Protein kinase CbetaII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo

Protein kinase C betaII (PKCbetaII) is induced early during colon carcinogenesis. Transgenic mice expressing elevated PKCbetaII in the colonic epithelium (transgenic PKCbetaII mice) exhibit hyperproliferation and enhanced colon carcinogenesis. Here we demonstrate that nullizygous PKCbeta (PKCbetaKO) mice are highly resistant to azoxymethane (AOM)-induced preneoplastic lesions, aberrant crypt foci. However, reexpression of PKCbetaII in the colon of PKCbetaKO mice by transgenesis restores susceptibility to AOM-induced colon carcinogenesis. Expression of human PKCbetaII in rat intestinal epithelial (RIE) cells induces expression of endogenous rat PKCbetaII mRNA and protein. Induction of PKCbetaII is dependent upon catalytically active PKCbetaII and does not appear to involve changes in alternative splicing of the PKCbeta gene. Two human PKCbeta promoter constructs are activated by expression of PKCbetaII in RIE cells. Both PKCbeta promoter activity and PKCbetaII mRNA levels are inhibited by the MEK1 and -2 inhibitor U0126, but not the Cox-2 inhibitor celecoxib in RIE/PKCbetaII cells. PKCbeta promoter activity correlates directly with expression of endogenous PKCbetaII mRNA and protein in HT29 and HCT116 human colon cancer cell lines. PKCbeta promoter activity and PKCbetaII mRNA expression in HCT116 cells are inhibited by the selective PKCbeta inhibitor LY317615 and by U0126, demonstrating autoregulation of PKCbetaII expression. Transgenic PKCbetaII mice exhibit specific induction of endogenous PKCbetaII, but not its splice variant PKCbetaI, in the colonic epithelium in vivo. Taken together, our results demonstrate that 1) expression of PKCbetaII in the colonic epithelium is both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in transgenic mice, 2) PKCbetaII regulates its own expression in RIE and human colon cancer cells in vitro and in the colonic epithelium in vivo, and 3) PKCbetaII autoregulation is mediated through a MEK-dependent signaling pathway in RIE/PKCbetaII and HCT116 colon cancer cells.     

Gene expression induced by copper stress in the diatom Thalassiosira pseudonana

Utilizing a PCR-based subtractive cDNA approach, we demonstrated that the marine diatom Thalassiosira pseudonana exhibits a rapid response at the gene level to elevated concentrations of copper and that this response attenuates over 24 h of continuous exposure. A total of 16 copper-induced genes were identified, 11 of which were completely novel; however, many of the predicted amino acid sequences had characteristics suggestive of roles in ameliorating copper toxicity. Most of the novel genes were not equivalently induced by H2O2- or Cd-induced stress, indicating specificity in response. Two genes that could be assigned functions based on homology were also induced under conditions of general cellular stress. Half of the identified genes were located within two inverted repeats in the genome, and novel genes in one inverted repeat had mRNA levels induced by approximately 500- to 2,000-fold by exposure to copper for 1 h. Additionally, some of the inverted repeat genes demonstrated a dose-dependent response to Cu, but not Cd, and appear to belong to a multigene family. This multigene family may be the diatom functional homolog of metallothioneins.     

Recombinant antibodies: towards a new generation of antivenoms?

Poisoning by scorpion venoms is a major health hazard in tropical and subtropical regions and serum therapy, which was discovered in 1894, remains the only specific treatment. No real progress has been made since this time and the therapeutic use of antivenoms which still consists in polyclonal antibody fragments from the sera of immunized animals may be associated with major drawbacks. Protein engineering now allows to design novel recombinant antibody fragments which are superior to polyclonal antivenoms in homogeneity, specific activity and possibly safety. Several single-chain antibody fragments (scFvs) which neutralize scorpion toxins have been produced and characterized over the last few years. These scFvs can also be used as building blocks to engineer more complex structures including multivalent monospecific antibody fragments (diabodies, triabodies) and bispecific molecules (tandem-scFv). Some of these molecules neutralize scorpion neurotoxins and protect mice from experimental envenoming. Thus, research projects currently underway suggest that new strategies might soon be available to treat poisonings in the absence of socio-economic considerations.     

Functional groups show distinct differences in nitrogen cycling during early stand development: implications for forest management

Background and aims

Nutrient acquisition of forest stands is controlled by soil resource availability and belowground production, but tree species are rarely compared in this regard. Here, we examine ecological and management implications of nitrogen (N) dynamics during early forest stand development in productive commercial tree species with narrow (Populus deltoides Bartr. and Platanus occidentalis L.) and broad (Liquidambar styraciflua L. and Pinus taeda L.) site requirements while grown with a range of nutrient and water resources.

Methods

We constructed N budgets by measuring N concentration ([N]) and N content (N _C ) of above- and belowground perennial and ephemeral tissues, determined N uptake (N _UP ), and calculated N use efficiency (NUE).

Results

Forest stands regulated [N] within species-specific operating ranges without clear temporal or treatment patterns, thus demonstrating equilibrium between tissue [N] and biomass accumulation. Forest stand N _C and N _UP increased with stand development and paralleled treatment patterns of biomass accumulation, suggesting productivity is tightly linked to N _UP . Inclusion of above- and belowground ephemeral tissue turnover in N _UP calculations demonstrated that maximum N demand for narrow-sites adapted species exceeded 200?kg?N ha^?1?year^?1 while demand for broad-site adapted species was below this level. NUE was species dependent but not consistently influenced by N availability, suggesting relationships between NUE and resource availability were species dependent.

Conclusions

Based on early stand development, species with broad site adaptability are favored for woody cropping systems because they maintain high above- and belowground productivity with minimal fertilization requirements due to higher NUE than narrow site adapted species.     

In Vivo Quantification Reveals Extensive Natural Variation in Mitochondrial Form and Function in Caenorhabditis briggsae

We have analyzed natural variation in mitochondrial form and function among a set of Caenorhabditis briggsae isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic nad5 gene deletion (nad5Δ) that correlates negatively with organismal fitness. We performed in vivo quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant among-isolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with nad5Δ levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in C. briggsae. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives.     

Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

Tissue inhibitor of metalloproteinases-2 binding to membrane-type 1 matrix metalloproteinase induces MAPK activation and cell growth by a non-proteolytic mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.