Dependence of arbuscular-mycorrhizal fungi on their plant host for palmitic acid synthesis

Lipids are the major form of carbon storage in arbuscular-mycorrhizal fungi. We studied fatty acid synthesis by Glomus intraradices and Gigaspora rosea. [(14)C]Acetate and [(14)C]sucrose were incorporated into a synthetic culture medium to test fatty acid synthetic ability in germinating spores (G. intraradices and G. rosea), mycorrhized carrot roots, and extraradical fungal mycelium (G. intraradices). Germinating spores and extraradical hyphae could not synthesize 16-carbon fatty acids but could elongate and desaturate fatty acids already present. The growth stimulation of germinating spores by root exudates did not stimulate fatty acid synthesis. 16-Carbon fatty acids (16:0 and 16:1) were synthesized only by the fungi in the mycorrhized roots. Our data strongly suggest that the fatty acid synthase activity of arbuscular-mycorrhizal fungi is expressed exclusively in the intraradical mycelium and indicate that fatty acid metabolism may play a major role in the obligate biotrophism of arbuscular-mycorrhizal fungi.     

Activation and expression of proteins during synchronous germination of aerial spores of Streptomyces granaticolor

Synchronously germinating aerial spores of Streptomyces granaticolor were used to study protein activation and expression during the transition from dormant to metabolically active vegetative forms. The first phase of protein activation is associated with the solubility of proteins. Three major chaperones, DnaK, Trigger factor, and GroEL, were identified in spores. Enhancement in rate of protein synthesis during germination was accompanied by the association of TF and DnaK with ribosomes. During germination, the chaperones TF, GroEL, and DnaK undergo reversible phosphorylation. GroEL was phosphorylated on both Ser and Thr, whereas phosphorylation of DnaK and TF was detected on Thr only. A proteomic approach was used to gain more information on protein expression during germination on two types of media differing in the ability of cells to produce antibiotic granaticin. To obtain an overview of the metabolic activity of germinating spores, glycolytic enzymes, enzymes of citric acid cycle, metabolism of amino acids and nucleic acids, and components of the protein synthesis system were identified and analyzed using the proteomic database. The results were deposited on the SWICZ proteomic server and are accessible on http://proteom.biomed.cas.cz.     

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.     

Actin cross-linking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development

Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other amoebae stream, forming multicellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA, RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced. As a consequence, cell streaming and development are completely blocked. Expression of ACA-yellow fluorescent protein in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype, indicating that the primary chemotaxis and development defect is the inhibition of ACA synthesis and cAMP production. These results demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis, and development in Dictyostelium.     

Molecular biology of the germination of Bacillus spores

The review deals with recent results and problems of gene expression during germination of Bacillus spores. Three problems were selected: 1. The activation of metabolism as a prerequisite for the synthesis of nucleic acids and proteins. 2. The activation of nucleic acid and protein synthesis during germination. 3. The gene expression programme of germinating spores. Using the highly sensitive two-dimensional polyacrylamide gel analysis three major classes of proteins were distinguished, depending on the time of onset and duration of their syntheses: a) proteins made throughout germination (main class), b) proteins whose synthesis started only after a lag phase and then continued throughout germination, and c) proteins which are synthesized only during the early phases of germination. The programme of protein synthesis is an indicator for the control of gene expression during germination. The regulation of expression of these major gene groups during spore outgrowth is discussed.     

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.     

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.     

Dependence of Arbuscular-Mycorrhizal Fungi on Their Plant Host for Palmitic Acid Synthesis

Lipids are the major form of carbon storage in arbuscular-mycorrhizal fungi. We studied fatty acid synthesis by Glomus intraradices and Gigaspora rosea. [¹⁴C]Acetate and [¹⁴C]sucrose were incorporated into a synthetic culture medium to test fatty acid synthetic ability in germinating spores (G. intraradices and G. rosea), mycorrhized carrot roots, and extraradical fungal mycelium (G. intraradices). Germinating spores and extraradical hyphae could not synthesize 16-carbon fatty acids but could elongate and desaturate fatty acids already present. The growth stimulation of germinating spores by root exudates did not stimulate fatty acid synthesis. 16-Carbon fatty acids (16:0 and 16:1) were synthesized only by the fungi in the mycorrhized roots. Our data strongly suggest that the fatty acid synthase activity of arbuscular-mycorrhizal fungi is expressed exclusively in the intraradical mycelium and indicate that fatty acid metabolism may play a major role in the obligate biotrophism of arbuscular-mycorrhizal fungi.     

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Cell cycle traverse and macromolecular synthesis in BHK fibroblasts as affected by insulin

Insulin produced a 10-fold activation of cAMP phosphodiesterase activity and a 3-fold reduction in cAMP content in serum-deprived, growth-arrested BHK fibroblasts. Insulin did not promote growth of these cells, as judged by measurement of cell number, but it did promote reentry of cells into the cell cycle and progression into S phase, as determined by rates of macromolecular synthesis, and autoradiographic and microfluorometric analyses. Insulin stimulation of macromolecular synthesis can be blocked by the addition of a phosphodiesterase inhibitor, suggesting that these effects of insulin on the cell cycle may be mediated through its activation of cAMP phosphodiesterase.     

Parathyroid hormone induction of creatine kinase activity and DNA synthesis is mimicked by phospholipase C, diacylglycerol and phorbol ester

Parathyroid hormone (PTH), which increases cAMP levels, also induces an increase in the activity of the brain isozyme of creatine kinase and in DNA synthesis in osteoblast-enriched bone cell cultures by a cAMP-independent mechanism. The following results lead us to the conclusion that PTH induction of brain isozyme of creatine kinase activity and DNA synthesis occurs by activation of membranal phospholipid metabolism leading to increased protein kinase C activity and Ca2+ mobilization, a mechanism demonstrated for several growth factors and other hormones. (1) Binding of membranal phospholipids by agents such as gentamycin or antiphospholipid antibodies abolishes the stimulation by PTH of creatine kinase activity and DNA synthesis but not of cAMP production. (2) Treatment of cell cultures with exogenous phospholipase C increases brain isozyme of creatine kinase activity and DNA synthesis, but not cAMP production; these stimulations are also blocked by serum containing anti-phospholipid antibodies. PTH has no additional effect on stimulation of creatine kinase activity by phospholipase C (and only a slight effect on DNA synthesis). (3) A synthetic diacylglycerol (1-oleyl-2-acetyl glycerol) or phorbol ester (phorbol 12-myristate 13-acetate) or Ca2+ ionophore, A23187 induces creatine kinase activity and DNA synthesis in the cultures. However, this effect is not blocked by antiphospholipid sera and PTH has no additional effect. (4) Inhibition of protein kinase C activity by drugs reported to inhibit the enzyme (retinoic acid, quercetin) abolishes the stimulation of brain isozyme of creatine kinase activity and of DNA synthesis by PTH.     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

Uptake of arachidonic acid into membrane phospholipids: Effect on chloride transport across cornea

Summary We demonstrate that arachidonic acid (AA) stimulation of chloride transport across frog cornea is mediated via two independent pathways: (1) stimulation of prostaglandins and cAMP synthesis, and (2) a direct physical change in the membrane produced by substitution of different phospholipid acyl chains. AA is well known as a precursor in the synthesis of prostaglandins, which have been shown to stimulate cAMP synthesis and chloride transport in frog cornea. We show that frog cornea can convert exogenous AA to PGE₂, but that in the presence of 10^–5 m indomethacin both the conversion to PGE₂ and stimulation of cAMP are completely blocked. However, with indomethacin the action of AA to stimulate chloride transport (as measured by SCC) remains, but peak height of the response is reduced to 57% of that found when AA alone is given. Similarly, we show that propranolol completely blocks cAMP stimulation, but stimulation of SCC is reduced to 45% of the original response. Therefore, cAMP appears to be responsible for roughly half of the observed stimulation in SCC. By gas chromatographic analysis we show that significant quantities of AA can rapidly substitute into membrane phospholipids of corneal epithelium and L929 cells following the addition of AA to the medium. Modification of membrane phospholipid structure can affect membrane viscosity, membrane-bound enzyme activity, and the distribution and lateral mobility of integral proteins. It seems likely that such alterations in the properties of the membrane may modulate the rate of chloride transport, and this may constitute the second mechanism. Upon addition of AA, both mechanisms appear to stimulate chloride transport simultaneously, and are apparently additive. We show that prolonged exposure to AA results in a large incorporation of AA into phospholipid and consequently, a perturbation in the ratio of unsaturated to saturated fatty acids. We also find evidence of a compensatory cellular mechanism that alters the ratio of endogenously synthesized fatty acids and tends to reduce the membrane-perturbing effect of AA.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

Mechanism of stimulation of globin synthesis by adenosine-3',5'-monophosphate and guanosine 5'-triphosphate in a rabbit reticulocyte lysate system

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.     

Temporal control of autoactivator synthesis and secretion during spontaneous spore germination in Dictyostelium discoideum

SG mutant and aged wild type spores of the cellular slime mold Dictyostelium discoideum germinate in the absence of an externally applied activation treatment. This type of germination is referred to as autoactivation. During the swelling stage of autoactivation, spores release a factor, the autoactivator, capable of stimulating germination in subsequent spore populations. The autoactivator was not present in the dormant spore, but it or a precursor was produced internally during the first hour of autoactivation. This production was sensitive to moderately high temperatures (+31° C) and was completely destroyed by heat activation (45° C for 30 min). Internal production of the autoactivator was not sensitive to protein synthesis inhibitors. However, the release of the activator from the spore appeared to be regulated by protein synthesis. Internal autoactivator was also produced in the aged wild type strain during the postautoactivation lag phase. The activator could not be directly isolated from within the germinating spore. Its activity on the rest of the spore population was dependent upon its release from the germinating spore. A model is presented integrating the effects of heat, cycloheximide, autoinhibitor and autoactivator on spores of D. discoideum.     

Metabolic effects and cyclic AMP levels produced by glucagon, (1-N alpha-Trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes

[1-N alpha-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.     

Cyclic AMP modulation of human B cell proliferative responses: role of cAMP-dependent protein kinases in enhancing B cell responses to phorbol diesters and ionomycin.

The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.