Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase

Metal binding at the activating site of rabbit muscle phosphoglucomutase has been studied by 31P, 7Li, and 113Cd NMR spectroscopy. A 7Li NMR signal of the binary Li+ complex of the phosphoenzyme was not observed probably because of rapid transverse relaxation of the bound ion due to chemical exchange with free Li+. The phosphoenzyme-Li+-glucose 6-phosphate ternary complex is more stable, kinetically, and yields a well-resolved peak from bound Li+ at -0.24 ppm from LiCl with a line width of 5 Hz and a T1 relaxation time of 0.51 +/- 0.07 s at 78 MHz. When glucose 1-phosphate was bound, instead, the chemical shift of bound 7Li+ was -0.13 ppm; and in the Li+ complex of the dephosphoenzyme and glucose bisphosphate a partially broadened 7Li+ peak appeared at -0.08 ppm. Thus, the bound metal ion has a somewhat different environment in each of these three ternary complexes. The 113Cd NMR signal of the binary Cd2+ complex of the phosphoenzyme appears at 22 ppm relative to Cd(ClO4)2 with a line width of 20 Hz at 44.4 MHz. Binding of substrate and formation of the Cd2+ complex of the dephosphoenzyme and glucose bisphosphate broaden the 113Cd NMR signal to 70 Hz and shift it to 75 ppm. The 53 ppm downfield shift upon the addition of substrate along with 1H NMR data suggests that one oxygen ligand to Cd2+ in the binary complex is replaced by a nitrogen ligand at some intermediate point in the enzymic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of actinomycin-D on the fat body RNA content in Dysdercus koenigii (Heteroptera: Pyrrhocoridae)

The effect of actinomycin-D on the RNA content of the total fat body was studied on day 3, 4, 5 and 6 of the first reproductive cycle in the females. Actinomycin-D exhibited its highest inhibitory effect in insects injected on day 4 and a lower degree of the effect was seen in insects injected on day 5. These results suggest that by the end of day 4, a major quantity of DNA-dependent RNA, necessary for vitellogenin synthesis, has already been synthesised and accumulated.     

The Tetrahymena rRNA intron self-splices in E. coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function

We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E. coli as the host. A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor. Plaque phenotypes correlate well with levels of excised intron RNA. Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S. All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important. The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.     

Inactivation and reactivation of a variant-specific antigen gene in cyclically transmitted Trypanosoma brucei.

In Trypanosoma brucei, the activation of the variant-specific antigen gene AnTat 1.1 proceeds by the synthesis of an additional gene copy, the AnTat 1.1 ELC, which is transposed to a new location, the expression site, where it is transcribed. Using the AnTat 1.1 variant to infect flies, we investigated the fate of the AnTat 1.1 ELC during cyclic transmission of T. brucei. We show here that the AnTat 1.1 ELC is conserved in procyclic trypanosomes, obtained either from the midgut of infected Glossina or from cultures, and in metacyclic trypanosomes, although the AnTat 1.1 serotype is not detected among metacyclic antigen types. This same AnTat 1.1 ELC, which is thus silent as the parasite develops in the insect vector, can be reactivated without duplication during the first parasitemia wave following cyclical transmission. This re-expression of the conserved ELC accounts for the early appearance of the 'ingested' antigenic type after passage through the fly.     

The problem of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscular disorder. The biochemical defect remains unknown, but the gene responsible has been mapped to band Xp21. The gene has now been cloned in two laboratories solely from knowledge of its map location. L. M. Kunkel and his colleagues isolated genomic sequences (PERT 87) from within a large deletion causing DMD, whereas our group isolated genomic sequences (XJ) spanning the junction of an X-autosome translocation causing the disease. Chromosome walking by both groups has led to the isolation of over 400 kilobases of the PERT 87 and XJ region. Subclones of PERT 87 and XJ reveal restriction fragment length polymorphisms that segregate with the DMD gene in 95% of meioses, and fail to hybridize with DNA from about 8% of male patients. Selected subclones of PERT 87 and XJ contain exons that hybridize to muscle-derived complementary DNA (cDNA) clones. The cDNA clones detect a large (16 kilobase) message. Analysis of deletions, mutations and translocations suggests a DMD gene of between two million and three million base pairs. The clones obtained so far are useful for attempts to generate antibody against the gene product and for carrier identification and prenatal diagnosis.     

Allosteric modulation of Leishmania donovani plasma membrane Ca(2+)-ATPase by endogenous calmodulin.

The plasma membrane of the human pathogen Leishmania donovani possesses a high-affinity transmembrane Ca(2+)-ATPase that has its catalytic site oriented toward the cytoplasmic milieu (Ghosh, J., Ray, M., Sarkar, S., and Bhaduri, A. (1990) J. Biol. Chem. 265, 11345-11351). When the enzyme is studied in its more authentic, physiologically relevant, membrane-associated form, it exhibits pronounced sigmoidal kinetics with Ca2+ (K0.5 approximately 700 nM) in a trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid buffering system that effectively complexes all available Mg2+. Addition of exogenous Mg2+ (60 microM) completely abolishes sigmoidicity and establishes strictly hyperbolic kinetics, and the Km for Ca2+ reduces to 100 nM. Mg2+ can be replaced by heterologous calmodulin. The exclusive dependence of the enzyme on only Ca2+ for its activity and its positive allosteric modulation by Mg2+ distinguish this enzyme from other well-characterized plasma membrane Ca(2+)-ATPases. Employing this Ca(2+)-ATPase as the assay system, a soluble endogenous activating protein factor was purified that, by several criteria, corresponds to authentic calmodulin. The parasite calmodulin shifts the kinetics to hyperbolic kinetics, increases the Vmax 2-fold, and most important lowers the Km (approximately 100 nM) to a physiological level. The interaction with endogenous calmodulin thus converts the enzyme from a totally inactive to a fully active state.     

Stress ulcer modulation by limbic system structures.

A number of studies suggest that the telencephalic limbic system modulates stress ulcer development. The amygdala is assumed to connect sensory experiences, including stressful stimuli, with the emotional reactions and gastrointestinal effects normally produce. The hippocampal formation (entorhinal cortex, dentate gyrus, hippocampus) is part of a gating system, modulating the organism's coping ability. Changes in transmission in this temporal brain region are linked to individual differences in stress ulcer severity. Interactions among "classical" transmitters and several neuropeptides mediate these differences.     

Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H

N-terminal analysis of purified pediocin AcH produced a partial sequence of 23 amino acids. This sequence matched perfectly with a segment of 23 amino acids in a 62 amino acid molecule generated from the 186 nucleotide sequence open reading frame in a Hind III fragment in pSMB74 encoding pap-gene (pediocin AcH production). It is suggested that the molecule is translated as inactive prepediocin AcH of 62 amino acids. Then through enzymatic modifications the leader segment of 18 amino acids is removed from the NH2-terminal. The remaining segment of 44 amino acids is active pediocin AcH of 4628 M(r).