Proteasome activity imaging and profiling characterizes bacterial effector syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.     

Conjugates of ferrocene with biological compounds. Coordination to gold complexes and antitumoral properties

Several bioconjugates of ferrocene with biological compounds such as aminoacid esters and related species have been prepared by reaction of chlorocarbonyl ferrocene with the corresponding amino acid ester (histidine methyl ester, tryptophan methyl ester, methionine methyl ester and lysine ethyl ester) or histamine or prolinamide in the presence of NEt₃. The reaction of the tryptophan or prolinamide ferrocene conjugates with [Au(acac)(PR₃)] (acac = acetylacetonate) results in the substitution of the proton of the cyclic NH groups by the fragment AuPR₃⁺ affording the complexes [Au(FcCO-tryptophan-OMe)(PR₃)] or [Au(FcCO-prolinamide)(PR₃)] (Fc = ferrocenyl group). The reaction of FcCO-Met-OMe with [Au(OTf)(PR₃)] (OTF = trifluoromethysulfonate) or [Au(C₆F₅)₃(OEt₂)] yields the gold(I) or gold(III) derivatives [Au(FcCO-Met-OMe)(PR₃)]OTf or [Au(C₆F₅)₃(FcCO-Met-OMe)], respectively. Cytotoxicity studies towards several cancer lines such as MCF-7, HeLa or NIE-115 have been performed. The ferrocene bioconjugates show no activity whereas the gold complexes exhibit antiproliferative effect. Preliminary studies of interaction of compounds with cells were carried out with the goal of increasing our knowledge on the mechanism of action of these potential drugs.     

Impairment of the activity of the plasma membrane Ca²⁺-ATPase in Alzheimer's disease

AD (Alzheimer's disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aβ (amyloid β-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aβ neurotoxicity and Ca2+ dyshomoeostasis, but the molecular mechanisms involved are still under discussion. The PMCAs (plasma membrane Ca2+-ATPases) are a multi-isoform family of proteins highly expressed in brain that is implicated in the maintenance of low intraneural Ca2+ concentration. Therefore the malfunction of this pump may also be responsible for Ca2+ homoeostasis failure in AD. We have found that the Ca2+-dependence of PMCA activity is affected in human brains diagnosed with AD, being related to the enrichment of Aβ. The peptide produces an inhibitory effect on the activity of PMCA which is isoform-specific, with the greatest inhibition of PMCA4. Besides, cholesterol blocked the inhibitory effect of Aβ, which is consistent with the lack of any Aβ effect on PMCA4 found in cholesterol-enriched lipid rafts isolated from pig brain. These observations suggest that PMCAs are a functional component of the machinery that leads to Ca2+ dysregulation in AD and propose cholesterol enrichment in rafts as a protector of the Aβ-mediated inhibition on PMCA.     

Host-derived pericytes and Sca-1+ cells predominate in the MART-1- stroma fraction of experimentally induced melanoma

Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti-MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1-, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31-, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.     

Biphasic effects of copper on neurotransmission in rat hippocampal neurons

The importance of copper in the CNS is well documented, but the mechanisms related to its brain functions are poorly understood. Copper is released at the synaptic cleft, where it may modulate neurotransmission. To understand the functional impact of copper on the neuronal network, we have analyzed the synaptic activity of primary rat hippocampal neurons by using different approaches including whole cell patch clamp, recording of calcium transients, immunofluorescence and western blot. Here, we show that copper produces biphasic changes in neurotransmission. When copper is acutely applied to the plate it blocks neurotransmission. Interestingly, when it is applied for 3 h to hippocampal neurons it mainly increases the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)ergic currents (control: 0.21 ± 0.05 Hz/22.9 ± 1.3 pA; copper: 0.68 ± 0.16 Hz/30.5 ± 2.5 pA), intracellular calcium transients (control: 0.05 ± 0.013 Hz; copper: 0.11 ± 0.02 Hz) and evoked AMPA currents (control: EC50 8.3 ± 0.5?μM; copper: EC50 2.9 ± 0.2 μM). Moreover, our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. We also found that copper-treated neurons displayed an undistinguishable neurotransmission to control neurons after 24 h of treatment, indicating that changes in neurotransmission induced by copper at 3 h of incubation are homeostatically regulated after long-term exposure to the metal. Together, our data reveal an unexpected biphasic effect of copper on neurotransmission, which may be relevant to understand the effects of this ion in brain diseases that display copper dyshomeostasis such as that observed in Alzheimer's disease (AD).     

Invasive American Mink: Linking Pathogen Risk Between Domestic and Endangered Carnivores

Infectious diseases, in particular canine distemper virus (CDV), are an important threat to the viability of wild carnivore populations. CDV is thought to be transmitted by direct contact between individuals; therefore, the study of species interactions plays a pivotal role in understanding CDV transmission dynamics. However, CDV often appears to move between populations that are ecologically isolated, possibly through bridge hosts that interact with both species. This study investigated how an introduced species could alter multihost interactions and act as a bridge host in a novel carnivore assemblage of domestic dogs (Canis familiaris), invasive American mink (Neovison vison), and threatened river otters (Lontra provocax) in southern Chile. We found that rural dogs interact with mink near farms whereas in riparian habitats, minks and river otters shared the same latrines with both species visiting sites frequently within time intervals well within CDV environmental persistence. No interactions were observed between dogs and otters at either location. Both dog and mink populations were serologically positive for CDV, making the pathogen transfer risk to otters a conservation concern. Altogether, introduced mink in this ecosystem have the potential to act as bridge hosts between domestic dogs and endangered carnivores.     

One Solution for Two Challenges: The Lizard Microlophus atacamensis Avoids Overheating by Foraging in Intertidal Shores

In lizards, one of the most important behavioral mechanisms to cope with spatial and temporal variations in thermal resources observed is activity time. The longer a lizard can maintain activity, the more time it has to forage and reach larger adult body size. We studied the behavioral adjustments to different climatic regimens on daily and seasonal scales in three natural populations of the lizard Microlophus atacamensis along a latitudinal temperature and rainfall gradient. We also used Niche Mapper to determinate the amount of thermally suitable time for activity for this species. Abundance and daily activity patterns varied greatly over the year for the three populations. In summer and spring, the daily activity times were greater, and were reduced in fall and winter seasons. In summer, when stressful heat loads should prohibit activity over a midday gap, lizards did not show bimodal patterns of activity. Instead, they move to the cooler intertidal habitat. Abundance and thermal quality in the southernmost coolest site was lower, and the potential annual activity time decreases with latitude. Contrary to expectations, lizards from this locality showed the largest body sizes possibly due to diet and/or time to sexual maturation. Our results indicate that the intertidal habitat is a key factor that influences daily and seasonal activity of M. atacamensis lizards. While this habitat is not climatically optimal for lizards, it allows them to behaviorally extend their activity window and gain access to food in the intertidal areas.     

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

Background

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton''s jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton''s jelly (hWMSCs) can accelerate tissue repair in vivo.

Methods

Mesenchymal stem cells were isolated from human Wharton''s jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).

Results

hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.

Conclusion

These results demonstrate that mesenchymal stem cells isolated from Wharton''s jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.