Structure of a genomic clone encoding biologically active human relaxin

Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.     

DNA sequence and expression of the B95-8 Epstein-Barr virus genome

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.     

Isoguvacine,isonipecotic acid,muscimol and N-methyl isoguvacine on the GABA receptor in rat sympathetic ganglia

Summary The GABA-mimetic activities of 4 analogues muscimol, isonipecotic acid, isoguvacine and N-methyl isoguvacine have been examined at the GABA receptor in the rat isolated superior cervical ganglion. The depolarizing action of all 4 analogues could be selectively antagonized by bicuculline methochloride and isopropyl bicyclophosphate. Muscimol was the only analogue more potent than GABA (molar potency ratio=5.08±0.707). The potency of isoguvacine was 0.23±0.026 and isonipecotic acid 0.011±0.0028. N.-methyl isoguvacine was <0.001 GABA.     

The reversibility of mitotic exit in vertebrate cells

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.     

Interplay of magnetism and high-Tc superconductivity at individual Ni impurity atoms in Bi2Sr2CaCu2O8+delta.

Magnetic interactions and magnetic impurities are destructive to superconductivity in conventional superconductors. By contrast, in some unconventional macroscopic quantum systems (such as superfluid 3He and superconducting UGe2), the superconductivity (or superfluidity) is actually mediated by magnetic interactions. A magnetic mechanism has also been proposed for high-temperature superconductivity. Within this context, the fact that magnetic Ni impurity atoms have a weaker effect on superconductivity than non-magnetic Zn atoms in the high-Tc superconductors has been put forward as evidence supporting a magnetic mechanism. Here we use scanning tunnelling microscopy to determine directly the influence of individual Ni atoms on the local electronic structure of Bi2Sr2CaCu2O8+delta. At each Ni site we observe two d-wave impurity states of apparently opposite spin polarization, whose existence indicates that Ni retains a magnetic moment in the superconducting state. However, analysis of the impurity-state energies shows that quasiparticle scattering at Ni is predominantly non-magnetic. Furthermore, we show that the superconducting energy gap and correlations are unimpaired at Ni. This is in strong contrast to the effects of non-magnetic Zn impurities, which locally destroy superconductivity. These results are consistent with predictions for impurity atom phenomena derived from a magnetic mechanism.     

Changes in fisheries discard rates and seabird communities

It is clear that discards from commercial fisheries are a key food resource for many seabird species around the world. But predicting the response of seabird communities to changes in discard rates is problematic and requires historical data to elucidate the confounding effects of other, more 'natural' ecological processes. In the North Sea, declining stocks, changes in technical measures, changes in population structure and the establishment of a recovery programme for cod (Gadus morhua) will alter the amount of fish discarded. This region also supports internationally important populations of seabirds, some of which feed extensively, but facultatively, on discards, in particular on undersized haddock (Melanogrammus aeglefinus) and whiting (Merlangius merlangus). Here we use long-term data sets from the northern North Sea to show that there is a direct link between discard availability and discard use by a generalist predator and scavenger--the great skua (Stercorarius skua). Reduced rates of discarding, particularly when coupled with reduced availability of small shoaling pelagic fish such as sandeel (Ammodytes marinus), result in an increase in predation by great skuas on other birds. This switching of prey by a facultative scavenger presents a potentially serious threat to some seabird communities.     

Competition and mutualism among the gut helminths of a mammalian host

Most animal species are infected with multiple parasite species; however, the role of interspecific parasite interactions in influencing parasite dynamics and shaping parasite communities has been unclear. Although laboratory studies have found evidence of cross-immunity, immunosuppression and competition, analyses of hosts in the field have generally concluded that parasite communities are little more than random assemblages. Here we present evidence of consistent interspecific interactions in a natural mammalian system, revealed through the analysis of parasite intensity data collected from a free-ranging rabbit (Oryctolagus cuniculus) population, sampled monthly for a period of 23 yr. The wild rabbit plays host to a diverse gut helminth community that reflects the communities seen in other economically important domestic herbivores. These findings suggest that parasite interactions could have profound implications for the dynamics of parasite communities. The efficacy of parasite control programmes could be jeopardized if such interactions are not taken into account. In contrast, a clear understanding of such interactions may provide the basis for the development of more environmentally acceptable methods of parasite control.     

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.     

Enterotoxin residues determining T-cell receptor V beta binding specificity.

Superantigens such as the staphylococcal enterotoxins bind to major histocompatibility complex (MHC) class II molecules and activate T cells through a specific interaction between the V beta region of the T-cell antigen receptor (TCR) and the toxin. The TCR beta-chain alone is sufficient to produce the interaction with the enterotoxin-class II complex. Identification of the regions of enterotoxins that interact with TCR has so far proved equivocal because of difficulties in distinguishing between direct effects on T-cell recognition and indirect effects resulting from alteration of binding to class II. For example, amino-terminal truncations of SEB abrogated T-cell stimulation whereas carboxy-terminal truncation of SEA stopped its mitogenic activity. The most comprehensive study to date, accounting for both enterotoxin binding to class II and enterotoxin interactions with the TCR, identified two functionally important regions for SEB binding to TCR. Although the amino-acid sequences of staphylococcal enterotoxins A and E are 82% identical, they activate T cells bearing different V beta elements. We have assayed the binding of cells coated with these enterotoxins to soluble secreted TCR beta-chain protein and find that V beta 3 binds enterotoxin A but not E, whereas V beta 11 binds enterotoxin but not A. To map the amino-acid residues responsible for these different binding specificities, we prepared a series of hybrids between the two staphylococcal enterotoxins. We report that just two amino-acid residues near the carboxy terminus of the enterotoxins are responsible for the discrimination between these molecules by V beta 3 and V beta 11.(ABSTRACT TRUNCATED AT 250 WORDS)