RESUMEN
Dendrimers are synthetic macromolecules with unique structure, which are a potential scaffold for peptides. Elastin is one of the main components of extracellular matrix and a temperature-sensitive biomacromolecule. Previously, Val-Pro-Gly-Val-Gly peptides have been conjugated to a dendrimer for designing an elastin-mimetic dendrimer. In this study, various elastin-mimetic dendrimers using different length peptides and different dendrimer generations were synthesized to control the temperature dependency. The elastin-mimetic dendrimers formed ß-turn structure by heating, which was similar to the elastin-like peptides. The elastin-mimetic dendrimers exhibited an inverse phase transition, largely depending on the peptide length and slightly depending on the dendrimer generation. The elastin-mimetic dendrimers formed aggregates after the phase transition. The endothermal peak was observed in elastin-mimetic dendrimers with long peptides, but not with short ones. The peptide length and the dendrimer generation are important factors to tune the temperature dependency on the elastin-mimetic dendrimer.
Asunto(s)
Dendrímeros/química , Elastina/química , Péptidos/química , Temperatura , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Dendrímeros/síntesis química , Elastina/síntesis química , Elastina/ultraestructura , Datos de Secuencia Molecular , Transición de Fase , Estructura Secundaria de ProteínaRESUMEN
The yeast Saccharomyces cerevisiae CCH1 gene encodes a homologue of the pore-forming α1 subunit of mammalian voltage-gated calcium channels. Cch1 cooperates with Mid1, a candidate for a putative, functional homologue of the mammalian regulatory subunit α2/δ, and is essential for Ca(2+) influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2). The product of CCH1* displayed a marked increase in Ca(2+) uptake activity in the presence and absence of α-factor, and its increased activity was still dependent on Mid1. Mutations in CCH1* did not affect its susceptibility to regulation by calcineurin. In addition, not only was the N1066D mutation in the cytoplasmic loop between domains II and III responsible for the increased activity of Cch1*, but also substitution of another negatively charged amino acid Glu for Asn(1066) resulted in a significant increase in the Ca(2+) uptake activity of Cch1. This is the first report of a hyperactive mutation in Cch1. On the other hand, the cch1-2 allele possesses the P1228L mutation located in the extracellular S1-S2 linker of domain III. The Pro(1228) residue is highly conserved from fungi to humans, and the P1228L mutation led to a partial loss in Cch1 function, but did not affect the localization and expression of Cch1. The results extend our understanding of the structure-function relationship and functional regulation of Cch1.
Asunto(s)
Canales de Calcio/genética , Mutación Missense , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly ß-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to ß-strands, thereby forming sequential intermolecular linkages.
Asunto(s)
Amiloide/química , Ovalbúmina/química , Péptidos/química , Amiloide/genética , Animales , Pollos , Cisteína/química , Cisteína/genética , Disulfuros/química , Mutagénesis Sitio-Dirigida , Ovalbúmina/genética , Péptidos/genética , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.
RESUMEN
Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Dendritas/metabolismo , Proteínas de Unión al GTP/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Animales , Encéfalo/citología , Encéfalo/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Dendritas/ultraestructura , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/genética , Hipocampo/citología , Hipocampo/metabolismo , Interferencia de ARN , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
OBJECTIVE: Endoscopic sphincterotomy (ES) is a well-established standard method for treating common bile duct stones. However, biliary sphincter function is impaired after the treatment, and this may influence the long-term outcomes. In this study, we aimed to compare the long-term outcomes after ES with those after endoscopic papillary balloon dilation (EPBD) because the latter procedure is expected to preserve biliary sphincter function better than ES. DESIGN: A prospective follow-up of the original cohort in a previously randomized, controlled trial to compare the early outcomes after ES and EPBD. SETTING: Eleven centers, including 6 clinical practices and 5 academic institutions. PATIENTS: A total of 282 patients with common bile duct stones were randomly selected to undergo ES (n = 144) or EPBD (n = 138) in the previous study. INTERVENTIONS: ES or EPBD. MAIN OUTCOME MEASUREMENTS: Complications after ES or EPBD occurring during long-term follow-up. RESULTS: The patients were followed up annually after the treatment. The median duration of the follow-up was 6.7 years. Morbidity was observed in 36 (25.0%) and 14 (10.1%) of the patients who underwent ES and EPBD, respectively (P = .0016). Kaplan-Meier analysis revealed a significantly higher incidence of biliary complications in the ES group than in the EPBD group (P = .0011). Multivariate analysis showed that ES, periampullary diverticulum, and in situ gallbladder stones were independent risk factors for stone recurrence. CONCLUSIONS: During long-term follow-up, patients who underwent ES had significantly more biliary complications than those who underwent EPBD. The biliary sphincter dysfunction after ES results in additional late complications.
Asunto(s)
Cateterismo , Cálculos Biliares/terapia , Esfinterotomía Endoscópica , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Japón , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Estudios ProspectivosRESUMEN
BACKGROUND: Self-expandable metallic stents (SEMSs) are widely used for palliation of malignant gastric outlet obstruction (GOO). A common complication of their use, however, is stent obstruction caused by tumor ingrowth or hyperplasia. The covered SEMS was designed to prevent these problems. OBJECTIVE: We compared the performance of uncovered and covered SEMSs in patients with GOO. DESIGN: A retrospective study, single center. SETTING: A tertiary-referral center. PATIENTS: Sixty patients with symptomatic malignant GOO. INTERVENTIONS: All patients received an uncovered or covered knitted nitinol stent by using the over-the-wire placement procedure. MAIN OUTCOME MEASUREMENT: Comparison of the clinical outcome, complications, and the reintervention rate between uncovered and covered stents. RESULTS: Thirty-one patients (mean [+/-SEM] age 72.2 +/- 2.1 years; 16 men) received uncovered SEMSs, and 29 (mean [+/-SEM] age 70.6 +/- 1.7 years; 17 men) received covered SEMSs. The technical success rate was 100% in both groups. No difference in clinical success was seen (90.3% uncovered group vs 86.2% covered group). Regarding early complications (<1 week), one mild case of pancreatitis from the stent covering the papilla occurred in each group. Late complications included reobstruction, migration, bleeding, stent fracture, and perforation. The occurrence of reobstruction did not differ between the 2 groups (3.2% uncovered group vs 10.3% covered group). No difference in migration (0% uncovered group vs 6.9% covered group) was seen. The uncovered group required less frequent reinterventions for stent reobstruction, migration, or stent fracture (3.2% uncovered group vs 20.7% covered group, P = .0490). The uncovered group had 2 major late complications: bleeding and perforation. All 60 patients died, with a median survival time of 51 days and 62 days, respectively. LIMITATIONS: Small-sized, single-center, retrospective study. CONCLUSIONS: In palliation for malignant GOO, covered stents were associated with a more frequent need for reintervention than uncovered stents, despite similar outcomes and complications. These results require confirmation in a larger randomized comparison.
Asunto(s)
Obstrucción de la Salida Gástrica/cirugía , Stents , Anciano , Femenino , Humanos , Masculino , Diseño de Prótesis , Reoperación , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
This study aimed to assess efficacy and limitations of regional cerebral blood flow imaging using single-photon emission computed tomography (rCBF-SPECT) in the diagnosis of Alzheimer's disease (AD) with amyloid-positron emission tomography (amyloid-PET). Thirteen patients, who underwent both rCBF-SPECT and amyloid-PET after clinical diagnosis of AD or mild cognitive impairment, were retrospectively identified. The rCBF-SPECTs were classified into 4 grades, from typical AD pattern to no AD pattern of hypoperfusion; amyloid-beta (Aß) positivity was assessed by amyloid-PET. Four patients were categorized into a typical AD pattern on rCBF-SPECT, and all were Aß+. The other 9 patients did not exhibit a typical AD pattern; however, 4 were Aß+. The Mini-Mental State Examination score and Clinical Dementia Rating scale were not significantly different between Aß+ and Aß- patients. A typical AD pattern on rCBF-SPECT can reflect Aß+; however, if not, rCBF-SPECT has a limitation to predict amyloid pathology.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/metabolismo , Circulación Cerebrovascular , Disfunción Cognitiva/diagnóstico , Tomografía de Emisión de Positrones , Tomografía Computarizada de Emisión de Fotón Único/normas , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Compuestos de Anilina , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Glicoles de Etileno , Femenino , Humanos , Masculino , Imagen Multimodal , Estudios RetrospectivosRESUMEN
Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.
Asunto(s)
Dendritas/fisiología , Dendritas/ultraestructura , Animales , Modelos Neurológicos , Morfogénesis , Plasticidad Neuronal , Proteínas Asociadas a Matriz Nuclear/fisiologíaRESUMEN
BACKGROUND/AIMS: Palliative stenting for gastric outlet obstruction (GOO) offers a more rapid resumption of oral intake than surgical gastrojejunostomy. Clinically, delayed gastric emptying is observed less frequently in patients with enteral stenting. The aim of this study was to conduct a functional assessment of gastric emptying after stent placement for GOO using isotope scanning. METHODOLOGY: Gastric emptying was assessed in 14 patients with GOO (4 female, 10 male; mean age 67.9 years; 8 with gastric cancer; 4 with pancreatic cancer; 1 with biliary cancer; 1 with metastasis) and 10 healthy volunteers (2 female, 8 male; mean age 31.5 years). None of the patients had undergone previous stomach surgery. The patients were studied 1 week after stent placement. Scintigraphy was performed for 2 hours following the ingestion of a labeled liquid meal. Gastric retention was evaluated at 2 hours in both groups. RESULTS: All patients underwent successful placement of stents and were able to resume an oral diet. All stents were fully deployed and no migration was seen at the time of the investigation. Retained gastric activity at 120 minutes (RGA120) was significantly greater in patients than in controls (65.4% vs. 27.5%, p=0.0128). Median survival time was 179 days in patients with T1/2 of 120 min or less and 75 days in patients with T1/2 of over 120 min. CONCLUSIONS: The results of our study show that although patients with GOO have resumed oral intake 1 week after stent placement, restoration of gastric emptying is often still incomplete.
Asunto(s)
Vaciamiento Gástrico , Obstrucción de la Salida Gástrica/fisiopatología , Obstrucción de la Salida Gástrica/terapia , Stents , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Sistema Biliar/complicaciones , Progresión de la Enfermedad , Femenino , Obstrucción de la Salida Gástrica/etiología , Gastroparesia/etiología , Humanos , Masculino , Persona de Mediana Edad , Cuidados Paliativos , Neoplasias Pancreáticas/complicaciones , Neoplasias Gástricas/complicacionesRESUMEN
Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 degrees C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (DeltaV, DeltaG, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.
Asunto(s)
Ácido Aspártico Endopeptidasas/efectos de los fármacos , Ácido Aspártico Endopeptidasas/efectos de la radiación , Ácido Aspártico Endopeptidasas/química , Rastreo Diferencial de Calorimetría , Carboxipeptidasas/química , Dicroismo Circular , Fluorescencia , Calor , Modelos Moleculares , Presión , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Soluciones/química , Termodinámica , Rayos Ultravioleta , Urea/químicaRESUMEN
Age-related macular degeneration (AMD) is the most common cause of vision loss in elderly individuals throughout the developed world. Inhibitors of vascular endothelial growth factor have been successfully used to treat choroidal neovascularization in late-stage AMD. The pathogenesis of early-stage AMD, however, remains largely unknown, impairing efforts to develop effective therapies that prevent progression to late-stage AMD. To address this, we performed comparative transcriptomics of macular and extramacular retinal pigmented epithelium-choroid (RPE-choroid) tissue from early-stage AMD patients. We found that expression of fatty acid desaturase 1 (FADS1), FADS2, and acetyl-CoA acetyltransferase 2 (ACAT2) is increased in macular but not extramacular tissue, possibly through activation of sterol regulatory element binding factor 1 (SREBF1). Consistent with this, we also found that expression of Fads1 is increased in RPE-choroid in a mouse model of early-stage AMD. In zebrafish, deletion of fads2, which encodes a protein that functions as both Fads1 and Fads2 in other species, enhanced apoptosis in the retina upon exposure to intense light. Similarly, pharmacological inhibition of Srebf1 enhanced apoptosis and reduced fads2 expression in zebrafish exposed to intense light. These results suggest that the SREBF1-FADS1/2 axis may be activated in macular RPE-choroid as a protective response during early-stage AMD and could thus be a therapeutic target for early-stage AMD.
RESUMEN
The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Péptidos/metabolismo , Desnaturalización Proteica , Estructura Terciaria de Proteína , Guanidina/química , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Temperatura , TermodinámicaRESUMEN
BACKGROUND: In patients with gastric outlet obstruction (GOO), palliative enteral stenting is a less invasive procedure compared with gastroenterostomy. Most diseases analyzed in previous studies of such stenting were pancreaticobiliary malignancies. METHODS: We reviewed the medical records of patients with GOO secondary to gastric cancer who were admitted to our institution between September 1994 and September 2004. The outcome of stent placement for GOO was compared with the outcome in patients who underwent palliative open gastrojejunostomy during the same period. Enrolled patients from both groups displayed symptomatic GOO. Patients with recurrent gastric cancer were excluded from this study. RESULTS: Twenty-two patients underwent palliative enteral stenting, and 22 patients were subjected to surgical gastrojejunostomy (bypass). There were no significant differences between the two groups regarding patient baseline characteristics. Technical success and clinical success were obtained in 100% and 77.3%, respectively, of both groups. The operating time was shorter in the stent group (30 vs 118 min; P<0.0001). The time from the procedure to the resumption of food intake was shorter in the stent group than in the bypass group (2 days vs 8 days; P<0.0001). An improvement in performance score after the procedure was observed in both groups (stent group; P=0.0264; bypass group; P=0.0235). No significant differences were observed regarding the possibility of discharge. In patients discharged, the median postoperative hospital stays were 19 days and 28 days (P=0.0558). The median survival periods were 65 days and 90 days. Minor complications were observed in 1 patient in the stent group and in 4 in the bypass group. No mortality or severe complications were observed for either group. CONCLUSIONS: Self-expandable metallic stent placement is a safe and efficacious procedure for palliation, with shorter operating time and more prompt restoration of oral intake, compared to surgical alternatives in patients with GOO caused by gastric cancer.
Asunto(s)
Obstrucción de la Salida Gástrica/etiología , Obstrucción de la Salida Gástrica/cirugía , Gastrostomía , Yeyunostomía , Cuidados Paliativos , Stents , Neoplasias Gástricas/complicaciones , Neoplasias Gástricas/cirugía , Anciano , Diseño de Equipo , Femenino , Humanos , Masculino , Estudios RetrospectivosRESUMEN
This study described the genetic map of tandem genes (est1 and est119) encoding cutinase-type polyesterases in Thermobifida alba AHK119 and comparison of wild type and mutant enzymes of Est1 and Est119. Two genes were independently and constitutively expressed. The activity of Est1 was higher by approximately 1.6-1.7-fold than that of Est119 towards p-nitrophenyl butyrate, although both enzymes shared 95% sequence identity and 98% similarity and possessed similar 3D structures except that several amino acids in the probable substrate-docking loops were different from each other. Calcium ion enhanced the activity and the thermostability of both enzymes. Based on conserved sequences among Thermobifida cutinases, valine, proline and lysine were introduced into Est1 at Ala68, Thr253 and Met256, respectively. Among wild and mutant enzymes of Est119 and Est1, Est1 (A68V/T253P) possessed three prolines in the substrate-docking loops and displayed the highest thermostability that spotlighted the important effect of proline numbers in the loops. Est1 (A68V/T253P) was stable for 1 h below 60°C and even at 65°C, more than 70% and 50% activities were maintained after 30 and 60 min, respectively. Est1 (A68V/T253P) degraded various aliphatic and aliphatic-co-aromatic polyesters and hydrophilized an amorphous PET film. The enzyme hydrolyzed a PET trimer model compound, indicating its specificity towards an ester bond between terephthalic acid and ethylene glycol.
Asunto(s)
Actinomycetales/enzimología , Actinomycetales/genética , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Secuencia de Aminoácidos , Butiratos/metabolismo , Calcio/farmacología , Hidrolasas de Éster Carboxílico/química , Secuencia Conservada , Estabilidad de Enzimas/efectos de los fármacos , Genes Bacterianos/genética , Hidrólisis , Datos de Secuencia Molecular , Poliésteres/química , Poliésteres/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiología , Animales , Sitios de Unión , Endopeptidasa Clp , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Cinética , Malato Deshidrogenasa/metabolismo , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteínas/metabolismoRESUMEN
Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli. Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch-activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.
Asunto(s)
Canales de Calcio/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Transporte Biológico , Calcio/metabolismo , Supervivencia Celular , Clonación Molecular , Cartilla de ADN , ADN Recombinante/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Stent placement in palliation of unresectable colon cancer is an alternative to surgical treatment. The through-the-scope stent for the exclusive treatment of colorectal cancer is not available in Japan. This report describes the use of an esophageal stent and the technical modifications required for its success in the treatment of colorectal strictures. We describe various technical strategies for colorectal stent placement and report on the outcomes. METHODS: Medical records of patients who underwent palliative colonic stenting between June 1997 and March 2003 were reviewed retrospectively, and the clinical outcome was evaluated. RESULTS: Insertion of a metallic esophageal stent was attempted in 12 patients (mean age, 73.0 years; 5 male, 7 female). Location of the stricture was in the rectum in 4 patients and in the sigmoid, descending, or transverse segments of the colon in 5, 1, and 2 patients, respectively. Two patients had recurrent colon cancer after surgery. The remaining 10 patients did not undergo surgery. Stent placement was technically successful in 11 patients, giving a technical success rate of 92%. Following successful stent placement, all but 1 patient obtained clinical success, generating a clinical success rate of 83%. Late complications occurred in 4 patients and included 2 migrations, 2 bleeds, and 1 obstruction. The complication rate of the procedure was 33.3%. There was no mortality or severe complications. The median survival period was 120 days. CONCLUSIONS: Stent placement can be considered safe and effective palliation for unresectable colorectal cancer. With technical modification of an esophageal stent, this procedure is now feasible. Stent placement in palliation of unresectable colon cancer is an alternative to surgical treatment. The through-the-scope stent for the exclusive treatment of colorectal cancer is not available in Japan. This report describes the use of an esophageal stent and the technical modifications required for its success in the treatment of colorectal strictures. We describe various technical strategies for colorectal stent placement and report on the outcomes.
Asunto(s)
Carcinoma/terapia , Neoplasias Colorrectales/terapia , Obstrucción Intestinal/terapia , Cuidados Paliativos/métodos , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/complicaciones , Colonoscopía/métodos , Neoplasias Colorrectales/complicaciones , Estudios de Factibilidad , Femenino , Humanos , Obstrucción Intestinal/etiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Stents , Resultado del TratamientoRESUMEN
Although many cases of solid cystic tumor of the pancreas (SCT) have been reported, its nature and histogenesis remain controversial. We herein report six cases of SCT, including three cases of noncystic type. A review of 22 cases of noncystic type SCT, including our 3 cases, was carried out to compare their features with those of 173 cases of classic SCT reported in Japan. Noncystic type SCTs tend to occur in male patients and are smaller in size and less frequently symptomatic, although they show histological characteristics similar to those of classic SCTs. The developmental process might be a cause of cyst formation. The accumulation and analysis of many, at present, "atypical" cases for clarification of its nature, will, it is hoped, lead to a new nomenclature for this condition that adequately describes its biological origin.