Identification and partial characterization of discrete apolipoprotein B containing lipoprotein particles produced by human hepatoma cell line HepG2

The purpose of this study was to test the use of human hepatocarcinoma HepG2 cells as a model for studying the formation and secretion of human hepatic lipoproteins. To this end, we determined the rate of accumulation and percent composition of neutral lipids and apolipoproteins in the culture medium of HepG2 cells and isolated and partially characterized the apolipoprotein B (ApoB) containing lipoprotein particles. The rates of accumulation in the medium of HepG2 cells, grown in minimum essential medium during a 24-h incubation, of triglycerides, cholesterol, and cholesterol esters expressed as microgram/(g of cell protein X h) were 373 +/- 55, 167 +/- 14, and 79 +/- 10, respectively; the secretion rates for apolipoproteins B, A-I, E, A-II, and C-III were 372 +/- 36, 149 +/- 14, 104 +/- 13, 48 +/- 4, and 13 +/- 1 microgram/(g of cell protein X h), respectively. The major portion of ApoB was present in very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) (84%), with the remainder occurring in high-density lipoproteins (HDL) (16%). Approximately 10-13% of ApoA-I and ApoA-II were present in VLDL and LDL, while 60% of ApoE occurred in HDL and 40% in VLDL and LDL. To separate ApoB-containing lipoproteins, secreted lipoproteins were fractionated by either sequential immunoprecipitation or immunoaffinity chromatography with antibodies to ApoB and ApoE. Results showed that 60-70% of ApoB occurred in the culture medium as lipoprotein B (LP-B) and 30-40% as lipoprotein B:E (LP-B:E). Both ApoB-containing lipoproteins represent polydisperse systems of spherical particles ranging in size from 100 to 350 A for LP-B and from 200 to 500 A for LP-B:E. LP-B particles were identified in VLDL, LDL, and HDL, while LP-B:E particles were only present in VLDL and LDL. The major neutral lipid of both ApoB-containing lipoproteins was triglyceride (50-70% of the total neutral lipid content); cholesterol and cholesterol esters were present in equal amounts. The LP-B:E particles contained 70-90% ApoB and 10-30% ApoE. The ApoB was identified in both types of particles as B-100. A time study on the accumulation of ApoB-containing lipoproteins showed that LP-B particles were secreted independently of LP-B:E particles.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Validated spectrofluorimetric assay of two co-administered drug mixtures containing hydroxychloroquine with either moxifloxacin or ofloxacin as a drug regimen for hospital-acquired pneumonia in patients with COVID-19

Moxifloxacin and ofloxacin are two broad-spectrum quinolone antibiotics. They are among the most widely used antibiotics, at this time, applied to control the COVID-19 pandemic. Hydroxychloroquine is an FDA-approved drug for the treatment of COVID-19. This work describes a simple, green, selective, and sensitive spectrofluorimetric method for the assay of moxifloxacin and ofloxacin in the presence of hydroxychloroquine, two co-administered mixtures used in the treatment of hospital-acquired pneumonia in patients with COVID-19. Simultaneous assay of hydroxychloroquine and moxifloxacin was carried out in methanol using a direct spectrofluorimetric method (method I) at 375 and 550 nm, respectively, after excitation at 300 nm. The direct spectrofluorimetric assay was rectilinear over concentration ranges 50.0–400.0 and 300.0–2500.0 ng/ml for hydroxychloroquine and moxifloxacin, respectively, with limits of detection (LOD) of 6.4 and 33.64 ng/ml and limits of quantitation (LOQ) of 19.4 and 102.6 ng/ml, respectively, for the two drugs. The assay for hydroxychloroquine and ofloxacin was carried out by measuring the first derivative synchronous amplitude for hydroxychloroquine at the zero crossing point of ofloxacin and vice versa at Δλ = 140 nm (method II). Hydroxychloroquine was measured at 266 nm, while ofloxacin was measured at 340 nm over the concentration range 4–40 ng/ml for hydroxychloroquine and 200–2000 ng/ml for ofloxacin with LOD of 0.467 and 25.3 ng/ml and LOQ of 1.42 and 76.6 ng/ml, respectively, for the two drugs. The two methods were validated following International Conference on Harmonization guidelines and were applied to the analysis of the two drugs in plasma with good percentage recoveries (109.73–93.17%).     

Effect of mesenchymal stem cells on cytochrome-c release and inflammation in colon cancer induced by 1,2-dimethylhydrazine in Wistar albino rats

Colon cancer is one of the most common causes of deaths by cancer worldwide. Stem cells have immunosuppressive properties that promote tumor targeting and circumvent obstacles currently in gene therapy. Bone marrow stem cells are believed to have anticancer potential. The transplantation of mesenchymal stem cells (MSCs), a type of bone marrow stem cells, has been considered a potential therapy for patients with solid tumors due to their capability to enhance the immune response; MSC transplantation has received renewed interest in recent years. The present study aimed to evaluate the antiapoptotic effects of the MSCs on 1,2-dimethylhydrazine (DMH)-induced inflammation in the rat model of colorectal cancer. The rats were randomly allocated into four groups: control, treated with MSCs, induced by DMH, and induced by DMH and treated with MSCs. The MSCs were intra-rectally injected, and DMH was subcutaneously injected at 20 mg/kg body weight once a week for 15 weeks. The administration of MSCs into rats starting from day 0 of the DMH injection was found to enhance the histopathological picture. The MSC treatment resulted in fewer inflammatory cells than in the DMH group. Therefore, our findings suggest that BMCs have antitumor effects by modulating the cellular redox status and down-regulating the pro-inflammatory genes. Thus, BMCs may provide therapeutic value for colon cancer treatment.     

Effectiveness of naturally occurring Aphis gossypii on tomato plants as a bio-indicator for heavy metals in Riyadh and Hafar Al-Batin,Saudi Arabia

Although certain pollutants can be biologically degraded by microorganisms, rendering their impact short-term, others can not be impaired, such that their effect persists. The present study evaluates the effectiveness of using a field-collected aphid, Aphis gossypii, as a bio-indicator for heavy metals in tomato farms in Riyadh and Hafar Al-Batin, Saudi Arabia. Heavy metals were selected (Cd, Cu, Zn, and Pb) and measured for comparative screening in field-collected plants, soil, and aphids using inductively coupled plasma-mass spectrometry (ICP-MS). Field-collected aphids from both studied regions were identified as Aphis gossypii. In Riyadh, there was no significant difference observed for Cd, Cu, and Zn for all experimental samples, while, Pb was showed differences among samples especially tomato leaves None of the studied samples in Hafar Al-Batin were showed statistically significant differences in Cd, in reverse to significant differences in the other heavy metals. Comparing concentrations of selected heavy metals between the two studied regions was showed that neither region showed a significant difference in heavy metals except for Cu. This study demonstrates that tomato leaf samples showed the highest concentrations of most studied heavy metals, followed by soil, then aphids. Aphids were utilized as a bio-indicator of heavy metals in the studied regions.     

Cytotoxicity of Anthrax Lethal Toxin to Human Acute Myeloid Leukemia Cells Is Nonapoptotic and Dependent on Extracellular Signal-Regulated Kinase 1/2 Activity

In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx). LeTx consists of protective antigen (PrAg) and lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the MEK1/2-extracellular signal-regulated kinase (ERK1/2) branch of the MAPK pathway. The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LeTx and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LeTx-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LeTx-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.     

Melatonin activates the vascular elements,telocytes, and neuroimmune communication in the adrenal gland of Soay rams during the non-breeding season

Protoplasma - The adrenal glands of 15 adult Soay rams were used to study the effect of melatonin on their vascular elements and cellular organization. A significant increase in the cross-sectional...     

Expression profile of lncRNAs and miRNAs in esophageal cancer: Implications in diagnosis,prognosis, and therapeutic response

Esophageal cancer is the seventh most common cancer worldwide. Although a number of environmental and lifestyle-related risk factors have been identified for this kind of cancer, the exact molecular mechanisms of tumor evolution have not been clarified yet. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) as important regulators of gene expression and chromatin configuration have essential roles in the pathogenesis of esophageal cancer. They have been shown to alter the function of cancer-related signaling pathways such as phosphoinositide 3-kinase/protein kinase B and Wnt pathway, thus they might modulate the response of patients to pathway-targeted therapies. Moreover, a number of lncRNAs, such as AFAP1-AS1, UCA1, HOTAIR, LOC285194, and TUSC7, are involved in conferring chemoresistant/radioresistant in esophageal cancer cells. A complex network of interaction exists between lncRNAs and miRNAs in the context of esophageal cancer. Finally, various panels of lncRNAs and miRNAs have been introduced that can predict the survival of esophageal cancer patients. In this review article, we summarize the recent findings regarding the role of miRNAs and lncRNAs in the pathogenesis of esophageal cancer with the special focus on their regulatory roles on signaling pathways, their potential as diagnostic/prognostic markers, and their relevance with therapeutic response.     

A possible role for inducible arginase isoform (AI) in the pathogenesis of chronic venous leg ulcer

Chronic venous ulcer (CVU) is a major cause of chronic wounds of lower extremities and presents a significant financial and resource burden to health care systems worldwide. Defects in the vasculature, matrix deposition, and re-epithelialization are the main histopathological changes believed to impede healing. Supplementation of the amino acid arginine that plays a crucial role in the interactions that occur during inflammation and wound healing was proven clinically to improve acute wound healing probably through enhancing activity of inducible arginase (AI) locally in the wounds. However, the possible mechanism of arginine action and the potential beneficial effects of AI/arginine in human chronic wounds remain unclear. In the present study, using biopsies, taken under local anesthesia, from adult patients (n = 12, mean age 55 years old) with CVUs in lower extremities, we investigated the correlation between AI distribution in CVUs and the histopathological changes, mainly proliferative and vascular changes. Our results show a distinct spatial distribution of AI along the ulcer in the epidermis and in the dermis with the highest level of expression being at the ulcer edge and the least expression towards the ulcer base. The AI cellular immunoreactivity, enzymatic activity, and protein levels were significantly increased towards the ulcer edge. Interestingly, a similar pattern of expression was encountered in the proliferative and the vascular changes with strong correlations between AI and the proliferative activity and vascular changes. Furthermore, AI cellular distribution was associated with increased proliferative activity, inflammation, and vascular changes. Our findings of differential expression of AI along the CVU base, edge, and nearby surrounding skin and its associations with increased proliferative activity and vascular changes provide further support to the AI implication in CVU pathogenesis. The presence of high levels of AI in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention.