Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence.

The process of translational reinitiation in Escherichia coli was studied in a two cistron system where expression of the downstream reporter gene was dependent on translation of an upstream reading frame. The dependence was almost absolute. Upstream translation increased expression of the downstream gene by two to three orders of magnitude. This large difference allowed us to quantitate restarts in a meaningful manner. In the absence of a Shine and Dalgarno (SD) region reinitiation occurred but its efficiency was about 10% of that found in the SD carrying counterpart. We discuss three ways by which translational coupling between neighboring cistrons can be enforced.     

Isolation and structural studies of phosphate-containing oligosaccharides from alkaline and acid hydrolysates of Streptococcus pneumoniae type 6B capsular polysaccharide

The capsular polysaccharide of Streptococcus pneumoniae serotype 6B [----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-( 1----4)- D-RibOH-(5-P----]n was depolymerised under alkaline (NaOH) and acidic (HF) conditions. The former treatment yielded, as the major component, alpha-2-P-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-5- P-RibOH. The latter treatment at -16 degrees gave alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH-(5-P----2)- alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH and at 4 degrees gave alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH. These oligosaccharides were characterised by sugar analysis, f.a.b.-m.s., and 1H- and 13C-n.m.r. spectroscopy.     

The biological activity of interferon alpha is influenced by two distinct regions in the protein

With the aim to assign differences in activity between murine interferon-alpha 1 and -alpha 4 to specific amino acids, we have constructed hybrid genes and analysed the antiviral properties of the corresponding hybrid proteins. The hybrid genes were constructed by means of homologous recombination between the alpha 1 and alpha 4 genes in Escherichia coli. Hybrids in which the N-terminal part is derived from alpha 1 show that two regions have a major effect on the activity: amino acid 10-20 and 55-67. When comparing hybrids with N-terminal alpha 4 sequences, transitions in activity are found in the same regions. Interestingly, the curves for the two sets of hybrids are exactly each others mirror image.     

Collagens as multidomain proteins

The number of proteins known to contain collagen-like triple helical domains is rapidly increasing. The functions of these domains are to provide molecular rods that separate spatially non-triple helical domains with varied properties and structures and to permit lateral interactions between molecules. Two-thirds of the amino acids of the triple helical domains have their side-chains at the surface of the protein. The triple helix is also a structure that is easily predictable from the primary structure. The structure of several recently discovered collagens are discussed in terms of domains and functions. The triple helical domains have sizes varying from 33 to over 1,000 amino acid residues. The longest uninterrupted triple helices are involved in the formation of the classical quarter-staggered fibrils. Other triple helical domains permit varied molecular aggregates. A very broad spectrum of non-triple helical or globular domains are interspersed by triple helices. Only those located at the extremities of the molecules are large in size, sometimes several hundred kDa, while the domains separating 2 triple helices are small (less than 50 amino acids) and provide the molecules with hinges, proteolytic cleavage sites or other specialized functions like a glycosaminoglycan attachment site. If the assembly of the 3 chains required for the triple helix formation can be controlled in vitro, collagen-like molecules offer an as yet unexploited potential for protein engineering.     

Outer-membrane PhoE protein of Escherichia coli K-12 as an exposure vector: possibilities and limitations.

The phosphate-limitation-inducible outer-membrane protein (PhoE) of Escherichia coli K-12 can be used in an expression system as a carrier for foreign antigenic determinants, facilitating their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature can be made in different cell-surface-exposed regions of PhoE protein, without interfering with the assembly process into the outer membrane. Multiple insertions of an antigenic determinant can be made in the second and eighth exposed regions, resulting in a total insert length of up to 30 and 50 amino acid (aa) residues. Insertions can be made in two exposed regions, simultaneously. However, some limitations were encountered, e.g., insertion of eight or more hydrophobic aa residues affected both the translocation process across the inner membrane and the assembly process into the outer membrane. Also, the insertion of sequences containing many charged residues resulted in accumulation of precursor protein in the cytoplasm.     

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Iron status in the acute phase and six weeks after myocardial infarction

In a case-control study of 84 myocardial infarction patients and 84 population controls we investigated the association between iron status parameters and myocardial infarction during the acute phase and after six weeks. Immediately after the infarction mean ferritin levels were significantly higher, whereas iron levels and iron saturation of transferrin were significantly lower in cases than in controls. Six weeks after the infarction, serum iron levels were still significantly lower in cases than in controls. Neither serum ferritin levels nor serum iron levels did show a clear association with the size of the ischemic tissue damage as estimated by creatine phosphokinase levels. Our results indicate that serum ferritin and iron levels are influenced by the traumatic effects of the myocardial infarction. Possibly, these transient changes are an acute effect, as seen in infections. An increased uptake of iron in the reticulo-endothelial system for synthesis of ferritin, may account for the lowered serum iron level and the iron saturation of transferrin.     

The effect of gamma-linolenic acid, an in vitro cytostatic substance contained in evening primrose oil, on primary liver cancer. A double-blind placebo controlled trial

The cytostatic effects of essential fatty acid metabolic intermediates and of some prostaglandins and leukotrienes in vitro have been extensively documented. The essential fatty acids (EFAs) exhibit no side-effects when taken as a dietary supplement, even in large doses. Primary Liver Cancer (PLC) is a fatal disease in our area as it is always multifocal in nature. In vitro studies have shown a cytostatic effect of gamma-linolenic acid (GLA) on primary liver cancer cells. In a double-blind placebo controlled trial, using Evening Primrose Oil (as a source of GLA) as a dietary supplement in PLC patients, no statistically significant effect was observed on survival time or liver size. There was however a statistical significant beneficial effect on Gamma Glutamyl transferase values as a measure of liver function. No side-effects were observed. The large size of tumour and the low doses of GLA used in this trial probably explain the lack of significant effect on survival times.