An improved purification method for cytosolic malate dehydrogenase from several sources

A new purification method for cytosolic malate dehydrogenases from several sources has been developed. The procedure, employing chromatographies on 5'AMP-Sepharose, DEAE-Sephacel and Blue-Sepharose, allows for a rapid isolation of the enzyme (approximately 40 hours), in large quantities, with good yields (45-54%). The specific activity of final preparations were around 1300 I.U./mg and were judged homogeneous by polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size exclusion chromatography and isoelectric focusing.     

The ATP depletion process of human erythrocytes studied by freeze fracturing

By the freeze-fracture method it is shown that metabolic depletion of erythrocytes affects three levels of cell organization: the microstructural (erythrocyte form), microstructural (micro-relief of erythrocyte surface) and ultrastructural (ultrastructural state of erythrocyte plasma membranes) ones. As it is established, the size of spikes on the echinocyte surface and that of membrane vesicles budding from a cell coincide with each other. The structural modification of the membrane precedes the stage of erythrocyte crenation. The following model of vesicle budding process is suggested: reduction of ATP level and dephosphorylation of actin-spectrin network--structural modification of the protein and lipid membrane phases with the formation of regions disconnected from the spectrin framework--protrusion of these anomalous regions in the form of spikes--budding of spikes as spherical vesicles.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Two Amino-acid Carriers in Pea Chloroplasts

CHLOROPLASTS are more permeable to amino-acids^1,2 than are cell membranes³ and also discriminate between their optical isomers². This has led to the suggestion that the passage of amino-acids through the limiting membranes of chloroplasts is facilitated by carriers². Using the additional criteria of saturation and competition, we support this suggestion and show that at least two carriers are probably involved.     

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.     

Direct microsensor measurement of nitric oxide production by the osteoclast.

Nitric oxide (NO) triggers marked osteoclast retraction which closely resembles that due to Ca2+. The effect of Ca2+ has been attributed to a stimulated release of NO. Here, we show for the first time, by direct measurement with a microsensor, that osteoclasts do indeed produce NO and that this production is enhanced by a high Ca2+. We also show that the Ca2+ ionophore, A23187, mimics the latter. Furthermore, osteoclasts on dentine produce more NO than osteoclasts on glass and NO release from dentine-plated osteoclasts is much less sensitive to stimulation by Ca2+. Finally, the microsomal Ca2+ store-depleting agent, thapsigargin, attenuates NO release only from osteoclasts on glass, suggesting that stored Ca2+ has the dominant effect in modulating NO release from non-resorbing cells. NO is a powerful inhibitor of bone resorption: a direct demonstration of its production is therefore strong evidence for a role in modulating osteoclast function.