The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

In field damage of high and low cyanogenic cassava due to a generalist insect herbivore Cyrtomenus bergi (Hemiptera: Cydnidae)

The hypothesis that cyanogenic potential in cassava roots deters polyphagous insects in the field is relevant to current efforts to reduce or eliminate the cyanogenic potential in cassava. To test this hypothesis, experiments were conducted in the field under natural selection pressure of the polyphagous root feeder Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae). A number of cassava varieties (33) as well as 13 cassava siblings and their parental clone, each representing a determined level of cyanogenic potential (CNP), were scored for damage caused by C. bergi and related to CNP and nonglycosidic cyanogens, measured as hydrogen cyanide. Additionally, 161 low-CNP varieties (< 50 ppm hydrogen cyanide, fresh weight) from the cassava germplasm core collection at Centro Internacional de Agricultura Tropical (CIAT) were screened for resistance/tolerance to C. bergi. Low root damage scores were registered at all levels of CNP. Nevertheless, CNP and yield (or root size) partly explained the damage in cassava siblings (r2 = 0.82) and different cassava varieties (r2 = 0.42), but only when mean values of damage scores were used. This relation was only significant in one of two crop cycles. A logistic model describes the underlying negative relation between CNP and damage. An exponential model describes the underlying negative relation between root size and damage. Damage, caused by C. bergi feeding, released nonglycosidic cyanogens, and an exponential model fits the underlying positive relation. Fifteen low-CNP clones were selected for potential resistance/tolerance against C. bergi.     

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[¹²⁵I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac₂[¹²⁵I]IUdR), 5-[¹²⁵I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv₂[¹²⁵I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac₂[¹²⁵I]IUdR, Piv₂[¹²⁵I]IUdR and [¹²⁵I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [¹²⁵I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv₂[¹²⁵I]IUdR>Ac₂[¹²⁵I]IUdR>[¹²⁵I]IUdR. For Ac₂[¹²⁵I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [¹²⁵I]IUdR, Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.     

Mechanism of inactivation gating of human T-type (low-voltage activated) calcium channels

Recovery from inactivation of T-type Ca channels is slow and saturates at moderate hyperpolarizing voltage steps compared with Na channels. To explore this unique kinetic pattern we measured gating and ionic currents in two closely related isoforms of T-type Ca channels. Gating current recovers from inactivation much faster than ionic current, and recovery from inactivation is much more voltage dependent for gating current than for ionic current. There is a lag in the onset of gating current recovery at -80 mV, but no lag is discernible at -120 mV. The delay in recovery from inactivation of ionic current is much more evident at all voltages. The time constant for the decay of off gating current is very similar to the time constant of deactivation of open channels (ionic tail current), and both are strongly voltage dependent over a wide voltage range. Apparently, the development of inactivation has little influence on the initial deactivation step. These results suggest that movement of gating charge occurs for inactivated states very quickly. In contrast, the transitions from inactivated to available states are orders of magnitude slower, not voltage dependent, and are rate limiting for ionic recovery. These findings support a deactivation-first path for T-type Ca channel recovery from inactivation. We have integrated these concepts into an eight-state kinetic model, which can account for the major characteristics of T-type Ca channel inactivation.     

Augmented Reticular Thalamic Bursting and Seizures in Scn1a-Dravet Syndrome

1. Download high-res image (154KB)
2. Download full-size image

     

Cytosolic hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific role in early development and stress response

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.     

Invasive species can handle higher leaf temperature under water stress than Mediterranean natives

Thermal tolerance of Photosystem II (PSII) highly influences plant distribution worldwide because it allows for photosynthesis during periods of high temperatures and water stress, which are common in most terrestrial ecosystems and particularly in dry and semi-arid ones. However, there is a lack of information about how this tolerance influences invasiveness of exotic species in ecosystems with seasonal drought. To address this question for Mediterranean-type ecosystems (MTE) of the Iberian Peninsula, we carried out an experiment with fifteen phylogenetically related species (8 invasive and 7 native, Pinus pinaster Ait., Pinus radiata D. Don, Schinus molle Linn., Elaeagnus angustifolia L., Eucalyptus globulus Labill., Acacia melanoxylon R. Br., Gleditsia triacanthos L., Pistacia terebinthus L., Rhamnus alaternus L., Anagyris foetida L., Colutea arborescens L., Oenothera biennis L., Epilobium hirsutum L., Achillea filipendulina Lam. and Achillea millefolium L). Seedlings were grown and maximal photochemical efficiency of PSII (Fv/Fm) was measured at two water availabilities (well-watered and with water stress). PSII thermal tolerance measurements were related to specific leaf area (SLA), which varied significantly across the study species, and to the mean potential evapotranspiration (PET) of the month with the lowest precipitation in the native areas of both groups and in the invaded area of the Iberian Peninsula. Additionally, PSII thermal tolerance measurements under water stress were phylogenetically explored. Invasive and native species neither differed in SLA nor in their thermal tolerance under well-watered conditions. For well-watered plants, SLA was significantly and positively related to PSII thermal tolerance when all species were explored together regardless of their invasive nature. However, this relationship did not persist under water stress and invasive species had higher plastic responses than Mediterranean natives resulting in higher leaf temperatures. Higher PSII thermal tolerance could explain invasiveness because it allows for longer periods of carbon acquisition under water stress. In fact, PSII thermal tolerance was positively related to the PET of the invaded and native areas of the Iberian Peninsula. PSII thermal tolerance was not related to PET at the native range of the invasive species, suggesting that successful invasive species were plastic enough to cope with novel dry conditions of the Iberian Peninsula. Moreover, our phylogenetic results indicate that future scenarios of increased aridity in MTE associated to climate change will filter invasion success by taxonomic identity. This study reveals the importance of studying ecophysiological traits to understand and better predict future biological invasions.     

Toxic effects of zinc on anaerobic microbiota from Zimapán Reservoir (Mexico)

The toxic effects of heavy metals have been extensively documented in different organisms. Nevertheless, a lack of information exists with regard to this topic in the case of autochthonous microorganism communities. The aim of this study was to evaluate the toxic effects of zinc on the anaerobic microorganisms present in the sediment and anoxic water of Zimapán Reservoir (Mexico), with particular focus on dissimilatory sulphate reducing bacteria. In the laboratory, a system of enrichment microcosms was set up with sediment and water from the reservoir. ATP, protein, carbohydrates and lactate and alcohol dehydrogenase activity were determined. The physicochemical parameters of the reservoir were evaluated over the course of one year. Sulphate reduction occurred in the reservoir throughout the year, but was most pronounced at the end of the wet season and during winter. In the field, increases in the rate of sulphate reduction coincided with the lowest levels of total phosphorus and hydrosoluble organic carbon. Zinc enrichment was observed to modify protein and carbohydrate content as well as to affect lactate and alcohol dehydrogenase activity. All responses followed a zinc concentration-response relationship and were dependent on reservoir physicochemical parameters. ATP content was used as a biomarker to evaluate the sublethal toxic effects of zinc. The acceptable threshold concentration of zinc in the aquatic and sediment enrichment microcosms was determined to be 0.06mgZn/L and 711.1mgZn/kg, respectively.