Self-association of isolated large cytoplasmic domain of plasma membrane H+-ATPase from Saccharomyces cerevisiae: Role of the phosphorylation domain in a general dimeric model for P-ATPases

AbstractLarge cytoplasmic domain (LCD) plasma membrane H+-ATPase from S. cerevisiae was expressed as two fusion polypeptides in E. coli: a DNA sequence coding for Leu353–Ileu674 (LCDh), comprising both nucleotide (N) and phosphorylation (P) domains, and a DNA sequence coding for Leu353–Thr543 (LCDΔh, lacking the C-terminus of P domain), were inserted in expression vectors pDEST-17, yielding the respective recombinant plasmids. Overexpressed fusion polypeptides were solubilized with 6 M urea and purified on affinity columns, and urea was removed by dialysis. Their predicted secondary structure contents were confirmed by CD spectra. In addition, both recombinant polypeptides exhibited high-affinity 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP) binding (Kd=1.9 μM and 2.9 μM for LCDh and LCDΔh, respectively), suggesting that they have native-like folding. The gel filtration profile (HPLC) of purified LCDh showed two main peaks, with molecular weights of 95 kDa and 39 kDa, compatible with dimeric and monomeric forms, respectively. However, a single elution peak was observed for purified LCDΔh, with an estimated molecular weight of 29 kDa, as expected for a monomer. Together, these data suggest that LCDh exist in monomer–dimer equilibrium, and that the C-terminus of P domain is necessary for self-association. We propose that such association is due to interaction between vicinal P domains, which may be of functional relevance for H+-ATPase in native membranes. We discuss a general dimeric model for P-ATPases with interacting P domains, based on published crystallography and cryo-electron microscopy evidence

População e território : retrato e perspetivas na Beira Interior Sul

Comunicação oral da qual só estão disponíveis o resumo e a apresentação.O presente ensaio foi elaborado segundo dois objetivos essenciais: explorar as ligações entre as pessoas e a desertificação dos territórios; levantar um conjunto de questões que consideramos serem relevantes na discussão e abordagem ao processo de planeamento no contexto do combate à desertificação. A lógica de abordagem utilizada centra-se na necessidade e importância de se conhecer profundamente a população “detentora” de um determinado território, conhecer as suas características demográficas mas também os seus interesses, as suas motivações e atitude face à propriedade e uso da terra. Consideramos esta como uma questão crucial. Analisados e discutidos os dados referentes à população e suas actividades, e considerando o processo de desertificação um agente delapidador de um recurso natural com as especificidades reconhecidas ao solo, deixamos a nossa questão de fundo, que consiste em determinar se será o risco de delapidar o património fundiário privado suficientemente forte ou percecionado como capaz de mobilizar para comportamentos preconizados no contexto do planeamento?!..

Proteinase activity regulation by glycosaminoglycans

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term family is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.Universidade de Mogi das Cruzes Centro Interdisciplinar de Investigação BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUNIFESP, EPM, Depto. de BiofísicaSciEL

HIPE: HMC Instruction Predication Extension Applied on Database Processing

The recent Hybrid Memory Cube (HMC) is a smart memory which includes functional units inside one logic layer of the 3D stacked memory design. In order to execute instructions inside the Hybrid Memory Cube (HMC), the processor needs to send instructions to be executed near data, keeping most of the pipeline complexity inside the processor. Thus, control-flow and data-flow dependencies are all managed inside the processor, in such way that only update instructions are supported by the HMC. In order to solve data-flow dependencies inside the memory, previous work proposed HMC Instruction Vector Extensions (HIVE), which embeds a high number of functional units with a interlock register bank. In this work we propose HMC Instruction Prediction Extensions (HIPE), that supports predicated execution inside the memory, in order to transform control-flow dependencies into data-flow dependencies. Our mechanism focus on removing the high latency iteration between the processor and the smart memory during the execution of branches that depends on data processed inside the memory. In this paper we evaluate a balanced design of HIVE comparing to x86 and HMC executions. After we show the HIPE mechanism results when executing a database workload, which is a strong candidate to use smart memories. We show interesting trade-offs of performance when comparing our mechanism to previous work

Comparative photodynamic inactivation of bioluminescent E. coli by pyridinium and inverted pyridinium chlorins

Photodynamic inactivation (PDI) is a therapeutic approach in study due to the ability to reduce or completely eliminate the bacterial strains without the development of resistance mechanisms. In this therapeutic methodology the cationic chlorins (Chls) with pyridinium or inverted pyridinium moieties are one of the photosensitizers exploited in our biological approaches. In this context, we synthesized and characterized new free-base and zinc(II) complexes of pyridinium or inverted pyridinium Chl derivatives (1b, 2, 2a and 2b, respectively) for the inactivation of Escherichia coli (E. coli). The PDI assay was performed with white light irradiation delivered at a fluence rate of 25 mW cm−2. The obtained results of this study demonstrate high PDI efficiency of the zinc(II) metallated Chl 1b, reaching the detection limit of the bioluminescent method (5.2 log reduction) in 45 min of irradiation.publishe

Molecular diversity and the fate of biochemical fractions of eucalypt tissues in soil

The molecular diversity of the source substrate has been regarded as a significant controller of the proportion of plant material that is either mineralized or incorporated into soil organic matter (SOM). However, quantitative parameters to express substrate molecular diversity remain elusive. In this research, we fractionated leaves, twigs, bark, and root tissues of 13C-enriched eucalypt seedlings into hot water extractables (HWE), total solvent (acetone) extractables (TSE), a cellulosic fraction (CF), and the acid unhydrolyzable residue (AUR). We used 13C NMR spectroscopy to obtain a molecular diversity index (MDI) based on the relative abundance of carbohydrate, protein, lignin, lipid, and carbonyl functional groups within the biochemical fractions. Subsequently, we obtained artificial plant organs containing fixed proportions (25%) of their respective biochemical fractions to be incubated with soil material obtained from a Haplic Ferralsol for 200-days, under controlled temperature (25 ± 1 ◦C) and moisture adjusted to 70–80% of the soil water holding capacity. Our experimental design was a randomized complete block design, arranged according to a factorial scheme including 4 plant organs, 4 biochemical fractions, and 3 blocks as replicates. During the incubation, we assessed the evolution of CO2 from the microcosms after 1, 2, 3, 4, 7, 10, 13, 21, 28, 38, 45, 70, 80, 92, 112, 148, 178 and 200 days from the start of the incubation. After the incubation, soil subsamples were submitted to a density fractionation to separate the light fraction of SOM (LFOM) i.e., with density <1.8 g cm 3. The heavy fraction remaining was submitted to wetsieving yielding the sand-sized SOM (SSOM) and the mineral-associated SOM (MAOM), with particle-size greater and smaller than 53 μm, respectively. We found that HWE and AUR exhibited comparatively higher MDIs than the TSE and CF. During the incubation, HWE and CF were the primary sources of 13C-CO2 from all plant organs and after 92 days, the respiration of the TSE of bark and roots increased. Otherwise, the AUR contributed the least for the release of 13C-CO2. There were no significant relationships between the MDI and the amount of 13C transferred into the LFOM or SSOM. Otherwise, the transfer of 13C into the MAOM increased as a linear-quadratic function of MDI, which in turn was negatively correlated with the total 13C-CO2 loss. Overall, the MDI exerted a stronger control on the 13C-labeled MAOM than on 13C-CO2 emissions, highlighting the need to improve our ability to distinguish and quantify direct plant inputs from those of microbial origin entering soil C pools.publishedVersio

Life-cycle greenhouse gas emissions of portuguese olive oil

The main goal of this paper was to assess the greenhouse gas (GHG) intensity of olive oil production in Portugal. A life-cycle model and inventory were implemented for the entire production process, including a comprehensive analysis of olive cultivation, olive oil extraction, packaging, and distribution. Data originates from five differently-sized Portuguese olive growers and from a total of six olive oil mills, representing the three extraction processes in use: three-phase extraction, two-phase extraction, and traditional pressing. The results show that the GHG intensity lies in the range 1.8-8.2 kg CO2eq/liter and that the main contributors were fertilizers (production and field emissions). Efficient use of fertilizers thus seems to be a key factor for mitigating the GHG intensity of olive oil production.This research was supported by project ECODEEP (Eco-efficiency and Eco-management in the Agro Industrial sector, FCOMP–05–0128–FEDER–018643) and the Portuguese Scienc

Photodynamic inactivation of a RNA-virus model using water-soluble β-octa-Substituted pyridinium-pyrazolyl phthalocyanines

Among the various groups of microorganisms, viruses have generally a greater capacity for mutation, especially RNA viruses, as was demonstrated by SARS-CoV2 virus mutations. This high mutation rate promotes the development of their resistance to traditional antivirals and establishes the resistance behaviour in virus populations, decreasing their susceptibility to these drugs. In this context, the photodynamic treatment appears as a potentially effective method against microorganisms and, considering its mode of action is not likely to lead to the development of resistance. In this work, two newly zinc(II) phthalocyanines (ZnPcs) bearing pyridinium-pyrazolyl groups (2a and 3a) were synthesized, characterized, and applied in photodynamic inactivation (PDI) of bacteriophage Φ6 (or Phage Phi6) as a RNA-virus model. These quaternized dyes were applied at different concentrations (from 5.0 to 20 μM, and under white light irradiation in the irradiance range between 50 and 150 mW/cm2) to test their efficiency for possible clinical or environmental applications. The results showed that the new cationic ZnPcs 2a and 3a efficiently inactivate the RNA-virus model (bacteriophage Φ6), even at the lowest tested irradiance. These compounds are thus promising photosensitizers to be used in various contexts.info:eu-repo/semantics/publishedVersio