Lipid-free Medium Research Articles

Functionalized monodisperse microbubble production: microfluidic method for fast, controlled, and automated removal of excess coating material

Functionalized monodisperse microbubbles have the potential to boost the sensitivity and efficacy of molecular ultrasound imaging and targeted drug delivery using bubbles and ultrasound. Monodisperse bubbles can be produced in a microfluidic flow focusing device. However, their functionalization and sequential use require removal of the excess lipids from the bubble suspension to minimize the use of expensive ligands and to avoid competitive binding and blocking of the receptor molecules. To date, excess lipid removal is performed by centrifugation, which is labor intensive and challenging to automate. More importantly, as we show, the increased hydrostatic pressure during centrifugation can reduce bubble monodispersity. Here, we introduce a novel automated microfluidic ’washing’ method. First, bubbles are injected in a microfluidic chamber 1 mm in height where they are left to float against the top wall. Second, lipid-free medium is pumped through the chamber to remove excess lipids while the bubbles remain located at the top wall. Third, the washed bubbles are resuspended and removed from the device into a collection vial. We demonstrate that the present method can (i) reduce the excess lipid concentration by 4 orders of magnitude, (ii) be fully automated, and (iii) be performed in minutes while the size distribution, functionality, and acoustic response of the bubbles remain unaffected. Thus, the presented method is a gateway to the fully automated production of functionalized monodisperse microbubbles.

Intestinal absorption of sphingosine: new insights on generated ceramide species using stable isotope tracing in vitro

AbstractDietary sphingomyelin (SM) has been reported to favorably modulate postprandial lipemia. Mechanisms underlying these beneficial effects on cardiovascular risk markers are not fully elucidated. Rodent studies showed that tritiated SM was hydrolyzed in the intestinal lumen into ceramides (Cer) and further to sphingosine (SPH) and fatty acids (FA) that were absorbed by the intestine. Our objective was to investigate the uptake and metabolism of SPH and/or tricosanoic acid (C23:0), the main FA of milk SM, as well as lipid secretion in Caco-2/TC7 cells cultured on semipermeable inserts. Mixed micelles (MM) consisting of different digested lipids and taurocholate were prepared without or with SPH, SPH and C23:0 (SPH+C23:0), or C23:0. Triglycerides (TG) were quantified in the basolateral medium, and sphingolipids were analyzed by tandem mass spectrometry. TG secretion increased 11-fold in all MM-incubated cells compared with lipid-free medium. Apical supply of SPH-enriched MM led to increased concentrations of total Cer in cells, and coaddition of C23:0 in SPH-enriched MM led to a preferential increase of C23:0 Cer and C23:0 SM. Complementary experiments using deuterated SPH demonstrated that SPH-d9 was partly converted to sphingosine-1-phosphate-d9, Cer-d9, and SM-d9 within cells incubated with SPH-enriched MM. A few Cer-d9 (2% of added SPH-d9) was recovered in the basolateral medium of (MM+SPH)-incubated cells, especially C23:0 Cer-d9 in (MM+SPH+C23:0)-enriched cells. In conclusion, present results indicate that MM enriched with (SPH+C23:0), such as found in postprandial micelles formed after milk SM ingestion, directly impacts sphingolipid endogenous metabolism in enterocytes, resulting in the secretion of TG-rich particles enriched with C23:0 Cer.

Mapping the lipidomic secretome of the early equine embryo.

The lipidomic secretions of embryos provide a unique opportunity to examine the cellular processes of the early conceptus. In this study we profiled lipids released by the early equine conceptus, using high-resolution mass spectrometry to detect individual lipid species. This study examined the lipidomic profile in embryo-conditioned media from in vivo-produced, 8-9 day-old equine embryos (n = 3) cultured in vitro for 36 h, analyzed over 3 timepoints. A total of 1,077 lipid IDs were recorded across all samples, containing predominantly glycerolipids. Seventy-nine of these were significantly altered in embryo conditioned-media versus media only control (p < 0.05, fold-change >2 or < 0.5). Fifty-five lipids were found to be released into the embryo-conditioned media, of which 54.5% were triacylglycerols and 23.6% were ceramides. The sterol lipid, cholesterol, was also identified and secreted in significant amounts as embryos developed. Further, 24 lipids were found to be depleted from the media during culture, of which 70.8% were diacylglycerols, 16.7% were triacylglycerols and 12.5% were ceramides. As lipid-free media contained consistently detectable lipid peaks, a further profile analysis of the various components of non-embryo-conditioned media consistently showed the presence of 137 lipids. Lipid peaks in non-embryo-conditioned media increased in response to incubation under mineral oil, and contained ceramides, diacylglycerols and triacylglycerols. These results emphasize the importance of a defined embryo culture medium and a need to identify the lipid requirements of the embryo precisely. This study sheds light on early embryo lipid metabolism and the transfer of lipids during in vitro culture.

The Pharmacological or Genetic Blockade of Endogenous De Novo Fatty Acid Synthesis Does Not Increase the Uptake of Exogenous Lipids in Ovarian Cancer Cells.

Ovarian cancer(OC) is a serious threat to women worldwide. Peritoneal dissemination, ascites and omental metastasis are typical features for disease progression, which occurs in a micro-environment that is rich in high-energy lipids. OC cells require high amounts of lipids for survival and growth. Not only do they import lipids from the host, they also produce lipids de novo. Inhibitors of fatty acid(FA) synthase(FASN) – the rate-limiting enzyme of endogenous FA synthesis that is overexpressed in OC – induce growth-arrest and apoptosis, rendering them promising candidates for cancer drug development. However, cancer researchers have long hypothesized that the lipid deficiency caused by FASN inhibition can be circumvented by increasing the uptake of exogenous lipids from the host, which would confer resistance to FASN inhibitors. In contrast to a very recent report in colorectal cancer, we demonstrate in OC cells (A2780, OVCAR3, SKOV3) that neither FASN inhibitors (G28UCM, Fasnall) nor FASN-specific siRNAs can stimulate a relief pathway leading to enhanced uptake of extrinsic FAs or low density lipoproteins (LDLs). Instead, we observed that the growth-arrest due to FASN inhibition or FASN knock-down was associated with significant dose- and time-dependent reduction in the uptake of fluorescently labeled FAs and LDLs. Western blotting showed that the expression of the FA receptor CD36, the LDL receptor(LDLR) and the lipid transport proteins fatty acid binding proteins 1–9 (FABP1–9) was not affected by the treatment. Next, we compared experimental blockade of endogenous lipid production with physiologic depletion of exogenous lipids. Lipid-free media, similar to FASN inhibitors, caused growth-arrest. Although lipid-depleted cells have diminished amounts of CD36, LDLR and FABPs, they can still activate a restorative pathway that causes enhanced import of fluorophore-labeled FAs and LDLs. Overall, our data show that OC cells are strictly lipid-depend and exquisitely sensitive to FASN inhibitors, providing a strong rationale for developing anti-FASN strategies for clinical use against OC.

Abstract 1899: Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma

Abstract Melanoma is the most aggressive of all skin cancers, being the leading cause of death for cutaneous malignancy, with elevated metastatic spread and drug resistance. Because the deregulation of components of lipid metabolism may contribute to melanoma cell resistance, the aim of this study was to examine the dependence of melanoma cell growth on extracellular lipids, the associated lipid metabolism gene expression profiles and lipid impact on sensitivity to BRAF inhibitors. We applied preclinical pharmacology approaches including western blot analyses, growth inhibition assays quantitative Real-time PCR (qRT-PCR) in melanoma cell lines including variants with acquired resistance to BRAF inhibitors. Two matched pairs of sensitive and PLX4032-resistant cell lines (LM16 and LM36) were used. Both pairs carried V600E BRAF gene mutation and BRAF gene amplification, and LM36R displayed mutant NRAS. When compared to the parental sensitive cells, LM16R cells showed increased activation of MAPK-ERK and PI3K-Akt-mTOR pathways, while LM36R cells displayed increased activation of IGF1R and LKB1. Under standard culture conditions, when compared to the parental sensitive cells, both the resistant variants showed reduced expression of DHCR24 (24-Dehydrocholesterol Reductase), and LM16R showed reduced protein level of the lipogenic enzyme FASN (Fatty Acid Synthase) while LM36R cells displayed similar FASN protein levels. When grown in lipid-free medium, LM16 and LM16R cells increased the expression of both FASN and DHCR24 proteins. These changes were particularly evident for DHCR24 in LM16R cells after 48 h of growth in the absence of lipids. Lipid starvation markedly reduced cell growth of LM16-LM16R as well as LM36-LM36R pairs over time, albeit with a lower impact at higher cell density. Growth inhibition assays indicated that all the cells cultured in lipid-free medium were more sensitive to PLX4032 than those cultured in standard medium. An analysis of the expression of lipid metabolism genes by qRT-PCR showed that after 48 hours under lipid-free culture conditions the levels of FASN were slightly increased in LM16 and LM16R cells but not in the LM36 pair. Conversely, the mRNA levels of DHCR24 were enhanced upon lipid starvation only in LM16 and LM16R cell lines. Besides, under these conditions, HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase) mRNA levels increased in LM16, LM16R and LM36R cell lines. ACSS2 (Acetyl-coenzyme A synthetase 2) was increased particularly in the LM16 cells. These findings suggest that lipid starvation redirects lipid pathways toward cholesterol synthesis (acetate-cholesterol) by increasing ACSS2, HMGCR as well as DHCR24 levels. Thus, extracellular lipid availability may influence tumor cell response to treatment, a finding to be considered in the frame of a personalized therapy tailored to the specific characteristics of each patient. Citation Format: Giovanni L. Beretta, Elisabetta Vergani, Cristina Corno, Stella Tinelli, Monica Rodolfo, Laura Gatti, Paola Perego. Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1899.

A lipid-free and insulin-supplemented medium supports De Novo fatty acid synthesis gene activation in melanoma cells.

While investigating the role played by de novo lipid (DNL) biosynthesis in cancer cells, we sought a medium condition that would support cell proliferation without providing any serum lipids. Here we report that a defined serum free cell culture medium condition containing insulin, transferrin and selenium (ITS) supports controlled study of transcriptional regulation of de novo fatty acid (DNFA) production and de novo cholesterol synthesis (DNCS) in melanoma cell lines. This lipid-free ITS medium is able to support continuous proliferation of several melanoma cell lines that utilize DNL to support their lipid requirements. We show that the ITS medium stimulates gene transcription in support of both DNFA and DNCS, specifically mediated by SREBP1/2 in melanoma cells. We further found that the ITS medium promoted SREBP1 nuclear localization and occupancy on DNFA gene promoters. Our data show clear utility of this serum and lipid-free medium for melanoma cancer cell culture and lipid-related areas of investigation.

Lipid droplets participate in modulating innate immune genes in Ctenopharyngodon idella kidney cells

Lipid droplets (LDs) are increasingly being recognized as important immune modulators in mammals, in additional to their function of lipid ester deposition. However, the role of LDs in fish immunity remains poorly understood. In this study, the function of LDs in the innate immune response of Ctenopharyngodon idella kidney (CIK) cells, which are the equivalent of myeloid cells in vertebrates, was investigated. LD number and TG content significantly increased in the CIK cells following exposure to lipopolysaccharide (LPS), peptidoglycan (PGN), and polyriboinosinic-polyribocytidylic acid (Poly [I: C]) for 24 h, accompanied by increases in the relative expression of several innate immune genes. However, fatty acid compositions of the triglycerides were not changed after treatment with these three pathogenic mimics. LPS, PGN, and Poly (I: C) did not alter the relative expressions of lipogenic (FAS, SCD, and DGAT) and lipid catabolic (PPARα, ATGL, and CPT-1) genes. However, these treatments did increase the mRNA levels of lipid transportation genes (FATP/CD36, ACSL1, and ACSL4), and also decreased the non-esterified fatty acid level in the medium. To further explore the role of LDs in the immune response, CIK cells were incubated with different concentrations (0, 100, 200, 300, 400, 500 μM) of exogenous lipid mix (LM; oleic acid [OA]:linoleic acid [LA]:linolenic acid [LNA] = 2:1:1), and were then transferred to a lipid-free medium and incubated for 24 h. LD size and number increased with the increase in lipid levels, and this was accompanied by increased expression of innate immune genes, including MyD88, IRF3, and IL-1β, which were expressed at their highest levels in 300 μM exogenous lipid mix. Interestingly, after incubating with different fatty acids (LM, OA, LA, LNA, arachidonic acid [ARA], and docosahexaenoic acid [DHA]; 300 μM), ARA and DHA were more potent in inducing LD formation and innate immune gene expression in the CIK cells. Finally, atglistatin, an ATGL inhibitor, effectively attenuated the expression of most genes upregulated by ARA or DHA, suggesting that lipolysis may be involved in the regulation of immune genes at the transcriptional level. Overall, the findings of this study demonstrate that LDs are functional organelles that could act as modulators in the innate immune response of CIK cells. Additionally, long-chain polyunsaturated fatty acid enriched LDs play a unique role in regulating this process.

Cell type-selective pathways and clinical associations of lysophosphatidic acid biosynthesis and signaling in the ovarian cancer microenvironment.

The peritoneal fluid of ovarian carcinoma patients promotes cancer cell invasion and metastatic spread with lysophosphatidic acid (LPA) as a potentially crucial mediator. However, the origin of LPA in ascites and the clinical relevance of individual LPA species have not been addressed. Here, we show that the levels of multiple acyl‐LPA species are strongly elevated in ascites versus plasma and are associated with short relapse‐free survival. Data derived from transcriptome and secretome analyses of primary ascite‐derived cells indicate that (a) the major route of LPA synthesis is the consecutive action of a secretory phospholipase A2 (PLA2) and autotaxin, (b) that the components of this pathway are coordinately upregulated in ascites, and (c) that CD163+CD206+ tumor‐associated macrophages play an essential role as main producers of PLA2G7 and autotaxin. The latter conclusion is consistent with mass spectrometry‐based metabolomic analyses of conditioned medium from ascites cells, which showed that tumor‐associated macrophages, but not tumor cells, are able to produce 20:4 acyl‐LPA in lipid‐free medium. Furthermore, our transcriptomic data revealed that LPA receptor (LPAR) genes are expressed in a clearly cell type‐selective manner: While tumor cells express predominantly LPAR1‐3, macrophages and T cells also express LPAR5 and LPAR6 at high levels, pointing to cell type‐selective LPA signaling pathways. RNA profiling identified cytokines linked to cell motility and migration as the most conspicuous class of LPA‐induced genes in macrophages, suggesting that LPA exerts protumorigenic properties at least in part via the tumor secretome.

Loss of virulence in NAD(P)H cytochrome b5 oxidoreductase deficient Leishmania major

Leishmania promastigotes have the ability to synthesize essential polyunsaturated fatty acids de novo and can grow in lipid free media. Recently, we have shown that NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) enzyme in Leishmania acts as the redox partner for Δ12 fatty acid desaturase, which catalyses the conversion of oleate to linoleate. So far, the exact role of Leishmania derived linoleate synthesis is still incomplete in the literature. The viability assay by flow cytometry as well as microscopic studies suggests that linoleate is an absolute requirement for Leishmania promastigote survival in delipidated media. Western blot analysis suggested that infection with log phase linoleate deficient mutant (KO) results in increased level of NF-κBp65, IκB and IKKβ phosphorylation in RAW264.7 cells. Similarly, the log phase KO infected RAW264.7 cells show dramatic increment of COX-2 expression and TNF-α secretion, compared to control or Ncb5or complement (CM) cell lines. The activation of inflammatory signaling pathways by KO mutant is significantly reduced when the RAW264.7 cells are pre-treated with BSA bound linoleate. Together, these findings confirmed that the leishmanial linoleate inhibits both COX-2 and TNF-α expression in macrophage via the inactivation of NF-κB signaling pathway. The stationary phase of KO promastigotes shows avirulence after infection in macrophages as well as inoculation into BALB/c mice; whereas CM cell lines show virulence. Collectively, these data provide strong evidence that de novo linoleate synthesis in Leishmania is an essential for parasite survival at extracellular promastigote stage as well as intracellular amastigote stage.

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential to gaining further insight into the function of specific molecules or the entire entity. Studies of biological surface modification are generally limited to genetic engineering of the organism or the covalent attachment of chemical moieties to the cell surface1,2. However these traditional techniques expose the cell to chemical reactants, or they require significant manipulation to achieve the desired outcome, making them cumbersome, and they may also inadvertently affect the viability/functionality of the modified cell. A simple method to harmlessly modify the surface of cells is required. Recently a new technology, KODE Technology has introduced a range of novel constructs consisting of three components: a functional head group (F), a spacer (S) and a lipid tail (L) and are known as Function-Spacer-Lipid or FSL constructs3. The spacer (S) is selected to provide a construct that is dispersible in water, yet will spontaneously and stably incorporate into a membrane. FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides3-12. FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions3-12. The process of modifying cells/virions is generic and extremely simple. The most common procedure is incubation of cells (in lipid free media) with a solution for FSL constructs for 1-2 hours at 37°C4-10. During the incubation the FSL constructs spontaneously incorporate into the membrane, and the process is complete. Washing is optional. Cells modified by FSL constructs are known as kodecytes6-9, while virions are kodevirions10. FSL constructs as direct infusions and kodecytes/kodevirions have been used in experimental animal models7,8,10. All kodecytes/kodevirions appear to retain their normal vitality and functionality while gaining the new function of the F moiety7,8,10,11. The combination of dispersibility in biocompatible media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs valuable research tools for the study of cells and virions.

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential to gaining further insight into the function of specific molecules or the entire entity. Studies of biological surface modification are generally limited to genetic engineering of the organism or the covalent attachment of chemical moieties to the cell surface(1,2). However these traditional techniques expose the cell to chemical reactants, or they require significant manipulation to achieve the desired outcome, making them cumbersome, and they may also inadvertently affect the viability/functionality of the modified cell. A simple method to harmlessly modify the surface of cells is required. Recently a new technology, KODE Technology has introduced a range of novel constructs consisting of three components: a functional head group (F), a spacer (S) and a lipid tail (L) and are known as Function-Spacer-Lipid or FSL constructs3. The spacer (S) is selected to provide a construct that is dispersible in water, yet will spontaneously and stably incorporate into a membrane. FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides(3-12). FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions(3-12). The process of modifying cells/virions is generic and extremely simple. The most common procedure is incubation of cells (in lipid free media) with a solution for FSL constructs for 1-2 hours at 37°C(4-10). During the incubation the FSL constructs spontaneously incorporate into the membrane, and the process is complete. Washing is optional. Cells modified by FSL constructs are known as kodecytes(6-9), while virions are kodevirions(10). FSL constructs as direct infusions and kodecytes/kodevirions have been used in experimental animal models(7,8,10). All kodecytes/kodevirions appear to retain their normal vitality and functionality while gaining the new function of the F moiety(7,8,10,11). The combination of dispersibility in biocompatible media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs valuable research tools for the study of cells and virions.

Properties of mitochondria from mammalian cells cultured on lipid-free media.

Journal Article Properties of Mitochondria from Mammalian Cells Cultured on Lipid-Free Media Get access Nutrition Reviews, Volume 28, Issue 3, March 1970, Pages 68–72, https://doi.org/10.1111/j.1753-4887.1970.tb06187.x Published: 01 March 1970

SREBP-1 regulates the expression of heme oxygenase 1 and the phosphatidylinositol-3 kinase regulatory subunit p55γ

Sterol-regulatory element binding proteins (SREBPs) control the expression of genes involved in fatty acid and cholesterol biosynthesis. Using microarrays, we observed that mature SREBP-1 also induced the expression of genes unrelated to lipid metabolism, such as heme oxygenase 1 (HMOX1), plasma glutathione peroxidase, the phosphatidylinositol-3 kinase regulatory subunit p55 gamma, synaptic vesicle glycoprotein 2A, and COTE1. The expression of these genes was repressed upon addition of sterols, which block endogenous SREBP cleavage, and was induced by the statin drug mevinolin. Stimulation of fibroblasts with platelet-derived growth factor, which activates SREBP-1, had a similar effect. Fasted mice that were refed with a high-carbohydrate diet presented an increased expression of HMOX1 and p55 gamma in the liver. Overall, the transcriptional signature of SREBP-1 in fibroblasts stimulated by growth factors was very similar to that described in liver cells. We analyzed the HMOX1 promoter and found one SREBP binding site of the E-box type, which was required for regulation by SREBP-1a and SREBP-1c but was insensitive to SREBP-2. In conclusion, our data suggest that SREBP-1 regulates the expression of stress response and signaling genes, which could contribute to the metabolic response to insulin and growth factors in various tissues.

Premating Isolation is Determined by Larval Rearing Substrates in Cactophilic Drosophila mojavensis. VII. Effects of Larval Dietary Fatty Acids on Adult Epicuticular Hydrocarbons

Low concentrations of dietary triacylglycerols including tristearin, triolein, and tripalmitolein, were assessed to determine their effects during egg to pupal stages on adult epicuticular hydrocarbon (EHC) variation in cactophilic Drosophila mojavensis. Triacylglycerols were added singly and in combination at concentrations of 1%, 3%, and 9% to a lipid-free culture medium. Control diets included Carolina Drosophila and lipid-free media. Egg to adult viability was reduced at triacylglycerol concentrations greater than 1%, except for tristearin. Both triolein and tripalmitolein increased EHC amounts to levels similar to those in combination and control diets. Tristearin caused significantly lower quantities of EHCs in adult flies than triolein or tripalmitolein, consistent with previous studies on reduced tristearin assimilation into adult EHCs. We rejected the hypothesis that unsaturated and saturated triacylglycerols were assimilated into unsaturated and saturated adult EHCs, respectively. Since these triacylglycerols comprise a fraction of known lipids in the columnar cacti used for breeding in nature, and EHCs serve as contact pheromones in D. mojavensis, these and other naturally occurring triacylglycerols may provide a direct causal link between host plant use and patterns of chemically mediated mate choice.

Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols

Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host.

Viability, infectivity and fatty acid synthetic activity of Perkinsus marinus meront cells incubated in estuarine and artificial seawater

We investigated the viability and fatty acid synthetic activity of in vitro cultured Perkinsus marinus (Dermo) in lipid-free medium and estuarine water, and the infectivity of P. marinus maintained in artificial seawater (ASW). Viability and fatty acid synthetic activity in 7 d old P. marinus meronts maintained in lipid-free medium and estuarine water were tested. The infectivity of meronts incubated in ASW was examined by first incubating P. marinus meronts in ASW for 2, 3 or 7 d, and then inoculating viable ASW-incubated meronts into the shell cavity of individual oysters Crassostrea virginica. P. marinus infection prevalence and intensity in oysters were determined 9 wk post-inoculation. Heavy mortality occurred in meronts maintained in estuarine water, a drop from an initial value of 100% viable to 7.8 and 6.1% after 3 and 14 d incubation, respectively. Viability was 85 and 67% in meronts maintained in lipid-free medium for 3 and 24 d, respectively. Meronts kept in lipid-free medium for 14 d retained their ability to synthesize fatty acids. Viable meronts incubated in ASW remained infective for up to 7 d. The infection prevalences were 85, 48 and 100%, in the treatments inoculated with viable meronts that were incubated in ASW for 2, 3 and 7 d, respectively. Infection prevalence in the group inoculated with viable meronts immediately after they were transferred to ASW ranged from 61 to 85%. Our results suggest that in nature meronts can survive for at least 14 d outside the host. Viable meronts are not only infective, but are also able to replicate and retain their fatty acid synthetic ability for 7 d.

Human stratified squamous epithelia differ in cellular fatty acid composition

The phospholipid component of the cellular membrane is crucial to the structure and function of cells. Basal cells from three epithelial tissues, adult human skin epidermis, oral mucosa, and hair follicles, grow rapidly in serum- and lipid-free medium. Analysis of phospholipid extracts from the above three types of stratified squamous epithelium in both in vivo and in vitro was done to relate fatty acid cell composition to cell function. The fatty acid composition of hair follicles in vivo was analyzed in plucked scalp hairs, and those of skin epidermis and oral mucosa in vivo were analyzed after separating the tissue into suprabasal and basal layers. The fatty acid composition of the in vivo cells from hair follicles shows a partial essential fatty acid (EFA)-deficient state. There was no significant difference between the skin epidermis and the oral mucosa in the fatty acid composition of the in vivo cells from each basal layer. However, in the suprabasal layers, the percent of linoleic acid (18:2) from the skin epidermis was higher than that from the oral mucosa. This study shows that total fatty acid composition in cell membranes of stratified squamous epithelium varies with their keratinization pattern. When cultured, the three types of rapidly growing keratinocytes showed the same essential fatty acid deficient pattern in the membrane phospholipids.

Fatty acid and sterol composition of three phytomonas species.

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.

Saccharomyces cerevisiae cell fatty acid composition and release during fermentation without aeration and in absence of exogenous lipids

Medium-chain fatty-acids (MCFA) are among the main aroma compounds of fermented beverages. High concentrations of MCFA have been found in sluggish and stuck fermentations. It has been suggested that they arrest cell growth, as they may be toxic, but the causes of sluggish and stuck fermentations are still unclear. The aim of this work was to see whether the production of MCFA is related to fatty acid synthesis in the absence of exogenous lipids and aeration, and whether their increase can be regarded as a consequence, instead of the cause, of sluggish and stuck fermentations. Two possibilities were considered: (i) MCFA are produced to replace unsaturated fatty acids (UFA) for cell membrane fluidity when the lack of oxygen makes desaturation of saturated fatty acids (SFA) impossible; or (ii) MCFA are produced following the release of medium-chain acyl-CoA from the fatty acid synthetase complex (FAS) due to the accumulation of SFA, and their hydrolysis to recycle CoA-SH. In the first hypothesis, MCFA should be active in cell metabolism and be found in cell structures; in the second, MCFA should be a discard and prevalently found outside the cell. We carried out a Saccharomyces cerevisiae fermentation experiment in a synthetic, lipid-free medium without aeration. We measured the fatty acid composition of yeast cells and the amounts of MCFA and their ethyl esters in the medium throughout the fermentation. Cell growth and the oxygen content of the medium were also monitored. We found that MCFA are not immobilized in cell structures, but mainly released into the medium. Cell growth is arrested because fatty acid biosynthesis is prevented by the lack of oxygen. The higher MCFA concentrations found in sluggish and stuck fermentations can be thus regarded as an effect, and not the cause, of this arrest. Some suggestions for the prevention of these events are proposed.

Demonstration of Malassezia furfur and M. sympodialis together with M. pachydermatis in veterinary specimens.

In the present study, 47 wild-type isolates of the genus Malassezia were isolated from dog and cat specimens by means of a simple differentiating system recently published. The purpose was to determine whether any of the other seven Malassezia spp. apart from M. pachydermatis occur in carnivores. Of the 47 isolates, three had been obtained from cats (ear 2, skin 1) and 44 from dogs (ear 37, skin 3, faeces 2, claw and paw 2). After primary isolation, they were subcultured on mDixon agar and then purified and differentiated by means of assimilation of Cremophor EL, splitting of esculin, growth on lipid-free medium and formation of tryptophan-dependent pigments and fluorochromes. Thus, a total of 100 strains could be obtained from the 47 primary isolates. Referring to the source material, M. pachydermatis was found in 83%, M. furfur in 45% and M. sympodialis in 75%. More than 80% of cultures were mixed, comprising two or all three species; a single species was isolated in only nine cases. This shows that animals are not colonized by M. pachydermatis alone, as has been thought until now, but in nearly all cases by mixed cultures. Thus, (domestic) animals could well be a reservoir for other Malassezia species such as M. furfur and M. sympodialis. Surprisingly, Malassezia yeasts were also isolated from dog faeces, indicating that they apparently pass through the gastrointestinal tract in unchanged form after having been taken up by licking colonized areas. The survivability of Malassezia yeasts in highly acid milieu was also demonstrated in vitro. The study confirms the usefulness of the new test procedures and allows new statements concerning the epidemiology of Malassezia yeasts.