Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and downregulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.
The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.
Goniothalamin (GN), a styryl-lactone isolated from Goniothalamus andersonii, has been demonstrated to possess antirestenostic properties by inducing apoptosis on coronary artery smooth muscle cells (CASMCs). In this study, the molecular mechanisms of GN-induced CASMCs apoptosis were further elucidated. Apoptosis assessment based on the externalization of phosphatidylserine demonstrated that GN induces CASMCs apoptosis in a concentration-dependent manner. The GN-induced DNA damage occurred with concomitant elevation of p53 as early as 2 h, demonstrating an upstream signal for apoptosis. However, the p53 elevation in GN-treated CASMCs was independent of NAD(P)H: quinone oxidoreductase 1 and Mdm-2 expression. An increase in hydrogen peroxide and reduction in free thiols confirmed the role for oxidative stress in GN treatment. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) that significantly abrogated GN-induced CASMCs apoptosis suggested the involvement of caspase(s). The role of apical caspase-2, -8, and -9 was then investigated, and sequential activation of caspase-2 and -9 but not caspase-8 leading to downstream caspase-3 cleavage was observed in GN-treated CASMCs. Reduction of ATP level and decrease in oxygen consumption further confirmed the role of mitochondria in GN-induced apoptosis in CASMCs. The mitochondrial release of cytochrome c was seen without mitochondrial membrane potential loss and was independent of cardiolipin. These data provide insight into the mechanisms of GN-induced apoptosis, which may have important implications in the development of drug-eluting stents.
Asthma is associated with increased pulmonary inflammation and airway hyperresponsiveness. The interaction between airway epithelium and inflammatory mediators plays a key role in the pathogenesis of asthma. In vitro studies evaluated the inhibitory effects of 3-(2,5-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMPF-1), a synthetic chalcone analogue, upon inflammation in the A549 lung epithelial cell line. DMPF-1 selectively inhibited TNF-α-stimulated CC chemokine secretion (RANTES, eotaxin-1, and MCP-1) without any effect upon CXC chemokine (GRO-α and IL-8) secretion. Western blot analysis further demonstrated that the inhibitory activity resulted from disruption of p65NF-κB nuclear translocation without any effects on the mitogen-activated protein kinase (MAPK) pathway. Treatment of ovalbumin-sensitized and ovalbumin-challenged BALB/c mice with DMPF-1 (0.2-100 mg/kg) demonstrated significant reduction in the secretion and gene expression of CC chemokines (RANTES, eotaxin-1, and MCP-1) and Th2 cytokines (IL-4, IL-5, and IL-13). Furthermore, DMPF-1 treatment inhibited eosinophilia, goblet cell hyperplasia, peripheral blood total IgE, and airway hyperresponsiveness in ovalbumin-sensitized and ovalbumin-challenged mice. In conclusion, these findings demonstrate the potential of DMPF-1, a nonsteroidal compound, as an antiasthmatic agent for further pharmacological evaluation.
The hydroquinone and catechol like metabolites, NCQ344 and NCQ436 respectively, of the antipsychotic remoxipride have recently been demonstrated to induce apoptosis in myeloperoxidase (MPO)-rich human bone marrow progenitor and HL-60 cells [S.M. McGuinness, R. Johansson, J. Lundstrom, D. Ross, Induction of apoptosis by remoxipride metabolites in HL-60 and CD34+/CD19- human bone marrow progenitor cells: potential relevance to remoxipride-induced aplastic anemia, Chem. Biol. Interact. 121 (1999) 253-265]. In the present study, we determined the molecular mechanisms of apoptosis induced by these remoxipride metabolites in HL-60 cells. Our results show that apoptosis was accompanied by phosphatidylserine (PS) exposure, activation of caspases-9, -3, -7 and DNA cleavage. In HL-60 cells treated with the hydroquinone NCQ344 and catechol NCQ436, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp. fluoromethyl ketone (Z-VAD.FMK) blocked DNA cleavage and activation of caspases-9, -3/-7. In addition, PS exposure was significantly but not completely inhibited by Z-VAD.FMK. These results demonstrate that although Z-VAD.FMK inhibitable caspases are necessary for maximal apoptosis induced by NCQ344 and NCQ436, additional caspase-independent processes may orchestrate changes leading to PS exposure during apoptosis induced by the remoxipride polyphenolic metabolites.
High anion gap metabolic acidosis (HAGMA) is a hallmark of Diabetic Ketoacidosis (DKA). Occasionally, a Normal Anion Gap Metabolic Acidosis (NAGMA) can be seen, especially during the treatment phase. In this case report, a 55-year-old lady with diabetes mellitus who presented with a 2-day history of fever, lethargy and multiple episodes of vomiting and diarrhoea. Initial laboratory investigations revealed: capillary blood glucose as 27 mmol/L, urine ketone as 3+, blood ketone as 3.5 mmol/L, serum bicarbonate as 14 mmol/L, and serum chloride as 95 mmol/L. She was treated with intravenous normal saline fluid resuscitation and constant rate insulin infusion which was fortunately accompanied by stabilization of blood glucose and normalization of blood ketone to 0.2 mmol/L. However, despite normalization of her anion gap (25 to 14), she remained unwell with acidotic breathing due to refractory hyperchloraemic NAGMA with bicarbonate at 11 mol/L and chloride of 112 mmol/L. It was then decided to administer 100 mL of 8.4% Sodium Bicarbonate solution. The next day, she was no longer tachypneic as her bicarbonate and carbon dioxide improved to 21 mmol/L and 32 mmHg respectively. The presence of NAGMA in DKA should prompt clinicians to conduct a thorough search for possible underlying causes, such as gastrointestinal fluid loss, sepsis and chloride load from aggressive fluid resuscitation with normal saline. Sodium bicarbonate should only be considered in intractable cases to correct a NAGMA and not routinely used in the treatment of DKA.
Fulminant type 1 diabetes mellitus (FT1DM) is recognised as a novel subtype of type 1 diabetes mellitus characterised by the abrupt onset of insulin-deficient hyperglycaemia and ketoacidosis. Fulminant type 1 diabetes mellitus is known to be associated with pregnancy and had been associated with high fetal mortality. We report a case of a gestational diabetes mellitus (GDM) mother complicated with FT1DM immediately post-delivery. A 29-year-old Malay lady who was diagnosed with GDM at 19 weeks of pregnancy, underwent emergency lower segment caesarean section (EMLSCS) due to fetal distress at 36 weeks of gestation; 18 h post-EMLSCS, she developed abrupt onset Diabetic ketoacidosis (DKA) (blood glucose 33.5 mmol/L, pH 6.99, bicarbonate 3.6 mmol/L, ketone 4.4 mmol/L and HbA1c 6.1%). She received standard DKA treatment and discharged well. Her plasma C-peptide level 3 weeks later showed that she has no insulin reserve (C-peptide <33 pmol/L, fasting blood glucose (FBS) 28 mmol/L). Her pancreatic autoantibodies were negative. This case highlights that FT1DM not only can occur in pregnancy with normal glucose tolerance but can also complicate mother with GDM.
The caspase inhibitor benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and l-cysteine, whereas d-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.
The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose-response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis.
Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T-cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases-3 and -7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP-ribose) polymerase cleavage (PARP). Pre-treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin-induced apoptosis in Jurkat T-cells.
Limonene oxidation products (LOPs) have gained interest on their harmful health effects over time. Recently, studies have shown that the selected LOPs: 4-oxopentanal (4-OPA), 3-isopropenyl-6-oxo-heptanal (IPOH) and 4-acetyl-1-methylcyclohexene (4-AMCH) have sensory irritation effects in mice and inflammatory effects in human lung cells. This study was therefore undertaken to investigate the potential capacity of 4-OPA, IPOH and 4-AMCH to cause cell membrane damage, oxidative stress and inflammation in human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines. Overall results suggest that 4-OPA, IPOH have cytotoxic effects on human lung cells that might be mediated by ROS: the highest concentration applied of IPOH [500 μM] enhanced ROS generation by 100-fold ± 7.7 (A549) and 230-fold ± 19.9 (16HBE14o-) compared to the baseline. 4-OPA [500 μM] increased ROS levels by 1.4-fold ± 0.3 (A549) and by 127-fold ± 10.5 (16HBE14o-), while treatment with 4-AMCH [500 μM] led to 0.9-fold ± 0.2 (A549) and 49-fold ± 12.8 (16HBE14o-) increase. IPOH [500 μM] caused a decrease in the thiol-state balance (e.g. after 2 h, GSH:GSSG was reduced by 37% compared to the untreated 16HBE14o-cells). 4-OPA [500 μM] decreased the GSH:GSSG by 1.3-fold change in A549 cells and 1.4-fold change in 16HBE14o-cells. No statistically significant decrease in the GSH:GSSG in A549 and 16HBE14o-cell lines was observed for 4-AMCH [500 μM]. In addition, IPOH and 4-OPA [31.2 μM] increased the amount of the inflammatory markers: RANTES, VEGF and EGF. On the other hand, 4-AMCH [31.2 μM] did not show inflammatory effects in A549 or 16HBE14o-cells. The 4-OPA, IPOH and 4-AMCH treatment concentration and time-dependently induce oxidative stress and/or alteration of inflammatory markers on human bronchial and alveolar cell lines.
A series of hitherto unreported piperidone embedded α,β-unsaturated ketones were synthesized efficiently in ionic solvent and evaluated for cholinesterase inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Most of the synthesized compounds displayed good enzyme inhibition; therein compounds 7i and 7f displayed significant activity against AChE with IC50 values of 1.47 and 1.74 µM, respectively. Compound 6g showed the highest BChE inhibitory potency with IC50 value of 3.41 µM, being 5 times more potent than galanthamine. Molecular modeling simulation was performed using AChE and BChE receptors extracted from crystal structure of human AChE and human BChE to determine the amino acid residues involved in the binding interaction of synthesized compounds and their relevant receptors.
Fresh roselle are high in moisture and deteriorate easily, which makes drying important for extending shelf-life and increasing availability. This study investigated the influence of different drying methods (oven-drying, freeze-drying, vacuum-drying, and sun-drying) on the quality of roselle calyx expressed as physicochemical properties (moisture content, water activity, soluble solids, color), volatile compounds, and microstructure. Oven-drying and freeze-drying reduced moisture content most while vacuum-drying and sun-drying were not as efficient. All drying methods except sun-drying resulted in water activities low enough to ensure safety and quality. Vacuum-drying had no impact on color of the dry calyx and only small impact on color of water extract of calyx. Drying reduced terpenes, aldehydes, and esters but increased furans. This is expected to reduce fruity, floral, spicy, and green odors and increase caramel-like aroma. Sun-drying produced more ketones, alcohols, and esters. Scanning electron microscopy revealed that freeze-drying preserved the cell structure better, and freeze-dried samples resembled fresh samples most compared to other drying techniques. The study concludes that freeze-drying should be considered as a suitable drying method, especially with respect to preservation of structure.
Adamantyl-based compounds are commercially important in the treatments for neurological conditions and type-2 diabetes, aside from their anti-viral abilities. Their values in drug design are chronicled as multi-dimensional. In the present study, a series of 2-(adamantan-1-yl)-2-oxoethyl benzoates, 2(a-q), and 2-(adamantan-1-yl)-2-oxoethyl 2-pyridinecarboxylate, 2r, were synthesized by reacting 1-adamantyl bromomethyl ketone with various carboxylic acids using potassium carbonate in dimethylformamide medium at room temperature. Three-dimensional structures studied using X-ray diffraction suggest that the adamantyl moiety can serve as an efficient building block to synthesize 2-oxopropyl benzoate derivatives with synclinal conformation with a looser-packed crystal packing system. Compounds 2a, 2b, 2f, 2g, 2i, 2j, 2m, 2n, 2o, 2q and 2r exhibit strong antioxidant activities in the hydrogen peroxide radical scavenging test. Furthermore, three compounds, 2p, 2q and 2r, show good anti-inflammatory activities in the evaluation of albumin denaturation.
The impacts of rubber variations (clonal, seasonal, and pre-treatment) were investigated to assess changes in the composition of volatile organic compounds (VOCs) emitted during rubber processing. VOC emissions from 14 different rubber types were evaluated by headspace micro-chamber (μ-TEC) extraction coupled with gas chromatography-mass spectrometry (GC-MS). Headspace extracted at 120 °C, which is equivalent to the drying temperature during rubber processing, revealed a significant number of odorants in terms of concentrations as well as odorant type. Volatile fatty acids (VFAs) such as acetic, propanoic, butanoic, pentanoic and hexanoic acids, were frequently detected at concentrations greater than their odour detection thresholds. Other odorous compounds including trimethylamine, p-cresol, butanone, indole, and phenol, were also detected. Emissions collected at ambient conditions represent odorants released during material storage (or maturation) and were dominated by benzene derivatives followed by ketones, aldehydes, esters, and acids. Emission composition during storage appeared to be governed by specific rubber properties such as protein and rubber moisture content. Seasonal variations revealed greater impacts on the concentration of VOCs for all studied clones, compared to pre-treatment variations, suggesting that the VOCs composition was seasonally dependent and may represents the 'potential' emissions from rubber as they are processed. A combination of sensorial and analytical measurements were used to produce odour wheels which may be used as tool to identify key malodours in onsite rubber processing. The linking of odours and odorants can facilitate communication between receptors (the public) and plant operators inorder to minimise odour impact and develop effective abatement and on-site management practices.
Aromatic (ar)-turmerone is one of the aromatic constituents abundant in turmeric essential oil from Curcuma longa. Ar-turmerone exhibited anti-inflammatory properties. So far, antiplasmodial data for ar-turmerone is still not reported. The data showed the in vitro antiplasmodial effect of ar-turmerone against Plasmodium falciparum 3D7 (chloroquine-sensitive) via Plasmodium lactate dehydrogenase assay (pLDH) and cytotoxic effect against Vero mammalian kidney cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colourimetric assay. Selectivity indexes of ar-turmerone were calculated based on inhibition concentration at 50% of parasite growth (IC50) from MTT and pLDH assays and the effects of ar-turmerone were compared to the antimalarial reference drug chloroquine diphosphate. The inhibitory effect of ar-turmerone at the intraerythrocytic stages of plasmodial lifecycles was evaluated via a stage-dependant susceptibility test. The antiplasmodial and cytotoxic activities of ar-turmerone revealed IC50 values of 46.8 ± 2.4 μM and 820.4 ± 1.5 μM respectively. The selectivity index of ar-turmerone was 17.5. Ar-turmerone suppressed the ring-trophozoite transition stage of the intraerythrocytic life cycle of P. falciparum 3D7.
Probiotics may be used to enhance the functionality and nutritional values of fermented sausages. This study aims to evaluate the physicochemical and sensory properties of beef sausages fermented by lactic acid bacteria of Lactobacillus plantarum IIA-2C12 and L. acidophilus IIA-2B4. These strains were isolated from beef cattle and have shown to display probiotic features. While the nutrient contents were not affected by the probiotics, the pH, texture, and color varied among the sausages. Further analysis on fatty acids showed different profiles of saturated (C14:0, C17:0, and C20:0) and unsaturated (C14:1, C18:1n9c, C18:2n6c, and C22:6n3) fatty acids in sausages with probiotics. Gas chromatography-mass spectrometry further revealed some flavor development compounds including acid, alcohols, aldehydes, aromatic, ketones, sulfur, hydrocarbons and terpenes, varied among the sausages. Hedonic test showed no difference in the preference toward aroma, texture, and color for untrained panelists.
In this research work, we report the synthesis and biological evaluation of two new series of 1-benzyl-4-(benzylidenehydrazono)-3,4-dihydro-1H-benzo[c] [1,2]thiazine 2,2-dioxides and 1-benzyl-4-((1-phenylethylidene)hydrazono)-3,4-dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxides. The synthetic plan involves the mesylation of methyl anthranilate with subsequent N-benzylation of the product. The methyl 2-(N-benzylmethylsulfonamido)benzoate was subjected to cyclization reaction in the presence of sodium hydride to obtain 1-benzyl-1H-benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxide which was treated with hydrazine hydrate to get corresponding hydrazone precursor. Finally, the titled compounds were obtained by reaction of hydrazone with various substituted aldehydes and ketones. The synthesized derivatives were subjected to carry out their inhibition activities against monoamine oxidases along with modelling investigations to evaluate their binding interactions and dynamic stability during the docking studies. The inhibition profile of potent compounds was found as competitive for both the isozymes. The compounds were more selective inhibitors of MAO-A as compared to MAO-B. Moreover, drug likeness profile of the derivatives was evaluated to have an additional insight into the physicochemical properties. The molecular dynamic simulations predicted the behaviour of amino acids with the active site residues.
Synthesis of a series of benzimidazole-ornamented pyrazoles, 6a-6j has been obtained from arylhydrazine and aralkyl ketones via a multistep synthetic strategy. Among them, a hybrid-possessing para-nitrophenyl moiety connected to a pyrazole scaffold (6a) exerted the highest anti-inflammatory activity, which is superior to the standard, diclofenac sodium. While executing the 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, a hybrid-possessing para-bromophenyl unit integrated at the pyrazole structural motif (6i) exhibited the highest activity among the hybrids examined. Besides, evaluation of anticancer potency of the synthesized hybrids revealed that the one containing a para-fluorophenyl unit tethered at the pyrazole nucleus (6h) showed the highest activity against both the pancreatic cancer cells (SW1990 and AsPCl) investigated. Considerable binding affinity between B-cell lymphoma and the hybrid, 6h has been reflected while performing molecular docking studies (-8.65 kcal/mol). The outcomes of the investigation expose that these hybrids could be used as effective intermediates to construct more potent biological agents.