Cortical bone lamina approach for mandibular large cystic defect: a case report.

This study evaluated the bone healing of the residual cavity after enucleation of mandibular inflammatory cyst by using only a coverage with cortical bone barrier. This study was conducted at the dental clinic of the Medical, Oral and Biotechnology Sciences Department of Chieti in February 2015. A 36-year-old male patient, D.G.M., had a mandibular inflammatory cyst (with a diameter of about 33.9x20.3mm) treated by surgical enucleation, subsequent apicectomy of the elements involved (4.1, 3.1, 3.2, 3.3, 3.4) and coverage of residual defect with a porcine cortical bone barrier (Bone Lamina). Postoperative clinical and radiographic examinations were performed at 3 and 9 months (panoramic radiographs) and at 12 and 24 months (CBCT Dental scan) respectively after cystic enucleation surgery. Uneventful healing and filling of the residual cavity was carried out. The computed analysis of the postoperative radiographs showed bone regeneration of cortical in terms of thickness increase at 12 and 24 months and showed mean values of reduction in size of the residual cavity of 92.1% after 12 months and 96.53% after 24 months. The volume of residual cavity (3123 mm3 in 2015) was seen to decrease (243.6mm3 at 12 months and 108.5 mm3 at 24 months). In this case report the Bone Lamina prevented tissue collapse within the defect and could maintain structural integrity throughout the period required for bone regeneration; it also has the advantage of resorbable membranes, avoiding a second surgery for its removal.

Comprehensive genomic analysis of the dromedary T cell receptor gamma (TRG) locus and identification of a functional TRGC5 cassette.

The emergent availability in public databases of more complete genome assemblies allows us to improve genomic data obtained by classical molecular cloning. The main goal of this study was to refine the genomic map of the dromedary TRG locus by integrating our previous genomic data with the analysis of recent genomic assemblies. We identified an additional TRGC cassette, defined as a V-J-C recombination unit, located at the 5' of the locus and made up of five TRGV genes followed by three TRGJ genes and one TRGC gene. Hence, the complete dromedary TRG locus spans about 105 Kb and consists of three in tandem TRGC cassettes delimited by AMPH and STARD3NL genes at the 5' and 3' end, respectively. An expression assay carried out on peripheral blood showed the functional competency for the dromedary TRGC5 cassette and confirmed the presence of the somatic hypermutation mechanism able to enlarge the repertoire diversity of the dromedary γδ T cells.

Organization, structure and evolution of 41kb of genomic DNA spanning the D-J-C region of the sheep TRB locus.

A genomic region of 41,045 bp encompassing the 3'-end of the sheep T cell receptor beta chain was sequenced. Extensive molecular analysis has revealed that this region retains a unique structural feature for the presence of a third D-J-C cluster, never detected in any other mammalian species examined so far. A total of 3 TRBD, 18 TRBJ and 3 substantially identical TRBC genes were identified in about 28kb. At 13kb, downstream from the last TRBC gene, in an inverted transcriptional orientation, lies a TRBV gene. Sequence comparison and phylogenetic analyses have demonstrated that the extra D-J-C cluster originated from an unequal crossing over between the two ancestral TRBC genes. Interspersed repeats spanning 22.2% of the sequence, contribute to the wider size of the sheep TRB locus with respect to the other mammalian counterparts, without modifying the general genomic architecture. The nucleotide and predicted amino acid sequences from peripheral T cells cDNA clones indicated that the genes from cluster 3 are fully implicated in the beta chain recombination machinery. Closer inspections of the transcripts have also shown that inter-cluster rearrangements and splice variants, involving the additional cluster, increase the functional diversity of the sheep beta chain repertoire.

Comparative analyses of sheep and human TRG joining regions: evolution of J genes in Bovidae is driven by sequence conservation in their promoters for germline transcription.

The availability of genomic clones representative of the T cell receptor gamma (TRG1@ and TRG2@) ovine loci enabled us to compare the germline genomic organization and nucleotide diversity of joining (J) segments and reconstruct their evolutionary history by phylogenetic analysis of cattle, sheep and human expressed sequences. Expression profiling (RT-PCR data) in fetus and adult indicated that only the ovine J genes in which two or more of the key sequence features, such as recombination signal sequences (RSS), 3' splice sites, and core sequences, are missing or severely altered fail to be transcribed. Comparative genomic examination of the two human with the six sheep germline transcription promoters located at 5' of the relevant constant (C)-distal J segments showed a strong conservation of the redundant STAT consensus motifs, indicating that TRG1@ and TRG2@ loci are under the influence of IL-7 and STAT signalling. These findings support the phylogenetic analysis of human and Bovidae (cattle and sheep) that revealed a different grouping pattern of C-distal compared to C-proximal J segments. Likewise, the phylogenetic behaviour of either C-distal and C-proximal J segments is in accordance with the Bovidae TRG clusters evolution. Comparison of sheep and human structures of recombination signal sequences (RSS) has highlighted a greater conservation in sheep 12 RSS rather than 23 RSS thus suggesting that the initial recruitment of recombination activating genes (RAG) products requires at least one relatively high-affinity RSS per recombination event.

Genomic organization of the sheep TRG1@ locus and comparative analyses of Bovidae and human variable genes.

gammadelta T cells commonly account for 0.5%-5% of human (gammadelta low species) circulating T cells, whereas they are very common in chickens, and they may account for >70% of peripheral cells in ruminants (gammadelta high species). We have previously reported the ovine TRG2@ locus structure, the first complete physical map of any ruminant animal TCR locus. Here we determined the TRG1@ locus organization in sheep, reported all variable (V) gamma gene segments in their germline configuration and included human and cattle sequences in a three species comparison. The TRG1@ locus spans about 140 kb and consists of three clusters named TRG5, TRG3, and TRG1 according to the constant (C) genes. The predicted tertiary structure of cattle and sheep V proteins showed a remarkably high degree of conservation between the experimentally determined human Vgamma9 and the proteins belonging to TRG5 Vgamma subgroup. However systematic comparison of primary and tertiary structure highligthed that in Bovidae the overall conformation of the gammadelta TCR, is more similar to the Fab fragment of an antibody than any TCR heterodimer. Phylogenetic analysis showed that the evolution of cattle and sheep V genes is related to the rearrangement process of V segments with the relevant C, and consequentely to the appartenence of the V genes to a given cluster. The TRG cluster evolution in cattle and sheep pointed out the existence of a TRG5 ancient cluster and the occurrence of duplications of its minimal structural scheme of one V, two joining (J), and one C.

Evolution of T-cell receptor gamma and delta constant region and other T-cell-related proteins in the human-rodent-artiodactyl triplet.

In this paper we report a detailed comparative and evolutionary analysis of the sequences of constant T-cell receptor (Tcr) C gamma delta genes of artiodactyls compared to the homologous sequences of rodents and primates. Because of the frequency and physiological distribution of gamma delta T-cells in different animals, rodents and humans are defined as "gamma delta low" species and ruminants as "gamma delta high" species. Such a characteristic seems to be due to an adaptive role of gamma delta T-cell function. By analyzing the ruminant gene phylogeny of Tcr C gamma we were able to estimate the distance between cattle and sheep at 18 million years ago, a time that is in agreement with other nonmolecular estimates. For Tcr C gamma delta genes a peculiar phylogenetic relationship was found, with human and mouse clustering together and leaving artiodactyls apart. By using appropriate outgroups, the same phylogenetic pattern was obtained with other T-cell related sequences: namely, Tcr C alpha chain, CD3 gamma and delta invariant subunits. Interleukin-2. Interleukin-2 receptor alpha chain and Interleukin-1 beta with the exception of Tcr C beta chain and Interleukin-1 alpha. In contrast, the analysis of all other T-cell nonrelated genes, available in primary databases reveals a different tree, where primates and artiodactyls are sister taxa and rodents are apart in accordance with the current view of mammalian phylogeny. These data are relevant to important evolutionary issues. They show how misleading a phylogeny based on a single or on a few homologous genes may be. In addition they demonstrate that genes with correlated functions may evolve in a lineage specific manner probably in relation to environmental conditions.

A patchwork interspersed sequence is present in a high copy number in the sheep genome.

We have isolated a new interspersed sequence present in a high copy number in the ovine genome. This patchwork sequence, named 3.79 AS1, is part of a larger element encompassing similarities to constant region of reverse transcriptase and to art2 shared with the Bovine Dimer Driven Family (BDDF). The 3.79 AS1 sequence includes homologies to amplification promoting sequences (APS), to a potential origin of bidirectional DNA replication (OBR), to the Alu core sequence motif GGAGGC required for RNA polymerase III promoter function and to the ATGGCTGCCAT sequence that has been shown to be able to induce amplification-dependent transformation in murine cells. Fluorescent in situ hybridization experiments using probes derived from both ends of the 3.79 AS1 sequence showed a widespread signal over all sheep chromosomes, except the Y chromosome. We propose that the structural features of the 3.79 AS1 patchwork sequence, that is likely to be a subfamily of Bov B LINE that invaded the Artiodactyl genome prior to the separation of the Bovidae species, facilitated its massive amplification and dispersion in the ovine genome.

The deduced structure of the T cell receptor gamma locus in Canis lupus familiaris.

Analyzing the recent high-quality genome sequence of the domestic dog (Canis lupus familiaris), we deduced for the first time in a mammalian species belonging to Carnivora order, the genomic structure and the putative origin of the TRG locus. New variable (TRGV), joining (TRGJ) and constant (TRGC) genes for a total of 40 are organized into eight cassettes aligned in tandem in the same transcriptional orientation, each containing the basic recombinational unit V-J-J-C, except for a J-J-C cassette, that lacks the V gene and occupies the 3' end of the locus. Amphiphysin (AMPH) and related to steroidogenic acute regulatory protein D3-N-terminal like (STARD3NL) genes flank, respectively, the 5' and 3' ends of the canine TRG locus that spans about 460kb. Moreover LINE1 elements, evenly distributed along the entire sequence, significantly (20.59%) contribute to the architecture of the dog TRG locus. Eight of the 16 TRGV genes are functional and belong to 4 different subgroups. Canine TRGJ genes are two for each cassette and only seven out of 16 are functional. The germline configuration and the exon-intron organization of the 8 TRGC genes was determined, six of them resulting functional. The dot plot similarity genomic comparison of human, mouse and dog TRG loci highlighted the occurrence of reiterated duplications of the cassettes during the dog TRG locus evolution. On the other hand the low ratio of functional genes to the total number of canine TRG genes (21/40), suggest that there is no correlation between the extensive duplications of the cassettes and a need for new functional genes. Furthermore the comparison revealed that the TRGC6, C7 and C8 genes are highly related across species suggesting these existed before the primate-rodent-canidae lineages diverged.

Artiodactyl emergence is accompanied by the birth of an extensive pool of diverse germline TRDV1 genes.

Molecular cloning of cDNA from gamma/delta T cells has shown that in sheep, the variable domain of the delta chain is chiefly determined by the expression of the TRDV1 subgroup, apparently composed of a large number of genes. There are three other TRDV subgroups, but these include only one gene each. To evaluate the extent and the complexity of the genomic TRDV repertoire, we screened a sheep liver genomic library from a single individual of the Altamurana breed and sheep fibroblast genomic DNA from a single individual of the Gentile di Puglia breed. We identified a total of 22 TRDV1 genes and the TRDV4 gene. A sequence comparison between germline and the rearranged genes indicates that, in sheep, the TRDV repertoire is generated by the VDJ rearrangement of at least 40 distinct TRDV1 genes. All germline TRDV1 genes present a high degree of similarity in their coding as well as in 5' and 3' flanking regions. However, a systematic analysis of the translation products reveals that these genes present a broadly different and specific repertoire in the complementarity-determining regions or recognition loops, allowing us to organize the TRDV genes into sets. We assume that selection processes operating at the level of ligand recognition have shaped the sheep TRDV germline repertoire. A phylogenetic study based on a sequence analysis of the TRDV genes from different mammalian species shows that the diversification level of these genes is higher in artiodactyl species compared to humans and mice.

Two plasma membrane antigens of testicular sertoli cells and H-2-restricted versus unrestricted lysis by female T cells.

Sertoli cell-only seminiferous tubules of sterile XX,Sxrl-male mice served as an excellent source of pure Sertoli cells. When H-2-compatible female mice were immunized 3 times with these Sertoli cells, resulting antibodies recognized two antigens on the plasma membrane of testicular Sertoli cells. They were male-specific, but ubiquitously expressed H-Y antigen and the cell lineage-specific antigen which Sertoli cells shared with ovarian follicular cells. Doubly primed (2 or 3 times in vivo, and once in vitro) cytotoxic T cells from these females lysed target Sertoli cells in both H-2-restricted and nonrestricted manners. While H-2-restricted killings were attributable to H-Y antigen, further work is needed to identify the Sertoli follicular cell lineage-specific antigen as the cause of H-2-nonrestricted killings.

The identification of human H-Y antigen and testicular transformation induced by its interaction with the receptor site of bovine fetal ovarian cells.

beta 2m(-), HLA (-) Daudi human male Burkitt lymphoma cells excreted a group of protein subunits that shared three distinctive characteristics; their conspicuously longer half-lives compared to more hydrophilic Daudi excreted proteins, their tendency to form progressively larger polymers by means of interchain disulfide bridges, and the extreme hydrophobicity of these polymers. The plasma membrane of extragonadal somatic cells absorbed 1.2 to 2.8% of these hydrophobic proteins. The unoccupied H-Y receptor sites residing on the plasma membrane of bovine fetal ovarian cells, on the other hand, selectively absorbed polymers of 18,000 mol. wt. subunits, and this antigen-receptor interaction, if allowed to continue for five days, induced the formation of tunica albuginea and seminiferous tubules in bovine XX embryonic indifferent gonads. In this manner, human H-Y antigen excreted by Daudi cells has functionally been identified as a series of polymers derived from 18,000 mol. wt. subunits. While, the H-Y antigenic determinants were retained even by the largest polymeric form that became irreversibly water insoluble, the receptor binding activity was shown only by 36.8% of the available polymeric forms of 18,000 mol. wt. subunits, at the most. Nevertheless, once bound to the receptor site, these polymers were rapidly reduced to the monomeric form on the plasma membrane of bovine fetal ovarian cells. Accordingly, the 18,000 mol. wt. monomer might actually represent the functional form of H-Y antigen.

Testicular cells lysostripped of H-Y antigen organize ovarian follicle-like aggregates.

A suspension of free testicular cells were obtained by mild trypsin treatment from newborn BALB/c testes, and their plasma membrane H-Y antigen sites were blocked (lysostripped) by an excess of H-Y antibody of proven specificity and potency (45 min in ice). Upon 16 h of the Moscona-type rotation culture, these treated testicular cells yielded primarily spherical aggregates, more than half of which demonstrated a strong resemblance to ovarian follicles. The resemblance was particularly striking between the smallest testicular folliculoids and primordial ovarian follicles that abound in the newborn female gonad. Under the same condition, control serum-treated testicular cells primarily yielded cylindrical tubular structures that can be very long. Over a critical range, concentrations of H-Y antibody apparently influenced the frequency of testicular folliculoid formation. The above directly supports the proposed testis-organizing function of H-Y antigen and is certainly compatible with the genetic situation encountered in the wood lemming (Myopus schisticolor), that in the functional absence of H-Y antigen, XY gonadal cells readily organize an ovary.

Cloning and cDNA sequence of the rat X-chromosome linked phosphoglycerate kinase.

This paper reports the isolation and the sequence determination of rat phosphoglycerate kinase (PGK) cDNA clones. This cDNA, derived from an X-linked PGK gene transcript, contains a reading frame of 1254 nt and 5' and 3' non coding regions of 40 and 380 nt respectively. Analysis of the nucleotide sequence at the three codon position shows a biased codon usage with a prevalence of the triplet G non G N. Comparison of the inferred rat amino acid sequence with that of other organisms makes possible the calculation of the unit evolutionary period (UEP) for this enzyme, placing it at around 40 million years (My). Thus PGK is one of the oldest housekeeping enzymes.

[Structural analysis of the gene for the beta chain of the antigen-specific receptor of mammalian T-lymphocytes]. / Analisi strutturale dei geni per la catena beta del recettore antigene specifico dei T-linfociti nei mammiferi.

The antigen specific receptor of T cells (TCR) is composed of alpha and beta chains and is normally present on the T cell surface complexed with the components which make up T3. In the case of beta chain, multiple somatic DNA rearrangements bring together V beta (variable), D beta (diversity) and J beta (joining) gene segments before a mature messenger RNA can be transcribed. So far beta chain genes have been extensively studied in the human and in the mouse system and we have very little information on other mammals. Our aims were to obtain information that may provide a structural basis for understanding developmental as well as evolutionary aspects of the TCR gene system in mammals. In this study we compare the hybridization pattern between a human cDNA probe coding for the beta chain constant region and restricted genomic DNA extracted from lymphocytes deriving from human as well as from rat and lamb. The comparison of the hybridization data represent a first piece of information about the variation of the structure of the TCR beta chain genes in mammals.

H-Y antigen in testes of XX(BALB)/XY (C3H) chimaeric male mouse.

Among experimentally produced BALB/C3H aggregation (Blastocyst fusion) chimaeras of the mouse, one fertile XX (BALB)/XY (C3H) male was identified who maintained 50% or more female cells in many parts of his body. Results of H-Y antibody absorption tests revealed an XY to XX transfer of testis-organizing H-Y antigen among testicular Sertoli and Leydig cells, but not among spleen and epidermal cells.

Genomic organization of sheep TRDJ segments and their expression in the delta-chain repertoire in thymus.

cDNA sequences obtained from polymerase chain reaction products of reverse-transcribed RNA from sheep thymus showed the presence of a large number of members of the TRDV1 gene family. Some are TRDV1 genes also found in peripheral blood lymphocytes, while four genes had not been described so far. The cDNA sequences also showed extensive junctional diversity and a preferential usage of the three TRDJ elements. We characterized the genomic organization of the sheep TRDJ locus and detected a correlation between the nonrandom usage of TRDJ elements during development and their chromosomal order.

In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen.

In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S-F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors-the characteristic of fetal Leydig cells.

Free H-Y antigen induces in vitro testicular differentiation of human XX embryonic indifferent gonads.

In accordance with the anchorage site hypothesis, Daudi beta 2-microglobulin (-) HLA (-) human male Burkitt lymphoma cells are incapable of stably maintaining H-Y antigen on their plasma membrane; instead, they excrete it into the culture medium. The proposed testis-organizing function of Daudi H-Y antigen in solution, previously demonstrated in bovine ovarian embryonic cells, has been tested for the first time in human undifferentiated gonads. Cultured in the presence of Daudi excreted H-Y antigen, gonads obtained from early human embryos of 46, XX chromosomal constitution underwent precocious and complete testicular differentiation.

Exon-intron organization of TRGC genes in sheep.

A series of genomic clones derived from a sheep library were used to determine the germline configuration and the exon-intron organization of TRGC2, TRGC3, and TRGC4 genes. Based on the outcomes of molecular analysis, we compared and aligned the genomic sequences with the known complete cDNA sequences of sheep and deduced the exon-intron organization of TRGC genes in this ruminant animal, EX1, corresponding to the disulfide-linked constant domain, and EX3, corresponding to the transmembrane and cytoplasmatic domains, are similar in length in all genes. Conversely, the hinge-encoding EX2A, EX2B, and EX2C exons differ in number and length between genes, and EX2A contains the TTKPP motif irrespective of whether it occurs in single or triplicate form. The molecular data also indicate that at least one additional gene is present in sheep. Phylogenetic analysis grouped the ruminant TRGC genes in two clusters that could have emerged from two ancestral forms that underwent a series of duplications giving rise to the new sequences that were selected and then fixed in the ruminant lineages. A correlation between the cluster distribution in the phylogenetic tree of TRGC genes and their expression during fetal development is discussed.