Cadmium accumulation by a Citrobacter sp

Cadmium accumulation by a Citrobacter sp. growing in the presence of the metal occurred as a sharp peak during the mid-exponential phase of growth, but cultures showed considerable inhibition of growth compared to cadmium-free controls. This problem was overcome by pregrowing the cells in cadmium-free medium and subsequently exposing them to the metal in the resting state, under which conditions higher concentrations of cadmium were tolerated and metal uptake was enhanced. This ability was retained when the cells were immobilized and then challenged with a flow containing Cd2+; 65% of the metal presented was removed from solution. The influence on uptake of the composition of the exposure buffer and of various cell treatments were investigated and the results are discussed with respect to the anticipated speciation of the cadmium presented to the cells and also with respect to the probable mechanism of metal uptake. This is thought to occur through the activity of a cell-bound phosphatase, induced during pre-growth by the provision of glycerol 2-phosphate as sole phosphorus source. Continued enzyme function in resting cells would then precipitate the metal as cell-bound cadmium phosphate.     

Gravity-independent inequality in pulmonary blood flow in humans

Single-photon emission computerized tomography of the lung with 99mTc-labeled human albumin macroaggregates (99mTc-MAA) was used in six healthy subjects to study the three-dimensional distribution of pulmonary blood flow. 99mTc-MAA was injected while the subjects were resting in the supine position and holding their lung volume at normal end expiration. Tomography was performed on each subject from 120 projections of radioactivity in the lungs acquired with a rotating gamma camera. To minimize lung motion artifacts, the subjects were asked to hold their breath at end expiration during the 10-s duration of data acquisition in each projectional angle. Perfusion images of lung slices (11 mm thick) were reconstructed, and the radioactivity within each slice was expressed per unit lung volume of 3.7 X 3.7 X 11 mm. Perfusion images of a midcoronal slice from each subject manifested a concentric pattern of radioactivity that decreased significantly from the center to the periphery, suggesting that blood flow rate per unit lung volume was up to 10 times larger near the central region. This gradient in activity between the center and the periphery of the coronary slices was gravity independent as the subjects were supine. Images of sagittal slices from the middle of the right lung also manifested a similar pattern of concentric gradient in activity, with the vertical distribution (gravity related) almost comparable with the horizontal distribution (gravity independent). These results indicate that pulmonary blood flow in resting supine humans is spatially stratified with a marked central-to-peripheral gradient in all directions. It appears that zone 4 (reduced blood flow) is not a phenomenon limited to the dependent region of the lung as commonly thought but rather is a manifestation of this spatial distribution whereby blood flow is lowest in all peripheral regions of the lung.     

Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila.

We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest.     

A simple immunological detection method for the direct screening of genes from clone banks

A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.     

Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Assay for characterization of human follicular oocyte maturation inhibitor using Xenopus oocytes

Human follicular fluid from healthy mature Graafian follicles and from pathologic ovarian cyst fluid was found to be inhibitory to progesterone-induced meiotic maturation of oocytes from the South African clawed toad, Xenopus laevis. Human follicular fluid but not human serum, collected from the same individuals, demonstrated a linear dose-response inhibition on the maturation of oocytes in the Xenopus assay system. These findings indicate that the human follicular and cyst fluids contain oocyte maturation inhibitor (OMI). This human OMI was inactivated when subjected to a boiling water bath for 2 min. The OMI action was shown to be reversible in its inhibitory action. The fact that OMI can act directly on the oocyte was demonstrated by its inhibitory action on maturation in defolliculated oocytes. The findings demonstrate that the inhibitory action of human OMI is not species-specific. Xenopus oocytes provide a simple, readily available, year-round bioassay material for testing follicular oocyte maturation inhibitor.     

Effect of solubilization on adenosine 5'-triphosphate induced calcium release from purified sarcoplasmic reticulum calcium adenosinetriphosphatase

ATP-induced Ca2+ release from the purified sarcoplasmic reticulum Ca2+-ATPase has been monitored in several different ATPase environments. Arsenazo III was used as a Ca2+ indicator in stopped-flow experiments and was shown to detect the early burst in Ca2+ transport, slower steady-state transport, and release of Ca2+ from fragmented sarcoplasmic reticulum. ATP-induced rapid release of Ca2+ followed by a slower rebinding step could be demonstrated for purified Ca2+-ATPase in leaky vesicles if the reaction was slowed by lowering the pH to 6.1 and by including dimethyl sulfoxide in the reaction medium. At a dodecyl octaoxyethylene glycol monoether (C12E8) to protein weight ratio of 0.2, a detergent concentration too low for solubilization to occur, ATP-induced Ca2+ release occurred more rapidly than for native leaky membranes, whereas the rebinding step was slower. In contrast, no Ca2+ release was observed for any soluble preparation. The kinetics of Ca2+ release was studied under conditions where the ATPase was monomeric or aggregated, and also in the presence of added phospholipid. The ATPase was shown to be monomeric by sedimentation equilibrium measurements in the presence of Ca2+, ADP, and beta, gamma-methylene-ATP at a C12E8 to protein weight ratio of 2.0. It is concluded that solubilization of the Ca2+-ATPase may result in uncoupling of ATP hydrolysis from ATP-induced Ca2+ release.     

Buoyant density heterogeneity in spores of Bacillus subtilis: biochemical and physiological basis

The biochemical and physiological basis of density heterogeneity in Renografin of Bacillus subtilis W23 spores was determined by analysis of metals, macromolecules, and dipicolinic acid in the two density classes of the population. Germination rate and heat resistance were measured in both density classes. Atomic absorption spectrophotometry revealed that heavy spores (density = 1.335 g/ml) have 30% more calcium than light spores (density = 1.290 g/ml). Other metals found in greater amounts in heavy spores were manganese and potassium. However, light spores had more sodium than heavy spores. The amounts of carbohydrates, nucleic acids, and proteins were the same in both types of spores, but light spores contained more lipids, whereas heavy spores had 30% more dipicolinic acid than light spores. Calcium and lipid were excluded as causes of the heterogeneity in density in that alteration of their contents in spores did not detectably affect the density of these spores. Spores of two densities were genetically similar. Furthermore, light density spores arose earlier during sporulation than heavy spores as determined by releasing refractile forespores at various times during sporulation. We concluded that light spores represent an incomplete stage in development because they became heavy when reinoculated into spent sporulation medium. This must involve the additional accretion or synthesis of dipicolinic acid.