Characterization of a mouse model to study the relationship between apical periodontitis and atherosclerosis.

AIM: First, to determine the feasibility of using the low-density lipoprotein receptor knockout (LDLR KO) mouse model to study apical periodontitis (AP). Secondly, to investigate the causal relationship between AP and atherosclerosis. It was hypothesized that it would be feasible to induce AP and atherosclerosis in LDLR KO mice and find a difference in atherosclerosis between AP and Sham groups. METHODOLOGY: Using a published methodology, AP was induced in LDLR KO mice by exposing the dental pulp of the four first molars (Tx). Shams received only anaesthesia. Mice were fed a high fat, Western-type diet (WTD), to induce atherosclerosis. At 16 weeks, mice were euthanized and aortas collected to measure atherosclerosis lesion burden (oil red O staining). Periapical lesions were validated using micro-CT and histology. Systemic inflammation was measured using a cytokine array. RESULTS: Both groups developed a similar degree of atherosclerosis (mean lesion area 7.46 ± 0.44% in the Tx group compared with 7.65 ± 0.46%, in the Sham group, P = 0.77), and a similar degree of inflammation. Periapical lesions (PALs) in all four molars were only identified in a small subset of Tx mice. CONCLUSIONS: A novel mouse model, which combines AP and CVD, was created. This model allows investigation of the relationship between the two diseases, whilst avoiding other potential common confounders. Although no difference in the degree of atherosclerosis was found between the groups, more studies in which the number of periapical lesions, changes in systemic inflammation and the degree of atherosclerosis are correlated are necessary to ultimately determine the impact of AP on CVD.

Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization.

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin ( Amlx), ameloblastin ( Ambn), and matrix metalloproteinase ( Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 ( Dkk1) in the epithelium and sclerostin ( Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein ( Dspp) but not collagen 1 a1 ( Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II-like appearance. Micro-computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Urine selenium changes during pregnancy do not correlate with thyroid autoantibodies in a mildly iodine deficient population.

Selenium (Se) is a key component of iodinases; higher Se levels are associated with lower titers of antithyroid peroxidase antibodies (anti-TPO). Pregnancy exerts profound effects on thyroid function and autoimmunity. To assess the relationship of urine Se levels with thyroid function and autoimmunity in pregnant women residing in Athens, Greece, we studied prospectively 47 euthyroid women in uncomplicated singleton pregnancies (mean age + SD: 30 + 5 years) in each trimester, measuring urine Se levels, urine iodine, plasma thyrotropin (TSH), free thyroxine and triiodothyronine (FT4 and FT3), as well as levels of anti-TPO antibodies. Changes of the measured parameters were assessed over each trimester; thyroid parameters were assessed with relation to Se levels. Urine Se dropped by the third trimester, whereas urine iodine did not change appreciably during pregnancy. TSH and anti-TPO did not show appreciable changes; FT4 and FT3 gradually decreased as the pregnancy advanced. No relationship between urine Se levels and anti-TPO was found. During pregnancy, changes in urine Se levels accompany mild changes in thyroid function. However, we did not find some association between these changes and thyroid autoimmune activity over this period, probably because the effect of Se on thyroid autoimmunity may only become apparent in case of excess Se fortification.

The effect of fused 12-membered nickel metallacrowns on DNA and their antibacterial activity.

The synthesis, characterization and the biological study of a series of Ni(ll)(2)(carboxylato)(2) [12- MC(Ni(II)N(shi)2(pko)2)-4][12-MC(Ni(ii)N(sh03(pko))-4] (CH(3)OH)(3)(H(3)O) fused 12-membered metallacrowns with 10 metal ions and commercial available herbicides or anti-inflammatory drugs as carboxylato ligands are reported. All the compounds have a mixed ligand composition with salicylhydroxamic acid and di-2-pyridylketonoxime as chelate agents. The compounds construct metallacrown cores {[12-MC(Ni(n)N(sj02(pko)2)-4][12-MC(Ni(ll)N(shO3(pko))-4]}(2+) following the pattern [-Ni-O-N-](4). The neutral decanuclear [Ni(II)(A)](2)[12-MC(Ni(II)N(shi)2(pko)2)-4][12-MC(Ni(II)N(pko)3(pko))-4] fused metallacrown, consists of two [12-MC(M(ox)N(ligand))-4] units the {Ni(ll)(A)[12-MC(Ni(II)N(shi)2(pko)2)-4]} and {Ni(II)(A)[12-MC(Ni(II)N(shi)3(pko))-4]} with 1(+) and 1(-) charge, respectively. Each metallacrown unit has four ring Ni(II) ions and one additional encapsulated Ni(II) ion in planar arrangement. The anionic unit is bonded with cationic one creating binuclear moieties. The herbicide or antiiflammatory carboxylato ligands are bridging the central octahedral nickel atom with a ring metal ion in a bindetate fashion. The effect on DNA and their antibacterial activity was examined. The changes in the mobility can be attributed to the altered structures of the pDNA treated with Ni(II) complexes. Evaluating the data of the antibacterial activity of the compounds tested, we can conclude that nickel complexes present strong antibacterial activity.

Functional analysis of the highly conserved exon IV of XIST RNA.

X inactivation is effected by a large CIS-acting RNA molecule termed the X inactive specific transcript (XIST). Exon IV of XIST RNA is highly conserved at the primary sequence level and is predicted to form a stable stem-loop structure. These features suggest that it is important for XIST RNA function. We have used homologous recombination to delete exon IV of the mouse XIST gene. Surprisingly we found no detectable effects on X inactivation. Heterozygous female animals show normal random X inactivation and transcripts from the mutant allele were seen to localise IN CIS over the length of the inactive X chromosome. There was however a reduced steady state level of mutant relative to wild type XIST RNA. This effect was not attributable to decreased stability, suggesting that the deletion affects transcription or processing of XIST RNA.

Is glutathione-S-transferase-pi expression a reliable predictor of chemoradiation response in cancer of the head and neck?

PURPOSE: Concurrent radiation and chemotherapy is being evaluated as an alternative treatment to surgery for patients with advanced squamous cell carcinoma of the head and neck, because organ preservation maybe possible without compromising survival. However, the response to concurrent chemoradiation treatment varies from patient to patient, and there is currently no available molecular predictor of response for this particular treatment modality. There is some evidence to indicate that glutathione S-transferase-pi (GST-pi), which is one of the drug detoxifying enzymes, may decrease the effectiveness of platinum-based chemotherapy in the treatment of a variety of tumor types. This study was performed to investigate whether GST-pi expression was correlated with response to concurrent chemotherapy and radiotherapy in patients with advanced squamous cell carcinoma of the head and neck. MATERIALS AND METHODS: Diagnostic biopsy specimens of 36 patients who underwent concurrent chemoradiotherapy for the treatment of advanced squamous cell carcinoma of the head and neck were examined for GST-pi expression by using immunohistochemistry with polyclonal antihuman GST-pi antibodies. GST-pi expression scores were compared among responders and nonresponders. RESULTS: Although the staining rate with antiGST-pi was slightly lower in the responder group in comparison with the nonresponders (82% vs 100%), the difference was not statistically significant. CONCLUSION: GST-pi expression is unlikely to be a valuable predictor of response to concurrent chemotherapy and radiation treatment in patients with advanced squamous cell carcinoma of the head and neck.

Preparation of site-differentiated mixed ligand and mixed ligand/mixed metal metallacrowns.

Assembly reactions that can prepare reliably regioselective metallamacrocyclic complexes have been a target in the development of metallacrowns. To this end, a series of mixed ligand and mixed ligand/mixed metal metallacrowns have been synthesized in high yield and structurally characterized. Two distinct connectivities have been observed in these types of metallacrowns. The monomeric, vacant metallacrown with mixed ligand composition [12-MC(Ni(II)N(Hshi)2(pko)2-4)] (1a) shows the connectivity pattern [-O-Ni-O-N-Ni-N-]2 while the other Ni metallacrowns, [12-MC(Ni(II)N(shi)2(pko)2-4)] (2a) and the coupled [12-MC(Ni(II)N(shi))2(pko)2-4)][12-MC(Ni(II)N(shi))3(pko)-4)] (3a) fused metallacrowns as well as the mixed metal Mn-Ni metallacrown [12-MC(Ni(II)Mn(III)N(shi)2(pko)2-4)] (4a), follow the pattern [-Ni-O-N-]4. Also, three distinct arrangements of the chelate rings around the metal ions have been observed. The syntheses are completely general, allowing for the substitution of different ligands into the metallacrown core. Compounds 1 and 4 show the 6-5-6-5-6-5-6-5 arrangement, compounds 2 and 3(1) the 6-6-5-5-6-6-5-5, and the 3(2) component the 6-6-5-5-6-5-6-5. The obtained structures can be rationalized by balancing the charge at each metal site in the metallacrown. Variable temperature magnetic susceptibility measurements show that exchange interactions for all the compounds are weak and dominantly antiferromagnetic (e.g., 2a gives an exchange coupling of J = -1.2 cm(-1) with g = 2.2). In solution, the metallacrowns are shown to be stable both to decomposition and ligand exchange.

Primary non-random X inactivation associated with disruption of Xist promoter regulation.

In this report we demonstrate primary non-random X chromosome inactivation following targeted mutagenesis of a region immediately upstream of XIST promoter P(1). In heterozygous animals there is a preferential inactivation of the targeted X chromosome in 80--90% of cells. The phenotype correlates with inappropriate activation of XIST in a proportion of the mutant XY embryonic stem cells. Strand-specific analysis revealed increased sense transcription initiating upstream of XIST promoter P(1). There was, however, no discernible effect on transcription from the antisense Tsix gene. We demonstrate that the in vitro and in vivo phenotypes are specifically attributable to the presence of a PGKneo cassette at the targeted locus. These findings are discussed in the context of understanding mechanisms of XIST gene regulation in X inactivation.

Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon.

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development.

A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.

Tetrapod fore-and hindlimbs have evolved from the pectoral and pelvic fins of an ancient vertebrate ancestor. In this ancestor, the pectoral fin appears to have arisen following the rostral homeotic recapitulation of an existing pelvic appendage (Tabin and Laufer (1993), Nature 361, 692-693). Thus the basic appendage outgrowth program is reiterated in both tetrapod fore- and hindlimbs and the pectoral and pelvic fins of extant teleost fishes (Sordino et al. (1995) Nature 375, 678-681). Recently a novel family of putative transcription factors, which includes the T (Brachyury) locus, has been identified and dubbed the "T-box' family. In mice, all of these genes have expression patterns indicative of involvement in embryonic induction (Chapman et al. (1996) Dev. Dyn., in press), and four (Tbx2-Tbx5) are represented as two cognate, linked gene pairs (Agulnik et al., (1996), Genetics, in press). We now report that, whereas Tbx2 and Tbx3 are expressed in similar spatiotemporal patterns in both limbs, Tbx5 and Tbx4 expression is primarily restricted to the developing fore- and hindlimb buds, respectively. These observations suggest that T-box genes have played a role in the evolution of fin and limb morphogenesis, and that Tbx5 and Tbx4 may have been divergently selected to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb (fin) identity.

Programmed cell death in the absence of c-Fos and c-Jun.

Programmed cell death, or apoptosis, is a normal process in the development of a variety of embryonic and adult tissues, and is also observed in several pathological conditions. Several recent studies, using both expression and functional assays, have implicated the transcription factor, AP-1, in the regulation of programmed cell death, and specifically implicate the genes c-fos and c-jun, as well as some other family members. If the products of the c-fos and/or c-jun genes are essential components in the cascade of events that leads to programmed cell death in mammalian cells, it follows that cell death would not occur in mice lacking functional copies of these genes. We have made use of null mutations in the c-fos and c-jun genes that were produced by gene targeting (Johnson, R.S., Spiegelman, B.M. and Papaioannou, V.E. (1992). Cell 71, 577-586; Johnson, R.S., Van Lingen, B., Papaioannou, V.E. and Spiegelman, B.M. (1993). Genes Dev. 7, 1309-1317) to investigate this possibility. Cell death was assayed using an in situ apoptosis assay in c-fos null embryos and adults, c-jun null embryos, and c-fos/c-jun double null embryos compared with control mice. The occurrence of cell death in c-fos null mice was also assessed in two experimental conditions that normally lead to neuronal cell death. The first was unilateral section of the sciatic nerve in neonates, which leads to the death of anterior horn cells of the spinal cord on the operated side. The second was a genetic cross combining the weaver mutation, which causes death of cerebellar granule cells, with the c-fos mutation. Our results show that programmed cell death occurs normally in developing embryonic tissues and adult thymus and ovary, regardless of the absence of a functional c-fos gene. Furthermore, absence of c-fos had no effect on neuronal cell death in the spinal cord following sciatic nerve section, or in heterozygous weavers' cerebellae. Finally, the results show that programmed cell death can take place in embryos lacking both Fos and Jun.

Enzymes of purine salvage and catabolism in the mouse preimplantation embryo measured by high performance liquid chromatography.

The activities of five enzymes involved in purine salvage and catabolism--hypoxanthine phosphoribosyl transferase (HPRT), adenine phosphoribosyl transferase (APRT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and guanase--were measured in mouse embryo extracts, from the one-cell to the blastocyst stage. Xanthine oxidase activity was not detected. The analyses were performed using high performance liquid chromatography and the enzymes showed different patterns of activity during development. Activities of HPRT, APRT and PNP were low before morula formation, and then increased until the blastocyst stage. ADA and guanase showed high activities after fertilization; guanase activity decreased sharply after the two-cell stage and ADA activity decreased sharply after the morula stage. Blastocyst formation was accompanied by a further decline in activity of both enzymes. The methods used may be suitable for measuring these enzymes in single human embryos, or in biopsies derived from them.

Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development.

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a 'wave' of production of the 'glue' secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the 'glue' secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the 'glue' secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the 'glue' secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)

Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos.

The importance of de novo purine synthesis as opposed to the reutilisation of metabolites by salvage pathways, and the nature of the excretory product(s) of purine degradation, have been examined in cultured preimplantation mouse embryos. In the presence of azaserine and mycophenolic acid, which inhibit de novo purine synthesis, embryo cleavage was blocked prior to compaction, the precise stages at which this occurred depended on whether the cultures were established on day 1 or day 2 after fertilisation, and indicated that salvage pathways were insufficient to fulfil the demand for nucleotides during early preimplantation development. The end-product of purine degradation appeared to be xanthine, which was excreted in very small amounts on days 1, 2 and 3, with a pronounced rise from the early to late blastocyst. Uric acid formation or excretion could not be detected. Exogenous hypoxanthine and adenine, which partially inhibited development, were taken up by the embryos and converted to xanthine, most probably by salvage pathways, since the enzyme xanthine oxidase, which converts hypoxanthine directly to xanthine and then to uric acid, could not be detected. Exogenous guanine had little effect on development and was also converted to xanthine, but in this case, the conversion was probably in a single step, via the enzyme guanase.

Renal cell carcinoma presenting as skeletal muscle mass.

Solitary muscle tumor, representing the first manifestation of metastatic renal cell carcinoma, is rare. A case report of a tumor mass in the left posteromedial arm, which proved to be metastatic from the right kidney, is presented. The incidence of this condition among patients presenting as a soft tissue sarcoma may be about 1%.

Primary neuroendocrine carcinoma of the skin (Merkel's cell carcinoma).

Primary neuroendocrine carcinoma of the skin (Merkel's cell carcinoma) is a rare tumor. Until recently 86 patients with this tumor have been described. Two cases of this tumor are presented. This neoplasm has a high propensity for lymphatic as well as hematogenous metastases. It presents as a dermal or subcutaneous nodule. Awareness of this condition may lead to earlier diagnosis and improved survival.