Time Course Expression Analysis of 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole Induction of Cytoprotection in Human Endothelial Cells.

1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a synthetic derivative of oleanolic acid that exhibits antioxidant and anti-inflammatory activity in several animal and in vitro models, has been shown to be beneficial if given after injury. Although induction of heme oxygenase 1 appears to be a major effector of cytoprotection, the mechanism by which the overall effect is mediated is largely unknown. This study evaluated temporal gene expression profiles to better characterize the early transcriptional events and their relationship to the dynamics of the cytoprotective response in human umbilical vein endothelial cells (HUVEC) to CDDO-Im. Time-course gene expression profiling was performed on HUVEC treated with CDDO-Im for 0.5, 1, 3, 6, and 24 hours. More than 10 000 genes were statistically altered in their expression in at least 1 time point across the time course. Large alterations in immediate-early gene expression were readily detectable within 0.5 hour after administration of CDDO-Im

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as `markers' for the sequential events of synthesis and assembly of zonulae occludentes: (1) development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, (2) appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, (3) appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and (4) alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: (1) reshaping the cell surface to align future junction-containing regions with those of adjacent cells, (2) delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and (3) aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.To test the functional significance of these morphological features of junction-forming cells and to validate the interpretation that they are reliable indicators of the stages of tight junction genesis, primary cultures of bovine brain capillary endothelium were treated with 25 [mu]g/ml of Cytochalasin-D or 0.25 mg/ml of n-ethylmaleimide (Sigma Chemical Co.) in order to prevent cytoskeletal mediation of surface contouring (step 1) or to inhibit vesicle fusion with the plasmalemma (step 2) and thereby prevent junction formation as a consequence of failure of the vesicle fusions to insert tight junctional intramembrane particles into the plasma membrane. Examination of platinum replicas of freeze-fractured control and treated endothelial monolayer cultures confirmed the absence of surface contours in Cytochalasin-D-treated cells, which exhibited no zonulae occludentes, and also clearly showed that n-ethylmaleimide-treated cells, which lacked tight junctions, did not have the rich endowment of vesicle fusion sites (and IMPs) which were conspicuous in control cells. Demonstration of the failure of MDCK cells to form tight junctions when cultured in the presence of 5-10 [mu]g/ml of cycloheximide (Griepp et al., 1983) lends further support for the schemata proposed above.Advantages of this model include: (1) all stages of de novo tight junction formation are present in each monolayer culture, and (2) cultures possess vast areas of tight junction-containing membrane which are easily sampled by freeze-fracture. This model will provide the basis for future attempts to identify the signals that regulate tight junction formation, and will facilitate studies to characterize the protein(s) of the endothelial tight junctions, the messages (m-RNA) that code for them, and ultimately, the genes bearing their blueprint.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25900/1/0000463.pd

Cell proliferation after ischemic injury in gerbil brain

Tritiated thymidine autoradiography was used to measure cellular proliferation after ischemic injury in gerbil brain. Gerbils were subjected to bilateral occlusion of the common carotid arteries which resulted in areas of necrosis, or infarcts, in the posterior thalamus or midbrain. From 12 h to 10 days following the ischemia, gerbils were injected with 3 H thymidine, sacrificed 4 h later, and the brains sectioned. In order to identify astrocytes and monocytes/macrophages, immunocytochemistry was performed prior to autoradiography, using antisera against glial fibrillary acidic protein and endothelial-monocyte reticuloendothelial antigen, respectively. Immunocytochemistry was also used to visualize microvessel laminin, myelin, and leakage of serum albumin. Lastly, a histochemical procedure for acid phosphatase activity was employed to verify cellular phagocytic activity in the wound. A reproducible sequence of reactions took place during the first 10 days after ischemia. Early changes included leakage of albumin and myelin breakdown, followed by arrival of monocytes at 2 days and their differentiation into macrophages by 5 days. These cells exhibited intense proliferation from 2 to 6 days post-ischemia. Microvessel endothelial cells were maximally labeled at 4 days post-ischemia. Hypertrophied astrocytes were apparent at 2 days and proliferated from 3 to 7 days post-ischemia, and by 10 days the wound was replaced by a “glial scar”.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47681/1/441_2004_Article_BF00225558.pd

Primary culture of capillary endothelial cells from the spiral ligament and stria vascularis of bovine inner ear

Methods for isolation and culture of microvascular endothelial cells of the inner ear were devised to provide an in-vitro system for studying endothelial functions in this tissue. Capillaries from the stria vascularis and spiral ligament were treated enzymatically to free them from surrounding tissue. Contamination by extraneous tissue was minimized by banding capillary segments in Percoll gradients and culture in plasma-derived serum on a fibronectin-coated substrate. Although only small amounts of inner ear tissue were available, tritiated thymidine autoradiography demonstrated that considerable growth in culture was possible. Addition of heparin and endothelial cell growth supplement to the medium enhanced proliferation. The endothelial origin of the cultured cells was confirmed by immunofluorescent demonstration of the presence of Factor VIII-related antigen and angiotensin-converting enzyme. In addition, tight junctions between cells were observed in both thin sections and platinum replicas obtained by freezefracture techniques. Endothelial cells from neither the stria vascularis nor the spiral ligament allowed passage of horseradish peroxidase across the monolayer during a 5-min period. However, endothelial cells from the stria vascularis exhibited a greater amount of pinocytotic activity than those of the spiral ligament, a difference that is also observed in vivo. Methods for expanding a small population of endothelial cells with retention of specialized properties into one of sufficient size for morphologic and biochemical studies have been demonstrated for the inner ear.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47680/1/441_2004_Article_BF00214566.pd

Angiogenesis in developing rat brain: An in vivo and in vitro study

Brain capillary proliferation in postnatal rats was measured in vivo by [3H]thymidine autoradiography. Maximal capillary proliferation occurred between 5 and 9 postnatal days, and was 40 times greater than in the adult. To test the hypothesis that soluble angiogenesis factors play a role in this developmental vascularization of brain, we prepared extracts from the brains of 6-day-old rats at the peak of proliferative activity, and from adults when it was lowest. We assayed them using an in vitro growth system measuring [3H]thymidine incorporation into cultured brain capillary endothelial cells. Extracts prepared from either 6-day or adult rats and containing 150 [mu]g/ml protein caused more than a 4-fold stimulation of the endothelial cells, increasing to 8-fold at a concentration of 1500 [mu]g/ml. The presence of growth-promoting activity in brain extracts from both adult and immature rats suggests that soluble angiogenesis factors may be present in the brain throughout life, but are unavailable for stimulation of in vivo capillary growth unless released or activated by an appropriate stimulus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25473/1/0000013.pd

Presence of a novel epithelial antigen on rat cerebellar cell lines as detected by a monoclonal antibody

We have derived a monoclonal antibody, MCAb 51, following immunization of BALB/c mice with a Rous sarcoma virus-transformed rat cerebellar cell line. When assayed by immunofluorescence on primary rat cerebellar cultures MCAb 51 recognizes only islands of cells with an epitheloid morphology. Double-label immunofluorescence experiments with MCAb 51 and antisera to tetanus toxin, glial fibrillary acidic protein, galactocerebroside and fibronectin reveal that these cells do not appear to be neurons, astrocytes, oligodendrocytes, or fibroblasts, respectively. In contrast, cells from kidney, liver, tongue and choroid plexus epithelium are positive for the antigen. Of 12 Rous sarcoma virus-transformed cell lines, in contrast to 2 out of 9 chemically transformed lines, 11 exhibit the MCAb 51 antigen. These findings demonstrate that MCAb 51 recognizes an epithelial cell surface marker. Possible explanations for the difference in the expression of the antigen on Rous sarcoma virus and chemically transformed neural lines are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25625/1/0000175.pd

Accuracy of genomic breeding values in multi-breed dairy cattle populations

<p>Abstract</p> <p>Background</p> <p>Two key findings from genomic selection experiments are 1) the reference population used must be very large to subsequently predict accurate genomic estimated breeding values (GEBV), and 2) prediction equations derived in one breed do not predict accurate GEBV when applied to other breeds. Both findings are a problem for breeds where the number of individuals in the reference population is limited. A multi-breed reference population is a potential solution, and here we investigate the accuracies of GEBV in Holstein dairy cattle and Jersey dairy cattle when the reference population is single breed or multi-breed. The accuracies were obtained both as a function of elements of the inverse coefficient matrix and from the realised accuracies of GEBV.</p> <p>Methods</p> <p>Best linear unbiased prediction with a multi-breed genomic relationship matrix (GBLUP) and two Bayesian methods (BAYESA and BAYES_SSVS) which estimate individual SNP effects were used to predict GEBV for 400 and 77 young Holstein and Jersey bulls respectively, from a reference population of 781 and 287 Holstein and Jersey bulls, respectively. Genotypes of 39,048 SNP markers were used. Phenotypes in the reference population were de-regressed breeding values for production traits. For the GBLUP method, expected accuracies calculated from the diagonal of the inverse of coefficient matrix were compared to realised accuracies.</p> <p>Results</p> <p>When GBLUP was used, expected accuracies from a function of elements of the inverse coefficient matrix agreed reasonably well with realised accuracies calculated from the correlation between GEBV and EBV in single breed populations, but not in multi-breed populations. When the Bayesian methods were used, realised accuracies of GEBV were up to 13% higher when the multi-breed reference population was used than when a pure breed reference was used. However no consistent increase in accuracy across traits was obtained.</p> <p>Conclusion</p> <p>Predicting genomic breeding values using a genomic relationship matrix is an attractive approach to implement genomic selection as expected accuracies of GEBV can be readily derived. However in multi-breed populations, Bayesian approaches give higher accuracies for some traits. Finally, multi-breed reference populations will be a valuable resource to fine map QTL.</p

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN