The effect of nitric oxide inhibition and temporal expression patterns of the mRNA and protein products of nitric oxide synthase genes during in vitro development of bovine pre-implantation embryos.

This study was conducted to determine the effect of Nitric oxide (NO) inhibition in bovine in vitro development and expression analysis of the three Nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), mRNA and protein in bovine oocytes and embryos. Selective inhibitor of NOS, N-omega-nitro-l-arginine methyl ester (l-NAME) was applied at different doses (0, 0.1, 1 and 10 mm) in maturation (experiment 1A), culture medium (experiment 1B) and in both maturation and culture media (experiment 1C). No significant differences were observed in cleavage and blastocyst rates when oocytes were matured in the presence of l-NAME as long as the inhibitor was omitted during fertilization and culture. However, significantly lower blastocyst rates were observed when l-NAME was present at higher level (10 mm) in culture medium alone and in both maturation and culture media. In experiment 2, mRNA isolated from triplicate pools of oocytes and embryos (n = 15-20) was subjected to quantitative real time reverse transcription polymerase chain reaction to investigate the expression of eNOS, iNOS and nNOS mRNA in normal IVP bovine oocytes and embryos. While eNOS and iNOS transcripts were detected at higher level in oocytes (immature and mature), two-cell and four-cell stage embryos, the nNOS was detected only in immature oocyte, two-cell and morula stages. In experiment 3, eNOS and iNOS protein expression analysis was performed in IVP oocytes and embryos and both proteins were detected in the cytoplasm and the nuclei (weak) of oocytes and embryos. These data provide the first evidence for the role of NO production and the presence of mRNA and protein products of NOS isoforms during bovine embryogenesis.

Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy.

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 microm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50-300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of gamma-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.

Migration of epidermal keratinocytes: mechanisms, regulation, and biological significance.

Keratinocytes are the prevalent cell type of the epidermis, a multilayered cornified epithelium which provides the cellular basis of the outermost barrier between the organism and its environment. By this barrier function the epidermis protects the organism against a variety of environmental hazards such as dehydration and mechanical stress. Under normal conditions, keratinocytes of all layers are interconnected by desmosomes and anchored by hemidesmosomes to a specialised type of extracellular matrix, the basement membrane. When the epidermis is injured, a vitally important response is initiated with the aim to restore the protective function of the epithelium. A fast but provisional sealing is achieved by the deposition of the fibrin clot before within 24 h after wounding keratinocytes from the wound margins begin to migrate into the wound bed, where they start to proliferate and to form the new epithelium. The development of new high-resolution assays for the study of cell migration and motility has potentiated major progress in our understanding of keratinocyte migration in vitro and in situ. The data reviewed here point to a sophisticated cooperation between soluble motogenic growth factors, cell-matrix interactions, and cell-to-cell communications as major parts of the machinery regulating keratinocyte migration.

The biological role of the Alzheimer amyloid precursor protein in epithelial cells.

Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimer's disease and (2) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial growth factor. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as keratinocytes and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the growth factor function of sAPP.

Differential absorption and esterification of dietary long-chain fatty acids by larvae of the dragonfly, Aeshna cyanea.

In order to evaluate whether dietary long-chain fatty acids were differentially absorbed, Aeshna cyanea larvae received 5 microliters oral doses containing combinations of two radiolabeled fatty acids at nearly equal radioactive and nmolar concentrations: (1) 3H-oleic and 14C-palmitic acids; (2) 3H-oleic and 14C-stearic acids; and (3) 3H-palmitic and 14C-stearic acids. After 3 h or 1 day, hemolymph samples, midgut tissue, midgut contents and fat body tissue were collected and assayed for labeled fatty acids. The 3H/14C ratios indicated that there was a preference for absorption of the monounsaturated oleic acid over both saturated palmitic and stearic acids and that the shorter palmitic acid was absorbed at a higher rate than the longer stearic acid. There were also differences in the 3H/14C ratios of the various lipid classes of the midgut wall, hemolymph, and fat body that reflected differential esterifications and transport of these fatty acids.