Stress distribution and displacement in the maxillofacial complex during intrusion and distalization of the maxillary arch using miniplates versus mini-implants: a 3-dimensional finite element study.

OBJECTIVES: To three-dimensionally analyse the stress distribution and displacement pattern in the maxillofacial complex following intrusion and distalization of the maxillary arch using finite element analysis in skeletal class II malocclusion with prognathic maxilla and vertical maxillary excess using miniplates and mini-implants. MATERIALS AND METHODS: Finite Element models of a skull, Y-shaped stainless steel miniplate, mini-implant and a posted arch were generated. Three force levels (1) 200 g (2) 300 g and (3) 500 g per side were applied to the assembly. The models were pre-processed and the analysis was performed using ANSYS version 18.1 software. Alterations in von mises stress, principal maximum stress, principal minimum stress and compressive stress were analysed around the sutures and surface landmarks. RESULTS: With miniplates, there was a maximum stress concentration at the zygomatic buttress with even stress distribution at the fronto-maxillary, zygomatico-temporal, zygomatico-frontal and pterygomaxillary sutures along with anatomical landmarks such as frontal process of maxilla, ANS, Point A, prosthion and maxillary process of zygoma. First molars experienced greater distalization effects with buccal flaring when miniplates were used. With mini-implants, canine and premolars also exhibited greater distalization effects. In the root apices, lateral incisors showed increased lingual root movement with mini-implants. CONCLUSION: Miniplates provide a greater distalizing effect while mini-implants produce increased intrusive effect. The distalizing effect is greater when 500 g of force is applied using miniplates with significantly even stress distribution and displacement pattern.

Synthesis, characterization, molecular docking and in vitro antimalarial properties of new carboxamides bearing sulphonamide.

Sulphonamides and carboxamides have shown large number of pharmacological properties against different types of diseases among which is malaria. Twenty four new carboxamide derivatives bearing benzenesulphonamoyl alkanamides were synthesized and investigated for their in silico and in vitro antimalarial and antioxidant properties. The substituted benzenesulphonyl chlorides (1a-c) were treated with various amino acids (2a-h) to obtain the benzenesulphonamoyl alkanamides (3a-x) which were subsequently treated with benzoyl chloride to obtain the N-benzoylated derivatives (5a-f, i-n and q-v). Further reactions of the N-benzoylated derivatives or proline derivatives with 4-aminoacetophenone (6) using boric acid as a catalyst gave the sulphonamide carboxamide derivatives (7a-x) in excellent yields. The in vitro antimalarial studies showed that all synthesized compounds had antimalarial property. Compound 7k, 7c, 7l, 7s, and 7j had mean MIC value of 0.02, 0.03, 0.05, 0.06 and 0.08 µM respectively comparable with chloroquine 0.06 µM. Compound 7c was the most potent antioxidant agent with IC50 value of 0.045 mM comparable with 0.34 mM for ascorbic acid. In addition to the successful synthesis of the target molecules using boric acid catalysis, the compounds were found to have antimalarial and antioxidant activities comparable with known antimalarial and antioxidant drugs. The class of compounds reported herein have the potential of reducing oxidative stress arising from malaria parasite and chemotherapeutic agent used in the treatment of malaria.

Success of root fillings with zinc oxide-ozonated oil in primary molars: preliminary results.

AIM: To evaluate the success rate of mixture of ozonated oil and zinc oxide as a primary teeth root filling material. STUDY DESIGN: Prospective randomised clinical study. METHODS: The study included 60 infected primary mandibular molars which were equally divided into study group (ozonated oil-ZnO) and control group (zinc oxide-eugenol). Pulpectomy procedure was performed and the children were followed at regular intervals. All the children were available for evaluation at the end of 12 months. The teeth were evaluated for success or failure based on clinical and radiographic criteria by a blinded investigator. STATISTICS: The proportional values were compared using χ(2) test. RESULTS: Clinical and radiographic evaluation suggested that teeth obturated with ozonated oil-zinc oxide demonstrated good success rate (93.3%) as compared to zinc oxide eugenol (63.3%). However, no statistically significant variation (p = 0.408) was observed between the groups. CONCLUSION: Ozonated oil-ZnO demonstrated a good clinical and radiographic success at 12 months follow-up and it can be considered as an alternative obturating material in infected primary teeth.

Global microsatellite content distinguishes humans, primates, animals, and plants.

Microsatellites are highly mutable, repetitive sequences commonly used as genetic markers, but they have never been studied en masse. Using a custom microarray to measure hybridization intensities of every possible repetitive nucleotide motif from 1-mers to 6-mers, we examined 25 genomes. Here, we show that global microsatellite content varies predictably by species, as measured by array hybridization signal intensities, correlating with established taxonomic relationships, and particular motifs are characteristic of one species versus another. For instance, hominid-specific microsatellite motifs were identified despite alignment of the human reference, Celera, and Venter genomic sequences indicating substantial variation (30-50%) among individuals. Differential microsatellite motifs were mainly associated with genes involved in developmental processes, whereas those found in intergenic regions exhibited no discernible pattern. This is the first description of a method for evaluating microsatellite content to classify individual genomes.

The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation.

Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.

Sequence and phylogenetic analyses of 4 TMS junctional proteins of animals: connexins, innexins, claudins and occludins.

Connexins and probably innexins are the principal constituents of gap junctions, while claudins and occludins are principal tight junctional constituents. All have similar topologies with four alpha-helical transmembrane segments (TMSs), and all exhibit well-conserved extracytoplasmic cysteines that either are known to or potentially can form disulfide bridges. We have conducted sequence, topological and phylogenetic analyses of the proteins that comprise the connexin, innexin, claudin and occludin families. A multiple alignment of the sequences of each family was used to derive average hydropathy and similarity plots as well as phylogenetic trees. Analyses of the data generated led to the following evolutionary and functional suggestions: (1) In all four families, the most conserved regions of the proteins from each family are the four TMSs although the extracytoplasmic loops between TMSs 1 and 2, and TMSs 3 and 4 are usually well conserved. (2) The phylogenetic trees revealed sets of orthologues except for the innexins where phylogeny primarily reflects organismal source, probably due to a lack of relevant organismal sequence data. (3) The two halves of the connexins exhibit similarities suggesting that they were derived from a common origin by an internal gene duplication event. (4) Conserved cysteyl residues in the connexins and innexins may point to a similar extracellular structure involved in the docking of hemichannels to create intercellular communication channels. (5) We suggest a similar role in homomeric interactions for conserved extracellular residues in the claudins and occludins. The lack of sequence or motif similarity between the four different families indicates that, if they did evolve from a common ancestral gene, they have diverged considerably to fulfill separate, novel functions. We suggest that internal duplication was a general evolutionary strategy used to generate new families of channels and junctions with unique functions. These findings and suggestions should serve as guides for future studies concerning the structures, functions and evolutionary origins of junctional proteins.

Gap junctional coupling in lenses from alpha(8) connexin knockout mice.

Lens fiber cell gap junctions contain alpha(3) (Cx46) and alpha(8) (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either alpha(3) or alpha(8) connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from alpha(3) connexin knockout lenses. Here, we present original data from alpha(8) connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell-cell contact of approximately 1 S/cm(2), which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is approximately 0.4 S/cm(2), and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/-) was approximately 70% of normal and 30-40% of the normal for both -/- lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of alpha(3) +/- lenses. In contrast, the coupling of MF in the alpha(8) +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of alpha(3) -/- lenses. Together, these results suggest that alpha(3) connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and alpha(3) connexin -/- lenses. However, in alpha(8) -/- lenses, the pure alpha(3) connexin junctions did not gate closed in the response to acidification. Since alpha(3) connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears alpha(8) connexin regulates the gating of alpha(3) connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that alpha(3) connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears alpha(8) connexin is required for gating in DF.

Defining a link between gap junction communication, proteolysis, and cataract formation.

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

Characterization of the association of connexins and ZO-1 in the lens.

ZO-1 (Zona Occludens protein 1) has previously been shown to bind Cx43alpha1. This interaction involves the most C-terminal residues of Cx43alpha1 and the second PDZ-domain of ZO-1. The biological significance of this interaction is not well understood. The similarity of the C-terminal residues of the lens connexins Cx46alpha3 and Cx50alpha8 to Cx43alpha1 prompted us to examine if ZO-1 is expressed in the lens, and if ZO-1 interacts with lens connexins. A high level of ZO-1 expression was detected in the mouse lens. Lens connexins were shown to co-immunoprecipitate with ZO-1, and the interaction was found to involve similar domains as those previously demonstrated for the Cx43alpha1/ZO-1 interaction (Nielsen et al. manuscript in preparation). Futhermore, transient expression of Cx46alpha3 and Cx50alpha8 in cell culture showed colocalization of gap junction plaques with ZO-1, further suggesting that lens connexins interact with ZO-1. Sequence comparison suggests that a large number of connexins of the alpha subclass may interact with ZO-1. Using the lens as a system to study connexin/ZO-1 interactions may further our understanding of their biological significance in the lens, as well as in other organs.

Expression, two-dimensional crystallization, and electron cryo-crystallography of recombinant gap junction membrane channels.

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Molecular biology of the interactions between connexins.

The protein structural component of gap junctions is the connexin. Studies on the association properties of the connexins to form heteromeric connexons and heterotypic gap junctions are necessary for a complete understanding of the role of different connexins in gap junction function. The connexins are coded by a multigene family consisting of at least 16 members. Most cells express multiple types of connexin that can potentially associate to form gap junction channels containing more than one type of connexin. The permeability and gating characteristics of gap junction channels are dependent on the isoform and post-translational modifications present on the connexins and their association properties. Together with an observed selectivity in the association properties of the different connexins and the development of more specific perturbation approaches, these studies have provided insights into the significance of connexin diversity and the temporal expression patterns for connexins that have been determined in vivo in both developmental and differentiating systems.

Electron cryo-crystallography of a recombinant cardiac gap junction channel.

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin 43 (Cx43; alpha 1), the principal cardiac gap junction protein. The projection map at 7 A resolution revealed that each 30 kDa connexin subunit has a transmembrane alpha-helix that lines the aqueous pore and a second alpha-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by approximately 30 degrees. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of approximately 7.5 A and a vertical resolution of approximately 25 A. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an alpha-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated alpha 1 Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of alpha 1 Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.

Genetic factors influence cataract formation in alpha 3 connexin knockout mice.

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.

Three-dimensional structure of a recombinant gap junction membrane channel.

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Gap junctional coupling in lenses lacking alpha3 connexin.

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing alpha3 (Cx46) and alpha8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous alpha3 knockout (-/-), heterozygous (+/-), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that alpha8 remained at similar levels in the three classes of lenses, whereas alpha3 was approximately 50% of the normal level in the +/- lenses, and it was absent from the -/- lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In -/- lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (approximately -65 mV). The DF remained coupled, but the conductance was reduced to 30-35% of normal. However, the gap junctions in the DF of alpha3 -/- lenses remained sensitive to pH. We conclude that alpha3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

Disruption of alpha3 connexin gene leads to proteolysis and cataractogenesis in mice.

Gap junction channels formed by alpha3 (Cx46) and alpha8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of alpha3 connexin in vivo, the alpha3 connexin gene was disrupted in mice. Although the absence of alpha3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted alpha3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell-cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.