Combinatorial Roles of Heparan Sulfate Proteoglycans and Heparan Sulfates in Caenorhabditis elegans Neural Development

Heparan sulfate proteoglycans (HSPGs) play critical roles in the development and adult physiology of all metazoan organisms. Most of the known molecular interactions of HSPGs are attributed to the structurally highly complex heparan sulfate (HS) glycans. However, whether a specific HSPG (such as syndecan) contains HS modifications that differ from another HSPG (such as glypican) has remained largely unresolved. Here, a neural model in C. elegans is used to demonstrate for the first time the relationship between specific HSPGs and HS modifications in a defined biological process in vivo. HSPGs are critical for the migration of hermaphrodite specific neurons (HSNs) as genetic elimination of multiple HSPGs leads to 80% defect of HSN migration. The effects of genetic elimination of HSPGs are additive, suggesting that multiple HSPGs, present in the migrating neuron and in the matrix, act in parallel to support neuron migration. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a linear signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function together in a pathway that is parallel to sdn-1 and hst-6. These results suggest core protein specific HS modifications that are critical for HSN migration. In C. elegans, the core protein specificity of distinct HS modifications may be in part regulated at the level of tissue specific expression of genes encoding for HSPGs and HS modifying enzymes. Genetic analysis reveals that there is a delicate balance of HS modifications and eliminating one HS modifying enzyme in a compromised genetic background leads to significant changes in the overall phenotype. These findings are of importance with the view of HS as a critical regulator of cell signaling in normal development and disease

Functional Polymorphism of IL-1 Alpha and Its Potential Role in Obesity in Humans and Mice

Proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. IL-1α is one of the proinflammatory cytokines; however, it has not been clarified whether IL-1α may also cause obesity. In this study, we investigated whether polymorphisms in IL-1α contribute to human obesity. A total of 260 obese subjects were genotyped for IL-1α C-889T (rs1800587) and IL-1α G+4845T (rs17561). Analyses of genotype distributions revealed that both IL-1α polymorphisms C-889T (rs1800587) and G+4845T (rs17561) were associated with an increase in body mass index in obese healthy women. In addition, the effect of rs1800587 on the transcriptional activity of IL-1α was explored in pre-adipocyte 3T3-L1 cells. Significant difference was found between the rs1800587 polymorphism in the regulatory region of the IL-1α gene and transcriptional activity. We extended these observations in vivo to a high-fat diet-induced obese mouse model and in vitro to pre-adipocyte 3T3-L1 cells. IL-1α levels were dramatically augmented in obese mice, and triglyceride was increased 12 hours after IL-1α injection. Taken together, IL-1α treatment regulated the differentiation of preadipocytes. IL-1α C-889T (rs1800587) is a functional polymorphism of IL-1α associated with obesity. IL-1α may have a critical function in the development of obesity

Genome-wide association study of bronchopulmonary dysplasia : a potential role for variants near the CRP gene

Bronchopulmonary dysplasia (BPD), the main consequence of prematurity, has a significant heritability, but little is known about predisposing genes. The aim of this study was to identify gene loci predisposing infants to BPD. The initial genome-wide association study (GWAS) included 174 Finnish preterm infants of gestational age 24-30 weeks. Thereafter, the most promising single-nucleotide polymorphisms (SNPs) associated with BPD were genotyped in both Finnish (n = 555) and non-Finnish (n = 388) replication cohorts. Finally, plasma CRP levels from the first week of life and the risk of BPD were assessed. SNP rs11265269, flanking the CRP gene, showed the strongest signal in GWAS (odds ratio [ OR] 3.2, p = 3.4 x 10(-6)). This association was nominally replicated in Finnish and French African populations. A number of other SNPs in the CRP region, including rs3093059, had nominal associations with BPD. During the first week of life the elevated plasma levels of CRP predicted the risk of BPD (OR 3.4, p = 2.9 x 10(-4)) and the SNP rs3093059 associated nominally with plasma CRP levels. Finally, SNP rs11265269 was identified as a risk factor of BPD (OR 1.8, p = 5.3 x 10(-5)), independently of the robust antenatal risk factors. As such, in BPD, a potential role for variants near CRP gene is proposed.Peer reviewe

Association between LTA, TNF and AGER Polymorphisms and Late Diabetic Complications

BACKGROUND: Several candidate genes on the short arm of chromosome 6 including the HLA locus, TNF, LTA and AGER could be associated with late diabetic complications. The aim of our study was therefore to explore whether polymorphisms (TNF -308 G-->A, LTA T60N C-->A and AGER -374 T-->A) in these genes alone or together (as haplotypes) increased the risk for diabetic complications. METHODOLOGY/PRINCIPAL FINDINGS: The studied polymorphisms were genotyped in 742 type 1 and 2957 type 2 diabetic patients as well as in 206 non-diabetic control subjects. The Haploview program was used to analyze putative linkage disequilibrium between studied polymorphisms. The TNF, LTA and AGER polymorphisms were associated with the HLA-DQB1 risk genotypes. The AGER -374 A allele was more common in type 1 diabetic patients with than without diabetic nephropathy (31.2 vs. 28.4%, p = 0.007). In a logistic regression analysis, the LTA but not the AGER polymorphism was associated with diabetic nephropathy (OR 2.55[1.11-5.86], p = 0.03). The AGER -374 A allele was associated with increased risk of sight threatening retinopathy in type 2 diabetic patients (1.65[1.11-2.45], p = 0.01) and also with increased risk for macrovascular disease in type 1 diabetic patients (OR 2.05[1.19-3.54], p = 0.01), but with decreased risk for macrovascular disease in type 2 diabetic patients (OR 0.66[0.49-0.90], p = 0.009). The TNF A allele was associated with increased risk for macrovascular complications in type 2 (OR 1.53 [1.04-2.25], p = 0.03, but not in type 1 diabetic patients. CONCLUSIONS/SIGNIFICANCE: The association between diabetic complications and LTA, TNF and AGER polymorphisms is complex, with partly different alleles conferring susceptibility in type 1 and type 2 diabetic patients. We can not exclude the possibility that the genes are part of a large haplotype block that also includes HLA-DQB1 risk genotypes

A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1

Arctigenin Efficiently Enhanced Sedentary Mice Treadmill Endurance

Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases

P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells

<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer.</p> <p>Methods</p> <p>Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA.</p> <p>Results</p> <p>HMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition.</p> <p>Conclusions</p> <p>The results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial cell differentiation.</p

Diseased muscles that lack dystrophin or laminin-α2 have altered compositions and proliferation of mononuclear cell populations

BACKGROUND: Multiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-α2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells. METHODS: We used fluorescence-activated cell sorting combined with bromodeoxyuridine labeling to examine proliferation rates and compositions of mononuclear cells in diseased and healthy mouse skeletal muscle. We prepared mononucleate cells from muscles of mdx (dystrophin-deficient) or Lama2(-/- )(laminin-α2-deficient) mice and compared them to cells from healthy control muscles. We enumerated subsets of resident muscle cells based on Sca-1 and CD45 expression patterns and determined the proliferation of each cell subset in vivo by BrdU incorporation. RESULTS: We found that the proliferation and composition of the mononucleate cells in dystrophin-deficient and laminin-α2-deficient diseased muscles are different than in healthy muscle. The mdx and Lama2(-/- )muscles showed similar significant increases in CD45(+ )cells compared to healthy muscle. Changes in proliferation, however, differed between the two diseases with proliferation increased in mdx and decreased in Lama2(-/- )muscles compared to healthy muscles. In particular, the most abundant Sca-1(-)/CD45(- )subset, which contains muscle precursor cells, had increased proliferation in mdx muscle but decreased proliferation in Lama2(-/- )muscles. CONCLUSION: The similar increases in CD45(+ )cells, but opposite changes in proliferation of muscle precursor cells, may underlie aspects of the distinct pathologies in the two diseases

An Aqueous Extract of Fagonia cretica Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Breast Cancer Cells via FOXO3a and p53 Expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression

Tuberous Sclerosis Complex-1 Deficiency Attenuates Diet-Induced Hepatic Lipid Accumulation

Non-alcoholic fatty liver disease (NAFLD) is causally linked to type 2 diabetes, insulin resistance and dyslipidemia. In a normal liver, insulin suppresses gluconeogenesis and promotes lipogenesis. In type 2 diabetes, the liver exhibits selective insulin resistance by failing to inhibit hepatic glucose production while maintaining triglyceride synthesis. Evidence suggests that the insulin pathway bifurcates downstream of Akt to regulate these two processes. Specifically, mTORC1 has been implicated in lipogenesis, but its role on hepatic steatosis has not been examined. Here, we generated mice with hepatocyte-specific deletion of Tsc1 to study the effects of constitutive mTORC1 activation in the liver. These mice developed normally but displayed mild hepatomegaly and insulin resistance without obesity. Unexpectedly, the Tsc1-null livers showed minimal signs of steatosis even under high-fat diet condition. This ‘resistant’ phenotype was reversed by rapamycin and could be overcome by the expression of Myr-Akt. Moreover, rapamycin failed to reduce hepatic triglyceride levels in models of steatosis secondary to Pten ablation in hepatocytes or high-fat diet in wild-type mice. These observations suggest that mTORC1 is neither necessary nor sufficient for steatosis. Instead, Akt and mTORC1 have opposing effects on hepatic lipid accumulation such that mTORC1 protects against diet-induced steatosis. Specifically, mTORC1 activity induces a metabolic shift towards fat utilization and glucose production in the liver. These findings provide novel insights into the role of mTORC1 in hepatic lipid metabolism