ATR-FTIR difference spectroscopy of the P(M) intermediate of bovine cytochrome c oxidase

Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic P(M) intermediate. The transition was induced in a film of detergent-depleted 'fast' oxidase with a buffer containing CO and O(2). The extent of formation of the P(M) state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The P(M) minus O FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a(3) and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds.     

Genetic modulation of PPARgamma phosphorylation regulates insulin sensitivity

Obesity-associated diabetes is epidemic in industrialized societies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in adipose tissue and the presumed molecular target for antidiabetic thiazolidinedione drugs that reverse insulin resistance but also promote weight gain. Phosphorylation reduces the activity of PPARgamma in vitro, but physiological relevance has not been demonstrated. We have studied mice homozygous for a mutation (S112A) that prevents PPARgamma phosphorylation. Surprisingly, the weights and adipose mass of PPARgamma-S112A mice are not greater than wild-type. Remarkably, however, genetic prevention of PPARgamma phosphorylation preserves insulin sensitivity in the setting of diet-induced obesity. Underlying this protection are smaller fat cells, elevated serum adiponectin, and reduced free fatty acid levels. Thus, the phosphorylation state of PPARgamma modulates insulin sensitivity. Compounds that prevent PPARgamma phosphorylation or ligands that induce the conformation of nonphosphorylated PPARgamma may selectively enhance insulin sensitivity without increasing body weight.     

Cotton rat<Emphasis Type="Italic"> Sihi-</Emphasis>M3 is a minimally oligomorphic<Emphasis Type="Italic"> Mhc</Emphasis> I-b molecule that binds the chemotactic peptide fMLF under stringent conditions

The leading model for class I-b evolution suggests non-polymorphic I-b genes evolve by gene duplication from polymorphic I-a genes. We recently found N-formyl peptide-specific orthologs of the class I-b gene H2-M3 in the rodent subfamily Sigmodontinae. To test if sigmodont M3 is a I-b gene, we sequenced M3 from wild cotton rats (Sigmodon hispidus) diverse at the class II locus, Sihi-DQA. These haplotypes carry a single allele of M3 that closely resembles H2-M3. However, peptide-binding assays showed that cotton rat M3 bound the chemotactic N-formylpeptide fMLF better than did rat or mouse M3. The Ala116Lys substitution in cotton rat M3 might enhance binding of fMLF and is one of eight residues of M3 that interact with ligand residues P3 and P4 and that are positively selected, with a d_N/d_S ratio of 1.8. Thus, M3 is a class I-b gene in both sigmodontine and murine murids, but positive selection operates on a small subset of residues in the traditionally defined antigen recognition site.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.     

Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2

Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for –1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem–loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag–pol and HTLV-2 gag–pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem–loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication.     

The origin of antigenic diversity in Plasmodium falciparum

Most studies of genetic variability of Plasmodium falciparum have focused on protein antigens and the genes that encode them. The consensus is that populations exhibit high levels of genetic polymorphism, most notably the genes encoding surface proteins of the merozoite (Msp1, Msp2) and the sporozoite (Csp). The age and derivation of this variation is a subject that warrants further careful consideration, as discussed here by Stephen Rich, Marcelo Ferreira and Francisco Ayala.     

Survey of the 1999 surface plasmon resonance biosensor literature

The application of surface plasmon resonance biosensors in life sciences and pharmaceutical research continues to increase. This review provides a comprehensive list of the commercial 1999 SPR biosensor literature and highlights emerging applications that are of general interest to users of the technology. Given the variability in the quality of published biosensor data, we present some general guidelines to help increase confidence in the results reported from biosensor analyses.     

The reactions of hydrogen peroxide with bovine cytochrome c oxidase

Oxidised cytochrome c oxidase is known to react with two molecules of hydrogen peroxide to form consecutively 607 nm 'Peroxy' and 580-nm 'Ferryl' species. These are widely used as model compounds for the equivalent P and F intermediates of the catalytic cycle. However, kinetic analysis of the reaction with H(2)O(2) in the pH range 6.0-9.0 reveals a more complex situation. In particular, as the pH is lowered, a 580-nm compound can be formed by reaction with a single H(2)O(2). This species, termed F(&z.rad;), is spectrally similar, but not identical, to F. The reactions are equivalent to those previously reported for the bo type quinol oxidase from Escherichia coli (T. Brittain, R.H. Little, C. Greenwood, N.J. Watmough, FEBS Lett. 399 (1996) 21-25) where it was proposed that F(&z.rad;) is produced directly from P. However, in the bovine oxidase F(&z.rad;) does not appear in samples of the 607-nm form, P(M), produced by CO/O(2) treatment, even at low pH, although this form is shown to be identical to the H(2)O(2)-derived P state, P(H), on the basis of spectral characteristics and kinetics of reaction with H(2)O(2). Furthermore, lowering the pH of a sample of P(M) or P(H) generated at high pH results in F(&z.rad;) formation only on a minutes time scale. It is concluded that P and F(&z.rad;) are not in a rapid, pH-dependent equilibrium, but instead are formed by distinct pathways and cannot interconvert in a simple manner, and that the crucial difference between them lies in their patterns of protonation.     

Evidence for the presence of a component of the Mn complex of the photosystem II reaction centre which is exposed to water in the S(2) state of the water oxidation complex

The interaction of water oxidising photosystem II preparations with the aqueous environment has been investigated using electron spin echo envelope modulation spectroscopy in the presence of 2H(2)O. The spectra show interaction of 2H of 2H(2)O with the preparation in the S(2) state. The component interacting with water decays during 1-4 weeks storage at 77 K. No interaction of water with the classical multiline S(2) Mn signal, which is more stable on storage at 77 K, was detected. The results show that a component of the water oxidation complex, possibly involving the Mn centre, is accessible to water and may be the water binding site for photosynthetic water oxidation.