Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of β-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.     

Vitamins K interact with N-terminus α-synuclein and modulate the protein fibrillization in vitro. Exploring the interaction between quinones and α-synuclein

In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinson’s disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.     

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta^TM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression. Received: 25 September 1998 / Accepted: 27 October 1998     

Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis

Intersectin 1 (ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin-mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled-coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co-localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.     

Localization of Microfibrillar-Associated Protein 4 (MFAP4) in Human Tissues: Clinical Evaluation of Serum MFAP4 and Its Association with Various Cardiovascular Conditions

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease.     

MicroRNA-24 regulates the processing of latent TGFβ1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN

Cyclic mechanical stress (CMS) leads to alterations of cellular functions in the trabecular meshwork (TM), including the up-regulation of transforming growth factor beta 1 (TGFβ1), that can potentially contribute to the pathogenesis of glaucoma. Although microRNAs (miRNAs) are known to play important roles in many biological functions, little is known about their potential involvement in the cellular responses elicited by mechanical stress. Here we analyzed changes in miRNA expression induced by CMS, and examined the possible role of miR-24 in the response of human TM cells to CMS. CMS induced the expression of miR-24 that led to the down regulation of the subtilisin-like proprotein convertase FURIN, which is known to play a major role in the processing of TGFβ1. FURIN was confirmed as a novel target of miR-24 by 3' UTR luciferase assay and western blot. Overexpression of miR-24 resulted in a significant decrease in activated TGFβ1. This effect was mimicked by down regulation of FURIN by siRNA. Conversely, inhibition of miR-24 expression with a specific antagomir led to a small but significant increase in TGFβ1. Furthermore, the increase in active TGFβ1 induced by CMS in HTM cells was prevented by miR-24. Altogether, our results suggest that miRNAs might contribute to the regulation of responses to CMS in TM cells. Specifically, miR-24 might play an important role in modulating the induction of TGFβ1 mediated by CMS through direct targeting of FURIN.     

Anatomical and histological features of <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> leaves under different in vitro shoot culture systems

Ilex paraguariensis: (Aquifoliaceae) is an evergreen tree traditionally used to prepare a caffeine-rich infusion that has several medicinal properties. The in vitro propagation of this species has been studied as an alternative to conventional methods such as cuttings and seedlings. The in vitro culture environment consists of closed flasks with high relative humidity, reduced gas exchange and low luminosity conditions, which may induce physiological, anatomical and histological disruption in the cultivated plants. The occurrence of anatomical and histological alterations to the leaves of I. paraguariensis was examined in plants cultivated under greenhouse and different in vitro conditions. The variations in the microclimate inside the culture vessels generated by the different treatments tested affected the anatomy and histology of I. paraguariensis leaves by causing alterations in their architecture, thickness, vascularization and stomatal differentiation. A temporary immersion system was the best treatment for conserving the anatomical and histological features of the leaves. This treatment led to the lowest stomatal index and an extensive system of intercellular spaces that were similar to the characteristics developed under external environment conditions. Consequently, more than 80% of rooted plantlet transferred to pots were successfully rusticated. Plants from temporary immersion had greater photosynthetic rates due to a higher stomatal conductance.     

Raman spectroscopy and PCA‐SVM as a non‐invasive diagnostic tool to identify and classify qualitatively glycated hemoglobin levels in vivo

In this study we identify and classify high and low levels of glycated hemoglobin (HbA1c) in healthy volunteers (HV) and diabetic patients (DP). Overall, 86 subjects were evaluated. The Raman spectrum was measured in three anatomical regions of the body: index fingertip, right ear lobe, and forehead. The measurements were performed to compare the difference between the HV and DP (22 well controlled diabetic patients (WCDP) (HbA1c <6.5%), and 49 not controlled diabetic patients (NCDP) (HbA1c ≥6.5%)). Multivariable methods such as principal components analysis (PCA) combined with support vector machine (SVM) were used to develop effective diagnostic algorithms for classification among these groups. The forehead of HV versus WCDP showed the highest sensitivity (100%) and specificity (100%). Sensitivity (100%) and specificity (60%), were highest in the forehead of WCDP, versus NCDP. In HV versus NCDP, the fingertip had the highest sensitivity (100%) and specificity (80%). The efficacy of the diagnostic algorithm by receiver operating characteristic (ROC) curve was confirmed. Overall, our study demonstrated that the

combination of Raman spectroscopy and PCA‐SVM are feasible non‐invasive diagnostic tool in diabetes to classify qualitatively high and low levels of HbA1c in vivo.     

NEIL3-Dependent Regulation of Cardiac Fibroblast Proliferation Prevents Myocardial Rupture

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Cross-sectional study of height and weight in the population of Andalusia from age 3 to adulthood

Background and objectives

In Andalusia there were no studies including a representative sample of children and adolescent population assessing growth and weight increase. Our objectives were to develop reference standards for weight, height and BMI for the Andalusian pediatric population, from 3 to 18 years of age for both genders, and to identify the final adult height in Andalusia.

Subjects and methods

Two samples were collected. The first included individuals from 3 to 18 years of age (3592 girls and 3605 boys). They were stratified according type of study center, size of population of origin, age (32 categories of 0.5 years) and gender, using cluster sampling. Subjects from >18 to 23 years of age (947 women and 921 men) were sampled in 6 non-university educational centers and several university centers in Granada. Exclusion criteria included sons of non-Spanish mother or father, and individuals with chronic conditions and/or therapies affecting growth. Two trained fellows collected the data through February to December 2004, for the first sample, and through January to May 2005, for the second.Reference curves were adjusted using Cole's LMS method, and the quality of the adjustment was assessed using the tests proposed by Royston. In addition, a sensitivity analysis was applied to the final models obtained.

Results

Data for 9065 cases (4539 women and 4526 men) were obtained; 79.39% (n = 7197) in the up to 18 years of age group. In the first sampling only 0.07% (3 girls and 2 boys) refused to participate in the study. In addition, 327 students (4.5%) were absent when sampling was done. We present mean and standard deviation fort height, weight and BMI at 0.5 years intervals, from 3 to 23 years of age, for both genders. After adjustment with the different models, percentiles for height, weight (percentiles 3, 5, 10, 25, 50, 75, 90, 95, and 97) and BMI (percentiles 3, 5, 50, 85, 95, and 97) are presented for both genders.

Conclusion

This is the first study in Andalusia with a representative sample from the child-juvenile population to investigate weight, height and BMI in subjects from 3 to 23 years of age. The great variability observed in the values from sample of 18 to 23 years of age individuals, ensures the inclusion of extreme values, although random sampling was not used. There still is a lack of standard reference values for the Andalusian population younger done 3 years of age.