POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

Ultrasonic examination of the parathyroid glands, bone x-ray and calcium deposits of the eye in diagnosis of secondary hyperparathyroidism in light of 12 observed cases]

Twelve patients with chronic renal failure treated with repeated dialysis underwent subtotal parathyroidectomy. Histologic examination revealed hyperplasia parathyroidal adenoma in all patients. Diagnostic correlation between bone X-ray, ultrasound of parathyroid glands, calcium deposits in cornea and conjunctiva, and histological findings has been analysed. Radiological abnormalities have been noted in 8 patients (66%), parathyroid glands hyperplasia in 9 (75%), and corneal and conjunctival calcium deposits in 10 (83%) patients. These data confirm the value of techniques under study for the diagnosis of the secondary hyperparathyroidism in patients with chronic renal failure.     

Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP

Electron micrographs of RecA-DNA filaments, formed under several different conditions, have been analyzed and the filament images reconstructed in three dimensions. In the presence of ATP and a non-hydrolyzable ATP analog. ATP-gamma-S, the RecA protein forms with DNA a right-handed helical complex with a pitch of approximately 95 A. The most detailed view of the filament was obtained from analysis of RecA filaments on double-stranded DNA in the presence of ATP-gamma-S. There are approximately six subunits of RecA per turn of the helix, but both this number and the pitch are variable. From the examination of single filaments and filament-filament interactions, a picture of an extremely flexible protein structure emerges. The subunits of RecA protein are seen to be arranged in such a manner that the bound DNA must be partially exposed and able to come into contact with external DNA molecules. The RecA structure determined in the presence of ATP-gamma-S appears to be the same as the "pre-synaptic" state that occurs with ATP, in which there is recognition and pairing between homologous DNA molecules.     

A karyological study of Asphodelus L. (Asphodelaceae) from the Western Mediterranean

The following aspects of Asphodelus karyology are analysed: base number, polyploidy, chromosome size, chromosome morphology, satellited chromosomes, structural heteromorphism, karyotype asymmetry and karyotype evolution. The base number 0 ×= 14 is common to all species except for A. refractus , which has the derived ×= 13. Three ploidy levels occur, often in the same species; diploid, tetraploid and hexaploid, with 2n = 28, 56 and 84. Chromosomes are generally small to medium-small, with the occasional presence of medium-large chromosomes. The most frequent chromosome types are metacentric of type m and submetacentric. Metacentric chromosomes of type M occur only in sections. Verineopsis, Verinea and Plagiasphodelus ; subtelocentric chromosomes occur only in sections Asphodelus and Plagiasphodelus. There is a wide variability in relation to the number of satellited chromosomes, relative to ploidy level. There are usually two to four in diploids, four to eight in tetraploids and usually six, exceptionally up to 12, in the hexaploid. Satellites are present on the shortest arm, exceptionally on the longest arm. There is a high degree of structural heteromorphism in practically all the species which affects satellited and non satellited chromosomes. Karyotype asymmetry is generally of type 2B. Inter-and intra-chromosomal differences are estimated by the A1 and A2 indexes. Both indices vary in the karyotype evolution of the genus, with a decrease of A1 and an increase of A2. The role of polyploidy, hybridization, asymmetry and decrease of chromosome size in the evolution of Asphodelus is discussed.     

Elongation of duplex DNA by recA protein

recA protein, which is essential for the recombination process in Escherichia coli, was incubated in the presence of 5′-γ-thiotriphosphate with circular plasmid pBR_βG containing small single-stranded gaps. Stable complexes were formed which appear in the electron microscope as fibres with a diameter about five times that of naked DNA. Complex formation appears to be a co-operative process whereby the average rise per base-pair with respect to the fibre axis increases from 3·39 ± 0·08 Å to 5·20 ± 0·18 Å. The elongation of DNA by about 50% is compatible with an unwinding of the double helix and an intercalating mode of binding of recA and/or 5′-γ-thiotriphosphate to DNA.     

In Search of Functional Advantages of Knots in Proteins

We analysed the structure of deeply knotted proteins representing three unrelated families of knotted proteins. We looked at the correlation between positions of knotted cores in these proteins and such local structural characteristics as the number of intra-chain contacts, structural stability and solvent accessibility. We observed that the knotted cores and especially their borders showed strong enrichment in the number of contacts. These regions showed also increased thermal stability, whereas their solvent accessibility was decreased. Interestingly, the active sites within these knotted proteins preferentially located in the regions with increased number of contacts that also have increased thermal stability and decreased solvent accessibility. Our results suggest that knotting of polypeptide chains provides a favourable environment for the active sites observed in knotted proteins. Some knotted proteins have homologues without a knot. Interestingly, these unknotted homologues form local entanglements that retain structural characteristics of the knotted cores.     

Formation of knots in partially replicated DNA molecules

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.