RESUMO
By means of scanning probe microscopy we demonstrate that Au(+) on NaCl films adsorbs in an embedded, slightly off-centered Cl-Cl bridge position and can be switched between two equivalent mirror-symmetric configurations using the attractive force exerted by a scanning probe tip. Density functional theory calculations demonstrate that the displacement of the Au atom from the centered position of the bridge configuration is accompanied by a large lifting of the closest Cl atom leading to significant changes in the local electrostatic field. Our findings suggest that Au(+) can be used to toggle the local electrostatic field.
RESUMO
PURPOSE: To determine the maximum-tolerated dose (MTD) of paclitaxel administered by 96-hour continuous infusion in combination with cisplatin, to determine if the addition of granulocyte colony-stimulating factor (G-CSF) permits significant paclitaxel dose escalation, and to assess the toxicity and preliminary activity of this combination in patients with advanced lung cancer. PATIENTS AND METHODS: Fifty patients with untreated lung cancer were enrolled: 42 had advanced non-small-cell lung cancer (NSCLC) and eight had extensive-stage small-cell lung cancer (SCLC). Patients received paclitaxel doses of 100 to 180 mg/m2/96 hours and cisplatin doses of 60 to 80 mg/m2 as a single 30-minute bolus injection at the end of the paclitaxel infusion. RESULTS: Two of six patients experienced dose-limiting neutropenia at a dose of paclitaxel 140 mg/m2/96 hours and cisplatin 80 mg/m2. With G-CSF support, one of three patients experienced both dose-limiting mucositis and fatal neutropenic sepsis at a dose of paclitaxel 180 mg/m2/96 hours and cisplatin 80 mg/m2. Significant peripheral neuropathy developed in five patients and occurred after six or more cycles of therapy. Thirty-three of 42 patients with NSCLC had measurable disease; the objective response rate was 55%, with two complete responses and 16 partial responses. For all 42 patients with NSCLC, the median time to progression and median survival duration were 5 months and 10 months, respectively. The actuarial 1-year survival rate was 41%. Of eight SCLC patients, four responded to therapy, and the median survival duration for all SCLC patients was 11 months. CONCLUSION: The MTD without G-CSF is paclitaxel 120 mg/m2/96 hours and cisplatin 80 mg/m2, and the MTD with G-CSF is paclitaxel 160 mg/m2/96 hours and cisplatin 80 mg/m2. Infusional paclitaxel with cisplatin is well tolerated and active in patients with advanced NSCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma de Células Pequenas/sangue , Cisplatino/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Infusões Intravenosas , Injeções Intravenosas , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Resultado do TratamentoRESUMO
The folding of the trimeric phage P22 tailspike protein is influenced by amino acid substitutions of two types, virtually all of which affect residues in the central domain, a large parallel beta-helix. Temperature sensitive folding (tsf) mutations lead to drastically decreased folding yields at elevated temperature. Their phenotype can be alleviated by global suppressor (su) mutations. Both types of mutations appeared to have no influence on the stability of the native protein at the time of their first isolation and were thus suggested to carry information needed for the folding pathway exclusively. The monomeric beta-helix of tailspike, expressed as an isolated domain, exhibits freely reversible unfolding and refolding transitions, allowing us to analyse the effects of two well-characterised tsf and all four known su mutations on its thermodynamic stability. We find a marked decrease in stability for the tsf mutants and a striking increase in stability for all su mutants. This leads to the conception that the isolated beta-helix domain, although active in receptor-binding and native-like in its spectroscopic properties, is close in conformation to a crucial monomeric folding intermediate whose thermolability is responsible for the kinetic partitioning between productive folding and irreversible aggregation during the maturation process of P22 tailspike protein.
Assuntos
Bacteriófago P22/química , Glicosídeo Hidrolases/química , Dobramento de Proteína , Proteínas da Cauda Viral/química , Glicosídeo Hidrolases/genética , Mutação , Estrutura Secundária de Proteína , Termodinâmica , Proteínas da Cauda Viral/genéticaRESUMO
The purpose of the study was to examine inter- and intrapatient variation in 5-fluorouracil (5-FU) plasma concentrations in adult cancer patients receiving a 3-day drug infusion. Fourteen patients received 1266 mg/m2 N-(phosphonacetyl)-L-aspartate (PALA) infused i.v. over 15 min on day 1, followed immediately by a loading dose of 500 mg/m2 calcium leucovorin over 30 min. Then a prolonged infusion of leucovorin at 500 mg/m2/day and 5-FU at 1750 mg/m2/day was administered as either a constant rate or as a circadian infusion over 72 h. During constant rate infusions, 5-FU concentrations within individuals varied by 1.7-fold, but no uniform time of peak or trough concentration was observed. Transformation of these data by setting the time of peak to 0 h and by expressing concentrations as the percentage of the 24-h mean value revealed a nonrandom distribution of the time from peak to trough with a median time of 12 h (P = 0.027). These transformed data were also successfully fit to a circadian cosinor function (P < 0.001). During multiple constant rate 5-FU infusions, the intrapatient variability was high; the times of peak 5-FU concentration occurred at the same approximate sampling time 43% of the time, and troughs coincided 17% of the time. No difference in clinical toxicity was observed when matched constant rate and circadian infusions of 5-FU were compared. High inter- and intrapatient variability exists in 5-FU plasma concentrations in adult cancer patients during constant rate infusions with no evidence of a consistent circadian rhythm in untransformed data.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacocinética , Neoplasias do Sistema Digestório/tratamento farmacológico , Fluoruracila/administração & dosagem , Fluoruracila/farmacocinética , Adulto , Idoso , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/sangue , Cronoterapia , Neoplasias do Colo/sangue , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Sistema Digestório/sangue , Feminino , Fluoruracila/efeitos adversos , Fluoruracila/sangue , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Retais/sangue , Neoplasias Retais/tratamento farmacológico , Reprodutibilidade dos Testes , Neoplasias Gástricas/sangue , Neoplasias Gástricas/tratamento farmacológico , Fatores de TempoRESUMO
Our purpose was to determine the antitumor efficacy and safety profile of the combination of paclitaxel administered by 96-h continuous i.v. infusion followed by bolus cisplatin in patients with untreated advanced non-small cell lung cancer (NSCLC). Fifty-eight patients with untreated advanced or recurrent NSCLC were enrolled between October 1995 and December 1998. The median patient age was 60 years (age range, 34-75 years). Twenty-four patients were female. The majority of patients (n = 52) had an Eastern Cooperative Oncology Group performance status of 0/1. Twelve patients had stage IIIB NSCLC, 43 had stage IV disease, and 3 had recurrent disease after prior resection. Seven patients had received cranial irradiation for brain metastases, and 5 patients had received bone irradiation before enrollment. Patients were treated with paclitaxel (120 mg/m2/96 h) by continuous i.v. infusion followed by cisplatin (80 mg/m2) on day 5. Therapy was administered every 3 weeks as tolerated until disease progression or a maximum of six cycles. A total of 264 cycles of therapy were administered. Twenty-nine patients received all six cycles. Forty-six patients had measurable disease, with 20 patients achieving a partial response, and no complete responses were seen (overall response rate, 43%; 95% confidence interval, 29-60%). The median progression-free survival was 5.5 months. At a median potential follow-up of 27.2 months, the median survival for all 58 enrolled patients was 8.5 months, and the actuarial 1-year survival was 37% (95% confidence interval, 25.9-50.5%). This is the most extensive evaluation of prolonged continuous infusional paclitaxel in patients with advanced-stage cancer. In contrast to predictions from in vitro cytotoxicity models, the regimen does not appear to be obviously superior to shorter infusion times in the clinical setting. Additional trials of this regimen in patients with NSCLC are therefore of low priority.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/efeitos adversos , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel/efeitos adversos , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Intervalo Livre de Doença , Esquema de Medicação , Feminino , Seguimentos , Humanos , Infusões Intravenosas , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Paclitaxel/administração & dosagem , Taxa de SobrevidaRESUMO
A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.
Assuntos
Bacteriófago P22/enzimologia , Estabilidade Enzimática/genética , Glicosídeo Hidrolases/química , Desnaturação Proteica , Proteínas da Cauda Viral/química , Fluorescência , Glicosídeo Hidrolases/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Salmonella/virologia , Temperatura , Triptofano/química , Proteínas da Cauda Viral/genéticaRESUMO
Lung cancer cell lines are between seven and 1,000 times more sensitive to paclitaxel (Taxol; Bristol-Myers Squibb Co, Princeton, NJ) when exposed for 120 hours (5 days) compared with 3-hour exposure. A phase I study of 4-day infusion of paclitaxel plus bolus cisplatin for patients with lung cancer has defined the recommended phase II dose. In this study, paclitaxel infused at 30 mg/m2/d for 4 days followed by a cisplatin bolus of 80 mg/m2 after infusion completion was associated with acceptable hematologic toxicity. Nine of the 16 patients with non-small cell lung cancer treated with at least two cycles of this regimen attained an objective tumor response (one complete response and eight partial responses; overall response rate, 56%). The recommended phase II dose of a 4-day infusion of paclitaxel plus bolus cisplatin followed by the administration of granulocyte colony-stimulating factor has not yet been determined.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cisplatino/efeitos adversos , Relação Dose-Resposta a Droga , Seguimentos , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Infusões Intravenosas , Injeções Intravenosas , Estadiamento de Neoplasias , Neutropenia/induzido quimicamente , Paclitaxel/efeitos adversos , Indução de Remissão , Células Tumorais CultivadasRESUMO
Species-specific and species-common monoclonal antibodies (MAbs) to nerve-specific cell surface epitopes were used to compare pre-treatment techniques for nerve staining. Endogenous peroxidases were inactivated in four ways: (1) 0.3% hydrogen peroxide (H2O2); (2) 1% periodic acid (PA) (pH 1.85-1.95); (3) sodium meta-periodate (10-40 mM, pH 4.5); or (4) HCl (pH 1.80). Staining of chick and quail corneal nerves and dorsal root ganglia (DRG) nerves with the MAbs was species-specific. Staining of chick and quail corneal nerves was unaffected by pre-treatment with 0.3% H2O2, but was eliminated by pre-treatment with 1% PA. Chick and quail DRG nerve staining tolerated 0.3% H2O2, and at least one epitope also tolerated 1% PA. Corneal nerves of both chick and quail displayed concentration-dependent sensitivity to pre-treatment with sodium meta-periodate; DRG nerves were not sensitive to such pre-treatment. Corneal nerves tolerated pre-treatment with HCI (pH 1.80), whereas DRG nerves did not. These findings indicate sensitivity of corneal nerve epitopes to oxidation, in contrast with sensitivity of DRG nerve epitopes to low pH. Results also indicate that tissue trimming regulated whole-mount staining of corneal nerves, suggesting that antibodies cannot diffuse across corneal basement membranes, even after detergent extraction. However, antibodies are able to diffuse laterally into the stroma from any cut edge.
Assuntos
Substância Própria/anatomia & histologia , Gânglios Espinais/anatomia & histologia , Imuno-Histoquímica/métodos , Animais , Embrião de Galinha , Coturnix , Epitopos/química , Imunofluorescência , PeroxidasesRESUMO
The epileptic homozygotes of the Fayoumi strain of chickens (Fepi) are affected by photogenic reflex epilepsy with complete penetrance. Here we demonstrate that they are equally affected by audiogenic reflex epilepsy induced by intense sound stimulation. All the Fepi display sound-induced seizures from hatching to adulthood consisting of initial 'ictal arousal' and running fits usually followed by generalized clonico-tonic convulsions. A running fit is the preconvulsive motor symptom specifically induced by auditory stimulation while neck myoclonus is the preconvulsive motor symptom specifically induced by photic stimulation. The EEG interictal spikes and spike and waves are suppressed and replaced by a desynchronized trace during the seizures of both kinds. Viable neural chimeras were obtained by graft of embryonic brain vesicles from Fepi donors into normal chick embryos. Transfer of the complete audiogenic and photogenic phenotypes was obtained in chimeras resulting from embryonic substitution of both the prosencephalon and mesencephalon. The substitution of the prosencephalon alone resulted in transfer of interictal paroxysmal EEG activity accompanied by the sound and light-induced desynchronization and 'ictal arousal' with no motor seizures. Chimeras with embryonic substitution of the mesencephalon alone displayed running fits and convulsions induced by sound stimulation but only neck myoclonus following light stimulation. The conclusions are reached that: (i) the Fepi is a model of audiogenic and photogenic reflex epilepsy; (ii) in both types, the seizure initiator and the convulsion generator are localized in the brainstem, although reinforcement from telencephalic visual structures is needed to trigger photogenic generalized convulsions.
Assuntos
Encéfalo/fisiopatologia , Quimera/fisiologia , Epilepsia/genética , Estimulação Acústica , Animais , Comportamento Animal/fisiologia , Embrião de Galinha , Galinhas , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia/psicologia , Mutação , Mioclonia/fisiopatologia , Estimulação LuminosaRESUMO
Genetic epilepsy was studied in Fayoumi epileptic (F.Epi) chickens and in neural chimeras obtained by selective substitution of embryonic brain vesicles of F.Epi donors in normal recipient chickens. Typical motor seizures accompanied by convulsions were evoked by intermittent light stimulation in F.Epi and in chimeras having embryonic substitution of the prosencephalon and the mesencephalon. The motor seizure was less severe in chimeras receiving only the prosencephalon. In the F.Epi, as well as in all the chimeras, the EEG during seizures was characterized by a desynchronized (or a flattening) pattern of activity. F.Epi and chimeras had a lower threshold to Metrazol induced seizures than control chickens. The experimental animals show that, in this model, large prosencephalic and mesencephalic areas are involved in the epileptic disease. The epileptic character of this genetic dysfunction is discussed.
Assuntos
Quimera/fisiologia , Eletroencefalografia , Convulsões/fisiopatologia , Animais , Embrião de Galinha , Galinhas , Eletromiografia , Feminino , Masculino , Mesencéfalo/fisiologia , Pentilenotetrazol/farmacologia , Fenótipo , Estimulação Luminosa , Prosencéfalo/fisiologia , Convulsões/genéticaRESUMO
Sixteen patients with untreated locally advanced (n = 15) or recurrent (n = 1) non-small-cell lung cancer (NSCLC) were enrolled in this study between July 1996 and March 1999. Eight patients had stage IIIA NSCLC, seven had stage IIIB disease, and one had recurrent disease after prior resection of stage I disease. Patients were treated with paclitaxel 30 mg/m2/d for 4 days by continuous intravenous infusion followed by cisplatin 80 mg/m2 on day 5. Therapy was administered every 3 weeks until disease progression or a maximum of four cycles. Thoracic radiation was started within 3 to 4 weeks of day 1 of the last cycle of paclitaxel and cisplatin. Fourteen patients (87.5%) received all four cycles of chemotherapy and subsequent radiation therapy. Forty-four percent of patients achieved a partial response, and 1 patient complete response (overall response rate, 50%). The median progression-free survival was 8.8 months. At a median potential follow-up of 3.7 years, the median survival for all 16 enrolled patients was 13.2 months, and the actuarial 1-, 2-, and 3-year survivals were 62.5%, 43.8%, and 21.9%. In contrast to predictions from in vitro cytotoxicity models, the sequential use of prolonged infusional paclitaxel and bolus cisplatin followed by thoracic radiation does not appear to have a greater impact over shorter chemotherapy
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Cisplatino/administração & dosagem , Esquema de Medicação , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Paclitaxel/administração & dosagem , Radioterapia de Alta Energia , Análise de SobrevidaRESUMO
The present therapy experiments with two different transplantable mammary tumors were performed to compare the therapeutic efficacy in BD-VI rats of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) and hexadecylphosphocholine (HPC). Both compounds were administered orally, subcutaneously or intracutaneously at equimolar doses ranging from 4.8 to 88 mumol/kg/day five times per week for two weeks. Under the experimental conditions, both transplanted mammary carcinomas were moderately sensitive to the therapy with either HPC or ET-18-OCH3. Comparing both tumors, TMA2 was more sensitive than TMA1. The activity and toxicity of both compounds were dose-related in both tumor lines. Females seemed to be less sensitive with respect to antineoplastic activity and toxicity. Like ET-18-OCH3, HPC was active also at low, probably noncytotoxic doses associated with no detectable toxicity according to body weight development. This suggests that there are at least two different mechanisms of action that lead to tumor growth inhibition.
Assuntos
Antineoplásicos/uso terapêutico , Colina/análogos & derivados , Neoplasias Mamárias Experimentais/tratamento farmacológico , Éteres Fosfolipídicos/uso terapêutico , Fosforilcolina/análogos & derivados , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Injeções Subcutâneas , Masculino , Éteres Fosfolipídicos/administração & dosagem , Fosforilcolina/administração & dosagem , Fosforilcolina/uso terapêutico , RatosRESUMO
The study reports on the investigation of acute and subacute toxicity and on antineoplastic activity of hexadecylphosphocholine (HPC), the first compound of a new class of antineoplastic chemotherapeutics. In rats, the LD50 of HPC was 606 mumol/kg; the maximum tolerable dose over four weeks was 39 mumol/kg. Symptoms of toxicity were enteritis, spider cell activation in the liver, hemosiderosis in the spleen and reversible transaminase increase. The best therapeutic effect was observed on methylnitrosourea (MNU)-induced mammary carcinoma in the rat. Two transplantable mammary carcinomas in the rat and autochthonous benzo(a)pyrene-induced sarcomas exhibited low-grade sensitivity to HPC. The MXT mammary carcinoma of the mouse, the Walker 256 carcinosarcoma of the rat, and autochthonous acetoxymethylmethylnitrosamine-induced colonic tumors of the rat were not chemosensitive to HPC.
Assuntos
Antineoplásicos/uso terapêutico , Colina/análogos & derivados , Neoplasias Mamárias Experimentais/tratamento farmacológico , Éteres Fosfolipídicos/uso terapêutico , Fosforilcolina/análogos & derivados , Animais , Antineoplásicos/toxicidade , Carcinoma/tratamento farmacológico , Relação Dose-Resposta a Droga , Feminino , Masculino , Éteres Fosfolipídicos/toxicidade , Fosforilcolina/uso terapêutico , Fosforilcolina/toxicidade , Ratos , Ratos Endogâmicos , Fatores SexuaisRESUMO
The effect of human fibrin adhesive applied to the middle ear has been studied in guinea pig. Auditory function was measured using acoustically evoked brainstem responses. Middle and inner ear structures were studied with light, transmission and scanning electron microscopy. A transitory conductive hearing loss was observed, but after 8 weeks the auditory function appeared normal. Microscopy of the middle and inner ear failed to show any tissue damage.
Assuntos
Aprotinina/toxicidade , Orelha Interna/efeitos dos fármacos , Orelha Média/efeitos dos fármacos , Fator XIII/toxicidade , Fibrinogênio/toxicidade , Audição/efeitos dos fármacos , Trombina/toxicidade , Adesivos Teciduais/toxicidade , Animais , Audiometria de Resposta Evocada , Limiar Auditivo/efeitos dos fármacos , Tronco Encefálico/fisiologia , Combinação de Medicamentos/toxicidade , Orelha Interna/ultraestrutura , Orelha Média/ultraestrutura , Potenciais Evocados Auditivos/efeitos dos fármacos , Feminino , Adesivo Tecidual de Fibrina , Cobaias , Humanos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Mucosa/efeitos dos fármacos , Mucosa/ultraestrutura , Tempo de Reação/efeitos dos fármacosRESUMO
Microfluidic mixing devices are increasingly popular tools for probing the non-equilibrium dynamics of biomolecular systems. Commonly, hydrodynamic focusing is used to reduce the length scales that limit the time of diffusive mixing in the laminar flow regime, such that even sub-millisecond dead times for triggering a reaction have been achieved. Detection of a suitable signal at different points along the channel downstream of the mixing region, corresponding to different times after mixing, then allows the kinetics of the reaction to be obtained. However, the requisite accurate conversion of the positions in the channel to times after mixing is complicated by Taylor dispersion, the combined effect of diffusion and shear flow on the dispersion of the molecules in the microfluidic device. As a result, an accurate position-to-time conversion has only been possible in the limiting regimes, i.e. for very early times, where sample diffusion can be neglected, and for very long times, where the molecules have uniformly sampled the entire channel cross-section. Here, we use detailed three-dimensional, time-dependent finite-element calculations to obtain an accurate position-to-time conversion that bridges these two limits and allows us to quantify the effects of Taylor dispersion on the time resolution of a representative mixing device optimized for single-molecule fluorescence detection. The accuracy of the calculations is confirmed by direct comparison of the calculated velocity field with dual-focus fluorescence correlation spectroscopy measurements.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Difusão , Desenho de Equipamento , Análise de Elementos Finitos , Hidrodinâmica , Técnicas Analíticas Microfluídicas/métodosRESUMO
AIM: It is unknown how the heart distinguishes various overloads, such as exercise or hypertension, causing either physiological or pathological hypertrophy. We hypothesize that alpha-calcitonin-gene-related peptide (αCGRP), known to be released from contracting skeletal muscles, is key at this remodelling. METHODS: The hypertrophic effect of αCGRP was measured in vitro (cultured cardiac myocytes) and in vivo (magnetic resonance imaging) in mice. Exercise performance was assessed by determination of maximum oxygen consumption and time to exhaustion. Cardiac phenotype was defined by transcriptional analysis, cardiac histology and morphometry. Finally, we measured spontaneous activity, body fat content, blood volume, haemoglobin mass and skeletal muscle capillarization and fibre composition. RESULTS: While αCGRP exposure yielded larger cultured cardiac myocytes, exercise-induced heart hypertrophy was completely abrogated by treatment with the peptide antagonist CGRP(8-37). Exercise performance was attenuated in αCGRP(-/-) mice or CGRP(8-37) treated wild-type mice but improved in animals with higher density of cardiac CGRP receptors (CLR-tg). Spontaneous activity, body fat content, blood volume, haemoglobin mass, muscle capillarization and fibre composition were unaffected, whereas heart index and ventricular myocyte volume were reduced in αCGRP(-/-) mice and elevated in CLR-tg. Transcriptional changes seen in αCGRP(-/-) (but not CLR-tg) hearts resembled maladaptive cardiac phenotype. CONCLUSIONS: Alpha-calcitonin-gene-related peptide released by skeletal muscles during exercise is a hitherto unrecognized effector directing the strained heart into physiological instead of pathological adaptation. Thus, αCGRP agonists might be beneficial in heart failure patients.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cardiomegalia Induzida por Exercícios/fisiologia , Miócitos Cardíacos/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Consumo de Oxigênio/fisiologiaAssuntos
Compostos de Bifenilo/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/análise , Mitocôndrias Hepáticas/enzimologia , Animais , Antimicina A/farmacologia , Azidas/farmacologia , Cianetos/farmacologia , Citocromos/metabolismo , Dinitrofenóis/farmacologia , Histocitoquímica , Métodos , Microscopia Eletrônica , Mitocôndrias Hepáticas/metabolismo , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Coloração e RotulagemRESUMO
A bioluminescence-based assay for enumeration of lytic bacteriophage was developed. The assay consists of a bioluminescent Escherichia coli as the host bacterium, the lytic bacteriophage T4 and an automated luminometer measuring luminescence over time. The assay is based on the decrease in luminescence as the bioluminescent host cells are lysed by T4. The T4 concentration, bioluminescent E. coli concentration, phage suspension medium, and temperature (25 degrees C and 37 degrees C) were varied. There was a strong negative correlation between bioluminescence intensities and T4 phage concentrations at both room temperature (R(2)=0.993) and 37 degrees C (R(2)=0.970). Phage was detected more rapidly at 37 degrees C than at 25 degrees C. The detection limit was also lower when the assay was performed at 37 degrees C with a minimum detection level of 2.4 log CFU/ml compared to 3.4 log CFU/ml for 25 degrees C. The assay was used to determine thermal inactivation using T4 phages heated at 70 degrees C for 0 to 30 min, and phage concentrations were determined using the bioluminescence assay and a standard plaque assay. There was no significant difference between the two enumeration methods (P>0.01). This study suggests the bioluminescence-based assay can be used as an alternative for quantitatively monitoring phage infectivity, instead of conventional standard plaque assays.
Assuntos
Bacteriófago T4/isolamento & purificação , Escherichia coli/virologia , Luminescência , Automação , Sensibilidade e Especificidade , Temperatura , Ensaio de Placa ViralRESUMO
This study tested the hypothesis that maximal oxygen uptake (VO(2max)) and performance increase upon altitude acclimatization at moderate altitude. Eight elite cyclists were studied at sea level, and after 1 (Day 1), 7 (Day 7), 14 (Day 14) and 21 (Day 21) days of exposure to 2340 m. Capillary blood samples were taken on these days before performing two consecutive maximal exercise trials. Acclimatization increased hemoglobin concentration and arterial oxygen content. On Day 1, VO(2max) and time to exhaustion (at 80% of sea-level maximal power output) decreased by 12.8% (P<0.05) and 25.8% (P<0.05), respectively, compared with the corresponding sea-level values. Subsequently, these parameters increased by 3.2% (P<0.05) and 6.0% (P<0.05) from Days 1 to 7, by 4.8% (P<0.05) and 5.7% (P<0.05) from Days 7 to 14, followed by 0.7% (P>0.05) and 1.4% (P>0.05) from Days 14 to 21, respectively. These data suggest that endurance athletes competing at altitudes around 2340 m should expose themselves to this altitude at least 14 days before competition.
Assuntos
Altitude , Ciclismo/fisiologia , Exercício Físico/fisiologia , Hemoglobinas , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Resistência Física/fisiologia , Aclimatação , Adulto , Antropometria , Dopagem Esportivo , Eritropoetina , Humanos , Hipóxia , Masculino , Estudos Prospectivos , Análise e Desempenho de Tarefas , Fatores de TempoRESUMO
In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel beta-helix, is essentially structured in this species. We used the isolated beta-helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.