Extracellular matrix metalloproteinase inducer (CD147) and matrix metalloproteinase-2 (MMP-2) have been documented in various malignancies. CD147 is a member of the immunoglobulin superfamily, which promotes the production and release of MMPs in mesenchymal cells and tumor cells. MMP-2 has been extensively studied and is considered to be particularly important in cancer invasion and metastasis. However, studies investigating the expression and prognostic value of CD147 in endometrioid endometrial carcinoma (EEC) are limited. The present study analyzed the expression of CD147 and MMP-2 by immunohistochemistry in endometrial tissue samples from 107 patients with EEC and 30 patients with benign uterus myoma. The association between CD147 and MMP-2 expression and clinicopathological characteristics was evaluated. The results showed that the overexpression of MMP-2 was significantly associated with International Federation of Gynecology and Obstetrics stage (P=0.007), depth of invasion (P=0.037) and reduced expression of progesterone receptor (P=0.005). Kaplan-Meier analyses indicated that CD147 overexpression alone (P<0.05 for disease-specific survival) or in combination with MMP-2 (P<0.001 for disease-specific survival) was correlated with adverse prognosis in EEC patients. Multivariate analysis revealed that the combined overexpression of CD147 and MMP-2 was an independent prognostic factor for disease-specific survival (hazard ratio=5.141, P=0.001) in EEC patients. CD147 and MMP-2 overexpression was positively correlated with aggressive phenotypic features in EEC, however it was negatively correlated with hormone receptor expression. The combination of CD147 and MMP-2 overexpression in EEC further distinguished a subgroup of patients with poor prognosis. Thus, the results of present study indicate that the co-expression of CD147 and MMP-2 may be an independent prognostic factor in EEC patients.
endometrioid endometrial cancer; extracellular matrix metalloproteinase inducer; matrix metalloproteinase-2; immunohistochemistry; prognosis
Paroxysmal kinesigenic choreoathetosis (PKC) is an inherited disease of the nervous system. We previously identified PRRT2 as the causative gene of PKC. However, as little is known about the function of PRRT2, elucidating its function will benefit not only PKC studies, but also many other related disorders. Here, we reveal higher levels of glutamate in the plasma of PKC patients and the culture medium of neurons following knock-out Prrt2 expression. Using double immunostaining assays we confirm Prrt2 is located at the glutamatergic neurons in accordance with its function. Our co-immunoprecipitation assays reveal mutant PRRT2 interferes with SNAP25 and GRIA1 interactions, respectively. Furthermore, using live-labeling techniques, we confirmed co-transfection with mutant PRRT2 caused an increase in GRIA1 distribution on the cell surface. Therefore, our results suggest that mutant PRRT2, probably through its weakened interaction with SNAP25, affects glutamate signaling and glutamate receptor activity, resulting in the increase of glutamate release and subsequent neuronal hyperexcitability.
PRRT2; glutamate; SNAP25; GRIA1
To compare compensatory sweating after lowering or restricting the level of sympathectomy.
A systematic review and meta-analysis were conducted of all randomized controlled trials published in English that compared compensatory sweating after lowering or restricting the level of sympathectomy. The Cochrane collaboration tool was used to assess the risk of bias, and the Mantel-Haenszel odds ratio method was used for the meta-analysis.
A total of 11 randomized controlled trials were included, including a total of 1079 patients. Five of the randomized controlled trials studied restricting the level of sympathectomy, and the remaining six studied lowering the level of sympathectomy.
The compiled randomized controlled trial results published so far in the literature do not support the claims that lowering or restricting the level of sympathetic ablation results in less compensatory sweating.
Sympathectomy; Compensatory sweating; Meta-analysis
Frequent readmissions for acute exacerbations of COPD (AECOPD) are an independent
risk factor for increased mortality and use of health-care resources. Disease
severity and C-reactive protein (CRP) level are validated predictors of long-term
prognosis in such patients. This study investigated the utility of combining serum
CRP level with the Global Initiative for Chronic Obstructive Lung Disease (GOLD)
exacerbation risk classification for predicting readmission for AECOPD.
This was a prospective observational study of consecutive patients hospitalized
for AECOPD at Peking University Third Hospital, in Beijing, China. We assessed
patient age; gender; smoking status and history (pack-years); lung function;
AECOPD frequency during the last year; quality of life; GOLD risk category (A-D; D
indicating the greatest risk); and serum level of high-sensitivity CRP at
The final sample comprised 135 patients. Of those, 71 (52.6%) were readmitted at
least once during the 12-month follow-up period. The median (interquartile) time
to readmission was 78 days (42-178 days). Multivariate analysis revealed that
serum hsCRP-D ≥ 3 mg/L and GOLD category D were independent predictors of
readmission (hazard ratio = 3.486; 95% CI: 1.968-6.175; p < 0.001 and hazard
ratio = 2.201; 95% CI: 1.342-3.610; p = 0.002, respectively). The ordering of the
factor combinations by cumulative readmission risk, from highest to lowest, was as
follows: hsCRP-D ≥ 3 mg/L and GOLD category D; hsCRP-D ≥ 3 mg/L and GOLD
categories A-C; hsCRP-D < 3 mg/L and GOLD category D; hsCRP-D < 3 mg/L and
GOLD categories A-C.
Serum hsCRP-D and GOLD classification are independent predictors of readmission
for AECOPD, and their predictive value increases when they are used in
Pulmonary disease; chronic obstructive/epidemiology; Acute disease; Acute-phase proteins; Hospitalization; Patient readmission; Inflammation
The association between increases in both systemic and airway inflammation and acute exacerbation of COPD (AECOPD) has been reported by many studies. However, relatively little is known about the dynamics of inflammation resolution and their correlations with the improvement of clinical indices during treatment. In this study, a total of 93 consecutively hospitalized patients with AECOPD were recruited. Sputum and serum inflammatory markers were measured on the day of admission before treatment (day 0), day 4, 7 and 14 during treatment as well as 8 weeks after discharge. Clinical indices (lung function, dyspnea and COPD assessment test (CAT) scores) were also measured at those time points. By day 4, all airway inflammatory measures rapidly decreased and returned to baseline level. Notably, lung function and dyspnea improved to the baseline level by day 4 as well, consistent with the resolution of respiratory inflammation. However, despite the significant decrease by day 4, systemic inflammation did not reach baseline until day 14, concordant with the decrease in CAT score. In summary, we observed a time lag between the resolution of systemic and airway inflammation, which were correlated with the improvements of different clinical indices.
Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in colorectal cancer (CRC) to determine its potential as a prognosis and immunotherapeutic target. The expression of MAGE-D4 mRNA and protein was determined by RT-PCR and immunohistochemistry (IHC) in CRCs with paired adjacent non-tumor tissues, colorectal adenomas and normal colorectal tissues, respectively. Sera from 64 CRC patients were tested for MAGE-D4 antibody by ELISA. MAGE-D4 mRNA was more frequently expressed in CRCs (76.7%, 46/60) than in adjacent non-tumor tissues (15.0%, 9/60). MAGE-D4 protein was detected in all the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; P=0.050). Humoral immunity to MAGE-D4 was detected in 12 of 64 (18.8%) CRC patients’ sera but not in 77 healthy donors. There was no correlation between MAGE-D4 expression, serum antibody and clinicopathological parameters. These findings suggest MAGE-D4 may serve as a potentially prognostic biomarker and an attractive target of immunotherapy in CRC.
Melanoma-associated antigen; MAGE-D4; colorectal cancer; serum immunoreactivity
DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case study, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the 3D structure of DNA binding sites. Thus, local shape of target sites might play a widespread role in achieving regulatory specificity within TF families.
transcription factors; bHLH; DNA binding sites; protein binding microarrays; DNA shape; support vector regression
Loss of GGPPS from childhood mumps infection or deletion in mice results in constitutively activated MAPK and NF-kB signaling that induces spermatogonium apoptosis, macrophage invasion into seminiferous tubules, and sterility.
Mumps commonly affects children 5–9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell–only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients’ fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-κB signaling pathways were constitutively activated in GGPPS−/− Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS−/− Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility.
We have demonstrated the neuroprotection of hydrogen sulfide (H2S) against chemical hypoxia-induced injury by inhibiting p38MAPK pathway. The present study attempts to evaluate the effect of H2S on chemical hypoxia-induced inflammation responses and its mechanisms in PC12 cells. We found that treatment of PC12 cells with cobalt chloride (CoCl2, a hypoxia mimetic agent) enhanced IL-6 secretion, nitric oxide (NO) generation and expression levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). L-canavanine, a selective iNOS inhibitor, partly blocked CoCl2-induced cytotoxicity, apoptosis and mitochondrial insult. In addition, 7-Nitroindazole (7-NI), an inhibitor of nNOS, also partly attenuated the CoCl2-induced cytotoxicity. The inhibition of p38MAPK by SB203580 (a selective p38MAPK inhibitor) or genetic silencing of p38MAPK by RNAi (Si-p38) depressed not only CoCl2-induced iNOS expression, NO production, but also IL-6 secretion. In addition, N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, conferred a similar protective effect of SB203580 or Si-p38 against CoCl2-induced inflammatory responses. Importantly, pretreatment of PC12 cells with exogenous application of sodium hydrosulfide (a H2S donor, 400 μmol/l) for 30 min before exposure to CoCl2 markedly attenuated chemical hypoxia-stimulated iNOS and nNOS expression, NO generation and IL-6 secretion as well as p38MAPK phosphorylation in PC12 cells. Taken together, we demonstrated that p38MAPK-iNOS pathway contributes to chemical hypoxia-induced inflammation and that H2S produces an anti-inflammatory effect in chemical hypoxia-stimulated PC12 cells, which may be partly due to inhibition of ROS-activated p38MAPK-iNOS pathway.
Hydrogen sulfide; Hypoxia; Inflammation; p38 MAPK; Nitric oxide; Cobalt chloride
This study investigated the use of direct gradient analysis of bacterial 16S pyrosequencing surveys to identify relevant bacterial community signals in the midst of a "noisy" background, and to facilitate hypothesis-testing both within and beyond the realm of ecological surveys. The results, utilizing 3 different real world data sets, demonstrate the utility of adding direct gradient analysis to any analysis that draws conclusions from indirect methods such as Principal Component Analysis (PCA) and Principal Coordinates Analysis (PCoA). Direct gradient analysis produces testable models, and can identify significant patterns in the midst of noisy data. Additionally, we demonstrate that direct gradient analysis can be used with other kinds of multivariate data sets, such as flow cytometric data, to identify differentially expressed populations. The results of this study demonstrate the utility of direct gradient analysis in microbial ecology and in other areas of research where large multivariate data sets are involved.
Impaired mitochondrial fusion/fission plays a causal role in neuronal death. This study delineated a PKCδ-related signaling cascade in which excessive mitochondrial fission is induced during oxidative stress. Moreover, a selective peptide inhibitor of PKCδ inhibits impaired mitochondrial fission under these pathological conditions.
Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase Cδ (PKCδ) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKCδ binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKCδ, using a selective PKCδ peptide inhibitor (δV1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKCδ activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.
Specific activation of amino acids by aminoacyl-tRNA synthetases is essential for maintaining translational fidelity. Here, we present crystal structures of Saccharomyces cerevisiae tryptophanyl-tRNA synthetase (sTrpRS) in apo form and in complexes with various ligands. In each complex, there is a sulfate ion bound at the active site which mimics the α- or β-phosphate group of ATP during tryptophan activation. In particular, in one monomer of the sTrpRS–TrpNH2O complex, the sulfate ion appears to capture a snapshot of the α-phosphate of ATP during its movement towards tryptophan. Simulation study of a human TrpRS–Trp–ATP model shows that during the catalytic process the α-phosphate of ATP is driven to an intermediate position equivalent to that of the sulfate ion, then moves further and eventually fluctuates at around 2 Å from the nucleophile. A conserved Arg may interact with the oxygen in the scissile bond at the transition state, indicating its critical role in the nucleophilic substitution. Taken together, eukaryotic TrpRSs may adopt an associative mechanism for tryptophan activation in contrast to a dissociative mechanism proposed for bacterial TrpRSs. In addition, structural analysis of the apo sTrpRS reveals a unique feature of fungal TrpRSs, which could be exploited in rational antifungal drug design.
The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5′ AMP (TrpAMP) at 3.0 Å resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.
Human tryptophanyl-tRNA synthetase (hTrpRS) differs from its bacterial counterpart at several key positions of the catalytic active site and has an extra N-terminal domain, implying possibly a different catalytic mechanism. We report here the crystal structures of hTrpRS in complexes with Trp, tryptophanamide and ATP and tryptophanyl-AMP, respectively, which represent three different enzymatic states of the Trp activation reaction. Analyses of these structures reveal the molecular basis of the mechanisms of the substrate recognition and the activation reaction. The dimeric hTrpRS is structurally and functionally asymmetric with half-of-the-sites reactivity. Recognition of Trp is by an induced-fit mechanism involving conformational change of the AIDQ motif that creates a perfect pocket for the binding and activation of Trp and causes coupled movements of the N-terminal and C-terminal domains. The KMSAS loop appears to have an inherent flexibility and the binding of ATP stabilizes it in a closed conformation that secures the position of ATP for catalysis. Our structural data indicate that the catalytic mechanism of the Trp activation reaction by hTrpRS involves more moderate conformational changes of the structural elements at the active site to recognize and bind the substrates, which is more complex and fine-tuned than that of bacterial TrpRS.
Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNATrp and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3′ end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an α-helix of the unique N-terminal domain and the anticodon loop by an α-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.