Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein

The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined by automatic sequence analysis of the intact protein and of peptides isolated from tryptic and BNPS-skatole digests. This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. There is sufficient sequence homology between MGP and bone Gla protein (BGP) to indicate that these two bovine bone proteins arose by gene duplication and subsequent divergent evolution. Although MGP has a very low solubility in water compared to BGP, there is no hydrophobic domain in MGP which could account for its insolubility, and the overall fraction of hydrophobic residues is 32% for MGP compared to 43% for BGP. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues between the putative targeting domain for the gamma-carboxylase in the MGP leader sequence and the mid-molecule Gla residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation.     

Darwinian gradualism and its limits: The development of Darwin's views on the rate and pattern of evolutionary change

Conclusion The major tenets of the recent hypothesis of punctuated equilibrium are explicit in Darwin's writing. His notes from 1837–1838 contain references to stasis and rapid change. In the first edition of the Origin (1859), Darwin described the importance of isolation of local varieties in the process of speciation. His views on the tempo of speciation were influenced by Hugh Falconer and also, perhaps, by Edward Suess (1831–1914). It is paradoxical that, although both topics were recorded in his unpublished notes of 1837–1838, the second was not explicitly and fully discussed until the fourth edition of the Origin (1866). While no wholly satisfactory explanation of this paradox suggests itself, it seems probable that Falconer's work on the persistence of fossil species of elephant helped Darwin to see the wider significance of the tempo of evolution for his general theory.     

Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Effects of dexamethasone on carnitine metabolism in liver and extrahepatic tissues

The in vivo effects of dexamethasone administration on liver and extrahepatic tissue carnitine concentrations were assessed in 48-h-starved rats. In heart and kidney, but not in liver, dexamethasone significantly increased total carnitine concentration. Acute (2.5 h) treatment with 2-tetradecylglycidate (TDG), a specific inhibitor of carnitine palmitoyl transferase 1, not only increased total hepatic carnitine concentrations, but also permitted an effect of dexamethasone (a further increase in hepatic carnitine concentration). The results are discussed in terms of acute (substrate-mediated) and chronic (hormonal) control of carnitine turnover.     

The cDNA cloning and immunological characterization of hamster p53.

We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.     

Exposure to nuclear magnetic resonance imaging procedure attenuates morphine-induced analgesia in mice

Adult male mice exposed to a Nuclear Magnetic Resonance Imaging (NMRI) procedure during the mid-dark period and injected with morphine (10 mg/kg) failed to exhibit the normal nocturnally enhanced morphine analgesia response to a thermal stimulus that was displayed by mice exposed to a sham imaging procedure and treated with morphine (p less than .01). When tested during the mid-light period, animals exposed to the NMRI procedure and given morphine displayed attenuated analgesia levels relative to sham exposed mice (p less than .01) treated with morphine. However, the morphine induced analgesia was not totally abolished since the imaged mice still exhibited analgesia relative to saline treated mice (p less than .01). These results suggest that the magnetic and/or radio-frequency fields associated with the NMRI procedure alter both day- and night-time responses to morphine. These results may reflect magnetic field induced alterations in neuronal calcium binding and/or alterations in nocturnal pineal gland activity.     

Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.     

Spatial frequency tuned channels: implications for structure and function from psychophysical and computational studies of stereopsis

Various psychophysical experiments investigating the role of spatial frequency tuned channels in stereopsis are reviewed and a computational model of stereopsis deriving from these studies is described. The distinctive features of the model are: (1) it identifies edge locations in each monocular field by searching for zero crossings in non-orientated centre-surround convolution profiles; (2) it selects among all possible binocular point-for-point combinations of edge locations only those which satisfy a (quasi-) collinear figural grouping rule; (3) it presents a concept of the orientated and spatial frequency tuned channel as a nonlinear grouping operator. The success of the model is demonstrated both on a stereo pair of a natural scene and on a random-dot stereogram.     

Cloning and sequence analysis of two embryonic beta-like globin cDNAs (y and z) of hamster.

A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC.