Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection

Invertebrates lack adaptive immune systems homologous to those of vertebrates, yet it is becoming increasingly clear that they can produce diversified antigen recognition molecules. We have previously noted that the snail Biomphalaria glabrata produces a secreted lectin, fibrinogen-related protein 3 (FREP3), unusual among invertebrate defense molecules because it is somatically diversified by gene conversion and point mutation. Here we implicate FREP3 in playing a central role in resistance to a major group of snail pathogens, digenetic trematodes. FREP3 is up-regulated in three models of resistance of B. glabrata to infection with Schistosoma mansoni or Echinostoma paraensei, and functions as an opsonin favoring phagocytosis by hemocytes. Knock-down of FREP3 in resistant snails using siRNA-mediated interference resulted in increased susceptibility to E. paraensei, providing a direct link between a gastropod immune molecule and resistance to trematodes. FREP3 up-regulation is also associated with heightened responsiveness following priming with attenuated digenetic trematodes (acquired resistance) in this model invertebrate immune system.     

Monitoring minimal residual disease in acute myeloid leukaemia with NPM1 mutations by quantitative PCR: clonal evolution is a limiting factor

Nucleophosmin ( NPM1 ) mutations in exon 12 represent the most frequent molecular aberrations in adult patients with acute myeloid leukaemia (AML). Molecular detection of NPM1 mutation A could be a useful marker for routine monitoring of minimal residual disease (MRD). We established a calibrator-normalized relative quantification real-time polymerase chain reaction (PCR) assay for NPM1 mutation A . ABL1 was used as a reference housekeeping gene and the NPM1 mutation A-containing OCI/AML3 cell line as a calibrator. Relative quantification was performed by calculating the NPM1 mutation A /ABL1 ratio which was normalized to the NPM1 mutation A /ABL1 ratio of OCI/AML3 calibrator cDNA. The assay showed a sensitivity of 10⁻⁵. The clinical usefulness was evaluated by monitoring MRD in 51 AML patients with NPM1 mutation A. In 27 patients analysed at diagnosis and after induction treatment, NPM1 mutation A ratios showed a median log₁₀ reduction of 2·48, which correlated with response to therapy. Among the 51 patients, 21 relapsed and two lost the mutation. We established a sensitive, specific and reproducible assay for routine quantification and monitoring of NPM1 mutation A levels. However, clonal evolution was observed in 9·5% limiting the usefulness of the NPM1 mutation A mutation as a molecular marker in these patients.     

Homoserine and asparagine are host signals that trigger in planta expression of a pathogenesis gene in Nectria haematococca

Some pathogenesis-related genes are expressed in fungi only when the pathogen is in the host, but the host signals that trigger these gene expressions have not been identified. Virulent Nectria haematococca infects pea plants and requires either pelA, which is induced by pectin, or pelD, which is induced only in planta. However, the host signal(s) that trigger pelD expression was unknown. Here we report the isolation of the host signals and identify homoserine and asparagine, two free amino acids found in uniquely high levels in pea seedlings, as the pelD-inducing signals. N. haematococca has evolved a mechanism to sense the host tissue environment by using the high levels of two free amino acids in this plant, thereby triggering the expression of pelD to assist the pathogenic process.     

The maize disease resistance gene Htn1 against northern corn leaf blight encodes a wall-associated receptor-like kinase

Northern corn leaf blight (NCLB) caused by the hemibiotrophic fungus Exserohilum turcicum is an important foliar disease of maize that is mainly controlled by growing resistant maize cultivars. The Htn1 locus confers quantitative and partial NCLB resistance by delaying the onset of lesion formation. Htn1 represents an important source of genetic resistance that was originally introduced from a Mexican landrace into modern maize breeding lines in the 1970s. Using a high-resolution map-based cloning approach, we delimited Htn1 to a 131.7-kb physical interval on chromosome 8 that contained three candidate genes encoding two wall-associated receptor-like kinases (ZmWAK-RLK1 and ZmWAK-RLK2) and one wall-associated receptor-like protein (ZmWAK-RLP1). TILLING (targeting induced local lesions in genomes) mutants in ZmWAK-RLK1 were more susceptible to NCLB than wild-type plants, both in greenhouse experiments and in the field. ZmWAK-RLK1 contains a nonarginine-aspartate (non-RD) kinase domain, typically found in plant innate immune receptors. Sequence comparison showed that the extracellular domain of ZmWAK-RLK1 is highly diverse between different maize genotypes. Furthermore, an alternative splice variant resulting in a truncated protein was present at higher frequency in the susceptible parents of the mapping populations compared with in the resistant parents. Hence, the quantitative Htn1 disease resistance in maize is encoded by an unusual innate immune receptor with an extracellular wall-associated kinase domain. These results further highlight the importance of this protein family in resistance to adapted pathogens.Plants are constantly attacked by potential pathogenic microbes, specifically, viruses, bacteria, and fungi. Although lacking an adaptive immune system comparable to the one found in vertebrates, plants have evolved a plethora of strategies to fend off microbial pathogens. The first tier of defense is formed by plasma membrane-anchored pattern recognition receptors (PRRs). These receptor proteins monitor the extracellular space for the presence of microbial- or host-derived elicitors, also called pathogen-associated molecular patterns (PAMP) or danger-associated molecular patterns (DAMP), respectively. Perception of PAMPs and DAMPs triggers a signaling cascade that activates numerous defense responses, called PAMP-triggered immunity and DAMP-triggered immunity, respectively. PAMPs are often highly conserved microbial structures that are characteristic for entire pathogen classes (1, 2). Examples are the fungal cell wall component chitin or bacterial flagellin. Perception of highly conserved PAMPs therefore results in broad-spectrum resistance against whole groups of nonadapted microbes, also referred to as nonhost resistance. To avoid PAMP-triggered immunity and DAMP-triggered immunity, pathogens are equipped with specific effectors that are tailored to suppress the plant’s immune response. These virulence effectors are injected into the host cytoplasm, where they can then, in turn, be recognized by cytoplasmic receptor proteins, which results in effector-triggered immunity (ETI) (3). The intracellular recognition of effector molecules is mediated by structurally related proteins belonging to the nucleotide-binding site-leucine-rich repeat (NBS-LRR) family (4). ETI forms the second tier of the plant immune response. In contrast to nonhost resistance, the ETI response against adapted pathogens is much stronger and often results in the death of the infected cell through hypersensitive reaction. Hence, there is a strong selective pressure on the pathogen to avoid ETI by evolving new virulence effectors that escape recognition by NBS-LRR immune receptors. This coevolutionary arms race is the major reason for rapid breakdown of NBS-LRR-based disease resistance in crop plants, which often occurs within only a few years (5, 6).Cell surface PRRs are encoded by receptor-like kinase (RLK) and receptor-like protein (RLP) genes. Both RLKs and RLPs contain an extracellular elicitor-binding domain and a transmembrane domain. In contrast to RLKs, RLPs lack a cytoplasmic serine/threonine kinase domain (7). The best-studied PRR is the leucine-rich repeat (LRR) receptor-like kinase flagellin-sensitive-2 (FLS2) of Arabidopsis, which binds an epitope of bacterial flagellin. On flagellin perception, this kinase initiates a signaling cascade that results in nonhost resistance (8). Most PRRs described until today contain such an extracellular LRR domain. Not all PRRs, however, are involved in nonhost resistance. The rice LRR-RLK Xa21, for example, confers race-specific resistance against the bacterial rice blast pathogen Xanthomonas oryzae pv. oryzae (9). The effector recognized by Xa21 is still unknown. In addition, plants have evolved the ability to perceive endogenous molecules through PRRs that are produced during pathogen infection. For example, the Arabidopsis wall-associated kinase 1 (WAK1) binds cell wall-derived oligogalacturonides that are released during pathogen infection and serve as DAMPs (10). AtDORN1, an Arabidopsis lectin-RLK, binds plant-derived extracellular ATP likely produced during pathogen infection or wounding (11).Maize (Zea mays L.) is the most widely grown crop in the world and represents an important source of food, feed, biofuel, and industrial products. Fungal diseases are a major threat to maize production and can result in severe crop losses. Northern corn leaf blight (NCLB) is the most devastating foliar disease of maize. It is caused by the hemibiotrophic ascomycete fungus Exserohilum turcicum (teleomorph Setosphaeria turcica) (12). The fungus spreads biotrophically during the initial infection process before switching to a necrotrophic lifestyle. Infections manifest as local lesions and necrosis, which lead to reduced photosynthetically active leaf area and yield losses. The disease occurs prevalently under conditions of high humidity and moderate temperatures and can be found in most regions where maize is grown (13–15). The Htn1 locus confers quantitative and partial resistance against NCLB by delaying lesion formation. Htn1 was originally introgressed into modern maize cultivars from the Mexican landrace Pepitilla in the 1970s (16). The Htn1 resistance reaction is different from the other known major NCLB resistance genes Ht1, Ht2, and Ht3, which confer qualitative resistance, resulting in chlorotic-necrotic lesions. In contrast, Htn1 leads to a delay of sporulation without chlorotic lesions (17, 18). The dominant and race-specific Htn1 gene is effective against the most prevalent NCLB races, but virulent isolates have been found (18). The strength of the Htn1 resistance depends on environmental conditions and maize genotype (19). Htn1 has been mapped to maize chromosome 8 ∼10 cM distal to the NCLB resistance gene Ht2 (20–23).Here, we describe the map-based isolation of the Htn1 gene, which encodes for a pattern recognition receptor with a putative extracellular, wall-associated domain.     

罗格列酮对高脂饮食诱导非酒精性脂肪肝大鼠胰岛素抵抗及脂联素的影响

目的:探讨罗格列酮对高脂饮食所致的非酒精性脂肪肝大鼠模型胰岛素抵抗及脂联素的影响.方法:30只大鼠随机分为3组,即模型组(高脂饮食 蒸馏水ig),空白对照组(正常饲料 蒸馏水ig)和罗格列酮组[高脂饮食 罗格列酮3 mg/(kg·d)ig].观察各组血脂、肝功、胰岛素抵抗指数(HOMA-IR)的变化和各组肝组织HE染色与脂肪特异性的苏丹Ⅲ染色的变化.采用实时荧光定量聚合酶链式反应(PCR)和免疫印迹(western blot)检测各组肝组织的脂联素水平.结果:模型组与空白对照组相比,甘油三酯(TG)(1.51±0.37 mmol/L vs 0.98±0.51 mmol/L,P<0.01),总胆固醇(TC)(2.74±0.65 mmol/L vs 1.71±0.37 mmol/L,P<0.05),ALT(450.20±244.12 U/L vs 264.56±48.44 U/L,P<0.011,AST(460.30±310.13 U/L vs 196.11±52.23 U/L,P<0.01)和HOMA-IR(3.46±1.16 vs 1.07±0.26,P<0.01)均显著升高.罗格列酮治疗可使TG(1.27±0.50 mmol/L),ALT(360.26±244.37 U/L,AST(300.20±233.13 U/L)以及HOMR- IR(1.44±0.37)得到明显改善(均P<0.05).从组织病理学亦可得到证实.肝组织实时荧光定量PCR及免疫印迹显示罗格列酮组的Adiponectin mRNA(552.40±268.13 vs 215.95±135.87,P<0.05)和蛋白表达均较模型组升高.结论:高脂饮食可诱导大鼠NAFLD和IR发生,并使肝功,血脂异常升高.罗格列酮可以改善高脂饮食大鼠的脂肪肝及IR情况,可能与Adiponectin缓解IR和NAFLD改善有关.     

The evolution of severe steatosis after bariatric surgery is related to insulin resistance

BACKGROUND & AIMS: In severely obese patients, factors implicated in the evolution of severe steatosis after bariatric surgery remain unresolved. Our aim was to determine whether insulin resistance (IR) influences the histologic effects induced by bariatric surgery. METHODS: We prospectively included 185 severely obese patients (body mass index >/=35 kg/m(2)) referred for bariatric surgery. The evolution of IR (IR index = 1/quantitative insulin sensitivity check index) and liver injury with consecutive biopsy was concomitantly assessed before and 1 year after surgery. RESULTS: At preoperative biopsy, 27% of severely obese patients disclosed severe steatosis (>/=60%). The alanine aminotransferase (P = .01) and IR indexes (P = .04) were independent predictive factors of severe steatosis at baseline. One year after surgery, surgical treatment induced a decrease in body mass index (9.5 kg/m(2); P < .0001), steatosis score (8.5%; P < .0001), and IR index (0.29; P < .0001). The preoperative IR index (P = .01) and preoperative steatosis (P = .006) were independent predictive factors in the persistence of severe steatosis after surgery. Moderate or severe steatosis was more frequently observed in patients who had conserved a higher IR index after surgery than in patients who had improved their IR index (44% vs 20.2%; P = .04). CONCLUSIONS: IR was independently associated with severe steatosis and predicted its persistence after surgery. The amelioration of IR after surgery is associated with a decrease in the amount of fat. Taken together, the results of this prospective study in severely obese patients demonstrate that severe steatosis and its evolution after surgery are intimately connected with IR.     

Multivariate analysis of maize disease resistances suggests a pleiotropic genetic basis and implicates a GST gene

Plants are attacked by pathogens representing diverse taxonomic groups, such that genes providing multiple disease resistance (MDR) are expected to be under positive selection pressure. To address the hypothesis that naturally occurring allelic variation conditions MDR, we extended the framework of structured association mapping to allow for the analysis of correlated complex traits and the identification of pleiotropic genes. The multivariate analytical approach used here is directly applicable to any species and set of traits exhibiting correlation. From our analysis of a diverse panel of maize inbred lines, we discovered high positive genetic correlations between resistances to three globally threatening fungal diseases. The maize panel studied exhibits rapidly decaying linkage disequilibrium that generally occurs within 1 or 2 kb, which is less than the average length of a maize gene. The positive correlations therefore suggested that functional allelic variation at specific genes for MDR exists in maize. Using a multivariate test statistic, a glutathione S-transferase (GST) gene was found to be associated with modest levels of resistance to all three diseases. Resequencing analysis pinpointed the association to a histidine (basic amino acid) for aspartic acid (acidic amino acid) substitution in the encoded protein domain that defines GST substrate specificity and biochemical activity. The known functions of GSTs suggested that variability in detoxification pathways underlie natural variation in maize MDR.     

Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38

The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth.The codling moth Cydia pomonella L. is a worldwide occurring insect pest that infests apples, pears, and walnuts. The larvae of codling moth bore into the fruit and cause severe economic damage if not controlled. A number of chemical and biological agents are available for the control of codling moth. One of the most efficient biological control agents (1–5) is the Cydia pomonella granulovirus (CpGV), which belongs to the dsDNA virus family Baculoviridae (genus Betabaculovirus). CpGV was first discovered in Mexico (Mexican isolate, CpGV-M) in 1963 (6); this isolate was later developed to commercial products now registered in 34 countries worldwide. The genome sequence of the in vivo cloned strain CpGV-M1 is ∼123 kbp and encodes for 143 ORFs (7). Based on SNPs in highly conserved genes, different geographic CpGV isolates were classified into four genome types, A–D (8). CpGV exhibits an extremely narrow host range that is restricted to C. pomonella and a very few closely related tortricids (Lepidoptera) (9).The development of resistance to baculovirus infection was thought to be unlikely before 2005 (10, 11), which is when the first cases of resistant codling moth populations with a 1,000- to 100,000-fold reduced susceptibility to commercial CpGV products containing the isolate CpGV-M were reported from organic apple plantations in Germany and France, where CpGV products had been intensively applied (12, 13). Since then, 38 apple plantations with CpGV resistance have been identified in Austria (2 orchards), Czech Republic (1), France (3), Germany (22), Italy (6), Switzerland (2), and the Netherlands (2) (14). For the codling moth strain CpR, which originated from a resistant field population in south Germany, as well as the genetically homogenous laboratory strain CpRR1, which derived from CpR, the mode of inheritance was revealed to be incompletely dominant, monogenic, and linked to the Z (sex) chromosome (15, 16). A similar mode of inheritance was also observed in resistant Czech and French codling moth populations arguing for a more or less universal mode of resistance in Europe (17, 18). These data led to the hypothesis that a genetic adaptation of codling moth to CpGV-M infection had occurred and was selected for by the intensive use of products containing CpGV-M (15).Insects manifest miscellaneous strategies to resist pathogens but lack an adaptive immune system. Insect defense to viral infections involves nonspecific factors such as physical barriers, enzymatic responses, and increasing ejection of infected midgut cells as larvae age, as well as specific factors involving cellular and humoral immunity (19). Physical barriers to infection include the perithropic membrane (PM), an ultrafilter for particles with size exclusion greater than 30 nm (20). Melanization of the cuticula mediated by phenoloxidase enzymes is involved in the encapsulation reaction of pathogens, such as bacteria, fungi, or virus-infected cells (21, 22). Developmental resistance, the decreasing susceptibility of larvae with increasing age, is rather common, and is mediated by infected midgut cell-sloughing (23–25). Behavior modifications also count among insect defense strategies (19). Feeding behaviors, e.g., can strongly affect the risk of insects for baculovirus infection as shown for Lymantria dispar L., which exhibit heritable cadaver-avoidance behaviors (26).An alternative way for insects to get rid of virus-infected cells is via programmed cell death—the apoptosis pathway (27). Baculoviruses, however, are able to block apoptosis with the help of different virus encoded antiapoptotic proteins. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV), these proteins—namely P35 and P49—are inhibitors of the insect caspases. These inhibitors of apoptosis (IAPs), thereby, ensure a permissive virus infection (28).For codling moth, however, resistance based on the PM, the midgut, or the immune system has been excluded for CpGV (16, 29). Lack of CpGV DNA replication and a systemic resistance in all five instars indicated an early block to virus replication in resistant codling moth individuals (29, 30). At the same time, it was observed that certain CpGV isolates, such as I12 or NPP-R1/R4, were able to infect larvae from resistant codling moth strains (30, 31). Therefore, these naturally occurring CpGV isolates from different geographic origins were considered resistance-breaking isolates. Some of these isolates, meanwhile, replace CpGV-M in commercial biocontrol agents in Europe and demonstrate the importance of identifying resistance-breaking CpGV variants (30–32) for managing CpGV-resistant codling moth populations; however, their functional difference to CpGV-M allowing them to overcome CpGV resistance remained unknown.In this study we compared different naturally occurring geographic CpGV isolates representing all known CpGV genome types (8) in laboratory assays for their infectivity to susceptible (CpS) and resistant (CpRR1) codling moth strains. We found that all tested isolates—except CpGV-M—were able to overcome resistance in CpRR1 larvae. Whole-genome sequencing of these isolates revealed a single common difference in all resistance-breaking isolates, which was located in ORF24 (pe38). Therefore, occlusion bodies of bacmid-based recombinant CpGV (in the following termed pseudoviruses) knockout and recovery mutants of pe38 were established and tested for their activity in susceptible CpS and resistant CpRR1 larvae. The recovery of infectivity of a CpGV-M–based pseudovirus harboring the pe38 of the resistance-breaking CpGV-S in CpRR1 larvae demonstrated the key function of pe38 in overcoming baculovirus resistance in codling moth. In their entirety, these investigations demonstrate the significance of using multiple isolates of CpGV concurrently in field applications to avoid resistance in codling moth populations.     

A guardian of grasses: specific origin and conservation of a unique disease-resistance gene in the grass lineage

The maize Hm1 gene provides protection against a lethal leaf blight and ear mold disease caused by Cochliobolus carbonum race 1 (CCR1). Although it was the first disease-resistance (DR) gene to be cloned, it remains a novelty because, instead of participating in the plant recognition and response system as most DR genes do, Hm1 disarms the pathogen directly. It does so by encoding an NADPH-dependent reductase, whose function is to inactivate Helminthosporium carbonum (HC) toxin, an epoxide-containing cyclic tetrapeptide, which the pathogen produces as a key virulence factor to colonize maize. Although CCR1 is strictly a pathogen of maize, orthologs of Hm1 and the HC-toxin reductase activity are present in the grass family, suggesting an ancient and evolutionarily conserved role of this DR trait in plants. Here, we provide proof for such a role by demonstrating its involvement in nonhost resistance of barley to CCR1. Barley leaves in which expression of the Hm1 homologue was silenced became susceptible to infection by CCR1, but only if the pathogen was able to produce HC toxin. Phylogenetic analysis indicated that Hm1 evolved exclusively and early in the grass lineage. Given the devastating ability of CCR1 to kill maize, these findings imply that the evolution and/or geographical distribution of grasses may have been constrained if Hm1 did not emerge.     

From the Cover: A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria’s host is a “farmer” clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer’s spore production and depresses a nonfarmer’s spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a “domesticated” food source.     

Insulin resistance is accompanied by increased von Willebrand factor levels in nondiabetic women: a study of offspring of type 2 diabetic subjects compared to offspring of nondiabetic subjects

OBJECTIVES: To examine whether levels of von Willebrand factor (vWF), fibrinogen and fibronectin are related to a parental history of type 2 diabetes and to determine possible explanatory factors for high versus low vWF and fibrinogen. DESIGN: Cross-sectional study. SUBJECTS, MAIN OUTCOME MEASURES: We compared vWF, fibrinogen and fibronectin in 88 nondiabetic offspring of type 2 diabetic subjects (relatives) and 103 offspring of nondiabetic subjects (controls). Other measurements included urinary albumin excretion rate, blood pressure, lipid profile and insulin resistance using homeostasis model assessment (HOMAIR). RESULTS: There were no significant differences in vWF (1.12 vs. 1.06 IU x mL(-1), P = 0.296), fibrinogen (3.2 vs. 3.1 g x L(-1); P = 0.263) or fibronectin (0.39 vs. 0.40 g x L(-1), P = 0.448) between relatives and controls. With multiple logistic regression we determined explanatory factors for high versus low vWF and fibrinogen. Age (P < 0.01), urinary albumin excretion rate (P < 0.05), ischaemic heart disease (IHD) (P < 0.05) were found to be significant explanatory factors for vWF above the median (1.10 IU x mL(-1)). Interaction between insulin resistance and sex was found. Odds ratio for high versus low insulin resistance was 18.39 (P < 0.001) for women and 1.92 (P = 0.32) for men. Body mass index (BMI) (P < 0.05), sex (P < 0.01), smoking status (P < 0.05) and IHD (P < 0.01) were significant explanatory factors for fibrinogen above the median (3.1 g x L(-1)). CONCLUSIONS: Levels of vWF, fibrinogen and fibronectin were not influenced by a parental history of type 2 diabetes. Insulin resistance was found to be a significant risk indicator for high vWF only in women. This may indicate that insulin resistance is a higher risk factor for women than for men, when the outcome is endothelial dysfunction possibly resulting in overt cardiovascular disease.     

The metastasis-associated gene S100A4 is a novel target of beta-catenin/T-cell factor signaling in colon cancer

BACKGROUND & AIMS: Activation of the Wnt/beta-catenin pathway is frequently observed in colorectal cancers. Our aim was to elucidate the impact of gain-of-function beta-catenin on the metastasis-associated gene S100A4 in human colon cancer cell lines and tumors. METHODS: We analyzed cell lines heterozygous for gain-of-function and wild-type beta-catenin, and variants homozygous for gain- or loss-of-function mutation in beta-catenin, for S100A4 expression, cell motility, and in vivo metastasis. beta-catenin-mediated S100A4 promoter activation was tested by reporter assays. For human colon carcinomas, S100A4 expression, beta-catenin genotype, and metachronous metastasis were correlated. RESULTS: We identified S100A4 as the most regulated gene by gain-of-function beta-catenin using a 10K microarray. Cell lines with gain-of-function beta-catenin expressed up to 60-fold elevated S100A4 levels, displayed strongly increased migration and invasion in vitro, and induced metastasis in mice. S100A4 small interfering RNA, beta-catenin small interfering RNA, or dominant negative T-cell factor (TCF) knocked down S100A4 and blocked biological effects. S100A4 complementary DNA transfection increased migration and invasion. We identified a TCF binding site within the S100A4 promoter and demonstrated the direct binding of heterodimeric beta-catenin/TCF complexes. Reporter assays confirmed the beta-catenin-induced S100A4 promoter activity. Furthermore, S100A4 mRNA expression was increased in primary colon cancers, which later developed distant metastases, compared to non-metastasizing tumors. Colon tumors heterozygous for gain-of-function beta-catenin showed concomitant nuclear beta-catenin localization, high S100A4 expression, and metastases. CONCLUSIONS: S100A4 is a direct beta-catenin/TCF target, induces migration and invasion in vitro and metastasis in vivo, and has value for prognosis of metastasis formation in colon cancer patients.