RESUMO
Many steps in programmed cell death are evolutionarily conserved across different species. The Caenorhabditis elegans proteins CED-9, CED-4 and EGL-1 involved in apoptosis are respectively homologous to anti-apoptotic Bcl-2 proteins, Apaf-1 and the "BH3-only" pro-apototic proteins in mammals. In the linear apoptotic pathway of C. elegans, EGL-1 binding to CED-9 leads to the release of CED-4 from CED-9/CED-4 complex. The molecular events leading to this process are not clearly elucidated. While the structures of CED-9 apo, CED-9/EGL-1 and CED-9/CED-4 complexes are known, the CED-9/CED-4/EGL-1 ternary complex structure is not yet determined. In this work, we modeled this ternary complex and performed molecular dynamics simulations of six different systems involving CED-9. CED-9 displays differential dynamics depending upon whether it is bound to CED-4 and/or EGL-1. CED-4 exists as an asymmetric dimer (CED4a and CED4b) in CED-9/CED-4 complex. CED-4a exhibits higher conformational flexibility when simulated without CED-4b. Principal Component Analysis revealed that the direction of CED-4a's winged-helix domain motion differs in the ternary complex. Upon EGL-1 binding, majority of non-covalent interactions involving CARD domain in the CED-4a-CED-9 interface have weakened and only half of the contacts found in the crystal structure between α/ß domain of CED4a and CED-9 are found to be stable. Additional stable contacts in the ternary complex and differential dynamics indicate that winged-helix domain may play a key role in CED-4a's dissociation from CED-9. This study has provided a molecular level understanding of potential intermediate states that are likely to occur when CED-4a is released from CED-9.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Ligação ao Cálcio , Proteínas Repressoras , Animais , Apoptose , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Interactions between pro- and anti-apoptotic Bcl-2 proteins decide the fate of the cell. The BH3 domain of pro-apoptotic Bcl-2 proteins interacts with the exposed hydrophobic groove of their anti-apoptotic counterparts. Through their design and development, BH3 mimetics that target the hydrophobic groove of specific anti-apoptotic Bcl-2 proteins have the potential to become anticancer drugs. We have developed a novel computational method for designing sequences with BH3 domain features that can bind specifically to anti-apoptotic Mcl-1 or Bcl-XL. In this method, we retained the four highly conserved hydrophobic and aspartic residues of wild-type BH3 sequences and randomly substituted all other positions to generate a large number of BH3-like sequences. We modeled 20000 complex structures with Mcl-1 or Bcl-XL using the BH3-like sequences derived from five wild-type pro-apoptotic BH3 peptides. Peptide-protein interaction energies calculated from these models for each set of BH3-like sequences resulted in negatively skewed extreme value distributions. The selected BH3-like sequences from the extreme negative tail regions have highly favorable interaction energies with Mcl-1 or Bcl-XL. They are enriched in acidic and basic residues when they bind to Mcl-1 and Bcl-XL, respectively. With the charged residues often away from the binding interface, the overall electric field generated by the charged residues results in strong long-range electrostatic interaction energies between the peptide and the protein giving rise to high specificity. Cell viability studies of representative BH3-like peptides further validated the predicted specificity. This study has revealed the importance of non-hot spot residues in BH3-mimetic peptides in providing specificity to a particular anti-apoptotic protein.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo , Sequência de Aminoácidos , Humanos , Células MCF-7 , Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Proteína bcl-X/químicaRESUMO
Supervised field trials were conducted at the research farms of four agricultural universities located at different agro-climatic zones of India to find out the harvest time residues of flubendiamide and its des-iodo metabolite on pigeon pea (Cajanus cajan) during the year 2006-2007. Two spray applications of flubendiamide 20 WDG at 50 g (T(1)) and 100 g (T(2)) a.i./ha were given to the crop at 15-days interval. The foliage samples at different time intervals were drawn at only one location, however, the harvest time samples of pigeon pea grain, shell, and straw were drawn at all the four locations. The residues were estimated by HPLC coupled with UV-VIS variable detector. No residues of flubendiamide and its des-iodo metabolite were found at harvest of the crop at or above the LOQ level of 0.05 µg/g. On the basis of the data generated, a pre-harvest interval (PHI) of 28 days has been recommended and the flubendiamide 20 WDG has been registered for use on pigeon pea by Central Insecticide Board and Registration Committee, Ministry of Agriculture, Government of India and the MRL has been fixed by Ministry of Health and Family Welfare, Government of India under Prevention of Food and Adulteration as 0.05 µg/g on pigeon pea grains.
Assuntos
Benzamidas/análise , Cajanus/química , Monitoramento Ambiental , Inseticidas/análise , Resíduos de Praguicidas/análise , Sulfonas/análise , Agricultura , Cajanus/metabolismo , Clima , Contaminação de Alimentos/estatística & dados numéricos , Meia-Vida , ÍndiaRESUMO
Multi-location supervised field trials were conducted at four different agro climatic locations in India to evaluate the dissipation pattern of deltamethrin on chili. Deltamethrin 10 EC was applied on chili @17.5 and 35 g a.i. ha(-1), samples of green chili were drawn at different time intervals and that of red chili and soil at harvest time and quantified by gas liquid chromatography equipped with electron capture detector. The identity of residues were confirmed by Gas Chromatograph-Mass Spectrophotometer in selective ion monitoring mode in mass range 181, 253 m/z. Limit of quantification of the method was found to be 0.01 mg kg(-1). Half-life of deltamethrin at application rate of 17.5 g a.i. ha(-1) varied from 0.36 to 1.99 days and at double the application rate was found to range from 0.38 to 2.06 days. Residues of deltamethrin were found below its determination limit of 0.01 mg kg(-1) in red chili and soil. On the basis of the data generated, Deltamethrin 10 EC has been registered for use on chili in India and its Maximum Residue Limit has been fixed as 0.05 µg/g.
Assuntos
Capsicum/química , Inseticidas/análise , Nitrilas/análise , Piretrinas/análise , Agricultura , Clima , Monitoramento Ambiental , Poluentes Ambientais/análise , Índia , Cinética , Modelos Químicos , Resíduos de Praguicidas/análiseRESUMO
CONTEXT: Role of CD105(Endoglin) in Pathogenesis and progression of OLP. AIM: To assess the role of neoangiogenesis in the progression of OLP by determining the expression of CD105 in OLP and normal mucosa. SETTINGS AND DESIGN: The present study includes a total of 70 formalin-fixed paraffin-embedded blocks of which the study group comprises 50 tissue sections histopathologically confirmed as OLP. They were subdivided into two groups - Group I (Reticular OLP) and Group II (Erosive OLP) - 25 each. The control group (designated as Group III) included 20 sections of normal mucosa. MATERIALS AND METHODS: All the sections were 4 µm thick and stained with CD105 antibodies. After identifying areas of highest vascularity (hot spots) in low power (×10) magnification, individual microvessels were counted manually at high power (×40) magnification. STATISTICAL ANALYSIS USED: Analysis of variance test was used to determine the difference of microvessel density (MVD) between variants of OLP and normal mucosa and Cohen's kappa statistic was used to check interobserver variability. RESULTS: CD105 staining showed a mean MVD of 1.31 ± 1.8 in the normal mucosa compared to 1.68 ± 1.4 and 4.14 ± 2.7 in the reticular and erosive variants, respectively, with a P = 0.000*, which is statistically significant (*P < 0.05 is statistically significant). CONCLUSION: Based on our observations, it is evident that compared to normal mucosa, MVD is greater in lichen planus. Within the two variants of OLP, MVD is higher in Erosive variant compared with Reticular variant, foreshadowing the role of neoangiogenesis in the progression of OLP and its possible malignant transformation.