ABSTRACT
Studying aqueous solutions of complex (bio)polymers is essential from both theoretical and practical perspectives. To understand the principles that govern the properties of these solutions is pivotal for the study of biological processes, considering that the most distinguished components of the cells are polymers (proteins, nucleic acids). These macromolecular aqueous systems, known as colloids, has raise the interest of scientists in recent years. It is known that several physicochemical properties deviate from ideal behaviour in this kind of solutions and that the physical state of water is different compared to its pure state. Particularly, the surface tension of such mixtures often shows a peculiar profile at semi-dilute and concentrated conditions. Here, we joined the colloidal concept of water polarization (proposed in the Association-Induction Hypothesis) with Damodaran's formalism for surface tension to theoretically derive a compelling mathematical model that explains the behaviour of polymer solutions. We measured the surface tension and osmolarity of different polyethylene oxide solutions and we used the ACDAN fluorescence probe to assess the water dipolar relaxation (polarization) in these mixtures. As a proof of concept, we also studied the influence of these polymer solutions on lipid interfaces. Our isotherm model explains the experimental observations with a unifying view that correlates with other measured properties, such as osmolarity and water dipolar relaxation. This provides a link between interfacial and bulk physicochemical properties of polymer solutions, also giving a new framework for studying the interaction of colloidal systems with lipid membranes interfaces.
Subject(s)
Polymers , Water , Surface Tension , Fluorescence , LipidsABSTRACT
HIV-1 assembly occurs at the plasma membrane, with the Gag polyprotein playing a crucial role. Gag association with the membrane is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with anionic lipids. Several pieces of evidence suggest that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP2) highly influences this binding. Furthermore, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP2-containing membranes. It is hypothesized that RNA has a chaperone function by interacting with the MA domain, preventing Gag from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focusing on the specificity for PIP2 and on the possible effects of a Gag N-terminal peptide on impairing the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but has no effect on the selectivity for PIP2. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negatively charged compositions, where MA alone does not discriminate between membranes with or without PIP2. Therefore, we propose that the specificity of MA for PIP2-containing membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario provides a new understanding of the regulation mechanism, with a macromolecular view, rather than considering molecular interactions within a ligand-receptor model.
Subject(s)
HIV-1 , Phosphatidylinositol 4,5-Diphosphate , gag Gene Products, Human Immunodeficiency Virus , gag Gene Products, Human Immunodeficiency Virus/chemistry , HIV-1/metabolism , Lipids/chemistry , Peptides/metabolism , RNA/metabolismABSTRACT
The use of phasors to analyze fluorescence data was first introduced for time-resolved studies for a simpler mathematical analysis of the fluorescence-decay curves. Recently, this approach was extended to steady-state experiments with the introduction of the spectral phasors (SP), derived from the Fourier transform of the fluorescence emission spectrum. In this work, we revise key mathematical aspects that lead to an interpretation of SP as the characteristic function of a probability distribution. This formalism allows us to introduce a new tool, called multi-dimensional spectral phasor (MdSP) that seize, not only the information from the emission spectrum, but from the full excitation-emission matrix (EEM). In addition, we developed a homemade open-source Java software to facilitate the MdSP data processing. Due to this mathematical conceptualization, we settled a mechanism for the use of MdSP as a tool to tackle spectral signal unmixing problems in a more accurate way than SP. As a proof of principle, with the use of MdSP we approach two important biophysical questions: protein conformational changes and protein-ligand interactions. Specifically, we experimentally measure the EEM changes upon denaturation of human serum albumin (HSA) or during its association with the fluorescence dye 1,8-anilinonaphtalene sulphate (ANS) detected via tryptophan-ANS Förster Resonance Energy Transfer (FRET). In this sense, MdSP allows us to obtain information of the system in a simpler and finer way than the traditional SP. Specifically, understanding a protein's EEM as a molecular fingerprint opens new doors for the use of MdSP as a tool to analyze and comprehend protein conformational changes and interactions.
Subject(s)
Fluorescence Resonance Energy Transfer , Fluorescent Dyes , Fluorescence Resonance Energy Transfer/methods , Fourier Analysis , Humans , Serum Albumin, Human , Spectrometry, Fluorescence/methodsABSTRACT
The sea anemone venom contains pore-forming proteins (PFP) named actinoporins, due to their purification from organisms belonging to Actiniaria order and its ability to form pores in sphingomyelin-containing membranes. Actinoporins are generally basic, monomeric and single-domain small proteins (â¼20 kDa) that are classified as α-type PFP since the pore formation in membranes occur through α-helical elements. Different actinoporin isoforms have been isolated from most of the anemones species, as was analyzed in the first part of this review. Several actinoporin full-length genes have been identified from genomic-DNA libraries or messenger RNA. Since the actinoporins lack carbohydrates and disulfide bridges, their expression in bacterial systems is suitable. The actinoporins heterologous expression in Escherichia coli simplifies their production, replaces the natural source reducing the ecological damage in anemone populations, and allows the production of site-specific mutants for the study of the structure-function relationship. In this second part of the review, the strategies for heterologous production of actinoporins in Escherichia coli are analyzed, as well as the different approaches used for their purification. The activity of the recombinant proteins with respect to the wild-type is also reviewed.
Subject(s)
Cnidarian Venoms/metabolism , Multigene Family , Pore Forming Cytotoxic Proteins/metabolism , Recombinant Proteins/biosynthesis , Sea Anemones/metabolism , Animals , Cation Exchange Resins , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cnidarian Venoms/chemistry , Cnidarian Venoms/genetics , Cnidarian Venoms/toxicity , Escherichia coli/growth & development , Escherichia coli/metabolism , Hemolytic Agents/isolation & purification , Hemolytic Agents/metabolism , Hemolytic Agents/toxicity , Mutant Proteins/biosynthesis , Mutant Proteins/chemistry , Mutant Proteins/toxicity , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/isolation & purification , Pore Forming Cytotoxic Proteins/toxicity , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/toxicity , Recombinant Proteins/isolation & purification , Recombinant Proteins/toxicityABSTRACT
Anabolic androgenic steroids (AAS) are used illicitly at high doses by bodybuilders. The misuse of these drugs is associated with serious adverse effects to the liver, including cellular adenomas and adenocarcinomas. We report two very different cases of adult male bodybuilders who developed hepatocellular adenomas following AAS abuse. The first patient was asymptomatic but had two large liver lesions which were detected by ultrasound studies after routine medical examination. The second patient was admitted to our hospital with acute renal failure and ultrasound (US) studies showed mild hepatomegaly with several very close hyperecogenic nodules in liver, concordant with adenomas at first diagnosis. In both cases the patients have evolved favourably and the tumours have shown a tendency to regress after the withdrawal of AAS. The cases presented here are rare but may well be suggestive of the natural course of AAS induced hepatocellular adenomas. In conclusion, sportsmen taking AAS should be considered as a group at risk of developing hepatic sex hormone related tumours. Consequently, they should be carefully and periodically monitored with US studies. In any case, despite the size of the tumours detected in these two cases, the possibility of spontaneous tumour regression must also be taken in account.
Subject(s)
Adenoma, Liver Cell/chemically induced , Anabolic Agents/adverse effects , Liver Neoplasms/chemically induced , Methenolone/analogs & derivatives , Nandrolone/analogs & derivatives , Substance-Related Disorders/complications , Testosterone Propionate/analogs & derivatives , Testosterone/analogs & derivatives , Weight Lifting , Adenoma, Liver Cell/diagnostic imaging , Administration, Oral , Adult , Anabolic Agents/administration & dosage , Biopsy, Fine-Needle/methods , Humans , Liver Neoplasms/diagnostic imaging , Magnetic Resonance Imaging/methods , Male , Methenolone/administration & dosage , Methenolone/adverse effects , Nandrolone/administration & dosage , Nandrolone/adverse effects , Nandrolone Decanoate , Oxymetholone/administration & dosage , Oxymetholone/adverse effects , Stanozolol/administration & dosage , Stanozolol/adverse effects , Substance Abuse, Intravenous , Testosterone/administration & dosage , Testosterone/adverse effects , Testosterone Propionate/administration & dosage , Testosterone Propionate/adverse effects , UltrasonographyABSTRACT
Thirty-five patients who had undergone surgery for non-small cell bronchogenic carcinoma with isolated involvement of the chest wall were reviewed. The diagnosis was preoperatively suspected in 80% of cases. En-bloc resection of the invaded chest wall was performed in 25 cases and parietal pleurectomy in ten in which the pleura was easily dissectable from the costal plane. Of the eight patients with major complications in the early postoperative period, six, including the two who died perioperatively, had undergone en-block resection. The 5-year actuarial survival rate was 22% overall and 36% in the patients without lymph node involvement. No significant relationship between survival and type of operation or degree of chest wall invasion was found. Isolated involvement of the chest wall by non-small cell bronchogenic carcinoma does not necessarily contraindicate surgery with curative intent. Parietal pleurectomy is valid in selected cases. Long-term survival depends basically on node involvement.