The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in baker's yeast

An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375 000–400 000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3′,5′-ADP and 4′-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzesa reaction between 4′-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase. to reform CoA. This sequence of reactions, referred to as the CoA/4′-phosphopantethiene cycle, provides a mechanism by which the 4′-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3′,5′-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4′-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment is referred to as the 10 000–15 000 M_r subunit. The 5′-ADP-4′-pantothenic acid-synthetase, 5′-ADP-4′-pantothenylcysteine-synthetase, 5′-ADP-4′-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10 000–15 000 M_r subunit. Thus, the 10 000–15 000 M_r subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.     

Coenzyme a-synthesizing protein complex of Saccharomyces cerevisiae

The coenzyme A-synthesizing protein complex (CoA-SPC) is a multienzyme complex of Saccharomyces cerevisiae (Bakers' yeast), which has a molecular weight in excess of 200,000 as determined by Sephadex G-200 column chromatography. This multienzyme complex, which is insoluble in the crude yeast cell lysate, has been purified 229-fold. A cellular component of the yeast cell lysate, referred to as t-Factor, with a molecular weight of 400-1000 and chloride ion are involved in the solubilization of CoA-SPC. The CoA-SPC requires L-cysteine, D-pantothenic acid and ATP as substrates. The terminal CoA-SPC-bound intermediate is dephospho-CoA, which is subsequently phosphorylated and released from the complex as CoA. The sequence of reactions for the synthesis of CoA by the CoA-SPC differs significantly from those previously proposed for other systems. It could be that the reaction sequence is unique for the yeast cell.     

Bursts of CO2 released during freezing offer a new perspective on avoidance of winter embolism in trees

MethodsCO₂ efflux measurements were conducted during freezing experiments for saplings of three Scots pine (Pinus sylvestris) and three Norway spruce (Picea abies) trees under laboratory conditions, and the magnitudes of the freezing-related bursts of CO₂ released from the stems were analysed using a previously published mechanistic model of CO₂ production, storage, diffusion and efflux from a tree stem. The freezing-related bursts of CO₂ released from a mature Scots pine tree growing in field conditions were also measured and analysed.ConclusionsAll gases dissolved in the xylem sap are not trapped within the ice in the stem during freezing, as has previously been assumed, thus adding a new dimension to the understanding of winter embolism formation. The conduit water volume not only determines the volume of bubbles formed during freezing, but also the efficiency of gas efflux out of the conduit during the freezing process.     

Determining the disinfection of textiles in compressed carbon dioxide using various indicator microbes

Aims: This paper presents a research on the disinfection efficiency of inoculated textile swatches by compressed carbon dioxide, an environmental friendly way to disinfect textiles as opposed to the conventional laundering procedures using water. The disinfection efficiency was determined by using the following microbes inoculated on cotton test fabrics: Enterococcus faecium, Enterobacter aerogenes and Candida albicans. Methods and Results: The experiments were performed using the high pressure extraction device with a maximum pressure of 50 MPa and a small extraction vessel of 500 ml. Pure CO₂ and CO₂ with added disinfection agent or commercial detergent were used. The chosen disinfecting agent was hydrogen peroxide, a widespread disinfecting chemical. It was found that treatment with CO₂ for 25 min at 5 MPa and 40°C (313K) and the addition of 4 ml of specific detergent per litre of CO₂ assures at least a five log step reduction of Enterobacter aerogenes and C. albicans, whilst treatment at 50°C (323K) with CO₂ for 25 min at 5 MPa is sufficient for at least a five log step reduction for Enterococcus faecium. It was also found that a 15‐min CO₂ treatment at 7 MPa and 20°C (293K) was sufficient for the inactivation of the yeast C. albicans, whilst these conditions were not rigorous enough for the challenge bacteria. On the other hand, the labscale treatment with CO₂ for 25 min at pressure 4 and 6 MPa with the addition of detergent or hydrogen peroxide only yields a log step reduction of up to 4 log steps, thus proving the slightly disinfective properties of the CO₂ treatment with added agents, but not reaching efficient results as a 5 log step reduction was not reached. Conclusions: Addition of heat to the compressed CO₂ treatment of textiles inoculated with microorganisms proved more effective than the addition of detergent or disinfectant with compressed CO₂ treatment at temperature of 20°C. Significance and Impact of the Study: CO₂ treatment of textiles is a promising ecological alternative dry‐cleaning method for the disinfection of medical textiles.     

The composition and structure of lithium tetrakis(pentafluorophenyl)borate diethyletherate

Treatment of LiC₆F₅ with B(C₆F₅)₃ in equal volumes of light petroleum and diethyl ether at low temperature followed by slow warming to room temperature precipitates a microcrystalline solid which dries under vacuum to a material with the composition [Li(OEt₂)₃][B(C₆F₅)₄]. Crystallization from diethyl ether yields solvent dependent [Li(OEt₂)₄][B(C₆F₅)₄], which has been crystallographically characterised.     

How the formation process influences the structure of BChl c aggregates

The change of absorption spectra has been measured during the drying process of (3¹ R)bacteriochlorophyll (BChl) c from diethyl ether, dichloromethane (CH₂Cl₂) and carbon tetrachloride (CCl₄) solutions. Absorption maxima of the Q_y(0–0) transition of BChl c appear at 659 nm in diethyl ether, 680 nm in CH₂Cl₂ and 710 nm in CCl₄. All these peaks are red-shifted to about 740 nm with formation of solid high aggregates when the solutions are completely dried. Fourier transform infrared spectra of the three solid aggregates are almost identical. However, magnetic circular dichroism and circular dichroism spectra are different and can be explained in terms of variations in stacking size of the aggregates and molecular arrangement of BChl c. Small-angle X-ray diffraction has been observed only for the aggregates treated with CH₂Cl₂, and the same sample gave rise to highly resolved cross polarization/magic angle spinning ¹³C nuclear magnetic resonance spectrum. The results suggest that molecular ordering of the solid-state BChl c aggregates is highly dependent on the formation process which is largely determined by the solvent used.     

Inactivation of Viable Ascaris Eggs by Reagents during Enumeration

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H₂SO₄) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-μm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO₄, MgSO₄, and NaCl) or during incubation (0.1 N H₂SO₄ and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.     

Fluid dynamics of anaerobic fermentation

In beer fermentation, yeast cells are kept in suspension, despite their higher density, by natural agitation created by ascending CO₂ bubbles. Yeast cells are unable to nucleate bubbles but instead release CO₂ in a soluble form in such a way that the medium tends to become supersaturated. A higher concentration of yeast cells and the presence of solid particles cause the formation of bubbles at the bottom of the fermenter and practically only there. The rising bubbles grow and accelerate by sweeping the CO₂ formed throughout the fermenter by the suspended yeast cells, thereby creating a fluid regime. A mathematical expression relating the bubble agitation power to the fermentation parameters was obtained and used to design more efficient fermenter shapes.     

The specific radioactivity of glycolic acid in relation to the specific activity of carbon dioxide evolved in light in photosynthesizing sunflower leaves

Attached leaves of sunflower (Helianthus annuus L.) were exposed to ¹⁴CO₂ during steady-state photosynthesis for 2 to 30 min in 345 l/l CO₂ and 21% O₂ at 29° C and a light intensity of 1300 E m^-2s^-1. Glycolic acid was extracted with water and diethyl ether, and was determined in the aqueous residue by high-pressure liquid column chromatography. The relative specific radioactivity of the glycolic acid synthesized during photosynthesis reached about 100% after 30 min of photosynthesis and was almost equal to that of the CO₂ evolved during photorespiration, their ratio at all times being nearly one. These results provide strong in-vivo evidence that the glycolic acid is the substrate for CO₂ evolved by sunflower leaves in light.     

The effect of freezing and the influence of isolation medium on the recovery of pathogenic fungi from sputum

The primary objective of this study was to determine whether freezing sputa in dry ice had any effect on the recovery of pathogenic fungi. Sputa seeded with each of five fungi (Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Coccidioides immitis, and Aspergillus fumigatus) were frozen and stored for 24, 48, and 72 hours on dry ice. H. capsulatum was killed, and only a few colonies of B. dermatitidis and C. neoformans were isolated from these sputa. However, A. fumigatus and C. immitis withstood the effects of freezing. A second objective was to compare the recovery of all five fungi from seeded sputa stored at room temperature for 24, 48, and 72 hours, on yeast extract-phosphate agar with NH₄OH and on Sabhi agar. The yeast extract-phosphate agar with NH₄OH was superior to Sabhi agar, for the isolation of all fungi studied, except A. fumigatus.     

Structural basis for carbon dioxide binding by 2-ketopropyl coenzyme M oxidoreductase/carboxylase

The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been determined in a state in which CO₂ is observed providing insights into the mechanism of carboxylation. In the substrate encapsulated state of the enzyme, CO₂ is bound at the base of a narrow hydrophobic substrate access channel. The base of the channel is demarcated by a transition from a hydrophobic to hydrophilic environment where CO₂ is located in position for attack on the carbanion of the ketopropyl group of the substrate to ultimately produce acetoacetate. This binding mode effectively discriminates against H₂O and prevents protonation of the ketopropyl leaving group.

Structured summary

2-KPCCbinds to 2-KPCC by x-ray crystallography (View interaction)     

Source-sink relations affect growth but not the allocation pattern of birch (Betula pendula Roth) seedlings under elevated [CO2]

ABSTRACT

Birch (Betula pendula Roth.) seedlings were kept for two growing seasons under ambient (～350 µmol mol^-1) and elevated (～700 µmol mol^-1) [CO₂]. The present study was designed to examine the effects of [CO₂] and pot size on growth and carbon allocation under conditions of non-limiting water and nutrient supply, in order to separate the effects of source-sink interaction from the effects of nutrient deficiency. The manipulation of the source-sink relations had a strong influence on the growth response to elevated [CO₂]. When the rooting volume was inadequate, it resulted in a source-sink imbalance which constrained growth under elevated [CO₂]. When root exploration was unconstrained, total dry mass was significantly increased (by about 24%) under elevated [CO₂]. However, the allometric relationships in allocation pattern and in morphogenetic development were not affected by either [CO₂] or pot treatments when the saplings were of the same size. Thus, by constraining dry mass production, small sinks affected the magnitude of the growth responses to elevated [CO₂], but did not affect the plant allocation pattern and allometric relationships when nutrient supply was non-limiting. However, by slowing down growth, sink restrictions counteract the speed-up of ontogeny which is the main effect of elevated [CO₂] on tree growth.     

Carbon dioxide improves the growth of hairy roots cultured on solid medium and in nutrient mists

The effect of varying CO₂ concentrations on the growth of beet and safflower hairy roots was measured for tissues cultured in nutrient mists and on solid media in chambers fed mixtures of humidified air supplemented with different CO₂ concentrations. Hairy root tissue grown on solid media in air enriched with CO₂ showed increased growth, as measured by dry weight increases vs air-fed controls. Growth increased with CO₂ enrichment as much as 2.5 times more than the air-fed control for safflower at 1.0% CO₂ and 1.4 times more than the air-fed control for beets at 1.5% CO₂ over a 12-day period. Beet hairy root tissue was also cultured aeroponically in nutrient mists. Beet hairy root cultured aeroponically in nutrient mists enriched with 1.0% CO₂ showed a 15% increase in biomass over a 7-day period vs tissue cultured in nutrient mists (with ambient air) or in shake flasks. The stimulation of root growth via CO₂ enrichment reduced the time required for biomass accumulation. Correspondence to: A. A. DiIorio     

Response of Sphagnum fuscum to water levels and CO2 concentration

Abstract

Sphagnum fuscum samples collected from an ombrotrophic bog were grown in a greenhouse at six water levels (0, 5, 10, 15, 25 and 30 cm) below the capitulum level and in four concentrations of CO₂ (350, 700, 1000 and 2000 ppm). The cores of S. fuscum were treated for 87 days and length increment was measured by the plastic strip method and by innate time markers. Water content of the shoot, dry mass of the capitulum, dry mass per unit length of stem and production of dry mass were measured at the end of the experiment.

The water content, capitulum dry mass, dry mass per unit length of stem, length increment and dry mass production differed markedly for S. fuscum grown in different water levels. With lower water levels, the water content of the shoot decreased and the dry mass of both the capitulum and unit length of stem increased. The total length increment was highest when the water level was at or near the capitulum level (0–10 cm). No clear trend in dry mass production on an areal basis could be found due to uncoupled responses in length increment and stem dry mass at the experimental water levels.

Neither capitulum dry mass nor dry mass per unit length of stem showed distinct trends in S. fuscum grown at different ambient CO₂ concentrations. Some increase in length increment and in dry mass production was detected at CO₂ concentrations above 350 ppm, but this effect appeared only at high water levels. It is suggested that the low response in length increment and production to CO₂ concentration resulted in part from insufficient moisture for photosynthesis at the lower water levels. Also, the possibility of increased nonstructural production is discussed.     

Synthesis and Biological Evaluation of Some Acyclic Pyridine C-Nucleosides. Part Two

Abstract

3-Bromo-5-(2-hydroxyethylthiomethyl)pyridine (7) was synthesized by reaction of 3-bromo-5-chloromethylpyridine hydrochloride (6) with the mono sodium salt of 2-mercaptoethanol. 3-Bromo-5-hydroxymethylpyridine (10) was, after protection as a silyl ether, converted to the 3-carboxy analogue using BuLi and CO₂. After deprotection with NH₄F, the alcohol function was chlorinated using SOCl₂. Finally, attachment of the acyclic chain and ammonolysis gave the acyclic nicotinamide nucleosides. Treatment of the latter compounds with Lawesson's reagent gave the thioamide analogues. All compounds were identified by NMR and DCI-MS. The acyclic pyridine C-nucleosides were evaluated against a series of tumor-cell lines and a variety of viruses. No marked biological activity was found.     

Determination of Nucleic Acids in Yeast from the Viewpoint of Elution Behavior of Acid-Soluble Compounds

It was reported previously that Ogur and Rosen’s method for the determination of nucleic acids should be applied to fresh or durable yeast, after a freezing pretreatment with dry ice and ether. The fractionation of nucleic acids from the frozen yeast should precede with a complete elimination of alcohol-ether-soluble and acid-soluble compounds. It has been found that in Ogur and Rosen’s method, the elimination of the former compounds is complete, but the removal of acid-soluble compounds is unsatisfactory in the case of yeast.

In order to improve this defect, the best conditions for the elution of acid-soluble compounds have been investigated in detail, and it has been found that water is the most suitable solvent.     

Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis

 Sporulation in the yeast Saccharomyces cerevisiae is a meiotic developmental process that occurs in MAT a/MATα heterozygotes in response to nutrient deprivation. Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions. Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination. In addition, there is no requirement for peroxisome degradation prior to spore packaging. Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle. Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous Δpex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether. Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate. On solid oleate medium, homozygous pex5,Δpex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was. The results presented here are discussed in terms of the transmission of organelles to progeny cells. Accepted: 19 December 1997     

CO<Subscript>2</Subscript> enrichment,nitrogen fertilization and development of freezing tolerance in Norway spruce

Plant growth and adaptation to cold and freezing temperatures in a CO₂-enriched atmosphere have received little attention despite the predicted effects of elevated CO₂ on plant distribution and productivity. Norway spruce [Picea abies (L.) Karst.] seedlings from latitudinally distinct seed sources (66°N and 60°N) were grown for one simulated growth season under controlled conditions in an atmosphere enriched in CO₂ (70 Pa) and at ambient CO₂ (40 Pa), combined factorially with low (3.6 mM) or high (15.7 mM) concentrations of nitrogen fertilization. There was a clear difference between the two provenances in height growth, in the timing of bud set, and in freezing tolerance. Nitrogen fertilization increased height growth in both provenances, while CO₂ enrichment stimulated height growth only in the southern provenance. We found no significant effects of elevated CO₂ or nitrogen fertilization on the timing of bud set. During cold acclimation, freezing tolerance increased from –10°C to –35°C, and there was a marked increase in all soluble sugars except inositol. Elevated CO₂ in combination with high nitrogen led to a slight increased freezing tolerance in both provenances during the early stages of cold acclimation. However, towards the end of cold acclimation, elevated CO₂ and high nitrogen led to reduced freezing tolerance in the southern provenance, while elevated CO₂ and low nitrogen reduced freezing tolerance in the northern provenance. These results suggest that CO₂ enrichment influences the development of freezing tolerance, and that these responses differ with available nitrogen and between provenances.     

Enzyme denaturation in supercritical CO2: Stabilizing effect of S-S bonds during the depressurization step

Summary The stability of the monomeric enzymes -chymotrypsin and trypsin, and the oligomeric enzyme penicillin amidase in supercritical CO₂ has been studied. They were found to be partly denatured during the depressurization step. The degree of denaturation was larger in humid CO₂ than in dry CO₂. Enzymes with S-S bridges (-chymotrypsin; trypsin) were denatured to a lesser degree than the enzyme without cysteine (penicillin amidase). These results and electrophoretic and spectroscopic analysis indicated that the denaturation was caused by partial unfolding during the depressurization step.     

Reduced early growing season freezing resistance in alpine treeline plants under elevated atmospheric CO2

The frequency of freezing events during the early growing season and the vulnerability to freezing of plants in European high‐altitude environments could increase under future atmospheric and climate change. We tested early growing season freezing sensitivity in 10 species, from four plant functional types (PFTs) spanning three plant growth forms (PGFs), from a long‐term in situ CO₂ enrichment (566 vs. 370 ppm) and 2‐year soil warming (+4 K) experiment at treeline in the Swiss Alps (Stillberg, Davos). By additionally tracking plant phenology, we distinguished indirect phenology‐driven CO₂ and warming effects from direct physiology‐related effects on freezing sensitivity. The freezing damage threshold (lethal temperature 50) under ambient conditions of the 10 treeline species spanned from ?6.7±0.3 °C (Larix decidua) to ?9.9±0.6 °C (Vaccinium gaultherioides). PFT, but not PGF, explained a significant amount of this interspecific variation. Long‐term exposure to elevated CO₂ led to greater freezing sensitivity in multiple species but did not influence phenology, implying that physiological changes caused by CO₂ enrichment were responsible for the effect. The elevated CO₂ effect on freezing resistance was significant in leaves of Larix, Vaccinium myrtillus, and Gentiana punctata and marginally significant in leaves of Homogyne alpina and Avenella flexuosa. No significant CO₂ effect was found in new shoots of Empetrum hermaphroditum or in leaves of Pinus uncinata, Leontodon helveticus, Melampyrum pratense, and V. gaultherioides. Soil warming led to advanced leaf expansion and reduced freezing resistance in V. myrtillus only, whereas Avenella showed greater freezing resistance when exposed to warming. No effect of soil warming was found in any of the other species. Effects of elevated CO₂ and soil warming on freezing sensitivity were not consistent within PFTs or PGFs, suggesting that any future shifts in plant community composition due to increased damage from freezing events will likely occur at the individual species level.