A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.     

Differential interactions of the antimicrobial peptide,RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes

BackgroundAntimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus.MethodsA physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations.ResultsRQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces.ConclusionsRQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application.General significanceThese results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.     

Metabolic Basis for the Self-Referential Genetic Code

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine—this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.     

The gelling of κ-carrageenan in light and heavy water

Gels of κ-carrageenan were prepared in H₂O and D₂O. Rheological measurements revealed that in heavy water the elastic modulus is substantially higher. From the rheological view point, this result indicates the formation of a higher number of physical-crosslinking points in the structure of the gel. These crosslinking points are mainly associated with the aggregation of the double-helix of the κ-carrageenan, which is enhanced in D₂O. The thermal stability of the gels in both solvents (relative to the sol-gel transition) was also investigated by using polarimetric measurements (to measure the relative fraction of helix-coil) and micro-differential scanning calorimetry (micro-DSC). The results obtained using these techniques are very consistent and allow us to propose a model to explain the differences for the gels formed in light and heavy water based on the degree of double-helix aggregation.     

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid: A LINK TO TOXIC EFFECTS OF CALCIUM OVERLOAD IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)?*

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca²⁺ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca²⁺. We show that at physiological pH, Ca²⁺ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca²⁺ boosts the onset of SOD1 aggregation. In agreement, Ca²⁺ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca²⁺ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca²⁺-induced aggregates, thus indicating that Ca²⁺ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca²⁺ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca²⁺ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.     

Microanatomy and Development of the Dwarf Male of Symbion pandora (Phylum Cycliophora): New Insights from Ultrastructural Investigation Based on Serial Section Electron Microscopy

Cycliophorans have a complex life cycle that involves several sexual and asexual stages. One of the sexual stages is the 40 μm-long dwarf male, which is among the smallest free-living metazoans. Although the dwarf male has a highly complex body plan, this minute organism is composed of a very low number of somatic cells (~50). The developmental processes that give rise to this unique phenotype are largely unknown. Here we use high resolution serial block face—scanning electron microscopy to analyze the anatomy and morphogenesis of three cycliophoran dwarf males at different developmental stages ranging from internal bud to mature male. The anatomical and morphological features of the mature dwarf male stage reported here largely correspond to those reported in earlier studies. Interestingly, the organs that typically characterize the anatomy of the mature dwarf male, e.g., muscles, brain, testis and glands, are already formed in the young male. However, there are striking differences between the mature male and young male stages at the level of cellular architecture. Thus, while the young male stage, like the internal bud stage, possesses approximately 200 nucleated cells, the mature male stage comprises only around 50 nucleated cells; muscle and epidermal cells of the mature male lack nuclei. Moreover, the total body volume of the mature male is only 63% of the body of the young male implying that the maturation of the young male into a mature male involves a marked reduction of internal body volume, mainly by massive nuclei loss. Our comparative analysis of these dwarf male specimens reveals unprecedented insight into the striking morphological and developmental differences that characterize these highly miniaturized male stages both at the level of body organization and at the level of cellular ultrastructure.     

The Expanded mtDNA Phylogeny of the Franco-Cantabrian Region Upholds the Pre-Neolithic Genetic Substrate of Basques

The European genetic landscape has been shaped by several human migrations occurred since Paleolithic times. The accumulation of archaeological records and the concordance of different lines of genetic evidence during the last two decades have triggered an interesting debate concerning the role of ancient settlers from the Franco-Cantabrian region in the postglacial resettlement of Europe. Among the Franco-Cantabrian populations, Basques are regarded as one of the oldest and more intriguing human groups of Europe. Recent data on complete mitochondrial DNA genomes focused on macrohaplogroup R0 revealed that Basques harbor some autochthonous lineages, suggesting a genetic continuity since pre-Neolithic times. However, excluding haplogroup H, the most representative lineage of macrohaplogroup R0, the majority of maternal lineages of this area remains virtually unexplored, so that further refinement of the mtDNA phylogeny based on analyses at the highest level of resolution is crucial for a better understanding of the European prehistory. We thus explored the maternal ancestry of 548 autochthonous individuals from various Franco-Cantabrian populations and sequenced 76 mitogenomes of the most representative lineages. Interestingly, we identified three mtDNA haplogroups, U5b1f, J1c5c1 and V22, that proved to be representative of Franco-Cantabria, notably of the Basque population. The seclusion and diversity of these female genetic lineages support a local origin in the Franco-Cantabrian area during the Mesolithic of southwestern Europe, ∼10,000 years before present (YBP), with signals of expansions at ∼3,500 YBP. These findings provide robust evidence of a partial genetic continuity between contemporary autochthonous populations from the Franco-Cantabrian region, specifically the Basques, and Paleolithic/Mesolithic hunter-gatherer groups. Furthermore, our results raise the current proportion (≈15%) of the Franco-Cantabrian maternal gene pool with a putative pre-Neolithic origin to ≈35%, further supporting the notion of a predominant Paleolithic genetic substrate in extant European populations.