Mechanism of the irreversible inhibition of aspartate aminotransferase by the bacterial toxin L-2-amino-4-methoxy-trans-3-butenoic acid.

The naturally occurring toxin L-2-amino-4-methoxy-trans-3-butenoic (AMB) acid irreversibly inhibits pyridoxal phosphate-linked aspartate aminotransferase. The inhibitor is a substrate for the enzyme, and as such is converted into a highly reactive intermediate which chemically reacts with an active site residue, thus irreversibly inactivating the enzyme. Enzymological and model studies on AMB are presented which enable one to determine the precise mechanism of action of this toxin. The mechanism involves Schiff base formation between the enzyme and toxin followed by alpha-C--H bond cleavage and aldimine isomerization to generate a bifunctional Michael acceptor. This molecule alkylates an active site residue by an addition and elimination route.     

The Fate of Myoblasts Following Transplantation into Mature Muscle

Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme β-galactosidase (β-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (β-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and/β-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest β-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell trans-plantation and for the application of such studies to cell therapy and cell-mediated gene therapy.     

Sheep α-globin gene sequences: Implications for their concerted evolution and for the down-regulation of the 3′ genes

In sheep as in man and most other mammals, there are two -globin genes (^I and ^II), which are expressed at different levels, the upstream gene being the most efficient. In -globin gene triplication and quadruplication, this trend is confirmed, i.e., the -chain output of the downstream genes progressively decreases. In this study, we have determined the complete sequence of the cDNAs and of both the introns in a triple- haplotype in which each gene could be recognized for the presence of distinct alleles. The sequence analysis reveals that the bodies of the three -globin genes are essentially identical (99.9% homology) and moreover indicates that the down-regulation of additional -globin genes in sheep is not the effect of sequence variation from the Cap to the Poly(A) addition sites. This striking similarity among -genes is higher than that seen in other mammals and is probably sustained by particularly efficient mechanisms of gene conversion and cross-over fixation. Correspondence to: Dr. M.S. Ristaldi     

Unraveling regulatory networks in plant defense using microarrays

DNA microarrays are being used to comprehensively examine gene expression networks during the plant defense response that is triggered when a plant encounters a pathogen or an elicitor molecule. In addition to identifying new genes induced during defense, these studies are providing new insights into the complex pathways governing defense gene regulation.     

Is there an inner nose?

Although behavioral and neuropsychological data regarding the existence of images for odors are inconclusive, reconsideration of earlier EEG work provides reasonably clear evidence for an inner nose. However, further EEG studies and neuroimaging data seem essential for conclusive demonstration of an inner nose.      

Chemical modification of rhodopsin and its effect on regeneration and G protein activation

The studies reported are concerned with the functional consequences of the chemical modifications of the lysines and carboxyl-containing amino acids of bovine rhodopsin. The 10 non-active-site lysine residues of rhodopsin can be completely dimethylated and partially acetimidated (8-9 residues) with no loss in the ability of the proteins to activate the G protein when photolyzed or to regenerate with 11-cis-retinal. These modifications do not alter the net charge on the protein. Surprisingly, heavy acetylation of these lysines (eight to nine residues) with acetic anhydride, which neutralizes the positive charges of the lysine residues, yields a modified rhodopsin fully capable of activating the G protein and being regenerated. It is concluded that the non-active-site lysine residues of rhodopsin are not importantly and directly involved in interactions with the G protein during photolysis. However, this is not to say that they are unimportant in maintaining the tertiary structure of the protein because heavy modification of these residues by succinylation and trinitrophenylation produces proteins incapable of G protein activation, although the succinylated protein still regenerated. The active-site lysine of rhodopsin was readily modified and prevented from regenerating with 11-cis-retinal and with o-salicylaldehyde and o-phthalaldehyde/mercaptoethanol, two sterically similar aromatic aldehyde containing reagents which react by entirely different mechanisms. It is suggested that rhodopsin contains an aromatic binding site within its active-site region. Monoethylation, but not monomethylation, of the active-site lysine also prevented regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in the number of alpha-globin loci in sheep

Southern blot analysis was used to compare sheep and goat restriction- endonuclease maps of the DNA region containing the alpha-globin genes. The identical digestion patterns observed in both species with three endonucleases (BamHI, BstEII, and PstI) show that in sheep a single chromosome normally bears two nonallelic alpha-globin genes positioned at the same distance as in goat. Variant digestion patterns with enzymes that cleave outside (BamHI and HindIII) and within (EcoRI) the alpha-globin loci allowed us to infer that chromosomes with different numbers of alpha-globin loci are also present in sheep. In particular, in the 60 sheep considered, four individuals were heterozygous (alpha alpha/alpha alpha alpha) and one was homozygous (alpha alpha alpha/alpha alpha alpha) for chromosomes with three loci and one individual was heterozygous for a chromosome with four loci (alpha alpha/alpha alpha alpha alpha). This variation in the number of copies of alpha-globin loci can be explained by means of unequal crossovers.      

Heterogeneity in the muscle satellite cell population

Satellite cells, the adult stem cells responsible for skeletal muscle regeneration, are defined by their location between the basal lamina and the fiber sarcolemma. Increasing evidence suggests that satellite cells represent a heterogeneous population of cells with distinct embryological origin and multiple levels of biochemical and functional diversity. This review focuses on the rich diversity of the satellite cell population based on studies across species. Ultimately, a more complete characterization of the heterogeneity of satellite cells will be essential to understand the functional significance in terms of muscle growth, homeostasis, tissue repair, and aging.