Pharmacology of a traditional Chinese herb, Shuangren-Anshen capsule, in a hemorrhage mouse model by using an orthogonal array design.

Shuangren-Anshen capsule (SAC) is a traditional Chinese herb that was improved in our laboratory. An orthogonal experiment [L9(3)(4)] was used to optimize the extraction conditions. In vivo, a hemorrhage mouse model was established and the hemoglobin contents of normal control, model control, and treated mice were measured. Additionally, the sedative and hypnotic effects of SACs were assessed based on pharmacological parameters such as changes in locomotive activity, forelimb raising, sleep latency, sleep duration, and number of mice that fell asleep. Brain tissue was sectioned and stained to detect changes in cell morphology by microscopy. The optimum extraction was achieved with 3 cycles of decoction for 120 min each with a 10-fold volume of water added. In the model control group, hemoglobin content significantly decreased and pharmacological parameters increased (P < 0.01) relative to that in the normal control group. Compared to the model control group, the group treated with 0.9 g/kg SAC showed significant (P < 0.05) increase or decrease in hemoglobin content and all pharmacological parameters except sleep duration. The groups treated with 1.8 or 3.6 g/kg SAC and the positive control group also showed significant alterations in hemoglobin content and pharmacological parameters (P < 0.05). In addition, SAC exhibited a protective effect on the morphological structures of the damaged nerve cells in the mouse model. Thus, an optimal extraction process was successfully identified. The pharmacological data also suggests that the drug can improve sleep quality. SAC treatment was shown to cause changes in hemoglobin content and cell morphology in a mouse model.

Molecular characterization of the myosatin gene and the effect of fasting on its expression in Chinese perch (Siniperca chuatsi).

Myostatin (MSTN) is an important member of the transforming growth factor-ß (TGF-ß) superfamily and is a muscle growth inhibitor. In the present study, we cloned the Chinese perch MSTN cDNA sequence and analyzed its expression patterns under various conditions. The MSTN full cDNA sequence was 3347 bp long, including an open-reading frame of 1131 bp, which encoded 376 amino acids. Sequence analysis demonstrated that the MSTN shared a highly conserved signal peptide, a TGF-ß functional peptide, a hydrolytic site (RARR), and nine conservative cysteine residues with other members of the TGF-ß superfamily. Sequence alignment and phylogenetic tree analyses indicated that the MSTN had a close relationship with teleostean fish, but they are far separated from mammals. Real-time polymerase chain reaction analysis revealed that the MSTN was strongly expressed in the skeletal muscle and heart tissues. Temporal expression analysis demonstrated that the MSTN gene was expressed in very low levels, from 20 to 90 dph (post-hatching development), and was at its highest level at 150 dph (P < 0.05). The fasting-re-feeding experiment showed that the expression of the MSTN gene was initially decreased in response to a single meal, after seven days of fasting, and subsequently increased significantly, and finally decreased back to its original level. Together, our results provided valuable knowledge regarding the regulation of MSTN gene expression in Chinese perch.

Comprehensive identification and expression analysis of Hsp90s gene family in Solanum lycopersicum.

Heat shock protein 90 (Hsp90) is a protein produced by plants in response to adverse environmental stresses. In this study, we identified and analyzed Hsp90 gene family members using a bioinformatic method based on genomic data from tomato (Solanum lycopersicum L.). The results illustrated that tomato contains at least 7 Hsp90 genes distributed on 6 chromosomes; protein lengths ranged from 267-794 amino acids. Intron numbers ranged from 2-19 in the genes. The phylogenetic tree revealed that Hsp90 genes in tomato (Solanum lycopersicum L.), rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana L.) could be divided into 5 groups, which included 3 pairs of orthologous genes and 4 pairs of paralogous genes. Expression analysis of RNA-sequence data showed that the Hsp90-1 gene was specifically expressed in mature fruits, while Hsp90-5 and Hsp90-6 showed opposite expression patterns in various tissues of cultivated and wild tomatoes. The expression levels of the Hsp90-1, Hsp90-2, and Hsp90- 3 genes in various tissues of cultivated tomatoes were high, while both the expression levels of genes Hsp90-3 and Hsp90-4 were low. Additionally, quantitative real-time polymerase chain reaction showed that these genes were involved in the responses to yellow leaf curl virus in tomato plant leaves. Our results provide a foundation for identifying the function of the Hsp90 gene in tomato.

Correlation of plasma soluble cluster of differentiation 40 ligand, alpha fetoprotein A, and pregnancy-associated plasma protein A with carotid plaque in patients with ischemic stroke.

This study investigated the correlation of plama levels of inflammatory biomarkers [soluble cluster of differentiation 40 ligand (sCD40L), alpha fetoprotein A (fetuin-A), and pregnancy-associated protein A (PAPP-A)] with carotid plaque in patients with acute ischemic stroke. After undergoing color Doppler ultrasonography of the bilateral carotid arteries, 200 patients with acute ischemic stroke were grouped into plaque and non-plaque groups. The plaque group was further divided into stable and unstable plaque sub-groups by carotid plaque stability. Inter-group and -subgroup comparisons included demographic characteristics, current condition and medical history, and clinical laboratory and plama inflammatory biomarker data, and logistic regression explored the correlations between plama inflammatory biomarker levels and carotid plaques. Significantly higher sCD40L and fetuin-A levels were found in the plaque group than in the non-plaque group (all P < 0.05), with odds ratios (plaque vs non-plaque) of 6.372 and 4.101, respectively. Increased plama inflammatory biomarker levels were accompanied by a high risk of carotid plaque formation. Similarly, significantly higher plama sCD40L and PAPP-A levels were found in the unstable plaque subgroup than in the stable plaque subgroup (all P < 0.05), and the odds ratios (unstable vs stable) were 5.290 and 4.125, respectively. Increased plama inflammatory biomarker levels were accompanied by a high risk of carotid plaque instability. The study findings showed that plasma sCD40L, fetuin-A, and PAPP-A levels are associated with carotid plaque formation and instability. Fetuin-A and sCD40L might be predictors of carotid plaque formation, while PAPP-A and sCD40L might be predictors of carotid plaque instability.

Cloning and functional identification of a novel BCA3 splice.

The human breast cancer-associated gene (BCA3) was first discovered in breast and prostate cancer cells lines. In vivo studies have shown that BCA3 is mainly expressed in breast tumor cells and not in normal breast and prostate tissues. To date, 3 splice variants of BCA3 have been reported: a double-absent variant lacking exon 3 and exon 5 (BCA3-1), an exon 3-absent variant (BCA3-2), and full-length BCA3. In this study, we investigated whether a novel BCA3 splice variant exists that lacks only the exon 5-encoding sequence. BCA3 variant splices were subcloned and sequenced using reverse transcription-polymerase chain reaction. The preliminary biological functions of the splices were identified using confocal microscopy and a luciferase assay. The absence of exon 3 and exon 5 influenced the subcellular localization of BCA3 and nuclear factor kappa B (NF-kB)-dependent gene expression. Exon 3 and exon 5 of BCA3 may function together to provide a nuclear localization signal or transport sequence to enter the nucleus, and exon 3 may contain specific sequence(s) or domain(s) that influence the NF-κB signal cascade. The discovery of novel BCA3 splicing indicates a new cancer research area, which may increase the understanding of cancer generation and development.

Behavior of calf Sertoli cells and fibroblast cells transfected with the human HNP-1 gene.

Human neutrophil peptide-1 (HNP-1) is an important defense molecule in neutrophils and Sertoli cells and plays an important role in the blood-testis barrier. In this study, we investigated the behavior of Sertoli cells transfected with the HNP-1 gene and compared the ability of Sertoli cells and fibroblast cells to resist transfection. Total RNA was isolated from human blood. The DNA coding sequence of HNP-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the eukaryotic expression vector pEGFP-N3-HNP-1 was identified by PCR, endonuclease digestion, and sequencing. Bovine Sertoli cells and fibroblast cells were transfected with pEGFP-N3-HNP-1 using Liposome reagent. The transfection efficiency and the behavior of the transfected cells were evaluated at 24, 48 and 72 h as well as at other times after transfection. The plasmid pEGFP-N3-HNP-1 was successfully constructed. The cells achieved maximum transfection efficiency at 48 h. Two weeks after transfection, the cells began to stop dividing. The ability of Sertoli cells to resist transfection was higher compared to fibroblast cells. The ability of the 2 cell types to resist transfection was higher with plasmid pEGFP-N3-HNP-1 than with the plasmid pEGFP-N3. The injury to Sertoli cells caused by transfection with the HNP-1 gene was less pronounced than in fibroblast cells, which may be closely correlated with the physiological function of Sertoli cells.

Association between the EGF rs4444903 polymorphism and liver cancer susceptibility: a meta-analysis and meta-regression.

Emerging evidence suggests that a common functional polymorphism, rs4444903 (A>G), in the EGF gene might impact an individual's susceptibility to liver cancer; however, individually published results are inconclusive. This meta-analysis aimed to derive a more precise estimation of the relationship between the EGF rs4444903 polymorphism and liver cancer risk. A literature search was conducted in the PubMed, Embase, Web of Science, and CBM databases from inception through May 1st, 2013. Seven case-control studies were included with a total of 1408 liver cancer cases and 1343 healthy controls. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Our meta-analysis results indicated that the G variant of the rs4444903 polymorphism might be associated with an increased risk of liver cancer (G allele vs A allele: OR = 1.25, 95%CI = 1.01-1.56, P = 0.040; GG + AG vs AA: OR = 1.65, 95%CI = 1.27-2.15, P < 0.001; GG vs AA: OR = 1.77, 95%CI = 1.34-2.35, P < 0.001). Further subgroup analysis by ethnicity also showed significant associations between the G variant of the rs4444903 polymorphism and an increased risk of liver cancer among Asian, Caucasian, and African populations. No publication bias was detected in this meta-analysis. In conclusion, the current meta-analysis suggests that the G variant of the rs4444903 polymorphism may increase the risk of liver cancer. The EGF rs4444903 (A>G) polymorphism can be useful as a biomarker in predicting the development of liver cancer.

Molecular cloning and expression analysis of the GNAS gene in pig and porcine fibroblast cells.

The Alpha subunit of the stimulatory guanine nucleotide-binding protein (GNAS) is a complex imprinted gene. The major product of the GNAS gene is the α-subunit of the guanine nucleotide-binding protein (Gas), which plays a key role in multiple signal transduction pathways. Gas is required for the production of the receptor-stimulated intracellular cyclic adenosine monophosphate (cAMP). It has been demonstrated that an increase in the concentration of the intracellular second messenger cAMP promotes apoptosis in different tumor entities. Mutations of GNAS have also been identified in many tumors. This study aimed to investigate the expression pattern and the apoptosis effect in fibroblast cells for porcine GNAS. The results show that GNAS mRNA was detected in a wide range of tissues, especially in the longissimus dorsi muscle and thyroid gland. The developmental pattern of GNAS mRNA in the thyroid gland of Jinhua pigs was then examined; however, there was no significant difference (P > 0.05) among any of the stages. GNAS gene expression was relatively stable in the thyroid gland during the entire growth and development process. The developmental pattern of GNAS mRNA in the longissimus dorsi muscle was significantly different among the various developmental stages (P < 0.01). GNAS mRNA was strongly expressed at 60 days, 90 days, and 150 days after birth, whereas the expression level was very low during the embryo stages. Target RNA interference of GNAS in porcine fibroblast cells leads to lower mRNA expression of Bcl-2, Fas, and Caspase-3, which are recognized as apoptosis related markers.

Cytogenetic and molecular identification of a wheat-Leymus mollis alien multiple substitution line from octoploid Tritileymus x Triticum durum.

Leymus mollis (Trin.) Pilger (NsNsXmXm, 2n = 28), a wild relative of common wheat, possesses many traits that are potentially valuable for wheat improvement. In order to exploit and utilize the useful genes of L. mollis, we developed a multiple alien substitution line, 10DM50, from the progenies of octoploid Tritileymus M842-16 x Triticum durum cv. D4286. Genomic in situ hybridization analysis of mitosis and meiosis (metaphase I), using labeled total DNA of Psathyrostachys huashanica as probe, showed that the substitution line 10DM50 was a cytogenetically stable alien substitution line with 36 chromosomes from wheat and three pairs of Ns genome chromosomes from L. mollis. Simple sequence repeat analysis showed that the chromosomes 3D, 6D, and 7D were absent in 10DM50. Expressed sequence tag-sequence tagged sites analysis showed that new chromatin from 3Ns, 6Ns, and 7Ns of L. mollis were detected in 10DM50. We deduced that the substitution line 10DM50 was a multiple alien substitution line with the 3D, 6D, and 7D chromosomes replaced by 3Ns, 6Ns, and 7Ns from L. mollis. 10DM50 showed high resistance to leaf rust and significantly improved spike length, spikes per plant, and kernels per spike, which are correlated with higher wheat yield. These results suggest that line 10DM50 could be used as intermediate material for transferring desirable traits from L. mollis into common wheat in breeding programs.

QTL verification of grain protein content and its correlation with oil content by using connected RIL populations of high-oil maize.

Maize with high grain protein and oil contents offers great advantages for human food and animal feed. In this study, grain protein contents of 282 and 263 F7:8 recombinant inbred lines (RILs) of 2 crosses were evaluated in 4 environments within and between populations. The RILs were developed from crosses between an inbred high-oil maize line and 2 normal dent inbred maize lines. A total of 16 single-population QTLs and 19 joint-population QTLs were identified for protein content, and 21 QTLs were detected for protein-oil in each of the 4 environments tested and in combination. Most of the QTLs for protein content were greatly influenced by variation among populations and environments. Seven QTLs showed generational consistency compared with QTLs detected in the 2 F2:3 populations. However, 7 and 6 QTLs were detected in only the RIL and F2:3 populations, respectively. Protein and protein-oil QTLs with the same parental effects were detected at bins 3.03-3.05, 5.04-5.06, 6.03-6.05, 8.03-8.04, and 8.04-8.06, demonstrating that tightly linked and/or pleiotropic QTLs are controlling both traits at these bins. Four single-population QTLs and 11 joint-population QTLs identified at bins 3.02-3.03, 3.05, 7.01, 8.02, 8.03, 8.04-8.05, 8.05, 9.03, and 9.05 with intervals <5 cM could be used in marker-assisted selection. Along with the previously detected QTLs qPRO1-8-1 and qPRO1-5-1 at bins 8.03-8.04 and 5.02-5.04, the QTLs detected herein could be used to develop near isogenic lines and chromosome segment substitution lines in future studies.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells.

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.