Control of yeast neutral trehalase by distinct polyphosphates and ribonucleic acid

Summary The activity of yeast trehalase when assayed at pH 7 in a crude extract was found to increase 2- to 3-fold upon incubation with 0.1 % (v/v) polyethyleneimine or other polycations such as polylysine (0.075-mMol) and calf thymus histories (0.08 mMol). Incubation with 3 mM-Mn²⁺ and 5 mM-Ca²⁺ also led to 3- and 1.6-fold increases in trehalase activity, respectively. The activities of 11 other enzymes assayed in the crude yeast extract did not increase after addition of polyethylene imine. At concentrations of polyethylenemine that maximally stimulated trehalase activity, 97% of the total RNA present in the crude extract, 40% of total protein, and 60% of the polyphosphate (assayed as inorganic phosphate liberated during 7 min incubation at 95 °C and pH O) were found to be precipitated. A similar finding was made with trehalase-stimulating concentrations of Mn²⁺. Activation of trehalase by polyethylene imine rendered this enzyme susceptible to inhibition by a preparation of total yeast RNA, inorganic polyphosphates, and related polyanions. We present further evidence that the removal of a distinct RNA and/or polyphosphate is the basic principle of polyetyleneimine-induced activation of trehalase.A more pronounced stimulation of trehalase activity (4-fold) could be obtained by enzymatic phosphorylation with ATP in the presence of cyclic AMP and Mg²⁺ as described by van Solingen and van der Plaat (1975) [9]. Thus, trehalase 2-fold activated by polyethylene imine was further activated by another 2-fold increase by enzymatic phosphorylation. Conversely, no additional stimulation by polyethylene imine was obtained for the maximally active, phosphorylated enzyme. We conclude that phosphorylation-mediated activation of trehalase is a two-step event, one being the removal of an inhibitor, which can be achieved by polyethylene imine or related cations independent on phosphorylation. The most likely candidate for the inhibitor appears to be a distinct RNA.

---

Kontrolle der neutralen Hefen-Trehalase durch bestimmte Polyphosphate und RNA

Zusammenfassung Die in einem rohen Extrakt aus Hefe bei pH 7 gemessene Aktivität von Trehalase nimmt bei der Inkubation mit 0,1% Polyethylenimin oder mit anderen Polykationen, wie Polylysin (0,075 mmol) oder Histon aus Kalbsthymus (0,08 mmol) auf das zwei- bis dreifache zu. Auch die Inkubation mit 3 mmol Mn²⁺ oder 5 mmol Ca²⁺ führt zu einer 3-, bzw. 1,6fachen Zunahme der Trehalase-Aktivität. Die Aktivität von 11 anderen Enzymen, die im rohen Extrakt aus Hefe bestimmt wurden, nimmt nach Zusatz von Polyethylenimin nicht zu. Bei der Inkubation mit Polyethylenimin, das eine maximale Stimulierung der Trehalase-Aktivität bewirkt, werden 97% der gesamten Ribonucleinsäure, 40% des gesamten Proteins und 60% des Polyphosphats (bestimmt als anorganisches Phosphat, das in 7 min bei 95 °C und pH O freigesetzt wird) präcipitiert. Eine gleichartige Präcipitation wurde bei der Inkubation mit Trehalase-stimulierenden Konzentrationen von Mn²⁺ beobachtet. Mit Polyethylenimin aktivierte Trehalase wird durch Ribonucleinsäure aus Hefe, anorganisches Polyphosphat und verwandte Polyanionen gehemmt. Vermutlich führt die Entfernung einer bestimmten Ribonucleinsäure-Fraktion und/oder von Polyphosphat zu der Polyethylenimin-induzierten Aktivierung der Trehalase.Wie von van Solingen und van der Plaat 1975 beschrieben [9], wird eine 4fache Stimulierung von Trehalase durch enzymatische Phosphorylierung mit ATP in der Gegenwart von cyclischem AMP und Mg²⁺ erreicht. Das so durch Phosphorylierung maximal aktivierte Enzym kann durch Zusatz von Polyethylenimin nicht weiter aktiviert werden. Das mit Polyethylenimin 2fach aktivierte Enzym kann jedoch noch einmal 2fach weiter aktiviert werden durch enzymatische Phosphorylierung. Aus diesen Beobachtungen wird geschlossen, daß die durch enzymatische Phosphorylierung bewirkte Aktivierung der Trehalase ein zweistufiger Vorgang ist: Zuerst findet die Entfernung eines Inhibitors statt (dies kann auch mit Polyethylenimin oder mit verwandten Kationen unabhängig von der enzymatischen Phosphorylierung erreicht werden), hierauf folgt weitere Aktivierung durch Konformationsänderung des Enzyms. Wahrscheinlich handelt es sich bei dem durch Phosphorylierung bzw. Behandlung mit Polyethylenimin abgetrennten Inhibitor um eine gewisse Ribonucleinsäure-Fraktion.

---

Abbreviation PEI polyethylene imine The followingenzymes are mentioned in the text: Alcohol dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (1.1.1.12), Malate dehydrogenase (1.1.1.37), Glucose-6-phosphate dehydrogenase (1.1.1.49), Glutamate dehydrogenase (NAD) (1.4.1.2), Glutamate dehydrogenase (NADP) (1.4.1.4), Aspartate -ketoglutarate aminotransferase (2.6.1.1), 6-Phosphofructokinase (2.7.1.11), Neutral trehalase (3.2.1.28), Pyruvate decarboxylase (4.1.1.1), Phosphoenolpyruvate carboxykinase (4.1.1.49).This paper is dedicated to Prof. Dr. Karl Decker on the occasion of his 60th birthday     

Accumulation of nitrite and sulfite in yeast cells and synergistic depletion of the intracellular ATP content

Summary When nitrite or sulfite are applied to yeast cells below pH 5.0, an enormous intracellular accumulation occurs. It is assumed that nitrite and sulfite penetrate the cell membrane in their undissociated forms as nitrous acid (pK = 3.3) or sulfurous acid (pK =1.8), respectively. Due to the neutral intracellular pH they are trapped inside the cell in their anionic forms, which are impermeable to the cell membrane. It has previously been shown that sulfite causes a rapid depletion of the ATP content of yeast cells [Schimz, K. L. and Holzer, H. (1979) resp. Hinze et al. as above]. Similarly, millimolar concentrations of nitrite decrease the ATP level to less than 10% of the initial value. Nitrite and sulfite in combination deplete the ATP content of yeast cells much stronger than expected for the sum of the separate effects of these compounds (synergistic effect).

---

Akkumulation von Nitrit und Sulfit in Hefezellen und synergistischer Abfall des intrazellulären ATP-Gehalts

Zusammenfassung Wenn Hefezellen mit Nitrit oder Sulft bei pH-Werten unter 5,0 inkubiert werden, beobachtet man eine starke intracelluläre Akkumulation von Nitrit, bzw. Sulfit. Es ist anzunehmen, daß Nitrit und Sulfit in ihrer undissoziierten Form als salpetrige Säure (pK=3,3) bzw. schweflige Säure (pK=1,8) penetrieren und dann in den Zellen durch Neutralisation zu den anionischen Formen, die die Zellmembran nicht mehr permeieren können, abgefangen werden. Ähnlich dem früher beschriebenen raschen Abfall des ATP-Gehaltes nach Zusatz von Sulfit in Hefe [Schimz, KL und Holzer H (1979) Arch Microbiol 121:225–229] und in Bakterien [Hinze H, Maier K, Holzer H (1981) Z Lebensm Unters Forsch 172:389-392] verursacht auch Nitrit einen raschen Abfall des ATP-Gehaltes in Hefe auf weniger als 10 % des Anfangswertes. Werden Nitrit und Sulfit in Kombination verabreicht, so beobachtet man einen wesentlich stärkeren Abfall des ATP-Gehaltes als er aus der Summe der Einzeleffekte von Nitrit, bzw. Sulfit zu erwarten wäre (Synergistischer Effekt).

     

Dependence of the 12-methyltridecanal concentration in beef on the age of the animal

The content of 12-methyltridecanal (12-MT), which contributes greatly to the characteristic aroma of stewed beef, was determined by a stable isotope dilution assay in meat samples from nine bovine animals of different ages. The results, which were related to the amount of phospholipids (PL), indicate that 12-MT content increases with the age of beef, e. g. from 36 g·g^–1 PL in a 4-monthold calf to 810 g·g^–1 PL in an 8-year-old cow. The increase of 12-MT content in PL per month varied between 8.4 and 10.9 g·g^–1 PL (mean: 9.3 ± 0.78 g·g^–1 PL). Possibly 12-MT content is suitable as an indicator for the estimation of the age of a beef sample.

---

Abhängigkeit der 12-Methyltridecanal-Konzentration in Rindfleisch vom Alter des Tieres

Zusammenfassung 12-Methyltridecanal (12-MT), das zum charakteristischen Aroma von geschmortem Rindfleisch wesentlich beiträgt, wurde durch Isotopenverdünnungsanalyse in Proben aus neun Rindern unterschiedlichen Alters bestimmt. Die Ergebnisse, die auf den Phospholipid (PL)-Gehalt bezogen wurden, zeigen, daß das 12-MT mit dem Alter des Rindes ansteigt, z.B. von 36 g·g^–1 PL bei einem 4 Monate alten Kalb auf 810 g·g^–1 PL in einer 8 Jahre alten Kuh. Der Anstieg des 12-MT in den PL pro Monat schwankte zwischen 8,4 und 10,9 g·g^–1 (Mittelwert 9,3 ±0,78 g·g^–1). Möglicherweise ist das 12-MT als Indikator für die Altersbestimmung von Rindfleisch geeignet.

     

Teaching information systems to small business management

The availability of cheaper, more efficient small computers opened up the possibility for medium-sized firms to install their own EDP system. Nevertheless these firms do not make sufficient use of EDP. One major reason for this is the lack of management knowledge about good computer applications. The paper describes a way to close this gap.On the basis of pilot and empirical studies, a combination of introductory lectures, autodidacts studies, and concluding lectures is proposed.To support the small business management, a manual was first designed. This deals with the practical problems and decisions of small and medium sized firms. In order to achieve better training seminars were designed that combined information materials with other systems of learning. The methods and the contents of these seminars including various case studies is described.     

Processing and analytical characterisation of pulp-enriched cloudy apple juices

Pulp-enriched cloudy apple juices were manufactured from two apple varieties by blending of apple puree with natural cloudy apple juice in order to enrich bioactive secondary plant substances. Finishing of the puree with a 0.6-mm mesh screen and final product homogenisation revealed as the optimum processing technology for the novel 100% fruit beverage. The presence of large particles originating from the puree prevented long-term cloud stability, but due to the proportion of cloud-stable juice a complete phase separation did not occur. Optimal drinkability was achieved at a viscosity of 11.5 mPa s. Polyphenol compositions and concentrations were determined by means of RP-HPLC/PDA. The results ranged from 109 to 610 mg/l. All samples showed the typical polyphenol pattern of apples with dominating hydroxycinnamic acids followed by flavanols and flavonols. The puree addition to the cloudy apple juices increased the polyphenol concentrations by average 100%. Relatively, the highest increase could be observed for dimeric procyanidins. Different technological variants did not affect significantly the polyphenol concentrations. The dietary fibre contents of the pulp-enriched cloudy apple juices ranged from 5.8 to 9.4 g/l.     

Investigation of the stability of tablets prepared from sucrose and citric acid anhydride utilizing response surface methodology

Inversion of sucrose is a stability problem particularly of candies with acidic taste that contain sucrose and small amounts of organic acids such as citric acid, since the free d-fructose produced by hydrolysis is hygroscopic. The following possibilities were investigated for preventing the hydrolysis of sucrose in tablets containing sucrose and citric acid: Adding various amounts of tri-sodium citrate to the formulation to neutralize the citric acid, (Hot) melt coating of citric acid and tri-sodium citrate with a vegetable fat at different coating ratios, variation of the ratio of coated citric acid and tri-sodium citrate in formulations, and compressing the formulations with different compression forces. After tablet processing and storage of tablets, the concentration of d-fructose was determined on the basis of enzymatic reactions. A response surface central composite design was used. The above-mentioned variations were chosen as independent variables and the amount of d-fructose was chosen as response variable. The lowest rates of inversion could be achieved by increasing the content of tri-sodium citrate and the ratio of coating material and decreasing the ratio of coated citric acid and tri-sodium citrate in the tablet formulations. The compression force had no significant effect on the inversion of sucrose.     

Quantitative analysis of caffeic and ferulic acids in oatmeal

[¹³C]Caffeic acid and [¹³C]ferulic acid were synthesized and then used as internal standards for the determination of these acids (free and esterified) in oatmeal. A comparative study indicated that 84% of the ferulic acid, but only 32% of the caffeic acid, which is more susceptible to oxidation than the former, could be found by a conventional analytical approach.

---

Quantitative Analyse von Kaffee- und Ferulasäure in Hafermehl. Vergleich einer konventionellen Methode mit einem Stable Isotope Dilution Assay

Zusammenfassung [¹³C]-Kaffee- und [¹³C]-Ferulasäure wurden synthetisiert und als interne Standardsubstanzen für die Bestimmung dieser Säuren (frei und verestert) in Hafermehl angewandt. Ein Vergleich ergab, daß von der Ferulasäure 84%, aber von der leichter oxidablen Kaffeesäure nur 32% mit einer konventionellen Methode erfaßt werden.

     

Untersuchungen an Caramel Curiepunkt-Pyrolyse von Caramelzuckersirupen und andere strukturspezifische Untersuchungen

Zusammenfassung Es wird über die Curiepunkt-Pyrolyse handelsüblicher Caramelzuckersirup-Proben und die Identifizerung von über 80 Pyrolysefragmenten durch kombinierte Capillargaschromatographie/ Massenspektrometrie berichtet. Mit dieser Methode sind die Proben untereinander and gegenüber Couleuren differenzierbar. Mittels Ultrafiltration wurden die Komponenten einer intensiv braunen Probe in Fraktionen unterschiedlicher Molekülmasse getrennt and nach Pyrolyse miteinander verglichen. In einer niedermolekularen Fraktion wurden nach Permethylierung Laevoglucosan, 1,6-Anhydro--d-glucofuranose, Trehalose, Cellobiose, Maltose, Isomaltose und Gentiobiose Bowie einige Difructosedianhydride nachgewiesen.

---

Investigations of caramel. Curiepoint pyrolysis of caramel syrups and other investigations of structure

Summary A report is given on the investigation of commercial caramel syrups by Curiepoint pyrolysis and the identification of more than 80 pyrolysis products by capillary gas chromatography mass spectrometry. This method enables the differentiation between different caramel syrups and, additionally, between caramel syrups and caramel colours. Compounds of different molecular masses have been examined in the same manner after they have been isolated from an intense brown caramel sample by ultrafiltration. In a fraction consisting of compounds with low molecular masses laevoglucosan, 1,6-anhydro--d-glucofuranose, trehalose, cellobiose, maltose, isomaltose, gentiobiose and some difructosedianhydrides were identified by GC/MS after permethylation.

     

Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno- and genotypic criteria

This paper reviews the methodology applied for the identification and characterisation of enterococci and covers phenotypic, genotypic and phylogenetic techniques. Although conventional phenotypic typing schemes are useful for rapid and simple identification of enterococcal species for routine applications, other methods like standardised sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), multilocus enzyme electrophoresis (MLEE), antimicrobial susceptibility testing, serotyping, pyrolysis mass spectrometry (pyMS) and vibrational spectroscopic methods allow a more in-depth characterisation of enterococci. Many of the recently described enterococcal species exhibit deviations from hitherto so-called classical enterococci with regard to their phenotypical properties. Therefore, genotypic methods have to be used to clarify their possible assignment to the genus Enterococcus. In this review, special emphasis is given on recently developed polymerase chain reaction (PCR)-based typing methods such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), specific and random amplification (SARA) and modifications of PCR-ribotyping as well as pulsed-field gel electrophoresis (PFGE) and partial sequence analysis. The use of PCR and probes for genus and species identification of enterococci is also considered like the application of sequence data of conserved DNA regions (e.g., ribosomal ribonucleic acid (rRNA) genes) in the case of species identification.     

Trypsin inhibitor activity of soybean as affected by genotype and fertilisation

The presence of protease inhibitors in soybean prohibits the utilisation of the raw beans for food and feed. However, little information is available about environmental influences and the effects of nitrogen and sulphur supply on the antinutritional constituents of soybean. As these factors may influence protease inhibitors, soybean genotypes segregated according to the presence or absence of the Kunitz trypsin inhibitor have been evaluated for trypsin inhibitor activity (TIA) in field trials. TIA was affected significantly by environment (geographical location), fertilisation treatment and genotype. Environmental means of TIA were between 69.5 and 104.8 mg g^?1. Nitrogen application, which caused an increase in seed protein content, resulted in a reduction in TIA by about 15% as compared with the control. Remarkably, simultaneous application of nitrogen and sulphur in the form of ammonium sulphate had a similar reductive effect on TIA to that of nitrogen application alone, although soybean protease inhibitors are rich in sulphur amino acids. Significant genetic variation in TIA was found both within the genotype class with the Kunitz inhibitor present as well as within the class lacking this inhibitor. The results suggest that TIA of soybean may be modified considerably by genetic improvement and appropriate agronomic management. Copyright © 2003 Society of Chemical Industry