Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features

The prion protein (PrP^c) is a copper-binding glycoprotein that can misfold into a β-sheet-rich and pathogenic isoform (PrP^sc) leading to prion diseases. The first non-mammalian PrP^c was identified in chicken and it was found to keep many structural motifs present in mammalian PrP^c, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV–vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in June 2005 with corrections to the text. The author name LaMendola has been corrected to La Mendola.     

Ticks (Acari: Ixodidae) of the state of Amazonas,Brazil

The tick fauna of Brazil is currently composed by 72 species. The state of Amazonas is the largest of Brazil, with an area of ≈ 19% of the Brazilian land. Besides its vast geographic area, only 19 tick species have been reported for Amazonas. Herein, lots containing ticks from the state of Amazonas were examined in three major tick collections from Brazil. A total of 5933 tick specimens were examined and recorded, comprising 2693 males, 1247 females, 1509 nymphs, and 484 larvae. These ticks were identified into the following 22 species: Amblyomma cajennense sensu lato, Amblyomma calcaratum, Amblyomma coelebs, Amblyomma dissimile, Amblyomma dubitatum, Amblyomma geayi, Amblyomma goeldii, Amblyomma humerale, Amblyomma latepunctatun, Amblyomma longirostre, Amblyomma naponense, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma rotundatum, Amblyomma scalpturatum, Amblyomma varium, Dermacentor nitens, Haemaphysalis juxtakochi, Ixodes cf. Ixodes fuscipes, Ixodes luciae, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato. Ticks were collected from 17 (27.4%) out of the 62 municipalities that currently compose the state of Amazonas. The following four species are reported for the first time in the state of Amazonas: A. coelebs, A. dubitatum, H. juxtakochi, and Ixodes cf. I. fuscipes. The only tick species previously reported for Amazonas and not found in the present study is Amblyomma parvum. This study provides a great expansion of geographical and host records of ticks for the state of Amazonas, which is now considered to have a tick fauna composed by 23 species. It is noteworthy that we report 1391 Amblyomma nymphs that were identified to 13 different species.     

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Abstract

Tetracycline‐controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon‐γ in the RK‐13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon‐γ (rGPoIFN‐γ) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN‐γ cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN‐γ (up to 16 μg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of ³⁵S‐Methionine, labelled rGPoIFN‐γ with natural leukocytic IFN‐γ after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN‐γ species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N‐glycosydase F. The biological activity of rGPoIFN‐γ was in the same range as that of natural leukocytic PoIFN‐γ (2 × 10⁶ U/mg). Eventually, this recombinant mammalian IFN‐γ should constitute a very useful substitute for leukocyte PoIFN‐γ in in vitro or in vivo experiments.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.     

Regulation of microRNA-145 by growth arrest and differentiation

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.     

Influence of the mother's reproductive state on the hormonal status of daughters in marmosets (Callithrix kuhlii)

Behavioral and endocrine suppression of reproduction in subordinate females produces the high reproductive skew that characterizes callitrichid primate mating systems. Snowdon et al. [American Journal of Primatology 31:11-21, 1993] reported that the eldest daughters in tamarin families exhibit further endocrinological suppression immediately following the birth of siblings, and suggested that dominant females exert greater control over subordinate endocrinology during this energetically challenging phase of reproduction. We monitored the endocrine status of five Wied's black tufted-ear marmoset daughters before and after their mother delivered infants by measuring concentrations of urinary estradiol (E(2)), pregnanediol glucuronide (PdG), testosterone (T), and cortisol (CORT). Samples were collected from marmoset daughters 4 weeks prior to and 9 weeks following three consecutive sibling-litter births when the daughters were prepubertal (M=6.1 months of age), peripubertal (M=11.9 months), and postpubertal (M=17.6 months). The birth of infants was associated with reduced ovarian steroid excretion only in the prepubertal daughters. In contrast, ovarian steroid levels tended to increase in the postpubertal daughters. Urinary E(2) and T levels in the postpubertal daughters were 73.8% and 37.6% higher, respectively, in the 3 weeks following the birth of infants, relative to prepartum levels. In addition, peak urinary PdG concentrations in peri- and postpubertal daughters were equivalent to luteal phase concentrations in nonpregnant, breeding adult females, and all of the peri- and postpubertal daughters showed clear ovulatory cycles. Cortisol excretion did not change in response to the reproductive status of the mother, nor did the concentrations change across age. Our data suggest that marmoset daughters of potential breeding age are not hormonally suppressed during the mother's peripartum period or her return to fertility. These findings provide an additional example of species diversity in the social regulation of reproduction in callitrichid primates.     

Negative occurrence between hippocampal rhythmic slow activity and epileptiform spikes: influence of the striatum

The effects of caudate and septal stimulation on hippocampal activity were studied. Sodium penicillin was intravenously injected in order to induce a steady rate of interictal epileptic activity. Penicillin injection caused significant reduction of the rate of occurrence and duration of hippocampal rhythmic slow activity (RSA). Spontaneous RSA as well as RSA-eliciting caudate and septal stimulation induced a marked inhibition on frequency of epileptiform complexes. Lesions of the medial septal nucleus made it impossible to record RSA or to observe any sort of influence on hippocampal epileptiform activity by caudate stimulation. This result suggests that the septum is important for RSA genesis in the striato-hippocampal pathway or in the reciprocal septo-hippocampal connections.     

Assessment of Real-time PCR Based Methods for Quantification of Pollen-mediated Gene Flow from GM to Conventional Maize in a Field Study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.     

Canadians’ Perceptions of Food,Diet, and Health – A National Survey

Background

Poor nutrition is harmful to one’s health as it can lead to overweight and obesity and a number of chronic diseases. Understanding consumer perceptions toward diet and nutrition is critical to advancing nutrition-related population health interventions to address such issues. The purpose of this paper was to examine Canadians’ perceived health and diet status, compared to their actual health status, and general concern about their own diet and beliefs about health. Also analyzed were some of the perceived barriers to eating “healthy” foods, with a focus on the availability of “healthy” processed foods.

Methods

Two surveys were administered online to a group of Canadian panelists from all ten provinces during May 2010 to January 2011. Thirty thousand were invited; 6,665 completed the baseline survey and 5,494 completed the second survey. Panelists were selected to be nationally representative of the Canadian adult population by age, sex, province and education level, according to 2006 census data.

Results

Approximately one third of Canadians perceived their health or diet to be very good while very few Canadians perceived their health or diet to be very poor. While the majority of Canadians believed food and nutrition to be very important for improving one’s health, fewer Canadians were concerned about their own diets. The majority of Canadians reported difficulty finding “healthy” processed foods (low in salt and sugar and with sufficient vitamins and minerals). Many also reported difficulty finding healthy foods that are affordable.

Conclusion

Although consumers believe that nutrition is one of the most important factors for maintaining health, there are still a number of attitudinal and perceived environmental barriers to healthy eating.