Statistical evidence for remnants of the primordial code in the acceptor stem of prokaryotic transfer RNA

Summary The specificity of interaction of amino acids with triplets in the acceptor helix stem of tRNA was investigated by means of a statistical analysis of 1400 tRNA sequences. The imprint of a prototypic genetic code at position 3–5 of the acceptor helix was detected, but only for those major amino acids, glycine, alanine, aspartic acid, and valine, that are formed by spark discharges of simple gases in the laboratory. Although remnants of the code at position 3–5 are typical for tRNAs of archaebacteria, eubacteria, and chloroplasts, eukaryotes do not seem to contain this code, and mitochondria take up an intermediary position. A duplication mechanism for the transposition of the original 3–5 code toward its present position in the anticodon stern of tRNA is proposed. From this viewpoint, the mode of evolution of mRNA and functional ribosomes becomes more understandable.Offprint requests to: W. Moller     

The radiosensitivity of cultured human and mouse keratinocytes

Clonogenic survival assays after gamma-radiation in vitro were performed on freshly isolated and subcultured keratinocytes from mouse skin, mouse tongue and human skin. Survival curves were constructed by fitting the data to a multi-target model of cell survival. When subcultured, keratinocytes from all sites produced survival curves which showed a reduced shoulder region and an increased D0 when compared with their freshly isolated counterparts. Freshly isolated human skin keratinocytes were more radiosensitive than mouse keratinocytes from either skin or tongue.     

Influence of Nitrate and Sulfate on the Anaerobic Treatment of Pharmaceutical Wastewater

Anaerobic treatment of wastewater from the pharmaceutical industry, which contained about 3.2 g/L of sulfate, was carried out in an Upflow Anaerobic Sludge Blanket (UASB) reactor. After a startup period of 120 days, a chemical oxygen demand (COD) removal efficiency of more than 90 % was obtained along with an organic loading rate (OLR) of 1.5 g COD/(L day). During the same period, the sulfate removal was about 90 %. However, the performance of the reactor was affected when the loading rate was increased to 2.09 g COD/(L day). It was found that the accumulation of sulfides, combined with a decrease in the pH, affected the reactor performance. In batch reactor studies with pharmaceutical wastewater it was observed that methane production began only after the initiation of nitrate consumption. The denitrification process can inhibit sulfate reduction at high nitrate concentrations, but compared to reactors without nitrate, the sulfate reduction process and sulfide formation were quickly initiated at low nitrate concentrations. The methanogenic activity was however affected by the presence of more than 2 g/L of sulfate.     

Saccharomyces cerevisiae structural cell wall mannoprotein

A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.     

Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease beta A4 peptides.

The deposition of amyloid protein aggregates in brain is the main pathological feature of Alzheimer's disease. Their principal constituent is a peptide termed beta A4, which comprises up to 43 amino acid residues. It is highly insoluble under physiological conditions and aggregates into filaments that form very dense clusters in vivo and in vitro. Based on a beta A4 prototype sequence spanning residues 10 to 42 or 43, we have designed analogues in which hydrophobic amino acid residues in position 17 to 20 were substituted by more hydrophilic residues. Depending on the kind of newly introduced amino acids and their position within the sequence, the substitution of only two residues led to variants exhibiting a broad spectrum of different properties. Common to them was a reduced beta-sheet content after solubilization in water and in the solid state. Some of the variants showed significantly reduced amyloidogenicity: although still forming filaments, they did not aggregate into the highly condensed depositions that are typical for amyloid. In addition, they could be solubilized in 200 mM-NaCl and KCl. When mixed with beta A4 peptides bearing the natural sequence, two of the analogues could inhibit the formation of filaments in vitro. These results demonstrate that a well-preserved hydrophobic core around residues 17 to 20 of beta A4 is crucial for the formation of beta-sheet structure and the amyloid properties of beta A4. The introduction of structural alterations within this region may guide the development of reagents for the therapy of Alzheimer's disease.     

Parvalbumins from frog skeletal muscle (Rana temporaria L.). Isolation and characterization. Structural modifications associated with calcium binding.

Two parvalbumins have been isolated from the skeletal muscle of Rana temporaria L. Amino acid composition, tryptic peptide maps, isoelectric points, calcium content and ultraviolet as well as circular dichroism spectra have been determined. Investigation on antigenic properties revealed no antigenic determinants common to both components. The two protein molecules appear to belong to far related gene lineages. They are also different from the parvalbumins found in Rana esculenta muscle. Modifications of physical parameters, associated with calcium binding and dissociation are described. While antigenicity remained essentially unchanged, conformational changes were revealed by alterations of circular dichroism spectra.     

Packing of ribosomes in crystals from the lizard Lacerta sicula

The packing of ribosomes in the large crystalline sheets found in the lizard Lacerta sicula has been investigated by electron microscopy. The ribosomes in each of the two layers composing a sheet are organised as tetramers on a P4 space group lattice. The two layers face in opposite directions and tend to be related to one another crystallographically, generating a family of P422 crystals of different unit cell dimensions. The projected structure of one layer was determined from negatively stained, isolated sheets by separating the contributions from each layer in Fourier transforms computed from electron micrographs. Comparison of the projection map with other, low resolution, analyses of images of isolated eukaryotic ribosomes indicates that the large subunit- small subunit axis lies approximately parallel to the plane of the sheet.