Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Estrategia de superación profesoral sobre atención integral al adulto mayor / Teaching Improvement Strategy about Comprehensive Care of the Elderly

Introducción: El informe mundial sobre envejecimiento y salud tiene en cuenta la sociedad e incluye los cuidados integrales para un envejecimiento saludable, por lo que elevar la calidad de la atención al adulto mayor a través del modelo de gestión docente-asistencial contribuirá a elevar la calidad de vida en estas edades. Objetivo: Diseñar una estrategia de superación profesoral sobre atención integral al adulto mayor dirigida a tutores de la residencia de Medicina General Integral. Método: Investigación aplicada, que tuvo una fase descriptiva-evaluativa y una de evaluación de resultados. La población estudiada fue el universo de tutores de la residencia de Medicina General Integral (12). Se estudiaron diferentes variables y se utilizaron encuestas y guías de desempeño, se aplicó frecuencia absoluta, relativa porcentual y la prueba de Mc Nemar. Resultados: Predominaron las edades de 45-54 años (58,3 por ciento). De los tutores, 41,7 por ciento eran asistentes y el 91,7 por ciento master o poseían categoría de investigador, se incrementaron los buenos conocimientos al 100 por ciento después de la intervención y el desempeño muy satisfactorio al 91,7 por ciento. Conclusiones: La estrategia de superación contribuyó al incremento del nivel de conocimientos y del desempeño sobre atención integral al adulto mayor en los tutores, así como una mayor participación en las actividades de ciencia y técnica relacionadas con la temática(AU)

Introduction: The World Report on Aging and Health takes society into account and includes comprehensive care for healthy aging; therefore, raising the quality of care for the elderly through the teaching-care management model will contribute to raising the quality of life in these ages. Objective: To design a teaching improvement strategy about comprehensive care for the elderly aimed at tutors from the Family Medicine residency. Method: Applied research with a descriptive-evaluative phase and a results assessment phase. The population studied was the universe of tutors from the Family Medicine residency (12). Different variables were studied and surveys and performance guides were used. Absolute frequency, relative percentage and the McNemar test were applied. Results: The ages 45-54 years (58.3 percent) predominated. Of the tutors, 41.7 percent were assistants, while 91.7percent were masters or had the researcher category. Good knowledge increased to 100 percent after the intervention, while very satisfactory performance did so to 91.7 percent. Conclusions: The improvement strategy contributed to the increase in the level of knowledge and performance on comprehensive care for the elderly in the tutors, as well as a greater participation in science and technical activities related to the subject(AU)

Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats.

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.

Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins.

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn(2+)-, Cd(2+)- or Cu(+)-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd(2+) coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn(2+), they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu(+), although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd(2+) and for Cu(+), and although not optimally, it yielded the best result when coordinating Zn(2+). The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).

Metal dealing at the origin of the Chordata phylum: the metallothionein system and metal overload response in amphioxus.

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.

Fractionation of selenium-containing proteins in serum by multiaffinity liquid chromatography before size-exclusion chromatography-ICPMS.

Immunoaffinity chromatography has been investigated for fractionation of serum into selenoalbumin and true selenoproteins. Among several albumin-depletion kits tested, a multiaffinity column specifically binding albumin and five other major serum proteins provided the best results. It extracted ca 95% of both albumin and selenoalbumin, which enabled interference-free determination of glutathione peroxidase, selenoprotein P, and selenoalbumin by size-exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICPMS). The efficiency of the multiaffinity column did not vary over a period of 18 months. The purity of fractions separated by immunoaffinity LC was confirmed by elution-volume matching with standards in SEC-ICPMS and by selenopeptide mapping in capillary HPLC-ICPMS. Quantification of the selenium distribution among the different proteins in human serum from a control group and from a person on a selenium-rich diet revealed that 67% of the supplemented selenium was incorporated into albumin, 30% into glutathione peroxidase, and 3% into selenoprotein P.

Analysis of the selenium species distribution in cow blood by size exclusion liquid chromatography-inductively coupled plasma collision cell mass spectrometry (SEC-ICPccMS).

A method for performing rapid semiquantitative screening of the distribution of Se species in the blood of cows fed with a diet enriched in selenized yeast was optimized. The method was based on direct injection of a blood sample onto a high resolution size exclusion chromatographic column and fractionation of the selenium species. Selenium was detected on-line by ICP-MS with a collision cell. The concentrations of selenized haemoglobin and free selenomethionine were estimated using the chromatogram. The method was applied to a study involving 15 control and 15 treated dairy cows at four different supplementation time points. The increase in the selenomethionine and selenized haemoglobin was a linear function of the total selenium concentration. A threshold value of 600 ng ml(-1) of total Se was established beyond which selenomethionine could not be incorporated into the protein. No inorganic selenium was found to be present. The total selenium in cow blood correlated well with that in milk. The selenium supplementation did not change the protein distribution profiles for other essential elements (Cu, Fe, Mn, Zn).