Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.     

Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.     

Structure of prothrombin fragment 1 refined at 2.8 A resolution

The structure of prothrombin fragment 1, solved at 2.8 A resolution (1 A = 0.1 nm) by a combination of multiple and single isomorphous replacement methods utilizing solvent flattening, has been refined by restrained least-squares methods (R = 0.24), solvent not included, using fairly stringent restraints on the molecular geometry and individual thermal parameters. The inner kringle loop possesses significantly lower B-values than the outer loops even though the former also constitutes a surface of the folded kringle structure. This surface forms the Lys sub-site of the fibrin binding site of other kringles. The hydrogen bonding network and ion pair interactions of fragment 1 appear to maintain a compact folded structure among the various loops of the kringle structure. On the other hand, since there is only one hydrogen bond between the kringle and its preceding 30 residues, considerable flexibility is suggested for the Gla-domain consistent with its disorder in crystals. A chitobiose has been located at the Asn77 glycosylation site, but only a single N-acetyl-glucosamine is ordered at Asn101. The lysine binding site region of other kringles is not properly developed in fragment 1, accounting for its lack of Lys/fibrin affinity. Most of the conserved sequence among 11 different kringles is associated with either: (1) protecting the inner loop disulfides Cys87-127, Cys115-139 upon which the folding is based; or (2) a requirement of the lysine binding site. The remainder of the conservation is generally associated with the ten reverse turns of the folding; of these 40 residues, or about half the sequence, 14 are conserved among eight different turns. The intermolecular packing consists of infinite helical columns of fragment 1 molecules related by a crystallographic 4(3) screw axis, which are held together by van der Waals' interactions of aromatic clusters from different molecules related by a crystallographic 2-fold rotation axis.     

Effect of pressure on thermal insulation in humans wearing wet suits

The present work was undertaken to determine the critical water temperature (Tcw), defined as the lowest water temperature a subject can tolerate at rest for 3 h without shivering, of wet-suited subjects during water immersion at different ambient pressures. Nine healthy males wearing neoprene wet suits (5 mm thick) were subjected to immersion to the neck in water at 1, 2, and 2.5 ATA while resting for 3 h. Continuous measurements of esophageal (T(es)) and skin (Tsk) temperatures and heat loss from the skin (Htissue) and wet suits (Hsuit) were recorded. Insulation of the tissue (Itissue), wet suits (Isuit), and overall total (Itotal) were calculated from the temperature gradient and the heat loss. The Tcw increased curvilinearly as the pressure increased, whereas the metabolic heat production during rest and immersion was identical over the range of pressure tested. During the 3rd h of immersion, Tes was identical under all atmospheric pressures; however, Tsk was significantly higher (P less than 0.05) at 2 and 2.5 ATA compared with 1 ATA. A 42 (P less than 0.001) and 50% (P less than 0.001), reduction in Isuit from the 1 ATA value was detected at 2 and 2.5 ATA, respectively. However, overall mean Itissue was maximal and independent of the pressure during immersion at Tcw. The Itotal was also significantly smaller in 2 and 2.5 ATA compared with 1 ATA. The Itissue provided most insulation in the extremities, such as the hand and foot, and the contribution of Isuit in these body parts was relatively small. On the other hand, Itissue of the trunk areas, such as the chest, back, and thigh, was not high compared with the extremities, and Isuit played a major role in the protection of heat drain from these body parts.     

Electron Microscopic Studies of Spruce Needles in Connection with the Occurrence of Novel Forest Decline

Needles of four spruce trees showing different degrees of novel kinds of forest decline were investigated by electron microscopy. Green needles appearing at least superficially still intact were selected for the present investigation. Most of the mesophyll appeared to be undamaged. However, groups of atypical mesophyll cells were found close to the endodermis or the hypodermis. The chloroplasts of the apparently damaged cells were particularly affected. Changes in the matrix of the chloroplasts, i.e,. increased affinity to osmium, occurrence of extensive nests of plastoglobuli, as well as damage to the membranes, i.e. lesions in the envelope and abnormal thylakoid membranes, were observed. Signs of decomposition of other cellular structures including mitochondria were also detectable. There appeared to be a close correlation between the degree of damage at the whole tree level and the degree of damage occurring at the cellular level. It is concluded that particularly the lipids and the proteinsof, the membranes are affected by anthropogenic air pollutants and natural stressors. The altered membrane structure may for instance cause abnormal osmotic conditions for the cellular compartments and may impair transport processes and thus lead to lossof function not only of the cells but also of the whole needle.     

Isolation and identification of the principal siderophore of the plant pathogenic fungusBotrytis cinerea

Summary The plant pathogenic hyphomyceteBotrytis cinerea has been shown to produce several trihydroxamate siderophores under conditions of low-iron stress. The total siderophores amounted to approximately 30 mg/l culture filtrate after 5 days of incubation in an asparagine/salt/glucose medium. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) on a reversed phase indicated that ferrirhodin is the predominant siderophore of this fungus. Chemical characterization of the principal siderophore by fast-atom-bombardment (FAB) mass spectrometry, nuclear magnetic resonance (¹H-NMR,¹³C-NMR) and comparison with a reference revealed the identity with ferrirhodin. NMR studies performed on desferrirhodin (desferrirhodin) in dimethylsulfoxide and water revealed the existence of two conformers in D₂O resulting from acis-trans isomerization of the hydroxamic acid groups. Comparative iron-uptake studies showed the following order of uptake inB. cinerea: ferrichrysin (100%), ferrirubin (57%), ferrirhodin (45%), hexahydroferrirhodin (45%), coprogen 6%. Concentration-dependent uptake of ferrirhodin resulted in saturation kinetics only in the low concentration range of 0–30 M (K _m = 2.5 M,V _max = 80 pmol min^–1 mg(^–1). A non-saturable, linear uptake was observed in the high concentration range of 30–80 M. The low concentration range appears to be the physiologically significant range, where siderophore-mediated iron transport inB. cinerea occurs.     

Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

Scale Growth and Squamation Chronology for the Laboratory-Reared Hermaphroditic Fish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> (Cyprinodontidae)

Scale morphology, growth and the squarnation chronology are described for the hermaphroditic fish Rivulus marmoratus reared in the laboratory. The scales are round or oval shaped cycloid type, and their sizes are about 0.3–1.0 mm in diameter. The number of ridges increases more rapidly relative to the body growth of the fish in early stages, but this increase is proportionate to growth subsequently. Three loci of scale development have been identified. The scales first appeared on the center of the parietal region at 8 days after hatching. The second locus of scale formation was on the lateral line of the posterior end of the caudal peduncle. A third locus was later observed on the lower right corner of the operculum: The final squamation was completed at 6 weeks after hatching.