Characterization of air-liquid interface culture of A549 alveolar epithelial cells

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.

MicroRNA-143 inhibits cell growth by targeting ERK5 and MAP3K7 in breast cancer

This study aimed to investigate the function and mechanism of microRNA-143 (miR-143) in the occurrence and development of breast cancer (BC). A total of 30 BC tissues, 30 corresponding noncancerous tissues, and 10 normal control (NC) breast tissues were obtained to detect the levels of miR-143, extracellular signal-regulated kinase 5 (ERK5) and mitogen-activated protein 3 kinase 7 (MAP3K7) using RT-qPCR, western blotting or immunohistochemistry. The correlation of miR-143 with ERK5 or MAP3K7 was evaluated using Pearson correlation analysis. MCF-7 cells were transiently transfected with miR-143 mimic, miR-143 inhibitor, miR-143 mimic/inhibitor + si-ERK5, si-MAP3K7 or si-cyclin D1. Then, cell growth was evaluated by MTT assay and the expressions of phospho-ERK5 (p-ERK5), ERK5, p-MAP3K7, MAP3K7 and cyclin D1 were detected by western blotting. Results showed that, compared with noncancerous tissues or NC breast tissues, miR-143 level was decreased, while p-ERK5, ERK5, p-MAP3K7 and MAP3K7 expressions were increased in BC tissues (all P<0.01). The miR-143 level was negatively correlated with the mRNA level of ERK5 or MAP3K7 (r=-4.231 or r=-4.280, P<0.01). In addition, up-regulated miR-143 significantly decreased the expressions of p-ERK5, ERK5, p-MAP3K7, MAP3K7 and cyclin D1 (all P<0.01), as well as cell viability in MCF-7 cells (all P<0.05) while the effect of down-regulated miR-143 was the opposite. In conclusion, both ERK5 and MAP3K7 may be the target genes of miR-143. Increased expression of miR-143 can inhibit cell growth, which may be associated with ERK5 and MAP3K7 expressions in BC.

A recombinant plasmid of composite cysteine proteinase inhibitor/ glyceraldehyde‑3‑phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host.

Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine‑elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription‑polymerase chain reaction in muscle tissues. The stimulation index (SI) of T‑lymphocyte proliferation and the levels of interferon‑gamma (INF‑γ) and interleukin‑4 (IL‑4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme‑linked immunosorbent assays. Results: The pcDNA3.1(+)‑BmCPI/ BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF‑γ and IL‑4 of pcDNA3.1(+)‑BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF‑γ of pcDNA3.1(+)‑BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)‑BmCPI/CpG group (P < 0.05). Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Gene expression profiling analysis of lung adenocarcinoma

The present study screened potential genes related to lung adenocarcinoma, with the aim of further understanding disease pathogenesis. The GSE2514 dataset including 20 lung adenocarcinoma and 19 adjacent normal tissue samples from 10 patients with lung adenocarcinoma aged 45-73 years was downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between the two groups were screened using the t-test. Potential gene functions were predicted using functional and pathway enrichment analysis, and protein-protein interaction (PPI) networks obtained from the STRING database were constructed with Cytoscape. Module analysis of PPI networks was performed through MCODE in Cytoscape. In total, 535 upregulated and 465 downregulated DEGs were identified. These included ATP5D, UQCRC2, UQCR11 and genes encoding nicotinamide adenine dinucleotide (NADH), which are mainly associated with mitochondrial ATP synthesis coupled electron transport, and which were enriched in the oxidative phosphorylation pathway. Other DEGs were associated with DNA replication (PRIM1, MCM3, and RNASEH2A), cell surface receptor-linked signal transduction and the enzyme-linked receptor protein signaling pathway (MAPK1, STAT3, RAF1, and JAK1), and regulation of the cytoskeleton and phosphatidylinositol signaling system (PIP5K1B, PIP5K1C, and PIP4K2B). Our findings suggest that DEGs encoding subunits of NADH, PRIM1, MCM3, MAPK1, STAT3, RAF1, and JAK1 might be associated with the development of lung adenocarcinoma.

LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats

Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250–280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases.

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

Development of fetal nicotine and muscarinic receptors in utero

The role of acetylcholine in the central and peripheral nervous systems is well established in adults. Cholinergic modulation of vascular functions and body fluid balance has been extensively studied. In the embryo-fetus, cholinergic receptors are widespread in the peripheral and central systems, including smooth muscle and the epithelial lining of the cardiovascular, digestive, and urinary systems, as well as in the brain. Fetal nicotine and muscarinic receptors develop in a pattern (e.g., amount and distribution) related to gestational periods. Cholinergic mechanisms have been found to be relatively intact and functional in the control of vascular homeostasis during fetal life in utero at least during the last third of gestation. This review focuses on the development of fetal nicotine and muscarinic receptors, and provides information indicating that central cholinergic systems are well developed in the control of fetal blood pressure and body fluid balance before birth. Therefore, the development of cholinergic systems in utero plays an important role in fetal vascular regulation, gastrointestinal motility, and urinary control.

Genetic and genotype x environment interaction effects for the content of seven essential amino acids in indica rice.

It is necessary for rice breeders to understand the genetic basis of nutrient quality traits of rice. Essential amino acids are most important in determining the nutrient quality of rice grain and can affect the health of people who depend on rice as a staple food. In view of the paucity of genetic information available on essential amino acids in indica rice, we estimated the genetic main effects and genotype x environment (G x E) interaction effects on the content of essential amino acids. Nine cytoplasmic male sterile lines as females and five restorer lines as males were introduced in a North Carolina II design across environments. Estimates of the content of the essential amino acids valine, methionine, leucine and phenylalanine showed that they were mainly controlled by genetic main effects, while the contents of threonine, cysteine and isoleucine were mainly affected by G x E effects. In the case of genetic main effects, both cytoplasmic and maternal genetic effects were predominant for all essential amino acids, indicating that selection for improving essential amino acid content based on maternal performance would be more effective than that based on seeds. The total narrow-sense heritabilities were high and ranged from 0.72 to 0.83. Since general heritabilities for these essential amino acids (except for cysteine) were found to be much larger than G x E interaction heritability, the improvement of content of most essential amino acids under selection would be expected under various environments. Rice varieties such as Zhenan 3, Yinchao 1, T49, 26715, 102 and 1391 should be selected as optimal parents for increasing the content of most essential amino acids, while the total genetic effects from Zhexie 2, Xieqingzao, Gangchao 1, V20, Zuo 5 and Zhenshan 97 were mainly negative and these parents could decrease the contents of most essential amino acids.

Children poisoning in Taiwan.

Poisoning is a well known cause of morbidity and mortality in children. In Taiwan, little information has been published regarding the status of pediatric poisoning exposures. To provide more information on pediatric poisoning exposures for the purpose of poison prevention, a retrospective study was designed and conducted to analyse the data of National Poison Centre (NPC), Taiwan. All telephone inquiries concerning poisoning exposures in those under 19 years of age, received by NPC-Taiwan from July 1985 through December 1993, were included in this study. The age, sex, reason for exposure, route of exposure, substances involved and clinical outcome of those telephone calls were then analyzed. A total of 5,812 inquiries concerning poisoning exposures in children were recorded. Male exposures were more prevalent than females (59%) Vs. 41%) Accidental exposures accounted for 77.7% of the cases and most were exposed by the oral route. Substances most frequently ingested were household products, benzodiazepines and pesticides. The data revealed a mortality rate of 1.4%. Accidental poisoning exposures from household products and drugs remain a significant problem for those younger than 6 years of age. Further education of parents and care takers and the employment of child-resistant containers are needed to prevent cases of pediatric poisoning. Reduction of amphetamine abuse in adolescents is also of major concern and deserves more attention.

Acetylcholine receptor antibody in patients with myasthenia gravis.

The determination of acetylcholine receptor antibody (AChR Ab) titer by an enzyme-linked immunosorbent assay (ELISA) in patients with myasthenia gravis was introduced. The optimal conditions were determined by chequerboard determination. The specificity was confirmed by inhibition tests. The sensitivity is 9 p mole. The comparison of AChR Ab titers among 49 myasthenic patients, 19 non-myasthenic neurological patients and 20 healthy blood donors has shown that it is a highly sensitive, specific, reproducible, rapid, simple and inexpensive method for determining AChR Ab and that it is highly valuable for the diagnosis of myasthenia gravis.