Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (52 residues out of a total of 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.Abbreviations A absorbance - Bis N,N-methylene bisacrylamide - IEF isoelectric focusing - 2-ME 2-meroaptoethanol - mol wt molecular weight - 62 opaque-2 - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - PAS periodic acid-Schiff stain - SDS sodium dodecyl sulphate - ICA trichloroacetic acid - TEMED N,N,N,N-tetramethyl ethylene diamine - Z₁ zein extracted with 55% isopropanol - Z₂ zein extracted with 55% isopropanol and 0.6% 2-ME - Z 9.6 zein of mol wt 9600 - Z 13.5 zein of mol wt 13,500 - Z 21 zein of mol wt 21,000 - Z 23 zein of mol wt 23,000     

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

Perthamide C Inhibits eNOS and iNOS Expression and Has Immunomodulating Activity In Vivo

Here we have characterized perthamide C, a cyclopeptide from a Solomon Lithistid sponge Theonella swinhoei, which displays an anti-inflammatory/immunomodulatory activity. The study has been performed using the carragenan-induced mouse paw edema that displays an early (0–6 h) and a late phase (24–96 h). Perthamide C significantly inhibits neutrophils infiltration in tissue both in the early and late phases. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) in the early phase while cyclooxygenase-1 and 2 (COX-1, COX-2), and inducible NOS (iNOS) expression were unaffected. In the late phase perthamide C reduced expression of both NOS isoforms without affecting COXs expression. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation. We found that perthamide C inhibited the proliferation of peripheral lymphocytes, and that this effect was secondary to its metabolic activation in vivo. Indeed, in vitro perthamide C did not inhibit proliferation as opposite to its metabolite perthamide H.In conclusion, perthamide C selectively interferes with NO generation triggered by either eNOS or iNOS without affecting either COX-1 or COX-2. This in turn leads to modulation of the inflammatory response through a reduction of vascular permeability, neutrophil infiltration as well as lymphocyte proliferation.     

Navigating from live to virtual social interactions: looking at but not manipulating smartphones provokes a spontaneous mimicry response in the observers

Journal of Ethology - By gathering data on people during their ordinary daily activities, we tested if looking at, but not manipulating, smartphones led to a mimicry response in the observer....     

Aromatic and aliphatic mono- and bis-nitroxides: A study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV–Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2′-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.     

Modification of chromatin organisation at low water potential in cultured cells of Solanum tuberosum: Possible involvement of dehydrins

ABSTRACT

To understand the mechanisms which enable the nucleus to function under low water potential, the morphology and biochemistry of potato cell nuclei were studied. Conformational modifications were observed in the chromatin of nuclei of cells growing under low water potential. These modifications include a higher number of heterochromatic centres, enlargement of the nuclear diameter, and a different accessibility of DNA to the action of restriction enzymes. Biochemical analyses showed that these chromatin modifications may coincide with quantitative and qualitative variations of several nuclear proteins, some of which may belong to the dehydrin family. We especially focussed our attention on a 45-kDa protein that is heat-stable and is recognised by an antibody raised against the conserved domain of dehydrins. The survival of potato cells in an environment where water availability is low may depend on several simultaneous events regarding the nucleus. The accumulation in the nucleus of specific proteins such as dehydrins could be required to stabilise the chromatin by means of their molecule-salvation action. Further studies are in progress to check whether or not variations in chromatin organisation may be one of the numerous traits that a cell must acquire to become water-stress resistant.     

Detection and Genotyping of Campylobacter jejuni and Campylobacter coli by Use of DNA Oligonucleotide Arrays

Campylobacter have emerged as the most common bacterial food-borne illness in the developed world. The ability to reduce Campylobacter infections in humans is linked to the full comprehension of the principal key aspects of its infection cycle. A microbial diagnostic microarray detecting Campylobacter housekeeping, structural, and virulence associated genes was designed and validated using genomic DNA from reference and field strains of Campylobacter jejuni and coli isolated from human, chicken, and raw milk. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging Campylobacter pathotypes as well as for epidemiological, environmental, and phylogenetic studies including the evaluation of genome plasticity.     

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10–17 yr and 18–50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization–MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer''s disease, which is frequently associated with Down syndrome.Down syndrome (DS)¹ is a frequent genetic disorder in humans characterized by premature aging (1). A clinical feature of people with DS is the increased prevalence and severity of periodontal disease compared with age-matched subjects of similar levels of intellectual impairment and compared with the general population (2). Common conditions observed in DS are marginal gingivitis, acute and subacute necrotizing gingivitis, advanced periodontitis, gingival recession, and pocket formation (3, 4). It is known that saliva plays an important role in maintaining oral and dental health, because of the presence of a variety of antimicrobial peptides mainly derived from gland secretion, oral epithelial cells, and neutrophils (5). Several papers reported that neutrophils and T-lymphocyte function is impaired in people with DS (6–9). However, the salivary secretion of the antimicrobial LL-37 in young individuals with DS was found normal (10). A review of the literature (11, 12) reveals only sporadic and contradictory reports that attempt to explain the role of saliva in the oral health of subjects with DS, and on the whole, information on the biochemical composition of their saliva is scarce. On the basis of the above information, in the present study, we proposed to investigate the salivary proteome of DS subjects by an intact protein-based “top-down” approach. The spectrum of salivary peptides of DS subjects was compared with that of sex and age-matched healthy control groups to determine qualitative and quantitative differences. Interestingly, the results showed that several members of the S100A family, which possess different biological functions, and also described as potential markers of the Alzheimer Disease, were significantly increased in saliva of Down syndrome subjects with respect to controls.