operation.

Pasteurella multocida septicemia caused by close contact with a domestic cat: case report and literature review.

We report here a case of Pasteurella multocida infection caused by cat exposure presenting with septic shock, sinusitis, and pneumonia. The patient was a febrile 20-year-old woman who had been experiencing disturbed consciousness progressively. She had close contact with a domestic cat and had received some scratches on both arms. A magnetic resonance imaging (MRI) scan of the head showed a high intensity in the paranasal cavity, and a computed tomographic (CT) scan of the chest showed bilateral lung consolidations. The pathogen was identified as P. multocida by the cultures from blood and nasal discharge. She was given intensive antibiotic therapy with ceftriaxone and piperacillin, continuous hemodiafiltration (CHDF) therapy, and anticoagulation therapy. Owing to these therapeutic regimens, the septic shock was successfully treated without complications. We also review the literature on P. multocida septicemia.

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.

Cilostazol inhibits high glucose-mediated endothelial-neutrophil adhesion by decreasing adhesion molecule expression via NO production.

OBJECTIVE: Endothelial-neutrophil adhesion is crucial for vascular injury, the major cause of diabetic vascular complications. On the other hand, platelet aggregation inhibitors, frequently used for diabetic patients with intermittent claudication, have been shown to decrease the incidence of atherosclerosis-mediated diseases (acute myocardial infarction and stroke). However, whether these agents act directly on the endothelial reactions to hyperglycemia remains unclear. Therefore, we examined their direct effects on endothelial-neutrophil adhesion and expression of endothelial adhesion molecules induced by high glucose. METHODS AND RESULTS: After human endothelial cells were cultured in high glucose medium, neutrophils from healthy volunteers were added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring their myeloperoxidase (MPO) activities, and surface expression of endothelial adhesion molecules was determined with an enzyme immunoassay. Of the platelet aggregation inhibitors tested, only cilostazol significantly attenuated the adhesion through decreasing expression of intercellular adhesion molecule-1 (ICAM-1) and P-selectin. In addition, nitric oxide (NO) synthase inhibitors reduced the inhibitory effects of cilostazol, but a protein kinase C (PKC) activator did not. CONCLUSIONS: Cilostazol may act directly on endothelial cells to inhibit expression of adhesion molecules and neutrophil adhesion induced by high glucose through increasing NO production.

In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium.

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

Cerivastatin ameliorates high insulin-enhanced neutrophil-endothelial cell adhesion and endothelial intercellular adhesion molecule-1 expression by inhibiting mitogen-activated protein kinase activation.

BACKGROUND AND AIMS: There is growing evidence that hyperinsulinemia is linked to the development of atherosclerosis in patients with diabetes. We demonstrated previously that high insulin exacerbates neutrophil-endothelial cell adhesion and endothelial intercellular adhesion molecule (ICAM)-1 expression through activation of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase. Though 3-hydroxymethyl-3-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been employed as therapeutic agents in the treatment of dyslipidemia, which is frequently accompanied by diabetes mellitus; it is not known whether statins protect against leukocyte-endothelial interactions, especially in hyperinsulinemia. In this study, we determined which statin(s) could protect against endothelial reactions to high insulin. METHODS: Studies of adhesion between neutrophils from healthy volunteers and human umbilical vein endothelial cells incubated in regular insulin-rich medium with or without statins were performed. Adhered neutrophils were quantified by measuring their myeloperoxidase (MPO) activities, and endothelial expression of ICAM-1 was examined using an enzyme immunoassay. RESULTS: Both the increased neutrophil-endothelial cell adhesion and ICAM-1 expression caused by high insulin (100 microU/ml) for 48 h were significantly attenuated by pretreatment with cerivastatin (0.01 microM), but not by fluvastatin (0.5 microM) or pravastatin (0.05 microM). These protective actions of cerivastatin were attenuated by a key intermediate in the cholesterol biosynthesis pathway, mevalonate (400 microM). In addition, cerivastatin attenuated both neutrophil-endothelial cell adhesion and endothelial ICAM-1 expression enhanced by a MAP kinase activator, anisomycin (1 microM) but not by a PKC activator, PMA (10 nM). CONCLUSIONS: These results suggest that through inhibiting MAP kinase but not PKC activation therapy with cerivastatin would be promising strategy for inhibiting neutrophil-endothelial cell adhesion and endothelial ICAM-1 expression enhanced by high insulin, which is closely correlated with atherosclerosis.

In vitro and in vivo developmental potential of nuclear transfer embryos using bovine cumulus cells prepared in four different conditions.

We examined the effect of culture of donor cells on nuclear transfer efficiency using bovine cumulus cells treated with four different conditions: (1). group A, the cells removed from cumulus-oocyte complexes (COC) after aspiration of ovarian follicles; (2). group B, the cells removed from COC after in vitro maturation; (3). group C, the cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (4). group D, the cells cultured in DMEM with 0.5% FBS for an additional 5 days. Analysis of cell cycle using flow cytometry revealed that the relative proportion of donor cells at G0/G1 phase of cell cycle was 89.7% in group A, 89.5% in group B, 76.0% in group C, and 90.6% in group D. The developmental rates to blastocyst stage in groups C (45.3%) and D (46.4%) were significantly (p < 0.05) higher than in groups A (17.5%) and B (31.9%). After transfer of blastocysts produced in each group, nine of 24 recipients became pregnant on day 30. A total of five live calves were obtained from cumulus cells in all groups (group A [n = 1], group B [n = 1], group C [n = 2], and group D [n = 1]).

Statins inhibit high glucose-mediated neutrophil-endothelial cell adhesion through decreasing surface expression of endothelial adhesion molecules by stimulating production of endothelial nitric oxide.

Neutrophil-endothelial adhesion is a crucial step in vascular inflammation, which is recognized as the direct cause of atherosclerosis-mediated serious diseases. We demonstrated previously that high glucose increased adhesion in a protein kinase C (PKC)-dependent manner within 48 h through increasing surface expression of endothelial adhesion molecules. On the other hand, statins, used for patients with hypercholesterolemia, have been shown to decrease the incidence of atherosclerosis-mediated diseases, but direct effects of statins on endothelial cells remain unclear. In this study, we examined the effects of these compounds on high glucose-mediated neutrophil-endothelial adhesion with respect to the participation of PKC and nitric oxide (NO). After human endothelial cells were cultured for 48 h in high glucose medium, neutrophils from healthy volunteers were added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring their myeloperoxidase activities, and surface expression of endothelial adhesion molecules was determined with an enzyme immunoassay. Both pravastatin (0.05 microM) and fluvastatin (0.5 microM) significantly attenuated the adhesion mediated by 27.8 mM glucose for 48 h through decreasing surface expression of endothelial adhesion molecules (intercellular adhesion molecule-1, P-selectin, and E-selectin). NO synthase inhibitors reduced the inhibitory effects of statins, whereas statins did not affect the adhesion mediated by a PKC activator. These data suggest that statins act directly on endothelial cells to inhibit expression of adhesion molecules and neutrophil adhesion mediated by high glucose through increasing endothelial NO production, but not by inhibiting PKC.

Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

The mechanisms of inhibitory actions of gliclazide on neutrophils-endothelial cells adhesion and surface expression of endothelial adhesion molecules mediated by a high glucose concentration.

BACKGROUND: We previously reported that culture of endothelial cells in the presence of high glucose concentrations (27.8 and 55.5 mM) increase neutrophils adhesion because of the increase in endothelial adhesion molecules expression via activation of a protein kinase C (PKC) pathway. The antidiabetic sulfonylurea gliclazide, but not glibenclamide, inhibited these events, but the mechanisms involved were not clarified then. We present hereafter the results of further investigations of that effect with special reference to PKC activation. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured for 48 h in a glucose-rich medium and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activities and the surface expression of endothelial adhesion molecules was determined by enzyme immunoassay. RESULTS: Culture in the presence of a high glucose concentration (27.8 mM for 48 h) increased neutrophils-endothelial cells adhesion and the surface expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on the endothelial cells. These phenomena were significantly inhibited by gliclazide (20 microM). On the other hand, phorbol 12-myristate 13-acetate (PMA), a PKC activator, had an effect similar to a high glucose concentration and that effect was also inhibited by gliclazide. CONCLUSIONS: These data suggest that gliclazide inhibits high glucose-mediated neutrophils-endothelial cells adhesion and expression of endothelial adhesion molecules through inhibition of a PKC pathway.

Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture.

In vitro cell culture is a convenient tool for studying cellular mechanisms. In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined. Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production. Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells. But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium. In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-tau) supplementation. It was IFN-tau that inhibited the production of MMP-2. In addition, progesterone at a lower dose appeared to inhibit MMP-2 production. Both TNF-alpha and TNF-beta strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect. Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity.

Implantation and placental development in somatic cell clone recipient cows.

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.