immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.     

Defective replication activity of a dominant-lethal dnaB gene product from Escherichia coli.

dnaB protein of Escherichia coli is an essential replication protein. A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P. Shrimankar, L. Shortle, and R. Maurer, unpublished data). This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB. Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E. coli chromosomal origin. Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA. The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication. By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein. Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.     

3'-->5' exonuclease in Drosophila mitochondrial DNA polymerase. Substrate specificity and functional coordination of nucleotide polymerization and mispair hydrolysis.

A mispair-specific 3'-->5' exonuclease copurifies quantitatively with the near-homogeneous Drosophila gamma polymerase (Kaguni, L.S., and Olson, M.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6469-6473). The exonuclease and polymerase exhibit similar reaction requirements and optima, suggesting functional coordination of their activities. Under nonpolymerization conditions, the 3'-->5' exonuclease hydrolyzes 3'-terminal mispairs approximately 15-fold more efficiently than 3'-terminal base pairs on primed single-stranded DNA substrates, whereas it does not discriminate between any of three specific mispairs (dAMP:dAMP;dGMP:dGMP; dGMP:dAMP). Under polymerization conditions, gamma polymerase does not extend a 3'-terminal mispair from the "stationary" state, even in the presence of a large excess of the next correct nucleotide. Instead, 3'-terminal mispairs are hydrolyzed quantitatively by the 3'-->5' exonuclease over the reaction time course. During DNA synthesis by gamma polymerase in the "polymerization" mode, limited misincorporation and subsequent mispair extension do occur. Here, it appears that misincorporation and not mispair extension is rate-limiting. Template-primer challenge experiments suggest that the mechanism of template-primer transfer from the 3'-->5' exonuclease active site to the DNA polymerase active site is intermolecular; transfer from the exonuclease to polymerase mode appears to require dissociation and reassociation of mitochondrial DNA polymerase.     

Post-construction avian mortality monitoring at Project West Wind

Abstract

Post-construction avifauna investigations were undertaken at Project West Wind, Meridian Energy Limited's 62-turbine wind farm on the Wellington south coast. These investigations were required in accordance with the resource consent conditions to quantify the level of avian mortalities occurring at the wind farm, particularly in regard to New Zealand falcon (Falco novaeseelandiae), kākā (Nestor meridionalis) and kererū (Hemiphaga novaeseelandiae). This is the first comprehensive study at a New Zealand operating wind farm. The methods included three field components necessary to calculate annual estimates of mortalities across the wind farm site: routine turbine searches; carcass detection trials; and carcass removal trials. Results from years 1 and 2 of a three-year programme are presented. To date, mortalities have been recorded for 17 taxa at 18 of the 24 study turbines. There have been no recorded mortalities of falcon, kākā or kererū. Australasian harrier (Circus approximans) has been the species for which the most mortalities have been recorded. Overall estimated annual mortality rates for years 1 and 2 were calculated to be approximately six and five birds per turbine respectively.     

Genetic architecture of survival and fitness-related traits in two populations of Atlantic salmon

The additive genetic effects of traits can be used to predict evolutionary trajectories, such as responses to selection. Non-additive genetic and maternal environmental effects can also change evolutionary trajectories and influence phenotypes, but these effects have received less attention by researchers. We partitioned the phenotypic variance of survival and fitness-related traits into additive genetic, non-additive genetic and maternal environmental effects using a full-factorial breeding design within two allopatric populations of Atlantic salmon (Salmo salar). Maternal environmental effects were large at early life stages, but decreased during development, with non-additive genetic effects being most significant at later juvenile stages (alevin and fry). Non-additive genetic effects were also, on average, larger than additive genetic effects. The populations, generally, did not differ in the trait values or inferred genetic architecture of the traits. Any differences between the populations for trait values could be explained by maternal environmental effects. We discuss whether the similarities in architectures of these populations is the result of natural selection across a common juvenile environment.     

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

Selected anthropometric variables and aerobic fitness as predictors of cardiovascular disease risk in children

The aim of this study was to assess the suitability of body mass index, waist circumference, waist-to-height ratio and aerobic fitness as predictors of cardiovascular risk factor clustering in children. A cross-sectional study was conducted with 290 school boys and girls from 6 to 10 years old, randomly selected. Blood was collected after a 12-hour fasting period. Blood pressure, waist circumference (WC), height and weight were evaluated according to international standards. Aerobic fitness (AF) was assessed by the 20-metre shuttle-run test. Clustering was considered when three of these factors were present: high systolic or diastolic blood pressure, high low-density lipoprotein (LDL) cholesterol, high triglycerides, high plasma glucose, high insulin concentrations and low high-density lipoprotein (HDL) cholesterol. A ROC curve identified the cut-off points of body mass index (BMI), WC, waist-to-height ratio (WHtR) and AF as predictors of risk factor clustering. BMI, WC and WHR resulted in significant areas under the ROC curves, which was not observed for AF. The anthropometric variables were good predictors of cardiovascular risk factor clustering in both sexes, whereas aerobic fitness should not be used to identify cardiovascular risk factor clustering in these children.     

Replication initiation at the Escherichia coli chromosomal origin

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.