Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel,Hyriopsis schlegelii

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5′-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.     

Construction of a genetic linkage map in Fenneropenaeus chinensis using SNP markers

Genetic linkage maps of Fenneropenaeus chinensis were constructed using a “double pseudo-testcross” strategy with 200 single nucleotide polymorphisms (SNPs) markers. This study represents the first SNP genetic linkage map for F. chinensis. The parents and F ₁ progeny of 100 individuals were used as mapping populations. 21 genetic linkage groups in the male and female maps were identified. The male linkage map was composed of 115 loci and spanned 879.7 cM, with an average intermarker spacing of 9.4 cM, while the female map was composed of 119 loci and spanned 876.2 cM, with an average intermarker spacing of 8.9 cM. The estimated coverage of the linkage maps was 51.94% for the male and 53.77% for the female, based on two estimates of genome length. The integrated map contains 180 markers distributed in 16 linkage groups, and spans 899.3 cM with an average marker interval of 5.2 cM. This SNP genetic map lays the foundation for future shrimp genomics and genetic breeding studies, especially the discovery of gene or regions for economically important traits in Chinese shrimp.     

Rapid Severing and Motility of Chloroplast-Actin Filaments Are Required for the Chloroplast Avoidance Response in Arabidopsis

Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light–induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light’s intensity and position.     

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-α-mediated apoptosis

Tumour necrosis factor-α(TNF-α) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-α increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-α induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-α (50ng/ml), a slow rise in intracellular free Ca²⁺ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3h after the addition of TNF-α) were observed in fluo-3 and furared or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-α in the presence of BAPTA/AM, an intracellular Ca²⁺ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-α-mediated Ca²⁺ rise. Moreover, when cells were exposed to TNF-α with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca²⁺ are two cross-talk messengers important in TNF-α-mediated apoptosis.     

Silencing of FABP3 Inhibits Proliferation and Promotes Apoptosis in Embryonic Carcinoma Cells

Fatty acid–binding protein 3 (FABP3) facilitates the movement of fatty acids in cardiac muscle. Previously, we reported that FABP3 is highly upregulated in the myocardium of ventricular septal defect patients and overexpression of FABP3 inhibited proliferation and promoted apoptosis in embryonic carcinoma cells (P19 cells). In this study, we aimed to investigate the effect of FABP3 gene silencing on P19 cell differentiation, proliferation and apoptosis. We used RNA interference and a lentiviral-based vector system to create a stable FABP3-silenced P19 cell line; knockdown of FABP3 was confirmed by quantitative real-time PCR. Expression analysis of specific differentiation marker genes using quantitative real-time PCR and observation of morphological changes using an inverted microscope revealed that knockdown of FABP3 did not significantly affect the differentiation of P19 cells into cardiomyocytes. CCK-8 proliferation assays and cell cycle analysis demonstrated that FABP3 gene silencing significantly inhibited P19 cell proliferation. Furthermore, Annexin V-FITC/propidium iodide staining and the caspase-3 activity assay revealed that FABP3 gene silencing significantly promoted serum starvation–induced apoptosis in P19 cells. In agreement with our previous research, these results demonstrate that FABP3 may play an important role during embryonic heart development, and that either overexpression or silencing of FABP3 will lead to an imbalance between proliferation and apoptosis, which may result in embryonic cardiac malformations.     

A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

mtDNA was isolated from cytoplasmic male sterility (CMS) line P3A and its maintainer P3B of kenaf (Hibiscus cannabinus L.). The atp9 gene and its two flanking sequences were obtained using homology cloning and high-efficiency thermal asymmetric interlaced PCR methods. The coding sequences showed only two base pairs difference between the CMS and its maintainer, and shared a homology of over 87 % with atp9 genes from other species in GenBank. However, when comparing the flanking sequences, a 47-bp deletion was characterized at the 3′ flanking sequence of atp9 in the CMS line. Quantitative PCR analysis indicated that the expression level of atp9 in the CMS line was 0.937-fold that of its maintainer. Furthermore, the respiratory rate of anthers in the CMS line was markedly lower than that of its maintainer. The results indicated that the 47-bp deletion at the 3′ flanking sequence of atp9 and/or down-regulated expression of the atp9 gene in the CMS line might be closely related to CMS in kenaf. To confirm whether the 47-bp deletion was specific to cytoplasm of male sterile lines, another 21 varieties were used for further analysis. The results showed that the 47-bp deletion was specific to male sterile cytoplasm (MSC) of kenaf. Based on these, a specific molecular marker was developed to distinguish the MSC from male fertile cytoplasm of kenaf.     

The association between genetic damage in peripheral blood lymphocytes and polymorphisms of three glutathione S-transferases in Chinese workers exposed to 1,3-butadiene

1,3-Butadiene (BD) has been classified as a human carcinogen, group I; however, the relationship between polymorphisms of glutathione S-transferases that metabolize BD and chromosomal damage is not clear. The present study used sister chromatid exchange (SCE) and cytokinesis-block micronucleus (CBMN) assays to detect chromosomal damage in peripheral lymphocytes of 44 BD-exposed workers and 39 non-exposed healthy controls. PCR and PCR-RFLP were employed to detect three known glutathione S-transferase polymorphisms GSTT1, GSTM1, and GSTP1 (Ile105Val). The data demonstrated that the micronucleus (CBMN) frequency in BD-exposed workers was significantly higher than that in controls (frequency ratio (FR) = 1.48, 95% CI: 1.14–1.91, P < 0.01), and the CBMN frequency was higher in workers exposed to higher cumulative BD levels (FR = 1.70, 95% CI: 1.28–2.27, P < 0.01). However, differences in SCE frequency were not observed (FR = 1.14, 95% CI: 0.81–1.61, P > 0.05). Among exposed workers, chromosomal damage was related to BD exposure levels (FR = 1.35, 95% CI: 1.02–1.80, P < 0.05); age, older workers exhibited higher MN frequencies than younger workers (FR = 1.45, 95% CI: 1.14–1.84, P < 0.05); and years of work, those with more years of work exhibited higher MN frequencies than those with fewer years (FR = 1.40, 95% CI: 1.10–1.77, P < 0.05). Multivariate Poisson regression analysis showed that those who carried GSTM1 (?) (FR = 1.48, 95% CI: 1.14–1.92) or GSTT1 (?) (FR = 1.42, 95% CI: 1.10–1.83) genotypes, and especially those who carried both (FR = 2.10, 95% CI: 1.43–3.09) exhibited significantly higher MN frequencies than those carrying GSTM1 (+), GSTT1 (+) genotypes or their combination. The GSTP1 Val genotype did not affect MN frequency (P > 0.05). Our results suggested that higher levels of BD exposure in the workplace resulted in increased chromosomal damage, and that polymorphisms in GSTT1 and GSTM1 genes might modulate the genotoxic effects of BD exposure. Furthermore, the GSTT1 and GSTM1 polymorphisms exhibited an additive effect. Finally, urinary DHBMA was found to provide a biomarker that correlated with airborne BD levels.     

Modified McKeown Minimally Invasive Esophagectomy for Esophageal Cancer: A 5-Year Retrospective Study of 142 Patients in a Single Institution

Background

To achieve decreased invasiveness and lower morbidity, minimally invasive esophagectomy (MIE) was introduced in 1997 for localized esophageal cancer. The combined thoracoscopic-laparoscopic esophagectomy (left neck anastomosis, defined as the McKeown MIE procedure) has been performed since 2007 at our institution. From 2007 to 2011, our institution subsequently evolved as a high-volume MIE center in China. We aim to share our experience with MIE, and have evaluated the outcomes of 142 patients.

Methods

We retrospectively reviewed 142 consecutive patients who had presented with esophageal cancer undergoing McKeown MIE from July 2007 to December 2011. The procedure, surgical outcomes, disease-free and overall survival of these cases were assessed.

Results

The average total procedure time was 270.5±28.1 min. The median operation time for thoracoscopy was 81.5±14.6 min and for laparoscopy was 63.8±9.1 min. The average blood loss associated with thoracoscopy was 123.8±39.2 ml, and for laparoscopic procedures was 49.9±14.3 ml. The median number of lymph nodes retrieved was 22.8. The 30 day mortality rate was 0.7%. Major surgical complications occurred in 24.6% and major non-surgical complications occurred in 18.3% of these patients. The median DFS and OS were 36.0±2.6 months and 43.0±3.4 months respectively.

Conclusions

Surgical and oncological outcomes following McKeown MIE for esophageal cancer were acceptable and comparable with those of open-McKeown esophagectomy. The procedure was both feasible and safe – properties that can be consolidated by experience.     

13-Methyltetradecanoic Acid Exhibits Anti-Tumor Activity on T-Cell Lymphomas In Vitro and In Vivo by Down-Regulating p-AKT and Activating Caspase-3

13-Methyltetradecanoic acid (13-MTD), a saturated branched-chain fatty acid purified from soy fermentation products, induces apoptosis in human cancer cells. We investigated the inhibitory effects and mechanism of action of 13-MTD on T-cell non-Hodgkin’s lymphoma (T-NHL) cell lines both in vitro and in vivo. Growth inhibition in response to 13-MTD was evaluated by the cell counting kit-8 (CCK-8) assay in three T-NHL cell lines (Jurkat, Hut78, EL4 cells). Flow cytometry analyses were used to monitor the cell cycle and apoptosis. Proteins involved in 13-MTD-induced apoptosis were examined in Jurkat cells by western blotting. We found that 13-MTD inhibited proliferation and induced the apoptosis of T-NHL cell lines. 13-MTD treatment also induced a concentration-dependent arrest of Jurkat cells in the G₁-phase. During 13-MTD-induced apoptosis in Jurkat cells, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP, a caspase enzymolysis product) were detected after incubation for 2 h, and increased after extending the incubation time. However, there was no change in the expression of Bcl-2 or c-myc proteins. The appearance of apoptotic Jurkat cells was accompanied by the inhibition of AKT and nuclear factor-kappa B (NF-κB) phosphorylation. In addition, 13-MTD could also effectively inhibit the growth of T-NHL tumors in vivo in a xenograft model. The tumor inhibition rate in the experimental group was 40%. These data indicate that 13-MTD inhibits proliferation and induces apoptosis through the down-regulation of AKT phosphorylation followed by caspase activation, which may provide a new approach for treating T-cell lymphomas.