cytosine deaminase (CD) converts 5-fluorocytosine (5-FC), a prodrug, into 5-fluorouracil (5-FU), a chemotherapeutic drug. However, the poor binding affinity of CD towards 5-FC as compared to the natural substrate cytosine, limits its application towards a successful suicide gene therapy. Although F186W mutant was developed to enhance the effect of wild-type CD, still scope for its improvement remains to further minimize the dose-dependent cytotoxicity of the drugs. Hence, in this study, we employ the anti-tumour attribute of the gap junction forming protein connexin-43 (Cx43) in conjunction with CD or F186W mutant. Lipofectamine was used to co-transfect CD/F186W-pVITRO2 and Cx43-pEGFP-N1 plasmids construct into MCF-7 cells. Comparative analysis of cell viability was observed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) and trypan blue-based assays. To further confirm the mode of cell death was apoptosis, propidium iodide and annexin V/7-aminoactinomycin D (7-AAD)-based apoptosis assays were performed. Semi-quantitative polymerase chain reaction (PCR) confirmed the expression of both Cx43 and CD/F186W genes after transfection. Furthermore, cell viability assays revealed the enhanced activity of F186W-Cx43 compared with CD-Cx43 and F186W alone. The trend of the reduction in cell viability was also reflected in the flow cytometry-based apoptosis analyses. Overall, F186W-Cx43 combination demonstrated its superiority over the CD-Cx43 and F186W mutant alone. The enhanced cytotoxic activity of F186W mutant was further amplified by gap junction protein Cx43.
The acclaimed explanation for mitochondrial oxidative phosphorylation (mOxPhos, or cellular respiration) is a deterministic proton-centric scheme involving four components: Rotary adenosine triphosphate (ATP)-synthesis, Chemiosmosis principle, Proton pumps, and Electron transport chain (abbreviated as RCPE hypothesis). Within this write-up, the RCPE scheme is critically analyzed with respect to mitochondrial architecture, proteins’ distribution, structure-function correlations and their interactive dynamics, overall reaction chemistry, kinetics, thermodynamics, evolutionary logic, and so on. It is found that the RCPE proposal fails to explain key physiological aspects of mOxPhos in several specific issues and also in holistic perspectives. Therefore, it is imperative to look for new explanations for mOxPhos.
Based on a previous study, glabridin displayed a dose-dependent increase in estrogenic activity and cell proliferative activity in Ishikawa cells. However, when treated in combination with 17β-E2, synergistic estrogenic effect was observed but without the same synergistic increase in cell proliferative effect. This study aimed to identify the estrogen and nonestrogen-regulated activities induced by glabridin and in combination with 17β-E2 in comparison with 17β-E2. The results showed that 10 µM glabridin and the combination treatment of 100 nM glabridin with 1 nM 17β-E2 regulated both the genomic and nongenomic estrogen pathways to possibly provide benefits of estrogens in cardiovascular, circulatory, and vasculature systems. Meanwhile, the combination of 100 nM glabridin with 1 nM 17β-E2 seems to be more suitable to be used as an estrogen replacement. Finally, the results of this study have added on to the present knowledge of glabridin’s function as a phytoestrogen and suggested new ideas for the usage of glabridin.
Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world.
Africa is rich in a wide range of flora that are exploited as herbal medicines and remedies. Several diseases such as diabetes, diarrhea, dysentery and jaundice have been successfully managed using herbal medicines. Herbal decoctions or concoctions have been used as pain killers, antibiotics, and hematinics. This study evaluated the hematopoietic and biochemical properties of the stem bark of Sterculia setigera Del. in Wistar rats. Results showed that S. setigera decoction has copiously high tannin and cardiac glycoside levels. Ingestion of the decoction by rats over a 16-day period significantly (P < 0.05) increased the body weights of rats by 22.4% in the S. setigera-treated group. Hematological profiles showed raised levels of red blood cells, hemoglobin, packed cell volume, mean corpuscular volume, mean cell hemoglobin, mean cell hemoglobin concentration, and platelets, while biochemical parameters showed lower levels of alanine aminotransferase and aspartate aminotransferase, and slight increase in albumin and TP levels. We posit that the results justify the use of the stem bark of S. setigera as a hematinic by traditional medical practitioners and show its relative safety. Further experiments are needed to evaluate its safety.
Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread.
A 112-amino-acid protein irisin (IRI) is widely expressed in many organs, but we currently do not know whether appendix tissue and blood cells express it. If appendix tissue and neutrophil cells express IRI, measuring its concentration in biological fluids might be helpful in the diagnosis of acute appendicitis (AA), since neutrophil cells are the currently gold-standard laboratory parameters for the diagnosis of AA. Therefore, the purpose of this study was to investigate the suitability of enzyme-linked immunosorbent assay-based measurements of the proposed myokine IRI for the discrimination of patients with AA from those with acute abdominal pain (AP) and healthy controls. Moreover, immunoreactivity to IRI was investigated in appendix tissues and blood cells. Samples were collected on admission (T1), 24 hours (T2), and 72 hours (T3) postoperatively from patients with suspected AA and from patients with AP corresponding to T1-T3, whereas control subject blood was once corresponding to T1. IRI was measured in serum, saliva, and urine by using enzyme-linked immunosorbent assay, whereas in appendix tissue and blood cells, IRI was detected by immunohistohcemistry. Appendix tissue and blood cells (except for erythrocytes) are new sources of IRI. Basal saliva, urine, and serum levels were higher in children with AA compared with postoperative levels (T2) that start to decline after surgery. This is in line with the finding that IRI levels are higher in children with AA when compared with those with AP or control subject levels, most likely due to a large infiltration of neutrophil cells in AA that release its IRI into body fluids. Measurement of IRI in children with AA parallels the increase or decrease in the neutrophil count. This new finding shows that the measurement of IRI and neutrophil count can together improve the diagnosis of AA, and it can distinguish it from AP. IRI can be a candidate marker for the diagnosis of AA and offers an additional parameter to neutrophil count. The promising receiving operating curve results indicate the following sensitivities and specificities, respectively, for IRI: serum 90% and 55%, saliva 90% and 60%, and urine 90% and 50%. Serum neutrophil count gave a sensitivity of 90% and a specificity of 90%. This promising result now needs to be confirmed in a larger group of patients.
Africa is rich in a wide range of flora that are exploited as herbal medicines and remedies. Several diseases such as diabetes, diarrhea, dysentery and jaundice have been successfully managed using herbal medicines. Herbal decoctions or concoctions have been used as pain killers, antibiotics, and hematinics. This study evaluated the hematopoietic and biochemical properties of the stem bark of Sterculia setigera Del. in Wistar rats. Results showed that S. setigera decoction has copiously high tannin and cardiac glycoside levels. Ingestion of the decoction by rats over a 16-day period significantly ( P < 0.05) increased the body weights of rats by 22.4% in the S. setigera -treated group. Hematological profiles showed raised levels of red blood cells, hemoglobin, packed cell volume, mean corpuscular volume, mean cell hemoglobin, mean cell hemoglobin concentration, and platelets, while biochemical parameters showed lower levels of alanine aminotransferase and aspartate aminotransferase, and slight increase in albumin and TP levels. We posit that the results justify the use of the stem bark of S. setigera as a hematinic by traditional medical practitioners and show its relative safety. Further experiments are needed to evaluate its safety.
Although dental pain is a serious health issue with high incidence among the human population, its cellular and molecular mechanisms are still unclear. Transient receptor potential (TRP) channels are assumed to be involved in the generation of dental pain. However, most of the studies were conducted with molecular biological or histological methods. In vivo functional studies on the role of TRP channels in the mechanisms of dental pain are lacking. This study uses in vivo cellular electrophysiological and neuropharmacological method to directly disclose the effect of LaCl3, a broad spectrum TRP channel blocker, on the response properties of neurons in the mouse primary somatosensory cortex to low-temperature noxious stimulation of the dental pulp. It was found that LaCl3 suppresses the high-firing-rate responses of all nociceptive neurons to noxious low-temperature stimulation and also inhibits the spontaneous activities in some nonnociceptive neurons. The effect of LaCl3 is reversible. Furthermore, this effect is persistent and stable unless LaCl3 is washed out. Washout of LaCl3 quickly revitalized the responsiveness of neurons to low-temperature noxious stimulation. This study adds direct evidence for the hypothesis that TRP channels are involved in the generation of dental pain and sensation. Blockade of TRP channels may provide a novel therapeutic treatment for dental pain.
Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monoga-lactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully dearabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.
Gallic acid is an organic acid known for its antioxidant and anticancer properties. The present study is focused on evaluating the role of gallic acid in providing better therapeutic outcomes against arsenic-induced toxicity. Animals pre-exposed to arsenic were treated with monoisoamyl meso-2,3-dimercaptosuccinic acid (MiADMSA), a new chelating drug, alone and in combination with gallic acid, consecutively for 10 days. The study suggests that (1) gallic acid in presence of MiADMSA is only moderately beneficial against arsenic, (2) monotherapy with gallic acid is more effective than in combination with MiADMSA after arsenic exposure in reducing oxidative injury, and (3) MiADMSA monotherapy as reported previously provides significant therapeutic efficacy against arsenic. Thus, based on the present results, we conclude that gallic acid is effective against arsenic-induced oxidative stress but provides limited additional beneficial effects when administered in combination with MiADMSA. We still recommend that lower doses of gallic acid be evaluated both individually and in combination with MiADMSA, as it might not exhibit the shortcomings we observed with higher doses in this study.