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1.
Microsc Microanal ; 30(1): 66-76, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38180779

RESUMO

In this study, a conjugate radiation/conduction multimode heat transfer analysis of cryogenic focused ion beam (FIB) milling steps necessary for producing ex situ lift out specimens under cryogenic conditions (cryo-EXLO) is performed. Using finite volume for transient heat conduction and enclosure theory for radiation heat transfer, the analysis shows that as long as the specimen is attached or touching the FIB side wall trenches, the specimen will remain vitreous indefinitely, while actively cooled at liquid nitrogen (LN2) temperatures. To simulate the time needed to perform a transfer step to move the bulk sample containing the FIB-thinned specimen from the cryo-FIB to the cryo-EXLO cryostat, the LN2 temperature active cooling is turned off after steady-state conditions are reached and the specimen is monitored over time until the critical devitrification temperature is reached. Under these conditions, the sample will remain vitreous for >3 min, which is more than enough time needed to perform the cryo-transfer step from the FIB to the cryostat, which takes only ∼10 s. Cryo-transmission electron microscopy images of a manipulated cryo-EXLO yeast specimen prepared with cryo-FIB corroborates the heat transfer analysis.

4.
Protein Expr Purif ; 107: 62-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25462810

RESUMO

DNA gyrase is a type IIA topoisomerase found in bacteria but not in humans. The enzyme is required for bacterial DNA replication and transcription, and is an important antibacterial target that is sensitive to the widely-used fluoroquinolone drugs. Due to the emergence of fluoroquinolone resistance, the discovery of new classes of drugs that target DNA gyrase is urgent. The DNA gyrase holoenzyme is a heterodimer of subunit pairs (A2B2). The 90 kDa A subunits bind, cleave, and rejoin double stranded DNA. The enzyme introduces negative supercoils into closed circular bacterial DNA using ATP hydrolysis catalysed by the 70 kDa B subunits. Subdomains of DNA gyrase subunits have been crystallised for structural analysis and the resulting models used to improve drugs that target the DNA binding region and active site. While crystal structures are available for topoisomerase IV complexes with cleaved DNA, there is none for the complete DNA gyrase complex with substrate DNA bound. Thermophiles offer significant advantages in obtaining stable enzymes for structural and functional studies. In order to develop a capability for drug screening and structure-directed drug discovery we have reconstituted a functional and drug-sensitive DNA gyrase complex using heterologously expressed subunits from the thermophile Thermus thermophilus.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Girase/química , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Estabilidade Enzimática , Hidrólise , Modelos Moleculares , Thermus thermophilus/química , Thermus thermophilus/genética
5.
PLoS One ; 6(11): e26809, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22096499

RESUMO

BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-ß activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-ß activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.


Assuntos
Sinalização do Cálcio/fisiologia , Receptores de Amina Biogênica/classificação , Receptores de Amina Biogênica/metabolismo , Receptores Dopaminérgicos/classificação , Receptores Dopaminérgicos/metabolismo , Sequência de Aminoácidos , Animais , Abelhas , Sítios de Ligação , Cálcio/metabolismo , Sinalização do Cálcio/genética , Linhagem Celular , AMP Cíclico/metabolismo , Dibenzazepinas/metabolismo , Flupentixol/farmacologia , Humanos , Imidazóis/metabolismo , Dados de Sequência Molecular , Filogenia , Receptores de Amina Biogênica/química , Receptores de Amina Biogênica/genética , Receptores Dopaminérgicos/química , Receptores Dopaminérgicos/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Espiperona/farmacologia
6.
Curr Biol ; 19(14): 1206-9, 2009 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-19523830

RESUMO

Queen mandibular pheromone (QMP) is produced by honey bee queens and used to regulate the behavior and physiology of their nestmates. QMP has recently been shown to block aversive learning in young worker bees, an effect that can be mimicked by treating bees with one of QMP's key components, homovanillyl alcohol (HVA). Although the mechanisms underlying this blockade remain unclear, HVA has been found to lower brain dopamine levels and to alter intracellular levels of cAMP in brain centers involved in learning and memory. These findings led to the hypothesis that HVA targets dopamine pathways in the brain, which are known to play a critical role in the formation of aversive olfactory memories. Here, we investigate the possibility that HVA interacts directly with dopamine receptors in the bee. We show that HVA selectively activates the D2-like dopamine receptor AmDOP3 but has neither agonist nor antagonist activity on the D1-like receptors AmDOP1 or AmDOP2 nor agonist activity on the octopamine receptor AmOA1. These results suggest a direct molecular mechanism by which queen pheromone can modulate dopamine signaling pathways. They also implicate the dopamine receptor AmDOP3 in HVA-induced blockade of aversive learning in young worker bees.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Abelhas/fisiologia , Comportamento Animal/fisiologia , Hierarquia Social , Feromônios/metabolismo , Receptores Dopaminérgicos/metabolismo , Transdução de Sinais/fisiologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Análise de Variância , Animais , Abelhas/metabolismo , Técnicas Imunoenzimáticas , Aprendizagem/fisiologia , Luciferases
7.
J Periodontal Res ; 43(3): 343-51, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18086169

RESUMO

BACKGROUND AND OBJECTIVE: A role for cytokines and growth factors in mediating the cellular and molecular events involved in orthodontic tooth movement is well established. The focus to date, however, has been largely on individual mediators, rather than to study cytokines in terms of complex interacting networks. Our objective was to expand our knowledge of the cytokines and growth factors expressed by human periodontal ligament (PDL) cells and to identify new genes that are responsive to mechanical deformation. MATERIAL AND METHODS: Human PDL cells were strained with a cyclic deformation of 12% for 6-24 h, and the differential expression of 79 cytokine and growth factor genes was quantified using real-time RT-PCR arrays. For statistical comparison, t-tests were used with mean critical threshold (CT) values derived from triplicate samples. RESULTS: Forty-one genes were detected at CT values < 35 and, of these, 15 showed a significant change in relative expression. These included seven interleukins (IL): IL1A, IL1F7, IL6 and IL7 (down), IL8, IL11 and IL12A (up). Eight genes representing other cytokine and growth factor families showed comparable mechanical sensitivity, including VEGFD and OPG (down) and PDGFA, INHBA, GDF8 and two transforming growth factor beta genes, TGFB1 and TGFB3 (up). The genes CSF2/GMCSF and IL11 were found to be consistently stimulated across all three time points. Genes that were not expressed included: (1) the immunoregulatory lymphokines (IL2-IL5), IL17 and IL17B; (2) IL10 and other members of the IL-10 family of anti-inflammatory cytokines (IL19, IL20, IL22 and IL24); and (3) TNF and RANKL. CONCLUSION: Human PDL cells constitutively express numerous osteotropic cytokines and growth factors, many of which are mechanoresponsive.


Assuntos
Remodelação Óssea/genética , Citocinas/biossíntese , Análise do Estresse Dentário , Substâncias de Crescimento/biossíntese , Ligamento Periodontal/metabolismo , Forma Celular , Células Cultivadas , Citocinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Substâncias de Crescimento/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resistência à Tração
8.
Proc Natl Acad Sci U S A ; 104(7): 2460-4, 2007 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-17287354

RESUMO

Honey bee queens produce a sophisticated array of chemical signals (pheromones) that influence both the behavior and physiology of their nest mates. Most striking are the effects of queen mandibular pheromone (QMP), a chemical blend that induces young workers to feed and groom the queen and primes bees to perform colony-related tasks. But how does this pheromone operate at the cellular level? This study reveals that QMP has profound effects on dopamine pathways in the brain, pathways that play a central role in behavioral regulation and motor control. In young worker bees, dopamine levels, levels of dopamine receptor gene expression, and cellular responses to this amine are all affected by QMP. We identify homovanillyl alcohol as a key contributor to these effects and provide evidence linking QMP-induced changes in the brain to changes at a behavioral level. This study offers exciting insights into the mechanisms through which QMP operates and a deeper understanding of the queen's ability to regulate the behavior of her offspring.


Assuntos
Abelhas/fisiologia , Encéfalo/efeitos dos fármacos , Dopamina/metabolismo , Feromônios/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Álcoois Benzílicos , Encéfalo/fisiologia , Dopamina/análise , Dopamina/genética , Dopamina/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos
9.
Insect Biochem Mol Biol ; 35(8): 873-82, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15944083

RESUMO

Dopamine is an important neurotransmitter in vertebrate and invertebrate nervous systems and is widely distributed in the brain of the honey bee, Apis mellifera. We report here the functional characterization and cellular localization of the putative dopamine receptor gene, Amdop3, a cDNA clone isolated and identified in previous studies as AmBAR3 (Apis mellifera Biogenic Amine Receptor 3). The Amdop3 cDNA encodes a 694 amino acid protein, AmDOP3. Comparison of AmDOP3 to Drosophila melanogaster sequences indicates that it is orthologous to the D2-like dopamine receptor, DD2R. Using AmDOP3 receptors expressed in HEK293 cells we show that of the endogenous biogenic amines, dopamine is the most potent AmDOP3 agonist, and that activation of AmDOP3 receptors results in down regulation of intracellular levels of cAMP, a property characteristic of D2-like dopamine receptors. In situ hybridization reveals that Amdop3 is widely expressed in the brain but shows a pattern of expression that differs from that of either Amdop1 or Amdop2, both of which encode D1-like dopamine receptors. Nonetheless, overlaps in the distribution of cells expressing Amdop1, Amdop2 and Amdop3 mRNAs suggest the likelihood of D1:D2 receptor interactions in some cells, including subpopulations of mushroom body neurons.


Assuntos
Abelhas/genética , Abelhas/fisiologia , Receptores de Dopamina D2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/fisiologia , AMP Cíclico/metabolismo , DNA Complementar/análise , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Análise de Sequência de DNA
10.
Arch Insect Biochem Physiol ; 59(3): 103-17, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15986382

RESUMO

Dopamine is found in the nervous systems of both vertebrates and invertebrates. However, the specific actions of dopamine depend on the dopamine receptor type that is expressed in the target cell. As in mammals, different subtypes of dopamine receptors have been cloned and characterized from invertebrates, and these receptor subtypes have different structural and functional properties. Understanding how these receptors respond to dopamine and in which cells each receptor type is expressed is key to our understanding of the role of dopamine signaling. Comparison of the amino acid sequences and experimentally determined functional properties suggest that there are at least three distinct types of dopamine receptors in invertebrates. This review focuses on invertebrate dopamine receptors for which the genes have been isolated and identified, and examines our current knowledge of the functional and structural properties of these receptors, and their pharmacology and expression.


Assuntos
Dopamina/metabolismo , Expressão Gênica , Invertebrados/metabolismo , Filogenia , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Componentes do Gene , Invertebrados/genética , Dados de Sequência Molecular , Receptores Dopaminérgicos/classificação , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência
11.
J Clin Microbiol ; 42(5): 2275-8, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15131211

RESUMO

The feasibility of detecting Candida albicans mRNA in formalin-fixed paraffin-embedded archival human histopathology specimens by reverse transcription-PCR (RT-PCR) was investigated. RT with gene-specific primers was used to detect five single-copy C. albicans gene transcripts, including those of two housekeeping genes, in oral candidiasis samples up to 8 years of age.


Assuntos
Candida albicans/genética , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Sequência de Bases , Candida albicans/isolamento & purificação , Candidíase Bucal/diagnóstico , Candidíase Bucal/patologia , Candidíase Bucal/virologia , Primers do DNA/genética , Formaldeído , Dosagem de Genes , Genes Fúngicos , Humanos , Inclusão em Parafina , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fixação de Tecidos , Virologia/métodos
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