Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

AIM： To explore the relationship between DNA methyltransferase 1 （DNMT1） and hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） and its biological significance in primary HCC. METHODS： We carried out an immunohistochemical examination of DNMT1 in both HCC and paired nonneoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC celt line SMMC-7721. RESULTS： DNMT1 protein expression was increased in HCCs compared to histologically normal nonneoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation （P = 0.014）. There were more cases with DNMT1 overexpression in HCC with HBV （42.85%） than in HCC without HBV （28.57%）. However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen （PCNA）, even in HCC cell lines where DNMT1 was stably decreased. CONCLUSION： The findings implied that DNMT1 plays a key role in HBV-retated hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.

Induction of chemotaxis to sodium chloride and diacetyl and thermotaxis defects by microcystin-LR exposure in nematode Caenorhabditis elegans

Apart from the liver disruption, embryotoxicity and genotoxicity, microcystin （MC）-LR also could cause neurotoxicity. Nematode Caenorhabditis elegans was explored as a model to study the neurotoxicity. In the present study, we provided evidence to indicate the neurotoxicity on chemotaxis to NaCl and diacetyl, and thermotaxis from MC-LR exposure to C. elegans. As a result, higher concentrations of MC-LR caused significantly severe defects of chemotaxis to NaCl and diacetyl, and thermotaxis. The neurotoxicity on chemotaxis to NaCl and diacetyl, and thermotaxis from MC-LR exposure might be largely mediated by the damage on the corresponding sensory neurons （ASE, AWA, and AFD） and interneuron AIY. The expression levels ofche-1 and odr-7 were significantly decreased （P 〈 0.01） in animals exposed to MC-LR at concentrations lower than 10 μg/L, whereas the expression levels of ttx-1 and ttx-3 could be significantly （P 〈 0.01） lowered in animals even exposed to 1 μg/L of MC-LR. Moreover, both the chemotaxis to NaCl and diacetyl and the thermotaxis were more significantly reduced in MC-LR exposed mutants of che-1（p674）, odr-7（ky4）, ttx-1（p767）, and ttx-3（ks5） than those in exposed wild-type N2 animals at the same concentrations.

Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice

Our previous studies have demonstrated that Fam20 C promotes differentiation and mineralization of odontoblasts,ameloblasts,osteoblasts and osteocytes during tooth and bone development.Ablation of the Fam20 C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice.However,control and regulation of the expression of Fam20 C are still unknown.In this study,we generated a transgenic reporter model which expresses green fluorescence protein（GFP） driven by the Fam20 C promoter.Recombineering was used to insert a 16 kb fragment of the mouse Fam20 C gene（containing the 15 kb promoter and 1.1 kb of exon 1） intoa pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence.GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice.Fluorescence was evident in the bone and teeth as early as E17.5.The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth.The expression of GFP was significantly reduced in teeth,alveolar bone and muscle by 8 weeks of age.We also observed colocalization of the GFP signal with the Fam20 C antibody in postnatal 1- and 7-day-old animals.Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20 C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans

Locomotion behaviors are susceptible to disruption by a broad spectrum of chemicals and environmental stresses. However, no systematic testing of locomotion behavior defects induced by metal exposure has been conducted in the model organism of nematode Caenorhabditis elegans. In this study, the acute toxicity from heavy metal exposure on the locomotion behaviors was analyzed in nematodes. Endpoints of head thrash, body bend, forward turn, backward turn, and Omega/U turn were chosen to evaluate the locomotio...

Prolonged manganese exposure induces severe deficits in lifespan,development and reproduction possibly by altering oxidative stress response in Caenorhabditis elegans

We examined the possible multiple defects induced by acute and prolonged exposure to high levels of manganese（Mn） solution by monitoring the endpoints of lifespan,development,reproduction,and stress response.Our data suggest that acute exposure（6 h） to Mn did not cause severe defects of life span,development,and reproduction,similarly,no significant defect could be found in animals exposed to a low concentration of Mn（2.5 μmol/L） for 48 h.In contrast,prolonged exposure（48 h） to high Mn concentrations（75 and 200 μmol/L） resulted in significant defects of life span,development,and reproduction,as well as the increase of the percentage of population with hsp-16.2：：gfp expression indicating the obvious induction of stress responses in exposed animals.Moreover,prolonged exposure（48 h） to high concentrations（75 and 200 μmol/L） of Mn decreased the expression levels of antioxidant genes of sod-1,sod-2,sod-3,and sod-4 compared to control.Therefore,prolonged exposure to high concentrations of Mn will induce the severe defects of life span,development,and reproduction in nematodes possibly by affecting the stress response and expression of antioxidant genes in Caenorhabditis elegans.

Toxicity evaluation in a paper recycling mill effluent by coupling bioindicator of aging with the toxicity identification evaluation method in nematode Caenorhabditis elegans

Toxicity identification evaluation (TIE) can be used to determine the specific toxicant(s) in industrial effluents.In the current study,the authors have attempted to combine the advantages of the model organism,Caenorhabditis elegans,with the virtues of the TIE technique,to evaluate and identify the toxicity on aging from a paper recycling mill effluent.The results indicate that only the toxicities from mixed cellulose (MC) filtration and EDTA treatment are similar to the baseline aging toxicity,suggesting ...

Phenotypic and Behavioral Defects Induced by Iron Exposure Can Be Transferred to Progeny in Caenorhabditis elegans

Objective Previous work has showed that excess iron accumulation is harrnftd to reproduction and even promotes death; however, whether the multiple biological toxicity of iron （Fe） exposure could be transferred to progeny remains unknown. The present study used Caenorhabditis elegans to analyze the multiple toxicities of iron exposure and their possible transferable properties. Methods Three concentrations of iron sulfate solution （2.5μmol/L, 75μmol/L, and 200 μmol/L） were used. The endpoints of lifespan, body size, generation time, brood size, head thrash and body bend frequencies, and chemotaxis plasticity were selected to investigate Fe toxicity and its effect on progeny in Caenorhabditis elegans. Results The Fe toxicity could cause multiple biological defects in a dose-dependent manner by affecting different endpoints in nematodes. Most of the multiple biological defects and behavior toxicities could be transferred from Fe-exposed Caenorhabditis elegans to their progeny. Compared to the parents, no recovery phenotypes were observed for some of the defects in the progeny, such as body bend frequency and life span. We further summarized the defects caused by Fe exposure into 2 groups according to their transferable properties. Conclusion Our results suggest that Fe exposure could cause multiple biological defects, and most of these severe defects could be transferred from Fe exposed nematodes to their progeny.

Evaluation of the long-term memory for thermosensation regulated by neuronal calcium sensor-1 in Caenorhabditis elegans

Objective To evaluate whether the thermotaxis tracking model is suitable for assessing long-term memory （LTM） in the nematode Caenorhabditis elegans. Methods Animals were trained at 20℃ overnight in presence of food. The percentage of animals performing isothermal tracking （IT） behavior was measured at different time intervals after the training. Results The percentage of animals performing IT behavior, the numbers of body bends inside and outside the training temperature, and the expression patterns of AFD and AIY neurons were similar to those in control animals at 36 and 48 h after training; whereas when extending to 60, 72, and 84 h, locomotory behavior defects were observed in the assayed animals, suggesting that this thermal tracking model is feasible for analyzing LTM at 36 and 48 h after training. Moreover, the percent-age of animals performing IT behavior was reduced at 18, 36, and 48 h after training in neuronal calcium sensor-1 gene （nsc-1） mutant animals compared with that in wild-type N2 animals. In addition, exposure to plumbum （Pb） significantly repressed the LTM at 18, 36, and 48 h after training in both wild-type N2 and ncs-1 mutant animals. Conclusion The thermotaxis tracking model is suitable for evaluating the LTM regulated by NCS-1, and can be employed for elucidating regulatory functions of specific genes or effects of stimuli on memory in C. elegans.

Molecular control of memory in nematode Caenorhabditis elegans

Model invertebrate organism Caenorhabditis elegans has become an ideal model to unravel the complex processes of memory. C. elegans has three simple forms of memory： memory for thermosensation, memory for chemosensation, and memory for mechanosensation. In the form of memory for mechanosensation, short-term memory, intermediate-term memory, and long-term memory have been extensively studied. The short-term memory and intermediate-term memory may occur in the presynaptic sensory neurons, whereas the long-term memory may occur in the postsynaptic interneurons. This review will discuss the recent progress on genetic and molecular regulation of memory in C. elegans.

Influence of berberine on protein tyrosine kinase of erythrocyte insulin receptors from type 2 diabetes mellitus

Objective： Bererine has been used to treat type 2 diabetes mellitus in Chinese traditional medicine because of its hypoglycemic effect. In this report, we compared the intrinsic tyrosine kinase activities of erythrocyte insulin receptors from type 2 diabetes mellitus with or without stimulation by berberine in vitro. Methods- Preparations containing insulin receptors were obtained from soluble human erythrocytes, and the insulin receptors were partially purified by affinity chromatography. The tyrosine kinase activity was measured by the exogenous substrate phosphorylation. Results： Both the membrane tyrosine kinase activity and the purified receptor tyrosine kinase activity from diabetics decreased significantly compared with those of normal individuals （reduced by 67.4% and 47.2%, respectively）. After incubation with berbefine, there is a statistical difference in the activity of membrane tyrosine kinase for diabetic patients （ a 150% increase）. Berefine had no effect on the tyrosine kinase activity of purified insulin receptors. Conclusion： We concluded from these results that berbefine was able to improve the insulin sensitivity by increasing the protein tyrosine kinase activity of membrane-bound insulin receptors from type 2 diabetes mellitus.

Multi-biological defects caused by lead exposure exhibit transferable properties from exposed parents to their progeny in Caenorhabditis elegans

Whether the multi-biological toxicity from lead exposure could be transferred to progeny has not been clarified. In the present study, we explored the Caenorhabditis elegans to analyze the multiple toxicities from lead exposure and their possibly transferable properties. The lead exposure could cause series of severe multi-biological defects with a concentration-dependent manner by affecting the endpoints of life span, development, reproduction and locomotion behaviors in nematodes. Moreover, most of these toxicities could be transferred to progeny from lead exposed animals and some of the defects in progeny appeared even more severe than in their parents, such as the body sizes and mean life spans. We summarized the defects caused by lead exposure into three groups according to their transferable properties or rescue patterns. That is, the defects caused by lead exposure could be largely, or partially, or became even more severe in progeny animals. Therefore, our results suggest that lead exposure can cause severely multi-biological defects, and most of these multiple toxicities can be considered as transferable for exposed animals in C. elegans.

Using the nematode Caenorhabditis elegans as a model animal for assessing the toxicity induced by microcystin-LR

Among more than 75 variants of microcystin （MC）, microcystin-LR （MC-LR） is one of the most common toxins. In this study, the feasibility of using Caenorhabditis elegans to evaluate MC-LR toxicity was studied. C. elegans was treated with MC-LR at different concentrations ranging from 0.1 to 80 μg/L. The results showed that MC-LR could reduce lifespan, delay development, lengthen generation time, decrease brood size, suppress locomotion behavior, and decreases hsp-16-2-gfp expression. The endpoints of generation time, brood size, and percentage of the population expressing hsp-16-2-gfp were very sensitive to 1.0μg/L of MC-LR, and would be more useful for the evaluation of MC-LR toxicity. Furthermore, the tissue-specific hsp-16-2-gfp expressions were investigated in MC-LR-exposed animals, and the nervous system and intestine were primarily affected by MC-LR. Therefore, the generation time, brood size, and hsp-16-2-gfp expression in C. elegans can be explored to serve as valuable endpoints for evaluating the potential toxicity from MC-LR exposure.

Transcriptome sequencing and annotation of the halophytic microalga Dunaliella salina

The unicellular green alga Dunaliella salina is well adapted to salt stress and contains compounds（including β-carotene and vitamins） with potential commercial value.A large transcriptome database of D.salina during the adjustment, exponential and stationary growth phases was generated using a high throughput sequencing platform.We characterized the metabolic processes in D.salina with a focus on valuable metabolites, with the aim of manipulating D.salina to achieve greater economic value in large-scale production through a bioengineering strategy.Gene expression profiles under salt stress verified using quantitative polymerase chain reaction（qPCR） implied that salt can regulate the expression of key genes.This study generated a substantial fraction of D.salina transcriptional sequences for the entire growth cycle, providing a basis for the discovery of novel genes.This first full-scale transcriptome study of D.salina establishes a foundation for further comparative genomic studies.

Occurrence of the Transition of Apical Architecture and Expression Patterns of Related Genes during Conversion of Apical Meristem Identity in G2 Pea

G2 pea exhibits an apical senescence delaying phenotype under short-day （SD） conditions; however, the structural basis for its apical development is still largely unknown. In the present study, the apical meristem of SD-grown G2 pea plants underwent a transition from vegetative to indeterminate inflorescence meristem, but the apical meristem of long-day （LD）-grown G2 pea plants would be further converted to determinate floral meristem. Both SD signal and GA3 treatment enhanced expression of the putative calcium transporter PPF1, and pea homologs of TFL1 （LF and DET）, whereas LD signal suppressed their expression at 60 d post-flowering compared with those at 40 d post-flowering. Both PPF1 and LF expressed at the vegetative and reproductive phases in SD-grown apical buds, but floral initiation obviously increased the expression level of PPF1 compared with the unchanged expression level of LF from 40 to 60 d post-flowering. In addition, although the floral initiation significantly enhanced the expression levels of PPF1 and DET, DET was mainly expressed after floral initiation in SD-grown apil buds. Therefore, the main structural difference between LD- and SD-grown apical meristem in G2 pea lies in whether their apical indeterminate inflorescence meristem could be converted to the determinate structure.

Can we treat neurodegenerative diseases by preventing an age-related decline in microRNA expression?

MicroRNA pathway is down-regulated in aged dopaminergic neurons： Parkinson＇s disease （PD） is the most frequent motor neuro- degenerative disorder and is morphologically mainly associated with progressive dopaminergic neuronal loss in the ventral midbrain.

Gibberellin Is Involved in the Regulation of Cell Death-mediated Apical Senescence in G2 Pea

Senescence is the process of programmed degradation. The G2 line of pea exhibits apical senescence-delaying phenotype under short-day （SD） conditions, but the mechanism regulating the apical senescence is still largely unknown. Gibberellin （GA） was proved to be able to delay this apical senescence phenotype in G2 pea grown under long-day （LD） conditions. Here we show that the initiation of cell death signals in the terminal floral meristem was involved in the regulation of apical senescence in pea plants. SD signals prevented the formation of the cell death region in the apical mersitem. Moreover, GA3 treatment could effectively inhibit the occurrence of cell death-mediated apical senescence in LD-grown apical buds. Therefore, our data suggest that the prevention of apical senescence in SD-grown G2 pea through GA3 treatment may be largely responsible for the regulation of occurrence of the DNA fragmentation in apical meristem.

Administration with Bushenkangshuai Tang alleviates UV irradiation- and oxidative stress-induced lifespan defects in nematode Caenorhabditis elegans

During normal metabolism,oxidative bypro-ducts will inevitably generate and damage molecules thereby impairing their biological functions,including the aging process.Bushenkangshuai Tang(补肾抗衰方,BT)is a traditional Chinese medicine widely used for clini-cally treating premature ovarian failure.In the present study,BT administration at high concentrations signifi-cantly increased lifespan,slowed aging-related decline,and delayed accumulation of aging-related cellular damage in wild-type Caenorhabditis elegans.BT admin-istration could further largely alleviate the aging defects induced by UV and oxidative stresses,and BT administra-tion at different concentrations could largely rescue the aging defects in mev-1 mutant animals.The protective effects of BT administration on aging process were at least partially dependent on the Ins/IGF-like signaling pathway.Moreover,BT administration at different concentrations obviously altered the expression patterns of antioxidant genes and suppressed the severe stress responses induced by UV and oxidative stresses,suggesting that BT-induced tolerance to UV or oxidative stress might result from reactive oxygen species scavenging.BT administration during development was not necessarily a requirement for UV and oxidative stress resistance,and the concentrations of administrated BT examined were not toxic for nematodes.Therefore,BT administration could effectively retrieve the aging defects induced by UV irradiation and oxidative stress in Caenorhabditis elegans.

PPF1 May Suppress Plant Senescence via Activating TFL1 in Transgenic Arabidopsis Plants

Senescence, a sequence of biochemical and physiological events, constitutes the final stage of development in higher plants and is modulated by a variety of environmental factors and intsmal factors. PPF1 possesses an important biological function in plant development by controlling the Ca^2＋ storage capacity within chloroplasts. Here we show that the expression of PPF1 might play a pivotal role in negatively regulating plant senescence as revealed by the regulation of overaxpression and suppression of PPF1 on plant development. Moreover, TFL1, a key regulator in the floral repression pathway, was screened out as one of the downstream targets for PPF1 in the senescence-signaling pathway. Investigation of the senescence-ralatsd phenotypes in PPFI（-） till-1 and PPFI（＋） till-1 double mutants confirmed and further highlighted the relation of PPF1 with TFL1 in transgenic plants. The activation of TFL1 expression by PPF1 defines an important pathway possibly essential for the negative regulation of plant senescence in transgenic Arabidopsis.

Acid Phosphatase Activity May Affect the Tuber Swelling by Partially Regulating Sucrose-mediated Sugar Resorption in Potato

APase activity is involved in regulating many physiological and developmental events by affecting the resorption process. In this study, we investigate the role of APase activity in tuber development in potato. APase activities were mainly localized in cytoplasm, gaps among cells and stroma of amyloplasts of parenchyma cells at the stage of tuber swelling. AP1, encoding a putative APase, was also highly expressed in swelling tubers and a low level of expression was observed in elongated stolons and matured tubers. Inhibition of APase activity by applying Brefeldin A, an inhibitor of APase production and secretion, significantly suppressed the tuber swelling and moderately affected the stolon elongation and the tuberization frequency. During tuber development, sucrose serves as the main soluble sugar for long-distance transportation and resorption. Moreover, inhibition of APase activity by Brefeldin A markedly reduced the sucrose content in tubers and further decreased the starch accumulation, suggesting that the function of APase in regulating the tuber swelling might be at least partially mediated by the sugar resorption. Exogenous sucrose treatments further indicate the important role of sucrose-mediated sugar resorption in tuber swelling. These results suggest that the APase activity might affect the tuber swelling by partially regulating the sucrose-mediated sugar resorption.