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PubMed1999

A molecular basis for the different local anesthetic affinities of resting versus open and inactivated states of the sodium channel.

Voltage-gated sodium channels are inhibited by local anesthetic drugs. This inhibition has complex voltage- and frequency-dependent properties, consistent with a model in which the sodium channel has low affinity for local anesthetics when it is in resting states and higher affinity when it is in open or inactivated states. Two residues, a phenylalanine (F1710) and a tyrosine (Y1717), in transmembrane segment IVS6 of the channel alpha subunit are critical for state-dependent block. We examined how these residues determine channel sensitivity to local anesthetics by introducing mutations that varied their size, hydrophobicity, and aromaticity. Block of resting channels by tetracaine was correlated with hydrophobicity at position 1710, as if hydrophobic drug-receptor interactions stabilize binding to resting states. In contrast, drug action on open or inactivated channels required an aromatic residue at this position. We propose that the native phenylalanine at position 1710 stabilizes drug binding to open or inactivated states by either cation-pi or aromatic-aromatic interactions between the aromatic side chain of the amino acid and charged or aromatic moieties on the drug molecule. We also consider the alternative possibility that mutations at this position affect drug action by either altering access to the receptor or by allosteric changes in receptor conformation. Mutations at position 1717 also altered drug action; however, these effects were not well-correlated with the size, hydrophobicity, or aromaticity of the substituted amino acid. These results suggest that the residue at this position does not contribute directly to the drug receptor.

Anesthetics, Local↗
PubMed1994

Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerization.

Poliovirus RNA replication is known to be inhibited by millimolar concentrations of guanidine. A variety of guanidine-resistant (gr) and guanidine-dependent (gd) poliovirus strains were selected, and mutations responsible for the phenotypic alterations were mapped to distinct loci of the viral NTP-binding pattern containing protein 2C. Together with already published results, our data have demonstrated that the overwhelming majority of guanidine mutants of poliovirus 2C can be assigned to one of the two classes, N (with a change in Asn179) or M (with a change in Met187). As inferred from the structure/function relations in other NTP-binding proteins, both these "main" mutations should reside in a loop adjoining the so-called B motif known to interact with the Mg2+ involved in the NTP splitting. In classes M (always) and N (not infrequently), these B motif mutations were combined with mutations in, or close to, motif A (involved in binding of the NTP phosphate moieties) and/or motif C (another conserved element of a subset of NTP-binding proteins). These data strongly support the notion that the region of polypeptide 2C involved in the NTP utilization is affected by the guanidine mutations and by the presence of the drug itself. The mutations, however, never altered highly conserved amino acid residues assumed to be essential for the NTP binding or splitting. These facts and some other considerations led us to propose that guanidine affects coupling between the NTP binding and/or splitting, on the one hand, and the 2C function (related to conformational changes), on the other. Both N and M classes of mutants contain gr and gd variants, and the gr/gd interconversion as well as modulations of the guanidine phenotype can be caused by additional mutations within each class; sometimes, these additional substitutions are located far away from the "main" mutations. It is suggested that the target for guanidine action involves long-range tertiary interactions. Under conditions restrictive for the individual growth of each parent, efficient reciprocal intra-allelic complementation between guanidine-sensitive (gs) and gd strains (of M or N classes) was observed. The complementation occurred at the level of viral RNA synthesis. These data allowed us to propose that oligomerization of polypeptide 2C is an essential step in the replication of viral genome.

Acid Anhydride Hydrolases↗
PubMed1998

Multiple negative elements contribute to repression of the HOX11 proto-oncogene.

The HOX11 proto-oncogene is normally expressed in embryogenesis where it directs the synthesis of the spleen. In adult tissues, HOX11 expression is silenced by an unknown mechanism. Aberrant expression of HOX11 occurs in T-cell acute lymphoblastic leukaemia (T-ALL), where it is thought to be involved in T-cell immortalization. The deregulated expression of HOX11 is frequently associated with chromosomal translocations which juxtapose a T-cell receptor (TCR) gene upstream of the HOX11 gene. In these cases, it is presumed that the activation of HOX11 expression results from bringing the gene under the control of TCR enhancer elements. However, activation of HOX11 also occurs in the absence of an associated translocation in both T-ALL and erythroleukaemia cells, implying that an alternative activation mechanism may exist. We hypothesized that HOX11 may be repressed in normal T-cells and erythroid cells by the action of negative elements which may be deleted or mutated in leukaemia. We therefore conducted a search for negative elements in the human HOX11 promoter which may function to silence its expression in normal cells of the haematopoietic lineages. Since little sequence of the HOX11 promoter was available, we began our investigation by sequencing over 4.5 kilobases of untranslated DNA from upstream of HOX11. The human sequence that overlaps with the 2.1 kb of murine Hox11 is highly conserved, suggesting that a large region of DNA upstream of HOX11 may have a regulatory function. We then used transfection assays to test the ability of portions of the promoter to drive transcription of a reporter gene. These studies identified four negative elements. Two of them (NRE2 and NRE4) function in all cell lines tested, while the remaining two (NRE1 and NRE3) appear to be cell-type specific. The DNA sequences of three elements are conserved between the human and mouse HOX11/Hox11 promoters. We propose a model in which the combined action of these negative elements contributes to the overall repression of HOX11 expression in normal blood cells.

Animals↗
PubMed2002

Sequence analysis and phenotypes of five temperature sensitive mutator alleles of dnaE, encoding modified alpha-catalytic subunits of Escherichia coli DNA polymerase III holoenzyme.

In the 1970s, several thermosensitive alleles of dnaE (encoding the alpha-catalytic subunit of pol III) were isolated. Genetic characterization of these dnaE mutants revealed that some are mutator alleles at permissive temperature. We have determined the nucleotide changes of five such temperature sensitive mutator alleles (dnaE9, dnaE74, dnaE486, dnaE511, and dnaE1026) and find that most are single missense mutations. The exception is dnaE1026 which is a compound allele consisting of multiple missense mutations. When the previously characterized mutator alleles were moved into a lexA51(Def) recA730 strain, dnaE486, dnaE1026 and dnaE74 conferred a modest approximately two-six-fold increase in spontaneous mutagenesis when grown at the permissive temperature of 28 degrees C, while dnaE9 and dnaE511 actually resulted in a slight decrease in spontaneous mutagenesis. In isogenic DeltaumuDC derivatives, the level of spontaneous mutagenesis dropped significantly, although in each case, the overall mutator effect conferred by the dnaE allele was relatively larger, with all five dnaE alleles conferring an increased spontaneous mutation rate approximately 5-22-fold over the isogenic dnaE+ DeltaumuDC strain. Interestingly, the temperature sensitivity conferred by each allele varied considerably in the lexA51(Def) recA730 background and in many cases, this phenotype was dependent upon the presence of functional pol V (UmuD'2C). Our data suggest that pol V can compete effectively with the impaired alpha-subunit for a 3' primer terminus and as a result, a large proportion of the phenotypic effects observed with strains carrying missense temperature sensitive mutations in dnaE can, in fact, be attributed to the actions of pol V rather than pol III.

Bacterial Proteins↗
PubMed1976

Coupling of rates of transcription, translation, and messenger ribonucleic acid degradation in streptomycin-dependent mutants of Escherichia coli.

The growth rates of streptomycin-dependent mutants varied in proportion to the level of streptomycin supplied; growth also varied characteristically from one dependent strain to another at a given streptomycin concentration. When cells growing at different rates (over a threefold range) were treated with rifampin, direct proportionality was observed for three parameters: (i) the rates of shutoff of transcription of total ribonucleic acid (RNA) and ribosomal RNA, as measured by pulse labeling at later times; (ii) the translation time for molecules of beta-galactosidase; and (iii) the rate of chemical degradation of messenger RNA. In contrast, the rate of functional inactivation of both total and beta-galactosidase messenger RNA was about the same at all growth rates. None of the variations of growth or other parameters were observed in an otherwise isogenic streptomycin-resistant strain treated with streptomycin. Since the mutational change in strd mutants and the site of action of streptomycin are in the 30S ribosomal subunits, it is suggested that the rate of ribosome function is set by the dependent lesion (and the level of streptomycin). One possibility is that the other correlated effects are mechanistically "coupled" to ribosome function, but the apparent coupling could also be an indirect result of differential effects of streptomycin on variables such as ribosomal miscoding and nucleotide pool size. However, since the rate of functional inactivation of messenger RNA is constant even when the RNA is broken down two- to fourfold more slowly, translation yield tends to be proportional to the growth rate of the dependent strains.

Escherichia coli↗
PubMed2026

Molecular Profiling Across 80,000 Patients With Lung Cancer.

INTRODUCTION: Biomarker testing is an essential component of optimal therapeutic management in NSCLC, enabling the use of both Food and Drug Administration-approved and emerging targeted therapies. Despite well-established biomarker testing guidelines and the availability of many approved targeted therapies, a substantial proportion of patients with advanced NSCLC are not benefiting from precision oncology. In this study, we analyze the distribution of actionable genomic alterations across histologic subtypes and clinicodemographic subgroups of NSCLC using data in 82,328 samples profiled with a single comprehensive genomic profiling assay, aiming to support universal molecular testing across all NSCLC subtypes to ensure equitable access to available therapeutics. METHODS: This is an observational retrospective analysis on histologically confirmed NSCLC cases tested with comprehensive genomic profiling by next-generation sequencing between 2014 and 2022 using Foundation One/Foundation CDx. All cases were centrally reviewed by board-certified anatomic pathologist to determine histologic type and subtype. RESULTS: A total of 82,328 patients with NSCLC were included. An actionable genomic alteration (GA) was found in 35.1% of the cases. Lung adenocarcinoma (LUAD) and adenosquamous carcinoma were more frequently associated with actionable GA (45.8% and 40.9%, respectively) as compared with sarcomatoid (29.1%), not otherwise specified (27.6%), large cell (21.1%), and squamous cell (6.5%) histologies. Sarcomatoid histology had the highest METex14 skipping mutation (mut) frequency (9.95% versus 2.43% in LUAD). Tumor mutation burden more than or equal to 10 mut/Mb was associated with histology (50.91% in large cell, 40.79% in not otherwise specified, 39.08% in squamous cell, and 36.30% in sarcomatoid versus 31.22% in LUAD and 29.22% in adenosquamous carcinoma). Patients with actionable GA had usually a low tumor mutation burden (80.88%). A significant correlation (p < 0.005) between age and actionable GA was reported for BRAF/ERBB2 muts, ALK/RET/ROS1 rearrangements, and MET amplification. EGFR actionable muts and KRAS G12C were more frequently observed in females, whereas no significant correlation between sex and other GA was observed. Finally, genetic ancestry analyses revealed a strong correlation for EGFR actionable muts and South/East Asia and America, but not for other GA. CONCLUSIONS: This is the largest NSCLC data set analyzed for biomarker distribution across histologies, age, sex, and genetic ancestry. This data set confirms sufficient enough biomarker prevalence across many histologic subtypes of NSCLC, providing reassurance that all NSCLC cases should be considered for biomarker workup.

Humans↗
PubMed2002

Identification of essential residues in the Erm(B) rRNA methyltransferase of Clostridium perfringens.

Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.

Amino Acid Sequence↗
PubMed1982

Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.

Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.

DNA Polymerase I↗
PubMed2002

Intrafamilial phenotype variability in nephrogenic diabetes insipidus.

X-Linked nephrogenic diabetes insipidus (NDI), which accounts for 90% of inherited cases of NDI, is caused by mutations in the AVPR2 gene that encodes the arginine vasopressin (AVP) receptor type 2 (V2R). The V2R mediates the antidiuretic action of AVP in principal cells of the collecting duct. To date, only three AVPR2 mutations (P322S, D85N, and G201D) have been associated with a mild NDI phenotype, and intrafamilial phenotype variability has not been reported in affected males. We describe a novel Belgian family with X-linked NDI caused by substitution of a histidine for an arginine at position 137 (R137H) of AVPR2. This mutation has been identified in two brothers and their mother. The R137H mutation results in a failure of V2R to stimulate adenylate cyclase and has been associated consistently with severe NDI and the inability to increase urinary osmolality to greater than plasma osmolality during water deprivation and/or infusion of 1-desamino-8-d-arginine vasopressin. Detailed examination of the two affected brothers showed the typical NDI phenotype in the 45-year-old proband, whereas a milder clinical phenotype associated with significant urinary concentrating ability during water deprivation was documented in the 33-year-old brother. Thus, in this family, the R137H mutation is associated with either a mild or severe NDI phenotype. Mechanisms that might account for these findings include genetic and/or environmental modifiers.

Amino Acid Sequence↗
PubMed1996

Syndrome of resistance to thyroid hormone: insights into thyroid hormone action.

Thyroid hormones (T3, T4) exert multiple cellular effects through nuclear thyroid hormone receptors (TR alpha, TR beta). Thyroid hormone receptors are transcription factors that act by altering patterns of gene expression. Resistance to thyroid hormone (RTH) is a rare disorder caused by mutations in the TR beta gene. Biochemically, the syndrome is defined by elevated circulating levels of free thyroid hormones due to reduced target tissue responsiveness and normal, or elevated, levels of thyroid-stimulating hormone (TSH). This "inappropriate" TSH elevation contrasts with the situation in hyperthyroidism, where the pituitary secretion of TSH is suppressed. Patients with RTH usually present with goiter and an euthyroid or mildly hypothyroid metabolic state. Thus, pituitary resistance results in hypersecretion of TSH, which compensates, at least in part, for hormone resistance in peripheral tissues. Despite this compensation, clinical effects of RTH can include short stature, delayed bone maturation, hyperactivity, learning disabilities, and hearing defects, as well as variable features of hyper- and hypothyroidism. With the exception of a single sibship, which harbored a deletion of the entire coding sequence of the TR beta gene and a recessive pattern of inheritance, all other cases of RTH have been inherited in an autosomal dominant manner or have been de novo heterozygous mutations of the TR beta gene. The dominant pattern of inheritance is explained by the functional properties of the mutant receptors which act in a dominant negative manner to block the activity of normal TR alpha and TR beta receptors. Now that a large number of different RTH mutations have been identified, it is striking that the mutations are clustered within restricted domains in the carboxyterminal region of the receptor. Mutations in these regions have been shown to preserve critical receptor functions such as dimerization and DNA binding, while inactivating other activites such as T3 binding and transcriptional activation. The examination of patients with RTH and their mutated receptors has provided important insights into the mechanisms of thyroid hormone action, the structure-function relationship of the receptors, and the molecular mechanisms of dominant negative activity.

Amino Acid Sequence↗
PubMed1995

Tobacco and cancer: epidemiology and the laboratory.

Tobacco smoke contains many mutagenic and carcinogenic chemicals. Both whole tobacco smoke and extracts induce tumors in experimental animals. Work with carcinogen-macromolecule adducts provided evidence for the action of specific chemicals. Molecular epidemiology studies suggested that point mutations in tumor-suppressor genes (e.g., p53) and oncogenes (e.g., ras) may be specific both for the type of tumor and for the critical environmental exposure. The consistency among investigations on oncogene/tumor-suppressor gene mutations in lung cancer (and other tobacco-related cancers) in smokers is highly suggestive, although we still lack information about the time sequence between exposure, gene mutation, and cancer onset. Current work that deserves emphasis includes investigations revealing that lungs of smokers contain benzo[a]pyrene diol-epoxide-guanine DNA adducts, which are in accordance with the type of mutations found in K-ras or p53 genes (G to T transversions). In addition, DNA in human exfoliated bladder cells showed a derivative of 4-aminobiphenyl as a main adduct; there was also an association between smoking habits (amount and type of tobacco) and the levels of both DNA adducts and hemoglobin adducts formed by aromatic amines. Increasing evidence indicates that genetically based metabolic polymorphisms exert a role in modulating individual susceptibility to the action of tobacco carcinogens. Overall, the weight of evidence strongly supports the causal nature of the association between smoking and cancer and falsifies Fisher's hypothesis that the association was due to confounding by genetic predisposition.

Confounding Factors, Epidemiologic↗
PubMed1990

Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.

Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A-type K+ channels (IA) in muscle. The molecular basis of three exemplary Sh alleles (ShKS133, ShE62 and Sh5) has been identified. They are point mutations in the Sh transcription unit expressing aberrant voltage-gated A-type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of IA in ShKS133 is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage-gated K+ channel forming proteins. The reduction of IA in ShE62 is due to a mutation in an AG acceptor site. The intervening sequence between exons 19 and 20 is not spliced in ShE62 RNA. As a consequence, ShE62 flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage-gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A-type K+ channels in modulating action potential profiles.

Action Potentials↗
PubMed1998

The role of IL-II in hematopoiesis as revealed by a targeted mutation of its receptor.

Interleukin 11 (IL-11) is a pleiotropic growth factor with several actions in common with members of the IL-6 family. IL-11 utilizes a specific receptor chain encoded by two genes, IL-11Ra, which is expressed in hematopoietic and other tissues and, IL-11Ra2, which has a restricted pattern of expression. The actions of IL-11 in the hematopoietic compartment include support of multilineage and committed progenitors contributing to myeloid, erythroid, megakaryocyte, and lymphoid lineages. IL-11 demonstrates a prominent thrombopoietic activity which is being evaluated in clinical trials. In contrast to the multiple in vitro and in vivo effects of IL-11, mice with a targeted mutation of the IL-11Ra gene (IL-11Ra-/-) did not exhibit an overt hematological phenotype. Generation of a null phenotype was confirmed by independent assays. The numbers of progenitor cells of various lineages as well as their terminally differentiated progeny were undisturbed in the IL-11Ra-/- mice. In addition, the mutant mice were able to respond appropriately to increased demand in situations of hematopoietic stress. This study has highlighted the growth factor redundancy operative in the hematopoietic compartment, and in addition, has served to identify a critical action of IL-11 in nonhematopoietic organs.

Animals↗
PubMed2026

The potential clinical benefit of routine comprehensive genomic profiling in non-small cell lung cancer for the detection of prognostic co-mutations - A multicenter next generation sequencing study.

INTRODUCTION: Non-driver mutations such as TP53, STK11 and KEAP1 are clinically relevant in determining immunotherapy efficacy in patients with non-small cell lung cancer (NSCLC). The aim of this study is to determine the prevalence and clinical relevance of variations in TP53, STK11 and KEAP1 in patients in the analysis of NSCLC, using targeted next-generation sequencing. METHODS: This real-life prospective multicenter cohort study from July 2022 until October 2023 utilized samples of patients in the analysis of NSCLC. The samples were subjected to a targeted DNA NGS panel and, if indicated, RNA sequencing. The outcome of the molecular diagnostics was retrieved, including driver alterations and more in-depth analysis of TP53, STK11 and KEAP1. RESULTS: In 134 of the 437 samples an actionable genomic alteration (AGA) was detected. Of the remaining samples, 213 carried a mutation in either TP53, STK11 and/or KEAP1, while 90 harbored either variants of unknown significance (VUS) (16) or no variant (74). In-depth analysis showed 77 alterations of STK11, with 56 pathogenic and 21 VUS. Most STK11 variants were identified in exon 1, which is hypothesized to be correlated to an oncogenic isoform. Moreover, variants in KEAP1 were mostly VUS, with 48 VUS and 24 mutations. Lastly, 264 TP53 alterations, of which 249 pathogenic and 15 VUS, occurred, with an even spread in the DNA-binding domain. CONCLUSION: This study demonstrated the broad spectrum of variants in STK11, KEAP1 and TP53 in routine panel-based DNA NGS, with 70.3% of the samples without AGA showing a potential clinically relevant mutation in TP53, STK11 and/or KEAP1.

Humans↗
PubMed1992

A molecular strategy to control tamoxifen resistant breast cancer.

Our research goal is to develop possible strategies that could have therapeutic implications for the control of breast cancer. Although tamoxifen therapy is successful for some patients, it does not provide adequate benefit for the majority, who have ER negative disease. There is also both laboratory and clinical evidence to support the position that initially responsive tumours will eventually develop resistance to tamoxifen therapy. Since the ER mechanism is the key to the successful control of tumour growth with anti-oestrogens, we have taken the strategic step of determining whether the ER will reassert growth control in breast tumour cells. We have demonstrated that this is feasible, and it might now be appropriate to plan a "gene therapy" approach to cancer control that is based on reactivation of the ER or the development of a targeted vector. Since the concept of growth control in refractory breast cancer has become a reality, there can now be enthusiasm about developing a means to achieve this therapeutic goal. Indeed, the implications for cancer therapy could be enormous. There is every reason to suppose that other types of cancer cells transfected with steroid receptor genes will respond with growth suppression to the appropriate ligand. We have further broadened our studies of anti-oestrogens to describe a laboratory model to dissect the molecular pharmacology of hormone and anti-hormone action. In practical terms, the model has provided an insight into tamoxifen stimulated growth as a mechanism of tamoxifen resistance. The pharmacology of a model compound, fixed ring 4-hydroxytamoxifen, was changed by a mutant ER. It is possible to envisage the clonal selection of cells with mutated ER or ERE that will thrive on the partial agonist actions of tamoxifen. These cells would, as is observed in the MCF-7 TAM and EnCa101 laboratory models, also respond to oestradiol stimulation. Future studies of mutant receptors in the laboratory and clinic might provide support for a novel mechanism of tamoxifen resistance. What is most encouraging, though, is the finding that the pharmacology of new pure anti-oestrogens is not affected by the mutation in the ER and that the pure anti-oestrogens can control tamoxifen stimulated growth. These observations provide additional support for the development of pure anti-oestrogens as a therapy for breast cancer. A clinically acceptable compound could be used as a therapy after tamoxifen failure or perhaps the compound may prevent the development of receptor mutants if it is used as a first line therapy.

Breast Neoplasms↗
PubMed1997

Craniofacial abnormalities induced by the ectopic expression of homeobox genes.

In this paper I have tried to bring together work that highlights the role of homeobox genes in generating craniofacial form. I review both normal and disrupted embryogenesis and ask whether mis-expression of the homeobox genes outside their normal domains could be contributing to congenital facial abnormalities arising from either genetic or teratogenic actions. Experimentally generated transgenic mice carrying loss- or gain-of-function mutations in homeobox genes, in combination with their normal expression patterns, have allowed us to compile and test models of embryonic specification based around a Hox/homeobox code. These models form the basis on which the functional questions are considered. There are four major sections covering different experimental approaches designed to ectopically induce homeobox genes in the head. Transgenic mice, where heterologous promoters drive a given Hox gene in the head, have shown that the more posteriorly expressed Hox genes tend to have a significant effect only on the skull bones of mesodermal origin whereas those normally expressed more anteriorly, in the hindbrain and branchial arches, can affect more anterior branchial arch and neural crest-derived structures. Manipulation experiments which can induce homeobox genes in small, localised regions of the facial precursors show clear and dramatic effects of this expression on facial development. Null mutations in predicted repressors of Hox gene expression, however, do not appear to give rise to substantial craniofacial abnormalities. Retinoic acid, on the other hand, is well known for its teratogenic actions and its ability to induce Hox gene expression. Evidence is now accumulating that at least some of its teratogenic actions may be mediated by its regulation of the Hox and other homeobox genes in the head.

Abnormalities, Drug-Induced↗
PubMed2004

Mutational analysis of mesentericin y105, an anti-Listeria bacteriocin, for determination of impact on bactericidal activity, in vitro secondary structure, and membrane interaction.

Mesentericin Y105 is a 37-residue bacteriocin produced by Leuconostoc mesenteroides Y105 that displays antagonistic activity against gram-positive bacteria such as Enterococcus faecalis and Listeria monocytogenes. It is closely related to leucocin A, an antimicrobial peptide containing beta-sheet and alpha-helical structures. To analyze structure-function relationships and the mode of action of this bacteriocin, we generated a collection of mesentericin derivatives. Mutations were obtained mostly by PCR random mutagenesis, and the peptides were produced by an original system of heterologous expression recently described. Ten derivatives were obtained displaying modifications at eight different positions in the mesentericin Y105 sequence. Purified peptides were incorporated into lysophosphatidylcholine micelles and analyzed by circular dichroism. The alpha-helical contents of these peptides were compared and related to their respective bactericidal activities. Moreover, studies of the intrinsic fluorescence of tryptophan residues naturally occurring at positions 18 and 37 revealed information about insertion of the peptides in micelles. A model for the mode of action of mesentericin Y105 and related bacteriocins is proposed.

Amino Acid Sequence↗
PubMed2005

A dominant negative peroxisome proliferator-activated receptor-gamma knock-in mouse exhibits features of the metabolic syndrome.

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARgamma mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARgamma L466A mutation, shown previously to inhibit wild-type PPARgamma action in vitro. Homozygous PPARgamma L466A knock-in mice die in utero. Heterozygous PPARgamma L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARgamma function and the metabolic syndrome.

Adipocytes↗