Quick, painless, and atraumatic gingival retraction: An overview of advanced materials.

The success of any fixed prosthesis depends on the accuracy of impressions. Finish line exposure has to be adequate during impression making. The goal of gingival retraction is to atraumatically displace gingival tissues to allow access for impression material to record the finish line and provide sufficient thickness of gingival sulcus so that the impression does not tear off during removal. Numerous advanced materials are available for gingival retraction. This article describes the different advanced materials available.

Analysis of fracture resistance of endodontically treated teeth restored with different post and core system of variable diameters: an in vitro study.

The restoration of endodontically treated teeth requires the fabrication of a post and core; to provide retention and support for the final crowns. The purpose of this study was to evaluate fracture resistance of endodontically treated teeth restored with prefabricated zirconia post (CP), milled zirconia post (MZ), pressable ceramic post (PC) and cast metal post (Ni-Cr) of 1.4 and 1.7 mm diameter. 48 freshly extracted human maxillary central incisors were used for this study. The teeth were distributed in four groups of 12 teeth each. From each group, 6 teeth were selected for 1.4 mm diameter post and rest of the 6 teeth, is selected for 1.7 mm diameter post. All teeth were restored with metal crowns. Each specimen from the group was subjected to "load to fracture" in universal testing machine at 130° angle and the maximum load at failure was recorded. Statistically significant difference was found between the failure load of the groups studied. In group I (Ni-Cr)-1.4 mm diameter post and core recorded a maximum fracture load of 534.83 ± 1.28 N and 1.7 mm diameter post and core showed 294.33 ± 1.02 N. In group II (PC)-1.4 mm diameter post and core recorded a maximum fracture load of 205.33 ± 1.61 N and 1.7 mm post and core showed 375.00 ± 1.57 N. In group III (CP)-1.4 mm diameter post and cores recorded a maximum fracture load of 313.00 ± 0.73 N and 1.7 mm post and core showed 638.67 ± 0.81 N. In group IV (MZ)-1.4 mm diameter post and cores recorded a maximum fracture load of 312.00 ± 0.86 N and 1.7 mm post and core showed 415.00 ± 0.89 N. Prefabricated zirconia post (1.7 mm) with pressable ceramic core (Cosmo post)-exhibited higher fracture resistance. Milled zirconia and prefabricated zirconia post-showed same value with 1.4 mm diameter post. Pressable ceramic post and core showed satisfactory result with 1.7 mm post, but showed lesser values with 1.4 mm diameter post.

Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase.

Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.

Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase.

A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2alpha:1beta:1gamma. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa alpha subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino-terminal sequencing reveals that these peptides arise by cleavage of the Val262-His263 bond of the alpha subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the alpha subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates alpha as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase.

Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase.

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis. The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.

Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli.

Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xa between the maltose binding protein and isocitrate dehydrogenase; however, since Factor Xa was also found to digest isocitrate dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E. coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a Km for DL-isocitrate of 7.74 +/- 0.18 microM and a Km for NADP+ of 6.63 +/- 1.34 microM. These values are similar to the Kms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

Identification of metal-isocitrate binding site of pig heart NADP-specific isocitrate dehydrogenase by affinity cleavage of the enzyme by Fe(2+)-isocitrate.

The divalent metal-isocitrate site of pig heart NADP-specific isocitrate dehydrogenase can be located by affinity cleavage of the enzyme by Fe(2+)-isocitrate in the presence of O2, in analogy to the "chemical nuclease" action of DNA-binding drugs linked to Fe-EDTA. The enzyme is irreversibly inactivated and cleaved by Fe(2+)-isocitrate more rapidly than by Fe2+. Mn2+ prevents inactivation and cleavage by Fe(2+)-isocitrate or by Fe2+. Furthermore, other tri- or dicarboxylates (such as citrate, tricarballylate, or malate), which are not effective substrates of the enzyme, fail to promote inactivation and cleavage of the enzyme by Fe2+. These results indicate that the oxidative inactivation and cleavage reactions are specific. Two pairs of major peptides are generated during Fe(2+)-isocitrate inactivation: 30 + 17 kDa and 35 + 11 kDa, as compared with 46 kDa for the intact enzyme. NH2-terminal sequencing revealed that these peptides arise by a mutually exclusive cleavage at either Asp253-Met254 or His309-Gly310, suggesting Asp253 and His309 as coordination sites for Fe(2+)-isocitrate and, by implication, for Mn(2+)-isocitrate. Fe2+ alone produces peptides (32 + 15 kDa) by an alternate specific cleavage between Tyr272 and Asp273, consistent with free metal ion occupying a different site from metal-isocitrate in NADP-dependent isocitrate dehydrogenase. Affinity cleavage may be a generally useful method for locating metal and metal-substrate sites in enzymes.

Biogenic amine status in acute fulminant hepatocellular failure in children.

This study involved pediatric cases with Acute fulminant hepatocellular failure (AFHF) put on conventional therapy at the Hospital for children, Madras. In these cases, the biogenic amine status was studied at the time of admission, during therapy and at the time of recovery in responders. The CSF 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and Homovanillic acid (HVA), blood 5-HT and 5-HIAA, and urinary 5-HIAA followed almost a similar pattern of changes during the course of AFHF: increase at precoma, further increase at coma, return towards control at recovery. In striking contrast, urinary 3-methoxy-4-hydroxyphenyl glycol (MHPG) and 3-methoxy-4-hydroxymandelic acid (VMA) registered a decrease at precoma, a further fall at coma and a value closer to control at recovery. The results suggest the usefulness of assay of these parameters in monitoring cases of AFHF during therapy and in offering prognosis for these cases.