Initiation of glucan synthesis in yeast

Abstract Fractions of the glucan synthesized in vitro by glucan synthase preparations obtained by mechanical breakage of whole cells of Sacchromyces cerevisiae and Candida albicans were solubilized by sodium dodecyl sulphate (SDS). Part of this material migrated in denaturing electrophoresis at the level of a Coomassie Blue-stained area. In addition, it was solubilized by both an exoglucanase and papain. These results suggest that some yeast glucan is built, associated with a protein.     

Synthesis of yeast wall glucan.

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.     

Promotion of wound healing by yeast glucan evaluated on single animals

The effectiveness of yeast glucan in the acceleration of wound healing was evaluated in mice, rats and guinea pigs. In all experiments comparison between glucan treatment in one hind leg and saline treatment as control on the other leg was made on identical wounds. The degree of healing in the two legs was evaluated macroscopically and classified as follows: 1. healing more advanced in glucan treated wound marked by (+). 2. No significant difference between the two legs marked by (0). 3. Healing more advanced in the control wound, marked by (-). During the days when the differences were most obvious, 60% to 80% of the animals showed more advanced healing in the glucan treated wound, 20% to 40% showed no significant difference; and 0 to 15% showed more advanced healing in the control, saline treated wound. The average time for complete wound healing was reduced by about 18% as a result of glucan treatment. The histological analysis shows that the acceleration of wound healing was mediated by early arrival of macrophages to the wound area in the glucan treated wounds.     

Endogenous factors that modulate yeast glucan synthetase in cell-free extracts

Yeast 1,3-beta-D-glucan synthetase (EC 2.4.1.34) activity is modulated by endogenous factors obtained by the extraction of different subcellular fractions with hot water. Cell wall fractions were enriched in activators while supernatant fractions also contained appreciable amounts of inhibitors. The action of these compounds requires the presence of EDTA. Maximal activation by the stimulatory material was reached when assayed in sonicated enzymatic preparations that had been obtained by mechanical breakage of cells in water. The activating material derived from cell wall fractions contained a mixture of low molecular weight compounds. They were found to be different from GTP as deduced from their resistance to alkaline phosphatase and different elution profile in gel filtration. The supernatant material was also heterogeneous with regard to both activators and inhibitors. The combined effect of GTP and activating material derived from cell wall fractions was supraadditive. The polymers synthesized in the absence and in the presence of the endogenous activator were characterized as beta-1,3-glucans on the base of their resistance to periodate and susceptibility to beta-glucanases. However, the length of the radioactive chains was greater when synthesized in the presence of the activator. This was mediated by an increase in the Vmax of the synthetase.     

Iron absorption by small intestine of chickens

Iron (Fe) absorption by three segments (duodenum, jejunum, and ileum) of the small intestine of chickens was studied by a perfusion technique in vivo in closed circuit using⁵⁹Fe Cl₃ and was related to the histological characteristics of each segment. The serosal transfers of Fe for the duodenum and jejunum were the same (14%/cm), but significantly different (p<0.05) from those of the ileum (9%/cm), which may be explained by the morphological and histological properties of the gut of chickens. However, the presence of Fe in blood and in liver was significantly lower after perfusion of the jejunum and ileum than after perfusion of the duodenum. It is concluded that chickens show an early adaptation of small intestine to Fe absorption in response to the considerable loss of Fe suffered during the laying process.     

Iron metabolism in the yeast

Current data concerning transport, storage and utilization of iron in the yeast cells, particularly Saccharomyces cerevisiae are summarized in the paper. It has been marked that iron uptake in the cells provides by high affinity system, it function is carried out by protein complex Fet3-Ftr1, and Fet4, protein with low affinity to iron ion. The both systems utilize Fe(II). Furthermore, the active site of the protein Fre1 is exposed on the outer side of plasmalemma. This protein, due to ferrireductase activity, provides availability of Fe(III) to the cell. The information regards to participation of siderophores and metal-proton plasma membrane exchangers Smf1 in iron transport is brought. Particular attention is given to regulation of expression of the genes, coding the iron metabolic systems. Some aspects of iron utilization for Fe-S-containing enzymes synthesis are lighted. It has been concluded that the yeast is a perspective subject for studying balance of living organisms between iron essentiality and its ability to trigger free radical reactions.     

Preliminary evidence for a glucan acceptor in the yeast Candida albicans.

Two membrane preparation containing glucan synthase activity were obtained by lysis of regenerating sphaeroplasts (enzyme A) or mechanical breakage (enzyme B) of yeast (Candida albicans) cells. The reaction products of both enzymes (glucans A and B respectively) were characterized as linear beta-1,3-linked glucans on the basis of chemical and enzymic analysis. In addition, two pools of glucan could be distinguished in glucan A preparations on the basis of their susceptibility to an exoglucanase. In no case were the reaction products synthesized de novo; rather the radioactive chains were added to the non-reducing end of non-radioactive preformed glucan chains or to an acceptor of a different nature. At least some of the performed chains of glucan A, but not those of glucan B, showed a free reducing terminal. Glucan A preparations were endowed with endoglucanase activity, which, under appropriate conditions, released glucose, laminaribiose and laminaritriose. These sugars were also found in cell-wall autolysates. On the basis of the origin of both enzyme preparations it is suggested that glucan molecules are synthesized while they are bound to a non-glucan acceptor that is subsequently excised, presumably by cell-wall-associated glucanases.     

Iron absorption from concentrated hemoglobin hydrolysate by rat

Although heme iron is highly bioavailable, the low iron content of hemoglobin prevents its use for dietary fortification; on the other hand, purified heme has low solubility and absorption rate. The present study was designed to assess the interactions between concentrated heme iron and peptides released during globin hydrolysis and cysteine and their relation with iron absorption. Hemoglobin was hydrolyzed by pepsin or subtilisin, and then, heme iron was concentrated by ultrafiltration. Iron absorption was studied in a Ussing chamber; gluconate was used as control. Iron uptake from nonconcentrated pepsin hydrolysate and gluconate was lower than from other groups. Cysteine significantly enhanced iron uptake except from the concentrated subtilisin hydrolysate. There was no significant difference between cysteine-supplemented groups. According to the different hydrolysis pathways of enzymes, it is assumed that the presence of hydrophobic peptides and the strength of heme-peptide interactions are both determining factors of heme iron absorption. These interactions occur mainly before iron uptake, as emphasized by the effect of cysteine.     

Iron absorption by Belgrade rat pups during lactation

Divalent metal transporter-1 (DMT1) mediates dietary nonheme iron absorption. Belgrade (b) rats have defective iron metabolism due to a mutation in the DMT1 gene. To examine the role of DMT1 in neonatal iron assimilation, b/b and b/+ pups were cross-fostered to F344 Fischer dams injected with (59)FeCl(3) twice weekly during lactation. Tissue distribution of the radioisotope in the pups was determined at weaning (day 21). The b/b pups had blood (59)Fe levels significantly lower than b/+ controls but significantly higher (59)Fe tissue levels in heart, bone marrow, skeletal muscle, kidney, liver, spleen, stomach, and intestines. To study the pharmacokinetics of nonheme iron absorption at the time of weaning, (59)FeCl(3) was administered to 21-day-old b/b and b/+ rats by intragastric gavage. Blood (59)Fe levels measured 5 min to 4 h postgavage were significantly lower in b/b rats, consistent with impaired DMT1 function in intestinal iron absorption. Tissue (59)Fe levels were also lower in b/b rats postgavage. Combined, these data suggest that DMT1 function is not essential for iron assimilation from milk during early development in the rat.     

Iron absorption in the iron-deficient rat

Iron absorption in the iron-deficient rat was compared with that in the normal rat to better understand the regulation of this dynamic process. It was found that: Iron uptake by the iron-deficient intestinal mucosa was prolonged as a result of slower gastric release, particularly when larger doses of iron were employed. The increased mucosal uptake of ionized iron was not the result of increased adsorption, but instead appeared related to a metabolically active uptake process, whereas the increased mucosal uptake of transferrin iron was associated with increased numbers of mucosal cell membrane transferrin receptors. Mucosal ferritin acted as an iron storage protein, but its iron uptake did not explain the lower iron absorption in the normal rat. Iron loading the mucosal cell (by presenting a large iron dose to the intestinal lumen) decreased absorption for 3 to 4 days. Iron loading of the mucosal cell from circulating plasma transferrin was proportionate to the plasma iron concentration. Mucosal iron content was the composite of iron loading from the lumen and loading from plasma transferrin versus release of iron into the body. These studies imply that an enhanced uptake-throughout mechanism causes the increased iron absorption in the iron-deficient rat. Results were consistent with the existence of a regulating mechanism for iron absorption that responds to change in mucosal cell iron, which is best reflected by mucosal ferritin.     

Increase in glucan formation by Botrytis cinerea and analysis of the adherent glucan

Summary Glucan production by Botrytis cinerea increased from 1 g/l to 3 g/l when KNO₃ or urea replaced asparagine as the nitrogen source. A further enhancement up to 5 g/l was obtained with nitrogen-limited medium or non-growing cells. Under these conditions an extracellular glucan layer was attached to the mycelium. The adherent glucan made up 60% of the total amount of glucan produced and thus increased the total glucan yield to 13 g/l. An enzymatic analysis of the adherent glucan indicated that only about every fifth molecule of the main chain was substituted by a glucose unit. In contrast, in the free glucan of culture filtrates glucose units were distributed at approximately every second to third residue of the main chain. Offprint requests to: P. Stahmann     

Immune-enhancing effect of water-soluble beta-glucan derived from enzymatic hydrolysis of yeast glucan

Immunostimulants play an important role in the treatment of immunodeficiency. Macrophages are the first line in our immune defense system and play a critical role in the immune response. Therefore, finding new and better substances to induce an immune response by activating macrophages is an attractive research topic, especially in the fields of immunopharmacology and cancer prevention. Keratinocytes actively crosstalk with immune cells during wound repair, so enhancing the function of keratinocytes is also an important part of improving immunity. Beta-glucans are naturally occurring polysaccharides, consisting of d-glucose monomers linked by beta-glycosidic bonds. Several studies have investigated the immunomodulatory effects of beta-glucan, such as its anti-inflammatory and antibacterial properties. However, the use of yeast cell wall glucan has been limited because it is not soluble in water. In this study, we produced low-molecular-weight water-soluble yeast glucan (WSY glucan) and confirmed various aspects of its immune-enhancing effect. The structure of the beta-(1→3) and (1→6) bonds of WSY glucan were confirmed by nuclear magnetic resonance spectroscopy (¹H-NMR) analysis. Our results showed that treatment with WSY glucan significantly and dose-dependently induced the production of inflammatory mediators (prostaglandin E₂ (PGE₂) and nitric oxide (NO)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) in macrophages. In addition, WSY glucan treatment showed changes in the morphological structure of the macrophages and promoted phagocytic activity of the macrophages and wound healing in keratinocytes. Based on these results, WSY glucan is considered as a potential candidate for the treatment of diseases related to the weakening of the immune system without the limitation of insolubility.     

Coordination between fission yeast glucan formation and growth requires a sphingolipase activity

css1 mutants display a novel defect in Schizosaccharomyces pombe cell wall formation. The mutant cells are temperature-sensitive and accumulate large deposits of material that stain with calcofluor and aniline blue in their periplasmic space. Biochemical analyses of this material indicate that it consists of alpha- and beta-glucans in the same ratio as found in cell walls of wild-type S. pombe. Strikingly, the glucan deposits in css1 mutant cells do not affect their overall morphology. The cells remain rod shaped, and the thickness of their walls is unaltered. Css1p is an essential protein related to mammalian neutral sphingomyelinase and is responsible for the inositolphosphosphingolipid-phospholipase C activity observed in S. pombe membranes. Furthermore, expression of css1(+) can compensate for loss of ISC1, the enzyme responsible for this activity in Saccharomyces cerevisiae membranes. Css1p localizes to the entire plasma membrane and secretory pathway; a C-terminal fragment of Css1p, predicted to encode a single membrane-spanning segment, is sufficient to direct membrane localization of the heterologous protein, GFP. Our results predict the existence of an enzyme(s) or process(es) essential for the coordination of S. pombe cell wall formation and division that is, in turn, regulated by a sphingolipid metabolite.