In vitro killing action of auranofin on Taenia crassiceps metacestode (cysticerci) and inactivation of thioredoxin–glutathione reductase (TGR)

Control of cellular redox homeostasis is a central issue for all living organisms. Glutathione and thioredoxin enzymatic redox systems are the usual mean used to achieve such a control. However, parasitic platyhelminths studied to date possess a nicotinamide adenine dinucleotide phosphate-dependent thioredoxin–glutathione reductase (TGR) as the sole redox control system. Thus, TGR is considered as a potential therapeutic target of parasitic platyhelminths, and based on this assumption, the gold compound auranofin is a potent inhibitor of TGR. The aim of this research was to investigate the effect of auranofin on metacestode (cysticerci) of Taenia crassiceps in culture. Accordingly, the time course for viability and respiration of cysticerci in culture was evaluated in the presence of this compound. After 4 h at 10 μM auranofin, 90% of cysticerci were alive, but respiration activity had declined by 50%. After 12 h, neither survivors nor respiration was detected; a LD₅₀ for auranofin of 3.8 μM was calculated. Interestingly, crude extracts of cysticerci pretreated with 3 μM auranofin nearly nil TGR activity (IC₅₀ = 0.6 μM). Zymography for TGR in polyacrylamide gel electrophoresis was conducted because the previously mentioned extracts clearly showed a dose–response inactivation of TGR toward auranofin. The killing of cysticerci by this gold compound is most likely related with TGR inactivation. Therefore, further research on the suitability of auranofin as a therapeutic tool in the treatment of cysticercosis in animals and humans is sustained.     

Effect of γ-PGA on the formation of collagen fibrils in vitro

The effect of γ-poly(glutamic acid) (γ-PGA) on the self-assembly of collagen was studied. Under physiological conditions, the kinetic curves for fibril formation showed that the turbidity of collagen/γ-PGA blends at 313 nm was increased with the addition of γ-PGA. Furthermore, it was shown using both field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) that fibrils with a larger diameter were obtained following the addition of γ-PGA, probably due to the electrostatic and hydrogen bond interactions between collagen and γ-PGA, which promoted the lateral association of collagen molecules. In addition, both the thermal stability and viscoelastic properties of the hybrid hydrogels, which were evaluated by differential scanning calorimetry and rheological measurements, respectively, were improved by the addition of γ-PGA.     

In vitro inhibitory effects of plant-based foods and their combinations on intestinal ¿-glucosidase and pancreatic ¿-amylase

ABSTRACT: BACKGROUND: Plant-based foods have been used in traditional health systems to treat diabetes mellitus. The successful prevention of the onset of diabetes consists in controlling postprandial hyperglycemia by the inhibition of alpha-glucosidase and pancreatic alpha-amylase activities, resulting in aggressive delay of carbohydrate digestion to absorbable monosaccharide. In this study, five plant-based foods were investigated for intestinal alpha-glucosidase and pancreatic alpha-amylase. The combined inhibitory effects of plant-based foods were also evaluated. Preliminary phytochemical analysis of plant-based foods was performed in order to determine the total phenolic and flavonoid content. METHODS: The dried plants of Hibiscus sabdariffa (Roselle), Chrysanthemum indicum (chrysanthemum), Morus alba (mulberry), Aegle marmelos (bael), and Clitoria ternatea (butterfly pea) were extracted with distilled water and dried using spray drying process. The dried extracts were determined for the total phenolic and flavonoid content by using Folin-Ciocateu's reagent and AlCl3 assay, respectively. The dried extract of plant-based food was further quantified with respect to intestinal alpha-glucosidase (maltase and sucrase) inhibition and pancreatic alpha-amylase inhibition by glucose oxidase method and dinitrosalicylic (DNS) reagent, respectively. RESULTS: The phytochemical analysis revealed that the total phenolic content of the dried extracts were in the range of 460.0-230.3 mg gallic acid equivalent/ g dried extract. The dried extracts contained flavonoid in the range of 50.3-114.8 mg quercetin equivalent/g dried extract. It was noted that the IC50 values of chrysanthemum, mulberry and butterfly pea extracts were 4.24+/-0.12 mg/ml, 0.59+/-0.06 mg/ml, and 3.15+/-0.19 mg/ml, respectively. In addition, the IC50 values of chrysanthemum, mulberry and butterfly pea extracts against intestinal sucrase were 3.85+/-0.41 mg/ml, 0.94+/-0.11 mg/ml, and 4.41+/-0.15 mg/ml, respectively. Furthermore, the IC50 values of roselle and butterfly pea extracts against pancreatic alpha-amylase occurred at concentration of 3.52+/-0.15 mg/ml and 4.05+/-0.32 mg/ml, respectively. Combining roselle, chrysanthemum, and butterfly pea extracts with mulberry extract showed additive interaction on intestinal maltase inhibition. The results also demonstrated that the combination of chrysanthemum, mulberry, or bael extracts together with roselle extract produced synergistic inhibition, whereas roselle extract showed additive inhibition when combined with butterfly pea extract against pancreatic alpha-amylase. CONCLUSIONS: The present study presents data from five plant-based foods evaluating the intestinal alpha-glucosidase and pancreatic alpha-amylase inhibitory activities and their additive and synergistic interactions. These results could be useful for developing functional foods by combination of plant-based foods for treatment and prevention of diabetes mellitus.     

In vitro effects of bone- and platelet-derived transforming growth factor-β on the growth of Walker 256 carcinosarcoma cells

Conditioned media from fetal rat calvarial cultures has previously been shown to stimulate the growth of the bone-metastasizing Walker 256 carcinosarcoma cell line. In the current investigation we looked at the possibility that transforming growth factor- (TGF-), present in conditioned media, and positively correlated with resorption in vitro, may be responsible for the enhanced proliferation of Walker cells cultured in these conditioned media. Purified platelet-derived TGF- produced a dose-dependent growth response in Walker cells with an ED₅₀ equal to 0·05 ng/ml. Bone-derived TGF- activity in conditioned media, measured by NRK fibroblast colony formation, correlated well with percentage resorption in bone cultures, and growth activity in Walker cell culture. In addition to this, the growth response normally seen with conditioned media cultures of Walker cells was significantly inhibited by the addition of anti-TGF-1 neutralizing antibody. We conclude that TGF- is an important growth stimulatory component from fetal rat calvaria.     

Evaluation of the in vitro and in vivo proinflammatory activities of gold (+) and gold (−) nanoparticles

Objective and design

The aim of this study was to determine potential effects of gold (+) and gold (?) nanoparticles, AuNP(+) and AuNP(?), on neutrophil biology.

Material or subjects

Freshly isolated human neutrophils were used for the in vitro aspects and CD-1 mice were used in the in vivo murine air pouch model of acute neutrophilic inflammation.

Treatment

Human neutrophils were treated with the indicated concentrations of AuNP(+) or AuNP(?) in vitro and mice received 100 or 500 µg/ml AuNP(+) or AuNP(?) into air pouches.

Methods

Cellular uptake of AuNP by neutrophils was confirmed by transmission electron microscopy and the ability of the NP to modulate apoptosis, gelatinase activity, and chemokine production and chemotaxis was determined by cytology, zymography, ELISArray, antibody array, and ELISA and by a micro-chemotaxis chamber, respectively. In vivo, exudates were harvested after 6 h to determine the leukocyte infiltration to detect the production of several cytokines by an antibody array approach and ELISA. One-way analysis of variance was used for statistical analysis.

Results

AuNP possess proinflammatory activities in vitro and induce mainly a neutrophil influx in vivo, albeit at different degrees.

Conclusions

AuNP(+) and AuNP(?) should be added as new candidates into a growing list of NP having proinflammatory activities by themselves.

     

Distinctive effects of arachidonic acid and docosahexaenoic acid on neural stem /progenitor cells

Arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids in the brain, have crucial roles in brain development and function. Recent studies have shown that ARA and DHA promote postnatal neurogenesis. However, the direct effects of ARA on neural stem/progenitor cells (NSPCs) and the effects of ARA and DHA on NSPCs at the neurogenic and subsequent gliogenic stages are still unknown. Here, we analyzed the effects of ARA and DHA on neurogenesis, specifically maintenance and differentiation, using neurosphere assays. We confirmed that primary neurospheres are neurogenic NSPCs and that tertiary neurospheres are gliogenic NSPCs. Regarding the effects of ARA and DHA on neurogenic NSPCs, ARA and DHA increased the number of neurospheres, whereas neither ARA nor DHA had a detectable effect on NSPCs in the differentiation condition. In gliogenic NSPCs, DHA increased the number of neurospheres, whereas ARA had no such effect. In contrast, ARA increased the number of astrocytes, whereas DHA increased the number of neurons in the differentiation condition. These results suggest that ARA promotes the maintenance of neurogenic NSPCs and might induce the glial differentiation of gliogenic NSPCs and that DHA promotes the maintenance of both neurogenic and gliogenic NSPCs and might lead to the neuronal differentiation of gliogenic NSPCs.     

The effect of stereoisomers of cycloserine on the formation and transformation of free γ-aminobutyric acid in brain tissue

Summary A study was made of the effect produced by the stereoisomers of cycloserine on decarboxylization of glutamic acid and transamination of -aminobutyric acid with -ketoglutaric acid in the homogenates of the rat brain at pH 7.5. As revealed. D.L-cycloserine in a concentration of 10^–3 M depressed the first process by 40% and the second one by 45%. In the same conditions D-cycloserine depressed glutamic acid decarboxylation by 9%; it somewhat activated -aminobutyric acid transamination. A discussion is presented on the possible effect of cycloserine isomers on the content of free -aminobutyric acid in the brain (in vivo) following the administration of isoniazid in large doses.(Presented by Active Member AMN SSSR V. V. Zakusov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 10, pp. 67–69, October, 1962     

Expression of ALDH1 and TGFβ2 in benign and malignant breast tumors and their prognostic implications

The specific mechanism underlying the role of putative stem cell marker aldehyde dehydrogenase 1 (ALDH1) playing in development and progression of breast cancer is currently unclear. Transforming growth factor β (TGFβ) signaling pathway is reported to be activated in most cancers. Thus a study was initiated to explore possible differences and correlation of ALDH1 and TGFβ2 expression in the most common malignant and benign tumors of the breast in Chinese women. Samples of 75 breast cancer tissues, 30 paracancerous normal tissues, and 39 fibroadenoma breast tissues were investigated for the expression of ALDH1 and TGFβ2 using immunohistochemistry. The positive rates of ALDH1 and TGFβ2 protein were 62.67% and 66.67%, respectively, in breast cancer tissues, which were significantly higher than that in normal fibroadenoma breast (P<0.05) and paracancerous tissues (P<0.01). ALDH1 and TGFβ2 status were significantly associated with tumor histological grade and receptor status (P<0.05). Expression of ALDH1 was found to be positively correlative to TGFβ2 in breast cancer (r = 0.33, P<0.01). Expression of both proteins remained significantly associated with reduced overall survival (OS) by univariate analysis (P<0.05). Multivariate Cox regression analysis showed that ALDH1 expression, tumor stage, and lymph node status are independent prognostic factors in invasive breast cancer patients. Thus ALDH1 and TGFβ2 play important roles in the development of breast cancer. The ALDH1 phenotype is an independent predictor of poor prognosis, and TGFβ2 signaling pathway activation might be involved in the pathological regulation of ALDH1 in breast cancer.     

Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro北大核心

BACKGROUND: Atorvastatin has a cardiovascular protective effect that significantly improves endothelial function and promotes the mobilization, migration, and differentiation of endothelial progenitor cells. However, the screening of atorvastatin concentration for in vitro cell culture is not well documented. OBJECTIVE: To investigate the effects of different concentrations of atorvastatin on rat bone marrow-derived EPCs growth characteristics. METHODS: Bone marrow mononuclear cells from Sprague-Dawley rats were induced in selective culture fluid to culture EPCs. Immunofluorescence staining was used to identify cell surface markers. Harvested EPCs were divided into control group and atorvastatin groups with four different concentrations (0.01, 0.1, 1, and 10 µmol/L) for culture. The growth and proliferation of EPCs were observed under light microscope and MTT assay. Flow cytometry was used to detect apoptosis in EPCs. Nitric oxide and endothelial nitric oxide synthase levels in the culture fluid were measured by nitrate reductase method. RESULTS AND CONCLUSION: The number of cells tended to increase in the control and atorvastatin groups, and it was highest in the 1 µmol/L atorvastatin group. The cell number in the 10 µmol/L atorvastatin group began to decrease at 7 days of culture. Among the five groups, the apoptotic rate of cells was lowest in the 1 µmol/L atorvastatin group and highest in the 10 µmol/L atorvastatin group. The levels of nitric oxide and endothelial nitric oxide synthase were significantly higher in the 0.01, 0.1 and 1.0 µmol/L atorvastatin groups compared with the control group (P < 0.01), but lower in the 10 µmol/L atorvastatin group compare with the other groups (P < 0.01). Overall, atorvastatin can promote the proliferation of endothelial progenitor cells and reduce apoptosis by increasing the production of endothelial nitric oxide synthase and nitric oxide, and 1 µmol/L atorvastatin is most suitable for the EPCs culture. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

In Vitro Effects of Folic Acid on γ-Glutamyltransferase and Glutathione Reductase Activities in Malignant Lung and Thymus Tumors

In vitro effects of folic acid (10^-5, 10^-4, and 10^-3 M) on activities of -glutamyltransferase and glutathione reductase, the enzymes involved in glutathione metabolism, were studied in tissue samples obtained after surgical treatment of the lungs and thymus. Folic acid did not change -glutamyltransferase activity in lung cancer tissue, but in thymoma tissue this substance in a concentration of 10^-3 M inhibited it by 16%. Folic acid had no effects on glutathione reductase activity in benign tumors and normal lung and thymus tissues, but increased this activity in thymoma and lung cancer tissues. Activation of glutathione reductase was probably related to binding of folic acid in the allosteric center of the enzyme, which probably induced conformational changes in the catalytic center, acceleration of electron transport from NADPH₂ to oxidized glutathione via flavin adenine nucleotide, and intense production of reduced glutathione.     

Studies on hematoporphyrin-photosensitized effects on human cancer cells in vitro——TEM and SEM observation

The ECa109 esophageal cancer epithelial cell line cells was treated with hematoporphyrin one hour and followed by irrediation with 20W black light at a distance of two cm for ten minutes. The cancer cells were examined by TEM and SEM after treatment for one, three and five days. By exposure to hematoporphyrin derivative (HpD) and photoirradiation, the proportion of dark-cells with high electron density were decreased and the proportion of empty cells with low electron density was increased. The latter showed distruction of cancer cells. In addition to the change of electron density the cell killing effects were shewn by enlargement     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Developing mouse Sertoli cells in vitro: effects on developing ovaries in co-culture and production of anti-Müllerian hormone

Previous work in our laboratory showed that pre-Sertoli cells adopt an epithelial phenotype when cultured in the presence of reconstituted basement membrane (RBM), and so cultures were established with and without this substrate. Biological activity of isolated developing mouse Sertoli cells maintained in vitro was assessed in the current study by utilising a co-culture approach, to determine whether the cells were capable of affecting ovarian differentiation. Developing Sertoli cells isolated at embryonic day (E) 12.5 exerted a deleterious effect on E12.5 ovaries in co-culture, inducing a loss of germ cells. However, when cells were isolated a day later and co-cultured with E13.5 ovaries (after entry to meiosis has begun), germ cells survived and showed evidence of meiosis, although ovigerous cords in co-cultures were masculinized compared to those of control cultured ovaries. Thus, both stages examined showed biological effects; cultured pre-Sertoli cells explanted at E12.5 showed a negative effect on female germ cells, whereas those explanted at E13.5 masculinized ovigerous cords. The functional status of isolated developing mouse Sertoli cells in vitro was further assessed by immunocytochemistry to investigate the expression of anti-Müllerian hormone, an early product of pre-Sertoli cells. Positive immunostaining was seen in developing Sertoli cells in vitro, particularly where cells had been explanted to an RBM substrate, demonstrating that good epithelial morphology is associated with improved function. Our culture system is therefore well suited for investigating factors produced by developing Sertoli cells, their role in influencing testicular morphogenesis and their potential to perturb ovarian differentiation. We believe that this in vitro approach provides a more physiological assessment compared with the knockout mouse model, where global effects of genes with housekeeping functions can compromise overall development.     

Reduced DNA repair in progeria cells and effects of γ-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33–50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by γ-ray irradiation to the cells before UV irradiation. The dose of γ-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, γ-ray irradiation further reduced UDS with a lower γ-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a ‘built-in’ defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by γ-ray irradiation.     

Specific effects of neuregulin-1β on the communication between DRG neurons and skeletal muscle cells in vitro

The communication between primary afferent neuron and skeletal muscle (SKM) is one of the important factors on maintaining the structure and function of SKM cells. Neuregulin-1β (NRG-1β) signaling is essential for regulating synaptic neurotransmission. Here, we established a neuromuscular coculture model of dorsal root ganglion (DRG) sensory neurons and SKM cells to explore the nerve-muscle communication in the presence of exogenous NRG-1β. The expression of three distinct subtypes (TrkA, TrkB, and TrkC) of tyrosine kinase receptors was monitored for the phenotypical alterations of the neurons. The aggregation extent of acetylcholine receptor (AChR) represents the specific changes of SKM cells after NRG-1β incubation in this neuromuscular coculture model. The results showed that NRG-1β not only enhanced neurite outgrowth of DRG neurons but also increased the length and branches of SKM cells. NRG-1β treatment not only induced expression of all the three subtypes of Trk receptors in neurons but also promoted AChR aggregation on the surface of SKM cells. The effects of NRG-1β could be blocked by administration of ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and JAK2 inhibitor AG490, respectively. These data imply that NRG-1β is essential for the nerve-muscle communication by enhancing growth and modifying phenotypes of the two different kinds of cells. The specific effects produced by NRG-1β add novel interpretation for nerve-muscle communication between sensory neurons and SKM cells.     

Identification of N-carboxyethyl γ-aminobutyric acid in bovine brain and human cerebrospinal fluid

The di-carboxylated derivative of spermidine, N-carboxyethyl γ-aminobutyric acid (CEGABA) has been identified in bovine brain and human cerebrospinal fluid by HPLC. This discovery strongly suggests the existence of a metabolic pathway connecting polyamines and GABA via putreanine and CEGABA through progressive oxidative deamination of the amino terminal groups in spermidine.