Conservation Of Physicochemical Properties Research Articles

Preparation, characterization and in vitro evaluation of sterically stabilized liposome containing a naphthalenediimide derivative as anticancer agent

The aim of this study was to incorporate a new naphthalenediimide derivative (AN169) with a promising anticancer activity into pegylated liposomes to an extent that allows its in vitro and in vivo testing without use of toxic solvent. AN169-loaded liposomes were prepared using the thin-film hydration method and characterized for size, polydispersity index, drug content and drug release. We examined their lyophilization ability in the presence of cryoprotectants (trehalose, sucrose and lysine) and the long-term stability of the lyophilized products stored at 4 °C for 3 and 6 months by particle size changes and drug leakage. AN169 was successfully loaded into liposomes with an entrapment efficiency of 87.3 ± 2.5%. The hydrodynamic diameter of these liposomes after sonication was ∼145 nm with a high degree of monodispersity. Trehalose was found to be superior to the other lyoprotectants. In particular, trehalose 1:10 lipid:cryoprotectant molar ratio may provide stable lyophilized liposomes with the conservation of physicochemical properties upon freeze-drying and long-term storage conditions. We also assessed their in vitro antitumor activity in human cancer cell lines (HTLA-230 neuroblastoma, Mel 3.0 melanoma, OVCAR-3 ovarian carcinoma and SV620 prostate cancer cells). However, only after 72 h incubation, loaded liposomes showed almost the same IC50 as free AN169. In conclusion, we developed a stable lyophilized liposomal formulation for intravenous administration of AN169 as anticancer drug, with the advantage of avoiding the use of potentially toxic solubilizing agents for future in vivo experiments.

Phylogeny and Polymorphism in the Long Control Region, E6, and L1 of Human Papillomavirus Types 53, 56, and 66 in Central Brazil

Several studies related that different human papillomavirus (HPV) types and intratype variants can present different oncogenic potential. In opposite to HPVs 16 and 18 variants, information about variants of other carcinogenic HPV types is still scarce. The aim of this study was to investigate the genetic variability of HPVs 53, 56, and 66 from Central Brazil isolates. The long control region (LCR), E6, and L1 genomic regions were amplified and sequenced. We evaluate for nucleotide variations in relation to the reference sequence of each HPV type and also the conservation of physicochemical properties of the deduced amino acid substitutions. In silico analysis was performed to locate binding sites for transcriptional factors within the LCR. Moreover, we performed a phylogenetic analysis with the Central Brazilian and worldwide sequences available at genomic databases. Gathering LCR, E6, and L1 genomic regions, the highest genetic variability was found among HPV-53 isolates with 52 nucleotide variations, followed by HPVs 56 and 66 with 24 and 16 nucleotide substitutions, respectively. The genetic analysis revealed 11 new molecular variants of all HPV types analyzed, totalizing 31 new nucleotide and 3 new amino acid variations. Eight nonconservative amino acid substitutions were detected, which may indicate a biological and pathogenic diversity among HPV types. Furthermore, 8 nucleotide substitutions were localized at putative binding sites for transcription factors in the LCR with a potential implication on viral oncogene expression. The HPVs 53, 56, and 66 phylogenetic analysis confirmed a dichotomic division only described to HPV subtypes and different from the patterns described for HPVs 16 and 18 variants. The high genetic variability observed emphasizes the importance of investigating polymorphisms in types other than HPVs 16 or 18 to better understand the molecular genomic profile of viral infection by different HPV types.

Flavitrack analysis of the structure and function of West Nile non-structural proteins

The Flavitrack database groups Flaviviruses (FV), evolutionarily related organisms with high subtype variability, according to their phenotypes. Here, PCPMer tools were used to calculate consensus sequences based on conservation of Physicochemical Properties (PCP) for 919 sequences of NS2a, a non-structural protein involved in preventing host interferon response to infection. Conserved PCP-motifs were detected, primarily in the N-terminal half of NS2a. One model structure, based on a nuclear receptor, groups residues essential for West Nile (WN) infectivity (I59, V61, and M103) in a pocket on the protein surface. These methods will aid in the design of vaccines and specific therapies against FV.

The Action of Key Factors in Protein Evolution at High Temporal Resolution

BackgroundProtein evolution is particularly shaped by the conservation of the amino acids' physico-chemical properties and the structure of the genetic code. While conservation is the result of negative selection against proteins with reduced functionality, the codon sequences determine the stochastic aspect of amino acid exchanges. Thus far, it is known that the genetic code is the dominant factor if little time has elapsed since the divergence of one gene into two, but physico-chemical forces gain importance at greater evolutionary distances. Further details, however, on how the influence of these factors varies with time are unknown to date.Methodology/Principal FindingsHere, we derive each 10,000 divergence specific substitution matrices for orthologues and paralogues from the Pfam collection of multiple protein alignments and quantify the action of three physico-chemical forces and of the structure of the genetic code at high resolution using correlation analysis. For closely related proteins, the codon sequence similarity is the most influential factor controlling protein evolution, but its influence decreases rapidly as divergence grows. From a protein sequence divergence of about 20 percent on the maintenance of the hydrophobic character of an amino acid is the most influential factor. All factors lose importance from about 40 percent divergence on. This suggests that the original protein structure often does no longer represent a constraint to the protein sequence. The proteins then become free to adopt new functions. We furthermore show that the constraints exerted by both physico-chemical forces and by the genetic code are quite comparable for orthologues and paralogues, however somewhat weaker for paralogues than for orthologues in weakly or moderately diverged proteins.Conclusion/SignificanceOur analysis substantiates earlier findings that protein evolution is mainly governed by the structure of the genetic code in the early phase after divergence and by the conservation of physico-chemical properties at the later phase. We determine the level of sequence divergence from which on the conservation of the hydrophobic character is gaining importance over the genetic code to be 20 percent. The evolution of orthologues and paralogues is shaped by evolutionary forces in quite comparable ways.

MULTIRESOLUTION ANALYSIS UNCOVERS HIDDEN CONSERVATION OF PROPERTIES IN STRUCTURALLY AND FUNCTIONALLY SIMILAR PROTEINS

Physicochemcial properties of amino acids are important factors in determining protein structure and function. Most approaches make use of averaged properties over entire domains or even proteins to analyze their structure or function. This level of coarseness tends to hide the richness of the variability in the different properties across functional domains. This paper studies the conservation of physicochemical properties in a functionally similar family of proteins using a novel wavelet-based technique known as multiresolution analysis. Such an analysis can help uncover characteristics that can otherwise remain hidden. We have studied the protein kinase family of sequences and our findings are as follows: (a) a number of different properties are conserved over the functional catalytic domain irrespective of the sequence identities; (b) conservation of properties can be observed at different frequency levels and they agree well with the known structural/functional properties of the subdomains for the protein kinase family; (c) structural differences between the different kinase family members are reflected in the waveforms; and (d) functionally important mutations show distortions in the waveforms of conserved properties. The potential usefulness of the above findings in identifying functionally similar sequences in the twilight and midnight zones is demonstrated through a simple prediction model for the protein kinase family which achieved a recall of 93.7% and a precision of 96.75% in cross-validation tests.

Amino acid substitutions preserve protein folding by conserving steric and hydrophobicity properties.

We present a comprehensive analysis of amino acid substitution patterns (sets of residues in a position of a multiple alignment) and conservation of physicochemical properties in alignments of protein sequences. Of the one million possible substitution patterns, only a few hundred account for the majority of aligned positions. Very similar distributions of substitution patterns are observed in all but one of the diverse databases of multiple alignments. In these substitution patterns we analyzed the conservation of 511 physicochemical and steric amino acid properties. Highest conservation was observed in those steric and transfer free energy-related properties that are crucial for folding. The best conserved steric properties include the minimal width of the side chains and their interactions with other residues. Among the hydrophobicity-related properties, charge and those properties that provide information on propensities to form secondary structures or side chain conformation, appear to be better conserved than pure hydrophobicity measures. Physicochemical sequence analysis based on the most conserved properties is expected to aid searching a protein sequence query against a database of multiple alignments, prediction of secondary and tertiary structures and protein engineering.