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Symbiotic dinoflagellates in the family Symbiodiniaceae release mobile compounds (e.g., glucose, glycerol, amino acids, and lipids) to their host's tissues. Little is known about how different symbionts affect quantitative and qualitative differences in these compounds. We tested how symbiont identity affects glycerol and glucose pools in the tissues of the sea anemone Exaiptasia pallida ('Aiptasia'). We infected symbiont-free anemones with three different symbiotic dinoflagellates: Breviolum minutum isolated from our Aiptasia stock, B. minutum isolated from a different Aiptasia population, and the free-living Effrenium voralum. We measured free glycerol and glucose levels in host tissues under photosynthetic conditions, as well as metabolite release by these algae when freshly isolated from Aiptasia and incubated in a host tissue homogenate. Anemone tissues containing the stock B. minutum accumulated glycerol at a higher symbiont cell-specific rate than those containing the alternative B. minutum or E. voratum (e.g., at 9 h of light incubation: stock B. minutum, 4.05 * [10.sup.-5] nmol per algal cell; alternative B. minutum, 0.90 * [10.sup.-5] nmol per algal cell; E. voratum: 1.14 * [10.sup.-5] nmol per algal cell). All symbiotic hosts accumulated glucose between 1 and 12 h of light incubation. At 12 h, the symbiont cell-specific glucose content was 6-fold higher in anemone tissues that contained stock B. minutum than in those containing the alternative B. minutum (1.73 * [10.sup.-6] vs. 0.30 * [10.sup.-6] nmol per algal cell, respectively). All freshly isolated algae released glycerol and glucose when incubated in host homogenate except E. voratum, which did not release glycerol. These trends mirrored those in hospite. Our results suggest that, on an algal cell-specific basis, B. minutum isolated from the same Aiptasia stock provided better nutritional support to the host than did the two other algae, though this was not reflected in colonization success, highlighting the underlying complexity of host-symbiont recognition and symbiosis establishment in the cnidariandinoflagellate partnership., Introduction Many cnidarians (e.g., corals, sea anemones) form symbioses with dinoflagellates in the family Symbiodiniaceae. This family is characterized by considerable genetic diversity, with multiple genera and species now recognized [...]

BACKGROUND. Postreceptor insulin resistance (IR) is associated with hyperglycemia and hepatic steatosis. However, receptor-level IR (e.g., insulin receptor pathogenic variants, INSR) causes hyperglycemia without steatosis. We examined 4 pathologic conditions of IR in humans to examine pathways controlling lipid metabolism and gluconeogenesis. METHODS. Cross-sectional study of severe receptor IR (INSR, n = 7) versus postreceptor IR that was severe (lipodystrophy, n = 14), moderate (type 2 diabetes, n = 9), or mild (obesity, n = 8). Lipolysis (glycerol turnover), hepatic glucose production (HGP), gluconeogenesis (deuterium incorporation from body water into glucose), hepatic triglyceride (magnetic resonance spectroscopy), and hepatic fat oxidation (plasma [beta]-hydroxybutyrate) were measured. RESULTS. Lipolysis was 2- to 3-fold higher in INSR versus all other groups, and HGP was 2-fold higher in INSR and lipodystrophy versus type 2 diabetes and obesity (P < 0.001), suggesting severe adipose and hepatic IR. INSR subjects had a higher contribution of gluconeogenesis to HGP, approximately 77%, versus 52% to 59% in other groups (P = 0.0001). Despite high lipolysis, INSR subjects had low hepatic triglycerides (0.5% [interquartile range 0.1%-0.5%]), in contrast to lipodystrophy (10.6% [interquartile range 2.8%-17.1%], P < 0.0001). [beta]-hydroxybutyrate was 2- to 7-fold higher in INSR versus all other groups (P < 0.0001), consistent with higher hepatic fat oxidation. CONCLUSION. These data support a key pathogenic role of adipose tissue IR to increase glycerol and FFA availability to the liver in both receptor and postreceptor IR. However, the fate of FFA diverges in these populations. In receptor-level IR, FFA oxidation drives gluconeogenesis rather than being reesterified to triglyceride. In contrast, in postreceptor IR, FFA contributes to both gluconeogenesis and hepatic steatosis. TRIAL REGISTRATION. ClinicalTrials.gov NCT01778556, NCT00001987, and NCT02457897. FUNDING. National Institute of Diabetes and Digestive and Kidney Diseases, US Department of Agriculture/Agricultural Research Service 58-3092-5-001., Introduction Insulin is a hormone that signals the fed state, promoting a switch from net glucose production to net glucose uptake and shifting lipids from lipid metabolism to lipid storage. [...]

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls blood phosphate levels by increasing renal phosphate excretion and reducing 1,25-dihydroxyvitamin [D.sub.3] [1,25[(OH).sub.2]D] production. Disorders of FGF23 homeostasis are associated with significant morbidity and mortality, but a fundamental understanding of what regulates FGF23 production is lacking. Because the kidney is the major end organ of FGF23 action, we hypothesized that it releases a factor that regulates FGF23 synthesis. Using aptamer-based proteomics and liquid chromatography-mass spectrometry-based (LC-MS-based) metabolomics, we profiled more than 1600 molecules in renal venous plasma obtained from human subjects. Renal vein glycerol-3-phosphate (G-3-P) had the strongest correlation with circulating FGF23. In mice, exogenous G-3-P stimulated bone and bone marrow FGF23 production through local G-3-P acyltransferase-mediated (GPAT-mediated) lysophosphatidic acid (LPA) synthesis. Further, the stimulatory effect of G-3-P and LPA on FGF23 required LPA receptor 1 (LPAR1). Acute kidney injury (AKI), which increases FGF23 levels, rapidly increased circulating G-3-P in humans and mice, and the effect of AKI on FGF23 was abrogated by GPAT inhibition or Lpar1 deletion. Together, our findings establish a role for kidney-derived G-3-P in mineral metabolism and outline potential targets to modulate FGF23 production during kidney injury., Introduction Fibroblast growth factor-23 (FGF23) levels increase with both acute and chronic kidney disease (CKD) (1) and are a strong independent risk factor for adverse renal outcomes and mortality in [...]

Stanniocalcin-1 and -2 belong to a family of molecules that exhibit both paracrine and autocrine effects in mammalian cells. Human stanniocalcin-1 (hSTC-1) is expressed in a wide range of tissues, including white adipose tissue. In fed rats, hSTC-1 increases carbon flux from glucose to lipids in retroperitoneal white adipose tissue. Human stanniocalcin-2 (hSTC- 2) is expressed in almost all tissues and regulates various biological processes. The aim of this work was to study the action of hSTC-1 and hSTC-2 in the lipid and glucose metabolism of epididymal white adipose tissue (eWAT) in rats in different nutritional states. This study shows for the first time an opposite effect of hSTC-1 and hSTC-2 on glyceride-glycerol generation from glucose in eWAT of fed rats. hSTC-1 stimulated the storage of triacylglycerol in eWAT in the postprandial period, increasing glucose uptake and glyceride-glycerol generation from [sup.14]C-glucose. hSTC-2 decreased triacylglycerol synthesis, reducing glyceride-glycerol generation from [sup.14]C-glucose, direct phosphorylation of glycerol, and fatty acid synthesis from [sup.14]C-glucose in eWAT of fed rats. However, both hormones increased glucose uptake in fed and fasting states. These findings provide evidence for a direct role of hSTC-1 and hSTC-2 in the regulation of lipid and glucose metabolism in eWAT of rats. Key words: stanniocalcin 1, stanniocalcin 2, white adipose tissue, metabolism, fed, fasting, glucose, glycerol. La stanniocalcine 1 et la stanniocalcine 2 appartiennent a une famille de molecules qui presentent des effets tant paracrines qu'autocrines dans les cellules de mammiferes. La stanniocalcine 1 humaine (hSTC-1) s'exprime dans une grande variete de tissus, y compris le tissu adipeux blanc. Chez les rats alimentes, la hSTC-1 entraine une augmentation du flux carbonique du glucose aux lipides dans le tissu adipeux blanc retroperitoneal. La stanniocalcine 2 humaine (hSTC- 2) s'exprime dans presque tous les tissus et participe a la regulation de divers processus biologiques. Ces travaux avaient pour but d'etudier l'action de la hSTC-1 et de la hSTC-2 sur le metabolisme lipidique et glucidique du tissu adipeux blanc d'epididyme (TABe) chez des rats soumis a differents etats nutritionnels. Cette etude est la premiere a montrer la presence d'effets opposes de la hSTC-1 et de la hSTC-2 sur la production de glyceride-glycerol a partir du glucose dans le TABe de rats alimentes. La hSTC-1 entrainait une stimulation du stockage du triacylglycerol dans le TABe pendant la periode postprandiale, par l'augmentation de la capture de glucose et de la production de glyceride-glycerol a partir de [sup.14]C-glucose. La hSTC-2 entrainait une diminution de la synthese de triacylglycerol, par la diminution de la production de glyceride-glycerol a partir de [sup.14]C-glucose, la phosphorylation directe du glycerol et la synthese d'acide gras a partir de [sup.14]C-glucose dans le TABe de rats alimentes. Cependant, les deux hormones entrainaient une augmentation de la capture du glucose dans les etats alimentes comme de jeune. Ces resultats apportent des donnees probantes quant a un role direct de la hSTC-1 et de la hSTC-2 dans la regulation du metabolisme lipidique et glucidique dans le TABe de rat. [Traduit par la Redaction] Mots-cles : stanniocalcine 1, stanniocalcine 2, tissu adipeux blanc, metabolisme, etat alimente, jeune, glucose, glycerol., Introduction Stanniocalcin (STC) was first isolated in fish where it was shown to regulate calcium/phosphate homeostasis (Chang et al. 1995; Olsen et al. 1996). Subsequently, 2 STC isoforms (STC-1 and [...]

Electronic nicotine delivery systems (ENDS) or e-cigarettes have emerged as a popular recreational tool among adolescents and adults. Although the use of ENDS is often promoted as a safer alternative to conventional cigarettes, few comprehensive studies have assessed the long-term effects of vaporized nicotine and its associated solvents, propylene glycol (PG) and vegetable glycerin (VG). Here, we show that compared with smoke exposure, mice receiving ENDS vapor for 4 months failed to develop pulmonary inflammation or emphysema. However, ENDS exposure, independent of nicotine, altered lung lipid homeostasis in alveolar macrophages and epithelial cells. Comprehensive lipidomic and structural analyses of the lungs revealed aberrant phospholipids in alveolar macrophages and increased surfactant- associated phospholipids in the airway. In addition to ENDS-induced lipid deposition, chronic ENDS vapor exposure downregulated innate immunity against viral pathogens in resident macrophages. Moreover, independent of nicotine, ENDS- exposed mice infected with influenza demonstrated enhanced lung inflammation and tissue damage. Together, our findings reveal that chronic e-cigarette vapor aberrantly alters the physiology of lung epithelial cells and resident immune cells and promotes poor response to infectious challenge. Notably, alterations in lipid homeostasis and immune impairment are independent of nicotine, thereby warranting more extensive investigations of the vehicle solvents used in e-cigarettes., Introduction The electronic nicotine delivery system (ENDS), or e-cigarette, entered the US market in the mid-2000s and rapidly gained popularity among tobacco smokers and never-smokers (1). Currently, an estimated 10 [...]

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric a-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10°C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed. Key words: aquaporins, detergent, glycerol facilitator, membrane protein, osmolyte, thermal stability, SDS, tetramer, TMAO. Les osmolytes sont des molecules naturelles utilisees par un large spectre d'organismes afin de stabiliser les proteines soumises a des conditions extremes de temperature, de salinite, de pression hydrostatique, de concentration d'agents denaturants et de dessiccation. Les auteurs rapportent les effets de l'oxyde de trimethylamine (TMAO), un osmolyte, de meme que l'influence de la tete polaire et de la longueur de la chaine acyle de differents detergents, sur la stabilite du tetramere du facilitateur du glycerol (GF) d'E. coli, une proteine membranaire, face a une denaturation thermique et chimique par le dodecylsulfate de sodium (SDS). Le TMAO favorise l'association de la proteine normalement a-helicoidale tetramerique en oligomeres d'ordre superieur dans le dodecyl maltoside (DDM), contrairement au tetradecyl-maltoside (TDM), a la lyso-lauroylphosphatidyl choline (LLPC) ou la lyso-myristoylphosphatidyl choline (LMPC), tel que determine par diffusion dynamique de la lumiere (DDL); le complexe octamerique est particulierement stable comme le montre l'electrophorese en gel de polyacrylamide SDS. Le TMAO augmente la stabilite thermique du tetramere de GF de 10°C en moyenne dans les quatre detergents et protege aussi la proteine de la denaturation par le SDS. Cependant, il ne favorise pas la reassociation du tetramere si on l'ajoute a la proteine dissociee par le SDS. Le TMAO favorise aussi la formation de micelles detergentes en batonnet, et le DLS s'est avere utile pour suivre la structure de la proteine et la redistribution du detergent durant la denaturation thermique de la proteine. La proteine est plus stable thermiquement dans les detergents possedant une tete polaire constituee de phosphatidylcholine (LLPC et LMPC) que dans les detergents comprenant un maltoside. Les implications de ces resultats dans les mecanismes osmolytes, la stabilite des proteines membranaires et les interactions proteine-proteine sont discutes. [Traduit par la Redaction] Mots-cles: aquaporines, detergent, facilitateur de glycerol, proteine membranaire, osmolyte, stabilite thermique, SDS, tetramere, TMAO., Introduction Many organisms use small organic osmolytes to combat dehydration (Tunnacliffe and Lapinski 2003), heat (Singer and Lindquist 1998), cold (Storey and Storey 1996), high hydrostatic pressure (Siebenaller and Somero [...]

The objective of this study was to evaluate the effects of crude glycerin on performance and carcass characteristics in finishing wether lambs. Thirty black-faced, Suffolk-cross wether lambs (44.1 [+ or -] 5.6 kg initial BW) were stratified and blocked by BW to 1 of 5 individually fed, isocaloric, isonitrogenous dietary treatments containing 0, 5, 10, 15, or 20% crude glycerin (88% pure) on a DM basis. Diets were fed once daily for ad libitum consumption and contained 15% chopped hay, approximately 25% dried distillers grains with solubles, and the specified treatment combination of cracked corn replaced with increasing amounts of crude glycerin. Wethers were weighed on 14-d intervals and were selected for slaughter when they reached an approximate 12th-rib fat depth of 0.51 cm (28 to 84 d on trial). Carcass characteristics were collected after a 48-h chill. Dry matter intake (linear, P = 0.004) and ADG (quadratic, P = 0.05) increased with increasing concentrations of glycerin in the diet during the first 14 d of the feeding period. Similarly, G:F tended to increase quadratically (P = 0.06) with increasing concentrations of crude glycerin in the diet during the first 14 d. However, there were no differences among treatments for final BW, days on feed, or cumulative DMI, cumulative ADG, and cumulative G:F (P [greater than or equal to] 0.11). Body wall thickness, dressing percent, HCW, LM area, flank streaking, leg score, conformation score, quality grade, yield grade, and percent boneless, closely trimmed retail cuts did not differ (P [greater than or equal to] 0.21). Adding up to 15% crude glycerin to finishing wether diets improved feedlot performance, particularly during the first 14 d, without any concomitant effect on carcass characteristics. Key words: dried distillers grains with solubles, glycerin, lamb, wether doi: 10.2527/jas.2009-2325

J Strength Cond Res 24(1): 74-78, 2010-Glycerol-induced hyperhydration (GIH) has been shown to increase endurance performance (EP). However, EP starts declining at a dehydration level >2% body weight (BW). It thus appears that the use of GIH is only required when athletes anticipate that their fluid intake during exercise would not be sufficient to prevent a loss of BW >2%. In such a scenario, the optimal GIH load to be ingested before exercise would correspond to the amount of fluid that cannot be drunk during exercise and that would be just sufficient to keep the dehydration level 2%. Step 3 consists of identifying, among those pre-selected, a GIH protocol that increases body water by at least the amount computed in step 2. This method will remove much of the guesswork involved in the decision-making process of the optimal amount of GIH that should be ingested before exercise by athletes for maximizing EP and will serve as a practical reference tool for all athletes using, and coaches, practitioners, and exercise physiologists recommending the utilization of, GIH as an ergogenic aid. KEY WORDS fluid overloading, hydration strategy, glycerin, dehydration, exercise performance

The polyunsaturated fatty acids (PUFAs) linoleic acid (18:2) and [alpha]-Iinolenic acid (18:3) in triacylglycerols (TAG) are major factors affecting the quality of plant oils for human health, as well as for biofuels and other renewable applications. These PUFAs are essential fatty acids for animals and plants, but also are the source of unhealthy trans fats during the processing of many foodstuffs. PUFAs 18:2 and 18:3 are synthesized in developing seeds by the desaturation of oleic acid (18:1) esterified on the membrane lipid phosphatidylcholine (PC) on the endoplasmic reticulum. The reactions and fluxes involved in this metabolism are incompletely understood, however. Here we show that a previously unrecognized enzyme, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), encoded by the Arabidopsis ROD1 gene, is a major reaction for the transfer of 18:1 into PC for desaturation and also for the reverse transfer of 18:2 and 18:3 into the TAG synthesis pathway. The PDCT enzyme catalyzes transfer of the phosphocholine headgroup from PC to diacylglycerol, and mutation of rod1 reduces 18:2 and 18:3 accumulation in seed TAG by 40%. Our discovery of PDCT is important for understanding glycerolipid metabolism in plants and other organisms, and provides tools to modify the fatty acid compositions of plant oils for improved nutrition, biofuel, and other purposes. Arabidopsis | lipid metabolism | oilseeds www.pnas.org/cgi/doi/10.1073/pnas.0909731106

The temperate phage [phi]SLT of Staphylococcus aureus carries genes for Panton-Valentine leukocidin. Here, we identify ORF636, a constituent of the phage tail tip structure, as a recognition/adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of S. aureus. ORF636 bound specifically to S. aureus; it did not bind to any other staphylococcal species or to several gram-positive bacteria.

During phenotypic characterization of various Escherichia coli mutants, we observed that [DELTA]phoA strains are capable of using glycerol-2-phosphate (G2P) as a sole source of phosphorus. Mutations in the ugpBAECQ operon eliminated this phenotype, suggesting that G2P is a previously unrecognized substrate for the binding protein-dependent Ugp transporter.

Solvent molecules play key roles in the conformational dynamics of proteins. Here we use single molecule force-clamp spectroscopy to probe the role played by the stabilizing osmolyte glycerol on the conformational ensembles visited by a single ubiquitin protein folding after mechanical extension. Using a variety of force-pulse protocols, we find that glycerol stabilizes the native state of ubiquitin, making it more resistant to mechanical unfolding. We also find that although glycerol enhanced the hydrophobic collapse of unfolded and highly extended ubiquitins, it had no effect on the resulting collapsed states that are essential precursors of the folded state. These disparate effects of glycerol may be the result of distinct structural roles played by solvent molecules at the transition state of each folding ensemble. Our results open the way for a detailed analysis of the transition state structures that form along the folding trajectory of a mechanically extended protein. force-clamp | glycerol | single molecule | solvent

Candida albicans accumulates large amounts of the polyols glycerol and D-arabitol when the cells are exposed to physiological conditions relevant to stress and virulence in animals. Intracellular concentrations of glycerol are determined by rates of glycerol production and catabolism and of glycerol uptake and efflux through the plasma membrane. We and others have studied glycerol production in C. albicans, but glycerol uptake by C. albicans has not been studied. In the present study, we found that [[sup.14]C]glycerol uptake by C. albicans SC5314 was (i) accumulative; (ii) dependent on proton-motive force; (iii) unaffected by carbon source; and (iv) unaffected by large molar excesses of D-arabitol or other polyols. The respective [K.sub.m] and [V.sub.max] values were 2.1 mM and 460 [micro]mol [h.sup.-1] [(g dry wt).sup.-1] in glucose medium and 2.6 mM and 268 [micro]mol [h.sup.-1] [(g dry wt).sup.-1] in glycerol medium. To identify the C. albicans glycerol uptake protein(s), we cloned the C. albicans homologues of the Saccharomyces cerevisiae genes GUP1 and STL1, both of which are known to be involved in glycerol transport. When multicopy plasmids encoding C. albicans STL1, C. albicans STL2 and C. albicans GUP1 were introduced into the corresponding S. cerevisiae null mutants, the transformants all acquired the ability to grow on minimal glycerol medium; however, only S. cerevisiae stl1 null mutants transformed with C. albicans STL1 actively took up extracellular [[sup.14]C]glycerol. When both chromosomal alleles of C. albicans STL1 were deleted from C. albicans BWP17, the resulting stl1 null mutants grew poorly on minimal glycerol medium, and their ability to transport [[sup.14]C]glycerol into the cell was markedly reduced. In contrast, deletion of both chromosomal alleles of C. albicans STL2 or of C. albicans GUP1 had no significant effects on [[sup.14]C]glycerol uptake or the ability to grow on minimal glycerol medium. Northern blot analysis indicated that C. albicans STL1 was expressed in both glucose and glycerol media, conditions under which we detected wild-type active glycerol uptake. Furthermore, STL1 was highly expressed in salt-stressed cells; however, the stl1 null mutant was no more sensitive to salt stress than wild-type controls. We also detected high levels of STL2 expression in glycerol-grown cells, even though deletion of this gene did not influence glycerol uptake activity in glycerol-grown cells. We conclude from the results above that a plasma-membrane [H.sup.+] symporter encoded by C. albicans STL1 actively transports glycerol into C. albicans cells.

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NAD[H.sub.2]. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

Elevated external solute stimulates a conserved MAPK cascade that elicits responses that maintain osmotic balance. The yeast high-osmolarity glycerol (HOG) pathway activates Hog1 MAPK (mammalian ortholog p38[alpha]/SAPK[alpha]), which enters the nucleus and induces expression of >50 genes, implying that transcriptional upregulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we show here that cells lacking the karyopherin required for Hog1 nuclear import or in which Hog1 is anchored at the plasma membrane (or both) can withstand longterm hyperosmotic challenge by ionic and nonionic solutes without exhibiting the normal change in transcriptional program (comparable with hog1[DELTA] cells), as judged by mRNA hybridization and microarray analysis. For such cells to survive hyperosmotic stress, systematic genetic analysis ruled out the need for any Hog1-dependent transcription factor, the Hog1-activated MAPKAP kinases, or ion, glycerol, and water channels. By contrast, enzymes needed for glycerol production were essential for viability. Thus, control of intracellular glycerol formation by Hog1 is critical for maintenance of osmotic balance but not transcriptional induction of any gene. hyperosmotic stress | mutants | protein kinase | signal transduction | yeast

Human and rodent erythrocytes are known to be highly permeable to glycerol. Aquaglyceroporin aquaporin (AQP)3 is the major glycerol channel in human and rat erythrocytes. However, AQP3 expression has not been observed in mouse erythrocytes. Here we report the presence of an aquaglyceroporin, AQP9, in mouse erythrocytes. AQP9 levels rise as reticulocytes mature into erythrocytes and as neonatal pups develop into adult mice. Mice bearing targeted disruption of both alleles encoding AQP9 have erythrocytes that appear morphologically normal. Compared with WT cells, erythrocytes from AQP9-null mice are defective in rapid glycerol transport across the cell membrane when measured by osmotic lysis, [[sup.14]C]glycerol uptake, or stopped-flow light scattering. In contrast, the water and urea permeabilities are intact. Although the physiological role of glycerol in the normal function of erythrocytes is not clear, plasma glycerol is an important substrate for lipid biosynthesis of intraerythrocytic malarial parasites. AQP9-null mice at the age of 4 months infected with Plasmodium berghei survive longer during the initial phase of infection compared with WT mice. We conclude that AQP9 is the major glycerol channel in mouse erythrocytes and suggest that this transport pathway may contribute to the virulence of intraerythrocytic stages of malarial infection. Plasrnodium berghei | aquaglyceroporin

Clinical procedures wherein supraphysiologic temperatures must be achieved in deep layers of tissue via light are often compromised by optical scattering and absorption. Optical clearing of tissue superficial to the target improves the efficacy of such procedures. Glycerol is an attractive chemical agent for achieving dramatic reductions in tissue turbidity, but its net effects on healthy tissue are not fully understood. In this paper, we investigate possible alterations of biaxial mechanical properties in a model collagenous tissue, bovine epicardium, induced by glycerol. Furthermore, we examine the effects of glycerol on the biaxiai thermomechanical properties of epicardium constrained at near-physiologic length. It is seen that mechanical changes induced by glycerol are fully reversed upon rehydration in normal saline. Moreover, glycerol protects cleared tissue by increasing its thermal stability and minimizing thermal alterations of mechanical properties. Index Terms--Biaxial mechanics, collagen, glycerol, isometric loading, optical clearing.

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [[U-.sup.13]C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.

For many years protein kinase C (PKC) has been the subject of extensive studies as a molecular target for the treatment of cancer and other diseases. To better define the role of PKC isozymes in the control of cell proliferation, survival and transformation, the examination of PKC-mediated signal transduction pathways by isozyme-specific intervention has become essential. However, issues related to the selectivity of activators and inhibitors of PKC isozymes, in addition to convoluted cross-talks between phorbol ester-regulated pathways, have greatly complicated our understanding of PKC-mediated responses. An additional level of complexity is provided by the fact diacylglycerol (DAG) signals can be transduced by phorbol ester receptors other than PKC. These receptors include chimaerins, RasGRPs, MUNC13s, PKD (PKC[micro]) and DAG kinases [beta] and [gamma]. Thus, it is conceivable that some of the effects that were originally attributed to PKC isozymes in response to phorbol esters might be mediated by PKC-independent pathways. A key issue for the design of novel therapeutic strategies that target PKC isozymes is a comprehensive analysis of isozyme-specific signal transduction pathways in different cell types and the development of pharmacological and molecular tools that can distinguish between the various PKC and 'non-PKC' phorbol ester receptors.

MALDI MS imaging and single-cell profiling are important new capabilities for mass spectrometry. The distribution of neuropeptides within a cell plays an important role in the functioning of the cells in a neuronal network. Protocols for subcellular MALDI MS are described that allow comparative peptide profiling of cell bodies and the neuronal processes (neurites) using single isolated neurons from the neuronal model Aplysia californica. The seawater surrounding the neurons is problematic for mass spectrometry and so must be removed in a manner that does not cause morphological changes or a redistribution of the neuropeptides. Several protocols have been investigated for subcellular spatial profiling, including the use of air-drying, replacement of the seawater with deionized water, and substitution of the cell matrix with flourinert, mineral oil and glycerol, as well as paraformaldehyde fixation. Glycerol stabilization offers the best combination of preservation of cell morphology and prevention of neuropeptide redistribution. The profiles of the peptides in specific neuronal processes and the cell bodies demonstrate a variety of differences that appear to be cell-specific. These methods are suitable for smaller cells and subcellular MS imaging.

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an 'all-transducer-knockout' strain of E. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

Research has been conducted on the ethanol fermentation by-product, glycerol. The kinetic model of the glycerol synthesis pathway used in evaluating parameters controlling glycerol synthesis rate has been constructed and is described.