Traction Forces of Endothelial Cells under Slow Shear Flow

Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress.     

Age estimation,growth and maturity of the Argentine hake (Merluccius hubbsi Marini, 1933) along the northernmost limit of its distribution in the south-western Atlantic

Age, growth and length-at-maturity of the Argentine hake (Merluccius hubbsi) were studied in the northernmost limit of the species distribution in the south-western Atlantic. A total of 351 otoliths and information from 1610 specimens sampled from the industrial double-rig trawl landings between May 2013 and April 2014 were used. Age and growth were estimated by counting and measuring increments in sectioned sagittae otoliths, and length at maturity was estimated based on macroscopic gonadal analysis. For both sexes, hepatosomatic index and condition index increased mainly during spring, reaching a maximum at the end of summer before the subsequent spawning season began. Gonadosomatic index was highest in April, believed to correspond with peak spawning. The annual periodicity of alternate opaque and translucent zones was validated by marginal increment analysis. Growth curves were fitted to back-calculated size at age by fitting the three-parameters von Bertalanffy growth function. The maximum age was 5 years in fish of either sex. Females attained larger sizes than males. The parameters of the von Bertalanffy growth equations were: L∞?=?533?mm, k?=?0.231 year^?1 and t₀?=??0.935 year for females; L∞?=?394?mm, k?=?0.405 year^?1 and t₀?=??0.463 year for males. The mean length and age at first maturity was 273?mm at 1.9 years for males and 274?mm at 2.0 years for females.     

Changes in the levels of jasmonates and free polyamines induced by Na<Subscript>2</Subscript>SO<Subscript>4</Subscript> and NaCl in roots and leaves of the halophyte <Emphasis Type="Italic">Prosopis strombulifera</Emphasis>

Prosopis strombulifera, a common legume in high-salinity soils of Argentina, is a useful model for elucidation of salt tolerance mechanisms and specific biochemical pathways in halophytes, since its NaCl tolerance exceeds the limit described for most halophytic plants. We analyzed the effects of the increasing concentration of two main soil salts, Na₂SO₄ and NaCl, on growth parameters of P. strombulifera, chlorophyll levels, and content of jasmonates (JAs) and polyamines (PAs), which are key molecules involved in stress responses. P. strombulifera showed a halophytic response (growth promotion) to NaCl, but strong growth inhibition by iso-osmotic solutions of Na₂SO₄. Chlorophyll levels, number of leaves and leaf area were also differentially affected. An important finding was the partial alleviation of SO₄²⁻ toxicity by treatment with two-salt mixture. JAs are not directly involved in salt tolerance in this species since its levels decrease under all salt treatments. Beneficial effects of Putrescine (Put) accumulation in NaCl treated plants maybe inferred probably associated with the antioxidative defense system. Another novel finding is the accumulation of the uncommon PA cadaverine in roots under high Na₂SO₄, which may be related to SO₄²⁻ toxicity.     

Apoptotic cell death of oestrogen activated lactotrophs induced by tamoxifen

Stimulation and inhibition of lactotroph cells cause remarkable morphological and functional changes. In keeping with these changes, the size of the lactotroph cell population undergoes striking alterations due to proliferation or cell death. Factors involved in the induction of apoptosis of pituitary cells are not well established. We demonstrated earlier that oestrogens prevent lactotroph cells of female rats to die by apoptosis induced by bromocryptine treatment, a fact that can be reversed in ovariectomised rats. In this study, we developed experimental models for in vivo and in vitro studies to gain further insight on the survival effect of oestrogens on lactotrophs. In rats pretreated with oestrogens, tamoxifen generates a massive cell death by apoptosis as validated by the TUNEL technique and DNA electrophoresis of pituitary gland. On electron microscope observations, numerous lactotrophs exhibited progressive morphological changes in the nuclei compatible with the apoptotic process. The cells remaining intact also exhibit signs of inhibition due to a significant transformation of regular lactotrophs in atypical subtypes. In pituitary cell cultures exposed to tamoxifen and oestrogen simultaneously, most of the lactotrophs displayed features of apoptosis in the nucleus. The present reports gathered new evidences on the apoptogenic potential of tamoxifen on lactotroph cells, and corroborates the contribution of oestrogens to sustain both a balanced population of lactotrophs and a competent secretory activity. The concept that opposed activities, such as inhibition and stimulation, can activate apoptosis is also strengthen by these observations.     

In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q Ca_V2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of Ca_V2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.     

Gold nanoparticles and bioconjugation: a pathway for proteomic applications

In the last few years gold nanoparticles (AuNPs) became extremely interesting materials due to their enhanced optical, chemical and electrical properties. With the intention of taking advantage of those properties, the use of AuNPs has spread into a wide variety of areas such as physics, chemistry, biology, industry and medicine. More interestingly, their ability to form robust conjugates with biomolecules has given proteomics a new tool to improve aspects where the current methods to study proteins and their interactions in living cells cannot achieve the success required. In this review we present some of the current methods for AuNPs synthesis, the tailoring of their surface with ligands to improve stability and strategies to conjugate with biomolecules. Lastly, we also discuss their application in proteomic methods and recent developments in clinical diagnosis.     

Increased light‐use efficiency sustains net primary productivity of shaded coffee plants in agroforestry system

In agroforestry systems, shade trees strongly affect the physiology of the undergrown crop. However, a major paradigm is that the reduction in absorbed photosynthetically active radiation is, to a certain extent, compensated by an increase in light‐use efficiency, thereby reducing the difference in net primary productivity between shaded and non‐shaded plants. Due to the large spatial heterogeneity in agroforestry systems and the lack of appropriate tools, the combined effects of such variables have seldom been analysed, even though they may help understand physiological processes underlying yield dynamics. In this study, we monitored net primary productivity, during two years, on scales ranging from individual coffee plants to the entire plot. Absorbed radiation was mapped with a 3D model (MAESPA). Light‐use efficiency and net assimilation rate were derived for each coffee plant individually. We found that although irradiance was reduced by 60% below crowns of shade trees, coffee light‐use efficiency increased by 50%, leaving net primary productivity fairly stable across all shade levels. Variability of aboveground net primary productivity of coffee plants was caused primarily by the age of the plants and by intraspecific competition among them (drivers usually overlooked in the agroforestry literature) rather than by the presence of shade trees.     

RNA as the stone guest of protein aggregation

The study of prions as infectious aggregates dates several decades. From its original formulation, the definition of a prion has progressively changed to the point that many aggregation-prone proteins are now considered bona fide prions. RNA molecules, not included in the original ‘protein-only hypothesis’, are also being recognized as important factors contributing to the ‘prion behaviour’, that implies the transmissibility of an aberrant fold. In particular, an association has recently emerged between aggregation and the assembly of prion-like proteins in RNA-rich complexes, associated with both physiological and pathological events. Here, we discuss the historical rising of the concept of prion-like domains, their relation to RNA and their role in protein aggregation. As a paradigmatic example, we present the case study of TDP-43, an RNA-binding prion-like protein associated with amyotrophic lateral sclerosis. Through this example, we demonstrate how the current definition of prions has incorporated quite different concepts making the meaning of the term richer and more stimulating. An important message that emerges from our analysis is the dual role of RNA in protein aggregation, making RNA, that has been considered for many years a ‘silent presence’ or the ‘stone guest’ of protein aggregation, an important component of the process.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae.