RESUMO
We present molecular dynamics simulation results of a liquid water/methane interface, with and without an oligomer of poly(methylaminoethylmethacrylate), PMAEMA. PMAEMA is an active component of a commercial low dosage hydrate inhibitor (LDHI). Simulations were performed in the constant NPT ensemble at temperatures of 220, 235, 240, 245, and 250 K and a pressure of 300 bar. The simulations show the onset of methane hydrate growth within 30 ns for temperatures below 245 K in the methane/water systems; at 240 K there is an induction period of ca. 20 ns, but at lower temperatures growth commences immediately. The simulations were analyzed to calculate hydrate content, the propensity for hydrogen bond formation, and how these were affected by both temperature and the presence of the LDHI. As expected, both the hydrogen bond number and hydrate content decreased with increasing temperature, though little difference was observed between the lowest two temperatures considered. In the presence of PMAEMA, the temperature below which sustained hydrate growth occurred was observed to decrease. Some of the implications for the role of PMAEMA in LDHIs are discussed.
Assuntos
Metano/química , Modelos Moleculares , Termodinâmica , Água/química , Simulação por Computador , Ligação de Hidrogênio , Ácidos Polimetacrílicos/química , TemperaturaRESUMO
Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Indóis/farmacologia , Pirróis/farmacologia , Células 3T3 , Inibidores da Angiogênese/química , Animais , Antineoplásicos/química , Divisão Celular/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indóis/química , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Oxindóis , Propionatos , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores Mitogênicos/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.
Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mutagênese , Espectrofotometria/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Cnidários/química , Escherichia coli/genética , Proteínas de Fluorescência Verde , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Cifozoários/química , Espectrometria de FluorescênciaRESUMO
Clathrate hydrates are important in both industrial and geological settings. They give rise to many technological and environmental applications, including energy production, gas transport, global warming and CO2 capture and sequestration. In all of these applications there is a need to exert a high degree of control on the crystallisation process, either to promote or inhibit it according to the application. This crystallisation process involves the formation of a tetrahedral hydrogen bonding network (as occurs with ice), but is complicated by mass transport limitations due to the poor mixing of the common guest molecules, such as methane, and the water that forms the host lattice. The net effect is that the mechanisms for hydrate formation and growth are still poorly understood, with the consequence that development of additives to control nucleation and growth is still largely governed by trial-and-error approaches. In this paper we show how classical molecular dynamics simulations can be used to provide a direct simulation of the nucleation process for methane hydrate and consequently to allow direct simulation of the effect of additives on the nucleation and growth process. Data are presented for oligomers of PVP and compared with existing data for PDMAEMA. The results show that the two additives work by very different mechanisms, with PVP increasing the surface energy of the interfacial region and PDMAEMA adsorbing to the surface of hydrate nanocrystals. The surface energy effect is a mechanism that has not previously been considered for hydrate inhibitors.
RESUMO
We examined the role of the Autographa californica nucleopolyhedrovirus (AcMNPV)-encoded chitinase in virus pathogenesis in Trichoplusia ni larvae. In conjunction with the AcMNPV-encoded cathepsin, it promotes liquefaction of the host in the latter stages of infection. Insects infected with virus mutants lacking either the chitinase A gene (chiA) or cathepsin gene (cath) remained intact several days after death. However, if both viruses were used to infect insects, liquefaction of the host was restored. Chitinase was readily detected in AcMNPV-infected insects using a chitinase-specific antibody, but it was absent from insects infected with a chiA deletion mutant (AcchiA-). The chitinase was also detected in polyhedra purified from AcMNPV-infected insects but not in those from AcchiA-. However, polyhedra derived from a virus lacking an intact chiA were no less effective in initiating an infection in second instar T. ni larvae than those of the unmodified AcMNPV. It was also demonstrated that the virus chitinase retained high levels of activity between pH 3.0 and 10.0. In contrast, chitinases isolated from Serratia marcescens, although active under acidic conditions, rapidly lost activity above pH 7.0 illustrating that despite 57% sequence identity, the two proteins have distinct enzymic activities.
Assuntos
Catepsinas/metabolismo , Quitinases/metabolismo , Mariposas/virologia , Nucleopoliedrovírus/enzimologia , Proteínas Virais/metabolismo , Animais , Catepsinas/genética , Quitinases/biossíntese , Quitinases/genética , Deleção de Genes , Genes Virais , Concentração de Íons de Hidrogênio , Insetos , Larva , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/patogenicidade , Proteínas Virais/genéticaRESUMO
A functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.