Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm^–3 6-benzyladenine and 0.5 mg dm^–3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm^–3 phosphinothricin (L-PPT) supplemented medium were confirmed by GUS assay and PCR analysis. The transformation rate was 58 % with AGL1/ pDM805 and 25 % with LB5-1. Rooted plantlets were acclimated and then again screened for Basta®-resistance by spraying with 15 – 60 mg dm^–3 L-PPT. Surviving plants were selfed and Basta®-resistance was demonstrated in T₁ progeny.     

Increased expression of PS1 is sufficient to elevate the level and activity of γ-secretase in vivo

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity

Epithelial–mesenchymal transition (EMT) of tubular cells is one of the mechanisms which contribute to renal fibrosis and transforming growth factor-β (TGF-β) is one of the main triggers. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan-sulfate thus regulating the bioavailability of growth factors (FGF-2, TGF-β). HPSE controls FGF-2-induced EMT in tubular cells and is necessary for the development of diabetic nephropathy in mice.The aim of this study was to investigate whether HPSE can modulate the expression and the effects of TGF-β in tubular cells.First we proved that the lack of HPSE or its inhibition prevents the increased synthesis of TGF-β by tubular cells in response to pro-fibrotic stimuli such as FGF-2, advanced glycosylation end products (AGE) and albumin overload.Second, since TGF-β may derive from sources different from tubular cells, we investigated whether HPSE modulates tubular cell response to exogenous TGF-β. HPSE does not prevent EMT induced by TGF-β although it slows its onset; indeed in HPSE-silenced cells the acquisition of a mesenchymal phenotype does not develop as quickly as in wt cells. Additionally, TGF-β induces an autocrine loop to sustain its signal, whereas the lack of HPSE partially interferes with this autocrine loop.Overall these data confirm that HPSE is a key player in renal fibrosis since it interacts with the regulation and the effects of TGF-β. HPSE is needed for pathological TGF-β overexpression in response to pro-fibrotic factors. Furthermore, HPSE modulates TGF-β-induced EMT: the lack of HPSE delays tubular cell transdifferentiation, and impairs the TGF-β autocrine loop.     

CARMA3 is overexpressed in colon cancer and regulates NF-κB activity and cyclin D1 expression

CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p=0.0383), lymph node metastasis (p=0.0091) and Ki67 proliferation index (p=0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-IκB levels and NF-κB activity and its overexpression increased p-IκB expression and NF-κB activity. NF-κB inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-κB mediated upregulation of cyclin D1.     

Glycosylation profile and biological activity of Remicade® compared with Flixabi® and Remsima®

As biosimilars enter the market, comparisons of product quality are needed. Manufacturing differences may lead to differences in critical quality attributes, which affect efficacy. Therefore, critical quality attributes (structure and biological activity) of Remicade® and of 2 biosimilar products (Flixabi®/Renflexis® and Remsima®/Inflectra®) were determined. We assessed binding to tumor necrosis factor in a fluorescence competitive binding assay; potency in a luciferase reporter gene assay; percentages of galactosylated glycan, afucose plus high mannosylated glycans, and charged glycan; FcγRIIIa (CD16) binding (assessed by 3 methods); and antibody-dependent cell-mediated cytotoxicity (ADCC) in the NK92-CD16a cell line and in peripheral blood mononuclear cells (PBMC). The results of Fab-related activity were similar for all products. Compared with Remicade®, Flixabi® had a lower percentage of charged glycan, and Remsima® had a higher percentage of galactosylated glycan and a lower percentage of afucose plus high mannosylated glycans. Whereas Remsima® and Remicade® are expressed in a Sp2/0 cell line, Flixabi® is expressed in a CHO cell line. Despite this difference, galactosylated glycans from the 3 products were not correlated with the expression system. The results of all 3 methods used in this study indicated that FcγRIIIa binding was lower with Remsima® than with Remicade®. The percentage of ADCC in NK92-CD16a cells was lower with Remsima® and higher with Flixabi® compared with Remicade®, but was similar for all 3 products in PBMC. Surface expression of CD16 was 5.7-fold greater on NK92-CD16a cells than on PBMC. Combined percentages of afucosylated and high mannosylated glycans were positively correlated with FcγRIIIa binding and ADCC in NK92-CD16 cells, while no correlation was observed in PBMC.     

Localization of β-glycerophosphatase activity in cotton fiber during differentiation

Summary The distribution of -glycerophosphatase activity in the outer integument of cotton (Gossypium hirsutum L.) ovules was determined histochemically at the electron microscope level. Both a linted cultivar and a lintless mutant line were examined from 1 day preanthesis to 3 days postanthesis. No enzyme activity was observed in the lintless line at any stage. In the linted cultivar there was no enzyme activity before anthesis, but as fibers were initiated on the day of anthesis, activity was observed in the expanding fiber cell wall and nucleus. As the fibers started elongating, enzyme activity was particularly concentrated in the cytoplasm and wall where directional growth towards the micropyle occurs. By 2 days postanthesis, -glycerophosphatase activity was decreasing in the cell wall and nucleus, but was increasing in the nucleolus. Enzyme activity in the nucleolus was highest at 3 days post-anthesis, but nuclear -glycerophosphatase activity was not observed then. These results indicate that -glycerophosphatase activity was associated with differentiating fiber cells, specifically with those sites where distinct anatomical, and perhaps catabolic, changes were occurring. The significance of the results is discussed in relation to possible mechanisms of cotton fiber differentiation.Abbreviations EM Electron microscopic - ER Endoplasmic reticulum - GP -Glycerophosphate - GPase -Glycerophosphatase - SEM Scanning electron microscopy     

Development of acetosyringone-inducible Gateway® and Golden Gate expression vectors for heterologous gene expression in Agrobacterium tumefaciens

In Vitro Cellular & Developmental Biology - Plant - As a plant genetic engineer, Agrobacterium tumefaciens utilizes phenolic compounds, such as acetosyringone, to activate its virulence genes...     

Inhibition of NF-κ B activity and cFLIP expression contribute to viral-induced apoptosis

Virus-induced activation of nuclear factor-kappa B (NF-B) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-B is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-B activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-B regulation, where NF-B activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-B activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-B is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-B also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-B-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-B activation in reovirus-infected cells. Further, inhibition of NF-B activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-B and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.     

IFN-λ1 in CHO cells: its expression and biological activity

Background

Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor.

Results

A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 10⁶ IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b.

Conclusion

The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

     

A cotton mitogen-activated protein kinase （GhMPK6） is involved in ABA-induced CAT1 expression and H2O2 production

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays a pivotal role in the regulation of stress and developmental signals in plants.Here,we identified one gene,GhMPK6,encoding an MAPK protein in cotton.GFP fluorescence assay demonstrated that GhMAPK6 is a cytoplasm localized protein.Quantitative RT-PCR analysis revealed that mRNA accumulation of GhMPK6 was significantly promoted by abscisic acid (ABA).Overexpression of GhMPK6 gene in the T-DNA insertion mutant atmkkl (SALK_015914) conferred a wild-type phenotype to the transgenic plants in response to ABA.Under ABA treatment,cotyledon greening/expansion in GhMPK6 transgenic lines and wild type was significantly inhibited,whereas the atmkkl mutant showed a relatively high cotyledon greening/expansion ratio.Furthermore,CAT1 expression and H2O2 levels in leaves of GhMPK6 transgenic lines and wild type were remarkably higher than those of atmkkl mutant with ABA treatment.Collectively,our results suggested that GhMPK6 may play an important role in ABA-induced CAT1 expression and H2O2 production.     

Inhibition of histone acetyltransferase by naringenin and hesperetin suppresses Txnip expression and protects pancreatic β cells in diabetic mice

BackgroundThe damage of pancreatic β cells is a major pathogenesis of the development and progression of type 2 diabetes and there is still no effective therapy to protect pancreatic β cells clinically. In our previous study, we found that Quzhou Fructus Aurantii (QFA), which is rich in flavanones, had the protective effect of pancreatic β cells in diabetic mice. However, the underlying mechanism is still unclear.PurposeIn the current study, we administered naringenin and hesperetin, two major active components of QFA, to protect pancreatic β cells and to investigate the underlying molecular mechanism focusing on the epigenetic modifications.MethodsWe used diabetic db/db mouse and INS-1 pancreatic β cell line as in vivo and in vitro models to investigate the protective effect of naringenin and hesperetin on pancreatic β cells under high glucose environment and the related mechanism. The phenotypic changes were evaluatedby immunostaining and the measurement of biochemical indexes. The molecular mechanism was explored by biological techniques such as western blotting, qPCR, ChIP-seq and ChIP-qPCR, flow cytometry and lentivirus infection.ResultsWe found that naringenin and hesperetin had an inhibitory effect on histone acetylation. We showed that naringenin and hesperetin protected pancreatic β cells in vivo and in vitro, and this effect was independent of their direct antioxidant capacity. The further study found that the inhibition of thioredoxin-interacting protein (Txnip) expression regulated by histone acetylation was critical for the protective role of naringenin and hesperetin. Mechanistically, the histone acetylation inhibition by naringenin and hesperetin was achieved through regulating AMPK-mediated p300 inactivation.ConclusionThese findings highlight flavanones and the phytomedicine rich in flavanones as important dietary supplements in protecting pancreatic β cells in advanced diabetes. In addition, targeting histone acetylation by phytomedicine is a potential strategy to delay the development and progression of diabetes.     

β-Lactamase activity and resistance to penicillins in Myxococcus xanthus

Several mutants and other variants of Myxococcus xanthus HP100 were obtained with differences in their sensitivity to carbenicillin and other penicillin derivatives. The specific activities of -lactamase in different resistant organisms varied from strain to strain but were consistently higher than in HP100. The relative molecular mass (M _r ) of the enzyme in M. xanthus HP100 was found to be 22,300. In certain carbenicillin resistant strains a second fraction of -lactamase activity of molecular weight 186,000 presumed to be an octamer of the other form was present. The enzyme was found in cell free extracts and also in culture supernatants of all carbenicillin resistant mutants but not in culture supernatants of strain HP100. In all the carbenicillin resistant mutants a part of the intracellular enzyme activity was released by osmotic shock and this activity may be periplasmic. The forms of the enzyme present in the culture supernatants and released by osmotic shock were monomeric. Carbenicillin resistance was not transferable between strains by conjugation. One resistance allele inhibited the transfer of the R factor Sa between myxococci.Non-standard abbreviations C^S C^R sensitivity and resistance to carbenicillin - C _u ^R C _S ^R unstable and stable resistance to carbenicillin     

Construction and expression of sTRAIL–melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL–melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85 % of total target protein which was much higher than that without the SUMO tag (approximately 10 %); sTRAIL–melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL–melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL–melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P?<?0.01). Furthermore, we found that sTRAIL–melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.     

Systemic induction of volatile release in cotton: How specific is the signal to herbivory?

Plants attacked by herbivorous insects release chemical signals that attract natural enemies of the herbivores to the damaged plants. Feeding of Spodoptera exigua larvae on the lower leaves of cotton (Gossypium hirsutum L.) for multiple feeding periods of 9–12 h with a 12 h, interval in between when the caterpillars are removed overnight, will induce a systemic release of volatile compounds that is comparable to the volatiles released in response to continuous feeding damage on the lower leaves for several days. The systemic volatile release in response to herbivory can be mimicked by mechanically damaging the lower leaves and applying caterpillar oral secretion to the injured leaves over 4 days. Cotton plants that are only mechanically damaged systemically release significantly less -pinene, myrcene, (Z)-3-hexenyl acetate, (E)--farnesene and (E,E)--farnesene after 4 days compared to plants damaged mechanically with application of caterpillar regurgitant. However, multiple 9–12 h mechanical damage alone induces a significantly higher systemic release of (Z)-3-hexenyl acetate, myrcene, (E)--ocimene, and (E)--farnesene after 4 days compared to undamaged control plants. This indicates that multiple mechanical damage alone cannot mimic completely the response induced by mechanically injuring the leaves and applying caterpillar regurgitant. A specific elicitor in the regurgitant of the caterpillar enhances the amount of several systemically released volatiles. Thus, the systemic release of volatile compounds by herbivore-damaged cotton plants appears to be regulated by at least two different mechanisms.     

Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum

The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenes Tü494 is located on a 0.8-kb BglII fragment [Strauch et al., Gene 63 (1988) 65-74]. By sequencing a 1.3-kb BglII-SstII fragment, an open reading frame representing the pat gene was found. It encodes a polypeptide of 183 amino acids with an Mr of 20,621. The base composition of the pat gene is typical for Streptomyces [70.1 mol% (G + C) in total and 93.5 mol% (G + C) in the third position]. Translation of pat is initiated by a GTG codon which was identified by frameshift mutations in Escherichia coli as well as in Streptomyces lividans. Significant homology of the pat gene was found to the bialaphos-resistance gene (bar) of Streptomyces hygroscopicus [Thompson et al., EMBO J. 9 (1987) 2519-2523]. However, variations were detected in the 5'-noncoding region of the two resistance genes which may reflect differences in regulation. Since Pt is a potent herbicide, the pat gene was modified and introduced into Nicotiana tabacum by Agrobacterium-mediated leaf-disc transformation. The GTG start codon of pat was replaced by ATG. Subsequently the modified pat-coding region was fused to the 35S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.     

Do We Need to Detect Isoniazid Resistance in Addition to Rifampicin Resistance in Diagnostic Tests for Tuberculosis?

Background

Multidrug-resistant tuberculosis (MDR-TB) is resistant to both rifampicin (RIF) and isoniazid (INH). Whereas many TB diagnostics detect RIF-resistance, few detect INH-monoresistance, which is common and may increase risk of acquired MDR-TB. Whether inclusion of INH-resistance in a first-line rapid test for TB would have an important impact on MDR-TB rates remains uncertain.

Methods

We developed a transmission model to evaluate three tests in a population similar to that of India: a rapid molecular test for TB, the same test plus RIF-resistance detection (“TB+RIF”), and detection of RIF and INH-resistance (“TB+RIF/INH”). Our primary outcome was the prevalence of INH-resistant and MDR-TB at ten years.

Results

Compared to the TB test alone and assuming treatment of all diagnosed MDR cases, the TB+RIF test reduced the prevalence of MDR-TB among all TB cases from 5.5% to 3.8% (30.6% reduction, 95% uncertainty range, UR: 17–54%). Despite using liberal assumptions about the impact of INH-monoresistance on treatment outcomes and MDR-TB acquisition, expansion from TB+RIF to TB+RIF/INH lowered this prevalence only from 3.8% to 3.6% further (4% reduction, 95% UR: 3–7%) and INH-monoresistant TB from 15.8% to 15.1% (4% reduction, 95% UR: (-8)-19%).

Conclusion

When added to a rapid test for TB plus RIF-resistance, detection of INH-resistance has minimal impact on transmission of TB, MDR-TB, and INH-monoresistant TB.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

The effects of quercetin protect cardiomyocytes from A/R injury is related to its capability to increasing expression and activity of PKCε protein

Quercetin is a ubiquitous flavonoid found in vegetable foods. Epidemiological and animal studies have reported an inverse association between quercetin intakes and occurrence and development of various cardiovascular diseases. Some researchers have inferred that the mechanisms of quercetin to protect cardiomyocytes from ischemia/reperfusion injury may be involved in modulation of intracellular signal pathways and regulation of proteins expression beyond its antioxidant activity. The aim of this study was to investigate whether quercetin protect cardiomyocytes from anoxia/reoxygenation injury through PKCε pathway. Neonatal rat primary cardiomyocytes were pretreated with quercetin or quercetin plus εV1-2, a selective PKCε inhibitor, prior to A/R treatment. Western blotting analysis showed that the level of PKCε and phosphor-PKCε Ser297 in the quercetin pretreatment group were all increased significantly compared to the control or A/R group. Subsequent assays showed that pretreated with quercetin could increase the viability of neonatal rat primary cardiomyocytes suffered A/R, decrease the apoptosis and ROS and alleviate the loss of mitochondrial membrane potential induced by A/R injury. However, the protective effects of quercetin disappeared in the group pretreated with εV1-2. Thus, for the first time, we revealed that one of the mechanisms of quercetin protecting cardiomyocytes from A/R injury might be increase the expression of PKCε protein and then enhance the activity of its downstream pathway.