Modulation of lipid metabolism and spiramycin biosynthesis in Streptomyces ambofaciens unstable mutants.

Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (orfPS), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205(+) Spi-). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205(-) Spi+). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.     

Large genomic rearrangements of the unstable region in Streptomyces ambofaciens are associated with major changes in global gene expression

Global gene expression is dramatically altered by genomic rearrangements in Streptomyces ambofaciens RP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse-labelled proteins separated by two-dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or over expressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their expression.     

Resistance to Mucosal Lysozyme Compensates for the Fitness Deficit of Peptidoglycan Modifications by Streptococcus pneumoniae

The abundance of lysozyme on mucosal surfaces suggests that successful colonizers must be able to evade its antimicrobial effects. Lysozyme has a muramidase activity that hydrolyzes bacterial peptidoglycan and a non-muramidase activity attributable to its function as a cationic antimicrobial peptide. Two enzymes (PgdA, a N-acetylglucosamine deacetylase, and Adr, an O-acetyl transferase) that modify different sites on the peptidoglycan of Streptococcus pneumoniae have been implicated in its resistance to lysozyme in vitro. Here we show that the antimicrobial effect of human lysozyme is due to its muramidase activity and that both peptidoglycan modifications are required for full resistance by pneumococci. To examine the contribution of lysozyme and peptidoglycan modifications during colonization of the upper respiratory tract, competition experiments were performed with wild-type and pgdAadr mutant pneumococci in lysozyme M-sufficient (LysM^+/+) and -deficient (LysM^−/−) mice. The wild-type strain out-competed the double mutant in LysM^+/+, but not LysM^−/− mice, indicating the importance of resistance to the muramidase activity of lysozyme during mucosal colonization. In contrast, strains containing single mutations in either pgdA or adr prevailed over the wild-type strain in both LysM^+/+ and LysM^−/− mice. Our findings demonstrate that individual peptidoglycan modifications diminish fitness during colonization. The competitive advantage of wild-type pneumococci in LysM^+/+ but not LysM^−/− mice suggests that the combination of peptidoglycan modifications reduces overall fitness, but that this is outweighed by the benefits of resistance to the peptidoglycan degrading activity of lysozyme.     

Altered Lipid Metabolism in Residual White Adipose Tissues of Bscl2 Deficient Mice

Mutations in BSCL2 underlie human congenital generalized lipodystrophy type 2 disease. We previously reported that Bscl2 ^−/− mice develop lipodystrophy of white adipose tissue (WAT) due to unbridled lipolysis. The residual epididymal WAT (EWAT) displays a browning phenotype with much smaller lipid droplets (LD) and higher expression of brown adipose tissue marker proteins. Here we used targeted lipidomics and gene expression profiling to analyze lipid profiles as well as genes involved in lipid metabolism in WAT of wild-type and Bscl2^−/− mice. Analysis of total saponified fatty acids revealed that the residual EWAT of Bscl2^−/− mice contained a much higher proportion of oleic_18:1n9 acid concomitant with a lower proportion of palmitic_16:0 acid, as well as increased n3- polyunsaturated fatty acids (PUFA) remodeling. The acyl chains in major species of triacylglyceride (TG) and diacylglyceride (DG) in the residual EWAT of Bscl2^−/− mice were also enriched with dietary fatty acids. These changes could be reflected by upregulation of several fatty acid elongases and desaturases. Meanwhile, Bscl2^−/− adipocytes from EWAT had increased gene expression in lipid uptake and TG synthesis but not de novo lipogenesis. Both mitochondria and peroxisomal β-oxidation genes were also markedly increased in Bscl2^−/− adipocytes, highlighting that these machineries were accelerated to shunt the lipolysis liberated fatty acids through uncoupling to dissipate energy. The residual subcutaneous white adipose tissue (ScWAT) was not browning but displays similar changes in lipid metabolism. Overall, our data emphasize that, other than being essential for adipocyte differentiation, Bscl2 is also important in fatty acid remodeling and energy homeostasis.     

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8⁺ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8⁺ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8⁺ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8⁺ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8⁺ T-cells segregated into IFNγ⁺ perforin (Pfn)^neg or IFNγ^negPfn⁺ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8⁺Pfn⁺ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8 ^−/− recipients showed that the CD8⁺ cells from infected ifnγ^−/− pfn ^+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8⁺ cells from ifnγ ^+/+ pfn ^−/− donors. Moreover, the reconstitution of naïve cd8 ^−/− mice with CD8⁺ cells from naïve ifnγ ^+/+ pfn ^−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ⁺ cells accumulation, whereas reconstitution with CD8⁺ cells from naïve ifnγ ^−/− pfn ^+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn⁺ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8⁺Pfn⁺ and CD8⁺IFNγ⁺ cells during CCC. CD8⁺IFNγ⁺ cells may exert a beneficial role, whereas CD8⁺Pfn⁺ may play a detrimental role in T. cruzi-elicited heart injury.     

Functional significance of glutamate–cysteine ligase modifier for erythrocyte survival in vitro and in vivo

Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate–cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm^−/−) mice. Erythrocytes from gclm^−/− mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm^−/− erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm^−/− mice showed dramatically increased hemolysis compared with gclm^+/+ controls. In addition, PHZ-treated gclm^−/− mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm^+/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm^−/− mice and none in gclm^+/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm^−/− mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm^+/+ mice survived, 90% of PHZ-treated gclm^−/− mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm^−/− erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm^+/+ erythrocytes, an effect at least partially due to increased cytosolic Ca²⁺ concentration. Under resting conditions, gclm^−/− mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.     

Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr ^−/− lung when compared to wild-type (Cftr ^+/+) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr ^−/− lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr ^−/− mice compared to Cftr ^+/+. Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr ^+/+ lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr ^−/− lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr ^−/− lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.     

In vivo hepatic lipid quantification using MRS at 7 Tesla in a mouse model of glycogen storage disease type 1a

The assessment of liver lipid content and composition is needed in preclinical research to investigate steatosis and steatosis-related disorders. The purpose of this study was to quantify in vivo hepatic fatty acid content and composition using a method based on short echo time proton magnetic resonance spectroscopy (MRS) at 7 Tesla. A mouse model of glycogen storage disease type 1a with inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc^−/−) mice and control mice were fed a standard diet or a high-fat/high-sucrose (HF/HS) diet for 9 months. In control mice, hepatic lipid content was found significantly higher with the HF/HS diet than with the standard diet. As expected, hepatic lipid content was already elevated in L-G6pc^−/− mice fed a standard diet compared with control mice. L-G6pc^−/− mice rapidly developed steatosis which was not modified by the HF/HS diet. On the standard diet, estimated amplitudes from olefinic protons were found significantly higher in L-G6pc^−/− mice compared with that in control mice. L-G6pc^−/− mice showed no noticeable polyunsaturation from diallylic protons. Total unsaturated fatty acid indexes measured by gas chromatography were in agreement with MRS measurements. These results showed the great potential of high magnetic field MRS to follow the diet impact and lipid alterations in mouse liver.     

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

The gene cassette (camA⁺ camB⁺ camC) encoding a cytochrome P-450_cam variant was integrated into the nonessential gene pcpM of the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723 by homologous recombination. The recombinant strain could degrade hexachlorobenzene at a rate of 0.67 nmol · mg (dry weight)⁻¹ · h⁻¹, and intermediate pentachlorophenol was also identified.     

Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH₃Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH₃Cl; then it catalyzed methyl transfer from CH₃Cl, CH₃Br, or CH₃I to the following acceptor ions (in order of decreasing efficacy): I⁻, HS⁻, Cl⁻, Br⁻, NO₂⁻, CN⁻, and SCN⁻. Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N₂O, and Hg²⁺ to affect the methyltransferase suggests significant differences. During CH₃Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS⁻, a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH₃Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899–2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.     

Tip30 controls differentiation of murine mammary luminal progenitor to estrogen receptor-positive luminal cell through regulating FoxA1 expression

Estrogen receptor-alpha positive (ER⁺) breast cancers comprise the majority of human breast cancers, but molecular mechanisms underlying this subtype of breast cancers remain poorly understood. Here, we show that ER⁺ mammary luminal tumors arising in Tip30^−/−MMTV-Neu mice exhibited increased enrichment of luminal progenitor gene signature. Deletion of the Tip30 gene increased proportion of mammary stem and progenitor cell populations, and raised susceptibility to ER⁺ mammary luminal tumors in female Balb/c mice. Moreover, Tip30^−/− luminal progenitors displayed increases in propensity to differentiate to mature ER⁺ luminal cells and FoxA1 expression. Knockdown of FoxA1 expression in Tip30^−/− progenitors by shRNA specific for FoxA1 reduced their differentiation toward ER⁺ mature luminal cells. Taken together, our results suggest that TIP30 is a key regulator for maintaining ER⁺ and ER⁻luminal pools in the mammary luminal lineage, and loss of it promotes expansion of ER⁺ luminal progenitors and mature cells and ER⁺ mammary tumorigenesis.     

Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in the liver and secreted as biliary glycoprotein 1 (BGP1) via bile canaliculi (BCs). CEACAM1-LF is a 72 amino acid cytoplasmic domain mRNA splice isoform with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ceacam1^−/− or Ser503Ala transgenic mice have been shown to develop insulin resistance and nonalcoholic fatty liver disease; however, the role of the human equivalent residue, Ser508, in lipid dysregulation is unknown. Human HepG2 hepatocytes that express CEACAM1 and form BC in vitro were compared with CEACAM1^−/− cells and CEACAM1^−/− cells expressing Ser508Ala null or Ser508Asp phosphorylation mimic mutations or to phosphorylation null mutations in the tyrosine ITIMs known to be phosphorylated by the tyrosine kinase Src. CEACAM1^−/− cells and the Ser508Asp and Tyr520Phe mutants strongly retained lipids, while Ser508Ala and Tyr493Phe mutants had low lipid levels compared with wild-type cells, indicating that the ITIM mutants phenocopied the Ser508 mutants. We found that the fatty acid transporter CD36 was upregulated in the S508A mutant, coexpressed in BCs with CEACAM1, co-IPed with CEACAM1 and Src, and when downregulated via RNAi, an increase in lipid droplet content was observed. Nuclear translocation of CD36 associated kinase LKB1 was increased sevenfold in the S508A mutant versus CEACAM1^−/− cells and correlated with increased activation of CD36-associated kinase AMPK in CEACAM1^−/− cells. Thus, while CEACAM1^−/− HepG2 cells upregulate lipid storage similar to Ceacam1^−/− in murine liver, the null mutation Ser508Ala led to decreased lipid storage, emphasizing evolutionary changes between the CEACAM1 genes in mouse and humans.     

atp Mutants of Escherichia coli Fail To Grow on Succinate Due to a Transport Deficiency

Escherichia coli atp mutants, which lack a functional H⁺-ATPase complex, are capable of growth on glucose but not on succinate or other C₄-dicarboxylates (Suc⁻ phenotype). Suc⁺ revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H⁺-ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc⁺ mutants on succinate, which implicates the yhiF gene product in the regulation of C₄-dicarboxylate metabolism. Indeed, when the E. coli C₄-dicarboxylate transporter (encoded by the dctA gene) was expressed in trans, the Suc⁻ phenotype of the atp deletion strain reverted to Suc⁺, which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C₄-dicarboxylates is insufficient transport capacity for these substrates.     

Diffusion of Single Cardiac Ryanodine Receptors in Lipid Bilayers Is Decreased by Annexin 12

Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca²⁺ flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were studied. RyR2 exhibited an apparent diffusion coefficient (D_RyR) of 1.2 × 10⁻⁸ cm² s⁻¹ and a mean path length of 5.0 μm. Optimal use of optical methods for analysis of RyR2 channel function requires that RyR2 diffusion be limited. Therefore, we tested the effect of annexin 12, which interacts with anionic phospholipids in a Ca²⁺-dependent manner. Addition of annexin 12 (0.25–4.0 μM) to the trans side of bilayers containing an 80:20 ratio of phosphatidylethanolamine/phosphatidylserine decreased RyR2 diffusion in a concentration-dependent manner. Annexin 12 (2 μM) decreased the apparent D_RyR 683-fold from 1.2–10⁻⁸ to 1.8 × 10⁻¹¹ cm² s⁻¹ and the mean path length 10-fold from 5.0 to 0.5 μm without obvious changes in the conductance of the native bilayer or in activation of RyR2 channels by Ca²⁺ or suramin. Thus, annexin 12 may provide a useful tool for optimizing optical analysis of RyR2 channels in lipid bilayers.     

Characterization of a Novel Intestinal Glycerol-3-phosphate Acyltransferase Pathway and Its Role in Lipid Homeostasis

Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3^−/−) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3^−/− mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3^−/− mice. Gpat3^−/− enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3^−/− mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.     

Cardiac Per2 Functions as Novel Link between Fatty Acid Metabolism and Myocardial Inflammation during Ischemia and Reperfusion Injury of the Heart

Disruption of peripheral circadian rhyme pathways dominantly leads to metabolic disorders. Studies on circadian rhythm proteins in the heart indicated a role for Clock or Per2 in cardiac metabolism. In contrast to Clock^−/−, Per2^−/− mice have larger infarct sizes with deficient lactate production during myocardial ischemia. To test the hypothesis that cardiac Per2 represents an important regulator of cardiac metabolism during myocardial ischemia, we measured lactate during reperfusion in Per1^−/−, Per2^−/− or wildtype mice. As lactate measurements in whole blood indicated an exclusive role of Per2 in controlling lactate production during myocardial ischemia, we next performed gene array studies using various ischemia-reperfusion protocols comparing wildtype and Per2^−/− mice. Surprisingly, high-throughput gene array analysis revealed dominantly lipid metabolism as the differentially regulated pathway in wildtype mice when compared to Per2^−/−. In all ischemia-reperfusion protocols used, the enzyme enoyl-CoA hydratase, which is essential in fatty acid beta-oxidation, was regulated in wildtype animals only. Studies using nuclear magnet resonance imaging (NMRI) confirmed altered fatty acid populations with higher mono-unsaturated fatty acid levels in hearts from Per2^−/− mice. Unexpectedly, studies on gene regulation during reperfusion revealed solely pro inflammatory genes as differentially regulated ‘Per2-genes’. Subsequent studies on inflammatory markers showed increasing IL-6 or TNFα levels during reperfusion in Per2^−/− mice. In summary, these studies reveal an important role of cardiac Per2 for fatty acid metabolism and inflammation during myocardial ischemia and reperfusion, respectively.     

Absence of Macrophage Inflammatory Protein-1α Prevents the Development of Blinding Herpes Stromal Keratitis

Prior studies in our laboratory have suggested that the CC chemokine macrophage inflammatory protein-1α (MIP-1α) may be an important mediator in the blinding ocular inflammation which develops following herpes simplex virus type 1 (HSV-1) infection of the murine cornea. To directly test this hypothesis, MIP-1α-deficient (−/−) mice and their wild-type (+/+) counterparts were infected topically on the scarified cornea with 2.5 × 10⁵ PFU of HSV-1 strain RE and subsequently graded for corneal opacity. Four weeks postinfection (p.i.), the mean corneal opacity score of −/− mice was 1.1 ± 0.3 while that of the +/+ mice was 3.7 ± 0.5. No detectable infiltrating CD4⁺ T cells were seen histologically at 14 or 21 days p.i. in −/− animals, whereas the mean CD4⁺ T-cell count per field (36 fields counted) in +/+ hosts was 26 ± 2 (P < 0.001). In addition, neutrophil counts in the −/− mouse corneas were reduced by >80% in comparison to the wild-type controls. At 2 weeks p.i., no interleukin-2 or gamma interferon could be detected in six of seven −/− mice, whereas both T-cell cytokines were readily demonstrable in +/+ mouse corneas. Also, MIP-2 and monocyte chemoattractant protein-1 protein levels were significantly lower in MIP-1α −/− mouse corneas than in +/+ host corneas, suggesting that MIP-1α directly, or more likely indirectly, influences the expression of other chemokines. Interestingly, despite the paucity of infiltrating cells, HSV-1 clearance from the eyes of −/− mice was not significantly different from that observed in +/+ hosts. We conclude that MIP-1α is not needed to control virus growth in the cornea but is essential for the development of severe stromal keratitis.     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Comparative Analyses of Three Chlorella Species in Response to Light and Sugar Reveal Distinctive Lipid Accumulation Patterns in the Microalga C. sorokiniana

While photosynthetic microalgae, such as Chlorella, serve as feedstocks for nutritional oils and biofuels, heterotrophic cultivation can augment growth rates, support high cell densities, and increase triacylglycerol (TAG) lipid content. However, these species differ significantly in their photoautotrophic and heterotrophic characteristics. In this study, the phylogeny of thirty Chlorella strains was determined in order to inform bioprospecting efforts and detailed physiological assessment of three species. The growth kinetics and lipid biochemistry of C. protothecoides UTEX 411, C. vulgaris UTEX 265, and C. sorokiniana UTEX 1230 were quantified during photoautotrophy in Bold''s basal medium (BBM) and heterotrophy in BBM supplemented with glucose (10 g L⁻¹). Heterotrophic growth rates of UTEX 411, 265, and 1230 were found to be 1.5-, 3.7-, and 5-fold higher than their respective autotrophic rates. With a rapid nine-hour heterotrophic doubling time, Chlorella sorokiniana UTEX 1230 maximally accumulated 39% total lipids by dry weight during heterotrophy compared to 18% autotrophically. Furthermore, the discrete fatty acid composition of each strain was examined in order to elucidate lipid accumulation patterns under the two trophic conditions. In both modes of growth, UTEX 411 and 265 produced 18∶1 as the principal fatty acid while UTEX 1230 exhibited a 2.5-fold enrichment in 18∶2 relative to 18∶1. Although the total lipid content was highest in UTEX 411 during heterotrophy, UTEX 1230 demonstrated a two-fold increase in its heterotrophic TAG fraction at a rate of 28.9 mg L⁻¹ d⁻¹ to reach 22% of the biomass, corresponding to as much as 90% of its total lipids. Interestingly, UTEX 1230 growth was restricted during mixotrophy and its TAG production rate was suppressed to 18.2 mg L⁻¹ d⁻¹. This constraint on carbon flow raises intriguing questions about the impact of sugar and light on the metabolic regulation of microalgal lipid biosynthesis.