Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study

<p>Abstract</p> <p>Background</p> <p>Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity.</p> <p>Results</p> <p>We show that DNA -bound salmon (salmine) and squid protamines contain <it>α</it>-helix, <it>β</it>-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No <it>β</it>-sheet was observed. In salmine, the <it>α</it>-helix amounted to ~20%, while in squid protamine it reached ~40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (~40%) than in squid protamine (~20%). Both protamines contained ~40% <it>β</it>-turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol.</p> <p>Conclusion</p> <p>DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of <it>β</it>-turns, but differ in the proportions of <it>α</it>-helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei.</p

Phosphorylation of the carboxy-terminal domain of histone H1: effects on secondary structure and DNA condensation

Linker histone H1 plays an important role in chromatin folding. Phosphorylation by cyclin-dependent kinases is the main post-translational modification of histone H1. We studied the effects of phosphorylation on the secondary structure of the DNA-bound H1 carboxy-terminal domain (CTD), which contains most of the phosphorylation sites of the molecule. The effects of phosphorylation on the secondary structure of the DNA-bound CTD were site-specific and depended on the number of phosphate groups. Full phosphorylation significantly increased the proportion of β-structure and decreased that of α-helix. Partial phosphorylation increased the amount of undefined structure and decreased that of α-helix without a significant increase in β-structure. Phosphorylation had a moderate effect on the affinity of the CTD for the DNA, which was proportional to the number of phosphate groups. Partial phosphorylation drastically reduced the aggregation of DNA fragments by the CTD, but full phosphorylation restored to a large extent the aggregation capacity of the unphosphorylated domain. These results support the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of H1 partial phosphorylation in interphase chromatin relaxation. More generally, our results suggest that the effects of phosphorylation are mediated by specific structural changes and are not simply a consequence of the net charge

Histone H1 Post-Translational Modifications : Update and Future Perspectives

Histone H1 is the most variable histone and its role at the epigenetic level is less characterized than that of core histones. In vertebrates, H1 is a multigene family, which can encode up to 11 subtypes. The H1 subtype composition is different among cell types during the cell cycle and differentiation. Mass spectrometry-based proteomics has added a new layer of complexity with the identification of a large number of post-translational modifications (PTMs) in H1. In this review, we summarize histone H1 PTMs from lower eukaryotes to humans, with a particular focus on mammalian PTMs. Special emphasis is made on PTMs, whose molecular function has been described. Post-translational modifications in H1 have been associated with the regulation of chromatin structure during the cell cycle as well as transcriptional activation, DNA damage response, and cellular differentiation. Additionally, PTMs in histone H1 that have been linked to diseases such as cancer, autoimmune disorders, and viral infection are examined. Future perspectives and challenges in the profiling of histone H1 PTMs are also discussed

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

<p>Abstract</p> <p>Background</p> <p>Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry.</p> <p>Results</p> <p>The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region) DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin.</p> <p>Conclusion</p> <p>H1 subtypes freely exchange <it>in vitro </it>between chromatin binding sites in physiological salt (0.14 M NaCl). The large differences in relative affinity of the H1 subtypes for chromatin suggest that differential affinity could be functionally relevant and thus contribute to the functional differentiation of the subtypes. The conservation of the relative affinities for SAR and non-SAR DNA, in spite of a strong preference for SAR sequences, indicates that differential affinity alone cannot be responsible for the heterogeneous distribution of some subtypes in cell nuclei.</p

Characterization of the secondary structure of histone h1 subtypes by circular dichroism (cd)

Introducción: Las histonas H1 modulan la estructura y la función de la cromatina. Las células somáticas de mamífero contienen los subtipos H1°, H1a, H1b, H1c, H1d y H1e; en células germinales de testículo y en ovocito, se encuentran respectivamente H1t y H1oo. Su estructura está conformada por un dominio central globular flanqueado por los dominios N-Terminal (DNT) y C-Terminal (DCT). Objetivo: Caracterizar la estructura secundaria de subtipos de la histona H1 mediante dicroísmo circular (DC). Materiales y Métodos: La histona H1 total se extrajo de núcleos de cerebro de rata por cromatografía de intercambio catiónico; la H1° se purificó por filtración en gel y las H1a, H1b, H1c y H1e por cromatografía líquida de alta resolución de fase reversa (RF-HPLC). Los espectros de DC se realizaron en tampón fosfato 10 mM; tampón fosfato 10 mM, 20% TFE (trifluoroetanol); tampón fosfato 10 mM, 40% TFE; tampón fosfato 10 mM, 60% TFE; tampón fosfato 10 mM, 150 mM NaCl y tampón fosfato 10 mM, 1 M NaCl. El análisis de los espectros se realizó con el programa Standard Analysis. Resultados: El porcentaje de hélice-alfa se calculó por diferentes métodos matemáticos teniendo en cuenta elipticidad molar a 193 nm y a 222 nm; con programa de deconvolución K2D y con relaciones cualitativas R1 y R2. El TFE induce la estructura en hélice-alfa en cada uno de los subtipos, mientras que NaCl no induce ningún cambio importante. Conclusión: Los subtipos con mayor contenido de hélice-alfa son H1a y H1c. Las diferencias observadas en el porcentaje de hélice-alfa entre los diferentes subtipos puede ser importante para su diferenciación funcional.H1 histones modulate the structure and function of chromatin. Mammalian somatic cells contain H1°, H1a, H1b, H1c, H1d and H1e subtypes; H1t and H1oo are found in testicular germ cells and oocyte, respectively. Its structure consists of a globular core domain flanked by N-terminal (DNT) and C-terminal (DCT) domains. Objective: To characterize the secondary structure of histone H1 subtypes through circular dichroism (CD). Materials and Methods: Total histone H1 was extracted for rat brain nuclei by cation exchange chromatography; histone H1° was purified by gel filtration and the histones H1a, H1b, H1c and H1e were purified by reversed phase high performance liquid chromatography (RP-HPLC). CD spectra were performed in 10 mM phosphate buffer; 10 mM, 20% TFE phosphate buffer (trifluoroethanol); 10 mM, 40% TFE; phosphate buffer 10 mM, 60% TFE; phosphate buffer 10 mM, 150 mM NaCl and phosphate buffer 10 mm, 1 M NaCl. The analysis of the spectra was performed with JASCO Standard Analysis. Results: The percentage of alpha-helix was calculated using different mathematical methods, taking into account the molar ellipticity at 193 nm, and 222 nm, with K2D deconvolution program and the R1 and R2 qualitative relationships. The results indicate that TFE induced the alpha-helix structure in each of the subtypes, whereas NaCl did not induce any significant change. Conclusion: H1a and H1c are subtypes with highest content of alpha-helix. The observed differences in the percentage of alpha-helix between different subtypes may be important for their functional differentiation

Senior managers in crisis situations. The use of LinkedIn by the CEO of Metro de Madrid during the storm Filomena

La intensa nevada provocada por la borrasca Filomena paralizó España los primeros días de enero de 2021 y provocó pérdidas millonarias en una economía ya de por sí muy debilitada por la crisis del COVID-19. Tan sólo en Madrid, el consistorio de la ciudad estimó que las pérdidas rondarían los 1.398 millones de euros. La ciudad sufrió un fuerte colapso, con problemas de movilidad y abastecimiento. Sólo algunos servicios, como Metro, siguieron funcionando. La consejera delegada de esta Compañía, Silvia Roldán, escribió un post diario en LinkedIn para explicar a los usuarios a qué problemas se enfrentaban día a día y cómo se solucionaban. El presente artículo hace una revisión teórica de la comunicación de crisis e investiga de forma empírica y semántica las publicaciones de Roldán. El objetivo de la investigación es doble. Primero: poner de manifiesto que, en un contexto de crisis, la comunicación directa entre un directivo/directiva con sus públicos reduce la incertidumbre y ayuda a recuperar el equilibro institucional. Segundo: hallar un modelo paradigmático en este tipo de comunicaciones / relaciones con stakeholders específicos.In January 2021, the storm Filomena paralysed Spain. One of the most affected cities was Madrid, which suffered a general collapse for several days. Metro de Madrid (underground railway company and one of the most popular public services in the city) continued to provide service to its users. Silvia Roldan (CEO of Metro de Madrid) wrote a daily post on LinkedIn to explain to users what problems they were facing, how they were being solved and how the service was ensured. Metro de Madrid is an underground railway company that was inaugurated in 1919. It extends over 294 kilometres distributed in 302 stations. It employs 7,000 persons. Other data of Metro de Madrid are: 2,400 cars, 8,758 security cameras, 1,263 vending machines, 2,424 tele-indicators, 1,703 escalators, 541 lifts and 5,386 intercoms. "LinkedIn is a social network that has a great attraction, you are addressing a professional audience, which judges you on the basis of the work you do", says the CEO of Metro de Madrid in an interview conducted to illustrate this research. This research is based on two hypotheses: 1) In a crisis context, direct communication between a company's executive and stakeholders reduces uncertainty and helps to restore institutional equilibrium; and 2) It is possible to find a paradigmatic model in this type of communication with specific stakeholders. This research carried out a quantitative and qualitative analysis using a content analysis of Roldan’s posts on LinkedIn during this crisis and an interview with the CEO of Metro de Madrid. This case gives us some lessons in communication as main results. LinkedIn is an optimal platform for crisis communication, especially if we want to transmit moderately long messages, with nuances, aimed at a professional audience willing to listen and dialogue. Roldan’s colloquial and friendly tone used in her posts are justified in this case (Filomena crisis). She is CEO of the company, direct witness of the facts and responsible for the solutions that are implemented. She uses the first-person narrative, which produces closeness and is a good remedy against uncertainty. It complements institutional messages, transmitted through institutional media and channels, which use a more aseptic and formal tone. The use of photographs in the published posts establishes an emotional links between the author of the post and readers. Readers feel like a direct witness of the facts, especially when the photograph is a subjective close-up. LinkedIn, despite not being Metro de Madrid's main communication channel, is a useful and valuable platform for communicating in emergency situations, even in a more personal and less institutional way than other platforms. Perhaps its greatest virtue is to target a professional and specialised audience, which can be proactive in the resolution of an emergency. The use of LinkedIn in emergency situations is still under-researched compared to other social platforms such as Twitter, Facebook, WhatsApp or Instagram. This article is part of the competitive research project: “Crisis and emergency communication in Social Media. Study of its use in Spain and design of information protocols for institutions and media (COMCRISIS - PID2019-109064GB-I00)” granted and funded by the Ministry of Science and Innovation of the Spanish Government.Gobierno de España. Ministerio de Ciencia e Innovació

San Francisco de Borja (1510-1572)

AntepA f. de lám. é fotogravado da figura de San Francisco de Borj

El Còmic i la Memòria Històrica: el cas de la Segona Guerra Mundial

Abstract not availabl