Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

?kologische untersuchungen an aphiden

Zusammenfassung In Temperaturkabinen wird die Fortpflanzungspotenz von Myzus persicae (Sulzer) (Herkunft Groß-Lüsewitz) bei Dauertemperaturen von 15 bis 30° auf Kohlrüben (Brassica napus subspec. rapifera) untersucht.Eine Populationsanalyse nach Birch (1948) (intrinsic rate of increase) ergab den höchsten Wachstumsfaktor bei Dauertemperaturen zwischen 20 und 23°.Dauertemperaturen >25° führten zu einer starken Minderung der. Fortpflanzungspotenz. 30° ist die obere Grenze der Fortpflanzung der untersuchten Myzus persicae-Population.

---

The reproductive potential of the peach-potato aphid (origin Gross-Lüsewitz) was studied at temperatures between 15° and 30° in constant temperature chambers. They were cultivated on Swede (Brassica napus spp. rapifera) which stood in Knop's nutrient under gauze cloches in petri dishes. The production of juvenile larvae and the mortality of the mothers was measured daily. The total of all larvae (including those which were dropped) and the total of larvae on the leaf were separately enumerated. The larvae on the leaf were designated as viable larvae. A population analysis using Birch's method showed a maximum value for the growth factor k (difference between birth and mortality rates) of 23° for the total of all larvae, and of 20° for the viable larvae (Fig. 6). The daily relative growth-ratio was at the same temperatures respectively 1.36 and 1.34 (Table IV). Optimum development of M. persicae on swedes occurs thus between 20° and 23°. The percentages of viable larvae which add to the net production of total larvae are 53, 61, 30, and 24 (Table III) at temperatures of 15, 20, 25, and 30° respectively. The average length of a generation was 18.5 days at 15° and less than 13 days at 28 to 30° (Fig. 5). The multiplication rate per generation was 38 at 15°, 48 at 20°, but only 5.5 at 30° (Fig. 4). The time of development from first-stage larva to adult was 12.5 days at 15°, 5 days at 28° and 6 days at 30° (Table VII). The upper limit, where a weak multiplication was still possible, was at 30°. It is concluded that in regions where such limiting temperatures occur during some part of the day, the temperature can be the major regulating factor of the insect populations.

     

Pre-moult foraging trips and moult locations of Emperor penguins at the Mawson Coast

The movements of nine breeding adult emperor penguins Aptenodytes forsteri from two colonies, Auster (67° 23S 64° 04E) and Taylor Glacier (67° 28S 60° 54E), were determined by satellite telemetry on their pre-moult foraging trips. While preparing for their annual moult the penguins travelled for 22–38 days and reached distances of up to 618 km from the colony. Six of the nine tracked penguins were followed to three different moult locations all to the west of their breeding colonies and near other known emperor penguins colonies, such as Kloa Point (66°38S, 59°23E) and Fold Island (67°17S, 59°23E). Sea-ice conditions changed throughout the tracking period; as the birds travelled north the sea-ice contracted south.     

Über Kernstrukturen in den endopolyploiden Antipoden von Clivia miniata

Zusammenfassung Die Antipoden vonClivia miniata wachsen vor der Anthese und in geringem Maß auch noch zu Beginn der Anthese endomitotisch heran und werden 32-ploid.Sie enthalten zum Teil Kerne, in denen die endomitotischen Tochterchromosomen gemeinsamer Herkunft nur in den heterochromatischen proximalen Teilen zu radiären oder stabförmigen, locker gebauten Endochromozentren zusammenschließen und in den euchromatischen Teilen anscheinend getrennt verlaufen; weiter kommen vor: typische pflanzliche Riesenchromosomen aus relativ eng und ihrer ganzen Länge nach gebündelten Einzelchromosomen, diese allerdings nur selten, häufiger dagegen etwas atypische aus lockeren Bündeln bestehende Riesenchromosomen ; zwischen den Kernen mit Endochromozentren und mit Riesenchromosomen gibt es alle Übergänge.Die Zahl der Riesenchromosomen entspricht erwartungsgemäß der haploiden Chromosomenzahl 11; einzelne von ihnen zeigen im Heterochromatin stellenweise Andeutungen eines Scheibenbaues und bei den meisten tritt die Spindelansatzstelle als Einschnürung oder Unterbrechung hervor.Auch Kerne mit einer gleichmäßig feinen körnigen Struktur, die wahrscheinlich dem endomitotischen Formwechsel angehört, wurden beobachtet.Als Arbeitshypothese kann man annehmen, daß das Vorhandensein von viel und von kompaktem Heterochromatin die Bildung von pflanzlichen Riesenchromosomen fördert und bei Clivia meistens nur atypische lockere Riesenchromosomen auftreten, weil kompaktes Heterochromatin völlig fehlt und das lockere hinter dem Euchromatin an Menge stark zurückbleibt.     

The temperature-dependent duration of development and parasitism of three cereal aphid parasitoids, Aphidius ervi, A. rhopalosiphi, and Praon volucre

Temperature dependencies were established for the egg-to-mummy and mummy-to-adult phases, for mummy mortality, and for parasitism of Aphidius ervi Haliday, Aphidius rhopalosiphi De Stefani-Perez, and Praon volucre (Haliday) (Hymenoptera, Aphidiidae), three parasitoids of Sitobion avenae (Fabricius) (Homoptera, Aphididae), at 8°C, 12°C, 16°C, 20°C, and 25°C on winter wheat (cv. Haven). A physiological model described temperature-dependent development over the full temperature range, whereas a linear model was fitted for data above 8°C and used to estimate the lower temperature thresholds and day-degrees (° D) required for development. The thresholds for A. ervi were 2.2°C for egg-mummy development and 6.6°C for mummy-adult development, those for A. rhopalosiphi were 4.5°C and 7.2°C, and those for P. volucre were 3.8°C and 5.5°C. The time to develop into mummies and adults differed significantly between the three species: A. ervi development into mummies required an average of 159 ° D, while development into adults took an average of 73 ° D. The corresponding average times required for A. rhopalosiphi and P. volucre to develop mummies were 124° D and 126° D, while their development into adults required an average of 70° D and 150° D, respectively. Mummy mortality was 25–35% at 8°C and less at the higher temperatures tested, but began to increase again at 25°C, showing a quadratic relationship between mortality and temperature. Parasitization was very low or, in the case of P. volucre, absent up to 12°C and thereafter increased with increasing temperature. The relationship between parasitization, recorded as percent aphids mummified, and temperature was linear at the temperatures tested and depended on species. A. ervisuperparasitized 11.1% aphids at 20°C and 16.6% aphids at 25°C, whereas superparasitism was low in A. rhopalosiphi and absent in P. volucre. From 16°C to 25°C the P. volucre sex ratio increased. For A. ervi and A. rhopalosiphi there was no trend with temperature, but at 20°C and 25°C it was close to even. Field data for 1996 and 1997 allowed for a comparison of actual and expected emergence of overwintering mummies. In both years, parasitoids were predicted to have emerged from overwintering mummies well in advance of the onset of aphid infestation, and more than a month earlier than the first parasitized aphids were found in winter wheat. Observations from trap plants in other crops supported the predictions of the models. Other factors that can affect biological control by cereal aphid parasitoids are discussed.     

Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced -amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55C. The -amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of -amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Plant regeneration from grape callus stored under a combination of low temperature and silicone treatment

Effects of the combination of low temperature and silicone treatment on the storage of grape callus (Vitis vinifera L. x V. labrusca L. cv. Kyoho; V. vinifera L. cv. Koshusanjaku) were examined. In Kyoho, the calli were stored at 10°C successfully for up to 360 days. Embryogenic calli of Koshusanjaku stored at 10°C retained the ability of embryogenesis after 360 days of storage. However, the color of both calli became brownish. This was improved by the combination of low temperature and silicone treatment. The calli of Kyoho survived by the storage under the combination of 15°C and silicone. Embryogenic calli stored at 10 and 15°C in combination with silicone survived for 360 days, and regenerated only after transfer onto a regeneration medium. Thus the combination of low temperature and silicone affects the longevity of the grape callus.     

Comparative studies of Feulgen hydrolysis for DNA

Summary We compared the Feulgen hydrolysis curves (37° C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20° C; ethanol-acetone, 11, 120 min at 4° C; ethanolglacial acetic acid mixture (31), containing 2% of formaldehyde (EAF), 90 min at 20° C; and ethanol-glacial acetic acid (31), 60 min followed by 5% chrome alum solution for 360 min at 20° C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest plateau region and the smallest scattering of DNA-Schiff amount values along the plateau. With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG₆₀₀₀ on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest plateau was obtained with PEG₆₀₀₀. The only effect of PEG₁₅₀₀ was a dramatic increase of DNA-Schiff amount at the peak point. PEG₂₀₀₀₀ had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's chain with a stable structure model of Feulgen hydrolysis.     

Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

The synthesis of oligosaccharides by the reversed hydrolysis reaction of β-glucosidase at high substrate concentration and at high temperature

Summary In the presence of -glucosidase from almond, a 90% glucose solution gave four kind of -linked glucose-disaccharides. The yield increased as the concentration of glucose was increased and as the reaction temperature was raised. The maximum yield of disaccharides from 90% glucose solution was 40% at 55°C.     

Continuous production of α,α-trehalose by immobilised fungal trehalose phosphorylase

Immobilisation of trehalose phosphorylase from Schizophyllum commune by adsorption onto anion-exchange materials stabilised the enzyme activity at 30°C by approx. 35-fold. Immobilised and free enzymes showed similar pH-dependence of activity but different inactivation behavior above 30°C. A fixed-bed enzyme reactor produced ,-trehalose at a stable substrate conversion of 80% with a productivity of 2.6 g l–1 h–1 for 72 h. Inhibition of trehalose phosphorylase by phosphate limited the productivity of a direct conversion of starch into ,-trehalose.     

The effect of ambient temperature on diurnal rhythm in heat production and activity in pigs kept in groups

The effect of ambient temperature on diurnal rhythm of metabolic rate and activity was studied in group housed pigs in two trials. Two climate controlled respiration chambers were used, one for each trial. Each chamber contained two groups of 8 pigs. During the experiment of 31 days the mean live weight of the animals increased from 28 to 40 kg. Feeding level was kept constant at 93 kg^–0.75. d^–1. Feed contained 12 kJ of metabolizable energy per g. Temperature treatment started with 20C for two days. After that it was lowered in stepwise fashion by 4K to 16, 12 and 8C. Each temperature treatment lasted two days. After that temperature it was increased in steps to 20C in 8 days. The whole procedure was repeated once. Metabolic rate and activity were measured continuously during the 24 hrs of every second day of the temperature treatment.Metabolic rate increased at temperatures below 16C. Activity was associated with ambient temperature. At low temperature, the total activity related heat production was increased, especially during day time. Variation in metabolic rate within a day was not reduced by low ambient temperature. Thermal requirement, calculated as lower critical temperature, was about 14C as averaged over the day, (16.1C during day time and 13.6C during night time). Extra thermal heat production below thermoneutrality was affected by time of day and activity.     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

“Hidden” popular illnesses in primary care: Residents' recognition and clinical implications

This study documents that ethnomedical beliefs and practices play an important role in primary care in a southern community. Thirty-three of 73 patients from a rural Appalachian area coming to a university primary care internal medicine practice presented 54 ethnomedical complaints such as high blood (24.1%), Weak 'n dizzy (22.2%), nerves (16.7%), sugar (5.6%) and fallin' out (3.7%). Thirty-three patients had both biomedical and ethnomedical complaints, 40 patients had biomedical complaints without ethnomedical complaints and no patients presented with ethnomedical complaints alone. Over two-thirds of all patients consulted non-medical personnel for their complaints, mostly family and friends, and 70 percent self-treated prior to clinic consultation. Patients presenting with ethnomedical complaints when compared with those presenting with biomedical complaints sought advice of non-physicians significantly more often (p < 0.02); no statistical difference, however, was found in their self-treatment practices. Ninety-two of 130 biomedical complaints were recorded by the patient's physician but none of the 54 ethnomedical complaints were formally recorded (p < 0.001). The high incidence of ethnomedical complaints in this population and the failure of physicians to recognize these complaints demand that primary care medicine residents be taught improved history-taking skills and the essentials of ethnomedical illnesses if they are to provide culturally-sensitive patient care. [ethnomedicine; physician education; Southern black and white Appalachian folk medicine; culturally-appropriate primary care.]     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Effects of high ambient temperatures on the metabolism of West African dwarf goats. II

32 West African dwarf goats were exposed in respiration chambers to temperature treatments of 20, 25, 30, 35, 35, 35, 30, 25, 20°C. Each treatment lasted three days. 16 goats were kept in individual pens (I); the others in two group pens of eight animals each (G). During each treatment, heat production and activity were recorded continuously over 48 hours. In addition, feed and water intake, rectal temperature, skin temperature and respiratory rate were measured during each treatment.Compared to 20°C, at 35°C rectal temperature increased from 39.0°C to 39.9°C, respiratory rate from 30 to 260 times. min^–1 and skin temperature from 37.1°C to 39.5°C. Hay intake decreased by 40%; concentrates (30 g. kg^–0.75. d^–1) were always completely consumed. Heat production was higher for the G animals at 20°C and higher for the I animals at 35°C. These differences in heat production between the two groups were reflected in differences in rectal and skin temperature and in respiratory rate but only very slightly in differences in hay intake.Tissue insulation was 0.014 K. m². W^–1 at 30°C and 35°C and 0.022 K. m². W^–1 at 20°C.It is concluded that the reactions of these dwarf goats to high ambient temperatures are not different in principle from those of other domestic ruminants and that they do not exhibit a specific suitability or unsuitability for ambient temperatures as prevailing in West Africa.     

Cloning and expression of β-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Summary The -galactosidase gene from Bacillus subtilis KL88 was cloned into Escherichia coli and the gene product characterized for its potential use in the dairy industry. The two recombinant plasmids that we obtanied encoded a -galactosidase with the same catalytic and thermal characteristics as the native -galactosidase from B. subtilis. The recombinant -galactosidases exhibited high activity at low temperature (10°C), with maximum activity at 50°C and an optimum pH of 6.0. Its molecular weight was estimated to be 90 Kd. The restriction maps of the recombinant plasmids were constructed. The -galactosidase gene was located in a 2.3 Kb fragment.     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide