New semidominant mutations that affect mouse development

Dominantly acting mutations that produce visible phenotypes are frequently recovered, either during routine maintenance of colonies or from mutagenesis experiments. We have studied 12 dominant mouse mutations that cause a tail dysmorphology, a coat spotting phenotype, or a combination of these. The majority of these mutations act in a semidominant manner with the homozygous state associated with embryonic lethality and a visible phenotype at or before midgestation. The homozygous phenotypes include axis truncation and neural crest cell defects, as may be expected from the heterozygous phenotypes. The majority of mutations, however, also produced other phenotypes that include neural tube closure defects and aberrant heart looping. In one coat spotting mutant the homozygous condition is lethal before neural crest cell production commences. The mutated genes often function in processes additional to those alluded to by the heterozygous phenotype.     

Aurora B regulates MCAK at the mitotic centromere

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.     

Double-stranded DNA bacteriophage prohead protease is homologous to herpesvirus protease

Double-stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre-formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid. A virus-encoded prohead or procapsid protease is often required in this maturation process. Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily. We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria-encoded hypothetical proteins. On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis. Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.     

Well-being: towards an integration of psychology, neurobiology and social science

Well-being: towards an integration of psychology, neurobiology and social science     

Bicyclic peptidomimetic tetrahydrofuro[3,2-b]pyrrol-3-one and hexahydrofuro[3,2-b]pyridine-3-one based scaffolds: synthesis and cysteinyl proteinase inhibition

A stereoselective synthesis of (3aS,6aR)-tetrahydrofuro[3,2-b]pyrrol-3-ones and (3aS,7aR)-hexahydrofuro[3,2-b]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13. A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis-fused geometry in comparison to that of the corresponding trans-fused. Since the bridgehead stereocentre situated beta to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10, 45a-e, 11 and 46. Both series were chirally stable with 5,5-series 10 and 45a-e exhibiting potent in vitro activity against a range of CAC1 cysteinyl proteinases. Compound 10, a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance

We present a novel method for the comparison of multiple protein alignments with assessment of statistical significance (COMPASS). The method derives numerical profiles from alignments, constructs optimal local profile-profile alignments and analytically estimates E-values for the detected similarities. The scoring system and E-value calculation are based on a generalization of the PSI-BLAST approach to profile-sequence comparison, which is adapted for the profile-profile case. Tested along with existing methods for profile-sequence (PSI-BLAST) and profile-profile (prof_sim) comparison, COMPASS shows increased abilities for sensitive and selective detection of remote sequence similarities, as well as improved quality of local alignments. The method allows prediction of relationships between protein families in the PFAM database beyond the range of conventional methods. Two predicted relations with high significance are similarities between various Rossmann-type folds and between various helix-turn-helix-containing families. The potential value of COMPASS for structure/function predictions is illustrated by the detection of an intricate homology between the DNA-binding domain of the CTF/NFI family and the MH1 domain of the Smad family.     

The speed of soil carbon throughput in an upland grassland is increased by liming

In situ (13)C pulse labelling was used to measure the temporal and spatial carbon flow through an upland grassland. The label was delivered as (13)C-CO(2) to vegetation in three replicate plots in each of two treatments: control and lime addition. Harvests occurred over a two month period and samples were taken along transects away from the label delivery area. The (13)C concentration of shoot, root, bulk soil, and soil-respired CO(2) was measured. There was no difference in the biomass and (13)C concentration of shoot and root material for the control and lime treatments meaning that the amount of (13)C-CO(2) assimilated by the vegetation and translocated below ground was the same in both treatments. The (13)C concentration of the bulk soil was lower in the lime treatment than in the control and, conversely, the (13)C concentration of the soil-respired CO(2) was higher in the lime. Unlike the difference in bulk soil (13)C concentration between treatments, the difference in the (13)C concentration of the soil-respired CO(2) was obvious only at the delivery site and primarily within 1 d after labelling. An observed increase in the abundance of mycorrhizal fungi in the lime treatment was a possible cause for this faster carbon throughput. The potential key role of mycorrhizas in the soil carbon cycle is discussed. The importance of a better understanding of soil processes, especially biological ones, in relation to the global carbon cycle and environmental change is highlighted.