The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Zebrafish Krüppel-Like Factor 4a Represses Intestinal Cell Proliferation and Promotes Differentiation of Intestinal Cell Lineages

BACKGROUND:Mouse krüppel-like factor 4 (Klf4) is a zinc finger-containing transcription factor required for terminal differentiation of goblet cells in the colon. However, studies using either Klf4(-/-) mice or mice with conditionally deleted Klf4 in their gastric epithelia showed different results in the role of Klf4 in epithelial cell proliferation. We used zebrafish as a model organism to gain further understanding of the role of Klf4 in the intestinal cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS:We characterized the function of klf4a, a mammalian klf4 homologue by antisense morpholino oligomer knockdown. Zebrafish Klf4a shared high amino acid similarities with human and mouse Klf4. Phylogenetic analysis grouped zebrafish Klf4a together with both human and mouse Klf4 in a branch with high bootstrap value. In zebrafish, we demonstrate that Klf4a represses intestinal cell proliferation based on results of BrdU incorporation, p-Histone 3 immunostaining, and transmission electron microscopy analyses. Decreased PepT1 expression was detected in intestinal bulbs of 80- and 102-hours post fertilization (hpf) klf4a morphants. Significant reduction of alcian blue-stained goblet cell number was identified in intestines of 102- and 120-hpf klf4a morphants. Embryos treated with γ-secretase inhibitor showed increased klf4a expression in the intestine, while decreased klf4a expression and reduction in goblet cell number were observed in embryos injected with Notch intracellular domain (NICD) mRNA. We were able to detect recovery of goblet cell number in 102-hpf embryos that had been co-injected with both klf4a and Notch 1a NICD mRNA. CONCLUSIONS/SIGNIFICANCE:This study provides in vivo evidence showing that zebrafih Klf4a is essential for the repression of intestinal cell proliferation. Zebrafish Klf4a is required for the differentiation of goblet cells and the terminal differentiation of enterocytes. Moreover, the regulation of differentiation of goblet cells in zebrafish intestine by Notch signaling at least partially mediated through Klf4a

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development

Contributions Made by CDC25 Phosphatases to Proliferation of Intestinal Epithelial Stem and Progenitor Cells

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells

Caspase Inhibition with XIAP as an Adjunct to AAV Vector Gene-Replacement Therapy: Improving Efficacy and Prolonging the Treatment Window

AAV-mediated gene therapy in the rd10 mouse, with retinal degeneration caused by mutation in the rod cyclic guanosine monophosphate phosphodiesterase β-subunit (PDEβ) gene, produces significant, but transient, rescue of photoreceptor structure and function. This study evaluates the ability of AAV-mediated delivery of X-linked inhibitor of apoptosis (XIAP) to enhance and prolong the efficacy of PDEβ gene-replacement therapy.Rd10 mice were bred and housed in darkness. Two groups of animals were generated: Group 1 received sub-retinal AAV5-XIAP or AAV5-GFP at postnatal age (P) 4 or 21 days; Group 2 received sub-retinal AAV5-XIAP plus AAV5- PDEβ, AAV5-GFP plus AAV5- PDEβ, or AAV- PDEβ alone at age P4 or P21. Animals were maintained for an additional 4 weeks in darkness before being moved to a cyclic-light environment. A subset of animals from Group 1 received a second sub-retinal injection of AAV8-733-PDEβ two weeks after being moved to the light. Histology, immunohistochemistry, Western blots, and electroretinograms were performed at different times after moving to the light.Injection of AAV5-XIAP alone at P4 and 21 resulted in significant slowing of light-induced retinal degeneration, as measured by outer nuclear thickness and cell counts, but did not result in improved outer segment structure and rhodopsin localization. In contrast, co-injection of AAV5-XIAP and AAV5-PDEβ resulted in increased levels of rescue and decreased rates of retinal degeneration compared to treatment with AAV5-PDEβ alone. Mice treated with AAV5-XIAP at P4, but not P21, remained responsive to subsequent rescue by AAV8-733-PDEβ when injected two weeks after moving to a light-cycling environment.Adjunctive treatment with the anti-apoptotic gene XIAP confers additive protective effect to gene-replacement therapy with AAV5-PDEβ in the rd10 mouse. In addition, AAV5-XIAP, when given early, can increase the age at which gene-replacement therapy remains effective, thus effectively prolonging the window of opportunity for therapeutic intervention

Evolution of somatic mutations in mammary tumors in transgenic mice is influenced by the inherited genotype

BACKGROUND: MMTV-Wnt1 transgenic mice develop mammary hyperplasia early in development, followed by the appearance of solitary mammary tumors with a high proportion of cells expressing early lineage markers and many myoepithelial cells. The occurrence of tumors is accelerated in experiments that activate FGF proto-oncogenes or remove the tumor suppressor genes Pten or P53, implying that secondary oncogenic events are required for progression from mammary hyperplasia to carcinoma. It is not known, however, which oncogenic pathways contribute to Wnt1-induced tumorigenesis – further experimental manipulation of these mice is needed. Secondary events also appear to be required for mammary tumorigenesis in MMTV-Neu transgenic mice because the transgene in the tumors usually contains an acquired mutation that activates the Neu protein-tyrosine kinase. METHODS: cDNA or DNA from the mammary glands and mammary tumors from MMTV-Wnt1, MMTV-Wnt1/p53(-/-), MMTV-Neu transgenic mice, and newly generated MMTV-Wnt1/MMTV-Neu bitransgenic mice, was sequenced to seek activating mutations in H-Ras, K-Ras, and N-Ras genes, or in the MMTV-Neu transgene. In addition, tumors from bitransgenic animals were examined to determine the cellular phenotype. RESULTS: We found activating mutations at codons 12, 13, and 61 of H-Ras in just over half of the mammary tumors in MMTV-Wnt1 transgenic mice, and we confirmed the high frequency of activating mutations of Neu in tumors in MMTV-Neu transgenic mice. Tumors appeared earlier in bitransgenic MMTV-Wnt1/MMTV-Neu mice, but no Ras or MMTV-Neu mutations were found in these tumors, which were phenotypically similar to those arising in MMTV-Wnt1 mice. In addition, no Ras mutations were found in the mammary tumors that arise in MMTV-Wnt1 transgenic mice lacking an intact P53 gene. CONCLUSIONS: Tumorigenic properties of cells undergoing functionally significant secondary mutations in H-Ras or the MMTV-Neu transgene allow selection of those cells in MMTV-Wnt1 and MMTV-Neu transgenic mice, respectively. Alternative sources of oncogenic potential, such as a second transgenic oncogene or deficiency of a tumor suppressor gene, can obviate the selective power of those secondary mutations. These observations are consistent with the notion that somatic evolution of mouse mammary tumors is influenced by the specific nature of the inherited cancer-promoting genotype

Sinusoidal Endothelial Dysfunction Precedes Inflammation and Fibrosis in a Model of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Most morbidity associated with the metabolic syndrome is related to vascular complications, in which endothelial dysfunction is a major pathogenic factor. However, whether NAFLD is associated with endothelial dysfunction within the hepatic vasculature is unknown. The aims of this study were to explore, in a model of diet-induced overweight that expresses most features of the metabolic syndrome, whether early NAFLD is associated with liver endothelial dysfunction. Wistar Kyoto rats were fed a cafeteria diet (CafD; 65% of fat, mostly saturated) or a control diet (CD) for 1 month. CafD rats developed features of the metabolic syndrome (overweight, arterial hypertension, hypertryglyceridemia, hyperglucemia and insulin resistance) and liver steatosis without inflammation or fibrosis. CafD rats had a significantly higher in vivo hepatic vascular resistance than CD. In liver perfusion livers from CafD rats had an increased portal perfusion pressure and decreased endothelium-dependent vasodilation. This was associated with a decreased Akt-dependent eNOS phosphorylation and NOS activity. In summary, we demonstrate in a rat model of the metabolic syndrome that shows features of NAFLD, that liver endothelial dysfunction occurs before the development of fibrosis or inflammation

Influence of Dll4 via HIF-1α-VEGF Signaling on the Angiogenesis of Choroidal Neovascularization under Hypoxic Conditions

Choroidal neovascularization (CNV) is the common pathological basis of irreversible visual impairment encountered in a variety of chorioretinal diseases; the pathogenesis of its development is complicated and still imperfectly understood. Recent studies indicated that delta-like ligand 4 (Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF pathway to regulate CNV angiogenesis. But little is known about the influence and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time RT-PCR, Western blotting were used to analyze the expression alteration of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a laser-induced rat CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules in vitro and in vivo. RPE-RF/6A cell co-culture systems were used to investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to the significantly faster proliferation and stronger tube forming ability, but inhibited cells migration and invasion across a monolayer of RPE cells in hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite effects. Pharmacological disruption of Notch signaling using gamma-secretase inhibitor (GSI) produced similar, but not identical effects, to that caused by the Dll4 siRNA. In addition, the expression of several key molecules involved in the angiogenesis of CNV was altered in RF/6A cells showing constitutively active Dll4 expression. These results suggest that Dll4 play an important role in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may facilitate further understanding of the mechanisms that underlie CNV angiogenesis

Occupation and skin cancer: the results of the HELIOS-I multicenter case-control study

<p>Abstract</p> <p>Background</p> <p>Non-melanoma skin cancer (NMSC) is the most frequent tumour among Caucasian populations worldwide. Among the risk factors associated with this tumour, there are host-related factors and several environmental agents. A greater likelihood of high exposure to physical agents (with the exception of solar radiation) and chemical agents depends on the work setting. Our objective is to evaluate the role of occupational exposures in NMSC, with special emphasis on risk factors other than solar radiation and skin type.</p> <p>Methods</p> <p>We analysed 1585 cases (1333 basal cell carcinoma (BCC) and 183 squamous cell carcinoma (SCC)) and 1507 controls drawn from the Helios-I multicenter study. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression mixed models.</p> <p>Results</p> <p>For NMSC as a whole (both <it>histological types</it>), miners and quarrymen, secondary education teachers, and masons registered excess risk, regardless of exposure to solar radiation and skin type (OR 7.04, 95% CI 2.44–20.31; OR 1.75, 95% CI 1.05–2.89 and OR 1.54, 95% CI 1.04–2.27, respectively). Frequency of BCC proved higher among railway engine drivers and firemen (OR 4.55; 95% CI 0.96–21.57), specialised farmers (OR 1.65; 95% CI 1.05–2.59) and salesmen (OR 3.02; 95% CI 1.05–2.86), in addition to miners and quarrymen and secondary education teachers (OR 7.96; 95% CI 2.72–23.23 and OR 1.76; 95% CI 1.05–2.94 respectively). The occupations that registered a higher risk of <it>SCC (though not of BCC</it>) were those involving direct contact with livestock, construction workers not elsewhere classified (OR 2.95, 95% CI 1.12–7.74), stationary engine and related equipment operators not elsewhere classified (OR 5.31, 95% CI 1.13–21.04) and masons (OR 2.55, 95% CI 1.36–4.78).</p> <p>Conclusion</p> <p>Exposure to hazardous air pollutants, arsenic, ionizing radiations and burns may explain a good part of the associations observed in this study. The Helios study affords an excellent opportunity for further in-depth study of physical and chemical agents and NMSC based on matrices of occupational exposure.</p