Nitroblue Tetrazolium Reduction Assay Research Articles

Saikosaponin a contributed to CCIN treatment by promoting neutrophil bactericidal activity via activation CBL-dependent ERK pathway

BackgroundDespite granulocyte colony-stimulating factor (GCSF) is widely used in clinical, cancer chemotherapy induced neutropenia (CCIN) infection and infection-related mortality is high for lack of functionally mature neutrophils. Generating functional neutrophils is new therapeutic approaches to reduce CCIN-associated infection and mortality. Saikosaponin a (SSA) is one of the major bioactive components of Radix Bupleuri (RB) and exerts immunoregulatory effects. PurposeThe present study aims to investigate the efficacy and mechanism of SSA in CCIN therapy. MethodsSSA was applied both in vitro and in vivo to assess the efficacy of CCIN therapy. The differentiation of neutrophils was measured by Nitroblue tetrazolium (NBT) reduction assay and Giemsa staining assay. The neutrophil differentiation related real-time transcription factors were detected by quantitative PCR (RT-qPCR) and Western Blot. Bacteria killing assay was used to assess the ability of fighting infection. Network pharmacology was employed to explore the mechanism network, and the predicted pathways were validated by Western Blot. ResultsWe found that SSA contributed to generate functional mature neutrophils which capable of fighting infection both in vitro and in vivo. Network pharmacology prediction showed 55 pathways were predicted involved in SSA against CCIN. Further validation showed that CBL-ERK1/2 pathway was activated by SSA, which could upregulate PU.1 and CEBPβ expression leading to neutrophil differentiation. ConclusionsOur findings suggest a natural regimen SSA regenerates microbicidal neutrophils to effectively reduce CCIN-associated infection via activating CBL-ERK1/2 pathway, providing a rationale for future therapeutic approaches.

Assessment of immunohematological, hematological and biochemical responses in cultivable fish Cyprinus carpio exposed to an antibiotic sulfamethoxazole (SMX)

Abstract Sulfamethoxazole (SMX) is a member of the sulfonamides group of antibiotics which is used extensively in aquaculture throughout the world. In this study, common carp (Cyprinus carpio) was used as the bioindicator to assess the toxicity potential of SMX. Effects were based on chronic toxicity of environmentally relevant dosages of 25, 50, 100, and 200 μg/L of SMX for 28 days. Cytotoxicity through hematology and biochemistry showed a dose–response relationship. Numerous variations were recorded in blood profile and biochemical parameters in SMX-exposed groups when compared to control. Hemoglobin, platelet, and erythrocyte levels were significantly decreased. Leukocyte level was significantly increased with values ranging from 131 to 303 (×103/μL). Changes in biochemical indices: glucose, total protein, and triglycerides showed biphasic trend, but alanine transaminase secretion was significantly increased from 25.13 to 204 U/L at higher concentration compared to control, suggesting liver damage. Spectrophotometric nitroblue tetrazolium reduction assay showed that respiratory burst activity increased as a function of SMX dose and exposure time (0.48–1.33 absorbance) ultimately leading to reduction in immunity. The present study highlights that prolonged exposure of SMX affects biochemistry, hematology, and immunohematology of fish and these biomarkers act as an effective tool for environmental risk assessment of drugs in the aquatic environment.

Photoperiodic manipulation modulates the innate and cell mediated immune functions in the fresh water snake, Natrix piscator

Objectives of the current work were to investigate the role of photoperiod and melatonin in the alteration of immune responses in a reptilian species. Animals were kept on a regimen of short or long days. Blood was obtained and leucocytes were isolated to study various innate immune responses. Lymphocytes were separated from blood by density gradient centrifugation and were used to study proliferation. Respiratory burst activity was measured through nitrobluetetrazolium reduction assay while nitric oxide production by leucocytes was assayed by nitrite assay. Lymphocytes were isolated and used to study proliferation with and without B and T cell mitogens. Photoperiodic manipulation acted differentially on leucocyte counts. Nitrite release was increased while superoxide production was decreased in cultures obtained from the snakes kept on the short day regimen. Significant enhancement of mitogen induced lymphocyte proliferation was observed in cultures from the animals kept in either long or short days compared to cultures from the animals kept in natural ambient day length. Use of in vitro melatonin showed that lymphocytes from the animals, kept in long days, were more reactive. Photoperiod induces changes in immune status which may permit adaptive functional responses in order to maintain seasonal energetic budgets of the animals. Physiological responses (like elevated immune status) are energetically expensive, therefore, animals have evolved a strategy to reduce immune functions at times when energy is invested in reproductive activities. Natrix piscator breeds from September to December and elevated pineal hormone in winter suppresses reproduction while immunity is stimulated.

Ingestion of organic acids and cinnamaldehyde improves tissue homeostasis of piglets exposed to enterotoxic Escherichia coli (ETEC).

Organic acids (OA) and phytogenic compounds have been used in pig feeding as alternatives to antibiotic growth promoters. However, few studies have evaluated the systemic effect of the combination of these additives. The aim of this study was to assess the impact of an organic acid-based feed additive (OAFA), containing a blend of OA and cinnamaldehyde, on the tissue integrity of bacterially challenged piglets. Thirty weaned piglets 21 d old were used in a 19-d trial. Pigs received a standard diet during the first 7 d and afterward were allotted to five treatments. Dietary treatments were: Control (basal diet), Escherichia coli (basal diet and challenge with E. coli), colistin (basal diet + 200 mg colistin/kg feed + challenge with E. coli), OAFA1 (basal diet + 1 kg OAFA/ton feed + challenge with E. coli), and OAFA2 (basal diet + 2 kg OAFA/ton feed + challenge with E. coli). Seven days after the beginning of the treatment, the animals were challenged with an enterotoxic strain of E. coli (K88) for pigs. Five days after the challenge, all animals were euthanized for tissue sampling for histological and oxidative stress (intestine and liver) analysis. The reduced glutathione (GSH), ferric-reducing ability potential (FRAP), and free-radical scavenging ability (ABTS) assays were used to evaluate the intestinal antioxidant defense. Lipid peroxidation and superoxide anion production were evaluated through the levels of thiobarbituric acid-reactive substances (TBARS) and nitroblue tetrazolium (NBT) reduction assay, respectively. Animals fed the OAFA (1 and 2) diets had a decrease (P < 0.05) on histological changes in the intestine, liver, mesenteric lymph nodes, and spleen. Greater villus height (VH) and a higher ratio of VH to crypt depth (CD) were observed in animals of the OAFA2 group compared with the control and E. coli groups. The colistin and OAFA groups decreased (P < 0.05) the number of inflammatory cells in intestinal lamina propria. OAFA2 group increased (P < 0.05) intestinal cell proliferation. Colistin and OAFA2 supplementation induced a decrease (P < 0.05) in the levels of TBARS in both the intestine and liver compared with the E. coli group. In addition, an increase (P < 0.05) in GSH and FRAP ileal levels was observed in the OAFA2 group compared with E. coli group. These results show that the supplementation with OAFA in the diet of weaned piglets, especially at a dose of 2 kg/ton (OAFA2) protected tissues against enterotoxigenic Escherichia coli (ETEC) damage.

Effect of heat stress on superoxide anion production in native and crossbred cattle under in vitro whole blood culture model.

Impact of global warming on the dairy industry has gained attention due to huge economic losses through low production and fertility caused by heat stress. Exposure to hyperthermia provokes a series of complex responses in mammals which are been related to morphological and physiological alterations including the production of reactive oxygen species (ROS). A quantitative spectrophotometric based nitroblue tetrazolium (NBT) reduction assay was used to estimate the superoxide anion (•O2-) level in heat stressed (at 42°C) whole blood cultures of native and crossbred bulls (Sahiwal and Frieswal), in vitro. The breed effect in the kinetics of •O2- production at different time periods of continual heat stress was analyzed by repeated measures ANOVA. Comparison between different time periods in reference to 37°C was analyzed by paired t-test. The •O2- level was significantly different (p<0.05) between cells at 37°C and 42°C at different periods of incubation. Kinetics study showed increment of •O2- production on the acute phase of stress followed by a reduction in both Sahiwal and Frieswal breeds. In Sahiwal breed, the inflated superoxide level continued abated till 4h and raised again at 6h, while in Frieswal •O2- level reverted to raise sooner with in 2h of incubation itself. Contrarily, kinetic of •O2- level in plasma showed a significant reduction (p<0.001) at 30min of 42°C incubation followed by increment of •O2- level. Further, the breed variation was significant (p<0.05) and a significant high reduction of •O2- level was observed in Sahiwal breed. Our finding indicates that, a better and longer •O2- production homeostasis and higher plasma scavenging ability of native breed may be one of the reasons for the higher thermal tolerance of these breeds in tropical climate.

Evaluation the Ozone Treatment To Control the Infection of Saprolegniasis in Cyprinus carpio L.

The objective of present study is to set up an alternative method of controlling of saprolegniasis, in Cyprinus carpio L. using ozone. For this purpose, six different treatments were applied of 120 fish (20 fish/treatment), of which three infected with Saprolegnia spp. (2×104 zoospors/l) and were treated with ozone (O3) in concentrations of 0.25, 0.50 and 0.75 mg l−1 (1hour), one reference treatment with formalin (0.15 ml l−1, 30 min), two treatments one as positive control (infected with Saprolegnia spp. without disinfectants) and one as negative control without any disinfectants. After 14 days of treatment with ozone and formalin, blood samples were collected from fish for hematological, biochemical and Immunological tests. Survival rate was also studied. Considerable changes have been recorded in the mean values of blood indices. RBCs count and Hemoglobin content, were found significantly increased (p<0.05) in all ozone treatments compared to C- group. Total protein and globulin were significantly increased (p<0.05) in all ozone and formalin treatments in comparison to positive and negative groups (C- and C+). Albumin showed a significant decrease (p<0.05) in all ozone and formalin treatments relative to C+ and C-group. The results of respiratory burst activity (measured by Nitro blue tetrazolium reduction assay) showed significantly different (p<0.05) in all ozone and formalin treatments respectively compared to control groups (C+ and C-); Highest respiratory activity was observed in ozone treatment at 0.50 mg/l. Among Ozone treatments 0.50 mg l−1 showed the highest survival rate (90%) followed by formalin treatment (80%). Survival rate of the control group (without disinfectant) was 20% and there was a significant difference (P<0.05) with other treatments. It was obvious that the obtained results were highly suggestive for the efficiency of ozone as an efficient antifungal disinfectant for fish.

Wood-Based Cellulose Nanofibrils: Haemocompatibility and Impact on the Development and Behaviour of Drosophila melanogaster.

Wood-based cellulose nanofibrils (CNF) offer an excellent scaffold for drug-delivery formulation development. However, toxicity and haemocompatibility of the drug carrier is always an important issue. In this study, toxicity-related issues of CNF were addressed. Different doses of CNF were orally administered to Drosophila and different tests like the developmental cycle, trypan blue exclusion assay, larva crawling assay, thermal sensitivity assay, cold sensitivity assay, larval light preference test, climbing behaviour, nitroblue tetrazolium (NBT) reduction assay, adult phenotype, and adult weight were conducted to observe the impact on its development and behaviour. A haemocompatibility assay was done on the blood taken from healthy Wistar rats. In Drosophila, the abnormalities in larval development and behaviour were observed in the behavioural assays. However, the cytotoxic effect could not be confirmed by the gut staining and level of reactive oxygen species. The larvae developed into an adult without any abnormality in the phenotype. The CNF did cause loss of weight in the adult flies and did not cause much toxicity within the body since there was no phenotypic defect. Hemolysis data also suggested that CNF was safe at lower doses, as the data was well within acceptable limits. All these results suggest that cellulose nanofibres have no significant cytotoxic effects on Drosophila. However, the developmental and behavioural abnormalities suggest that CNF may act as a behavioural teratogen.

Notopterol-induced apoptosis and differentiation in human acute myeloid leukemia HL-60 cells.

Purpose: This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms.Methods: Cell viability was assessed using sulforhodamine B assay. Cell proliferation was determined by the trypan blue dye exclusion test. Colony-forming units were assayed in methylcellulose. Apoptosis assays were carried out by annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) double staining, Hoechst 33342 staining, mitochondrial membrane potential, and Western blot. Wright–Giemsa staining, nitroblue tetrazolium (NBT) reduction assay, CD11b and CD14 and Western blot were detected for induction of differentiation. In addition, cell-cycle phase distribution was analyzed by flow cytometry and Western blot. The combination therapy of notopterol and all-trans retinoic acid (ATRA) on HL-60 cells was examined.Results: Notopterol obviously inhibited the growth of HL-60 cells with an IC50 value of 40.32 μM and remarkably reduced the number of colonies by 10, 20, and 40 µM. In addtion, notopterol induced the percentage of apoptotic HL-60 cells, reduced the mitochondrial membrane potential, decreased the protein expresstion of Bcl-2 and Mcl-1, and increased the expression of Bax, cleavage of caspase 9, caspase 3, and PARP. As for cell differentiation, notopterol clearly induced chromatin condensation; increased the nucleocytoplasmic ratio, nitroblue tetrazolium-positive cells, expression of CD14 and CD11b, and protein expression of c-Jun and Jun B, and decreased c-myc. Furthermore, notopterol induced the G0/G1 cell-cycle arrest as determined using flow cytometry, which may be related to the regulation of cell-cycle-related proteins p53, CDK2, CDK4, Cyclin D1, Cyclin E, and survivin. The combined use of notopterol and ATRA did not enhance the apoptotic effect as evidenced by cell viability test and Hoechst 33342. However, the combination of notopterol and ATRA enhanced the effect of inducing differentiation when compared with using either notopterol or ATRA alone, which can be evidenced by the increased nucleocytoplasmic ratio, NBT positive cells, and expression of CD14.Conclusion: This is the first time it has been demonstrated that notopterol could induce apoptosis, differentiation, and G0/G1 arrest in human AML HL-60 cells, suggesting that notopterol has potential therapeutic effects on AML. The combination application of notopterol (20 and 40 μM) and ATRA (2 μM) could augment differentiation of HL-60 cells.

Oxidative stress, DNA damage, and mutagenicity induced by the extractable organic matter of airborne particulates on bacterial models

The biological activity induced by the extractable organic matter (EOM) of size-segregated airborne Particulate Matter (PM) from two urban sites, urban traffic (UT) and urban background (UB), was assessed by using bacterial assays. The Gram-negative Escherichia coli (E. coli) coliform bacterium was used to measure the intracellular formation of Reactive Oxygen Species (ROS) by employing the Nitroblue tetrazolium (NBT) reduction assay and the lipid peroxidation by malondialdehyde (MDA) measurement. To the best of our knowledge, this is the first study using E. coli for assessing the bioactivity of ambient air in term of oxidative mechanism studies. E. coli BL21 cells were further used for DNA damage assessment by employing the reporter (β-galactosidase) gene expression assay. The bacterial strain S. typhimurium TA100 was used to assess the mutagenic potential of PM by employing the well-known mutation assay (Ames test).Four PM size fractions were assessed for bioactivity, specifically the quasi-ultrafine mode (<0.49 μm), the upper accumulation mode (0.49–0.97 μm), the upper fine mode (0.97–3 μm), and the coarse mode (>3.0 μm). The EOM of each PM sample included three organic fractions of successively increased polarity: the non-polar organic fraction (NPOF), the moderately polar organic fraction (MPOF), and the polar organic fraction (POF). The toxicological endpoints induced by each organic fraction were correlated with the concentrations of various organic chemical components determined in previous studies in an attempt to identify the chemical classes involved.

Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: In Vitro and In Vivo studies

The major feature that distinguishes acute promyelocytic leukemia (APL) cells from other malignant hematopoietic cells is the expression of promyelocytic leukemia-retinoicacid receptor α (PML-RARα) fusion protein, which contributes to the inhibition of RARα-regulated hematopoietic cellular differentiation. The complete remission rate of APL exceeded 90% with the application of all-trans retinoic acid (ATRA), arsenic trioxide (ATO), and anthracycline-based chemotherapy. However, the 7-year cumulative incidence of relapses was reported as 28.6% in APL maintenance with ATRA and daunorubicin and even reached 33% in the ATRA maintenance treatment group. The relapse/refractory patients showed resistance to ATRA and/or ATO, which has been identified as a clinically significant problem. Currently, no effective drugs are available to reverse the ATRA resistance.Matrine (MAT) is the main active component of Sophora flavescens. It has been used to treat chronic hepatitis for several years in China. Recently, the molecule has been proven to exhibit an anti-leukemic effect. Previous studies have revealed that MAT can reverse the ATRA resistance of NB4-LR1 cells when coupled with ATRA. The treatment with 0.1mmol/L MAT and 1μmol/L ATRA can restore the ability of NB4-LR1 cells to differentiate, which might be related to the increased level of cyclic adenosine monophosphate and protein kinase A activity in NB4-LR1 cells, reduced telomerase activity and downregulated expression of topoisomerase II beta (TopoIIβ) (Wu, et al. Planta Med 2014). Subsequently, this study aimed to investigate the mechanism underlying the degradation of the PML/RARα fusion protein in the presence of MAT and ATRA in NB4 and NB4-LR1 cell lines.ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator, RAPA), hydroxychloroquine (lysosomal inhibitor, HCQ) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results showed that MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein (Fig.A-C).The ubiquitin proteasome pathway plays a crucial role in protein degradation. MAT promoted the ubiquitylation level in NB4-LR1 by stabilized the 20S protein expression and enhanced the activity of the proteasome (Fig.D). ATRA inhibited the expression of RARα in NB4-LR1 cells, which was contradictory to that in NB4 cells. MAT can stabilize the expression of RARα in NB4-LR1 cells, whereas MG132 downregulated the expression of RARα in both cell lines, which hampered the differentiation of NB4 cells (Fig.E-G). In addition to UPP, the autophagy pathway also had a significant role in arsenious acid- or ATRA-mediated PML-RARα fusion protein degradation. MAT could promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62 (Fig.H-K). A similar phenomenon was observed in mouse xenografts (Fig.L-N).In summary, the present study revealed the difference in the chain reaction of sensitive and resistant APL cell lines (NB4 and NB4-LR1, respectively) to the treatment of ATRA, explaining the mechanism underlying the resistance to ATRA. ATRA decrease the level of 20S core subunit and the RARα in NB4-LR1 cells, but could not activate the autophagy process. These effects were reversed by the combination of MAT. The proteasome inhibitor might hamper the RARα stabilization and hence was not advantageous for the differentiation of cells. It also induced autophagy in NB4-LR1 cells. MAT induced the activation of UPP and mediated the autophagic degradation process, which synergistically induced the degradation of PML-RARα fusion protein and promote the differentiation of NB4-LR1 cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunomodulatory activities of Acacia catechu, a traditional thirst quencher of South India

BackgroundAcacia catechu has been widely used in Ayurveda for treating many diseases. Its heartwood extract is used in asthma, cough, bronchitis, colic, diarrhea, dysentery, boils, skin afflictions, sores and for stomatitis. The decoction of heartwood is used for drinking purpose in southern part of India especially in Kerala. ObjectiveThe current study was carried out to evaluate immunomodulatory effects of heartwood extracts of A. catechu in Swiss albino mice. Material and methodsIn vivo immunomodulatory activity was analyzed by hemagglutinating antibody (HA) titer, plaque forming cell assay and delayed type hypersensitivity (DTH). In vitro immunomodulatory potential of the extracts was studied using peritoneal macrophages and splenocytes from mice. Effect of extracts on phagocytic activity of macrophages was analyzed by nitroblue tetrazolium (NBT) reduction assay and cellular lysosomal enzyme assay. Anti-inflammatory activity was studied by nitric oxide (NO) assay and production of TNF-α and IL-10. ResultsA dose dependent increase in antibody titer was observed with extracts treatment. Treatment with extracts produced an enhancement in the number of antibody producing cells in the spleen. DTH reaction was significantly decreased with extracts treatment. An increased phagocytic response was shown by peritoneal macrophages on treatment with the extracts as evidenced by its effect on NBT reduction and cellular lysosomal enzyme activity. The extracts inhibited the release of pro-inflammatory cytokine TNF-α and the production of NO. IL-10 production was significantly increased after extract treatment. ConclusionThe results of the present study indicate the immunomodulatory effects of A. catechu extracts on humoral, cell mediated and non-specific immune functions.

Innate Immune System Status of Sulphur Mustard-Poisoned Iranian Veterans Three Decades after Exposure.

Sulphur mustard (SM) is an incapacitating chemical warfare agent which causes acute and chronic toxicities in different body organs of affected individuals. The aim of this study was to investigate the innate immune status of the Iranian veterans who were exposed to SM around 30 years earlier and had more than 25% disabilities. In this regard, most functional and non-functional parameters of innate immunity were evaluated in 35 veterans. Phagocytic activity, nitroblue tetrazolium (NBT) reduction assay and haemolytic complement activity (HCA) in addition to routine haematological parameters, serum protein electrophoresis, complements C3 and C4 levels were studied. Measures of haematological parameters, serum proteins, C3 and C4 were almost within the normal range. Functional experiments such as phagocytic activity, NBT reduction assay and HCA were normal as well. However, serum protein analysis revealed a fair decrease in percentages of α1 -globulin. Mean values of the parameters of innate immune system of the veterans three decades after SM poisoning were almost within the upper and lower normal limits. Reduced α1 -globulin - maybe subsequent to a chain of SM-induced genetic disorders - may have been the result of α1 -antitrypsin deficiency which may result in prevalent respiratory complications among these veterans. As a supplementary study, measurement of serum α1 -antitrypsin in SM-poisoned veterans could be beneficial. Further studies are required to prove this hypothesis. Further investigations on the evaluation of the acquired immunity parameters as the second line of defence may reveal a better understanding of SM veterans' immune system status.

Catechin rich butanol fraction extracted from Acacia catechu L. (a thirst quencher) exhibits immunostimulatory potential

Acacia catechu L., (Fabaceae) named as “catechu” is a plant, the decoction of heartwood of which is daily consumed as thirst quencher by a good percentage of the population in South India. The plant is mainly distributed in India and other Asian countries. It has been used in Indian traditional medicine for the treatment of asthma, bronchitis, colic, diarrhea, boils, skin afflictions, sores and stomatitis. The present investigation was aimed to study the immunomodulatory effects of different fractions of ethanol extract of A. catechu heartwood and HPLC analysis of the active fraction. Three fractions namely, butanol, chloroform and ethyl acetate were prepared from ethanol extract of A. catechu heartwood. Each of these fractions was assessed for its immunomodulatory activity. In vivo immunomodulatory activity was analyzed by sheep red blood cells (SRBC) specific hemagglutinating antibody titer, plaque-forming cell assay and delayed type hypersensitivity (DTH) reaction in Swiss albino mice. In vitro immunomodulating potential of the fractions was studied using murine peritoneal macrophages and splenocytes. Non-specific immune functions such as phagocytosis (nitroblue tetrazolium reduction assay and cellular lysosomal enzyme assay), nitric oxide (NO) production and cytokine release (TNF-α and IL-10) were studied in macrophages. In addition, splenocyte proliferation was also studied. In the in vivo experiments, butanol and chloroform fractions showed an increase in antibody titer dose-dependently. At higher dose (400 mg/kg b. w.) treatment the butanol fraction produced an enhancement in the number of plaque-forming cells (antibody producing cells) in the spleen. SRBC induced DTH reaction was significantly increased with butanol fraction in a dose-dependent manner. Peritoneal macrophages showed an increased phagocytic response on treatment with butanol fraction (100 μg/mL) as evidenced by its effect on nitroblue tetrazolium reduction and cellular lysosomal enzyme activity. All three fractions inhibited the production of NO and the release of TNF-α. Interleukin-10 production was significantly increased after treatment with butanol fraction. High-performance liquid chromatography analysis of the butanol fraction showed the presence of high concentration of catechin. The results suggested that butanol fraction of ethanol extract of A. catechu heartwood had immunomodulatory effects on non-specific, humoral, and cell-mediated immune functions. This study may be useful in validating the rationality of daily consumption of decoction of A. catechu and also its use in traditional medicine system. The study also suggests the possible use of A. catechu in the immunostimulatory herbal preparations.

Silver nanoparticle-mediated cellular responses in isolated primary Sertoli cells in vitro

The present study explored the mechanism of cytotoxic and genotoxic effects of AgNPs on a primary culture of mouse Sertoli cells in vitro. To understand the possible molecular mechanisms of testicular lesions following exposure to AgNPs, isolated Sertoli cells were exposed to 5, 10, or 15 μg/ml. DNA damage in the Comet assay and apoptosis in the TUNEL assay were evaluated. The mRNA expression of p53 and bcl-2 genes and their proteins involved in apoptosis was also investigated. The antioxidant status of treated Sertoli cells was determined by measuring superoxide dismutase (SOD-1), catalase (CAT) and glutathione peroxidase (GPX-1) using quantitative polymerase chain reaction (qPCR). The superoxide anions were detected using the nitroblue tetrazolium (NBT) reduction assay. Results indicated that AgNP exposure causes increased oxidative stress levels. The activation of p53, repression of bcl-2 and reduction of endogenous antioxidant enzymes were also involved in these mechanistic pathways, leading to reduced cell numbers and cell detachment.

Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: in Vitro and in Vivo Studies

Background/Aims: Although the cure rate of acute promyelocytic leukemia (APL) has exceeded 90%, the relapse/refractory APL that resistant to all-trans retinoic acid (ATRA) or ATO was still serious concern. Matrine (MAT) could improve the differentiation ability of ATRA-resistant APL cells. This study aimed to explore how the APL-specific fusion protein was degraded in ATRA-resistant APL with the application of MAT and ATRA. Methods: ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator), hydroxychloroquine (lysosomal inhibitor) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results: MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein. MAT promoted the ubiquitylation level in NB4-LR1. MG 132 induced the decrease in RARα in both cell lines, and hampered the differentiation of NB4 cells. MAT also promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62. The expression of LC3II increased significantly in the MAT and ATRA + MAT groups in combination with lysosomal inhibitors. A similar phenomenon was observed in mouse xenografts. MAT induced apoptosis and differentiation. Conclusions: Autophagy and ubiquitin-mediated proteolytic degradation of PML/RARα fusion protein are crucial in MAT-induced differentiation sensitivity recovery of NB4-LR1 cells.

The efficacy of Poly-β-Hydroxy Butyrate (PHB)/biosurfactant derived from Staphylococcus hominis against White Spot Syndrome Virus (WSSV) in Penaeus monodon

White Spot Syndrome Virus (WSSV) is one of the most important causative agents of Penaeid shrimps diseases that incur heavy losses to the shrimp aquaculture. It has severe impact on the sustainability and the production of Penaeus monodon. Hence, the present study focussed on the investigation of Poly-β-hydroxybutyrate/biosurfactant as immunostimulants against WSSV infected shrimps. Infection of WSSV was periodically checked in all the experimental shrimps using PCR diagnostic kit. After ensuring all shrimps were free of viral infection, experiments were carried out to analyze the nonspecific immune responses (prophenol oxidase, nitro blue tetrazolium reduction assay and total haemocyte count) both in control and experimental group. Further, gills and muscles of Penaeus monodon were subjected to proteome analysis after treated it with PHB/biosurfactant independently in the concentration of 2% and 5% each. Increase in the level of haemocytes was observed in both PHB (26 ± 2 × 10⁴ cells)/biosurfactant (28 ± 2 × 104 cells) treated shrimps, when compared with control (17 ± 2 × 10⁴ cells). proPhenolOxidase (proPO) activity was also enhanced in treated groups compared to WSSV infected shrimps. Less production of superoxide anion was observed in control and treated groups. Differences in the protein expression was analyzed in muscle tissue of control, WSSV infected and PHB/biosurfactant treated shrimps. Our finding suggested that partial substitution of feed with 2% PHB and biosurfactant showed increased rate on the survival of WSSV infected P. monodon which might be due to either the over expression/down regulation of proteins that play a vital role in enhancing the immune system/the progression of the disease respectively.

In Vitro Testing of Potential Entamoeba histolytica Pyruvate Phosphate Dikinase Inhibitors.

Adverse effects and resistance to metronidazole have motivated the search for new antiamoebic agents against Entamoeba histolytica. Control of amoeba growth may be achieved by inhibiting the function of the glycolytic enzyme and pyruvate phosphate dikinase (PPDK). In this study, we screened 10 compounds using an in vitro PPDK enzyme assay. These compounds were selected from a virtual screening of compounds in the National Cancer Institute database. The antiamoebic activity of the selected compounds was also evaluated by determining minimal inhibitory concentrations (MICs) and IC50 values using the nitro-blue tetrazolium reduction assay. Seven of the 10 compounds showed inhibitory activities against the adenosine triphosphate (ATP)/inorganic phosphate binding site of the ATP-grasp domain. Two compounds, NSC349156 (pancratistatin) and NSC228137 (7-ethoxy-4-[4-methylphenyl] sulfonyl-3-oxido-2, 1, 3-benzoxadiazol-3-ium), exhibited inhibitory effects on the growth of E. histolytica trophozoites with MIC values of 25 and 50 μM, and IC50 values of 14 and 20.7 μM, respectively.

Mechanism of antagonistic effects of Andrographis paniculata methanolic extract against Staphylococcus aureus.

To investigate the effects of Andrographis paniculata (Burm.f.) Wall. Ex Nees (A.paniculata) on expressions and activities of catalase, superoxide dismutase and alkylhydroperoxide reductase C in Staphylococcus aureus (S.aureus) with respect to its survival invitro. Antioxidative property of methanolic leaves extract of A.paniculata (0.06mg/mL). Minimum inhibitory concentration (MIC) was determined by its ability to reduce hydrogen peroxide (H2O2) toxicity against S.aureus ATCC 25923 [(3.8×108)cfu/mL]. Effects of the extract on expressions of katA (encoding catalase), sodA and sodM [encoding superoxide dismutases (SODs)], and ahpC [encoding alkylhydroperoxide reductase C (AhpC)] in S.aureus were determined by RT-qPCR and corresponding enzyme activity assays were performed. Nitroblue tetrazolium reduction (NBT) assay was performed to determine effects of the extract on intracellular and extracellular levels of O2- in S.aureus. Cells challenged with 7.5mmol/L H2O2 showed 0% survival in 30min whereas 25% survived after treatment with the extract and H2O2. Cells that were treated with the extract alone had 43% survival in the same exposure period. Expressions of sodA and sodM genes in extract-treated cells were lowered 0.8-fold and 0.7-fold, respectively with decrease in total SOD activity of 26.8U compared to untreated cells, 32.4U (P<0.05). In contrast, extract-treated S.aureus cells showed 3.3-fold increase in katA expression with corresponding increase in catalase activity of 1.828U compared to untreated cells which was 1.248U, (P<0.05). More profoundly, ahpC expression was increased 61-fold in extract-treated cells, (P<0.05) with corresponding increase in AhpC activity of 0.018U compared to untreated cells, 0.012U, (P<0.05). Extract-treated cells had significantly lower intra- and extracellular O2- levels with absorbance readings (A575nm) of 0.340 and 0.524 compared to untreated cells which were 0.516 and 0.928 (P<0.05), respectively. Taken together these results suggest that the low MIC of A.paniculata methanolic leaves extract (0.06mg/mL) reduce H2O2 toxicity and more importantly, was in itself effectively inhibitory against S.aureus. Further, our observations suggest that a probable mode of its inhibitory mechanism against S.aureus is by reducing total SOD activity through downregulation of sodA and sodM expressions.

Antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 on the Gram-negative bacteria Escherichia coli.

To investigate the antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 (HMGN2) on Escherichia coli K12, focusing on the antibacterial and antibiofilm formation effects. Its chemotactic activity on human neutrophils was also investigated. Human tissue-derived HMGN2 (tHMGN2) was extracted from fresh uterus fiber cystadenoma and purified by HP1100 reversed-phase high-performance liquid chromatography (RP-HPLC). Recombinant human HMGN2 (rHMGN2) was generated in E. coli DE3 carrying PET-32a-c(+)-HMGN2. Antibacterial activity of HMGN2 was determined using an agarose diffusion assay and minimum inhibitory concentration (MIC) of HMGN2 was determined by the microdilution broth method. Bacterial membrane permeability assay and DNA binding assay were performed. The antibiofilm effect of HMGN2 was investigated using a crystal violet assay and electron microscopy scanning. The activating effect and chemotactic activity of HMGN2 on neutrophils were determined using a nitroblue tetrazolium (NBT) reduction assay and Transwell chamber cell migration assay, respectively. HMGN2 showed a relatively high potency against Gram-negative bacteria E. coli and the MIC of HMGN2 was 16.25 μg/ml. Elevated bacterial membrane permeability was observed in HMGN2-treated E. coli K12. HMGN2 could also bind the bacterial plasmid and genomic DNA in a dose-dependent manner. The antibiofilm effect of HMGN2 on E. coli K12 was confirmed by crystal violet staining and scanning electron microscopy. However, the activating effects and chemotactic effects of HMGN2 on human neutrophils were not observed. As an antimicrobial peptide (AMP), HMGN2 possessed a good capacity for antibacterial and antibiofilm activities on E. coli K12. This capacity might be associated with disruption of the bacterial membrane and combination of DNA, which might affect the growth and viability of E. coli.

Killing of Staphylococcus aureus by allylpyrocatechol is potentiated by induction of intracellular oxidative stress and inhibition of catalase activity

ObjectiveThis study investigated the effects of allylpyrocatechol (APC), the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host. MethodsEffects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in S. aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay. ResultsAPC-treated S. aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A310nm) compared to untreated cells which was 0.394 (A310nm). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 × 10−4 (U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 × 10−4 U/L (P<0.05). Absorbance readings (A575nm) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S. aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05). ConclusionAPC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S. aureus. Higher sodA expression indicated stress induced intracellularly involving O2−, presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by O2− and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.