Craniodental variation in the African macaque, with reference to various Asian species

Based on twenty-seven craniodental measurements and ratios derived from them, the relationship between the African macaque (M. sylvanus) and the others in Asia were examined with principal components analyses (PCA) and Euclidean distance analysis based upon prior discriminant function analyses (DFA). Results based on analyses of raw measurements indicate that the variation between species lies in the first axis of PCA; the species are dispersed according to their differences in size. The variation between sexes (sexual dimorphism) lies in the second axis. In the analyses of ratio variables, though these two patterns of separation remain orthogonal, they lie at approximately forty-five degrees to each axis. Variables relating to anterior teeth were found to play an important role in variation analysis, and this may be related to the special food preferences of these monkeys: more frequent usage of the incisor teeth for processing frugivorous diets than in other primates that are mainly folivorous. The results from Euclidean distance analyses indicate that the average distance of species within the Asian group is shorter than that between Asian and African groups regardless of sex and variable type. In addition the variation between African and Asian groups is larger than that within Asian group. Thus, it is reasonable to suggest that the African macaque has a range of measurements and ratios quite distinct from the species found in Asia (though the greatest separations result from the analyses of ratio data). These results therefore support the view that M. sylvanus may be regarded as an independent species group in the genus Macaca as proposed by Delson [1980].     

Histidine 167 is the phosphate acceptor in glucose-6-phosphatase-beta forming a phosphohistidine enzyme intermediate during catalysis

The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-alpha and the ubiquitous Glc-6-Pase-beta. Both share similar kinetic properties. Sequence alignments predict the two proteins are structurally similar. During glucose 6-phosphate (Glc-6-P) hydrolysis, Glc-6-Pase-alpha, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His(176), which lies on the lumenal side of the ER membrane. We showed that Glc-6-Pase-beta is also a nine-transmembrane domain protein that forms a covalently bound phosphoryl enzyme intermediate during Glc-6-P hydrolysis. However, the intermediate was not detectable in Glc-6-Pase-beta active site mutants R79A, H114A, and H167A. Using [(32)P]Glc-6-P coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in Glc-6-Pase-beta is His(167) and that it lies inside the ER lumen with the active site residues, Arg(79) and His(114). Therefore Glc-6-Pase-alpha and Glc-6-Pase-beta share a similar active site structure, topology, and mechanism of action.     

Biochemical evidence for the presence of a single CD3delta and CD3gamma chain in the surface T cell receptor/CD3 complex

The T cell antigen receptor (TCR) consists of an alphabeta heterodimer and associated invariant CD3gamma, delta, epsilon, and zeta chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCRalpha, TCRbeta, CD3gamma, and CD3delta and two molecules each of CD3epsilon and CD3zeta, is not clearly understood. Although it has been shown that there are two chains of CD3epsilon and CD3zeta, the stoichiometry of CD3gamma or CD3delta chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3delta and CD3gamma were employed to show that the surface TCR complexes contain one molecule each of CD3delta and CD3gamma. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3delta and CD3gamma in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3delta and CD3gamma in the surface TCR/CD3 complex.     

An amperometric glucose biosensor based on poly(o-aminophenol) and Prussian blue films at platinum electrode

Prussian blue (PB), as a good catalyst for the reduction of hydrogen peroxide, has been combined with nonconducting poly(o-aminophenol) (POAP) film to assemble glucose biosensor. Compared with PB-modified enzymatic biosensor, the biosensor based on glucose oxidase immobilized in POAP film at PB-modified electrode shows much improved stability (78% remains after 30 days) in neutral medium. Additionally, the biosensor, at an applied potential of 0.0 V, exhibits other good characteristics, such as relative low detection limit (0.01 mM), short response time (within 5s), large current density (0.28 mA/cm2), high sensitivity (24 mAM(-1)cm(-2)), and good antiinterferent ability. The apparent activation energy of enzyme-catalyzed reaction and apparent Michaelis-Menten constant are 34.2 KJmol(-1) and 10.5 mM, respectively. In addition, effects of temperature, applied potential used in the determination, pH value of the detection solution, and electroactive interferents on the amperometric response of the sensor were investigated and are discussed.     

Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol

Previous work has shown that the endogenous cystathionine γ-synthase (CSE)/hydrogen sulfide (H₂S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H₂S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H₂S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H₂S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H₂S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.     

ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators

The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.     

Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity

TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.     

Epitope scanning reveals gain and loss of strain specific antibody binding epitopes associated with the conversion of normal cellular prion to scrapie prion

We used anti-prion (PrP) monoclonal antibodies (Mabs) in different combinations to scan changes in the availability of antibody binding epitopes--using an epitope scanning assay--in brain homogenates from normal mice, and from mice infected with either ME7 or 139 A strains of infectious scrapie prion (PrPSc). In ME7-infected brains, the epitope detected by the Mab pair 8B4/8H4 is reduced, while the epitope detected by the Mab pair 8F9/11G5 is increased. Mab 8F9/11G5 detect a conformational epitope on PrPSc because the rise in Mab 8F9/11G5 binding is sensitive to a denaturing agent but resistant to proteinase K (PK). While the increase in Mab 8F9/11G5 binding correlates with the presence of PK-resistant PrP and clinical signs of infection, the reduction in Mab 8B4/8H4 binding is detected earlier. Fractionation of the ME7-infected brain homogenate in sucrose gradient revealed that the PrPSc species detected by the epitope scanning assay are heterogeneous in size, with a molecular mass of approximately > or = 2000-kDa. We also investigated whether these findings were applicable to two other strains of PrPSc, namely 87 V and 22 L. We found that the decrease in Mab 8B4/8H4 binding detected in ME7-infected brains was also detected in 87 V-infected brains but not in 22 L-infected brains. In contrast, the increase in Mab 8F9/11G5 binding detected in ME7- and 139 A-infected brains was also detected in 22 L-infected brains but not in 87 V-infected brains. Therefore, each prion strain has its unique conformation, and we can monitor the conversion of normal cellular prion (PrPC) to PrPSc based on the changes in the antibody binding patterns. The epitope can be decreased or increased, linear or conformational, detected late or early during infection, in a strain specific manner.