Western Blot Procedure Research Articles

Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts.

Tau is a microtubule-associated protein enriched in the axonal compartment. Its most well-known function is to bind and stabilize microtubules. In Alzheimer's disease and other neurodegenerative diseases known as tauopathies, tau undergoes several abnormal post-translational modifications including hyperphosphorylation, conformational changes, oligomerization, and aggregation. Numerous mouse models of tauopathies have been developed, and Western blotting remains an invaluable tool in studying tau protein physiological and pathological changes in these models. However, many of the antibodies that have been developed to analyze tau post-translational modifications are mouse monoclonal, which are at risk of producing artifactual signals in Western blotting procedures. This risk does not arise due to their lack of specificity, but rather because the secondary antibodies used to detect them will also react with the heavy chain of endogenous mouse immunoglobulins (Igs), leading to a non-specific signal at the same molecular weight as tau protein (around 50kDa). Here, we present the use of anti-light-chain secondary antibodies as a simple and efficient technique to prevent non-specific Ig signals around 50kDa. We demonstrate the efficacy of this method by either eliminating or identifying artifactual signals when using monoclonal antibodies directed at non-phosphorylated epitopes (T49, Tau3R, Tau4R), phosphorylated epitopes (MC6, AT180, CP13), or an abnormal tau conformation (MC1), in wild-type (WT) mice with tau hyperphosphorylation (hypothermic), transgenic mice overexpressing human tau (hTau mice), and tau knockout (TKO) mice.

Need for speed: Human fast-twitch mitochondria favor power over efficiency

ObjectiveHuman skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation. While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic. MethodsHere, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity. ResultsMaximal complex I+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per mitochondrial volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for complex I+II, which was primarily mediated through elevated complex II respiration. Furthermore, the abundance of complex II protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid–or complex I respiration. ConclusionMitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.

Membrane Protein Fractionation and Analysis through Western Blot inAedes aegyptiMalpighian Tubules

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins. In this protocol, we describe use of the membrane fractionation kit to isolate membrane-bound proteins, followed by western blot analysis, to observe protein abundance. The protocol involves methods that require organ (or tissue) collection, followed by protein processing, and a 2-d western blot procedure.

HT22 cell differentiation reduces insulin receptor levels

Purpose: The brain is an insülin-sensitive organ and has widespread insulin receptor (IR) expression. IR signaling in the brain is essential for neuronal development, feeding behavior, body weight, and cognitive processes such as attention, learning, and memory. HT22 cells, which are derived from parent HT4 cells that are immortalized from primary mouse hippocampal neuronal cells are used in research related to insulin signaling. However, the role of these cells in insulin signaling is not known. In this study, we aimed to examine IR levels in cells differentiated using neurobasal medium. Material and methods: For the study, briefly, the cells were seeded in 6-well plates at 2x105 cells/well for 24 h. After the cells reached 80% confluence, the normal growth medium was replaced with a differentiation medium and the cells were incubated for 72 hours at 37 0C in 5% CO2. Western blot procedure was used to determine the expression of the IR. Result: Our results show that differentiation of HT22 cells stimulates neurite outgrowth. Furthermore, IR protein levels were significantly downregulated in differentiated HT22 cells. Conclusion: This finding may require careful consideration of the use of neurobasal medium in conditions where IR signaling is important.

Anti-inflammatory effect of Rhododendron brachycarpum D. Don ex G. Don leaves extract on dermatitis

Introduction: Rhododendron brachycarpum D. Don ex G. Don(RbGD) leaves have traditionally been used to treat diabetes, rheumatoid arthritis, hypertension, and skin ailments. Although RbGD leaves are used as a medicinal plant, there is no scientific evidence to support skin treatment. Therefore, the purpose of this study was to look into the anti-inflammatory effect of an ethanolic extract of RbGD on skin disease, specifically atopic dermatitis (AD). Methods: The anti-inflammatory effect of RbGD ethanol leaf extract (RbGDE) on tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-activated keratinocytes was evaluated using MTT, qPCR, ELISA, and Western blot Procedures. The therapeutic effects of RbGDE were evaluated in vivo inflammatory responses by histological observation, quantitative polymerase chain reaction (qPCR), and ELISA using the 1-chloro-2,4-dinitrobenzene (DNCB)/Dermatophagoides farina extract (DfE)-induced AD-like skin mouse model. Results: RbGDE showed the protective effect against irritating and stimulating substances (H2 O2 and TNF-α/IFN-γ) and inhibited TNF-α/IFN-γ-activated keratinocytes by inhibiting the p38 mitogen-activated protein kinase and nuclear factor-kappa B activation. Furthermore, topical RbGDE treatment reduced the AD features such as thickened skin, erythema, immune cells infiltration (eosinophils and mast cells), and AD-related cytokines (IL-12a, IL-1β, IL-4, and TSLP) in the ear tissues of DNCB/DfE-induced mice. The RbGDE also reduced histamine and immunoglobulins (Igs) levels in the serum, including DfE-specific IgE, total IgE, and IgG2a. Conclusion: RbGD leaf extract had an anti-inflammatory effect on dermatitis by reducing inflammatory mediators, indicating that it might be used to treat skin disease.

The relationship of antibodies detected in the Western Blot test with clinical and immunological stages in HIV-infected patients

Background/Aim: The laboratory diagnosis of human immunodeficiency virus (HIV) infection is based on detection of anti-HIV antibodies in an initial ELISA (enzyme-linked immunosorbent assay) test and its confirmation with the Western Blot (WB) procedure. Even though the specificity of WB is high for the detection of antibodies against various viral proteins, there are important differences in the timing of the appearance of antibody bands and intensities in different stages of HIV infection. The aim of this study was to evaluate antibodies detected in WB testing and the relationship to clinical and immunological stages in HIV-infected patients. Methods: Newly diagnosed 78 patients with HIV/AIDS (Acquired Immunodeficiency Syndrome) in our outpatient department between April 2009 and September 2012 were included in the study as a retrospective cohort. Age, gender, complaints, clinical signs, CD4+ T lymphocyte counts and HIV RNA level at diagnosis were collected retrospectively from medical records. WB band patterns obtained from the reference laboratory of the Istanbul Public Health Center were examined retrospectively. Results: Of the 78 HIV/AIDS cases, 68 (87.2%) were male. Mean age was 38.87(13.09) years (range, 17-83 years). Median CD4+ T lymphocyte count at diagnosis was 410.2 /mm³ (range, 3-1114). Mean HIV RNA level at diagnosis was 592.894 copies/ml. Rare band profiles were seen in 29.4% (23/78). According to World Health Organization (WHO) clinical staging, 59 (75.6%) patients were at stage I, 4 at stage II, 10 at stage III and 5 at stage IV. Gp120, gp160 and gp41, known as envelope glycoproteins in WB band antibodies, were seen in all patients. There was determined to be a decrease in the p17, p51, p55 and p39 bands in WB tests of the advanced grade (Grade IV) of HIV infection. Conclusions: Reduction of p17, p51, p55, p39 antibodies in advanced stages were related with the progression of HIV infection. This shows that WB test is an important parameter not only in the diagnosis of HIV infection, but also in the follow-up of clinical progression in the absence of these antibodies.

Abundance of estrogen receptors involved in non-canonical signaling in the dog testis

With estrogen regulation of the reproductive system, G-protein-coupled membrane estrogen receptor (GPER) and estrogen-related receptors (ERRs) are implicated. Non-canonical receptors can bind estrogens such as environmental and pharmacological chemicals. These compounds induce rapid non-genomic pathways or receptor interaction including autoactivation. Testicular tumors occur in dogs more frequently than in other domestic animals. Also, in recent decades there were increased occurrences of various tumor types in dogs. Using qRT-PCR, Western blot and immunohistochemistry procedures in the present study, there was determination of abundance pattern of GPER, ERRα, β and γ in dog tests when there were intratubular germ cell tumors. There was quantitation of estradiol, cyclic GMP and calcium ions (Ca2+). There were changes (P < 0.01; P < 0.001) in GPER, ERRα and β in both mRNA transcript and protein abundances including less (P < 0.001) co-abundance of ERRγ mRNA transcript and protein. Receptors were mainly located in Leydig cells with there being receptor delocalization to the cell cytoplasm or occasionally detections in the seminiferous tubule epithelia, especially of testicular tumor tissues. There were also greater estradiol (P < 0.05) and lesser cGMP and Ca2+ concentrations in testicular tumor tissues indicating there was a disrupted sex steroid milieu and tumor cell metastasis. Results from the present study provide further evidence that ERRγ has marked actions in testicular germ cell tumor initiation and development and in further structural-functional disruptions of dog testis. Concomitantly, abundance pattern of GPER and ERRs, relative to concentrations of cGMP and Ca2+, may be an additional indicator of intratubular germ cell tumors in dogs.

Lower Formalin-Induced Pain Responses in Painless Diabetic Neuropathy Rat Correlate with the Reduced Spinal Cord NR2B Subunit of N-Methyl-D-Aspartate Receptor Activation.

Diabetic neuropathy (DN) is a late complication of diabetic mellitus and may rise into painful and painless variants. Limited studies have looked at nociceptive mechanisms of painless DN variant. The study aimed to determine phosphorylation and total NR2B subunit of N-methyl-D-aspartate receptor in the spinal cord of painless DN rat during early phase following formalin injection. Thirty-six Sprague-Dawley male rats were randomly assigned into three groups: control, painful, and painless DN (n = 12). The rats were developed into the early phase of DN for 2weeks following diabetic induction. Two weeks later, the rats were injected with 5% formalin solution and flinching and licking responses were recorded for 60min. The rats were sacrificed 3days later, and the spinal cord enlargement region was collected. Immunohistochemistry and Western blot procedures were conducted to determine the phosphorylated and total NR2B subunit expressions. The results showed reduced flinching and licking responses in painless DN rats compared to control and painful DN groups, followed by a significant reduction in phosphorylated and total NR2B expression at both ipsilateral and contralateral regions of the spinal cord. In conclusion, reduced pain behavior responses in painless DN rats following formalin injection is possibly contributed by the reduced expression of phosphorylated and total NR2B subunit in the spinal cord.

PIA and rSesC Mixture Arisen Antibodies Could Inhibit the Biofilm-Formation in Staphylococcus aureus.

Staphylococcus aureus as a causative agent of hospital-acquired infections has been considered as the primary concern in biomaterial-related infections (BAIs). Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S. epidermidis surface-exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm-forming S. aureus strains was assessed by the semi-quantitative micro-plate procedure. Data showed that the total IgGs were boosted in mice immunized sera. By performing an inhibition assay, the biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing individual antigens in a biofilm inhibition assay. immunization of mice with mentioned antigens especially a mixture of them, could eliminate the biofilm formation process in S. aureus. Hopefully, this study corresponds to the suggestion that the immunization of mice with PIA and rSesC candidate vaccines could protect against S. aureus infection.

Effects of electromagnetic field (EMF) radiation on androgen synthesis and release from the pig endometrium during the fetal peri-implantation period

An electromagnetic field (EMF) may have effects on female reproduction. This study was conducted to determine whether EMF [50 and 120 Hz, 2 and 4 h of incubation in the presence or absence of progesterone (P4, 10−5 M)] affects androgen synthesis and release from the pig endometrium. Endometrial slices were collected from pigs (n = 5) during the fetal peri-implantation period (i.e., days 15–16 of gestation) and treated in vitro with EMF. The selected endometrial slices were treated with P4 to determine whether this hormone has effects on protection of the tissue from EMF radiation. The CYP17A1 and HSD3B1 mRNA transcript abundance, steroid 17αhydroxylase/17, 20-lyase (cytochrome P450c17) and hydroxyΔ5steroid dehydrogenase/3β and steroidΔisomerase (3βHSD) protein abundance were examined using Real-Time PCR and Western Blot procedures, respectively. In media collected after incubation, the concentrations of androstenedione (A4) and testosterone (T) were quantified used a RIA. When P4 was added to the culture medium, EMF radiation had suppressive effects on endometrial T release after 2 and 4 h of incubation when the EMF treatment was occurring and increased A4 release after 4 h of incubation with EMF at 120 Hz. When there was no inclusion of P4, release of A4 was decreased after 2 h of EMF treatment at 120 Hz and after 4 h of EMF treatment at 50 and 120 Hz. Progesterone did not have functions that protected the pig endometrium against EMF radiation during the fetal peri-implantation period.

Evaluating the Natrium Iodide Symporter Expressions in Thyroid Tumors

BACKGROUND: Decreased Natrium iodide symporter (NIS) expression levels or diminished NIS targeting thyroid cancer cells’ plasma membrane leads to radioiodine-refractory disease.&#x0D; AIM: The aim of this study was to analyze the NIS expression in thyroid tumors.&#x0D; MATERIALS AND METHODS: The samples were thyroid tissues of patients who underwent surgery for a thyroid tumor. The tissues were processed for NIS protein expressions by immunohistochemistry (IHC) and Western blot (WB). Graves’ disease samples were used as positive controls. The samples were incubated without the primary antibody, and they were used as negative controls for IHC examination. Na+/K+ ATPase was a plasma membrane protein marker in the WB procedure.&#x0D; RESULTS: Twenty-nine samples were assessed for NIS protein. All of them showed the expression in the cytoplasm with intensity 1+ to 3+ with Allred score 3-8. Fourteen out of 29 cases (48.2%) showed NIS cytoplasm staining intensity ≥2+ consist of 10 papillary thyroid cancer (PTC), three follicular thyroid cancer, and one adenoma. Membrane staining was found in 2 samples of PTC (6.9%). Six samples (adenoma 1 sample, PTC 5 samples) showed NIS expression at membrane very weak (1+); they were considered as negative. NIS protein has several bands of ~ 80 kDa, ~ 62 kDa, and ~ 49 kDa.&#x0D; CONCLUSION: NIS expression in thyroid cancer mostly expresses in the cytoplasm instead of the membrane. NIS will play a functional role in the membrane to bring iodine across the membrane against the concentration. It can be the main reason for the lack of response of radioiodine in some differentiated thyroid cancers.

Western Blotting of Membrane-Bound Proteins in Yarrowia lipolytica.

With the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane. Once immobilized on these membranes, the protein of interest can be detected and visualized by exploiting antigen-antibody interactions. However, not all proteins are amenable to easy detection by Western blotting. Integral membrane proteins are a class of proteins that are attached to a biological membrane through a series of transmembrane segments that span the width of the membrane. Due to the inherent hydrophobicity of these proteins and their tendency to aggregate, the characterization and detection of these proteins can be challenging. In this methods chapter, we present a protocol for the easy detection and quantification of these proteins in the industrially important oleaginous yeast Yarrowia lipolytica. The first protocol describes a Western blotting procedure to quantify soluble cytosolic proteins of interest in Yarrowia lipolytica from its total cell lysate. The second protocol describes modifications to the first that are done to enhance detection and quantification of membrane-bound proteins in Yarrowia lipolytica from its total cell lysate, without the need for isolating the membrane-bound proteins, for use in Western blotting. The immunoblotting strategies described here should serve as an efficient and simple guide to quantify both cytosolic and the intractable membrane-bound proteins in Yarrowia lipolytica.

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

Effects on the Post-translational Modification of H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 Levels in Cerebral Cortex, Hypothalamus and Pons of Rats after a Systemic Administration of Cannabidiol: A Preliminary Study.

Cannabidiol (CBD), a non-psychotropic constituent of Cannabis sativa, has shown therapeutic promises by modulating several pathological conditions, including pain, epilepsy autism, among others. However, the molecular mechanism of action of CBD remains unknown and recent data suggest the engagement on CBD´s effects of nuclear elements, such as histone activity. This study assessed the changes in the post-translational modification (PTM) on the histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 in several brain regions of rats after the administration of CBD (20 mg/Kg/i.p.). To evaluate the effects on the PTM of histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 levels in the cerebral cortex, hypothalamus and pons of CBD-treated rats. Ten adult rats were randomly assigned into 2 groups: Control or CBD (20 mg/Kg/i.p). Animals were sacrificed after treatments and brains were collected for dissections of the cerebral cortex, hypothalamus and pons. Samples were analyzed for PTM on the histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 levels by Western blot procedure. CBD increased the PTM levels on the histones H3K4Me3, H3K9ac, and H3K27Me3 in the cerebral cortex whereas no significant differences were found in H3K9Me2 and H3K36Me2. In addition, in the hypothalamus, CBD decreased the contents of H3K9ac while no significant effects were observed in H3K4Me3, H3K9Me2, H3K27Me3, and H3K36Me2. Lastly, in the pons, CBD- treated rats showed a significant decline on the PTM levels of H3K4Me3 whereas no statistical differences were found in H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2. The study showed that CBD induced differential effects in levels of PTM on the histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 in several brain regions.

Oxytocin receptor expression in stallion testes and epididymides

Endocrine, paracrine, and autocrine factors orchestrate the development and physiology of the stallion reproductive system. Oxytocin (OXT) is one of the critical endocrine, paracrine, and autocrine factors for the male reproductive system. Previous studies have investigated OXT receptor (OXTR) expression in testes and epididymides, including humans, marmosets, macaques, swine, and sheep. This study aimed to explore (1) OXTR localization in the testes and epididymides and (2) the seasonal modification of OXTR expression in the testes. Adult stallion testis and epididymis samples were prepared using routine castration procedures. Reverse-transcription PCR was performed to detect the presence of OXTR messenger RNA (mRNA) in the testes. Western blot procedure was performed to confirm the cross-reactivity of OXTR antibody to horse OXTR. Immunohistochemistry was performed to detect OXTR protein expression in the testes and epididymides. Oxytocin receptor mRNA was detected in the stallion testes. The OXTR protein band was observed at 55 kDa. Interestingly, the relative intensity of the OXTR protein band varied between nonbreeding and breeding season. The OXTR protein level in the testes collected during the breeding season was higher than that during the nonbreeding season. Oxytocin receptor localization was observed in the cytoplasm of Type A spermatogonia and spermatid. Oxytocin receptor protein expression was also observed in the cytoplasmic area of Leydig cells and the membrane of the seminiferous tubules. The cytoplasm of principal and basal cells in the caput, corpus, and cauda was also immunolabeled with OXTR antibody. In conclusion, based on the expression of OXTR in tissues of testes and epididymides, OXT–OXTR system may be a critical factor for stallion testicular and epididymal function. In addition, according to the seasonal alteration of intensity, the OXT–OXTR system may be associated with seasonal changes in the reproductive system in stallions.

Is The Hexosamine Biosynthesis Pathway Affected By Inflammation In Endothelial Cells?

Excessive addition of β-linked N-acetylglucosamine (O-GlcNAc) via the hexosamine biosynthesis pathway (HBP) occurs with diabetes and decreases in muscles with exercise. HBP flux increases inflammation and may be specifically important in the activation phase of the inflammatory response. Inflammation plays a key role in insulin resistance and hyperglycemia (HG), with the process contributing to the development of diabetes and associated complications. β-cell development and differentiation are affected by the HBP, and O-GlcNAc has been indicated in insulin secretion from the pancreas (1). PURPOSE: We explored whether in the absence of HG, an inflammatory insult, can affect key factors in this pathway and total O-GlcNAcylation within the endothelium. Because African Americans are overrepresented in conditions involving chronic inflammation, potential racial differences were also examined. METHODS: Human umbilical vein endothelial cells (HUVECs), n=6, were stimulated with 10ng/mL of tumor necrosis factor alpha (TNFα) for 4 hours, or treated as a control. Western blot procedures were used to measure expression of the HBP rate-limiting enzyme (GFAT) and the transferase that adds the end-product onto proteins (OGT). Total protein O-GlcNAcylation (O-GlcNAc) was also measured. RESULTS: With TNFα stimulation, there was no difference in GFAT (0.63 + .09 vs 0.62 + .08, p=.89), OGT (0.49 + .09 vs 0.41 + .08, p=.56), nor total O-GlcNAc (1.56 + .38 vs 1.77 + .42, p=.71) expression compared to controls. There were no racial differences found among the various conditions (p>.05) with either of the proteins examined. CONCLUSIONS: The HBP is not altered with acute inflammation in endothelial cells (ECs), with no racial differences additionally found. Low-grade inflammation vitally contributes to diabetes, atherosclerosis, and their complications, with the HBP playing a role in each. We conclude that HG may be necessary to work in conjunction with inflammation to increase protein O-GlcNAcylation in ECs. Although increased flux though the HBP has been shown to upregulate inflammation, the reverse relationship cannot be confirmed. Future research is needed to examine links between this pathway, various conditions, and the ability of exercise to alter it within the endothelium.

Serotonin transporter protein in autopsied brain of chronic users of cocaine.

The long-held speculation that the brain serotonin system mediates some behavioral effects of the psychostimulant cocaine is supported in part by the high affinity of cocaine for the serotonin transporter (SERT) and by reports that the serotonin transporter (SERT), estimated by SERT binding, is increased in brain of human chronic cocaine users. Excessive SERT activity and consequent synaptic serotonin deficiency might cause a behavioral (e.g., mood) abnormality in chronic users of the drug. Previous studies focused on changes in SERT binding, which might not necessarily reflect changes in SERT protein. Therefore, we compared levels of SERT protein, using a quantitative Western blot procedure, in autopsied brain (striatum, cerebral cortices) of chronic human cocaine users (n = 9), who all tested positive for the drug/metabolite in brain, to those in control subjects (n = 15) and, as a separate drug of abuse group, in chronic heroin users (n = 11). We found no significant difference in protein levels of SERT or the serotonin synthesizing enzyme tryptophan hydroxylase-2 among the control and drug abuse groups. In the cocaine users, no significant correlations were observed between SERT and brain levels of cocaine plus metabolites, or with levels of serotonin or its metabolite 5-hydroxyindoleacetic acid. Our postmortem data suggest that a robust increase in striatal/cerebral cortical SERT protein is not a common characteristic of chronic, human cocaine users.

Cortisol inhibits the Escherichia coli-induced endometrial inflammatory response through NF-κB and MAPK pathways in postpartum goats

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Proteomic characterization of arsenic and cadmium exposure in bladder cells.

Accumulating evidence has linked prolonged exposure to heavy metals to cancer occurrence in the urinary system. However, the specific biological mechanisms responsible for the association of heavy metals with the unusually high incidence of upper tract urothelial carcinoma in Taiwan are complex and incompletely understood. To elucidate the specific biological mechanism and identify molecular indicators of the unusually high association of upper tract urothelial carcinoma with heavy metal exposure, protein expression following the treatment of T24 human bladder carcinoma and RT4 human bladder papilloma cell line models with arsenic (As) and cadmium (Cd) was studied. Proteomic changes in these cell models were integrated with data from a human bladder cancer (BLCA) tissue proteome to identify possible protein indicators of heavy metal exposure. After mass spectrometry based proteomic analysis and verification by Western blotting procedures, we identified 66 proteins that were up-regulated and 92 proteins that were down-regulated in RT4 cell extracts after treatment with As or Cd. Some 52 proteins were up-regulated and 136 proteins were down-regulated in T24 cell extracts after treatment with Cd. We further confirmed that down-expression of the PML (promyelocytic leukemia) protein was sustained for at least 75 days after exposure of bladder cells to As. Dysregulation of these cellular proteins by As was associated with three biological pathways. Immunohistochemical analyses of paraffin-embedded BLCA tissue slides confirmed that PML protein expression was decreased in BLCA tumor cells compared with adjacent noncancerous epithelial cells. These data suggest that PML may play an important role in the pathogenesis of BLCA and may be an indicator of heavy metal exposure in bladder cells.

Autophagy-Inducing Effect of Compound Berberine on CNE2 NPC Cells via Interference with the Targets in P13K/AKT/mTOR Signaling Pathway

Objective: To investigate the autophagy-inducing effect of Compound Berberine (CBBR) on CNE2 nasopharyngeal carcinoma (NPC) cells and its possible targets in P13K/AKT/mTOR signaling pathway. Methods: CNE2 cells at exponential growth phase were taken as the target cells in this study. Firstly, IC50 concentration for CBBR was determined by MTT assay. Then, 3 different concentrations of CBBR, 0.25 mg⋅mL−1, 0.50 mg⋅mL−1 and 1.00 mg⋅mL−1, around the concentration of IC50, were taken for followed intervention experiments respectively. Fluorescein labeling method was utilized to assay the inducing effect of CBBR on the autophagic activity of CNE2 cells, followed by Western blot procedure to explore the changes of key messenger molecules in the autophagy-related signaling pathway of P13K/AKT/mTOR, both combined with 3-MA block test in a comparative way and carried out by detecting the expressive levels of Beclin 1, LC3-II and LC3-I as well as the ratio of LC3-II:LC3-I. Results: IC50 of CBBR was determined at the level of 0.5 mg⋅mL−1. The inducing effect of CBBR on autophagy of CNE2 cells was shown occurring in various modes, not a simple concentration-dependent tendency, with its effect minimal at the concentration of 0.25 mg⋅mL−1 and maximal at the concentration of 0.50 mg⋅mL−1, while only slightly higher at the concentration of 1.00 mg⋅mL−1 than that of 0.5 mg⋅mL−1. Although its inducing effect was weakened a little following the pretreatment by 3-MA, the effect combined with CBBR was still significantly higher than that of simply blocked by 3-MA. Moreover, changes in the expressive levels of Beclin1, LC3-II and LC3-I as well as LC3-II:LC3-I all showed a tendency corresponding to the changed autophagic features of CNE2 cells (P < 0.05 or P < 0.01), given more supporting evidences for the effect of CBBR on autophagy of CNE2 cells. Conclusions: CBBR can bring about inhibiting effect on the proliferating activity of CNE2 cells through inducing increased autophagic activity via intervening targets in P13K/AKT/mTOR signaling pathway, and this effect could not be completely blocked by the antagonist 3-MA.