Expression Levels Of Target Proteins Research Articles

Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer.

IntroductionExosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion.MethodsThe expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay.ResultsIn this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells.ConclusionTogether, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-256) contains supplementary material, which is available to authorized users.

MiR-143 and miR-145 synergistically regulate ERBB3 to suppress cell proliferation and invasion in breast cancer.

IntroductionERBB3, one of the four members of the ErbB family of receptor tyrosine kinases, plays an important role in breast cancer etiology and progression. In the present study, we aimed to identify novel miRNAs that can potentially target ERBB3 and their biological functions.MethodThe expression levels of miR-143/145 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. We used bioinformatic analyses to search for miRNAs that can potentially target ERBB3. Luciferase reporter plasmids were constructed to confirm direct targeting. Furthermore, the biological consequences of the targeting of ERBB3 by miR-143/145 were examined by cell proliferation and invasion assays in vitro and by the mouse xenograft tumor model in vivo.ResultsWe identified an inverse correlation between miR-143/145 levels and ERBB3 protein levels, but not between miR-143/145 levels and ERBB3 mRNA levels, in breast cancer tissue samples. We identified specific targeting sites for miR-143 and miR-145 (miR-143/145) in the 3’-untranslated region (3’-UTR) of the ERBB3 gene and regulate ERBB3 expression. We demonstrated that the repression of ERBB3 by miR-143/145 suppressed the proliferation and invasion of breast cancer cells, and that miR-143/145 showed an anti-tumor effect by negatively regulating ERBB3 in the xenograft mouse model. Interestingly, miR-143 and miR-145 showed a cooperative repression of ERBB3 expression and cell proliferation and invasion in breast cancer cells, such that the effects of the two miRNAs were greater than with either miR-143 or miR-145 alone.ConclusionTaken together, our findings provide the first clues regarding the role of the miR-143/145 cluster as a tumor suppressor in breast cancer through the inhibition of ERBB3 translation. These results also support the idea that different miRNAs in a cluster can synergistically repress a given target mRNA.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-220) contains supplementary material, which is available to authorized users.

MiR-181 family: regulators of myeloid differentiation and acute myeloid leukemia as well as potential therapeutic targets.

MicroRNAs have been shown to play an important role in normal hematopoisis and leukemogenesis. Here, we report function and mechanisms of miR-181 family in myeloid differentiation and acute myeloid leukemia (AML). The aberrant overexpression of all the miR-181 family members (miR-181a/b/c/d) was detected in French-American-British M1, M2 and M3 subtypes of adult AML patients. By conducting gain- and loss-of-function experiments, we demonstrated that miR-181a inhibits granulocytic and macrophage-like differentiation of HL-60 cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) by directly targeting and downregulating the expression of PRKCD (which then affected the PRKCD-P38-C/EBPα pathway), CTDSPL (which then affected the phosphorylation of retinoblastoma protein) and CAMKK1. The three genes were also demonstrated to be the targets of miR-181b, miR-181c and miR-181d, respectively. Significantly decreases in the expression levels of the target proteins were detected in AML patients. Inhibition of the expression of miR-181 family members owing to Lenti-miRZip-181a infection in bone marrow blasts of AML patients increased target protein expression levels and partially reversed myeloid differentiation blockage. In the mice implanted with AML CD34+ HSPCs, expression inhibition of the miR-181 family by Lenti-miRZip-181a injection improved myeloid differentiation, inhibited engraftment and infiltration of the leukemic CD34+ cells into the bone marrow and spleen, and released leukemic symptoms. In conclusion, our findings revealed new mechanism of miR-181 family in normal hematopoiesis and AML development, and suggested that expression inhibition of the miR-181 family could provide a new strategy for AML therapy.

Different sensitivity of germinal center B cell-like diffuse large B cell lymphoma cells towards ibrutinib treatment

BackgroundAlthough rituximab in the combination of CHOP chemotherapy has been widely used as the standard treatment for several kinds of B-cell non-Hodgkin lymphoma (B-NHL), a great number of B-NHL patients treated with this immunotherapy still develop primary and secondary resistance. Recently Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib showed promising therapeutic effect in relapsed/refractory CLL and B-cell NHL, which provided essential alternatives for these patients.MethodsThe proliferation and apoptosis induction of tumor cells were measured by cell viability assay and Annexin-V staining. Western Blotting analysis and real-time PCR were used to detect the expression level of target proteins and chemokines production.ResultsWe demonstrated that ibrutinib inhibited the proliferation and induced apoptosis of GCB-DLBCL cell lines through suppression of BCR signaling pathway and activation of caspase-3. Furthermore, the chemokines CCL3 and CCL4 production from tumor cells were also found to be attenuated by ibrutinib treatment. But different cell lines exhibited distinct sensitivity after ibrutinib treatment. Interestingly, the decreasing level of p-ERK after ibrutinib treatment, but not the basal expression level of Btk, correlated with different drug sensitivity.ConclusionsIbrutinib could be a potentially useful therapy for GCB-DLBCL and the decreasing level of p-ERK could become a useful biomarker to predict related therapeutic response.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

BackgroundCombination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.MethodsCell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.ResultsWe found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.ConclusionsThese results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Orthogonal Test Design for Optimization of the Expression of Thymosin α1 and Thymosin α1- iRGD Gene in Engineered <i>E.coli</i> BL21 Strain

In order to increase the expression of fusion protein of thymosin α1(Tα1) and thymosin α1-iRGD (Tα1-iRGD) by the engineered E. coli BL21 strain, containing pET32a-Trx-Tα1 and pET32a-Trx-Tα1-iRGD plasmid, respectively. The key parameters that influenced the expression of Tα1 and Tα1-iRGD were optimized by employing an orthogonal experiment [L25(5)3], including OD600nm before induction, lactose concentration and induction time, each with five levels. The intensity of target protein band was scanned as a quantitative measure method of the protein expression, after electrophoresis separation of total soluble protein on SDS-PAGE. For Tα1 fusion protein, the optimal conditions were OD600nm=0.6 before induction, 2.5 mmol/L lactose concentration, 4 hours induction time. Target protein expression levels could be achieved 32.8% of the total soluble proteins. For Tα1-iRGD fusion protein, the optimal conditions were OD600nm=0.8 before induction, 7.5mmol/L lactose concentration, 4 hours for induction. Under the condition, the amount of expressed protein could reach 33.8% of the total soluble proteins. Whereas before optimization, the expression level of Tα1 and Tα1-iRGD fusion proteins were 20.6% and 9.0% of the total soluble proteins, respectively. The orthogonal test was proved to be an effective method to optimize the expression of target proteins.

Hydroxysafflor yellow A of Carthamus tinctorius attenuates lung injury of aged rats exposed to gasoline engine exhaust by down-regulating platelet activation

Long-term inhalation of gasoline engine exhaust (GEE) increases the risk of respiratory disease. Studies have suggested involvement of platelets in the development of some lung diseases. Hydroxysafflor yellow A (HSYA), a flavonoid compound, prevents hemostasis. Therefore, we investigated its effects on GEE-induced lung injury, and role of platelets in injury. Sixty-week-old male Sprague-Dawley rats were exposed to GEE for 4h/day for 6 weeks, and then grouped as follows: control, GEE, GEE+HSYA, GEE+HSYA+GW9662, and GEE+GW9662. Arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2), pH, and the PaO2/fraction of inspired oxygen ratio (PaO2/FiO2) in the blood were detected using a blood gas analyzer. Wet/dry lung weight ratio, total protein in bronchoalveolar lavage fluid (BALF), and cytokine concentrations in serum and BALF were determined. Furthermore, cyclic adenosine monophosphate (cAMP) level and expression levels of target proteins were analyzed. Platelets were counted and their state was evaluated. HSYA attenuated GEE-mediated decreases in PaO2, PaO2/FiO2, platelet cAMP level, protein kinase A (PKA) activity, and peroxisome proliferator-activated receptor γ (PPARγ) expression. HSYA also attenuated GEE-mediated increases in lung permeability, cytokine levels in serum and BALF, plasma platelet count, and ADP-mediated platelet aggregation. Moreover, it suppressed GEE-induced increases in the expression of adhesion molecules and proinflammatory cytokines in platelets and lung tissue. Therefore, HSYA is therapeutically effective for GEE-mediated lung injury and acts by enhancing PKA activity and inhibiting platelet activation.

Abstract C239: Development of EphA10/CD3 bispecific tandem scFv as a drug candidate against breast cancer.

Abstract Background: Monoclonal antibodies (mAbs) are clinically used to treat several types of cancer due to their high specificity and potential to activate immune systems. The therapeutic effects of mAbs are unsatisfactory, however, in cases of aggressive cancer or low expression level of target protein. Recently, various antibody formats has been developed through genetic engineering technologies to improve therapeutic effect. Most notably bispecific antibodies (BsAb) are a highly promising form of target therapy. A BsAb that simultaneously targets the antigens on cancer cells and activated T-cells reportedly has the potential to show cytotoxicity more effectively than conventional mAbs. In our efforts to develop a new bispecific monoclonal antibody drug against breast cancer, we attempted to generate a tandem single-chain variable fragment (taFv) targeting both the T-cell CD3 antigen and Ephrin receptor A10 (EphA10), which were previously identified as breast cancer novel biomarker proteins. Methods: We constructed the taFv (EphA10/CD3) expression vector that contain two single chain Fv (scFv) derived from the anti-EphA10 antibody and anti-CD3 antibody joined by a flexible five amino acid linker (G4S). The taFv was expressed transiently in CHO-S cells and verified for identity by Western blot analysis, was purified via its C-terminal His tag by metal ion affinity chromatography (IMAC). Flow cytometry was used to confirm dual specificity of taFv against cells expressing the appropriate targets. The ability of cytotoxic for taFv from PBMC was analyzed by lactate dehydrogenase (LDH) release assay. Results: The taFv monomer was purified from CHO-S cells together with the taFv dimer. By flow cytometry analysis, each monomer and dimer bound both antigens EphA10 and CD3 while binding to CD3 was lower than full-body IgG. And each taFvs showed highly cytotoxicity specifically against EphA10 over-expression cancer cell line. Most notably, the cytotoxicity of taFv dimer was higher than taFv monomer at low Effector/Target ratio and low concentration. Conclusion: In this research, it's proved that this taFv (EphA10/CD3) could induce EphA10 specific cytotoxicity. So, we are now ongoing to evaluate the therapeutic effects of each taFvs (monomer and dimer) in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C239. Citation Format: Shintaro Taki. Development of EphA10/CD3 bispecific tandem scFv as a drug candidate against breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C239.

Long DNA passenger strand highly improves the activity of RNA/DNA hybrid siRNAs

Small interfering RNAs (siRNAs) are potent tools in biomedical research, which can reduce the expression level of target proteins through RNAi pathway. They are composed of 19-25 bp double strand RNA (dsRNAs), therefore, stimulate dsRNAs dependent interferon responses in a non-specific manner. This problem has prevented siRNAs from being applied as new therapeautic agents. In the present paper, we tried to circumvent interferon responses using RNA/DNA hetero siRNAs (HsiRNAs) composed of RNA guide and DNA passenger strands. It was previously reported that siRNAs which were partially substituted with DNA had RNAi activity and that DNA substitution often caused the activity loss. In our results, HsiRNAs, in which the passenger strand of siRNAs were exchanged with DNA also showed much lower activity than that of parental siRNAs. Here, we found that attachment of 5' flanking sequence to DNA passenger strand improved the activity of HsiRNAs. Furthermore, the effective HsiRNAs induced much lower interferon responses than parental siRNAs. Thus, HsiRNAs with 5' flanking sequence are expected to be novel siRNA drug candidates.

Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22phox expression

Intracellular reactive oxygen species (ROS) are derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Angiotensin II (Ang II) can cause endothelial dysfunction by promoting intracellular ROS generation. Safflor yellow B (SYB) effectively inhibits ROS generation by upregulating Bcl-2 expression. In this study, we examined the effects of SYB on Ang II-induced injury to human umbilical vein endothelial cells (HUVECs), and elucidated the roles of NADPH oxidase and Bcl-2. We treated cultured HUVECs with Ang II, SYB, and Bcl-2 siRNA, and determined NADPH oxidase activity and ROS levels. Furthermore, cellular and mitochondrial physiological states were evaluated, and the expression levels of target proteins were analyzed. Ang II significantly enhanced intracellular ROS levels, caused mitochondrial membrane dysfunction, and decreased cell viability, leading to apoptosis. This was associated with increased expression of AT1R and p22phox, increased NADPH oxidase activity, and an increased ratio of Bax/Bcl-2, leading to decreases in antioxidant enzyme activities, which were further strengthened after blocking Bcl-2. Compared to Ang II treatment alone, co-treatment with SYB significantly reversed HUVEC injury. Taken together, these results demonstrate that SYB could significantly protect endothelial cells from Ang II-induced cell damage, and that it does so by upregulating Bcl-2 expression and inhibiting ROS generation.

The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells

BackgroundIn eukaryotes, miR-16 is an important microRNA (miRNA) that is involved in numerous biological processes. However, it is not fully understood how miR-16 executes its physiological functions. In the present study, we aimed to identify novel miR-16 targets and study their biological functions.MethodsCandidate target genes of miR-16 were screened by microarray analysis of mRNA levels in several cancer cell lines with enhanced miR-16. Three bioinformatics algorithms, including TargetScan, PicTar, and miRanda, were used in combination to calculate the miR-16 targets. The expression levels of miR-16 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. The effect of miR-16 and target gene on cell viability was evaluated using MTT assays. The effects of miR-16 and target gene on apoptosis and cell cycle distribution were evaluated by flow cytometry analysis.ResultsBy overexpressing miR-16 in several cancer cell lines and measuring global mRNA levels using microarray analysis, we identified 27 genes that may be regulated by miR-16. After the bioinformatics filtering process, 18 genes were selected as candidate miR-16 targets. Furthermore, we experimentally validated three of these candidates, MAP7 (microtubule-associated protein 7), PRDM4 (PR domain containing 4) and CDS2 (CDP-diacylglycerol synthase 2), as direct targets of miR-16. Finally, we demonstrated that miR-16 targeting MAP7 played a critical role in regulating proliferation but not apoptosis and cell cycle progression in cancer cells.ConclusionIn summary, the present study identifies several novel miR-16 targets and illustrates a novel function of miR-16 targeting MAP7 in modulating proliferation in cancer cells.

Hybrid Viral Vectors for Vaccine and Antibody Production in Plants

Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

Grape-Derived Polyphenols Prevent Doxorubicin-Induced Blunted EDH-Mediated Relaxations in the Rat Mesenteric Artery: Role of ROS and Angiotensin II

This study determined whether doxorubicin, an anticancer agent, impairs endothelium-dependent relaxations mediated by nitric oxide (NO) and endothelium-derived hyperpolarization (EDH) in the mesenteric artery and, if so, the mechanism underlying the protective effect of red wine polyphenols (RWPs), a rich natural source of antioxidants. Male Wistar rats were assigned into 4 groups: control, RWPs, doxorubicin, and doxorubicin + RWPs. Vascular reactivity was assessed in organ chambers; the vascular formation of reactive oxygen species (ROS) using dihydroethidine and the expression levels of small and intermediate conductance calcium-activated potassium channels (SKCa, IKCa) and connexin 40 (Cx40), which are involved in EDH-type relaxations, endothelial NO synthase (eNOS), angiotensin II, and AT1 receptors by immunofluorescence. The doxorubicin treatment impaired EDH-mediated relaxations, whereas those mediated by NO were minimally affected. This effect was associated with reduced expression levels of SKCa, IKCa, and Cx40, increased expression levels of eNOS, angiotensin II, and AT1 receptors, and formation of ROS in mesenteric arteries. RWPs prevented both the doxorubicin-induced blunted EDH-type relaxations and the increased vascular oxidative stress, and they improved the expression levels of target proteins. These findings suggest that polyphenol-rich natural products might be of interest in the management of doxorubicin-induced vascular injury possibly by improving the vascular angiotensin system.

An ultra scale‐down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges

Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra-scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot-scale disk-type machines, was used in this work. Two recombinant P. pastoris strains, namely a X-33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot- and a large-scale disk-type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X-33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot-scale runs during early stages of process development for therapeutic products.

Analysis of miRNA regulation suggests an explanation for ‘unexpected’ increase in target protein levels

MicroRNA (miRNA) has been mostly associated with decrease in target protein expression levels. Recently, 'unexpected' observations of increase in target protein expression attributed to microRNA regulation have been reported. We formulate a comprehensive model for regulation by miRNA that includes both reversible mRNA-miRNA binding and selective return of RNA. We use this mathematical model incorporating multiple individual steps in the regulation process to study the simultaneous effects of these steps on the target protein level. We show that four dimensionless numbers obtained from 12 rate constants are sufficient to define the relative change in steady state target protein levels. We quantify the range of these numbers for which such pleiotropic increase in protein levels is possible, and interpret the experimental findings in the framework of our model such that the results are no longer unexpected. Finally, we show through stochastic simulation that the nature of the target protein distribution remains unchanged and the relative steady state noise levels are also completely defined by the values of these dimensionless numbers, irrespective of the individual reaction rate constants.

A rapid method to improve protein detection by indirect ELISA

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.

Transcription Factor Stat5 Knockdown Enhances Androgen Receptor Degradation and Delays Castration-Resistant Prostate Cancer Progression In vivo

Signal transducer and activator of transcription 5 (Stat5) plays an important role in the transition of prostate cancer (PCa) to its castrate-resistant state. Pharmacologic targeting of Stat5 is a rational approach to delay castrate-resistant progression, in part, because Stat5 cooperates with the androgen receptor (AR) to promote PCa progression. Immunostaining of tissue microarrays was used to correlate Stat5 expression with Gleason grade and to characterize changes in treatment-naive and androgen-deprived human PCa. Potency of a Stat5 antisense oligonucleotide (ASO) on Stat5 knockdown, cell growth, and apoptosis was assessed in LNCaP, C4-2, and DU145 cells. Effects of Stat5 knockdown on AR activity and stability was assessed using a PSA transactivation-luciferase assay and cyclohexamide plus MG132 treatment, respectively. LNCaP tumor-bearing mice were castrated and randomly assigned to treatment with Stat5-ASO or controls. Here, we show that the frequency of Stat5 expression is significantly increased in high Gleason grade as well as in hormone-treated PCa. Also, specific knockdown of Stat5 with ASO abrogates androgen-induced AR nuclear translocation and PSA transactivation despite R1881 stimulation. Moreover, Stat5 knockdown destabilizes AR, which leads to AR degradation via the proteasome. Shown for the first time as a preclinical proof-of-principle, Stat5 knockdown with Stat5-ASO significantly delays CRPC tumor progression in vivo. Thereby, we are able to recapitulate our in vitro results by reducing serum PSA and expression levels of target proteins in the xenograft tumors.

An economical single-sided antibody incubation method for Western blotting

A simple, single-sided antibody method for incubating primary and secondary antibodies in Western blotting was developed, which generates significant savings on the use of antibodies. Compared with the conventional immersion technique for antibody incubation, the present economical single-sided antibody incubation method resulted in a saving of 80% of antibody use. Besides, the present incubation method did not compromise the Western blot results and was not affected by the expression levels of target proteins.

Expression optimization of subunitfasGof 987P fimbriae in enterotoxigenicEscherichia coli(ETEC)

AbstractFour bioengineered bacteria having thefasGgene were constructed using twoEscherichia coliexpression strains (BL21 (DE3) and Rossetta (DE3)) with different tRNA profiles and twoE. coliexpression vectors (pET28a(+) and pGEX-KG) to examine the influence of rare codons on the expression of thefasGgene. The induction expression result of bioengineered bacteria showed that the expression levels of target proteins in Rossetta (DE3), which contained the tRNAs of the rare codons, were higher than in BL21 (DE3), either using pET-fasGor pKG-fasG.

Attenuation of Leakiness in Doxycycline-Inducible Expression Via Incorporation of 3′ AU-rich mRNA Destabilizing Elements

Tetracycline-regulated expression systems have been widely used for inducible protein expression in cultured mammalian cells. With these systems, however, leakiness in expression of the target gene in the absence of the inducing agent is a frequent problem. Here we describe a novel approach to overcome this problem that involves the incorporation of AU-rich mRNA destabilizing elements (AREs) into the 3' untranslated regions of the tetracycline-inducible constructs. Using the inducible expression of sphingosine kinase 1 and 2 in HEK293 cells as model systems, we found this ARE approach to be remarkably successful in ablating expression of these proteins in the absence of doxycycline through decreasing stability of their mRNAs. We show that this undemanding and flexible process results in a substantial decrease in the leakiness of the tetracycline-inducible expression system while maintaining a high level of target protein expression following induction.