Efrapeptin prevents modification by phenylglyoxal of an essential arginyl residue in mitochondrial adenosine triphosphatase.

Studies of phenylglyoxal incorporation by beef-heart mitochondrial ATPase reveal one fast-reacting arginyl residue/enzyme molecule. Modification of this group proceeds at a rate which parallels the loss of enzymatic activity. Efrapeptin protects the arginyl residue from reaction with phenylglyoxal. The data suggest that efrapeptin binds at the catalytic site and blocks accessibility of an essential arginine at the adenine nucleotide binding site. The detection of a single, fast-reacting, essential arginine on an enzyme with multiple copies of the catalytic subunit, provides further evidence in support of the alternating site mechanism for ATP synthesis proposed by Kayalar et al. (Kayalar, C., Rosing, J., and Boyer, P.D. (1977) J.Biol. Chem. 252, 2486--2491).     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Increased Resistance to ABT-378, a Novel Protease Inhibitor

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 μM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P₁′ residue Leu to Phe) and p7/p1 (P₂ residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 μM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 μM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763–3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662–6670, 1997).     

3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity.

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.