Irene-Eva Triantaphyllidou

Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content

In this short review, we summarize the latest research in the production of polyunsaturated microbial oils that are of interest in food technology. The current research targets the productivity of oleaginous microorganisms, as well as the biosynthesis of particular polyunsaturated fatty acids (PUFAs). The most important efforts target the efficiency of the oleaginous machinery, via overexpression of key-enzymes involved in lipid biosynthesis, as well as the minimization of lipid degradation, by repressing genes involved in the β-oxidation pathway. The production of specific PUFAs is approached by homologous or heterologous expression of specific desaturases and elongases involved in PUFA biosynthesis in oleaginous microorganisms. New perspectives, such as the production of triacylglycerols of specific structure and the employment of adaptive experimental evolution for creating robust oleaginous strains able to produce PUFAs are also discussed.

Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment

Yarrowia lipolytica, an ascomycete with biotechnological potential, is able to form either yeast cells or hyphae and pseudohyphae in response to environmental conditions. This study shows that the morphology of Y. lipolytica, cultivated in batch cultures on hydrophilic (glucose and glycerol) and hydrophobic (olive oil) media, was not affected by the nature of the carbon source, nor by the nature or the concentration of the nitrogen source. By contrast, dissolved oxygen concentration (DOC) should be considered as the major factor affecting yeast morphology. Specifically, when growth occurred at low or zero DOC the mycelial and/or pseudomycelial forms predominated over the yeast form independently of the carbon and nitrogen sources used. Experimental data obtained from a continuous culture of Y. lipolytica on glycerol, being used as carbon and energy source, demonstrated that the mycelium-to-yeast form transition occurs when DOC increases from 0.1 to 1.5 mg l(-1). DOC also affected the yeast physiology, as the activity of enzymes implicated in lipid biosynthesis (i.e. ATP-citrate lyase, malic enzyme) was upregulated at high DOC whereas the activity of enzymes implicated in glycerol assimilation (such as glycerol dehydrogenase and kinase) remained fundamentally unaffected in the cell-free extract.

Perturbations in the HDL metabolic pathway predispose to the development of osteoarthritis in mice following long-term exposure to western-type diet

OBJECTIVE Recent data suggest that obesity and related metabolic aberrations are associated with osteoarthritis (OA) development, a phenomenon that is attributed at least in part to the consumption of lipid-rich diets. To date, the molecular mechanisms that govern the lipid-OA connection remain largely unknown. Given the important role of high-density lipoprotein (HDL) in plasma and tissue lipid metabolism, the main purpose of the present study was to investigate the role of HDL metabolism in the pathobiology of OA. METHODS We used apolipoprotein A-I (apoA-I)(-/-) mice that lack classical apoA-I containing HDL, LCAT(-/-) mice that have only immature HDL and relatively reduced HDL-cholesterol levels and control C57BL/6 mice. Mice were placed on chow or western-type (WTD) and monitored for 24 weeks. Knee joints were removed and articular cartilage was isolated for further analyses. RESULTS The LCAT(-/-) mice were significantly more sensitive to the development of diet-induced obesity compared to the C57BL/6 and apoA-I(-/-) mice. Morphological, biochemical and molecular analyses revealed that the LCAT(-/-) obese mice developed OA, while the C57BL/6 mice that were fed WTD did not. Notably, apoA-I(-/-) mice that received WTD also developed OA although their body-weight gain was similar to their wild-type counterparts. Interestingly, bone marrow from LCAT(-/-) and apoA-I(-/-) mice contained significantly increased number of adipocytes, compared to the other groups. CONCLUSIONS Our findings suggest that perturbations in HDL metabolism predispose to OA following chronic insult with WTD and raise the challenging possibility that HDL has a causative relation to OA in patients with metabolic syndrome.

High lipid accumulation in Yarrowia lipolytica cultivated under double limitation of nitrogen and magnesium

Yarrowia lipolytica cultivated under double nitrogen and magnesium limitation, but not under single nitrogen or single magnesium limitation, produced 12.2g/l biomass containing 47.5% lipids, which corresponds to a lipid production 5.8g/l. These yields are the higher described in the literature for wild strains of Y. lipolytica. Transcription of ACL1 and ACL2, encoding for ATP-citrate lyase (ATP:CL) was observed even under non-oleaginous conditions but high activity of ATP:CL was only detected under oleaginous conditions induced by low or zero activity of NAD(+) dependent isocitrate dehydrogenase. The low activity of malic enzyme (ME), a NADPH donor in typical oleaginous microorganisms, indicated that ME may not be implicated in lipid biosynthesis in this yeast, and NADPH may be provided by the pentose phosphate pathway (PPP). These findings underline the essential role of magnesium in lipogenesis, which is currently quite unexplored. The presence of organic nitrogen in low concentrations during lipogenesis was also required, and this peculiarity was probably related with the PPP functioning, being the NADPH donor of lipogenic machinery in Y. lipolytica.

A proposed mechanism for the inhibitory effect of the anticancer agent titanocene dichloride on tumour gelatinases and other proteolytic enzymes

Titanocene dichloride, the most studied metallocene, exhibits antiproliferative activity in a wide spectrum of murine and human tumours. In this article it is demonstrated that titanocene dichloride inhibits tumour gelatinases in a dose-dependent manner. Substrate saturation experiments and the fact that the IC50 values were increased in correlation with collagen substrate concentrations indicate that the titanocene dichloride induced inhibition is of a competitive type. Titanocene dichloride also specifically inhibits clostridium collagenase and trypsin, particularly when collagens are used as substrates. Binding experiments demonstrate that cyclopentadiene–Ti(IV) moieties, resulting from titanocene dichloride at physiological pH, are bound mainly to different types of collagens and to a lesser extent to casein or bovine serum albumin, forming soluble and stable adducts. These results indicate that titanocene dichloride behaves as a competitive inhibitor against various proteolytic enzymes by binding to the substrate rather than to the enzyme active site. This property may be responsible for the antiangiogenic effect of titanocene dichloride and additionally contributes to its anticancer action.

Apolipoprotein A-I Modulates Processes Associated with Diet-Induced Nonalcoholic Fatty Liver Disease in Mice

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL). We investigated the involvement of apoA-I in diet-induced accumulation of triglycerides in hepatocytes and its potential role in the treatment of nonalcoholic fatty liver disease (NAFLD). ApoA-I-deficient (apoA-I−/−) mice showed increased diet-induced hepatic triglyceride deposition and disturbed hepatic histology while they exhibited reduced glucose tolerance and insulin sensitivity. Quantification of FASN (fatty acid synthase 1), DGAT-1 (diacylglycerol O-acyltransferase 1), and PPARγ (peroxisome proliferator-activated receptor γ) mRNA expression suggested that the increased hepatic triglyceride content of the apoA-I−/− mice was not due to de novo synthesis of triglycerides. Similarly, metabolic profiling did not reveal differences in the energy expenditure between the two mouse groups. However, apoA-I−/− mice exhibited enhanced intestinal absorption of dietary triglycerides (3.6 ± 0.5 mg/dL/min for apoA-I−/− versus 2.0 ± 0.7 mg/dL/min for C57BL/6 mice, P < 0.05), accelerated clearance of postprandial triglycerides and a reduced rate of hepatic very low density lipoprotein (VLDL) triglyceride secretion (9.8 ± 1.1 mg/dL/min for apoA-I−/− versus 12.5 ± 1.3 mg/dL/min for C57BL/6 mice, P < 0.05). In agreement with these findings, adenovirus-mediated gene transfer of apoA-IMilano in apoA-I−/− mice fed a Western-type diet for 12 wks resulted in a significant reduction in hepatic triglyceride content and an improvement of hepatic histology and architecture. Our data extend the current knowledge on the functions of apoA-I, indicating that in addition to its well-established properties in atheroprotection, it is also an important modulator of processes associated with diet-induced hepatic lipid deposition and NAFLD development in mice. Our findings raise the interesting possibility that expression of therapeutic forms of apoA-I by gene therapy approaches may have a beneficial effect on NAFLD.

Feasibility of Raw Glycerol Conversion into Single Cell Oil by Zygomycetes Under Non-Aseptic Conditions

The use of plant oils as feedstock for the biodiesel manufacture has many drawbacks, thus, the interest has turned to single cell oil (SCO) as an alternative. However, the production of SCO is still too expensive, mainly due to the low oil productivity and the high cost of medium sterilization required. In this work raw glycerol was converted into SCO by oleaginous Zygomycetes under non-aseptic conditions on selective (i.e., containing essential oils and/or antibiotics) nitrogen limited media. The obtained data showed that although bacterial populations inhibited the fungal growth, lipid accumulation remained unaffected by the presence of bacteria in the growth medium compared to control experiments (conducted under aseptic conditions). Therefore, a two-stage process was developed in which growth was performed under aseptic conditions (1st stage) followed by lipid accumulation performed under non-aseptic conditions (2nd stage) in the presence of thyme essential oil as an antibacterial agent. Large amounts of lipids were accumulated inside the mycelia, yielding around 13% wt/wt of oil per glycerol consumed.

Expression and distribution of aggrecanases in human larynx: ADAMTS-5/aggrecanase-2 is the main aggrecanase in laryngeal carcinoma

Members of the ADAMTS family of proteases degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular functions. Aggrecanases are the main enzymes responsible for aggrecan degradation, due to their specific cleavage pattern. In this study, the expression status, the macromolecular organization and localization of ADAMTS-1, ADAMTS-4/aggrecanase-1 and ADAMTS-5/aggrecanase-2 in human normal larynx and laryngeal squamous cell carcinoma (LSCC) were investigated. On mRNA level, the results showed that ADAMTS-4 was the highest expressed enzyme in normal larynx, whereas ADAMTS-5 was the main aggrecanase in LSCC presenting a stage-related increase up to stage III (8-fold higher expression compared to normal), and thereafter decreased in stage IV. Accordingly, immunohistochemical analysis showed that ADAMTS-5, but not ADAMTS-4, was highly expressed by carcinoma cells. Sequential extraction revealed an altered distribution and organization of multiple molecular forms (latent, activated and fragmented forms) of the enzymes within the cancerous and their corresponding macroscopically normal laryngeal tissues, compared to the normal ones. Importantly, these analyses indicated that critical macromolecular changes occurred from the earliest LSCC stages not only in malignant parts of the tissue but also in areas that were not in proximity to carcinoma cells and appeared otherwise normal. Overall, the results of the present study show that ADAMTS-5/aggrecanase-2 is the main aggrecanase present in laryngeal carcinoma suggesting a critical role for the enzyme in aggrecan degradation and laryngeal tissue destruction during tumor progression.

The chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cancer: Expressional and epigenetic studies

BackgroundSignificant biochemical changes are observed in glycosaminoglycans in squamous cell laryngeal carcinoma. The most characteristics are in chondroitin/dermatan sulfate fine structure and proportion, which might be due to differential expression of the enzymes involved in their biosynthesis. The aim of the present work was the investigation in expressional and epigenetic level of the enzymes involved in chondroitin/dermatan sulfate biosynthesis in laryngeal cancer.MethodsTissues subjected to total RNA and DNA isolation, and protein extraction. The techniques used in this study were RT-PCR analysis, western blotting and methylation specific PCR.ResultsWe identified that many enzymes were expressed in the cancerous specimens intensively. Dermatan sulfate epimerase was expressed exclusively in the cancerous parts and in minor amounts in healthy tissues; in the macroscopically normal samples it was not detected. Furthermore, chondroitin synthase I and chondroitin polymerizing factor were strongly expressed in the cancerous parts compared to the corresponding normal tissues. Sulfotransferases, like chondroitin 6 sulfotransferase 3, were highly expressed mainly in healthy specimens.ConclusionsThe study of the various chondroitin/dermatan synthesizing enzymes revealed that they were differentially expressed in cancer, in human laryngeal cartilage, leading to specific chondroitin/dermatan structures which contributed to proteoglycan formation with specific features. The expression of the examined enzymes correlated with the glycosaminoglycan profile observed in previous studies.

Advances in the analysis of chondroitin/dermatan sulfate.

Publisher Summary Chondroitin/dermatan sulfate (CS/DS) is a glycosaminoglycan (GAG) found in extracellular matrix (ECM) and cell surface and participates in various ECM and cell–ECM interactions. It is made from the alternate addition of glucuronic acid (GlcA) and N‐acetylgalactosamine (GalNAc). However, during biosynthesis, CS/DS is sulfated on C‐4 and/or C‐6 of GalNAc and on C‐2 of GlcA and a part of GlcA is epimerized on C‐5 to obtain iduronic acid (IdoA), all steps depend on the tissue or cell status, which makes the molecule more complex. Because of the complexity of CS/DS, its analysis is useful to characterize the molecules present in a sample and identify alterations with respect to their amounts and fine structural features in various pathological conditions. This chapter presents the various techniques developed to analyze and characterize CS/DS. The analysis of CS/DS is used for either quantitative purposes or its characterization. Very simple assays of high efficiency are developed for analytical determinations, with or without prior separation or degradation of the other GAGs, such as chromogenic, electrophoretic, and solid phase assays. In addition, sophisticated techniques are proposed in the chapter for CS/DS structural characterization in highly purified samples, such as high performance liquid chromatography, capillary electrophoresis (CE), and hyphenated CE.