IRAG working group 4. Cell cytotoxicity assays. Interagency Regulatory Alternatives Group.

Twenty-seven data sets from 12 cellular cytotoxicity assays, intended to predict ocular irritation, were submitted to the Interagency Regulatory Alternatives Group (IRAG) for review. These data consisted of paired in vivo (Draize) and in vitro responses to individual chemicals and formulations. In vivo data consisted of individual tissue scores so that the predictive value of the in vitro assay could be assessed for each tissue response normally measured in the standard Draize assay. Data were compiled and evaluated according to the IRAG Guidelines Document. The Pearson's linear correlation coefficient was used as the first step in assessing the relationship between the in vitro and in vivo responses. The majority of the data sets represented the study of surfactant-based materials. In many cases, there was good correlation between the in vitro scores and the in vivo tissue responses. Most pronounced were the particularly good correlations between the in vitro scores and conjunctival redness scores across most of the assays. Based on the data submitted, a number of the cell cytotoxicity assays show considerable promise as screens for ocular irritancy. None of the submitters recommended that their cell cytotoxicity assay be used as a sole replacement for in vivo assessment. For almost all of these assays, the materials being tested should be water-soluble/miscible. The toxicity of products with reserve acidity or alkalinity or with high reactivity may be underestimated. A given user may prefer certain assays depending on the types of materials to be tested, the expected range of toxicities and the resources available. The cell cytotoxicity assays can serve as a valuable component of a tiered or battery testing program. As with any assay, a sufficient number of replicate values, concurrent positive and negative controls, and a strict adherence to assay acceptance criteria are essential to produce credible data.

Quantitative and qualitative video analysis of infants feeding: angled- and straight-bottle feeding systems.

OBJECTIVE: To determine whether an angled-bottle feeding system is more effective than a straight-bottle feeding system in facilitating the medically recommended semiupright infant posture and to determine whether an angled bottle promotes improved ergonomic alignment and comfort for the feeder. STUDY DESIGN: On two consecutive days, 23 pairs of parent-infant teams, seated in an ergonomic chair, were videotaped at the same time of day, during which infants were fed by their parents with either an angled or a straight bottle (presented in balanced order) fitted with the infants' regular nipple. Infants were given their regular type and amount of formula. RESULTS: The angled bottle provided a higher level of satisfaction compared with the straight bottle. Less gastric discomfort occurred with the angled bottle versus the straight bottle when each was compared with the bottle used at home. In addition, feeders assumed ergonomically sound positions. CONCLUSION: This study indicates that an angled bottle is preferable to a straight bottle because it encourages more physiologic positioning of the infant, improves the comfort level of the feeder, improves ergonomic feeding performance, and decreases the need for burping.

Control of RNA and protein synthesis by the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2.

The effect of varying the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2 has been studied by varying the amount of exogenously added tryptophan (Trp) to cells bearing a mutation which results in a tryptophanyl-tRNA synthetase with a higher Km value for Trp. The phenotype of the mutant has been confirmed by measuring the level of tRNATrp which can be aminoacylated in vivo, and the mutant has also been shown to have elevated tRNATrp levels compared to wild-type. The growth of MS2 decreases continuously with decreasing Trp concentration (and hence, decreasing Trp-tRNATrp concentration). This appears to be due to reduced gene expression, since at later times in infection the amount of MS2 coat protein synthesized likewise decreases continuously with decreasing Trp concentration. However, there is little decrease in the amount of coat protein or replicase synthesized during the first few minutes after the Trp concentration shift. A continuous increase in the average polysome size distribution is seen as the Trp concentration is decreased. MS2 RNA synthesis also decreases continuously with decreasing Trp concentration, and is shut off in the absence of Trp. This does not seem to be due to ppGpp as these cells are functionally relaxed under these conditions, nor does it seem to be due to degradation of pre-existing template. Addition of chloramphenicol abolishes the effect of Trp concentration on RNA synthesis. The data are consistent with a model in which ribosomes pause at Trp codons in the absence of Trp-tRNATrp, while other ribosomes queue behind and continue to load onto the message. The reduction of RNA synthesis would then be a consequence of coupling to translation.

A covalent adduct between the uracil ring and the active site of an aminoacyl tRNA synthetase.

A covalent adduct of an aminoacyl tRNA synthetase and uracil nucleoside has been isolated. The enzyme adduct is catalytically inactive; one nucleoside is bound per catalytic site. The release of uridine restores enzyme activity. The nucleoside attaches to a protein segment required for tRNA interaction. The findings add support to concepts of a covalent component for some protein-nucleic acid complexes.

Coenzyme binding site of glutamate decarboxylase.

Dissociation constants have been measured for the binding of a variety of simple analogues of pyridoxal 5'-phosphate to apoglutamate decarboxylase. Compounds studied have a simple alkyl or aryl group and a negatively charged substituent (phosphate, phosphonate, phosphoramidate, sulfate, sulfonate, or carboxylate). Optimum binding to the phosphate binding site of the enzyme is achieved by compounds having a double negative charge and a tetrahedral geometry. Planar anions and monoanions bind considerably less well. These and previous data are used to to derive the magnitudes of the contributions of various coenzyme functional groups to the strength of the apoenzyme-coenzyme interaction.

Aminoacyl-tRNA synthetase-catalyzed cleavage of the glycosidic bond of 5-halogenated uridines.

Because of previous data suggesting that aminoacyl-tRNA synthetases make a transient Michael adduct with a specific uridine residue in the tRNA structure, (Schoemaker, H.J.P., and Schimmel, P.R. (1977) Biochemistry 16, 5454-5460) attempts were made to find simple model systems in which this reaction might be studied in more detail. In the course of these investigations, it was found that Escherichia coli Ile-tRNA synthetase catalyzes cleavage of the glycosidic bond of 5-bromouridine. At pH 7.5, ambient temperatures, the turnover number is roughly 5/h. 5-Fluoro-, 5-chloro-, and 5-iodouridine are also cleaved in an analogous way by Ile-tRNA synthetase. In the case of uridine, conversion of uridine to uracil and ribose was also detected, but with a smaller turnover number. Three other E. coli and one mammalian aminoacyl-tRNA synthetases were also examined and all were found to catalyze glycosidic bond cleavage of 5-bromouridine. The data indicate that, in general, synthetases have a catalytic center that shows an unusual reactivity for uridine.