Effects of substitutions in the binding surface of an antibody on antigen affinity

The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and scFv-neuraminidase complexes. To investigate the contribution to binding made by cavities, salt links and hydrogen bonds in the antibody- antigen interface, 14 single amino acid replacements were made at six contact residues in the scFv fragment by site-directed mutagenesis. The binding affinity of each mutant scFv antibody for neuraminidase was determined with a BIAcore optical biosensor. Four of the mutations resulted in large changes in the free energy of binding to neuraminidase (deltadeltaG > 1 kcal/mol) and together may account for approximately 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link between antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was demonstrated to be important for affinity, since substitution of (Asp)H56 with Asn caused a large reduction in the free energy of binding (deltadeltaG = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (deltadeltaG = +1.7 kcal/mol). Disruption of hydrophobic interactions (van der Waals contacts) led to significant reductions in affinity also ((Tyr)H99 to Ala, deltadeltaG = +1.5 kcal/mol; (Leu)L94 to Ala, deltadeltaG > +3.0 kcal/mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as the free energy gained by new antibody-antigen interactions did not compensate for the removal of cavity-bound water molecules.      

APIC position paper: hepatitis C exposure in the health care setting. Association for Professionals in Infection Control and Epidemiology, Inc

The Association for Professionals in Infection Control and Epidemiology, Inc (APIC), is a multidisciplinary, voluntary, international organization of professionals who practice infection control and the application of epidemiology in all health settings. APIC is an international leader in prevention and control of infection transmission.     

The venotonic drug hydroxyethylrutosiden does not prevent or reduce docetaxel-induced fluid retention: results of a comparative study

PURPOSE: Fluid retention, which includes peripheral edema, ascites, pleural or pericardial effusion, or a combination of these that is sometimes associated with significant weight gain, is one of the most troublesome cumulative side effects of docetaxel. A suggestive observation from the data base available at the manufacturer (Rhone-Poulenc Rorer) was that patients who received venotonic drugs appeared to tolerate more courses of docetaxel. This prompted a comparative study to investigate whether the venotonic drug hydroxyethylrutosiden could reduce or delay docetaxel-related fluid retention. METHODS: A total of 85 patients with metastatic breast cancer who were treated with docetaxel at a dose of 100 mg/m2 with corticoid comedication were allocated to receive either 300 mg hydroxyethylrutosiden given orally four times daily (group A) or no hydroxyethylrutosiden (group B). The end point for analysis was the development of fluid retention of > or = grade 2. RESULTS: Fluid retention of > or = grade 2 was reported in 14 of 42 patients (33%) in group A and in 15 of 43 patients (35%) in group B and occurred after a median of 4 cycles of docetaxel in both groups. Weight gain was similar in groups A and B. CONCLUSION: We conclude that hydroxyethylrutosiden does not reduce or delay the incidence and severity of docetaxel-related fluid retention.     

Dental microwear of Griphopithecus alpani

The examination of microscopic dental wear allows inferences to be made about diet in extinct species. This study reconstructs the diet of Griphopithecus alpani, a 15 Ma fossil hominoid from the Miocene site of Pa?alar in north-western Turkey, using scanning electron microscopy (SEM) to examine the microscopic wear on its molar teeth. The microwear patterns of Griphopithecus are compared with those of three extant hominoid taxa-Gorilla gorilla gorilla, Pan troglodytes verus, and Pongo pygmaeus pygmaeus. The microwear on three occlusal wear surfaces is examined in this study, and sex and age differences are also included. Analysis of variance is performed on the following microwear variables-feature density, pit density, striation density, the ratio of pits relative to striations, pit widths, and striation widths. Griphopithecus has significantly higher microwear feature densities and higher percentages of pits than Gorilla. It also has larger pit frequencies and narrower striations than both Pan and Gorilla. There are no significant differences between the microwear patterns of Griphopithecus and Pongo. This suggests that the diet of Gripho-pithecus was more similar to that of Pongo, which consumes mainly fruit, and occasionally hard and unripe fruits and nuts, than to that of Pan and Gorilla. In addition, the high percentage of pits displayed by Griphopithecus may indicate that it was ingesting harder fruits and/or objects than the extant hominoids, although this is not a significant difference. There also are consistent variations in the microwear present on the three different wear facets examined in the study-Phase II facets display more microwear and pitting than the Phase I surface examined. The results of this study do not indicate variation in dental microwear according to sex or age.     

Molecular cloning of mouse glycolate oxidase. High evolutionary conservation and presence of an iron-responsive element-like sequence in the mRNA

Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells.     

A hermeneutic analysis of the process of conducting clinical interviews

1. Although no right way to interview exists, an interviewer should develop a comfortable, personal style. 2. Allowing an interview to follow its own course while remembering the overall interview agenda will contribute to quality of the data. 3. Interviewing skills are enhanced as the interviewer develops a personal style and becomes comfortable as a practitioner.     

Prognostic significance of tumour markers in endometrial cancer

BACKGROUND: Steroid 5 alpha-reductase is implicated in the pathogenesis of benign prostatic hyperplasia (BPH). We studied the in vitro and in vivo effects of FR146687, a new inhibitor of 5 alpha-reductase. METHODS: Two isozymes of rat and human 5 alpha-reductases were expressed in 293 cells. In vivo effects of drugs were evaluated on rat and dog prostates. Castrated immature rats were injected with testosterone propionate (TP) or 5 alpha-dihydrotestosterone propionate (DHTP) to induce growth of the ventral prostates. Testosterone and 5 alpha-dihydrotestosterone (DHT) contents in rat and dog prostates were measured by gas chromatography-mass spectrophotometry (GC-MS). RESULTS: FR146687 showed noncompetitive inhibition in both isozymes and no inhibitory effects on other steroid oxidoreductases. In mature rats and castrated immature rats treated with TP, FR146687 dose-dependently reduced ventral prostate and seminal vesicle weight at doses above 0.1 mg/kg, while castrated immature rats treated with DHTP were not affected by FR146687. FR146687 showed more potent reduction of rat prostates than finasteride. DHT concentration in the prostates was significantly reduced when FR146687 was administered to rats and beagles. CONCLUSIONS: FR146687 is a dual inhibitor for 5 alpha-reductase isozymes and significantly reduced the growth and DHT content in the prostate.     

Compartment-ablation studies of GLUT4 distribution in adipocytes: evidence for multiple intracellular pools

The available data suggest that GLUT4 does populate the recycling endosomal system to some extent, but that a large proportion of the intracellular GLUT4 resides in a compartment that is devoid of transferrin receptors and may have properties more akin to specialized secretory vesicles. The study of the nature and biogenesis of this compartment will provide important insight into the mechanism by which insulin stimulates glucose transport. Further study of the role of the synaptobrevins in these distinct subcellular compartments will probably shed further light on the mechanism by which insulin stimulates GLUT4 translocation.