Effect of various concentrations of sodium hypochlorite on primary dentin: an in vitro scanning electron microscopic study.

BACKGROUND: Sodium hypochlorite solutions have been evaluated for their effects in bonding procedures as they are found to deplete or remove the organic portion of the dentin, particularly the collagen fibrils. The aim of this in vitro study was to assess and compare the efficacies of 1%, 2.5%, 5% and 10% NaOCl at 30, 60 and 120s on etched primary dentin. METHODS: 84 primary anterior teeth were ground to expose a flat dentin area on the buccal surface. The specimens were divided into fourteen groups of six each based on the dentin surface treatment (35% phosphoric acid etching for 7 seconds-AE and/or NaOCl application), NaOCl solution concentrations (1%, 2.5%, 5% and 10%) and time of application (0, 30, 60 and 120s). Specimens were prepared for SEM and photomicrographs were taken of the surface and were scored against a five point scale, based on the smear layer and amount of collagen removed. The scores were submitted to Kruskal-Wallis and Mann Whitney tests. RESULTS: This study showed the presence of smear layer in the control group. The group treated with Acid Etchant showed a demineralized pattern of dentin with exposure of dentin tubules and collagen fibrils network on the intertubular and peritubular dentin which was not significantly different from the groups treated with 1% and 2.5% NaOCl. Groups treated with 5% NaOCI were not statistically different from each other the surface was corroded but collagen fibrils were not completely depleted. Groups treated with 10% NaOCl were not statistically different from each other and showed complete removal of collagen fibrils with wider tubular apertures and several secondary tubules on peritubular and intertubular dentin. CONCLUSION: Higher concentrations of NaOCl solutions (5% and 10%) produced significant changes in the etched primary dentin. The higher the concentration of the NaOCI solution, the lower can be the time for the application of the solution for the complete removal of collagen fibrils.

Functional regulation of PVBV Nuclear Inclusion protein-a protease activity upon interaction with Viral Protein genome-linked and phosphorylation.

Regulation of NIa-Pro is crucial for polyprotein processing and hence, for successful infection of potyviruses. We have examined two novel mechanisms that could regulate NIa-Pro activity. Firstly, the influence of VPg domain on the proteolytic activity of NIa-Pro was investigated. It was shown that the turnover number of the protease increases when these two domains interact (cis: two-fold; trans: seven-fold) with each other. Secondly, the protease activity of NIa-Pro could also be modulated by phosphorylation at Ser129. A mutation of this residue either to aspartate (phosphorylation-mimic) or alanine (phosphorylation-deficient) drastically reduces the protease activity. Based on these observations and molecular modeling studies, we propose that interaction with VPg as well as phosphorylation of Ser129 could relay a signal through Trp143 present at the protein surface to the active site pocket by subtle conformational changes, thus modulating protease activity of NIa-Pro.

Foreign body ingestion in dental practice.

Most foreign bodies pass through the gastrointestinal tract uneventfully. The majority of the reported literature describes the management of ingested blunt objects. However, ingestion of sharp objects can still occur with a higher rate of perforation corresponding to treatment dilemmas. We report a case of inadvertently ingested sharp foreign body by a special child, which was retrieved by endoscopic guided forceps. Urgent endoscopic assessment and retrieval of recently ingested sharp dental foreign body is indicated and routine use of preventive measures such as rubber dam, gauze throat screens or floss ligatures is suggested.

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab).

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.

Antigenic determinants and reactive sites of a trypsin/chymotrypsin double-headed inhibitor from horse gram (Dolichos biflorus).

The major proteinase inhibitor in horse gram (Dolichos biflorus) is a low molecular weight (approximately 8500) Bowman-Birk inhibitor (BBI), HGI-III, that inhibits both trypsin and chymotrypsin simultaneously. Analysis of the reactivity of the polyclonal antibodies raised against native HGI-III, with tryptic, lysylendoproteinase-C and CNBr peptides, in dot-blot assays, revealed the presence of three sequential epitopes (Asp1-Lys14 (I), Leu37-Lys63 (II) and Asp64-Lys71 (III)). Of these, epitope II and III occur consecutively in the sequence of HGI-III. The reactive site peptide bonds were identified by cleavage with catalytic quantities of either trypsin or chymotrypsin at acidic pH. The reactive site peptide bond for trypsin was found to be Lys24-Ser25, whereas for chymotrypsin it was Phe51-Ser52. The highly conserved reactive site loop residues of the Bowman-Birk inhibitors are also conserved in HGI-III. The less immunogenic peptide sequence, Leu37-Lys63, also contains the chymotrypsin reactive site. The recognition of this polypeptide by the immune system provides for a new strategy in the design of ideal, smaller proteinase inhibitors as cancer preventive agents.

Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.

Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors.

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that of Macrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

The complete primary structure of a unique mannose/glucose-specific lectin from field bean (Dolichos lab lab).

The complete amino acid sequence of two non-identical subunits of the glucose/mannose-specific lectin from Dolichos lab lab (field bean) has been determined by sequential Edman analyses of the intact subunits and peptides derived by enzymatic and chemical cleavage. Peptides were purified by reverse phase high performance liquid chromatography and ion pair chromatography. The D. lab lab lectin is a glycoprotein having two polypeptide chains of 132 and 105 amino acid residues. The amino acid sequence of the D. lab lab lectin is compared with the various lectins of the family Leguminosae. The D. lab lab lectin is the only species of the tribe Phaseoleae that contains two nonidentical subunits of almost equal size and that shows a specificity to glucose/mannose. The lectin shows a greater homology to the glucose/mannose-specific lectins, especially concanavalin A. The unique subunit architecture of the D. lab lab lectin indicates the presence of new post-translational cleavage sites.

Detergent permeabilized yeast cells as the source of intracellular enzymes for estimation of biomolecules.

The feasibility of using permeabilized whole cells as a source of intracellular enzymes instead of isolated expensive enzymes for the estimation of biomolecules has been studied. Alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), and beta-galactosidase (beta-GAL) activities of cetyltrimethylammonium bromide (CTAB)-permeabilized whole yeast cells were employed to estimate ethyl alcohol, glucose, and lactose. The method using permeabilized cells was comparable to that of isolated enzymes and was applicable for the estimation of these analytes in complex samples such as blood, milk, and fermented samples. The usefulness of permeabilized cells as a single source of more than one enzyme required for coupled enzyme assays was demonstrated.

Preparation of NADH/NADPH using cetyltrimethylammonium bromide permeabilized baker's yeast cells.

Alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) activities of cetyltrimethylammonium bromide permeabilized baker's yeast whole cells were employed to prepare reduced nicotinamide nucleotides NADH and NADPH from their corresponding oxidised forms. Both NADH and NADPH were found to be stable in the presence of permeabilized cells under the conditions of preparation. No dephosphorylation of NADP+ to NAD+ or of NADPH to NADH was found. Reduction is complete and the prepared NADH and NADPH are chromatographically pure. Since readily available Baker's yeast cells were used instead of expensive isolated enzyme the method described here is simple, economical, and easy to scale up.

In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane.

The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin. The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells. The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min. The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.

Population Genetics of DROSOPHILA NASUTA NASUTA, DROSOPHILA NASUTA ALBOMICANA and Their Hybrids. I. Karyotypic Mosaicism in the Hybrid Populations.

D. n. nasuta and D. n. albomicana constitute a pair of chromosomal races with 2n=8 and 2n=6, respectively. The F(1) of these has 2n=7 and it is fertile. There exists a state of karyotypic mosaicism as evidenced by the presence of 26 types of chromosome combinations in F(2), F(3) and F(10) populations. In the midst of this karyotypic noise, the karyotype similar to that of F(1) reached 51% of the population. Implications of these findings are discussed.