α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Next-generation sequencing analyses of the emergence and maintenance of mutations in CTL epitopes in HIV controllers with differential viremia control

Background

Despite the low level of viral replication in HIV controllers (HICs), studies have reported viral mutations related to escape from cytotoxic T-lymphocyte (CTL) response in HIV-1 plasma sequences. Thus, evaluating the dynamics of the emergence of CTL-escape mutants in HICs reservoirs is important for understanding viremia control. To analyze the HIV-1 mutational profile and dynamics of CTL-escape mutants in HICs, we selected 11 long-term non-progressor individuals and divided them into the following groups: (1) viremic controllers (VCs; n?=?5) and (2) elite controllers (ECs; n?=?6). For each individual, we used HIV-1 proviral DNA from PBMCs related to earliest (V_E) and latest (V_L) visits to obtain gag and nef sequences using the Illumina HiSeq system. The consensus of each mapped gene was used to assess viral divergence, and next-generation sequencing data were employed to identify SNPs and variations within and flanking CTL epitopes.

Results

Divergence analysis showed higher values for nef compared to gag among the HICs. EC and VC groups showed similar divergence rates for both genes. Analysis of the number of SNPs showed that VCs present more variability in both genes. Synonymous/non-synonymous mutation ratios were?<?1 for gag among ECs and for nef among ECs and VCs, exhibiting a predominance of non-synonymous mutations. Such mutations were observed in regions encoding CTL-restricted epitopes in all individuals. All ECs presented non-synonymous mutations in CTL epitopes but generally at low frequency (<?1%); all VCs showed a high number of mutations, with significant frequency changes between V_E and V_L visits. A higher frequency of internal mutations was observed for gag epitopes, with significant changes across visits compared to Nef epitopes, indicating a pattern associated with differential genetic pressure.

Conclusions

The high genetic conservation of HIV-1 gag and nef among ECs indicates that the higher level of viremia control restricts the evolution of both genes. Although viral replication levels in HICs are low or undetectable, all individuals exhibited CTL epitope mutations in proviral gag and nef variants, indicating that potential CTL escape mutants are present in HIC reservoirs and that situations leading to a disequilibrium of the host-virus relationship can result in the spread of CTL-escape variants.

     

EFFECT OF COPPER ON ALGAL COMMUNITIES FROM OLIGOTROPHIC CALCAREOUS STREAMS1

Two sets of experiments were done to quantify the effects of chronic copper exposure on natural peri‐ phyton in a nonpolluted calcareous river. The results of short‐term (up to 6 h exposure) experiments corroborated the significance of pH on copper toxicity. Copper toxicity increased when pH was reduced from 8.6 to 7.7, and this was related to the effect of pH on copper speciation (free copper concentration increased from 0.2% to 2.3% of total copper). Longer term experiments demonstrated that periphyton communities exposed to copper under pH variation (8.2–8.6) were already affected at 10 μg·L ^{? 1} (20–80 ng·L ^{? 1} Cu^{2 +} ) after 12 days of exposure. Copper exposure caused stronger effects on structural (algal biomass and community structure) than on functional (photosynthetic efficiency) parameters of peri‐ phyton. Changes in community composition included the enhancement of some taxa (Gomphonema gracile), the inhibition of others (Fragilaria capucina and Phormidium sp.), and the appearance of filament malformations (Mougeotia sp.). The results of our study demonstrated that several weeks of exposure to copper (10–20 μg·L ^{? 1}) were sufficient to cause chronic changes in the periphyton of oligotrophic calcareous rivers. This degree of copper pollution can be commonly found in the Mediterranean region as a result of agricultural practices and farming activities.     

ENVIRONMENTAL FACTORS CORRELATED WITH CHRYSOPHYTE CYST ASSEMBLAGES IN LOW ARCTIC LAKES OF SOUTHWEST GREENLAND1

We analyzed the relationship between chrysophyte cyst assemblages in surface sediment samples and limnological and geographical variables for 70 lakes located along Søndre Strømfjord in southwest Greenland. Over 247 stomatocysts were identified and of these, 153 were sufficiently abundant for use in statistical analyses. Eight stomatocysts were considered to be new and are described formally. Canonical correspondence analysis indicated that conductivity was the dominant variable explaining cyst distribution, reflecting the large conductivity gradient in lake water chemistry in this area. High conductivity lakes had distinctive cyst assemblages with lower diversity than low alkalinity lakes, where assemblages were similar to alpine soft‐water lakes elsewhere. The high conductivity lakes, however, had similar cysts to other saline lakes elsewhere in the arctic. Additionally, pH, calcium, maximum depth, longitude, sulfate, total phosphorus, and altitude all explained significant amounts of variability of cyst assemblages. Longitude was the only geographical variable that explained cyst variability independently of other variables (i.e. had a unique effect), which suggests that the climatic gradient from the coast to the head of the fjord has a structuring effect on cyst assemblages. Conductivity (weighted‐averaging partial least squares, r²=0.917; root mean square error=0.142; r²_jack=0.861, root mean square error of prediction=0.191) and pH inference models (weighted averaging, r²=0.924; root mean square error=0.158; r²_jack=0.826, root mean square error of prediction=0.240) were developed. For the pH model, high conductivity lakes (>800 μ S₂₀·cm^?1) were removed. Both models are statistically robust and could be applied to lakes in west Greenland to reconstruct conductivity and/or pH. Such paleolimnological reconstructions provide the means of acquiring long‐term data for use in the evaluation of, for example, regional paleoclimatic models.     

Ethanol administration and the relationship of malonyl-coenzyme A concentrations to the rate of fatty acid synthesis in rat liver

1. The effect of ethanol on liver fatty acid synthesis was studied in vivo in 24h-starved and ;meal-fed' rats (i.e. fed for 3h per day and not ad libitum). 2. In the fed animal (3)H(2)O was incorporated into fat at a rate of 0.46mumol of C(2) units/min per g wet wt. of liver. Administration of either ethanol (3.2g/kg) or equicaloric amounts of glucose had no effect on the rate of (3)H(2)O incorporation into lipid. 3. In the 24h-starved animal, administration of the same dose of ethanol produced an increase in the rate of (3)H(2)O incorporation from 0.06 to 0.12mumol of C(2) units/min per g fresh wt. after 3h whereas [malonyl-CoA] increased from 0.006 to 0.009mumol/g. Glucose given in amounts equicaloric to ethanol was significantly more lipogenic, increasing both the (3)H(2)O incorporation from 0.06 to 0.20mumol of C(2) units/min per g and the malonyl-CoA content from 0.006 to 0.013 mumol/g wet wt. at 3h. 4. The decrease in the redox state of free cytoplasm NAD or NADP couples or the changes in content of citrate, glucose 6-phosphate and pyruvate of liver after ethanol administration had no measurable effect on the rate of fatty acid synthesis in vivo. 5. Under the conditions of the experiments there was no significant difference, among any of the groups, in the activity of liver fatty acid synthetase measured in vitro. A double-reciprocal plot of the rate of (3)H(2)O incorporation and the total tissue malonyl-CoA concentrations showed a striking relationship. It has been concluded that the rate of fatty acid synthesis in vivo is determined principally by the V(max.) of fatty acid synthetase and the concentration of free malonyl-CoA. 6. It has also been concluded that under the conditions of the present study, the synthesis of fatty acids de novo is unlikely to be an important factor in the increased liver lipid content associated with ethanol administration.     

Impacts of forestry planting on primary production in upland lakes from north‐west Ireland

Planted forests are increasing in many upland regions worldwide, but knowledge about their potential effects on algal communities of catchment lakes is relatively unknown. Here, the effects of afforestation were investigated using palaeolimnology at six upland lake sites in the north‐west of Ireland subject to different extents of forest plantation cover (4–64% of catchment area). ²¹⁰Pb‐dated sediment cores were analysed for carotenoid pigments from algae, stable isotopes of bulk carbon (δ¹³C) and nitrogen (δ¹⁵N), and C/N ratios. In lakes with >50% of their catchment area covered by plantations, there were two‐ to sixfold increases in pigments from cryptophytes (alloxanthin) and significant but lower increases (39–116%) in those from colonial cyanobacteria (canthaxanthin), but no response from biomarkers of total algal abundance (β‐carotene). In contrast, lakes in catchments with <20% afforestation exhibited no consistent response to forestry practices, although all lakes exhibited fluctuations in pigments and geochemical variables due to peat cutting and upland grazing prior to forest plantation. Taken together, patterns suggest that increases in cyanobacteria and cryptophyte abundance reflect a combination of mineral and nutrient enrichment associated with forest fertilization and organic matter influx which may have facilitated growth of mixotrophic taxa. This study demonstrates that planted forests can alter the abundance and community structure of algae in upland humic lakes of Ireland and Northern Ireland, despite long histories of prior catchment disturbance.     

Neoadjuvant Sequential Docetaxel Followed by High‐Dose Epirubicin in Combination With Cyclophosphamide Administered Concurrently With Trastuzumab. The DECT Trial

To report the results of the DECT trial, a phase II study of locally advanced or operable HER2‐positive breast cancer (BC) treated with taxanes and concurrent anthracyclines and trastuzumab. Eligible patients (stage IIA‐IIIB HER2‐positive BC, 18–75 years, normal organ functions, ECOG ≤1, and left ventricular ejection fraction (LVEF) ≥55%) received four cycles of neoadjuvant docetaxel, 100 mg/m² intravenously, plus trastuzumab 6 mg/kg (loading dose 8 mg/kg) every 3 weeks, followed by four 3‐weekly cycles of epirubicin 120 mg/m² and cyclophosphamide, 600 mg/m², plus trastuzumab. Primary objective was pathologic complete response (pCR) rate, defined as ypT0/is ypN0 at definitive surgery. We enrolled 45 consecutive patients. All but six patients (13.3%) completed chemotherapy and all underwent surgery. pCR was observed in 28 patients (62.2%) overall and in 6 (66.7%) from the inflammatory subgroup. The classification and regression tree analysis showed a 100% pCR rate in patients with BMI ≥25 and with hormone negative disease. The median follow up was 46 months (8–78). Four‐year recurrence‐free survival was 74.7% (95%CI, 58.2–91.2). Seven patients (15.6%) recurred and one died. Treatment was well tolerated, with limiting toxicity being neutropenia. No clinical cardiotoxicity was observed. Six patients (13.4%) showed a transient LVEF decrease (<10%). In one patient we observed a ≥10% asymptomatic LVEF decrease persisting after surgery. Notwithstanding their limited applicability due to the current guidelines, our findings support the efficacy of the regimen of interest in the neoadjuvant setting along with a fairly acceptable toxicity profile, including cardiotoxicity. Results on BMI may invite further assessment in future studies. J. Cell. Physiol. 231: 2541–2547, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Higher plants possess two different types of ATX1-like copper chaperones

Copper (Cu) chaperones constitute a family of small Cu+-binding proteins required for Cu homeostasis in eukaryotes. The ATX1 family of Cu chaperones specifically delivers Cu to heavy metal P-type ATPases. The plant Arabidopsis thaliana expresses the ATX1-like Cu chaperone CCH, which exhibits a plant-specific carboxy-terminal domain (CTD) with unique structural properties. We show that CCH homologues from other higher plants contain CTDs with structural properties similar to Arabidopsis CCH. Furthermore, we identify a new ATX1-like Cu chaperone in Arabidopsis, AtATX1, which functionally complements yeast atx1Delta and sod1Delta associated phenotypes, and localizes to the cytosol of Arabidopsis cells. Interestingly, AtATX1, but not full-length CCH, interacts in vivo with the Arabidopsis RAN1 Cu-transporting P-type ATPase by yeast two-hybrid. We propose that higher plants express two types of ATX1-like Cu chaperones: the ATX1-type with a predominant function in Cu delivery to P-type ATPases, and the CCH-type with additional CTD-mediated plant-specific functions.     

Genetic study of the population of Tenerife (Canary Islands,Spain): Protein markers and review of classical polymorphisms

Data on six protein polymorphisms (19 alleles) from the human population of Tenerife are presented and discussed along with other classical markers in relation to the origin of the Canarians. Genetic influences from three population groups were considered: the Iberians, and the Berbers and non-Berbers (Arabs) from north Africa. The systems examined show the Tenerife population lies within the limits of variation described for various Iberian groups, with a slight tendency towards the characteristics of north African populations. When blood groups, red cell enzymes and serum protein data were considered, the similarity of the Canary population to Iberians seems strengthened (70% estimated contribution of Iberian peninsula genes to the present-day Canarian pool), while some relation with north African groups is shown. Genetic distances between Canarians and Arabs and Canarians and Berbers are lower than those between the two north African groups, indicating a relative and comparable contribution of each to the present-day gene pool of the Canarian population. The Arab contribution could be attributable to the slaves who were introduced to these islands after the conquest in the 15th century, while the Berber contribution could be the remnants of the extinct aboriginal peoples of the islands (Guanches) or a more recent immigration due to slavery. Genetic data do not allow us to distinguish between these two possibilities. Am. J. Phys. Anthropol. 102:337–349, 1997. © 1997 Wiley-Liss, Inc.