Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Calcium-activated adenosine triphosphatase activity of pellicles from Paramecium caudatum.

Pellicles were isolated from Paramecium caudatum for a study of the properties of its insoluble ATPase [EC 3.6.1.3] activity. Pellicular ATPase was solubilized by sonication and fractionated by sucrose density gradient centrifugation. The sedimentation coefficient of the ATPase was about 9S. The ATPase required Ca2+ for maximum activation. Addition of neutral salts to the assay medium inhibited the activity. Substrate specificity for ATP was low; other nucleoside triphosphates were hydrolyzed at about the same rate as ATP; AMP, pyrophosphate, and p-nitrophenyl phosphate were not hydrolyzed. The ATPase activity of the pellicle preparation had a pH optimum at pH 6.5, and a Michaelis constant of 9 micrometer. On the other hand, the enzymatic properties of the ATPase were somewhat modified by the procedure of solubilization and fractionation. The pellicular ATPase does not resemble ciliary dynein ATPase or the soluble ATPase of Tetrahymena.     

Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.     

Lactate dehydrogenase isoenzymes are present in matrix vesicles

Matrix vesicles were isolated from epiphyseal growth plates of young rabbits. Lactate dehydrogenase activity was detected in the isolated matrix vesicles only in the presence of detergents, suggesting that NADH, the cofactor for the assay, does not penetrate the membrane of matrix vesicles. In contrast, the activity of alkaline phosphatase, a marker enzyme of the outer surface of matrix vesicles, was detected in the matrix vesicles using p-nitrophenyl phosphate as the substrate both in the presence and absence of detergents. Lactate dehydrogenase activity was detected only in the cytosol of chondrocytes of the epiphyseal growth plates but not in other subcellular fractions, showing that lactate dehydrogenase is not from the plasma membrane and membranes of intracellular organelles of chondrocytes. The isolated matrix vesicles contained all five lactate dehydrogenase isoenzymes but did not possess other cytosolic enzymes. These results show that lactate dehydrogenase is located in the matrix vesicles and suggest the presence of a mechanism for the specific uptake of cytosolic lactate dehydrogenase and the possibility of enzymatic quantification of the matrix vesicles at various calcification sites.     

Membrane flow in plants: preparation and kinetics of labelling of plasma membranes from growing pollen tubes of tobacco

Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [³H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system.     

Characterization of insulin receptors in primary cultures of quail (Coturnix coturnix japonica) oviduct cells. The level of insulin receptor is regulated by steroid and peptide hormones

1. We have characterized the insulin receptor in primary cultured quail oviduct cells and examined the hormonal regulation of its level. 2. We have also shown the recycling pathway of insulin receptors in the cultured cells using specific inhibitors (tunicamycin, chloroquine, monensin, and brefeldin A). 3. Our data suggest that glucocorticoids play important physiological roles in egg-white protein synthesis through increasing the number of insulin receptors and insulin through enhancing the transport of amino acids.     

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.     

Adaptive response of Schizosaccharomyces pombe to hydrogen peroxide

Abstract Schizosaccharomyces pombe becomes resistant to killing by high concentration of hydrogen peroxide and other severe stresses including oxidants, high temperature and high concentration of ethanol when pretreated with nonlethal levels of hydrogen peroxide. In the presence of the protein synthesis inhibitor, cycloheximide, during hydrogen peroxide pretreatment, the cell obtained partial resistance to a higher level of hydrogen peroxide. The partial resistance to hydrogen peroxide in the presence of cycloheximide was acquired within 30 min of pretreatment but complete resistance obtained with de novo protein synthesis was not attained before 45 min of pretreatment. During adaptation to hydrogen peroxide, at least 15 polypeptides are induced, as analyzed by two-dimensional gel electrophoresis. Catalase activity is induced eight-fold by treatment with a nonlethal level of hydrogen peroxide.