Affinity chromatography on immobilised nucleotides. Some applications to the purification of thermophilic dehydrogenases and kinases.

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6-(6-aminohexyl)-5'-AMP-Sepharose has been examined. Specific elution from the substituted AMP-Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP-Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6-(6-aminohexyl)-5'-AMP-Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant. The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde-3-phosphate dehydrogenase to 6-aminohexanoyl-NAD+-Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde-3-phosphate dehydrogenase from partially purified extracts of this organism was achieved.     

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

Separation of a proteoglycan fraction from Kurloff cells stimulating protein synthesis in macrophages.

Proteoglycan from Kurloff cells, when present in the medium in low concentrations, increased the incorporation of (3-H)leucine into trichloracetic acid-precipitable material by macrophages from peritoneal exudates, in addition to inhibiting their migration from capillary tubes, as observed previously. After treatment with 0.5 M-NaOH, followed by dialysis or ultrafiltration, material with the distinctive u.v. and i.r. spectra of the whole proteoglycan appeared in the diffusate, and biological activity was lost from the proteoglycan which remained in the dialysis residue. The diffusible material absorbed near 260 nm and had i.r. bands at 805 cm-minus-1 and 1260 cm-minus-1, but did not display the i.r. bands characteristic of chondrotin 4-sulphate. It contained little sulphate, no hexosamine and less than 1% of the uronic acid present in the whole proteoglycan, and there were only trace amounts of amino acids, xylose and galactose. However, significant amounts of ribose and organic phosphate were present, each representing about 1% of the whole proteoglycan. After proteolysis and chondroitanase digestion of the proteoglycan, a fraction with absorbance at 260 mn was eluted from Dowex 1 with water which stimulated the incorporation of (3H)leucine by macrophages and inhibited their migration from capillary tubes.     

Connective tissue growth factor and cardiac fibrosis after myocardial infarction.

The temporal and spatial expression of transforming growth factor (TGF)-beta(1) and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-beta(1), CTGF, and procollagen alpha1(I) mRNA were localized by in situ hybridization, and TGF-beta(1) and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-beta(1), CTGF, and collagen after MI. Procollagen alpha1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-beta(1) mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-beta(1) or CTGF. TGF-beta(1) is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.     

Bats and moths: what is there left to learn?

Abstract.  Over 14 families of moths have ears that are adapted to detect the ultrasonic echolocation calls of bats. On hearing a bat, these moths respond with an escape response that reduces their chances of being caught. As an evolutionary response, bats may then have evolved behavioural strategies or changes in call design to overcome the moth's hearing. The nature of this interaction is reviewed. In particular, the role of the echolocation calls of bats in the shaping of the structure, neurophysiology and behavioural responses of moths is discussed. Unresolved issues, such as the structural complexity of the moth's auditory system, the nature of temporal integration and the role of the non-auditory B cell, are described. Issues in which the interactions between bats and moths may be of more general interest to biologists, such as noise filtering within the central nervous system, protean behaviours and coevolution between predator and prey, are also discussed. The interaction between bats and moths has much to interest general biologists, and may provide a useful model in understanding the neurophysiological basis of behaviour, including protean escape behaviours. The validity of the term coevolution as applied to this system is discussed, as there is no doubt that the auditory system of moths is a response to the echolocation calls of bats, although the evolutionary response of bats to moths is more ambiguous.     

Mitogenomic phylogeny of the Percichthyidae and Centrarchiformes (Percomorphaceae): comparison with recent nuclear gene-based studies and simultaneous analysis

Delineation of the fish family Percichthyidae (Percomorphaceae) has a long and convoluted history, with recent morphological-based studies restricting species members to South American and Australian freshwater and catadromous temperate perches. Four recent nuclear gene-based phylogenetic studies, however, found that the Percichthyidae was not monophyletic and was nested within a newly discovered inter-familial clade of Percomorphaceae, the Centrarchiformes, which comprises the Centrarchidae and 12 other families. Here, we reexamined the systematics of the Percichthyidae and Centrarchiformes based on new mitogenomic information. Our mitogenomic results are globally congruent with the recent nuclear gene-based studies although the overall amount of phylogenetic signal of the mitogenome is lower. They do not support the monophyly of the Percichthyidae, because the catadromous genus Percalates is not exclusively related to the freshwater percichthyids. The Percichthyidae (minus Percalates) and Percalates belong to a larger clade, equivalent to the Centrarchiformes, but their respective sister groups are unresolved. Because all recent analyses recover a monophyletic Centrarchiformes but with substantially different intra-relationships, we performed a simultaneous analysis for a character set combining the mitogenome and 19 nuclear genes previously published, for 22 centrarchiform taxa. This analysis furthermore indicates that the Centrarchiformes are divided into three lineages and the superfamily Cirrhitoidea is monophyletic as well as the temperate and freshwater centrarchiform perch-like fishes. It also clarifies some of the relationships within the freshwater Percichthyidae.     

Recombination in the ompA gene but not the omcB gene of Chlamydia contributes to serovar-specific differences in tissue tropism, immune surveillance, and persistence of the organism

Sequences of the major outer membrane protein (MOMP) gene (ompA) and the outer membrane complex B protein gene (omcB) from Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci were analyzed for evidence of intragenic recombination and for linkage equilibrium. The Sawyer runs test, compatibility matrices, and index of association analyses provided substantial evidence that there has been a history of intragenic recombination at ompA including one instance of interspecies recombination between the C. trachomatis mouse pneumonitis strain and the C. pneumoniae horse N16 strain. Although none of these methods detected intragenic recombination within omcB, differences in divergence reported in earlier studies suggested that there has been intergenic recombination involving omcB, and the analyses presented in this study are consistent with this. For C. trachomatis, index-of-association analyses suggested a higher degree of recombination for C class than for B class strains and a higher degree of recombination in the downstream half of ompA. In concordance with these findings, many significant breakpoints were found in variable segments 3 and 4 of MOMP for the recombinant strains D/B120, G/UW-57, E/Bour, and LGV-98 identified in this study. We provide examples of how genetic diversity generated by repeated recombination in these regions may be associated with evasion of immune surveillance, serovar-specific differences in tissue tropism, and persistence.