Directing phage selections towards specific epitopes

It is possible to direct selections from antibody repertoires displayed on filamentous phage towards unique epitopes on protein antigens by competing with related molecules. A phage display repertoire of human single chain Fvs (scFvs) was panned three times against foetal haemoglobin (HbF). The selection was dominated by one clone with a Kd of 10 nM but yielded at least 17 others, all of which bound HbF but crossreacted with adult haemoglobin (HbA). To direct selection towards HbF-specific epitopes, the repertoire was preincubated with HbA in solution before each panning. Crossreactive scFvs can form complexes with the soluble HbA and thereby be prevented from binding the immobilized HbF. Four clones with preferential binding to HbF emerged under these conditions. One of these (Hb-1), with a Kd of 6 microM, had exquisite specificity for HbF and could distinguish cells expressing HbF from those expressing HbA by immunocytochemistry and flow cytometry. This antibody has an affinity that is 600-fold lower than the dominant crossreactive clone, and so only emerged under conditions of 'competitive deselection'. Thus, competitive deselection is a viable means for directing selections towards useful epitopes. It permits a more effective 'search' of phage display repertoires and allows the emergence of lower affinity clones with useful specificities. These clones may be useful in themselves or may serve as leads for in vitro affinity maturation.     

Clinical evaluation of sodium fluoride chewable tablets in dental caries

Chewable tablets containing low dosage fluoride content were prepared using two varieties of celluloses and their in vitro parameters were evaluated. An eighteen month clinical trial revealed that both these formulations were effective in controlling the caries. However, ethyl cellulose is proved to be superior to methylcellulose as a controlled release matrix material in controlling caries. Thus this study recommends ethylcellulose matrix tablets containing low fluoride content is an efficacious and cost effective drug device in controlling dental caries.     

Indirect mitogenic effect of transforming growth factor-beta on cell proliferation of subconjunctival fibroblasts

Single amino acid replacement analogs of Manduca adipokinetic hormone (M-AKH) pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH2 were tested for activity in bioassays as well as receptor binding assays. Amino acids were replaced by Ala and by D-analogs. In addition an extended M-AKH and analogs containing photo affinity labels were tested. All analogs had reduced activity. All the peptides which had enough activity to allow a full dose response curve reached the same maximal activity as native M-AKH. The use of analogs, in which L-Phe4 was replaced by Ala or by D-Phe and of L-Thr3 replaced by D-Thr, as competitors led to improved binding of M-AKH in our competitive receptor binding assay. In the bioassay an inactive concentration of Ala4 M-AKH increased the activity of a half optimal concentration of native M-AKH.     

Perforation repair comparing mineral trioxide aggregate and amalgam using an anaerobic bacterial leakage model

The purpose of this study was to evaluate the ability of mineral trioxide aggregate (MTA) and amalgam to seal furcal perforations in extracted human molars using an anaerobic bacterial leakage model. Furcal perforations were made in 39 maxillary and mandibular human molars with a high-speed bur. These were randomly divided into two experimental groups of 18, with the remaining three teeth used as positive controls. Experimental group 1 was repaired with MTA and group 2 with amalgam. Three additional teeth without perforations served as negative controls. A dual chamber anaerobic bacterial leakage model was assembled. Brain heart infusion broth with yeast extract, hemin, menadione, and the chromogenic indicator bromcresol purple was used as the culture broth for Fusobacterium nucleatum. Eight of 18 amalgam samples leaked, whereas none of the 18 MTA samples leaked. MTA was significantly better than amalgam in preventing leakage of F. nucleatum past furcal perforation repairs.     

Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects

To define the mechanism of insulin's anticatabolic action, the effects of three different dosages of insulin (0.25, 0.5, and 1.0 mU x kg(-1) x min(-1)) versus saline on protein dynamics across splanchnic and skeletal muscle (leg) beds were determined using stable isotopes of phenylalanine, tyrosine, and leucine in 24 healthy subjects. After an overnight fast, protein breakdown in muscle exceeded protein synthesis, causing a net release of amino acids from muscle bed, while in the splanchnic bed protein synthesis exceeded protein breakdown, resulting in a net uptake of these amino acids. Insulin decreased (P < 0.003) muscle protein breakdown in a dose-dependent manner with no effect on muscle protein synthesis, thus decreasing the net amino acid release from the muscle bed. In contrast, insulin decreased protein synthesis (P < 0.03) in the splanchnic region with no effect on protein breakdown, thereby decreasing the net uptake of the amino acids. In addition, insulin also decreased (P < 0.001) leucine nitrogen flux substantially more than leucine carbon flux, indicating increased leucine transamination (an important biochemical process for nitrogen transfer between amino acids and across the organs), in a dose-dependent manner, with the magnitude of effect being greater on skeletal muscle than on the splanchnic bed. In conclusion, muscle is in a catabolic state in human subjects after an overnight fast and provides amino acids for synthesis of essential proteins in the splanchnic bed. Insulin achieves amino acid balance across splanchnic and skeletal muscle beds through its differential effects on protein dynamics in these tissue beds.