Methoxyflurane: a review with emphasis on its role in dental practice

Methoxyflurane was developed as an anaesthetic agent and introduced into clinical practice in 1960. It soon became evident that it possessed analgesic properties that other drugs did not. Due to toxicity concerns, it lost favour in general anaesthesia and had been largely abandoned by the late 1970's. The manufacturer withdrew it in 1999, and the Food and Drug Administration in the United States did not renew its license in 2005. It has also been withdrawn by the European Union. However, it continues to be used in Australasia, primarily as an inhaled self-administered analgesic by emergency services immediately following trauma. It has become attractive for use in dental practice, likely due to its effectiveness as an analgesic and its additional sedative qualities. Its acceptance is controversial as its use in dentistry is largely elective. Despite its good safety record in analgesic doses, adverse reactions have been recorded. Practitioners should be well aware of risks associated with its use before considering administration, and carefully assess whether or not there are equally good alternative options that do not the carry the same risks. Methoxyflurane is reviewed below with an emphasis on its use in dental practice.Angus Kingon, Tami Yap, Carmelo Bonanno, Paul Sambrook, and Michael McCulloug

Genetic Analysis of Pathways Regulated by the von Hippel-Lindau Tumor Suppressor in Caenorhabditis elegans

The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated α subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1–independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants (dpy-18, let-268, gon-1, mig-17, and unc-6), with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1–independent function of VHL-1 that is connected with extracellular matrix function

Examination of determinants for intranuclear localization and transactivation within the RING finger of herpes simplex virus type 1 IE110k protein

The herpesvirus regulatory protein IE110k possesses a cysteine-rich, RING finger motif required for its role in transactivation and virus replication. IE110k also localizes to subnuclear compartments termed PODs (PML oncogenic domains). Localization to PODs induces redistribution of the proteins associated with this nuclear compartment, including the cellular RING finger protein, PML. Here we construct a series of deletions, RING domain swaps and point mutations to analyse specific requirements within the IE110k RING finger for subnuclear localization, redistribution of PML and transactivation and we examine the relationship between these activities. We find that IE110k localizes to distinct nuclear subdomains that are more numerous than the cellular PODs and that mutation of two residues within a predicted loop of the RING finger, or replacing the IE110k RING finger with a RING finger from a cellular gene abrogates the ability of IE110k to localize to these extra compartments and traps IE110k in the original PODs. We further demonstrate that RING fingers from the cellular genes mdm-2 and Bmi I, when placed within IE110k, alter the nuclear distribution of IE110k, do not transactivate, and do not redistribute PML. We also demonstrate that the majority of wild-type IE110k, like PML, is associated with the nuclear matrix. Although substitutions and deletions within the RING finger abolish transactivation, these mutant proteins remain tightly associated with the matrix. These results further dissect the determinants involved in different aspects of nuclear compartmentalization of IE110k and are discussed in relation to PML, PODs and the IE110k RING finger

Glycosylation Genes Expressed in Seam Cells Determine Complex Surface Properties and Bacterial Adhesion to the Cuticle of Caenorhabditis elegans

The surface of the nematode Caenorhabditis elegans is poorly understood but critical for its interactions with the environment and with pathogens. We show here that six genes (bus-2, bus-4, and bus-12, together with the previously cloned srf-3, bus-8, and bus-17) encode proteins predicted to act in surface glycosylation, thereby affecting disease susceptibility, locomotory competence, and sexual recognition. Mutations in all six genes cause resistance to the bacterial pathogen Microbacterium nematophilum, and most of these mutations also affect bacterial adhesion and biofilm formation by Yersinia species, demonstrating that both infection and biofilm formation depend on interaction with complex surface carbohydrates. A new bacterial interaction, involving locomotory inhibition by a strain of Bacillus pumilus, reveals diversity in the surface properties of these mutants. Another biological property—contact recognition of hermaphrodites by males during mating—was also found to be impaired in mutants of all six genes. An important common feature is that all are expressed most strongly in seam cells, rather than in the main hypodermal syncytium, indicating that seam cells play the major role in secreting surface coat and consequently in determining environmental interactions. To test for possible redundancies in gene action, the 15 double mutants for this set of genes were constructed and examined, but no synthetic phenotypes were observed. Comparison of the six genes shows that each has distinctive properties, suggesting that they do not act in a linear pathway

Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions

Multiple Genes Affect Sensitivity of Caenorhabditis elegans to the Bacterial Pathogen Microbacterium nematophilum

Interactions with bacteria play a major role in immune responses, ecology, and evolution of all animals, but they have been neglected until recently in the case of C. elegans. We report a genetic investigation of the interaction of C. elegans with the nematode-specific pathogen Microbacterium nematophilum, which colonizes the rectum and causes distinctive tail swelling in its host. A total of 121 mutants with altered response to infection were isolated from selections or screens for a bacterially unswollen (Bus) phenotype, using both chemical and transposon mutagenesis. Some of these correspond to known genes, affecting either bacterial adhesion or colonization (srf-2, srf-3, srf-5) or host swelling response (sur-2, egl-5). Most mutants define 15 new genes (bus-1–bus-6, bus-8, bus-10, bus-12–bus-18). The majority of these mutants exhibit little or no rectal infection when challenged with the pathogen and are probably altered in surface properties such that the bacteria can no longer infect worms. A number have corresponding alterations in lectin staining and cuticle fragility. Most of the uninfectable mutants grow better than wild type in the presence of the pathogen, but the sur-2 mutant is hypersensitive, indicating that the tail-swelling response is associated with a specific defense mechanism against this pathogen

Isolation and molecular identification of nematode surface mutants with resistance to bacterial pathogens

Numerous mutants of the nematode Caenorhabditis elegans with surface abnormalities have been isolated by utilizing their resistance to a variety of bacterial pathogens (Microbacterium nematophilum, Yersinia pseudotuberculosis, and 2 Leucobacter strains), all of which are able to cause disease or death when worms are grown on bacterial lawns containing these pathogens. Previous work led to the identification of 9 srf or bus genes; here, we report molecular identification and characterization of a further 10 surface-affecting genes. Three of these were found to encode factors implicated in glycosylation (srf-2, bus-5, and bus-22), like several of those previously reported; srf-2 belongs to the GT92 family of putative galactosyltransferases, and bus-5 is homologous to human dTDP-D-glucose 4,6-dehydratase, which is implicated in Catel–Manzke syndrome. Other genes encoded proteins with sequence similarity to phosphatidylinositol phosphatases (bus-6), Patched-related receptors (ptr-15/bus-13), steroid dehydrogenases (dhs-5/bus-21), or glypiation factors (bus-24). Three genes appeared to be nematode-specific (srf-5, bus-10, and bus-28). Many mutants exhibited cuticle fragility as revealed by bleach and detergent sensitivity; this fragility was correlated with increased drug sensitivity, as well as with abnormal skiddy locomotion. Most of the genes examined were found to be expressed in epidermal seam cells, which appear to be important for synthesizing nematode surface coat. The results reveal the genetic and biochemical complexity of this critical surface layer, and provide new tools for its analysis

Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4).

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host-pathogen interactions