Pc-pis is a novel piscidin-like antimicrobial polypeptide that was identified in Pseudosciaena crocea. Although active against most bacteria tested, Pc-pis was inactive against Aeromonas hydrophila and Pseudomonas aeruginosa. The Pc-pis analogue Pc-pis-His was designed by adding a histidine residue at the carboxyl terminal. Pc-pis-His demonstrated a more broad-spectrum and stronger antimicrobial activity against a representative set of microorganisms and more potent antiparasitic activity against Cryptocaryon irritans trophonts than Pc-pis. The stability assay revealed that Pc-pis-His was active against Staphylococcus aureus not only in acidic (pH 5.5–7.3) and relatively low concentration monovalent cation (0–160 mM NaCl) environments but also in alkaline (pH 7.5–9.5), divalent cation (1.25–160 mM MgCl2 and 1.25–40 mM CaCl2) and high concentration monovalent cation (320–2560 mM NaCl) environments, which indicates that the added histidine residue conferred better salt-, acid- and alkali-tolerance to Pc-pis-His. Pc-pis-His also possessed the desired heat-tolerance, which was reflected by the antimicrobial activity of the peptide after being boiled for 10–60 minutes. Hemolytic activity analysis revealed that Pc-pis-His at concentrations up to 6 µM exhibited no hemolysis against human erythrocytes, with 6 µM being a concentration that is highly active against most of the microorganisms tested, although the hemolytic activity of Pc-pis-His was enhanced compared to Pc-pis. These results provide a unique, reasonable basis for designing novel piscidins with potent, broad-spectrum and stable antimicrobial activity and new insight into the future development of piscidins as potential therapeutic agents against microbial and external protozoan parasite infections.
DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR.
AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1−/−) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1−/− mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1−/− mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1−/− mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1−/− mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS). ADMA is eliminated largely by the action of dimethylarginine dimethylaminohydrolase1 (DDAH1). Decreased DDAH activity is found in several pathological conditions and is associated with increased risk of vascular disease. Overexpression of DDAH1 has been shown to augment endothelial proliferation and angiogenesis. To better understand the mechanism by which DDAH1 influences endothelial proliferation, this study examined the effect of DDAH1 deficiency on cell cycle progression and the expression of some cell cycle master regulatory proteins. DDAH1 KO decreased in vivo Matrigel angiogenesis and depressed endothelial repair in a mouse model of carotid artery wire injury. DDAH1 deficiency decreased VEGF expression in HUVEC and increased NF1 expression in both HUVEC and DDAH1 KO mice. The expression of active Ras could overcome the decreased VEGF expression caused by the DDAH1 depletion. The addition of VEGF and knockdown NF1 could both restore proliferation in cells with DDAH1 depletion. Flow cytometry analysis revealed that DDAH1 sRNAi knockdown in HUVEC caused G1 and G2/M arrest that was associated with decreased expression of CDC2, CDC25C, cyclin D1 and cyclin E. MEF cells from DDAH1 KO mice also demonstrated G2/M arrest that was associated with decreased cyclin D1 expression and Akt activity. Our findings indicate that DDAH1 exerts effects on cyclin D1 and cyclin E expression through multiple mechanisms, including VEGF, the NO/cGMP/PKG pathway, the Ras/PI3K/Akt pathway, and NF1 expression. Loss of DDAH1 effects on these pathways results in impaired endothelial cell proliferation and decreased angiogenesis. The findings provide background information that may be useful in the development of therapeutic strategies to manipulate DDAH1 expression in cardiovascular diseases or tumor angiogenesis.
Low-penetrance alleles associated with breast cancer risk have been identified in population-based studies. Most risk loci contain either no or multiple potential candidate genes. Rat mammary carcinoma susceptibility 1b (Mcs1b) is a quantitative trait locus (QTL) on RN02 that confers decreased susceptibility when Copenhagen (COP) resistant alleles are introgressed into a Wistar Furth (WF) susceptible genome. Five WF.COP congenic lines containing COP RN02 segments were compared. One line developed an average of 3.4 ± 2.0 and 5.5 ± 3.6 mammary carcinomas per rat ± SD when females were Mcs1b resistant homozygous and Mcs1b heterozygous, respectively. These phenotypes were significantly different from susceptible genotype littermates (7.8 ± 3.1 mean mammary carcinomas per rat ± SD, P = 0.0001 and P = 0.0413, respectively). All other congenic lines tested were susceptible. Thus, Mcs1b was narrowed to 1.8 Mb of RN02 between genetic markers ENSRNOSNP2740854 and g2UL2-27. Mammary-gland-graft carcinoma-susceptibility assays were used to determine that donor (P = 0.0019), but not recipient Mcs1b genotype (P = 0.9381), was associated with ectopic mammary carcinoma outcome. Rat Mcs1b contains sequence orthologous to human 5q11.2, a breast cancer susceptibility locus identified in multiple genome-wide association studies. Human/rat MAP3K1/Map3k1 and MIER3/Mier3 are within these orthologous segments. We identified Mier3 as a candidate Mcs1b gene based on 4.5-fold higher mammary gland levels of Mier3 transcripts in susceptible compared to Mcs1b resistant females. These data suggest that the human 5q11.2 breast cancer risk allele marked by rs889312 is mammary-gland autonomous, and MIER3 is a candidate breast cancer susceptibility gene.
breast cancer susceptibility; rat Mcs1b; rat mammary cancer; complex disease genetics; comparative genetics
Thousands of kilometers of shelterbelt plantations of Casuarina equisetifolia have been planted to protect the southeast coastline of China. These plantations also play an important role in the regional carbon (C) cycling. In this study, we examined plant biomass increment and C accumulation in four different aged C. equisetifolia plantations in sandy beaches in South China. The C accumulated in the C. equisetifolia plant biomass increased markedly with stand age. The annual rate of C accumulation in the C. equisetifolia plant biomass during 0–3, 3–6, 6–13 and 13–18 years stage was 2.9, 8.2, 4.2 and 1.0 Mg C ha−1 yr−1, respectively. Soil organic C (SOC) at the top 1 m soil layer in these plantations was 17.74, 5.14, 6.93, and 11.87 Mg C ha−1, respectively, with SOC density decreasing with increasing soil depth. Total C storage in the plantation ecosystem averaged 26.57, 38.50, 69.78, and 79.79 Mg C ha−1 in the 3, 6, 13 and 18- yrs plantation, with most of the C accumulated in the aboveground biomass rather than in the belowground root biomass and soil organic C. Though our results suggest that C. equisetifolia plantations have the characteristics of fast growth, high biomass accumulation, and the potential of high C sequestration despite planting in poor soil conditions, the interactive effects of soil condition, natural disturbance, and human policies on the ecosystem health of the plantation need to be further studied to fully realize the ecological and social benefits of the C equisetifolia shelterbelt forests in South China.
GABA is the major inhibitory neurotransmitter in the mammalian central nervous system that has been strongly implicated in the regulation of sleep. GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA and regulates GABAergic transmission in the brain. However, the role of GAT1 in sleep-wake regulation remains elusive. In the current study, we characterized the spontaneous sleep-wake cycle and responses to sleep deprivation in GAT1 knock-out (KO) mice. GAT1 KO mice exhibited dominant theta-activity and a remarkable reduction of EEG power in low frequencies across all vigilance stages. Under baseline conditions, spontaneous rapid eye movement (REM) sleep of KO mice was elevated both during the light and dark periods, and non-REM (NREM) sleep was reduced during the light period only. KO mice also showed more state transitions from NREM to REM sleep and from REM sleep to wakefulness, as well as more number of REM and NREM sleep bouts than WT mice. During the dark period, KO mice exhibited more REM sleep bouts only. Six hours of sleep deprivation induced rebound increases in NREM and REM sleep in both genotypes. However, slow wave activity, the intensity component of NREM sleep was briefly elevated in WT mice but remained completely unchanged in KO mice, compared with their respective baselines. These results indicate that GAT1 plays a critical role in the regulation of REM sleep and homeostasis of NREM sleep.
Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.
Autism is a pervasive neurodevelopmental disorder,thought to be caused by a combination of genetic heritability and environmental risk factors. Some autistic-like traits have been reported in mothers of autistic children. We hypothesized that dysregulation of oxytocin (OXT), Arg-vasopressin (AVP) and sex hormones, found in autistic children, may also exist in their mothers.
We determined plasma levels of OXT (40 in autism vs. 26 in control group), AVP (40 vs. 17) and sex hormones (61 vs. 47) in mothers of autistic and normal children by enzyme immunoassay and radioimmunoassay, respectively and investigated their relationships with the children’s autistic behavior scores (Childhood Autism Rating Scale (CARS) and Autism Behavior Checklist (ABC)).
Significantly lower plasma concentrations of OXT (p<0.001) and AVP (p<0.001), as well as a higher level of plasma testosterone (p<0.05), were found in mothers of autistic children vs. those of control. The children’s autistic behavior scores were negatively associated with maternal plasma levels of OXT and AVP.
These results suggest that dysregulation of OXT, AVP and/or testosterone systems exist in mothers of autistic children, which may impact children’s susceptibility to autism.
Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l−1 for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.
With the advancement of biotechnology in the last two decades, optimized and novel modalities and platforms of biologic moieties have emerged rapidly in drug discovery pipelines. In addition, new technologies for delivering therapeutic biologics (e.g., needle-free devices, nanoparticle complexes), as well as novel approaches for disease treatments (e.g., stem cell therapy, individualized medicine), continue to be developed. While pharmacokinetic studies are routinely carried out for therapeutic biologics, experiments that elucidate underlying mechanisms for clearance and biodistribution or identify key factors that govern absorption, distribution, metabolism, and excretion (ADME) of biologics often are not thoroughly conducted. Realizing the importance of biologics as therapeutic agents, pharmaceutical industry has recently begun to move the research focus from small molecules only to a blended portfolio consisting of both small molecules and biologics. This trend brings many opportunities for scientists working in the drug disposition research field. In anticipation of these opportunities and associated challenges, this review highlights impact of ADME studies on clinical and commercial success of biologics, with a particular focus on emerging applications and technologies and linkage with mechanistic pharmacokinetic/pharmacodynamic modeling and biomarker research.
ADME; biodistribution; pharmacokinetics; therapeutic biologics
The aim of this study was to determine the expression of miR-21, miR-31, miR-96 and miR-135b in 52 paired colorectal cancer (CRC) tissues and to analyze the correlation between microRNAs (miRNAs) and clinicopathological features. We developed a quantification method that relies on a standard plot, constructed from known concentrations of standards, in order to measure the number of miRNAs. In addition to this, we analyzed the expression levels of miR-21, miR-31, miR-96 and miR-135b in 52 cases of primary CRC and corresponding normal mucosal tissue using real-time PCR with SYBR-Green I. An independent sample t-test was used to compare the differential expression between tumor tissues and normal mucosal tissues. The Mann-Whitney U and Kruskall-Wallis tests were used to compare the correlation between miRNA expression levels and clinicopathological features. The expression of miR-21, miR-31, miR-96 and miR-135b was upregulated in the CRC tissues compared to normal mucosal tissues (P<0.05). Furthermore, miR-21 and miR-135b were positively correlated with the clinical stage (P=0.048 and P=0.029, respectively), while miR-96 and miR-135b were correlated with liver metastasis (P=0.006 and P=0.013, respectively). Our results suggest that miR-21, miR-31, miR-96 and miR-135b may function in the process of CRC development and progression. miR-135b levels in particular may correlate with the degree of malignancy.
microRNA; colorectal cancer; real-time RT-PCR
Stem cells, such as adult stem cells or embryonic stem cells, are the most important seed cells employed in tooth tissue engineering. Even though dental-derived stem cells are a good source of seed cells for such procedures, they are not often used in clinical applications because of the limited supply. Induced pluripotent stem (iPS) cells, with their high proliferation and differentiation ability, are now considered a promising alternative. The objectives of this study were to assess the role of iPS cells in tooth tissue engineering. We used real-time polymerase chain reaction to confirm that mouse iPS (miPS) cells can be induced to express both odontogenic and osteogenic gene profiles. We then established a tooth germ model and transplanted the recombinant tooth germ into a mouse subrenal capsule for 4 weeks to reproduce early-tooth organogenesis. After 4 weeks, hematoxylin and eosin staining results showed newly formed bone-like and dental pulp-like areas. Further immunohistochemical staining confirmed that osteopontin was present in the apical part of the tooth-like structure. These results demonstrate that miPS cells have the potential to differentiate into odontogenic cells, confirming that they could be a new source of seed cells for use in tooth tissue engineering.
Objective: High body mass index (BMI) is considered as the most important risk factor for elevated serum alanine aminotransferase (ALT) concentration. This study examined an array of factors, including waist circumference (WC) and folate deficiency, which may mediate the association of BMI with serum ALT concentration in Chinese hypertensive adults without known hepatic diseases. Methods: A multicenter, cross-sectional study was carried out. A total of 378 patients with mild or moderate hypertension and without known hepatic diseases were recruited from five hospitals in Harbin, Shanghai, Beijing, Xi’an, and Nanjing. Results: Of the 360 hypertensive patients with complete data in our final analysis, 13.6% had high ALT concentrations (>40 IU/L). Factors including BMI, WC, triglyceride level, and folate concentration were associated with ALT concentration in univariate analysis. Consistently higher prevalence rates of elevated ALT were observed in subjects with lower folate concentrations (≥12 vs. <12 nmol/L, 9.9% vs. 17.8%, P=0.03), with higher BMI (≥28 vs. <28 kg/m2, 21.5% vs. 11.4%, P=0.02) or higher WC (≥90 vs. <90 cm, 18.5% vs. 10.0%, P=0.02). However, in multivariate analysis, the association between BMI and ALT concentration disappeared (P=0.802 in males and 0.369 in females), while WC in females (P<0.001) and folate concentration (P=0.036 in males and 0.044 in females) remained as significant predictors for ALT concentration. Conclusions: This multicenter study demonstrated that WC and low folate concentration were important factors underlying the association between BMI and ALT concentrations in Chinese hypertensive adults without known hepatic diseases.
Alanine aminotransferase (ALT); Body mass index (BMI); Waist circumference (WC); Folate
To explore the feasibility of complete video-assisted thoracoscopic surgery (c-VATS) following neoadjuvant therapy (chemotherapy, targeted therapy and radiotherapy, either alone or in combination) for the treatment of patients with non-small cell lung cancer (NSCLC).
The clinical data of 43 NSCLC patients undergoing c-VATS following neoadjuvant therapy were retrospectively analyzed, including the preoperative functional indicators, staging, concurrent diseases, surgical techniques, operation time, number of lymph nodes dissected and postoperative drainage time and quantity, postoperative hospital stay, postoperative complications, and survival.
From January 2006 to March 2012, a total of 43 patients with stage IIA-IIIB NSCLC were included in this study (IIIA: 27 cases, 62.8%; IIIB: 11 cases, 25.6%), including 32 males (74.4%) and 11 females (25.6%). Forty-two patients were operated successfully, 28 underwent pulmonary lobectomies (including 9 bronchial sleeve resections), 5 had double lobectomies, 5 had wedge resections, and 4 had total pneumonectomies. Seven patients were referred to undergo Hybrid VATS (7/42, 16.7%). The mean length of the operation was 160.48±16.52 min (range, 130-180 min); the intraoperative blood loss was 253.57±117.08 mL; the number of lymph nodes dissected was 16.88±10.93; the postoperative drainage time was 1-7 d (mean: 2.62±0.96 d); and the postoperative hospital stay was 3-7 d (mean: 5.45±1.30 d). The incidence of postoperative complications was 9.5% (4/42), and the perioperative mortality was 2.4% (1/42). The 1-, 2-, and 3-year overall survival rates were 94%, 79%, and 65%, respectively.
c-VATS following neoadjuvant therapy is safe and feasible for the treatment of locally advanced NSCLC.
Non-small-cell lung cancer (NSCLC); neoadjuvant chemotherapy; targeted therapy; complete video-assisted thoracoscopic surgery (c-VATS)
To assess the feasibility, safety and long-term outcomes of video-assisted thoracic surgery (VATS) lobectomy for the treatment of non-small cell lung cancer (NSCLC) in patients with severe chronic obstructive pulmonary disease (COPD).
The clinical data of patients with NSCLC and severe COPD (preoperative FEV1% <50%) who underwent VATS lobectomy from January 2000 to January 2011 were retrospectively analyzed to identify their demographic parameters, postoperative complications and outcomes.
The preoperative FEV1/FVC was <70% and FEV1% <50% in all 61 patients in this study, with a mean preoperative FEV1 of 0.99 L (0.54-1.58 L) and mean FEV1% of 38.4% (22-49.82%). All of the 61 patients underwent the VATS lobectomy or sleeve resection plus systemic lymph node dissection. The mean operative time was 218 minutes (120-355 minutes), with a mean intraoperative blood loss of 342 mL (50-1,600 mL). None of the patients converted to thoracotomy. Multivariate statistical analysis revealed that age and TNM staging after tumor resection were independent predictive factors for the 5-year survival in those patients (P=0.014 and 0.013).
With preoperative imaging studies, pulmonary function assessment and target positioning, VATS lobectomy can be safely and effectively performed for patients with NSCLC and severe COPD to achieve a satisfying long-term survival outcome.
Non-small cell lung cancer (NSCLC); video-assisted thoracic surgery (VATS); lobectomy; chronic obstructive pulmonary disease (COPD)
Biotherapeutics are becoming an increasingly common drug class used to treat autoimmune and other inflammatory conditions. Optimization of absorption, distribution, metabolism, and excretion (ADME) profiles of biotherapeutics is crucial for clinical, as well as commercial, success of these drugs. This review focuses on the common questions and challenges in ADME optimization of biotherapeutics for inflammatory conditions. For these immunomodulatory and/or immunosuppressive biotherapeutics, special consideration should be given to the assessment of the interdependency of ADME profiles, pharmacokinetic/pharmacodynamic (PK/PD) relationships, and immunogenicity profiles across various preclinical species and humans, including the interdependencies both in biology and in assay readouts. The context of usage, such as dosing regimens, extent of disease, concomitant medications, and drug product characteristics may have a direct or indirect (via modulation of immunogenicity) impact on ADME profiles of biotherapeutics. Along these lines, emerging topics include assessments of preexisting reactivity to a biotherapeutic agent, impact of immunogenicity on tissue exposure, and analysis of penetration to normal versus inflamed tissues. Because of the above complexities and interdependences, it is essential to interpret PK, PD, and anti-drug antibody results in an integrated manner. In addition, because of the competitive landscape in autoimmune and inflammatory markets, many pioneering ADME-centric protein engineering and subsequent in vivo testing (such as optimization of novel modalities to extend serum and tissue exposures and to improve bioavailability) are being conducted with biotherapeutics in this therapeutic area. However, the ultimate challenge is demonstration of the clinical relevance (or lack thereof) of modified ADME and immunogenicity profiles.
ADME; autoimmune disease; immunogenicity; pharmacokinetics; therapeutic proteins
Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of iNOS, arginase, and NADPH oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C+Ly-6G− and down-modulated T cell activation, recruited Treg cells, and dramatically down-regulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C−Ly-6G+ and expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C+Ly-6G+ cells also suppressed T cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.
In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.
AIM: To investigate whether the reduction of stem cell factor (SCF) is mediated by decreased endogenous insulin-like growth factor (IGF)-1 in diabetic rat colon smooth muscle.
METHODS: Sixteen Sprague-Dawley rats were randomly divided into two groups: control group and streptozotocin-induced diabetic group. After 8 wk of streptozotocin administration, colonic motility function and contractility of circular muscle strips were measured. The expression of endogenous IGF-1 and SCF was tested in colonic tissues. Colonic smooth muscle cells were cultured from normal adult rats. IGF-1 siRNA transfection was used to investigate whether SCF expression was affected by endogenous IGF-1 expression in smooth muscle cells, and IGF-1 induced SCF expression effects were studied. The effect of high glucose on the expression of endogenous IGF-1 and SCF was also investigated.
RESULTS: Diabetic rats showed prolonged colonic transit time (252 ± 16 min vs 168 ± 9 min, P < 0.01) and weakness of circular muscle contraction (0.81 ± 0.09 g vs 2.48 ± 0.23 g, P < 0.01) compared with the control group. Endogenous IGF-1 and SCF protein expression was significantly reduced in the diabetic colonic muscle tissues. IGF-1 and SCF mRNA expression also showed a paralleled reduction in diabetic rats. In the IGF-1 siRNA transfected smooth muscle cells, SCF mRNA and protein expression was significantly decreased. IGF-1 could induce SCF expression in a concentration and time-dependent manner, mainly through the extracellular-signal-regulated kinase 1/2 signal pathway. High glucose inhibited endogenous IGF-1 and SCF expression and the addition of IGF-1 to the medium reversed the SCF expression.
CONCLUSION: Myopathy may resolve in colonic motility dysfunction in diabetic rats. Deficiency of endogenous IGF-1 in colonic smooth muscle cells leads to reduction of SCF expression.
Diabetes; Gastrointestinal motility function; Insulin-like growth factor-1; Stem cell factor; Smooth muscle cell
Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized germinal centers were observed in 6 of the 12 RA synovial samples, and AID+ cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID+ cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (p<0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10+IgM+CD20+ B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (p < 0.01). Aicda expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (p ≤ 0.0001) and anti-cyclic citrullinated peptide (CCP) (p = 0.0005). Serum levels of IFN-γ (p = 0.0005) and IL-17 (p = 0.007), but not IL-4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN-γ and IL-17, leading to the development of anti-CCP and RF.
The tumour stroma is an active participant during cancer progression. Stromal cells promote tumour progression and metastasis through multiple mechanisms including enhancing tumour invasiveness and angiogenesis, and suppressing immune surveillance. We report here that miR-126/miR-126*, a microRNA pair derived from a single precursor, independently suppress the sequential recruitment of mesenchymal stem cells and inflammatory monocytes into the tumour stroma to inhibit lung metastasis by breast tumour cells in a mouse xenograft model. miR-126/miR-126* directly inhibit stromal cell-derived factor-1 alpha (Sdf-1α) expression, and indirectly suppress the expression of chemokine (C–C motif) ligand 2 (Ccl2) by cancer cells in an Sdf-1α-dependent manner. miR-126/miR-126* expression is downregulated in cancer cells by promoter methylation of their host gene Egfl7. These findings determine how this microRNA pair alters the composition of the primary tumour microenvironment to favour breast cancer metastasis, and demonstrate a correlation between miR-126/126* downregulation and poor metastasis-free survival of breast cancer patients.
Chronic left ventricular failure causes pulmonary congestion with increased lung weight and type-2 pulmonary hypertension. Understanding the molecular mechanisms for type-2 pulmonary hypertension and the development of novel treatments for this condition requires a robust experimental animal model and a good understanding of the nature of the resultant pulmonary remodeling. Here we demonstrate that chronic transverse aortic constriction causes massive pulmonary fibrosis and remodeling, and type-2 pulmonary hypertension in mice. Thus, aortic constriction-induced left ventricular dysfunction and increased left ventricular end-diastolic pressure is associated with up to 5.3-fold increase in lung wet weight and dry weight, pulmonary hypertension and right ventricular hypertrophy. Interestingly, the aortic constriction-induced increase in lung weight was not associated with pulmonary edema, but resulted from profound pulmonary remodeling with a dramatic increase in the percentage of fully muscularized lung vessels, marked vascular and lung fibrosis, myofibroblast proliferation, and leukocyte infiltration. The aortic constriction-induced left ventricular dysfunction was also associated with right ventricular hypertrophy, increased right ventricular end-diastolic pressure and right atrial hypertrophy. The massive lung fibrosis, leukocyte infiltration and pulmonary hypertension in mice after transverse aortic constriction clearly indicate that congestive heart failure also causes severe lung disease. The lung fibrosis and leukocyte infiltration may be important mechanisms in the poor clinical outcome in patients with end-stage heart failure. Thus, the effective treatment of left ventricular failure may require additional efforts to reduce lung fibrosis and the inflammatory response.
pulmonary hypertension; transverse aortic constriction; pulmonary vascular morphology
Apigenin (4’,5,7-trihydroxyflavone) was recently shown effective in inhibiting several cancers. The aim of this study was to investigate the effect and mechanism of apigenin in the human bladder cancer cell line T24 for the first time.
T24 cells were treated with varying concentrations and time of apigenin. Cell viability was evaluated by MTT assay. Cell motility and invasiveness were assayed by Matrigel migration and invasion assay. Flow cytometry and western blot analysis were used to detect cell apoptosis, cell cycle and signaling pathway.
The results demonstrated that apigenin suppressed proliferation and inhibited the migration and invasion potential of T24 bladder cancer cells in a dose- and time-dependent manner, which was associated with induced G2/M Phase cell cycle arrest and apoptosis. The mechanism of action is like to involve PI3K/Akt pathway and Bcl-2 family proteins. Apigenin increased caspase-3 activity and PARP cleavage, indicating that apigenin induced apoptosis in a caspase-dependent way.
These findings suggest that apigenin may be an effective way for treating human bladder cancer.
Apigenin; Bladder cancer; Cell cycle arrest; Apoptosis; Invasion