Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Human-modified landscapes driving the global primate extinction crisis

The world's primates have been severely impacted in diverse and profound ways by anthropogenic pressures. Here, we evaluate the impact of various infrastructures and human-modified landscapes on spatial patterns of primate species richness, at both global and regional scales. We overlaid the International Union for the Conservation of Nature (IUCN) range maps of 520 primate species and applied a global 100 km² grid. We used structural equation modeling and simultaneous autoregressive models to evaluate direct and indirect effects of six human-altered landscapes variables (i.e., human footprint [HFP], croplands [CROP], road density [ROAD], pasture lands [PAST], protected areas [PAs], and Indigenous Peoples' lands [IPLs]) on global primate species richness, threatened and non-threatened species, as well as on species with decreasing and non-decreasing populations. Two-thirds of all primate species are classified as threatened (i.e., Critically Endangered, Endangered, and Vulnerable), with ~86% experiencing population declines, and ~84% impacted by domestic or international trade. We found that the expansion of PAST, HFP, CROP, and road infrastructure had the most direct negative effects on primate richness. In contrast, forested habitat within IPLs and PAs was positively associated in safeguarding primate species diversity globally, with an even stronger effect at the regional level. Our results show that IPLs and PAs play a critical role in primate species conservation, helping to prevent their extinction; in contrast, HFP growth and expansion has a dramatically negative effect on primate species worldwide. Our findings support predictions that the continued negative impact of anthropogenic pressures on natural habitats may lead to a significant decline in global primate species richness, and likely, species extirpations. We advocate for stronger national and international policy frameworks promoting alternative/sustainable livelihoods and reducing persistent anthropogenic pressures to help mitigate the extinction risk of the world's primate species.     

Evolutionary analysis of the picornavirus family

An exhaustive evolutionary analysis of the picornavirus family has been carried out using the amino acid sequences of several proteins of the viruses including: the capsid proteins (1D, 1B, and 1C) situated at the 5 end of the genome and responsible for the serotype of the viruses, and the viral polymerase (3D), located at the 3 end of the genome. The evolutionary relationships found among the viruses studied support the new classification, recently suggested, in contrast to the classical one, and the existence of a new genus for the picornavirus family. In the new taxonomic organization, five genera form the picornavirus family: (1) aphthoviruses, (2) cardioviruses, (3) hepatoviruses (previously classified as enteroviruses), (4) renteroviruses (which mainly constitute a combination of the previous genera rhinovirus and enterovirus), and (5) a new genus, with a new and unique representative: the echovirus 22. Our analysis also allowed us, for the first time, to propose the most probable sequence of speciation events to have given rise to the current picornavirus family.The bootstrap procedure was used to check the reliability of the phylogenetic trees obtained. The application of the method of the statistical geometry in distance space to internal branches of the tree revealed a high degree of evolutionary noise, which makes the resolution of some internal branching points difficult. Correspondence to: J. Dopazo     

Contribution of Mutation and RNA Recombination to the Evolution of a Plant Pathogenic RNA

The nucleotide sequence of 17 variants of the satellite RNA of cucumber mosaic virus (CMV-satRNA) isolated from field-infected tomato plants in the springs of 1989, 1990, and 1991 was determined. The sequence of each of the 17 satRNAs was unique and was between 334 and 340 nucleotides in length; 57 positions were polymorphic. There was much genetic divergence, ranging from 0.006 to 0.141 nucleotide substitutions per site for pairwise comparisons, and averaging 0.074 for any pair. When the polymorphic positions were analyzed relative to a secondary structure model proposed for CMV-satRNAs, it was found that there were significantly different numbers of changes in base-paired and non–base-paired positions, and that mutations that did not disrupt base pairing were preferred at the putatively paired sites. This supports the concept that the need to maintain a functional structure may limit genetic divergence of CMV-satRNA. Phylogenetic analyses showed that the 17 CMV-satRNA variants clustered into two subgroups, I and II, and evolutionary lines proceeding by the sequential accumulation of mutations were apparent. Three satRNA variants were outliers for these two phylogenetic groups. They were shown to be recombinants of subgroup I and II satRNAs by calculating phylogenies for different molecular regions and by using Sawyer's test for gene conversion. At least two recombination events were required to produce these three recombinant satRNAs. Thus, recombinants were found to be frequent (∼17%) in natural populations of CMV-satRNA, and recombination may make an important contribution to the generation of new variants. To our knowledge this is the first report of data allowing the frequency of recombinant isolates in natural populations of an RNA replicon to be estimated. Received: 14 May 1996 / Accepted: 17 July 1996     

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments. Composition and growth performance of ten filamentous heterocystous strains

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growth performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent type being C-phycocyanin, followed by alloppycocyanin, with levels of 17 and 11% d.wt, respectively, in some strains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10% d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains tolerated an irradiance of ca 2000 μmol photon m^-2 s^-1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited the widest optimum range of both temperature (30–45 and 25–40 °C) and pH (6.5–9.5 and 6.0–9.0) for growth, the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of two phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m^-2 d^-1 during summer. The growth performance of the allophycocyanin-rich Anabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m^-2 d^-1.     

Lethality and mutagenicity inHalobacterium mediterranei caused by N-methyl-N′-nitro-N-nitrosoguanidine

Compared withEscherichia coli, Halobacterium mediterranei was highly resistant to the lethal effect of N-methyl-N-nitro-N-nitrosoguanidine (nitrosoguanidine), but it was sensitive to the mutagenic action of this chemical agent. Nitrosoguanidine at 500 g ml^–1 gave a cell survival level between 1% and 10%, and this allowed us to obtain more Josamycin-resistant mutants compared with lower concentrations, which gave higher survival rates but fewer mutants. The efficiency of the mutagenicity obtained with the nitrosoguanidine treatment was examined under a variety of conditions. The optimal conditions for obtaining Josamycinresistant mutants were achieved by exposing, in darkness and without shaking, a suspension of about 10⁸ log-phase cells to 500 g nitrosoguanidine in 1 ml of 50 mM modified saline Tris-maleate buffer at pH 7.5, or in 1 ml of 5 mM modified saline Tris-citrate-maleate for 30 min at 37°C.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

Impacto del ejercicio físico en variables relacionadas con el bienestar emocional y funcional en adultos mayores

Background and aimThe benefits of the physical exercise in aging, and specially in frailty, have been associated with reduced risk of mortality, chronic disease, and cognitive and functional impairments. Multi-component training, which combines strength, endurance, balance, and gait training, represents the most beneficial kind of physical exercise in older adults.MethodsGiven the effectiveness of the multi-component training, a physical exercise program «Actívate» (based on the methodology Vivifrail), with the focus on «active aging», was conducted in the present study. Forty-nine older adults over 60 years participated in this program.ResultsThe physical exercise intervention led to a reduction in diastolic blood pressure, pain threshold and sleep disturbances (e. g. hypersomnia) (t ≥ 2.72, p < 0.01), as well as an increase of walking speed (t = 7.84, p ≤ 0.001). Further, quality of life factors (GENCAT scale), like emotional well-being, personal development, physical well-being, self-determination, and social inclusion, were greater after intervention (t ≥ ?2.06, p < 0.05).ConclusionsThese findings underline the benefits of multi-component training in functionality of older adults, and further, provide relevant aspects about the modulation of pain perception, sleep disturbances, social factors and physical and emotional well-being. Physical exercise programs such as «Actívate» should be promoted, in order to encourage healthy lifestyle habits, in the older adults’ population.