RESUMO
Water-soluble upconversion nanoparticles (UCNPs), based on polyvinylpyrrolidone (PVP)-coated NaYF4:Er3+,Yb3+,Gd3+, with various concentrations of Gd3+ ions and relatively high upconversion efficiencies, were synthesized. The internalization and cytotoxicity of the thus obtained UCNPs were evaluated in three cell lines (HeLa, HEK293 and astrocytes). No cytotoxicity was observed even at concentrations of UCNPs up to 50 µg ml-1. The fate of the UCNPs within the cells was studied by examining their upconversion emission spectra with confocal microscopy and confirming these observations with transmission electron microscopy. It was found that the cellular uptake of the UCNPs occurred primarily by clathrin-mediated endocytosis, whereas they were secreted from the cells via lysosomal exocytosis. The results of this study, focused on the mechanisms of the cellular uptake, localization and secretion of UCNPs, demonstrate, for the first time, the co-localization of UCNPs within discrete cell organelles.
RESUMO
Analysis of the inhibition of gelation of deoxyhemoglobin S by the alkylureas and alkylamides, based on multiple equilibria formulation of the interactions of the inhibiting reagent and protein, is described and applied to minimum gelling concentration and saturation-solubility measurements in the presence of these inhibitors. Our principal expression which describes the influence of a given inhibiting reagent on the hemoglobin activity at the critical gelling concentration, (a)D = (a)w exp(N'KB[D]) has been used to predict the parameters expected of a given inhibitor, and also to provide information concerning the nature and the number of amino acids at the areas of contact of the hemoglobin tetramers that form the microtubular fibers of the gel phase of deoxyhemoglobin S. In this expression N' represents the effective number of amino acid sites per hemoglobin tetramer or the pregelation unit that enters the gelation reaction, KB is the interaction constant characterizing the interaction between the average amino acid and the inhibiting reagent, and [D] is the concentration of the inhibiting reagent. The analysis of our data includes correction for non-ideality. Our calculated gelling concentrations and saturation-solubilities are in good accord with the experimental data obtained with the various alkylureas and amides of increasing hydrophobicity or hydrocarbon content, demonstrating the utility of such analysis for the rational design of pharmacologically significant new reagents. In this regard it is to be noted that the interaction parameter, KB, used to predict the effectiveness of a given reagent is evaluated by assuming additivity of the group values of the chemical constituents of the reagent. Analysis of our data obtained as a function of concentration of several ureas (ethyl-, propyl-, butyl-, and n-pentylurea) gave N' estimates in the neighborhood of 80--100, suggesting that a substantial fraction of the surface groups of hemoglobin must be removed from contact with solvent in the course of the polymerization reaction that leads to the formation hemoglobin S gels.
Assuntos
Amidas/farmacologia , Hemoglobina Falciforme , Ureia/análogos & derivados , Relação Dose-Resposta a Droga , Géis , Humanos , Substâncias Macromoleculares , Matemática , Solubilidade , Termodinâmica , Ureia/farmacologiaRESUMO
The kinetics of surface pressure change (deltapi vs. t isotherms) were determined for several single point mutations of the human hemoglobin system. It was observed that hemoglobin S and hemoglobin CHarlem (both containing beta6 Glu leads to Val substitutions) have a specific behavior at the water-air interface: their extent of surface pressure change is larger than for hemoglobin A, hemoglobin C and hemoglobin Korle Bu (beta73 Asp leads to Asn). In addition, hemoglobin S seems to occupy a larger area per molecule than hemoglobin A. The conformational requirements for this property, in addition to the beta6 Val substitution, appear to be the liganded state of the betas chain in the tetramer. Electrostatic, hydrogen bonding and hydrophobic interactions are involved in determining the surface activity of a hemoglobin molecule. The differences between the surface activity of oxyhemoglobin S and oxyhemoglobin A could be the basis for their differences in mechanical precipitability, although other factors may play a role.
Assuntos
Hemoglobina Falciforme , Hemoglobinas Anormais , Asparagina , Ácido Aspártico , Glutamatos , Hemoglobinas , Humanos , Cinética , Lisina , Cidade de Nova Iorque , Concentração Osmolar , Oxigênio/sangue , Pressão , Conformação Proteica , Cloreto de Sódio/sangue , Propriedades de Superfície , Termodinâmica , ValinaRESUMO
Using a light scattering (turbidity) method to estimate the delay time of gelation of deoxy hemoglobin S hemolysates, we have examined the effects of various alkylureas, variations in ionic strength by addition of NaCl, and admixture with other hemoglobins on gelation kinetics. Each of these factors substantially prolonged the delay times to different extents, but the dependence of the delay times on a high power of the hemoglobin concentration varied only slightly. These findings suggest that the events preceding gelation, most likely the formation of nuclei, are affected by these factors, but the critical nuclear size for gelling is fairly constant. Parallel changes indicated good qualitative correlation between gelation, delay times, minimum gelling concentrations of solutions of mixed hemoglobins and kinetics of sickling of red cells containing these mixtures, with the exception of hemoglobin Charlem trait cells the sickling kinetics of which were slower than predicted by the solution properties.
Assuntos
Hemoglobina Falciforme , Hemoglobinas , Ureia/análogos & derivados , Géis , Humanos , Cinética , Compostos de Metilureia , Concentração Osmolar , Conformação ProteicaRESUMO
Corpora lutea from pregnant rats were incubated to determine their ability to produce 17beta-estradiol and to aromatize testosterone in vitro. Corpora lutea and non-luteal ovarian tissues were removed from rats on days 7, 15, and 22 of pregnancy, and these tissues were immediately frozen or incubated separately in medium 199 at 37 C in an atmosphere of 95% O2-5% CO2 for 4 h. 17Beta-Estradiol in tissue and medium were quantified by a highly specific radioimmunoassay. The estradiol content ivnariably increased in non-luteal tissues during incubation, while it decreased or remained the same in incubated corpora lutea. The synthesis in non-luteal tissues, which was 18 to 400-fold greter. The incubation of corpora lutea (5 to 25 mg of tissue) with testosterone (200 ng) on days 7, 15, and 22 of pregnancy resulted in a mean accumulation of 17beta-estradiol in medium of 2.5 x 103 pg/mg tissue, compared with a mean value of 6 pg/mg for luteal tissue removed from the same ovaries and incubated without testosterone. The incubation of corpora lutea from 15-day pregnant rats with (7alpha-3H)-testosterone resulted in 15% conversion to presumptive (7alpha-3H)17beta-estradiol, which was isolated identically to estradiol isolated for radioimmunoassay. Recrystallization to constant specific activity revealed a high degree of radiochemical purity (75%) of the isolated (3H)estradiol. Rat diaphragm muscle and rabbit corpora lutea did not aromatize testosterone to 17beta-estradiol in amounts detectable by radioirpora lutea in vitro is virtually diol by non-luteal ovarian tissues. However,the corpora lutea show a striking capacity to aromatize testosterone, which might explain the high estradiol content of the rat corpora lutea during pregnancy. The physiological significance of this aromatizing system and of 17beta-estradiol in the corpus luteum is unknown but may be related to the luteotropic action of estradiol in the pregnant rat.
Assuntos
Corpo Lúteo/metabolismo , Estradiol/biossíntese , Testosterona/metabolismo , Animais , Castração , Feminino , Idade Gestacional , Ovário/metabolismo , Gravidez , Prenhez , Radioimunoensaio , RatosRESUMO
Normal human plasma contains "inactive renin," whose ability to generate angiotensin I increases after exposure to pH 3.3. Big renin is a partially inactive enzyme of larger molecular weight, which is also activated at pH 3.3, and is found of pregnant women, and in amniotic fluid, but not in normal plasma. We have compared the effects of acid exposure and storage at 4 and -4 C on normal plasma and plasma containing big renin. The concentration of inactive renin in normal plasma was approximately equal to that of normal active renin, and its activity increased slowly on prolonged standing at -4 but not 4 C. In contrast, the activity of big renin increased by 50% as early as 1-3 days at 4 C and increased even more quickly at -4 C. Acid treatment of plasma containing big renin caused 4-10 times greater increase in active renin than similar treatment of normal plasma. During gel filtration, both cold-activated and previously acidified big renin coeluted with unactivated big renin. These data indicate that big renin is highly susceptible to cold or acid activation and that such activation of big renin does not result in a detectable decrease in its molecular weight of 60,000 daltons. Furthermore, acid and cold seem to activate the same pool of inactive renin in normal plasma. Although both normal and big renin are stable for long periods below -20 C, a serious overestimate of plasma renin activity can occur if plasma is stored just above its freezing point before assay.
Assuntos
Temperatura Baixa , Precursores Enzimáticos/sangue , Renina/sangue , Líquido Amniótico/enzimologia , Cromatografia em Gel , Nefropatias Diabéticas/sangue , Ativação Enzimática , Feminino , Humanos , Concentração de Íons de Hidrogênio , Gravidez , Renina/metabolismoRESUMO
The protein kinase family represents both a huge opportunity and a challenge for drug development. The conservation of structural features within the ATP binding cleft initially led to the belief that specificity would be difficult to achieve. This dogma has now been clearly dispelled with the discovery and clinical testing of a group of first generation compounds, which are characterized by a high degree of selectivity towards a variety of oncology targets. The structural basis for selectivity and potency has now been clarified with the crystallization of a number of such targets in complex with inhibitors. The protein kinase inhibitor field is now ripe for the structure based exploitation of additional highly validated targets from a variety of therapeutic areas.
Assuntos
Trifosfato de Adenosina/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Adenilil Imidodifosfato/química , Antraquinonas/química , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Quinases Ciclina-Dependentes/química , Inibidores Enzimáticos/química , Humanos , Cinetina , Piperazinas/química , Proteínas Tirosina Quinases/efeitos dos fármacos , Purinas/química , Piridinas/química , Pirimidinas/química , Pirróis/química , Quinazolinas/química , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/efeitos dos fármacos , Roscovitina , Estaurosporina/química , Relação Estrutura-AtividadeRESUMO
Several tests are currently available to assist in the diagnosis of the hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Tests that actually detect or quantify these viruses are based on the polymerase chain reaction (PCR) technique. However, the application of PCR is limited by the cost, labor, time-consumption, and potential for contamination. In this article we describe some procedures developed to reduce these limitations. We have developed and validated simultaneous detection methods for HIV RNA and HCV RNA in single serum samples using Amplicor PCR tests. The sensitivity and specificity of this method are comparable with the results obtained with commercial reverse transcription polymerase chain reaction (RT-PCR) techniques for HIV and HCV RNA detection. In addition we have modified the HIV Amplicor test for the RT-PCR procedure and the Chomczynski's method of RNA isolation. We hope that our method can find same applications in HIV and HCV coinfection research, blood screening, and medical diagnosis.
Assuntos
HIV/genética , Hepacivirus/genética , RNA Viral/sangue , Infecções por HIV/sangue , Infecções por HIV/virologia , Hepatite C/sangue , Hepatite C/virologia , Humanos , Reação em Cadeia da Polimerase/métodos , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA , Distribuição Aleatória , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A facile sol-gel synthesis of novel ZnO/MgO/Fe2O3 nanoparticles (NPs) is reported and their performance is compared to that of ZnO/MgO. Powder x-ray diffraction (XRD) patterns reveal the crystal structure of the prepared samples. The average particle size of the sample was found to be 4.8 nm. The optical properties were determined by UV-vis absorption and fluorescence measurements. The NPs are stable in biologically relevant solutions (phosphate buffered saline (PBS), 20 mM, pH = 7.0) contrary to ZnO/MgO NPs which degrade in the presence of inorganic phosphate. Superparamagnetic properties were determined with a superconducting quantum interference device (SQUID). Biocompatible and stable in PBS ZnO/MgO/Fe2O3 core/shell composite nanocrystals show luminescent and magnetic properties confined to a single NP at room temperature (19-24 ° C), which may render the material to be potentially useful for biomedical applications.
Assuntos
Compostos Férricos/química , Medições Luminescentes/métodos , Óxido de Magnésio/química , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Óxido de Zinco/química , Cristalização/métodos , Impedância Elétrica , Substâncias Macromoleculares/química , Campos Magnéticos , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Propriedades de SuperfícieAssuntos
Angina Pectoris/fisiopatologia , Pulso Arterial , Adulto , Pressão Sanguínea , Eletrocardiografia , Feminino , Humanos , Contração MiocárdicaRESUMO
After 4 hours of exposure to incandescent light, 80% of 0.05% topical isotretinoin and 60% of 0.05% topical tretinoin preparations remained in their original form. In contrast, after 2 hours of exposure to fluorescent light only 25% of topical tretinoin and possibly 60% of topical isotretinoin remained in their original forms. Longer exposure to fluorescent light did not result in further breakdown, and the final breakdown of both preparations was similar. A 12-week, double-blind clinical trial comparing isotretinoin (0.05%) with tretinoin (0.05%) applied topically to patients with moderate acne was carried out. Both preparations caused significant reductions in papules and pustules. However, neither treatment was significantly superior to the other in the reduction of acne lesions. This may be because both preparations are rapidly broken down to similar products when exposed to fluorescent light.
Assuntos
Acne Vulgar/tratamento farmacológico , Isotretinoína/administração & dosagem , Tretinoína/administração & dosagem , Administração Cutânea , Cromatografia Líquida de Alta Pressão , Método Duplo-Cego , Estabilidade de Medicamentos , Humanos , Isotretinoína/análise , Luz , Distribuição Aleatória , Tretinoína/análiseRESUMO
Interactions between human red cell's band 4.1 and spectrin were studied by fluorescence resonance energy transfer and batch microcalorimetry techniques. The association constant (Ka = 8.6 X 10(7) M-1), the stoichiometry (one molecule of band 4.1 to one molecule of spectrin), the reversibility, and the enthalpy (delta H = -6 kcal/mol) were determined. A proton uptake was observed to take place as a result of the spectrin-band 4.1 complex formation. In addition to the protonation of the reaction products, the entropic contribution (-T delta S) has been observed to be responsible for approximately 50% of the binding free energy. We concluded that the environment plays a significant role in the stabilization of the complex. Since band 4.1 has been required for the maintenance of the cytoskeletal stability, small alterations of the binding energies or the degree of interaction could have a pronounced effect on the structure of the erythrocyte membrane.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas do Citoesqueleto , Membrana Eritrocítica/metabolismo , Proteínas de Membrana/sangue , Neuropeptídeos , Espectrina/metabolismo , Corantes Fluorescentes , Humanos , Cinética , EspectrofotometriaRESUMO
The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.
Assuntos
Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Hemoglobina A/metabolismo , Hemoglobina Falciforme/metabolismo , Trifosfato de Adenosina/metabolismo , Endocitose , Humanos , Concentração Osmolar , Oxiemoglobinas/metabolismoRESUMO
Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.
Assuntos
Anquirinas , Proteínas do Citoesqueleto , Membrana Eritrocítica/metabolismo , Proteínas de Membrana/sangue , Neuropeptídeos , Proteínas Sanguíneas/metabolismo , Fluoresceína-5-Isotiocianato , Fluoresceínas , Corantes Fluorescentes , Glicoforinas/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Espectrina/metabolismo , TiocianatosRESUMO
beta-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733-738]. In the present work we compare the ability of beta-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of beta-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human beta-amyloid (1-40), containing three amino-acid substitutions (Arg5-->Gly, Tyr10-->Phe and His13-->Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that beta-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of beta-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to beta-amyloid toxicity.