Is there a role for community pharmacists in promoting oral health?

AIM: The main aim of the present study was to investigate whether pharmacists recognised that they have a role in the promotion of oral health advice within the community. METHODS: A cross sectional survey was conducted using a structured questionnaire which was distributed to randomly selected pharmacies (n = 1,500) in the London area. RESULTS: Six hundred and forty-five pharmacies (43%) responded to the initial invitation and 589 (39%) of pharmacy participants acknowledged that pharmacists should have a role in oral health promotion. Participants from 354 pharmacies (23.6%) subsequently agreed to complete the questionnaire. Of those pharmacies completing the questionnaire, 99.4% of the pharmacy participants recognised that there was a role for pharmacists in oral health promotion. Although 91.5% of the pharmacists reported a fairly high level of knowledge for most of the common oral conditions, they also indicated that they were interested in receiving further training on oral conditions through continuing professional development (CPD) courses. A number of the pharmacies (72.5%) expressed a willingness to incorporate oral health promotion within the NHS pharmacy contract. CONCLUSION: Pharmacies may be used effectively in oral health promotion by virtue of their frequent contact with members of public. As a result of their established role in promoting and improving the health within the community, it may possible to incorporate oral health within the existing NHS contract.

Characterization of five CYP4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus-infected and uninfected psyllids.

Previously, we reported that Candidatus Liberibacter asiaticus (Las)-infected Diaphorina citri are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In the present study, we investigated expression levels of family 4 cytochrome P450 (CYP4) genes in Las-infected and uninfected D.citri adults. Five novel CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) were identified. Four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las-infected males, whereas only one was expressed at significantly higher levels in uninfected than Las-infected females. These results suggest that levels of cytochrome P450 monooxygenases in D.citri may be linked to expression levels of these CYP4 genes. Expression of all five CYP4 genes was induced by exposure of D.citri to imidacloprid, suggesting their possible involvement in metabolism of this toxin. Higher expression of the five CYP4 genes was found in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA-interference to silence overexpression of genes associated with insecticide resistance in D.citri. These newly identified genes may also serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of D.citri.

Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), response to citrus volatiles.

The Asian citrus psyllid, Diaphorina citri Kuwayama, vectors Candidatus Liberibacter asiaticus (Las) and Candidatus Liberibacter americanus (Lam), the presumed causal agents of huanglongbing. D. citri generally rely on olfaction and vision for detection of host cues. Plant volatiles from Allium spp. (Alliaceae) are known to repel several arthropod species. We examined the effect of garlic chive (A. tuberosum Rottl.) and wild onion (A. canadense L.) volatiles on D. citri behaviour in a two-port divided T-olfactometer. Citrus leaf volatiles attracted significantly more D. citri adults than clean air. Volatiles from crushed garlic chive leaves, garlic chive essential oil, garlic chive plants, wild onion plants and crushed wild onion leaves all repelled D. citri adults when compared with clean air, with the first two being significantly more repellent than the others. However, when tested with citrus volatiles, only crushed garlic chive leaves and garlic chive essential oil were repellent, and crushed wild onions leaves were not. Analysis of the headspace components of crushed garlic chive leaves and garlic chive essential oil by gas chromatography-mass spectrometry revealed that monosulfides, disulfides and trisulfides were the primary sulfur volatiles present. In general, trisulfides (dimethyl trisulfide) inhibited the response of D. citri to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behaviour of D. citri adults. A blend of dimethyl trisulfide and dimethyl disulfide in 1:1 ratio showed an additive effect on inhibition of D. citri response to citrus volatiles. The plant volatiles from Allium spp. did not affect the behaviour of the D. citri ecto-parasitoid Tamarixia radiata (Waterston). Thus, Allium spp. or the tri- and di-sulphides could be integrated into management programmes for D. citri without affecting natural enemies.

The ground state of the ventral appendage in Drosophila.

In Drosophila melanogaster, the antennae, legs, genitalia, and analia make up a serially homologous set of ventral appendages that depend on different selector genes for their unique identities. The diversity among these structures implies that there is a common ground state that selector genes modify to generate these different appendage morphologies. Here we show that the ventral appendage that forms in the absence of selector gene activity is leglike but consists of only two segments along its proximo-distal axis: a proximal segment and a distal tarsus. These results raise the possibility that, during evolution, leglike appendages could have developed without selector gene activity.

The developmental and molecular biology of genes that subdivide the body of Drosophila.

During the past decade, much progress has been made in understanding how the adult fly is built. Some old concepts such as those of compartments and selector genes have been revitalized. In addition, recent work suggests the existence of genes involved in the regionalization of the adult that do not have all the features of selector genes. Nevertheless, they generate morphological distinctions within the body plan. Here we re-examine some of the defining criteria of selector genes and suggest that these newly characterized genes fulfill many, but not all, of these criteria. Further, we propose that these genes can be classified according to the domains in which they function. Finally, we discuss experiments that address the molecular mechanisms by which selector and selector-like gene products function in the fly.

A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila.

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. Here we show that wg activates the gene homothorax (hth) in the hinge and that hth is necessary for hinge development. Further, we demonstrate that hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. We also show that the gene teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. Our results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary.

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex.

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

The control of trunk Hox specificity and activity by Extradenticle.

We characterize a 37-bp element (fkh[250]) derived from the fork head (fkh) gene, a natural target of the Hox gene Sex combs reduced (Scr). In vitro, Scr cooperatively binds to this DNA with the Hox cofactor Extradenticle (Exd), and the activation of this enhancer in vivo requires Scr and exd. Other Hox/Exd heterodimers do not activate this element in vivo and do not bind this element with high affinity in vitro. The amino-terminal arm of the Scr homeodomain is crucial for the specific activation of this element in vivo. By mutating two base pairs within this element, we can convert the Scr/Exd-binding site to a Hox/Exd consensus site that binds several different Hox/Exd heterodimers. This element, fkh[250(con)], is activated by Scr, Antennapedia (Antp), and Ultrabithorax (Ubx) but repressed by abdominal-A (abd-A). We also show that Scr and Exd are only able to activate the fkh[250] element during the early stages of embryogenesis because, by stage 11, Scr negatively regulates the gene homothorax (hth), which is required for the nuclear localization of Exd. These results suggest that Exd is a specificity cofactor for the trunk Hox genes, and that the control of Exd subcellular localization is a mechanism to regulate Hox activity during development.

Expression pattern of notch1, 2 and 3 and Jagged1 and 2 in lymphoid and stromal thymus components: distinct ligand-receptor interactions in intrathymic T cell development.

The suggested role of Notch1 or its mutants in thymocyte differentiation and T cell tumorigenesis raises the question of how the different members of the Notch family influence distinct steps in T cell development and the role played by Notch ligands in the thymus. We report here that different Notch receptor-ligand partnerships may occur inside the thymus, as we observed differential expression of Notch1, 2 and 3 receptors, their ligands Jagged1 and 2, and downstream intracellular effectors hairy and Enhancer of Split homolog 1 (HES-1) and hairy and Enhancer of Split homolog 5 (HES-5), depending on ontogenetic stage and thymic cell populations. Indeed, while Jagged2 is expressed in both stromal cells and thymocytes, Jagged1 expression is restricted to stromal cells. Moreover, a differential distribution of Notch3, with respect to Notch1, was observed in distinct age-related thymocyte subsets. Finally, Notch3 was preferentially up-regulated in thymocytes, following the induction of their differentiation by interaction with thymic epithelial cells expressing the cognate Jagged1 and 2 ligands, suggesting that, besides Notch1, Notch3 may also be involved in distinct steps of thymocyte development. Our results suggest that the Notch signaling pathway is involved in a complex interplay of T cell developmental stages, as a consequence of the heterogeneity and specific expression of members of the Notch receptor family and their cognate ligands, in distinct thymic cell compartments.

Control of the nuclear localization of Extradenticle by competing nuclear import and export signals.

The Drosophila PBC protein Extradenticle (Exd) is regulated at the level of its subcellular distribution: It is cytoplasmic in the absence of Homothorax (Hth), a Meis family member, and nuclear in the presence of Hth. Here we present evidence that, in the absence of Hth, Exd is exported from nuclei due to the activity of a nuclear export signal (NES). The activity of this NES is inhibited by the antibiotic Leptomycin B, suggesting that Exd is exported by a CRM1/exportin1-related export pathway. By analyzing the subcellular localization of Exd deletion mutants in imaginal discs and cultured cells, we identified three elements in Exd, a putative NES, a nuclear localization sequence (NLS), and a region required for Hth-mediated nuclear localization. This latter region coincides with a domain in Exd that binds Hth protein in vitro. When Exd is uncomplexed with Hth, the NES dominates over the NLS. When Exd is expressed together with Hth, or when the NES is deleted, Exd is nuclear. Thus, Hth is required to overcome the influence of the NES, possibly by inducing a conformational change in Exd. Finally, we provide evidence that Hth and Exd normally interact in the cytoplasm, and that Hth also has an NLS. We propose that in Exd there exists a balance between the activities of an NES and an NLS, and that Hth alters this balance in favor of the NLS.

Structure of a DNA-bound Ultrabithorax-Extradenticle homeodomain complex.

During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbxl in mammals). To understand the structural basis of Hox-Extradenticle pairing, we determine here the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 A resolution, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax-located amino-terminally to its homeodomain-which forms a reverse turn and inserts into a hydrophobic pocket on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals.

Human ligands of the Notch receptor.

During development, the Notch signaling pathway is essential for the appropriate differentiation of many cell types in organisms across the phylogenetic scale, including humans. Notch signaling is also implicated in human diseases, including a leukemia and two hereditary syndromes known as Alagille and CADASIL. To generate tools for pursuing the role of the Notch pathway in human disease and development, we have cloned and analyzed the expression of three human homologues of the Notch ligands Delta and Serrate, human Jagged1 (HJ1), human Jagged2 (HJ2), and human Delta1 (H-Delta-1), and determined their chromosomal localizations. We have also raised antibodies to HJ1, and used these antibodies in conjunction with in situ hybridization to examine the expression of these ligands in normal and cancerous cervical tissue. We find that, as reported previously for Notch, the ligands are up-regulated in certain neoplastic tissues. This observation is consistent with the notion that Notch signaling is an important element in these pathogenic conditions, raising the possibility that modulation of Notch activity could be used to influence the fate of the cells and offering a conceivable therapeutic avenue.

Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development.

homothorax (hth) is a Drosophila member of the Meis family of homeobox genes. hth function is required for the nuclear localization of the Hox cofactor Extradenticle (EXD). We show here that there is also a post-transcriptional control of HTH by exd: exd activity is required for the apparent stability of the HTH protein. In leg imaginal discs, hth expression is limited to the domain of exd function and this domain is complementary to the domain in which the Wingless (WG) and Decapentaplegic (DPP) signals are active. We demonstrate that WG and DPP act together through their targets Distal-less (Dll) and dachshund (dac) to restrict hth expression, and therefore EXD's nuclear localization, to the most proximal regions of the leg disc. Furthermore, there is a reciprocal repression exerted by HTH on these and other DPP and WG downstream targets that restricts their expression to non-hth-expressing cells. Thus, there exists in the leg disc a set of mutually antagonistic interactions between proximal cells, which we define as those that express hth, and distal cells, or those that do not express hth. In addition, we show that dac negatively regulates Dll. We suggest that these antagonistic relationships help to convert the WG and DPP activity gradients into discreet domains of gene expression along the proximodistal axis.

Hox proteins meet more partners.

The Hox genes are clustered sets of homeobox-containing genes that play a central role in animal development. Recent genetic and molecular data suggest that Hox proteins interact with pre-existing homeodomain protein complexes. These complexes may help to regulate Hox activity and Hox specificity, and help cells to interpret signaling cascades during development.

Antagonism between extradenticle function and Hedgehog signalling in the developing limb.

The Drosophila homeobox gene extradenticle (exd) encodes a highly conserved cofactor of Hox proteins. exd activity is regulated post-translationally by a mechanism involving nuclear translocation; only nuclear Exd protein is functional. The exd gene is required for patterning of the proximal region of the leg, whereas patterning of the distal region requires signalling by the Wingless (Wg) and Decapentaplegic (Dpp) proteins, which are in turn activated by Hedgehog (Hh). Here we show that exd function and Dpp/Wg signalling are antagonistic and divide the leg into two mutually exclusive domains. In the proximal domain, exd activity prevents cells from responding to Dpp and Wg. Conversely, in the distal domain, exd function is suppressed by the Dpp/Wg response gene Distal-less (Dll), which prevents the nuclear transport of Exd. We also found that the product of a murine homologue of exd (Pbx1) is regulated at the subcellular level, and that its pattern of nuclear localization in the mouse limb resembles that of Exd in the Drosophila leg. These findings suggest that the division of the limb into two antagonistic domains, as defined by exd (Pbx1) function and Hh signalling, may be a general feature of limb development.

Control of antennal versus leg development in Drosophila.

During the evolution of insects from a millipede-like ancestor, the Hox genes are thought to have promoted the diversification of originally identical body structures. In Drosophila melanogaster, antennae and legs are homologous structures that differ from each other as a result of the Hox gene Antennapedia (Antp), which promotes leg identities by repressing unknown antennal-determining genes. Here we present four lines of evidence that identify extradenticle (exd) and homothorax (hth) as antennal-determining genes. First, removing the function of exd or hth, which is required for the nuclear localization of Exd protein, transforms the antenna into leg; such transformations occur without activation of Antp. Second, hth is expressed and Exd is nuclear in most antennal cells, whereas both are restricted to proximal cells of the leg. Third, Antp is a repressor of hth. Fourth, ectopic expression of Meis1, a murine hth homologue, can trigger antennal development elsewhere in the fly. Taken together, these data indicate that hth is an antennal selector gene, and that Antp promotes leg development by repressing hth and consequently nuclear Exd.

Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein.

We show that homothorax (hth) is required for the Hox genes to pattern the body of the fruit fly, Drosophila melanogaster. hth is necessary for the nuclear localization of an essential HOX cofactor, Extradenticle (EXD), and encodes a homeodomain protein that shares extensive identity with the product of Meis1, a murine proto-oncogene. MEIS1 is able to rescue hth mutant phenotypes and can induce the cytoplasmic-to-nuclear translocation of EXD in cell culture and Drosophila embryos. Thus, Meis1 is a murine homolog of hth. MEIS1/HTH also specifically binds to EXD with high affinity in vitro. These data suggest a novel and evolutionarily conserved mechanism for regulating HOX activity in which a direct protein-protein interaction between EXD and HTH results in EXD's nuclear translocation.

Why are Hox genes clustered?

The evolutionarily conserved genomic organization of the Hox genes has been a puzzle ever since it was discovered that their order along the chromosome is similar to the order of their functional domains along the antero-posterior axis. Why has this colinearity been maintained throughout evolution? A close look at regulatory sequences from the mouse Hox clusters suggests that enhancer sharing between adjacent Hox genes may be one reason. Moreover, characterizing the activity of one of these mouse enhancers in Drosophila illustrates that despite many similarities, not all Hox clusters are built in the same way.

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

Switching the in vivo specificity of a minimal Hox-responsive element.

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein extradenticle or its vertebrate homologs, the pbx proteins (together, the PBC family). Here we show that a two basepair change in a Hox-PBC binding site switches the Hox-dependent expression pattern generated in vivo, from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Further, we identify similar Deformed-PBC binding sites in the Deformed and Hoxb-4 genes and show that they generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites.