Accumulation Of Droplets Research Articles

The role of vitamin D in weight-related metabolic derangements, inflammation and insulin resistance

T role of vitamin D in maintaining calcium homeostasis and bone mineralization is well-established. We investigated the effect of vitamin D on weight-related inflammation, metabolic derangements and associated histological changes of the live. Vitamin D treatment induced a significant gradual decrease in animals’ weight under high fat diet fed conditions compared to a steady increase in the weights of controls. Furthermore, vitamin D reduced the levels of inflammatory markers including TNF-α, CRP and IL-6. There were also lower concentrations of insulin and C-peptide, which indicates improvement in insulin resistance. Liver histology showed a marked accumulation of fat droplets in approximately 60-70% of hepatocytes of animals while vitamin D rendered the whole structure more normal. Transcriptional regulation of genes in tissues primarily involved in glucose homeostasis in response to vitamin D treatment revealed a general trend of up-regulation in the expression levels of insulin receptor (IR), glucose transporter-4 (Glut4), insulin receptor substrate (IRS) and vitamin D receptor (VDR) in particular under conditions resembling obesity (high fat fed animal model). These data signify an important effect of vitamin D on weightrelated insulin resistance and a protective consequence on the liver structure.

Establishment and evaluation of a new model for studying lipogenesis in grass carp (Ctenopharyngodon idella) preadipocytes

The objective of this study was to establish and evaluate a new model for studying lipogenesis in grass carp preadipocytes. The morphology characteristic from preadipocytes to mature adipocytes was observed with the microscopic morphology, and the proliferation kinetics of cells was tested by cell counting. In addition, the nature and differentiation degree of cells were evaluated using Oil Red O staining, lipase activity determination, and reverse transcription PCR (RT-PCR) assay. Morphologically, grass carp preadipocytes started to attach and grow on day 3, then they resembled fibroblasts, and most underwent attachment, proliferation, and growth arrest with subsequent accumulation of intracellular lipid droplets before becoming mature adipocytes. Glycerol-3-phosphate dehydrogenase (GPDH) activity was increased gradually during the progress of culture. Analysis of RT-PCR confirmed that peroxisome proliferator-activated receptor-γ expression patterns were consistent with my observations regarding GPDH activity. In summary, grass carp preadipocytes cultured with 10% FBS at 28°C in a humidified 5% CO2 atmosphere have high proliferation potential. Furthermore, the cells synthesize a range of markers that are consistent with this cell type. We conclude therefore that the grass carp preadipocytes described here have high capacity for lipogenesis and may, therefore, represent a unique tool for studying fish fat cell development and metabolism.

Abstract B69: Cellular neutral lipid content is a novel biomarker of cancer preventive activity

Abstract Introduction: Commonly used biomarkers to assess the cancer preventive activity of cancer preventive agents include markers of cell proliferation or apoptosis. However, the expression of proliferation or apoptosis markers is highly variable, and measurement is often done using semi-quantitative assays. In addition, cancer preventive agents may act through mechanisms that do not depend on proliferation or apoptosis. Therefore, identification of new biomarkers indicative of the cancer preventive activity of chemopreventive agents is essential for the development of new cancer preventive drugs. Methods: We applied high-content microscopic analysis to assess the phenotypic changes occurring in normal mammary epithelial cells (HMECs) after treatment with cancer preventive drugs by measuring cell proliferation, DNA and neutral lipid content and localization, as well as cell cycle distribution of the cells. Gene specific knock-down was achieved by transfections with siRNA duplexes. Measurement of transcript levels was performed using quantitative real-time RT-PCR. Results: Multiparametric analysis of high-throughput image based studies have revealed that anti-proliferative activity of cancer preventive rexinoids is associated with increased formation of cytoplasmic lipid droplets. Accumulation of cytoplasmic neutral lipids was restricted to cell types that undergo significant growth suppression by bexarotene (HMECs, SUM225, and ZR75.1), but not observed in retinoid-resistant cells (MCF10A, HCC1937, and HCC1143). Other growth suppressive drugs including the kinase inhibitor lapatinib, vitamin D, and certain natural compounds, including epicatechine monogallate, duartin, actinonin and homoisoflavonoids, were also found to increase cellular neutral lipid content in breast cells. The addition of external fatty acids, such as oleic and palmitoleic acids did not result in dose-dependent growth suppression or accumulation of neutral esterified lipid droplets. Lipid accumulation and growth suppression were associated with the up-regulation of genes involved in regulating the formation of neutral lipids, triglycerides and phospholipids, but not genes involved in de novo fatty acid synthesis. Conversely, knock-down of key proteins controlling cellular triglyceride synthesis and storage, diacylglycerol acyltransferase 1 (DGAT1) and adipophyllin (Adfp) increased cell proliferation whereas their induction was associated with growth suppression in HMECs. Conclusion: These results suggest that increased accumulation of cellular triglycerides may be a useful biomarker to assess the preventive effect of putative cancer preventive agents. These results support the systematic testing of cellular neutral lipid levels in preclinical and clinical models testing novel chemopreventive agents. This work was supported by the grant RO1 CA 78480 (to P.H.B.). Citation Information: Cancer Prev Res 2011;4(10 Suppl):B69.

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.

Antitumor effects of tetrazanbigen may be related to its interference with lipid metabolism of human hepatocellular carcinoma cells

Tetrazanbigen (TNBG), a novel synthetic antitumor drug, may induce the death of cancer cells through a tumor-associated lipoidosis mechanism and result in accumulation of lipid droplets (LDs) in the cytoplasm. In this study, the mechanism underlying the antitumor effects of TNBG was investigated in human hepatocellular carcinoma cell line (QGY-7701 cells). The subcellular proteome of QGY-7701 cells after TNBG treatment was isolated using an established two-dimensional gel electrophoresis (2-DE) technique. Gel images were analyzed by the 2-DE PDQuest software. The differentially expressed proteins were identified by matrix-associated laser desorption ionization time-of-flight mass (MALDI-TOF-MS) spectrometry. Two proteins of interest, microsomal triglyceride transfer protein (MTTP) and sterol regulatory element binding protein 1(SREBP-1), the levels of which were significantly changed in the TNBG-treated cells, were further characterized by western blot assay. A total of 155 proteins, including 56 cytoplasm proteins, 65 membrane proteins and 34 nucleus proteins, were detected to be differentially expressed, and 33 proteins were successfully identified, of which 11 proteins were related to lipid synthesis, 4 proteins to lipid degradation and 3 proteins to lipid transport. Results from Western Blot assay were consistent with proteome analysis. The antitumor effects of TNBG may be related to its interference with lipid metabolism of cancer cells, resulting in accumulation of LDs. MTTP may be a main cause of TNBG induced accumulationof LDs. Key words: Tetrazanbigen, antitumor, subcellular proteomics, lipid metabolism, microsomal triglyceride transfer protein.

Cytoplasmic Accumulation of Liquid-Crystal Like Droplets in Post-Infection Sputum Generated by Gram-Positive Bacteria

Massive liquid crystal droplets (LCDs) have been reported in early embryogenesis and implicated in pathological progression of human diseases. The presence of LCDs has even been established as an effective diagnostic hallmark of Fabry-Anderson's disease. In this study, we report the presence of LCDs, identified by established thermal stage phase transition methods, in sputum collected during the recovery phase of respiratory infection by gram-positive bacteria. This finding provides additional insight on the breadth of liquid crystal presence in human pathology. Further study on the formation of these LCDs may lead to new perspectives on post-infection removal of infectious agents.

Increased uptake of low density lipoprotein by SV40-transformed smooth muscle cells

We compared the metabolism of low density lipoprotein (LDL) in SV40-transformed smooth muscle cells (TSMCs) to that in nontransformed smooth muscle cells (SMCs). When SMCs were incubated in medium with 100 μg/ml LDL for 24 hours, they did not accumulate sudanophilic lipid droplets. On the other hand, when TSMCs were incubated in medium containing more than 100 μg/ml LDL, they accumulated a large amount of lipid droplets in their cytoplasm. When cells were incubated with 200 μg/ml LDL for 24 hours, cholesteryl ester levels significantly increased in TSMCs (18.3±3.53 μg/mg protein), as compared with SMCs (2.40±0.85 μg/mg protein). However, there was no difference in the cellular level of free cholesterol between the TSMCs and SMCs. Although the TSMCs and SMCs had a similar number of binding sites for LDL, the TSMCs demonstrated a markedly higher uptake of LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (Dil-LDL), compared with the SMCs. SMCs that had been pretreated with 100 μg/ml of unlabeled LDL for 24 hours showed a decreased uptake of Dil-LDL. In contrast, TSMCs incorporated Dil-LDL independently of the preincubation with 100 μg/ml LDL. The presence of brefeldin A, which may block the transport of glycoproteins from the ER to Golgi apparatus, had less of an effect on the uptake of LDL in the TSMCs than in the SMCs. These results suggest that SV40-transformed smooth muscle cells show an increased uptake of LDL independent of the cellular cholesterol level, which may induce the accumulation of lipid droplets in their cytoplasm. A LDL receptor-independent pathway may be related to the increased uptake of LDL in SV40-transformed smooth muscle cells.

Predicting the preferential sites to liquid droplet erosion of the bellows assemblies by CFD

Bellows assemblies are placed within extraction steam lines to absorb the differential displacement (driven primarily by thermal growth) between the turbine lower casing and the condenser shell in nuclear power plants. The thin wall thickness and service environmental conditions of the bellows predetermine their vulnerable endurance to the liquid droplet erosion (LDE). A three-dimensional computational fluid dynamics (CFD) bellows model proposed in this paper consists of a hydraulic model and a LDE model aiming to investigate the LDE on the external surface of the bellows assemblies. The result shows that the most prone site to LDE locates at the upwind side of the first bellows convolution (USFBC). The droplet diameter has significant effect on the LDE extent, rather than the LDE distribution. Moreover, the addition of the weld seam before the bellows can mitigate the LDE of the bellows downstream. In addition, the protecting cover (PC) effectively prevents the bellows from the LDE, but causes the droplet accumulation on the convolution, which possible leads to corrosive damage. The PC structure was designed optimization according to the requirements of the displacement compensation, the manufacture cost and the LDE resistance.

Theoretical and Numerical Analysis of the Mechanical Erosion in Steam Turbine Blades. Part I

The methodology of calculation of the velocity distribution for the stream frictionless and the drops in the flow line, on the basis of the frictionless, two-dimensional, stationary, transonic and homogenous flow is established. The knowledge of conditions that govern the low pressure section of steam turbines in the last stage to have an approximate movement of the droplets in the blade cascades and the accumulation of droplets on the stator blades, flowing through the steam, is presented. This study is used for developing a code in Fortran about the velocity distribution in the output of stator blades that have flow conditions of wet steam, in order to understand the causes that originate the erosion on the blades of the last stages in the low pressure section of steam turbines.

Abstract 50: Comparison of NMR lipid profiles in mitotic arrest and apoptosis as indicators of drug resistance

Abstract Aim: To compare NMR-visible lipids in paclitaxel exposed cells undergoing apoptosis or mitotic arrest in order to explore their utility as a biomarker of drug resistance. Methods: Cultured cervical cell lines (HeLa, C33A and Me180, ATCC, USA) were exposed to 1 µM paclitaxel (Sigma, UK) for 8, 16, 24, and 48 hours (n=3). Diffusion-weighted (DW) spectra were acquired using a 11.74T spectrometer (Avance Bruker BioSpin, Germany) using a stimulated echo sequence with bipolar gradients. Cellular morphology was assessed with transmission electron microscopy (TEM) using uranyl acetate followed by lead citrate staining. Following TOPRO-3 and nile red co-staining, flow cytometry and confocal microscopy verified cell size and viability, visualized cell cycle phase distribution, apoptotic features, and accumulation of cytoplasmic lipid droplets. Western blots were used to assess activation (by phosphorylation) of cytoplasmic phospholipase A2 (P-cPLA2) and expression of fatty acid synthase (FAS) at each stage. Results: 24 h after exposure to paclitaxel, all lines showed >65% mitotic arrest, and HeLa showed apoptosis as well. At 48 h HeLa cells progressed to apoptosis (due to mitotic catastrophe) while C33A and Me180 cells progressed beyond mitotic arrest to normal morphology or multinucleation indicating resistance. There was significant increase in saturated and unsaturated lipids at 24 h (mitotic arrest) followed by a further striking increase at 48 h in all lines, especially in the 5.3ppm peak (unsaturated) in HeLa at 48 h. Unsaturated lipids increased more than saturated lipids in all lines, triglycerides increase the most in HeLa cells. Resistant lines showed lower increased methylene / methyl (1.3 ppm / 0.9 ppm) ratio (from 1.3 to 2.8, 1.7 to 2.8 compared with 1.5 to 3.8 in HeLa cells at 48 h). The levels of P-cPLA2 and FAS were unchanged at 24 h followed by a drop at 48 h in HeLa cells. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines with increase in droplet size only in HeLa cells at 24 and 48 h. This implies that the larger droplets are associated with apoptosis but that smaller ones are less specific. Apoptotic cells showed 3-4 x the number of droplets compared to cells arrested in mitosis. Conclusion: After exposure of cells to paclitaxel, increase in lipids on NMR results from increased visibility not synthesis (more droplets, FAS not increased). In apoptosis predominant increases are in unsaturated fatty acids in larger droplets, whereas paclitaxel resistant lines accumulate smaller droplets with significantly less triglycerides. Acknowledgements: This work was funded by the EC FP6 Marie Curie Action: Early Stage Training (contact No. 020718). We also acknowledge the support received for the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) (grant C1060/ A10334) and NHS Funding to the NIHR Biomedical Research Centre. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 50.

Current concepts of the pathogenesis of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) - common chronic liver disease, characterized by pathological accumulation of fat droplets, not associatedwith alcohol. NAFLD is often a component of other diseases such as metabolic syndrome, diabetes, obesity, and contributes to the prevalence of CVDamong the population.Asymptomatic disease, the difficulty of diagnosis, the lack of a unified concept of treatment NAFLD - topical issues that require more in-depth studyand worthy of attention a wide range of specialists.

Do Cytoplasmic Lipid Droplets Accumulate in Immature Oocytes from Over-Conditioned Repeat Breeder Dairy Heifers?

One of the main sources of repeat breeding in dairy cattle, caused by fertilization failure or early embryonic death, is metabolic stress during lactation. Nutrition seems also to play a role when the condition is seen in heifers, where oocyte cytoplasmic maturation is impaired. To determine whether over conditioning affects oocyte morphology, immature oocytes were collected by ovum pick-up (OPU) twice weekly during 5 weeks from three over-conditioned repeat breeder dairy heifers (RBH) and two normal virgin heifers (VH, controls) of the Swedish Red breed, monitored by body weight and condition. Oocyte quality was assessed under stereomicroscope and further examined by transmission electron microscope for accumulation of cytoplasmic lipid deposits. After OPU, the RBH yielded more low quality oocytes (60% vs 52% for VH, p = 0.14). The relative occupancy of osmophilic lipid droplets in the cytoplasm was higher in oocytes of bad quality compared with good ones, especially in RBH (p = 0.08) but also in VH (p = 0.11). Moreover, the oocytes from over-conditioned RBH showed higher amounts of cytoplasmic lipid deposits both in good (p = 0.14) and, even more prominent, in bad quality oocytes (p = 0.06). Such accumulation of lipid droplets may imply increased sensitivity to oxidative stress, hinder cytoplasmic maturation and lead to subfertility, as accounted in over-conditioned repeat breeders of the Swedish Red breed.

Signalling pathways involved in 1-nitropyrene (1-NP)-induced and 3-nitrofluoranthene (3-NF)-induced cell death in Hepa1c1c7 cells

We previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin-alpha reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca(2+). These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features.

A potential role for free fatty acids in the pathogenesis of preeclampsia

Pregnant women with the vascular complication of preeclampsia show altered lipid metabolism characterized by elevated circulating triglycerides and nonesterified free fatty acids. We have compared the effect of maternal plasma from women with and without preeclampsia on cultured vascular endothelial cells and determined whether these plasma-induced changes were reproduced with free fatty acid solutions of palmitic, oleic and linoleic acid, representative of circulating levels reported in preeclampsia. Lipid accumulation was quantified by oil-red O staining, apoptosis by terminal dUTP nick-end labelling (TUNEL) and the measurement of mitochondrial redox capacity, and membrane potential recorded using MTT reduction and JC-1 accumulation for human umbilical vein endothelial cells (HUVECs) exposed to plasma and free fatty acids. Lipid droplet accumulation was significantly increased in cultured HUVECs conditioned with maternal plasma from pregnancies with preeclampsia compared with normal uncomplicated controls. This increase was replicated following exposure to free fatty acids at the combined concentrations defined in preeclampsia. Plasma from these women also caused a significant decrease in mitochondrial dehydrogenase activity, a marked reduction in mitochondrial membrane potential and an increase in apoptosis compared with normal pregnancy. Again these effects were reproduced using free fatty acids in combination at the levels previously associated with preeclampsia. These findings support the concept of a circulating pathogenic factor for preeclampsia and highlight the possibility that this factor is not a single compound but perhaps the combined elevation of the free fatty acids palmitic, oleic and linoleic acid in the maternal circulation.

Effects of tetrazanbigen on the protein expression in human hepatocellular carcinoma cell line QGY-7701

Tetrazanbigen (TNBG) is a novel synthetic antitumor drug with significant antitumor effects on common solid tumors in vitro and in vivo. It may lead to death of cancer cells through a tumor-associated lipoidosis mechanism, and result in lipid droplets (LDs) accumulation at the cytoplasm. In this study, the effects of TNBG on protein expression in human hepatocellular carcinoma cell line QGY-7701 were studied for elucidating its antitumor mechanism. The proteins extracted from TNBG-treated human hepatocellular carcinoma cell line QGY-7701 were analyzed and compared with control cells by two-dimensional gel electrophoresis. The differential proteins were identified by matrix-associated laser desorption ionization time-of-flight mass (MALDI-TOF-MS) spectrometry. Two proteins of interest, the levels of which were significantly increased in TNBG-treated cells, were further characterized by Western blot analysis. The results showed a total of 846+/-23 spots in control cells and 853+/-30 spots in TNBG-treated cells. Twenty-six up-regulated or down-regulated proteins were found by analyzing differential proteomic 2-DE map. Eleven of them were identified by mass spectrometry. They were protein disulfide-isomerase precursor, 94 kD glucose-regulated protein, heat shock protein (HSP) 90-alpha, ATP-citrate lyase, HMG-CoA reductase, glucose-6-phosphate 1-dehydrogenase, very-long-chain specific acyl-CoA dehydrogenase, squalene synthetase, sterol regulatory element-binding protein 1, fructose-bisphosphate aldolase A, and peroxiredoxin-1. These up-regulated or down-regulated proteins are mostly related to lipid metabolism. The TNBG antitumor mechanism is probably to influence tumor lipid metabolism, resulting in accumulation of LDs in tumor cells.

Pomegranate flower ameliorates fatty liver in an animal model of type 2 diabetes and obesity

Aims of the study Fatty liver is the most common cause of abnormal liver function tests. We investigated the effect and its underlying mechanism of pomegranate flower (PGF), a traditional antidiabetic medicine, on fatty liver. Materials and methods At the endpoint of treatment of male Zucker diabetic fatty (ZDF) rats with PGF extract (500 mg/kg, p.o. × 6 weeks), liver weight index, hepatic lipid contents (enzymatic colorimetric methods) and droplet accumulation (Oil Red O staining) were determined. Gene profiles (RT-PCR) were analyzed in the liver of ZDF rats and in human liver-derived HepG2 cell line. Results PGF-treated ZDF rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. These effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase (ACO), and reduced stearoyl-CoA desaturase-1. In contrast, PGF showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. PGF treatment also increased PPAR-alpha and ACO mRNA levels in HepG2 cells. Conclusion Our findings suggest that this Unani medicine ameliorates diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation.

Methyl isobutyl ketone (MIBK) induction of α2u-globulin nephropathy in male, but not female rats

Male F-344 rats were administered corn oil (vehicle control), d-limonene (positive control, 300 mg/kg), or MIBK (1000 mg/kg) and female F-344 rats corn oil (vehicle control) or MIBK for 10 consecutive days by oral gavage. Approximately 24 h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohistochemical staining for α2u-globulin (α2u), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and α2u using an ELISA. MIBK elicited an increase in protein droplets, accumulation of α2u, and renal cell proliferation in male, but not female rats, responses characteristic of α2u-mediated nephropathy. MIBK produced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of α2u-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats. Therefore, renal histopathology, along with the other measures of α2u accumulation, provides additional weight of evidence to support the inclusion of MIBK in the category of chemicals exerting renal effects through a α2u-nephropathy-mediated mode-of-action.

Ultrastructural Features of Atretic Follicles in the Sexually Immature Ostrich (Struthio Camelus)

The aim of this study was to describe the ultrastructural features of atresia in follicles of the immature ostrich (12-14 months old); a ratite that displays seasonal, precocious ovarian activity. The early stage of atresia in primordial, pre-vitellogenic and vitellogenic follicles was characterized by the accumulation of lipid droplets in the granulosa cells. Granulosa cells with condensed cytoplasm and nuclei were a prominent feature during the intermediate phase of atresia. The degenerating follicles were then infiltrated by stroma during the terminal stages of atresia. The results of this study provide further information on the morphology of atretic ovarian follicles in the immature ostrich.

120 EFFECTS OF BOVINE PLASMA LIPOPROTEINS ON ACCUMULATION OF CYTOPLASMIC LIPID DROPLETS AND MITOCHONDRIAL MORPHOLOGY IN BOVINE EMBRYOS

The excessive accumulation of cytoplasmic lipid droplets (LD) may affect the embryo quality such as the sensitivity of embryos to chilling and freezing. Previous studies demonstrated that the presence of serum in embryo culture medium caused the abnormal accumulation of cytoplasmic LD. However, the mechanism and source of cytoplasmic LD accumulation in bovine embryos cultured in serum-containing medium is unclear. In this study, the effects of bovine plasma lipoproteins (BPL) on the accumulation of cytoplasmic LD were investigated in bovine embryos. Bovine oocytes were matured in IVMD101 serum-free medium and inseminated in BO-based medium. After insemination, embryos were cultured in IVMD101 supplemented BPL (Sigma) at various concentrations (60, 150, 300, 600 μg mL–1) or 5% calf serum (CS) in a humidified atmosphere of 5% CO2 in air at 38.5°C. The embryos of 2-, 8-cell, morula, and blastocyst stages were used for experiments. Embryos were fixed and stained with Sudan black B (SBB) for histochemical evaluation. Some embryos were prepared for transmission electron microscopy. The effect of media supplemented with BPL on in vitro development of bovine embryos is shown in Table 1. The proportion of embryos developing to morula and blastocyst stages decreased with increased levels of BPL (Table 1). Histochemical examinations demonstrated that embryos cultured in BPLcontaining media had numerous lipid droplets in their cytoplasm as observed in the embryos developed in CS-supplemented medium. The increase in number of LD was dose-dependent. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of morulae and blastocysts cultured in medium supplemented with BPL (300 μg mL–1). Many elongated mitochondria were found in embryos developed in IVMD101 medium at the morula and blastocyst stages, whereas many of the mitochondria in the morulae and blastocysts developed in IVMD101 medium supplemented with BPL or CS were of an immature form such as spherical or ovoid shape. These results indicate that bovine embryos cultured in BPL-containing media abnormally accumulated LD in their cytoplasm, suggesting that the BPL directly affects the accumulation of cytoplasmic LD. Furthermore, the excessive accumulation of cytoplasmic LD in embryos may affect the mitochondrial development of bovine embryos. Table 1. Effects of BPL on in vitro development of IVM/IVF bovine embryos This work was parially supported by Grant-in-Aid for Scientific Research (17380164), on Priority Areas ‘Lifesurveyor’(19021006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Special Coordination Funds for Promoting Science and Technology of Japan, and the Japan Livestock Technology Association.

Surface kinetics and thermal instability of N-face InN grown by plasma-assisted molecular beam epitaxy

The role of thermal instability and In surface coverages on the growth kinetics has been investigated for N-face InN films grown by plasma-assisted molecular beam epitaxy. Film thickness analysis using scanning electron microscopy combined with In desorption measurements by quadrupole mass spectrometry demonstrated significant thermal decomposition starting at ∼560°C and inhibiting growth completely beyond ∼635°C. Within this temperature region two decomposition pathways were identified: a low-temperature regime characterized by In droplet accumulation and a high-temperature regime with direct desorption from bulk InN. A growth diagram has been constructed, exhibiting three characteristic growth structures for different In∕N flux ratios and growth temperatures: a dry no-adlayer terminated surface under N-rich conditions, an In adlayer terminated surface, and a surface, consisting of an In adlayer and droplets under In-rich conditions. Smooth step-flow growth terraces were observed in films grown under In-rich and surprisingly also under N-rich conditions at temperatures of thermal decomposition. Such high adatom diffusivity resulted from the autosurfactant action of the In adlayer, with a saturated coverage of 1 ML as determined from the reflection high energy electron diffraction patterns during the consumption of adsorbed In by active nitrogen.