An effect-specific track-length model for radiations of intermediate to high LET

A model for the induction of transformation, mutation, and cell killing by radiations of intermediate to high linear energy transfer (LET) is presented. The mathematical formulation presupposes a constant probability per unit path length for damaging multiple subcellular targets by radiation of a fixed LET. The coupling between effects is accounted for through an explicit calculation of the probability that any specific combination of effects occurs in a given cell. This feature avoids the false assumption that cell killing and mutation (or transformation) are independent events. The resulting model then is applied to data on the in vitro survival, mutation, and transformation of cells by radiations of varying LET. A summary of estimated parameter values is provided and calculations of the effect of cellular flattening on transformation are presented.     

Isolation and characterization of a diuretic peptide from Acheta domesticus. Evidence for a family of insect diuretic peptides.

A diuretic peptide (Acheta-DP) has been isolated from extracts of whole heads of the house cricket, Acheta domesticus. The native peptide increases both cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro to an extent comparable with those responses obtained with supra-maximal amounts of crude extracts of corpora cardiaca. The primary structure of Acheta-DP was established as a 46-residue amidated peptide: TGAQSLSIVAPLDVLRQRLMNELNRRRMRELQGSRIQQNRQLLTSI-NH2. Acheta-DP has 41% sequence identity with a diuretic peptide isolated from Manduca sexta, providing direct evidence for the presence of a family of diuretic peptides in insects.     

cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region.     

A eukaryotic genome of 660 kb: electrophoretic karyotype of nucleomorph and cell nucleus of the cryptomonad alga, Pyrenomonas salina.

Cryptomonads are unicellular algae with chloroplasts surrounded by four membranes. Between the inner and the outer pairs of membranes is a narrow plasmatic compartment which contains a nucleus-like organelle called the nucleomorph. Using pulsed field gel electrophoresis it is shown that the nucleomorph of the cryptomonad Pyrenomonas salina contains three linear chromosomes of 195 kb, 225 kb and 240 kb all of which encode rRNAs. Thus, this vestigial nucleus has a haploid genome size of 660 kb, harboring the smallest eukaryotic genome known so far. From the cell nucleus of P. salina at least 20 chromosomes ranging from 230 kb to 3.000 kb were fractionated. Here, the rDNA was detected on a single chromosome of about 2.500 kb.     

Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine-binding sites

Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non-radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, and 33 kDa. Incorporation into the 55-kDa protein was blocked by 10--50-fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165-kDa protein, suggesting that the 165-kDa and 55-kDa proteins contain a high and a low-affinity verapamil-binding site respectively. The photoaffinity-labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165-kDa protein bound to wheat-germ-agglutinin(WGA)--Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55-kDa membrane protein did not bind to the WGA-Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165-kDa protein. The 165-kDa but not the 55-kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55-kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55-kDa protein of the membrane is unrelated to the purified high-affinity dihydropyridine receptor.     

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.