Semi-quantitative Western Blot Analysis Research Articles

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance.

The incidence of infertility is significantly higher in women with diseases linked to impaired glucose homeostasis, such as insulin resistance. Defective glucose metabolism interferes with fertilization; however, the molecular mechanism underlying this interference is unclear. Smoothelin-like protein 1 (SMTNL1) was isolated from muscle and steroid hormone-responsive tissues and regulates the contractile functions of various cell types through the inhibition of myosin phosphatase (MP) holoenzyme. In addition, SMTNL-1 after phosphorylation at Ser301 by protein kinase A translocates to the nucleus and functions as a transcriptional co-activator of the progesterone receptor-B. SMTNL1 null mice exhibit reduced reproductive fitness and are more prone to type 2 diabetes mellitus. However, the role of SMTNL1 in endometrial epithelial cells is not known. The effect of SMTNL1 overexpression was investigated in pregnancy and in gestational diabetic endometrial epithelial cell models by immunofluorescent staining, cell migration, and semi quantitative Western blot analysis and glucose uptake assay. We show that SMTNL1 promotes the differentiation of endometrial epithelial cells in a progesterone-dependent manner to attenuate insulin resistance. Furthermore, SMTNL1 hampers the migration capacity of epithelial cells in a gestational diabetes model by inhibiting the expression of MYPT1, the regulatory subunit of MP, and the activity of the holoenzyme, resulting in increased phosphorylation of the 20 kDa regulatory myosin light chain. SMTNL1 also acts as an insulin-sensitizing agent by increasing the gene expression of PP2A and DUPS9 protein phosphatases, resulting in decreased ERK1/2 activity and, hence, decreasing the phosphorylation of IRS-1 at Ser612 under gestational diabetes conditions. SMTNL1 may have therapeutic relevance to the progesterone-dependent inhibition of endometrial epithelial cell migration under hyperglycemic conditions and insulin sensitivity in the endometrium in gestational diabetes or other metabolic disorders.

High-intensity green light potentially activates the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2).

The development of practices that enhance the potential of actinomycetes as major antibiotic producers is a challenge in discovering new secondary metabolites. Light, an essential external stimulus for most microorganisms, could be exploited to manipulate their physiological processes. However, the effects of monochromatic green light on the production of secondary metabolites in actinomycetes have not yet been reported. In this paper, we report a novel and simple method that uses high-intensity monochromatic green light to potentially induce the production of cryptic secondary metabolites in the model actinomycete Streptomyces coelicolor A3(2). Using actinorhodin (ACT), a blue-pigmented antibiotic, and undecylprodigiosin (RED), a red-pigmented antibiotic, as indicators, we found that irradiation with high-intensity monochromatic green light-emitting diodes promoted sporulation, significantly decreased RED production, and increased ACT production. Semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses revealed, for the first time, that stimulation with green light accelerated the expression of ActII-ORF4, a pathway-specific regulator of ACT biosynthesis in S. coelicolor A3(2). This approach of stimulating secondary metabolite biosynthesis pathways in actinomycetes by irradiation with high-intensity monochromatic green light is expected to facilitate the discovery of cryptic antibiotics that are not typically produced under conventional dark culture conditions. However, the effective intensity and duration of irradiation with green light that are required to activate these metabolite pathways may vary markedly among actinomycetes.

Developmental Inhibitory Changes in the Primary Somatosensory Cortex of the Stargazer Mouse Model of Absence Epilepsy.

Childhood absence epilepsy seizures arise in the cortico-thalamocortical network due to multiple cellular and molecular mechanisms, which are still under investigation. Understanding the precise mechanisms is imperative given that treatment fails in ~30% of patients while adverse neurological sequelae remain common. Impaired GABAergic neurotransmission is commonly reported in research models investigating these mechanisms. Recently, we reported a region-specific reduction in the whole-tissue and synaptic GABAA receptor (GABAAR) α1 subunit and an increase in whole-tissue GAD65 in the primary somatosensory cortex (SoCx) of the adult epileptic stargazer mouse compared with its non-epileptic (NE) littermate. The current study investigated whether these changes occurred prior to the onset of seizures on postnatal days (PN) 17-18, suggesting a causative role. Synaptic and cytosolic fractions were biochemically isolated from primary SoCx lysates followed by semiquantitative Western blot analyses for GABAAR α1 and GAD65. We found no significant changes in synaptic GABAAR α1 and cytosolic GAD65 in the primary SoCx of the stargazer mice at the critical developmental stages of PN 7-9, 13-15, and 17-18. This indicates that altered levels of GABAAR α1 and GAD65 in adult mice do not directly contribute to the initial onset of absence seizures but are a later consequence of seizure activity.

Targeting Artemisinin-Resistant Malaria by Repurposing the Anti-Hepatitis C Virus Drug Alisporivir.

The emergence of Plasmodium falciparum resistance raises an urgent need to find new antimalarial drugs. Here, we report the rational repurposing of the anti-hepatitis C virus drug, alisporivir, a nonimmunosuppressive analog of cyclosporin A, against artemisinin-resistant strains of P. falciparum. In silico docking studies and molecular dynamic simulation predicted strong interaction of alisporivir with PfCyclophilin 19B, confirmed through biophysical assays with a Kd value of 354.3 nM. Alisporivir showed potent antimalarial activity against chloroquine-resistant (PfRKL-9 with resistance index [Ri] 2.14 ± 0.23) and artemisinin-resistant (PfKelch13R539T with Ri 1.15 ± 0.04) parasites. The Ri is defined as the ratio between the IC50 values of the resistant line to that of the sensitive line. To further investigate the mechanism involved, we analyzed the expression level of PfCyclophilin 19B in artemisinin-resistant P. falciparum (PfKelch13R539T). Semiquantitative real-time transcript, Western blot, and immunofluorescence analyses confirmed the overexpression of PfCyclophilin 19B in PfKelch13R539T. A 50% inhibitory concentration in the nanomolar range, together with the targeting of PfCyclophilin 19B, suggests that alisporivir can be used in combination with artemisinin. Since artemisinin resistance slows the clearance of ring-stage parasites, we performed a ring survival assay on artemisinin-resistant strain PfKelch13R539T and found significant decrease in parasite survival with alisporivir. Alisporivir was found to act synergistically with dihydroartemisinin and increase its efficacy. Furthermore, alisporivir exhibited antimalarial activity in vivo. Altogether, with the rational target-based Repurposing of alisporivir against malaria, our results support the hypothesis that targeting resistance mechanisms is a viable approach toward dealing with drug-resistant parasite.

Target Mechanisms of the Cyanotoxin Cylindrospermopsin in Immortalized Human Airway Epithelial Cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs in aquatic environments worldwide. It is known for its delayed effects in animals and humans such as inhibition of protein synthesis or genotoxicity. The molecular targets and the cell physiological mechanisms of CYN, however, are not well studied. As inhalation of CYN-containing aerosols has been identified as a relevant route of CYN uptake, we analyzed the effects of CYN on protein expression in cultures of immortalized human bronchial epithelial cells (16HBE14o-) using a proteomic approach. Proteins whose expression levels were affected by CYN belonged to several functional clusters, mainly regulation of protein stability, cellular adhesion and integration in the extracellular matrix, cell proliferation, cell cycle regulation, and completion of cytokinesis. With a few exceptions of upregulated proteins (e.g., ITI inhibitor of serine endopeptidases and mRNA stabilizer PABPC1), CYN mediated the downregulation of many proteins. Among these, centrosomal protein 55 (CEP55) and osteonectin (SPARC) were significantly reduced in their abundance. Results of the detailed semi-quantitative Western blot analyses of SPARC, claudin-6, and CEP55 supported the findings from the proteomic study that epithelial cell adhesion, attenuation of cell proliferation, delayed completion of mitosis, as well as induction of genomic instability are major effects of CYN in eukaryotic cells.

In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer‐related death worldwide. Alterations in proteins of the p53‐family are a common event in CRC. ΔNp73, a p53‐family member, shows oncogenic properties and its effectors are largely unknown. We performed an in‐depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high‐density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot‐blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy‐negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5‐foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain‐derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex‐interacting multifunctional protein 1 (EMAP‐II)–vascular endothelial growth factor C–vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity.

Personalized Disease Monitoring in Pediatric Onset Multiple Sclerosis Using the Saliva Free Light Chain Test.

BackgroundDevelopment of new safe methods of monitoring disease activity in the pediatric onset multiple sclerosis (POMS) is a challenging task, especially when trying to refrain from frequent MRI usage. In our recent study, the saliva immunoglobulin free light chains (FLC) were suggested as biomarkers to discriminate between remission and active MS in adults.ObjectivesTo assess utility of saliva FLC measurements for monitoring disease activity in POMS.MethodsWe used semiquantitative Western blot analysis to detect immunoreactive FLC monomers and dimers and to calculate the intensity of their bands. Statistical tests included Firth logistic regression analysis suitable for small sample sizes, and Spearman’s non-parametric correlation.ResultsIn naive POMS patients, the saliva levels of FLC in relapse were significantly higher than those in remission. Significant correlation was found between FLC levels (monomers, dimers or both) and the load of enhanced lesions in MRI scans. FLC levels may be reduced under treatment, especially as result of corticosteroids therapy. Follow-up of individual patients showed the correspondence of changes in the FLC levels to MRI findings.ConclusionsOur results show the potential of the non-invasive saliva FLC test, as a new tool for monitoring the disease activity in POMS.

Smoothelin-Like Protein 1 Regulates the Thyroid Hormone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase.

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

Connexin43 in Germ Cells Seems to Be Dispensable for Murine Spermatogenesis.

Testicular Connexin43 (Cx43) connects adjacent Sertoli cells (SC) and SC to germ cells (GC) in the seminiferous epithelium and plays a crucial role in spermatogenesis. However, the distinction whether this results from impaired inter-SC communication or between GC and SC is not possible, so far. Thus, the question arises, whether a GC-specific Cx43 KO has similar effects on spermatogenesis as it is general or SC-specific KO. Using the Cre/loxP recombinase system, two conditional KO mouse lines lacking Cx43 in premeiotic (pGCCx43KO) or meiotic GC (mGCCx43KO) were generated. It was demonstrated by qRT-PCR that Cx43 mRNA was significantly decreased in adult pGCCx43KO mice, while it was also reduced in mGCCx43KO mice, yet not statistically significant. Body and testis weights, testicular histology, tubular diameter, numbers of intratubular cells and Cx43 protein synthesis and localization did not show any significant differences in semi-quantitative Western blot analysis and immunohistochemistry comparing adult male KO and WT mice of both mouse lines. Male KO mice were fertile. These results indicate that Cx43 in spermatogonia/spermatids does not seem to be essential for successful termination of spermatogenesis and fertility as it is known for Cx43 in somatic SC, but SC-GC communication might rather occur via heterotypic GJ channels.

Korean Red Ginseng exerts anti-inflammatory and autophagy-promoting activities in aged mice

BackgroundKorean Red Ginseng (KRG) is a traditional herb that has several beneficial properties including anti-aging, anti-inflammatory, and autophagy regulatory effects. However, the mechanisms of these effects are not well understood. In this report, the underlying mechanisms of anti-inflammatory and autophagy-promoting effects were investigated in aged mice treated with KRG-water extract (WE) over a long period. MethodsThe mechanisms of anti-inflammatory and autophagy-promoting activities of KRG-WE were evaluated in kidney, lung, liver, stomach, and colon of aged mice using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and western blot analysis. ResultsKRG-WE significantly suppressed the mRNA expression levels of inflammation-related genes such as interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 in kidney, lung, liver, stomach, and colon of the aged mice. Furthermore, KRG-WE downregulated the expression of transcription factors and their protein levels associated with inflammation in lung and kidney of aged mice. KRG-WE also increased the expression of autophagy-related genes and their protein levels in colon, liver, and stomach. ConclusionThe results suggest that KRG can suppress inflammatory responses and recover autophagy activity in aged mice.

The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice

PurposeCerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. MethodsIschemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood–brain barrier function on day 1 after tMCAO. ResultsAfter tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton’s Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood–brain barrier integrity. ConclusionsOur data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood–brain barrier.

H2S-producing enzymes in anoxia-tolerant vertebrates: Effects of cold acclimation, anoxia exposure and reoxygenation on gene and protein expression.

To lend insight into the potential role of the gasotransmitter hydrogen sulfide (H2S) in facilitating anoxia survival of anoxia-tolerant vertebrates, we quantified the gene expression of the primary H2S-synthesizing enzymes, 3-mercaptopyruvate sulfurtransferase (3MST), cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), in ventricle and brain of normoxic, anoxic and reoxygenated 21°C- and 5°C-acclimated freshwater turtles (Trachemys scripta) and 10°C-acclimated crucian carp (Carassius carassius). Semi-quantitative Western blotting analysis was also conducted to assess 3MST and CBS protein abundance in ventricle and brain of 5°C turtles and 10°C crucian carp subjected to normoxia, anoxia and reoxygenation. We hypothesized that if H2S was advantageous for anoxia survival, expression levels would remain unchanged or be upregulated with anoxia and/or reoxygenation. Indeed, for both species, gene and protein expression were largely maintained with anoxia exposure (24h, 21°C; 5 d, 10°C; 14 d, 5°C). With reoxygenation, 3MST expression was increased in turtle and crucian carp brain at the protein and gene level, respectively. Additionally, the effect of cold acclimation on gene expression was assessed in several tissues of the turtle. Expression levels were maintained in most tissues, but decreased in others. The maintenance of gene and protein expression of the H2S-producing enzymes with anoxia exposure and the up-regulation of 3MST with reoxygenation suggests that H2S may facilitate anoxic survival of the two champions of vertebrate anoxia survival. The differential effects of cold acclimation on H2S enzyme expression may influence blood flow to different tissues during winter anoxia.

Regulation of epithelial mesenchymal transition by the renin-angiotensin system: a role for klotho in renal tubular epithelial cells.

Klotho is a putative aging suppressor gene that is primarily expressed in renal tubular epithelial cells. Its expression has been reported to protect against fibrosis in human chronic kidney disease. However, the roles of klotho in epithelial-mesenchymal transition (EMT) and renal fibrosis are yet to be elucidated. The present study aimed to investigate the putative roles of klotho in angiotensin (Ang) II-induced damage of renal tubular epithelial cells. NRK-52E rat cells were treated with various combinations of Ang II, the Ang-converting enzyme inhibitor fosinopril (Fos) and the Ang II receptor antagonist valsartan (Val). The levels of transforming growth factor (TGF)-β1, soluble klotho, α-smooth muscle actin (α-SMA) and E-cadherin in NRK-52E culture supernatants were measured using enzyme-linked immunosorbent assays. Furthermore, the mRNA and protein expression of TGF-β1, klotho, α-SMA and E-cadherin was detected using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemistry and Western blot analysis. The results demonstrated that Ang II inhibited the expression of klotho and E-cadherin, while it upregulated the expression of TGF-β1 and α-SMA, in NRK52E cells. Fos and/or Val were revealed to enhance klotho and E-cadherin expression, and suppress the expression of TGF-β1 and α-SMA, compared with the Ang II-only group. Furthermore, a positive linear correlation was detected between the expression of klotho and E-cadherin, while negative linear correlations with klotho expression were detected for TGF-β1 and α-SMA expression. In conclusion, the expression of klotho was demonstrated to be enhanced following treatment with Fos and Val in Ang II-treated NRK-52E cells. The present results indicate that klotho may be involved in the inhibition of Ang II-induced EMT in renal tubular epithelial cells. Therefore, klotho may serve as a protective factor in renal tubulointerstitial fibrosis and aid the treatment of chronic kidney disease (CKD) patients using precision therapy.

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco.

Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin‐binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD‐DrsB1 and pGSA/DrsB1‐CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi‐quantitative RT‐PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD‐DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1‐CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

Eukaryotic translation initiation factor 5A2 is highly expressed in prostate cancer and predicts poor prognosis.

Eukaryotic translation initiation factor (EIF) 5A2 exerts important functions that regulate the development and progression of cancers. The present study aimed to investigate the expression of EIF5A2 in prostate cancer (PCa) and its association with biological and prognostic significance. EIF5A2 mRNA and protein levels were analyzed in three paired samples of freshly resected PCa and adjacent non-tumor tissues. Immunohistochemical staining was used to detect the expression of EIF5A2 protein levels in 72 paraffin-embedded PCa tumor specimens. Subsequently, the association between EIF5A2 protein expression and clinicopathological parameters was assessed. Semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses showed both EIF5A2 mRNA and protein levels were elevated in PCa compared with adjacent non-tumor tissues. Elevated EIF5A2 protein levels were observed in 73.6% (53/72) of the clinical PCa tissues using immunohistochemical staining. EIF5A2 expression was significantly associated with tumor stage (P=0.011) and biochemical recurrence status (P=0.032). Additionally, high levels of EIF5A2 predicted worse progression-free survival (P=0.007). Multivariate Cox regression analysis indicated that high expression of EIF5A2 was an independent prognostic factor for poor progression-free survival (hazard ratios, 0.366; 95% confidence interval, 0.349-0.460; P=0.021). The present study demonstrated that EIF5A2 is overexpressed in prostate cancer and may be a potential predictor and therapeutic target in PCa patients.

Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation.

Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-β, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.

EVALUATION OF SIRTUIN 3 BIOMARKER BEFORE AND AFTER EXERCISE REGIMEN IN CHRONIC UNPREDICTABLE MILD STRESS-INDUCED DEPRESSED RATS

Objective: Depression is one of the most common psychiatric illnesses that cause disability and public health problems. Physical exercise has been shown to reduce the stress impairment and ameliorate depressive symptoms. The objectives of the present study were to confirm, whether swimming exercise alleviates the depression-like behavior induced by chronic unpredictable mild stress (CUMS) through the alteration in the expression of sirtuin 3 (sirt 3) in brain.Methods: This study was undertaken to evaluate the possible antidepressant activity of exercise. Female Wistar rats weighing about 120–150 g were divided into four groups of six animals in each: Control, CUMS-induced, venlafaxine-treated, and exercise-treated groups. The rat model of depression was established using CUMS protocol. Behavioral assessment was performed by sucrose consumption test and forced swim test. Semi-quantitative reverse transcription-polymerase chain reaction and Western blot analysis were performed to study the gene expression and the protein activity of SIRT3.Results: Our results showed that the gene expression and protein activity of sirt3 were found to be significantly increased in the exercise-treated group when compared to the depressed group.Conclusion: The interesting fact from this study is that swimming exercise increases sirt3 expression and possesses a beneficial effect in curing depression.

EVALUATION OF SIRTUIN 3 BIOMARKER BEFORE AND AFTER EXERCISE REGIMEN IN CHRONIC UNPREDICTABLE MILD STRESS-INDUCED DEPRESSED RATS

Objective: Depression is one of the most common psychiatric illnesses that cause disability and public health problems. Physical exercise has been shown to reduce the stress impairment and ameliorate depressive symptoms. The objectives of the present study were to confirm, whether swimming exercise alleviates the depression-like behavior induced by chronic unpredictable mild stress (CUMS) through the alteration in the expression of sirtuin 3 (sirt 3) in brain.Methods: This study was undertaken to evaluate the possible antidepressant activity of exercise. Female Wistar rats weighing about 120–150 g were divided into four groups of six animals in each: Control, CUMS-induced, venlafaxine-treated, and exercise-treated groups. The rat model of depression was established using CUMS protocol. Behavioral assessment was performed by sucrose consumption test and forced swim test. Semi-quantitative reverse transcription-polymerase chain reaction and Western blot analysis were performed to study the gene expression and the protein activity of SIRT3.Results: Our results showed that the gene expression and protein activity of sirt3 were found to be significantly increased in the exercise-treated group when compared to the depressed group.Conclusion: The interesting fact from this study is that swimming exercise increases sirt3 expression and possesses a beneficial effect in curing depression.

Protective role of H2S on acute renal damages in urinary-derived sepsis

The aim of this study was to investigate the protective roles of H2S on acute renal damage in urinary-derived sepsis. Totally 40 male rabbits were randomly divided into the Control, Sham, Sepsis, NaHS and the dl-propargylglycine (PAG) groups. White blood cell counts (WBC), C-reactive protein (CRP), creatinine (Cr) and blood urea nitrogen were determined in peripheral blood samples. Hematoxylin and eosin staining was performed to detect the morphological changes. The mRNA and protein expression levels were detected by semi-quantitative reverse transcriptase PCR (sqRT-PCR) and Western blot analysis. The results showed that, 72 h after surgery, the WBC and CRP levels in the Sepsis group were significantly elevated, and the expression levels of inducible nitric oxide synthase (iNOS) and HO-1 were significantly up-regulated, compared with the Control and Sham groups, indicating renal damage induced by urinary-derived sepsis. Compared with the Sepsis group, the expression levels of iNOS in the renal tissues of the PAG group were significantly up-regulated, whereas the expression levels of HO-1 in the renal tissue were significantly down-regulated, indicating aggravated renal damage. On the other hand, compared with the Sepsis group, the expression levels of iNOS in the renal tissue of the NaHS group were declined, whereas the expression levels of HO-1 were elevated, together with the declined WBC and CRP levels, indicating alleviated renal functions. We could conclude that iNOS and HO-1 expression levels are elevated in the renal tissue of urinary-derived sepsis. H2S could exert protective effects on the urinary-derived sepsis-associated renal damages by down-regulating iNOS expression and up-regulating HO-1 expression.

Expression of OsTPX Gene Improves Cellular Redox Homeostasis and Photosynthesis Efficiency in Synechococcus elongatus PCC 7942.

Cyanobacterial 2-Cys peroxiredoxin (thioredoxin peroxidase, TPX) comprises a family of thiol antioxidant enzymes critically involved in cell survival under oxidative stress. In our previous study, a putative TPX was identified using a proteomics analysis of rice (Oryza sativa L. japonica, OsTPX) seedlings exposed to oxidative stress. This OsTPX gene is structurally similar to the Synechococcus elongatus TPX gene in the highly conserved redox-active disulfide bridge (Cys114, Cys236) and other highly conserved regions. In the present study, the OsTPX gene was cloned into rice plants and S. elongatus PCC 7942 strain to study hydrogen peroxide (H2O2) stress responses. The OsTPX gene expression was confirmed using semi-quantitative RT-PCR and western blot analysis. The OsTPX gene expression increased growth under oxidative stress by decreasing reactive oxygen species and malondialdehyde level. Additionally, the OsTPX gene expression in S. elongatus PCC 7942 (OT) strain exhibited a reduced loss of chlorophyll and enhanced photosynthesis efficiency under H2O2 stress, thereby increasing biomass yields twofold compared with that of the control wild type (WT) strain. Furthermore, redox balance, ion homeostasis, molecular chaperone, and photosynthetic systems showed upregulation of some genes in the OT strain than in the WT strain by RNA-Seq analysis. Thus, OsTPX gene expression enhances oxidative stress tolerance by increasing cell defense regulatory networks through the cellular redox homeostasis in the rice plants and S. elongatus PCC 7942.