Evaluation of 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as a new sulfurizing reagent in combination with labile exocyclic amino protecting groups for solid-phase oligonucleotide synthesis.

3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) was recently introduced as an efficient sulfurizing reagent for solid-phase oligonucleotide synthesis. The successful syntheses were performed using standard base protecting groups (i.e. benzoyl for A and C, isobutyryl for G), which required deprotection in concentrated ammonium hydroxide at 55 degrees C for 15-18 h. We have explored the possibility of using EDITH in combination with fast deprotection chemistry(e.g. Expedite Chemistry using tert -butylphenoxy acetyl as a base protecting group). Surprisingly, poor synthesis performance was observed when syntheses were conducted with EDITH, Expedite Chemistry and standard synthesis cycle (i.e. Coupling-Thio-Cap). Potential G modification seemed to be the source of incompatibility since sequences containing no G or carrying isobutyryl- protected G residues could be synthesized with high efficiency. However, the deleterious G modification can be readily eliminated by inserting a capping step before the sulfurization reaction. Oligomers prepared with the Coupling-Cap-Thio-Cap cycle contained few phosphodiester contaminants as measured by31P-NMR, anion-exchange HPLC and MALDI-TOF mass spectrometry. In addition to reducing deprotection time, this new combination also provides a mild method for the preparation of certain phosphorothioate oligomers that may be sensitive to prolonged ammonia treatment (e.g. thioated RNAs).     

DNA markers linked to a T10 loose smut resistance gene in wheat (Triticum aestivum L.).

Screening for loose smut resistance in wheat is difficult. Selecting lines with DNA markers linked to loose smut resistance would be more reliable and less costly. Molecular markers linked to a race T10 loose smut resistance gene were identified using a F6 single seed descent segregating population. A RAPD marker and a RFLP marker were located on opposite flanks of the resistance gene and were shown to be loosely linked. The RAPD marker was converted to a user friendly polymorphic SCAR marker that represented a single genetically defined locus in hexaploid wheat. Using these two bracketing markers simultaneously, the error rate for T10 resistance selection due to crossing-over was reduced to 4%. These markers can be used for a faster and more reliable selection of T10 resistant plants than previous conventional loose smut ratings.     

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10⁶ cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Developmental expression of hexokinase 1 and 3 in rats

 Mammalian hexokinase types one and three (HK1 and HK3) are 100 kDa isozymes that phosphorylate glucose to glucose-6-phosphate. HK1 is present in most tissues but is especially prominent in brain and kidney. HK3 is less well studied, but may be most prominent in the spleen and lymphocytes. In this study, we determined the ontogeny of the expression of these isoforms in the rat. Using immunohistochemistry, we identified HK1 and HK3 immunoreactivity in the brain, heart, kidney, liver, skeletal muscle and spleen from gestational day 14 (E14) to 45 days after birth (P45). With the exception of the liver and spleen, we observed a similar age- and cell-dependent staining pattern for both isoforms in all organs studied. The brain and spleen were analyzed in more detail to identify specific regions of immunoreactivity during maturation. A transient expression of HK1 and HK3 was noted in the cell bodies of mature neurons, including layers V and VI of the cerebral cortex and the cerebellar Purkinje cells followed by localization to the white matter of the cerebrum and cerebellum. In the spleen, HK3 immunoreactivity was detected postnatally and appeared to track with the infiltration of B cells. Our demonstration of changing patterns of immunoreactivity for HK1 and HK3 in fetal and postnatal organs suggests that these HK isoforms are involved the process of development. We speculate that HK1 and HK3 share a complex interaction during development of these organs and regulate glucose metabolism at multiple levels during development. Accepted: 16 May 1997     

Guidelines for drug donations.

Drug donations are usually given in response to acute emergencies, but they can also be part of development aid. Donations may be given directly by governments, by non-governmental organisations, as corporate donations (direct or through private voluntary organisations), or as private donations to single health facilities. Although there are legitimate differences between these donations, basic rules should apply to them all. This common core of "good donation practice" is the basis for new guidelines which have recently been issued by the World Health Organisation after consultation with all relevant United Nations agencies, the Red Cross, and other major international agencies active in humanitarian emergency relief. This article summarises the need for such guidelines, the development process, the core principles, and the guidelines themselves and gives practical advice to recipients and donor agencies.     

Azide-sensitive thylakoid membrane insertion of chimeric cytochrome f polypeptides imported by isolated pea chloroplasts

Post-translational integration of cytochrome f into thylakoid membranes was observed after import by isolated pea chloroplasts of a chimeric protein consisting of the presequence of the small subunit of ribulose 1,5-bisphosphate carboxylase fused to the cytochrome f precursor. Import of a similar chimeric protein lacking the C-terminal 33 amino acid residues resulted in a soluble cytochrome f protein in the thylakoid lumen, indicating that the C-terminal region contains a stop-transfer sequence for membrane integration. Azide inhibited the insertion of cytochrome f into the thylakoid membrane, whereas the ionophores nigericin and valinomycin had little effect on membrane insertion. The precursor of the 33 kDa protein, but not the 23 kDa protein, of the photosystem II oxygen-evolving complex inhibited the thylakoid insertion of cytochrome f , suggesting competition for a component of the transport pathway. These experiments suggest that the post-translational insertion of cytochrome f into the thylakoid membrane uses a SecA-dependent pathway.     

Marine biodiversity: patterns, threats and conservation needs

Marine biodiversity is higher in benthic rather than pelagic systems, and in coasts rather than the open ocean since there is a greater range of habitats near the coast. The highest species diversity occurs in the Indonesian archipelago and decreases radially from there. The terrestrial pattern of increasing diversity from poles to tropics occurs from the Arctic to the tropics but does not seem to occur in the southern hemisphere where diversity is high at high latitides. Losses of marine diversity are highest in coastal areas largely as a result of conflicting uses of coastal habitats. The best way to conserve marine diversity is to conserve habitat and landscape diversity in the coastal area. Marine protected areas are only a part of the conservation strategy needed. It is suggested that a framework for coastal conservation is integrated coastal area management where one of the primary goals is sustainable use of coastal biodiversity.     

Structural studies on membrane-embedded influenza hemagglutinin and its fragments.

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.