RESUMO
Extradural motor cortex stimulation has been employed in cases of Parkinson's disease (PD), fixed dystonia (FD) and spastic hemiparesis (SH) following cerebral stroke. Symptoms of PD are improved by EMCS: results were evaluated on the basis of the UPDRS and statistically analysed. In PD EMCS is less efficacious than bilateral subthalamic nucleus (STN) stimulation, but it may be safely employed in patients not eligible for deep brain stimulation (DBS). The most rewarding effect is the improvement, in severely affected patients, of posture and gait. FD, unresponsive to bilateral pallidal stimulation, has been relieved by EDMS. In SH reduction of spasticiy by EMCS allows improvement of the motor function.
Assuntos
Estimulação Encefálica Profunda/métodos , Distúrbios Distônicos/terapia , Córtex Motor/fisiopatologia , Espasticidade Muscular/terapia , Doença de Parkinson/terapia , Idoso , Idoso de 80 Anos ou mais , Estimulação Encefálica Profunda/estatística & dados numéricos , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Feminino , Seguimentos , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Fatores de Tempo , Resultado do TratamentoRESUMO
Dioxins and dioxin-like PCBs are given toxic equivalency factors (TEFs) in order to calculate the combined toxic equivalence (TEQ) of these contaminants in a sample of food. This study calculates the probability of an average consumer exceeding the recommended tolerable daily intake of 1-4 pg WHO-TEQ kg(-1) bw day(-1) as the amount of salmon in the diet is increased. Probabilistic risk analysis is used to account for the known uncertainties in this calculation. When the TEF uncertainty was excluded with no salmon consumption, the background dietary intake ranged from 1.36 to 1.78 pg TEQ kg(-1) bw day(-1). A weekly consumption of three standard salmon portions resulted in a 36% chance of exceeding the upper limit of the TDI. Inclusion of the TEF uncertainty increased the background dietary intake range from 2.1 to 4.4 pg TEQ kg(-1) bw day(-1), and the weekly consumption of one salmon portion resulted in a 79% chance of the average consumer exceeding the upper TDI. The most important factors contributing to the uncertainty in these results were, in order of magnitude, the TEF for PCB 126 and the sampling uncertainty (sample size) followed by the measurement uncertainty of PCB 126. We recommend that it is more important to increase sample size and produce more precise estimates in the TEF than to improve analytical accuracy.
Assuntos
Dioxinas/análise , Contaminação de Alimentos/análise , Dibenzodioxinas Policloradas/análogos & derivados , Salmão , Animais , Benzofuranos/análise , Dieta , Humanos , Concentração Máxima Permitida , Carne , Método de Monte Carlo , Bifenilos Policlorados/análise , Dibenzodioxinas Policloradas/análise , Medição de Risco/métodosRESUMO
Field study of the locomotor behavior of sympatric woolly monkeys (Lagothrix lagothricha) and spider monkeys (Ateles belzebuth) in undisturbed rainforest of northern Ecuador revealed similar patterns in use of plant forms (categorized tree and liana structure), and substantial differences in the frequencies of use of different grouped modes (aggregates of kinematically similar specific modes). Lagothrix progressed more than Ateles by leaping/dropping and quadrupedal walking/running, whereas Ateles exhibited more suspensory locomotion. Grouped modes are associated with different plant forms in similar ways in the two species. In contrast, the species differed in use of tree zone (trunk/bole, major branches, intermediate branches, and terminal branches), with Lagothrix using intermediate branches and Ateles terminal branches more. Correlated with this difference was greater use by Lagothrix of quadrupedal movement, especially on intermediate branches, and greater use of suspensory modes by Ateles, especially in the terminal zone. Further research is needed to determine how these patterns are facilitated and constrained by morphological mechanisms. Analysis of specific locomotor modes within groups shows several interspecific differences in relative frequencies.
Assuntos
Comportamento Animal/fisiologia , Cebidae/fisiologia , Locomoção/fisiologia , Animais , Fenômenos Biomecânicos , Cebidae/psicologia , Equador , Feminino , Masculino , Método de Monte Carlo , Estatísticas não Paramétricas , Árvores , Gravação de VideoteipeRESUMO
Centrin/Cdc31p is a Ca2+-binding protein related to calmodulin found in the MTOC of diverse organisms. In yeast, Cdc31p localizes to the SPB where it interacts with Kar1p and is required for SPB duplication. Recent findings suggest that centrin also functions elsewhere in the cell. To dissect the functions of Cdc31p, we generated cdc31 mutations chosen only for temperature sensitivity, but otherwise unbiased as to phenotype. Three phenotypes of the cdc31 mutants, temperature sensitivity, G2/M arrest, and cell lysis, were not well correlated, indicating that the mutations may differentially affect Cdc31p's interactions with other proteins. Alleles near the C-terminal region exhibited high G2/M arrest and genetic interactions with kar1-Delta17, suggesting that this region modulates an SPB-related function. Alleles causing high lysis and reduced Kic1p kinase activity mapped to the middle of the gene, suggesting disruption of a KIC1-like function and defects in activating Kic1p. A third region conferred temperature sensitivity without affecting cell lysis or G2/M arrest, suggesting that it defines a third function. Mutations in the C-terminal region were also defective for interaction with Kic1p. Mapping the alleles onto a predicted structure of Cdc31p, we have identified surfaces likely to be important for interacting with both Kar1p and Kic1p.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Mapeamento Cromossômico , Proteínas de Saccharomyces cerevisiae , Alelos , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Proteínas Fúngicas/genética , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/genética , Fenótipo , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , Software , Fuso Acromático/metabolismo , Supressão Genética , Temperatura , Técnicas do Sistema de Duplo-HíbridoRESUMO
The machinery that mediates membrane fusion during yeast mating has remained elusive. But now a post-genomics approach has provided a powerful wedge into this difficult problem: a pheromone-induced multimembrane spanning protein has been identified as a key part of the mating machine.
Assuntos
Saccharomyces cerevisiae/fisiologia , Adesão Celular , Membrana Celular , Proteínas Fúngicas/fisiologia , Fusão de MembranaRESUMO
Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.
Assuntos
Alelos , Inibidores Enzimáticos/farmacologia , Inibidores de Proteínas Quinases , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Proteína Quinase CDC28 de Saccharomyces cerevisiae/antagonistas & inibidores , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Carbazóis/farmacologia , Ciclo Celular , Proteínas Fúngicas/antagonistas & inibidores , Expressão Gênica , Humanos , Alcaloides Indólicos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Proteínas/farmacologia , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, and KAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion of BIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein-Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/química , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genótipo , Humanos , Proteínas dos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno , Proteína Quinase C , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/genética , Western Blotting , Proteínas de Ligação a Calmodulina , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/metabolismo , Fase G2 , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana/genética , Mitose , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Fosforilação , Saccharomyces cerevisiae/genética , Temperatura , Fatores de Tempo , Ubiquitinas/genéticaRESUMO
We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein. Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants. Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts. While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop. These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/química , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de Sequência , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMO
Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication. Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events. At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate. Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate. The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell. We propose that SLG1 plays a regulatory role in bud emergence or stationary phase.
Assuntos
Fase G1/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Sequência de Bases , Ciclo Celular/genética , Ciclo Celular/fisiologia , Primers do DNA/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Fase G1/genética , Genes Fúngicos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/fisiologia , Saccharomyces cerevisiae/genética , TemperaturaRESUMO
Most food containing drug residues is consumed after cooking or processing, yet surveillance for these residues is almost always conducted on raw tissue. This investigation was to establish the effect of cooking on residues of the nitroimidazole drugs dimetridazole and ronidazole in chicken muscle and egg, to enable dietary intake calculations based on surveillance results. In model aqueous and lipid solutions, dimetridazole and its 2-hydroxy metabolite, which is usually found when residues are present, were relatively stable for times and temperatures normally encountered during standard cooking methods. Ronidazole in hot aqueous solutions, except at acidic pH, was converted into the 2-hydroxy metabolite. Chicken meat and eggs from birds treated with dimetridazole or ronidazole were used to investigate the effects of cooking on food containing these residues. It was apparent that these residues were not destroyed by cooking chicken meat although some of the residue leached from the sample with juices which exuded as it cooked. Residue concentration in egg reduced during cooking by between 14% and 32% of the original concentration.
Assuntos
Antiprotozoários/química , Culinária , Resíduos de Drogas , Contaminação de Alimentos , Nitroimidazóis/química , Animais , HumanosRESUMO
Pendular motion during brachiation of captive Lagothrix lagothricha lugens and Ateles fusciceps robustus was analyzed to demonstrate similarities, and differences, between these two closely related large bodied atelines. This is the first captive study of the kinematics of brachiation in Lagothrix. Videorecordings of one adult male of each species were made in a specially designed cage constructed at the DuMond Conservancy/Monkey Jungle, Miami, FL. Java software (Jandel Scientific Inc., San Rafael, CA) was used for frame-by-frame kinematic analysis of individual strides/steps. Results demonstrate that the sequence of hand and tail contacts differ significantly between the two species with Lagothrix using a new tail hold with every hand hold, while Ateles generally utilizes a new tail hold with only every other hand hold. Stride length and stride frequency, even after adjusting for limb length, also differ significantly between the two species. Lagothrix brachiation utilizes short, choppy strides with quick hand holds, while Ateles uses long, fluid strides with longer hand holds. During brachiation not only is Lagothrix's body significantly less horizontal than that of Ateles but also, within Ateles, there are significant differences between steps depending on tail use. Because of the unique nature of tail use in Ateles, many aspects of body positioning in Lagothrix more closely resemble Ateles steps without a simultaneous tail hold rather than those with one. Overall pendulum length in Lagothrix is shorter than in Ateles. Tail use in Ateles has a significant effect on maximum pendulum length during a step. Although neither species achieves the extreme pendulum effect and long period of free-flight of hylobatids in fast ricochetal brachiation, in captivity both consistently demonstrate effective brachiation with brief periods of free-flight and pendular motion. Morphological similarities between ateline brachiators and hylobatids are fewer and less pronounced in Lagothrix than in Ateles. This study demonstrates that Lagothrix brachiation is also less hylobatid-like than that of Ateles.
Assuntos
Cebidae/fisiologia , Locomoção , Animais , Fenômenos Biomecânicos , Mãos , Masculino , Especificidade da Espécie , Cauda , Gravação em VídeoRESUMO
The use of antibiotic therapy to treat and prevent udder infections of cows during the dry period is a key component of mastitis control in many countries. At the same time, the general public is becoming increasingly aware of potential hazards from antibiotic residues in foods. Consequently, Delvotest Cow Test (Royal Gist-brocades NV, Delft, The Netherlands), an on-farm version of Delvotest P, a microbial inhibitor test for antimicrobials, is being increasingly used by farmers to assess that milk from individual cows is fit for consignment to the bulk tank. Occasional reports of unexplained positive test results have led to suggestions of possible false-positive reactions in milk from individual cows. To investigate the potential causes of such positive test results, three separate investigations were undertaken. In a field survey of unexplained positive reports from farmers, 14 milk samples from six farms that tested positive were all found to contain antibiotic residues. In more formal investigations of individual quarter milk samples from an experimental herd, none of 134 milk samples from midlactation cows yielded positive reactions; for cows that had just calved, 16 of 144 milk samples were positive, and, of those, 13 had somatic cell counts > 4,000,000/ml. Natural inhibitors were responsible for 1 positive reaction, 8 positive reactions were related to incomplete milking, and 7 samples contained beta-lactam antibiotics. Positive reactions caused by antibiotic persisted in individual quarter samples for up to 7 d postcalving compared with 4 d for milk samples from the whole udder. Delvotest was sensitive to cephalonium, the active ingredient of Cepravin Dry Cow (Mallinckrodt Veterinary Ltd., Uxbridge, United Kingdom), which is the market-leading product in the United Kingdom. Test results yielded a partial purple color reaction in the presence of 8 micrograms/kg of cephalonium and a completely purple reaction at 16 micrograms/kg. These results confirm the validity of Delvotest when used to examine composite milk samples from individual cows supplying the United Kingdom dairy industry and suggest that, with proper attention to milk withdrawal periods and complete milking, there is no obvious risk of antibiotic contamination of milk.
Assuntos
Antibacterianos/análise , Bovinos , Resíduos de Drogas/análise , Testes de Sensibilidade Microbiana , Leite/química , Animais , Contagem de Células , Cefalosporinas/análise , Reações Falso-Positivas , Feminino , Contaminação de Alimentos , Lactação , Mastite Bovina/prevenção & controle , Leite/citologiaRESUMO
In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.
Assuntos
Actinas/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Sequência de Bases , Núcleo Celular/metabolismo , Primers do DNA , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas dos Microtúbulos/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
Assuntos
Núcleo Celular/genética , Proteínas Fúngicas/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Transporte Biológico , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fusão de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Chaperonas Moleculares , Mutação , Membrana Nuclear/genética , Proteínas Nucleares/metabolismo , Canais de Translocação SEC , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Supressão GenéticaRESUMO
Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis. Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone. Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them. Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons. Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately. At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating. However, the two forms of Kar4p could also be expressed separately from the regulatable GAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels. Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells. In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition. Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic. Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Transcrição Gênica , Mapeamento Cromossômico , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Meiose , Mitose , Mutagênese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
A method for the determination of nine compounds closely related to oxfendazole has been developed for the monitoring of residues in food. The method is based on a multi-residue procedure for basic drug residues and used strong cation exchange solid phase extraction for sample clean-up. These nine compounds include fenbendazole, which is itself a licensed veterinary product. The pro-drug febantel converts quickly to fenbendazole or oxfendazole soon after administration. The method is therefore suitable for monitoring residues following the use of any of these compounds. Some of these analytes have been shown to be present as residues following the treatment of farm animals with oxfendazole. Average recoveries for the nine compounds from tissue fortified with 100 micrograms kg-1 were between 34% and 96% with relative standard deviations between 3% and 22%.
Assuntos
Anti-Helmínticos/metabolismo , Benzimidazóis/metabolismo , Resíduos de Drogas/análise , Contaminação de Alimentos/análise , Fígado/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Rim/química , Fígado/metabolismo , Leite/química , Músculo Esquelético/química , Aves Domésticas , Ovinos , SuínosRESUMO
Galvanic corrosion is an electrochemical reaction between dissimilar metals that has the potential to cause unpleasant and even painful biological effects intra-orally. A case is presented where a full gold crown underwent galvanic change after being placed in contact with an amalgam restoration.
Assuntos
Amálgama Dentário/efeitos adversos , Eletrogalvanismo Intrabucal , Ligas de Ouro/efeitos adversos , Adulto , Corrosão , Humanos , Masculino , Dente MolarRESUMO
The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.