Expression of metastasis-associated protein 1 (MTA1) gene correlates with the degree of invasion and metastasis in hepatocellular carcinoma (HCC). Expression of MTA1 is induced by hepatitis B virus X protein (HBx); however, little is known about the transcriptional regulation of MTA1 gene expression. Here, we report that the 5′-flanking region of the human MTA1 promoter contains two CpG islands. Transient expression of HBx in Chang liver cells increased the methylation of the CpG island1 from 18 to 49% when measured by bisulfite-modified direct sequencing. Chromatin immunoprecipitation showed that HBx recruited DNA methyltransferase 3a (DNMT3a) and DNMT3b to the CpG island1. In silico analysis of CpG island1 predicted the existence of putative p53-binding sequences. p53 was pulled down by a DNA probe encoding the p53-binding sequences but not by the methylated DNA probe. The mouse MTA1 promoter also contains a CpG island encoding a p53-binding sequence of which p53 binding was decreased in the presence of HBx, and the expression of MTA1 and DNMT3 was increased in the liver of HBx-transgenic mice. Comparison of MTA1 and DNMT3a expression in the human normal liver and HCC specimens produced a significant correlation coefficient >0.5 (r=0.5686, P=0.0001) for DNMT3a, and a marginally significant coefficient (r=0.3162, P=0.0103) for DNMT3b. These data show that HBx induces methylation of CpG island in the MTA1 promoter, which interferes with DNA binding of p53 in the specific DNA region. This result may explain the molecular mechanism responsible for the induction of MTA1 gene expression by HBx.
MTA1; p53; HBx; DNA methylation
Recently apoptotic cell death has been reported in differentiated skeletal muscle, where apoptosis was generally assumed not to occur. To investigate whether apoptosis may contribute to the steroid-induced myopathy, rats treated with triamcinolone acetonide (TA) for 9 days were sacrificed for detecting apoptosis by in situ end labeling (ISEL) and electron microscopy in the soleus muscles. Immunohistochemical stainings of Fas antigen and p53 protein were performed to examine whether apoptosis-related proteins were present in the myopathy. Muscle fiber necrosis and apoptotic myonuclei appeared in the soleus muscles following administration of TA, while control muscles showed no evidences for apoptosis. Fas antigen was not detected in control muscles, but expressed in the soleus muscles of steroid-induced myopathy. Some of the Fas antigen-expressing muscle fibers were positive for ISEL. p53 protein was not detected in any muscle fibers. These findings indicate that TA can induce apoptosis in differentiated skeletal muscles, and Fas antigen might be partly related to apoptotic muscle death in steroid-induced myopathy.
We report three autopsy cases of congenital cytomegalovirus (CMV) infection in fetuses with a review of literature. The clinical manifestations in these cases of congenital CMV infection include intrauterine fetal death, hydrops fetalis, and CMV pneumonia associated with cardiovascular defect. The pathological characteristics were as follows: 1) the kidney was the most frequently involved organ, followed by lung and liver, 2) CMV inclusions were found predominantly in epithelial cells and to a lesser degree in endothelial cells, 3) intrahepatic bile duct epithelial cells were frequently involved, and 4) inflammatory reaction around CMV inclusions was not prominent in the early stage of pregnancy. Diagnostic confirmation was obtained by in situ hybridization (ISH) using a biotinylated CMV-DNA probe, which demonstrated intranuclear inclusions and sometimes recognized cells that did not show intranuclear inclusion.
The cytokine pattern on viral antigen recognition is believed to exert a profound influence on the resolution of viral infections and viral clearance. This study was initiated to investigate whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analysed by RT-PCR. Upon HBsAg stimulation, expression of IFN-gamma, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of IFN-gamma was associated with high levels of serum AST/ALT. However, we could not prove that Th2 type cytokines had a protective effect on hepatocytes. Upon HBxAg stimulation, there was no recognizable association of cytokine patterns with AST/ALT levels. In conclusion, production of a Th1 cytokine, IFN-gamma, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B, while no counteracting effect of Th2 cytokines produced by those cells was observed.
Multiple symmetric lipomatosis (MSL) is an extremely uncommon disorder. In the medical literatures about 200 cases have been reported. MSL is not associated with other generalized lipomatous disorders, nor are these patient to be necessarily obese. The cause of MSL is unknown. The disorder usually occurs in middle-aged males and there is frequently a history of alcoholism. Some instances of familial occurrence have been reported, but the majority of cases are sporadic. Two cases of MSL are presented.
Automated data processing and quality control of radioimmunoassays offer not only increased speed but also a more thorough and statistically rigorous analysis of results. An external quality assessment scheme for serum thyroxine, triiodothyronine and thyroid stimulating hormone (TSH) assays was performed in five nuclear medicine laboratories in Korea to compare with the assay performances of the World Health Organization Radioimmunoassay Program. The required radioimmunoassay kits were supplied through the International Atomic Energy Agency (IAEA). We have determined the weighted root mean squared error, and variance ratio as the indices of standard curve and also the average batch coefficient of variation (ABCV) as the parameters of response error relationship curve and precision profile. There was a good fit for the triiodothyronine assay, but 3 of 5 laboratories showed possible bad fit in the T4 and TSH assay systems. The ABCV was less than 5 percent for the T3 and T4 assay system, however for the TSH system, only 1 laboratory showed the ABCV value of less than 5 percent. We have also calculated the within batch variation (drift) and between laboratory variations.
Facultative or “secondary” symbionts are common in eukaryotes, particularly insects. While not essential for host survival, they often provide significant fitness benefits [1-5]. It has been hypothesized that secondary symbionts form a “horizontal gene pool” shuttling adaptive genes among host lineages in an analogous manner to plasmids and other mobile genetic elements in bacteria [6, 7]. However, we do not know whether the distributions of symbionts across host populations reflect random acquisitions followed by vertical inheritance or whether the associations have occurred repeatedly in a manner consistent with a dynamic horizontal gene pool. Here we explore these questions using the phylogenetic and ecological distributions of secondary symbionts carried by 1,104 pea aphids, Acyrthosiphon pisum. We find that not only is horizontal transfer common, but it is also associated with aphid lineages colonizing new ecological niches, including novel plant species and climatic regions. Moreover, aphids that share the same ecologies worldwide have independently acquired related symbiont genotypes, suggesting significant involvement of symbionts in their host’s adaptation to different niches. We conclude that the secondary symbiont community forms a horizontal gene pool that influences the adaptation and distribution of their insect hosts. These findings highlight the importance of symbiotic microorganisms in the radiation of eukaryotes.
The molecular mechanisms controlling post-translational modifications of p21 have been pursued assiduously in recent years. Here, utilizing mass-spectrometry analysis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at amino-acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for the first time. Detection of adriamycin-induced p21 acetylation, which disappeared after Tip60 depletion with concomitant destabilization of p21 and disruption of G1 arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA damage-induced cell-cycle regulation. The ability of 2KQ, a mimetic of acetylated p21, to induce cell-cycle arrest and senescence was significantly enhanced in p21 null MEFs compared with those of cells expressing wild-type p21. Together, these observations demonstrate that Tip60-mediated p21 acetylation is a novel and essential regulatory process required for p21-dependent DNA damage-induced cell-cycle arrest.
p21 (Cip1/WAF1); Tip60; acetylation; cell-cycle arrest; DNA damage
We investigate whether differences in breast cancer survival in six high-income countries can be explained by differences in stage at diagnosis using routine data from population-based cancer registries.
We analysed the data on 257 362 women diagnosed with breast cancer during 2000–7 and registered in 13 population-based cancer registries in Australia, Canada, Denmark, Norway, Sweden and the UK. Flexible parametric hazard models were used to estimate net survival and the excess hazard of dying from breast cancer up to 3 years after diagnosis.
Age-standardised 3-year net survival was 87–89% in the UK and Denmark, and 91–94% in the other four countries. Stage at diagnosis was relatively advanced in Denmark: only 30% of women had Tumour, Nodes, Metastasis (TNM) stage I disease, compared with 42–45% elsewhere. Women in the UK had low survival for TNM stage III–IV disease compared with other countries.
International differences in breast cancer survival are partly explained by differences in stage at diagnosis, and partly by differences in stage-specific survival. Low overall survival arises if the stage distribution is adverse (e.g. Denmark) but stage-specific survival is normal; or if the stage distribution is typical but stage-specific survival is low (e.g. UK). International differences in staging diagnostics and stage-specific cancer therapies should be investigated.
breast cancer; survival; stage; population-based
ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.
Pregnancy alters the function of many body systems, including the immune system. However, little is known regarding the affects of pregnancy on maternal immunoglobulin E (IgE) levels or atopy.
To determine whether pregnancy consistently influences serum levels of total- or allergen-specific IgE.
Blood samples were obtained from 470 women during the third trimester of pregnancy and 1 month post partum. A third sample was obtained from 103 of these women 1 year post partum. Samples were analyzed for total and specific IgE to 8 regionally common allergens using the Phadia UniCAP system. Sensitization was defined as an allergen-specific IgE ≥0.35 kU/L to any allergen.
Total IgE increased significantly postpartum, both at 1 month (34.7 versus 39.8 IU/ml, P = 0.001) and at 1 year (38.0 versus 46.4 IU/ml, P = 0.01). Allergen-specific IgE decreased significantly at 1 month for cat, dog, cockroach, ragweed, timothy grass, Alternaria and egg (P = 0.001–0.012), but not for dust mite (D. farinae, P = 0.569). Similar patterns of change in total and specific IgE were seen at 1 year. However, on average, only 3.5% of subjects changed sensitization status to the individual allergens studied over the 1 year of observation.
Compared to intrapartum levels, total IgE levels increased significantly at one month and one year postpartum. Conversely, at the same time points, IgE levels specific for common allergens significantly declined to most but not all allergens. Few women changed their sensitization status over one year.
total IgE; allergen-specific IgE; pregnancy
In a crowded environment, how do we hear a single talker while ignoring everyone else? In this issue of Neuron, Zion Golumbic et al. record from the surface of the human brain to show how speech tracking arises through multiple neural frequency channels, both within and beyond auditory cortex.
cocktail party; attention; speech; ECoG
Obesity is associated with insulin resistance, a major risk factor for type 2 diabetes and cardiovascular disease. However, not all obese individuals are insulin resistant, which confounds our understanding of the mechanistic link between these conditions. We conducted transcriptome analyses on 835 obese subjects with mean BMI of 48.8, on which we have previously reported genetic associations of gene expression. Here, we selected ∼320 nondiabetic (HbA1c <7.0) subjects and further stratified the cohort into insulin-resistant versus insulin-sensitive subgroups based on homeostasis model assessment–insulin resistance. An unsupervised informatics analysis revealed that immune response and inflammation-related genes were significantly downregulated in the omental adipose tissue of obese individuals with extreme insulin sensitivity and, to a much lesser extent, in subcutaneous adipose tissue. In contrast, genes related to β-oxidation and the citric acid cycle were relatively overexpressed in adipose of insulin-sensitive patients. These observations were verified by querying an independent cohort of our published dataset of 37 subjects whose subcutaneous adipose tissue was sampled before and after treatment with thiazolidinediones. Whereas the immune response and inflammation pathway genes were downregulated by thiazolidinedione treatment, β-oxidation and citric acid cycle genes were upregulated. This work highlights the critical role that omental adipose inflammatory pathways might play in the pathophysiology of insulin resistance, independent of body weight.
Peritoneal dialysis catheter; spontaneous catheter fracture; drainage failure
Endoplasmic reticulum (ER) stress is considered one of the pathological mechanisms of idiopathic pulmonary fibrosis (IPF). Therefore, we examined whether an ER stress regulator, Bax inhibitor-1 (BI-1), regulates collagen accumulation, which is both a marker of fibrosis and a pathological mechanism of fibrosis. The presence of BI-1 inhibited the transforming growth factor-β1-induced epithelial–mesenchymal transition of epithelial pulmonary cells and bleomycin-induced pulmonary fibrosis in a mouse model by enhancing collagen degradation, most likely by enhanced activation of the lysosomal V-ATPase through glycosylation. We also found a correlation between post-translational glycosylation of the V-ATPase and its associated chaperone, calnexin, in BI-1-overexpressing cells. BI-1-induced degradation of collagen through lysosomal V-ATPase glycosylation and the involvement of calnexin were confirmed in a bleomycin-induced fibrosis mouse model. These results highlight the regulatory role of BI-1 in IPF and reveal for the first time the role of lysosomal V-ATPase glycosylation in IPF.
idiopathic pulmonary fibrosis; BI-1; epithelial—mesenchymal transition; ER stress; lysosome; V-ATPase glycosylation
Transitional 2 B cells home to gut-associated lymphoid tissue and present an activated phenotype in healthy subjects, but gut immune compartments are depleted in SLE.
We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells, the transitional 2 (T2) B cells, homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire, a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus, activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals, this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans.
Quantitative ultrasound (QUS) traits are correlated with bone mineral density (BMD), but predict risk for future fracture independent of BMD. Only a few studies, however, have sought to identify specific genes influencing calcaneal QUS measures. The aim of this study was to conduct a genome-wide linkage scan to identify quantitative trait loci (QTL) influencing normal variation in QUS traits. QUS measures were collected from a total of 719 individuals (336 males and 383 females) from the Fels Longitudinal Study who have been genotyped and have at least one set of QUS measurements. Participants ranged in age from 18.0 to 96.6 years and were distributed across 110 nuclear and extended families. Using the Sahara ® bone sonometer, broadband ultrasound attenuation (BUA), speed of sound (SOS) and stiffness index (QUI) were collected from the right heel. Variance components based linkage analysis was performed on the three traits using 400 polymorphic short tandem repeat (STR) markers spaced approximately 10 cM apart across the autosomes to identify QTL influencing the QUS traits. Age, sex, and other significant covariates were simultaneously adjusted. Heritability estimates (h2) for the QUS traits ranged from 0.42 to 0.57. Significant evidence for a QTL influencing BUA was found on chromosome 11p15 near marker D11S902 (LOD = 3.11). Our results provide additional evidence for a QTL on chromosome 11p that harbors a potential candidate gene(s) related to BUA and bone metabolism.
Genetic linkage; quantitative ultrasound; calcaneus; family studies; bone
We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive cells co-localized with CRC cells in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC conditioned medium or blockade of ADAM17 activity. ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.
Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease (PD), an infection-driven chronic inflammatory disease that leads to the resorption of tooth supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis-induced alveolar bone loss in vivo and our in vitro work implicated TNF as a key downstream mediator. Here we show that TNF-deficient (Tnf−/−) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental PD. Using bone marrow-derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naïve macrophages is MyD88-dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed HEK cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. While both TLR2 and TLR4 contributed to TNF production in naïve macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis-stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.
Although Roux-en-Y gastric bypass (RYGB) is a generally effective treatment for severe obesity, weight loss (WL) after this operation is highly variable. Accurate predictors of outcome would thus be useful in identifying those patients who would most benefit from this invasive therapy. WL has been characterized using several different metrics, including the number of BMI units lost (ΔBMI), percent baseline WL (%WL), and percent excess body WL (%EBWL). To identify clinically relevant predictors most sensitively it is necessary to avoid confounding by other factors, including preoperative BMI (pBMI), the strongest known predictor of RYGB-induced WL.
Design and Methods
To determine the WL measure least associated with pBMI, we analyzed outcomes of 846 patients undergoing RYGB.
Patients in this cohort had an average pBMI of 50.0 kg/m2. At weight nadir, they lost an average 19.4 kg/m2, 38.7% WL, and 81.2% EBWL. pBMI was strongly and positively associated with ΔBMI at both one year (r=0.56, p=4.7×10−51) and nadir (r=0.58, p=2.8×10−77) and strongly but negatively associated with %EBWL at one year (r=−0.52, p=3.8×10−44) and nadir (r=−0.45, p=7.2×10−43). In contrast, pBMI was not significantly associated with %WL at one year (r=0.04, p=0.33), and only weakly associated at nadir (r=0.13, p=0.0002).
Of the metrics examined, %WL is the parameter describing WL after RYGB least influenced by pBMI. It thus improves comparison of WL outcomes across studies of patients undergoing surgery and facilitates the most sensitive identification of novel predictors of surgery-induced WL. We therefore recommend that %WL be adopted more broadly in reporting weight loss after RYGB.
The International Tamoxifen Pharmacogenomics Consortium was established to address the
controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes
in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated
patients (12 globally distributed sites). Using strict eligibility requirements
(postmenopausal women with estrogen receptor–positive breast cancer, receiving
20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status
was associated with poorer invasive disease–free survival (IDFS: hazard ratio =
1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However,
CYP2D6 status was not statistically significant when tamoxifen duration,
menopausal status, and annual follow-up were not specified (criterion 2, n =
2,443; P = 0.25) or when no exclusions were applied (criterion 3, n
= 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS
using strict inclusion criteria, because the results are not robust to inclusion criteria
(these were not defined a priori), prospective studies are necessary to fully
establish the value of CYP2D6 genotyping in tamoxifen therapy.
Pegylated interferon-α (PEG-IFN-α or PEG-IFN 2a and 2b)– and ribavirin (RBV)-based regimens are the mainstay for treatment of hepatitis C virus (HCV) genotype 1. IFNL3 (IL28B) genotype is the strongest baseline predictor of response to PEG-IFN-α and RBV therapy in previously untreated patients and can be used by patients and clinicians as part of the shared decision-making process for initiating treatment for HCV infection. We provide information regarding the clinical use of PEG-IFN-α- and RBV-containing regimens based on IFNL3 genotype.