Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.     

Bioemulsifier production by Aspergillus niger MYA 135: presumptive role of iron and phosphate on emulsifying ability

Microbial emulsifiers are compounds employed in primary mechanisms for bioremediation of petroleum and other hydrocarbon pollutants from the environment. Although emulsifiers of biological origin are produced by microorganisms generally in response to growth on hydrocarbons, Aspergillus niger MYA 135 produced a bioemulsifier during fermentation in a sucrose-based culture medium at an initial pH of 5.0 and at 30°C. The production of bioemulsifiers can be strongly influenced by environmental factors. In this connection, a study of the effect of initial pH, the incubation temperature and presence of CaCl₂ or FeCl₃ in the culture medium was conducted. Emulsification index was increased by 112 and 206% at an initial pH 2.0 or in medium supplemented with FeCl₃, respectively. On the other hand, emulsifying ability of Aspergillus niger supernatants was detected during the exponential phase, suggesting that bioemulsifiers accumulation and microbial growth would be related. Interestingly, this study suggests that iron and/or phosphate ions would play a key role in maintaining the emulsifying ability. Finally, factorial design was also employed to study the effects of the initial pH, the presence of FeCl₃ and the concentration of KH₂PO₄ on the emulsification index.     

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBF_nn) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N⁸-acetylated spermidine, inhibit the hydrolytic activity of TanBF_nn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBF_nn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.     

P2X7 receptors and glioma cells

Purinergic Signalling -     

Sexual selection of anther trichomes and sexual dimorphism in Ibervillea lindheimeri (Cucurbitaceae: Melothrieae)

Ibervillea lindheimeri is a dioecious member of the Cucurbitaceae whose male flowers possess a number of sex-specific characteristics including eglandular trichomes on the anther connective. A 2-year study (1989 and 1990) was undertaken to assess the potential for sexual selection of anther trichome number as a male floral trait that enhances pollen removal (a male fitness component). Correlation analysis of ten reproductive and floral traits showed that only the mean number of anther trichomes per flower was significantly associated with mean pollen removal per flower. Anther trichomes are two-celled, consisting of a small, caplike, apical cell and a large, bulbous basal cell. The basal cell is under tension and produces a sticky polysaccharide that is forcefully ejected upward when the apical cell is broken off by visiting bees. The sticky polysaccharide accumulates on the bees and aids in adherence of pollen. We propose that there is directional selection on individual males for number of anther trichomes per flower. The more anther trichomes per flower, the more pollen exported, and the greater the likelihood of siring seeds. Males differed significantly in mean number of anther trichomes per flower and were relatively consistent in their rank order over a 2-year period; both are prerequisites for sexual selection to operate. Selection coefficients and differentials for 1989 and 1990 were 0.005 and 0.007, and 0.22 and 0.32, respectively, and suggest moderate to strong directional selection on anther trichome number.     

Hypoxia increases susceptibility of non-small cell lung cancer cells to complement attack

The complement system can be specifically targeted to tumor cells due to molecular changes on their surfaces that are recognized by complement directly or via naturally occurring antibodies. However, tumor cells often overexpress membrane-bound complement inhibitors protecting them from complement attack. We have previously shown that non-small cell lung cancer (NSCLC) cells, additionally to membrane-bound inhibitors, produce substantial amounts of soluble regulators such as factor I (FI) and factor H (FH). Since low oxygen concentration is associated with rapidly growing solid tumors, we studied how NSCLC cells protect themselves from complement attack under hypoxic conditions. Unexpectedly, mRNA levels and secretion of both FI and FH were significantly decreased already after 24 h exposure to hypoxia while cell viability measured by XTT assay and annexin V/7-AAD staining was affected only marginally. Furthermore, we observed decrease of mRNA level and loss of membrane-bound complement inhibitor CD46 and increased deposition of early (C3b) and terminal (C9) complement components on hypoxic NSCLC cells. All three complement pathways (classical, lectin and alternative) were employed to deposit C3b on cell surface. Taken together, our results imply that under hypoxic conditions NSCLC give up some of their available defense mechanisms and become more prone to complement attack.