Expression analysis of the human adducin gene family and evidence of ADD2 beta4 multiple splicing variants

Adducin is a cytoskeleton heterodimeric protein. Its subunits are encoded by three related genes (ADD1, ADD2, and ADD3) which show alternative spliced variants. Adducin polymorphisms are involved in blood pressure regulation in humans and rats. We have analyzed mRNA distribution of ADD gene family in human tissues and cells with Real-Time TaqMan RT-PCR. Whereas ADD1 is ubiquitously distributed, ADD3 is more expressed in kidney medulla and cortex than in fetal kidney, while in adult liver it is less abundant than in fetal liver. ADD2 beta1 and beta4 variants show the same pattern of distribution with the highest expression in brain, fetal liver, and kidney. Conventional RT-PCR identified new beta4 variants. Beta4a is characterized by an in-frame insertion of 21 nucleotides upstream exon 15 predicting a 7 amino acids longer protein with a similar C-terminus region. It is coexpressed with beta1 and beta4 in several tissues. Fetal kidney shows further beta4b, beta4c and beta4d variants containing internal exon deletions that enormously modify the predicted NH(2) and central regions. Our findings could help one to understand the functional role of adducin variants in specific tissues and cells.     

PKCdelta requires p53 for suppression of the transformed phenotype in human colon cancer cells

We have previously demonstrated that the delta isoform of Protein Kinase C (PKCdelta) acts as a tumor suppressor in HCT116 human colon cancer cells, and that p21(waf1/cip1) is an essential downstream effector of PKCdelta. Our data suggested that p53 might also be involved in the suppression of the neoplastic phenotype induced by PKCdelta. Here we show that homozygous knockout of p53 renders the HCT116 cell line unresponsive to PKCdelta overexpression. Whereas reconstitution of p53 alone did not modify the morphology and growth properties of HCT116/p53null cells, overexpression of both p53 and PKCdelta induced a number of alterations indicating suppression of the transformed phenotype. Interestingly, PKCdelta was ineffective when overexpressed in HT29 cells, a human colon cancer line characterized by the Arg273His dominant-negative mutation of p53. Thus, our data indicate that wild-type p53 is an essential effector of PKCdelta in human colon cancer cells.     

Equine and canine Anaplasma phagocytophilum strains isolated on the island of Sardinia (Italy) are phylogenetically related to pathogenic strains from the United States

The presence of Anaplasma phagocytophilum, a tick-transmitted zoonotic pathogen, was investigated in Sardinia using a molecular approach. Phylogenetic analysis revealed that Sardinian strains are genetically distinct from the two lineages previously described in Europe and are closely related to strains isolated in different areas of the United States.     

Structural damage induced by peroxidation may account for functional impairment of heavy synaptic mitochondria

Coenzyme Q distribution, as well as respiratory chain features, in rat brain mitochondria depend on mitochondrial subpopulation, brain region and age. Heavy mitochondria (HM) usually display the lowest content of respiratory components and the lowest enzymatic activities and it has been suggested that they represent the oldest mitochondrial population. In this study, we confirmed that HM are considerably compromised in their structure. In fact, HM showed to have the highest hydroperoxide content and the most consistent modifications in their fatty acid pattern with wide loss of fatty acids (or part of them) in the phospholipid moiety. Such situation could explain the typical impairment of HM and could support the hypothesis that they represent an old mitochondrial population.     

Topological investigation of amyloid fibrils obtained from beta2-microglobulin

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.     

Silk fibroin film from golden‐yellow Bombyx mori is a biocomposite that contains lutein and promotes axonal growth of primary neurons

The use of doped silk fibroin (SF) films and substrates from Bombyx mori cocoons for green nanotechnology and biomedical applications has been recently highlighted. Cocoons from coloured strains of B. mori, such as Golden‐Yellow, contain high levels of pigments that could have a huge potential for the fabrication of SF based biomaterials targeted to photonics, optoelectronics and neuroregenerative medicine. However, the features of extracted and regenerated SF from cocoons of B. mori Golden‐Yellow strain have never been reported. Here we provide a chemophysical characterization of regenerated silk fibroin (RSF) fibers, solution, and films obtained from cocoons of a Golden‐Yellow strain of B. mori, by SEM, ¹H‐NMR, HPLC, FT‐IR, Raman and UV‐Vis spectroscopy. We found that the extracted solution and films from B. mori Golden‐Yellow fibroin displayed typical Raman spectroscopic and optical features of carotenoids. HPLC‐analyses revealed that lutein was the carotenoid contained in the fiber and RSF biopolymer from yellow cocoons. Notably, primary neurons cultured on yellow SF displayed a threefold higher neurite length than those grown of white SF films. The results we report pave the way to expand the potential use of yellow SF in the field of neuroregenerative medicine and provide green chemistry approaches in biomedicine. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 287–299, 2016.     

Light effects on in vitro rooting of pear cultivars of different rhizogenic ability

The effect of varying light regimes on in vitro rooting of microcuttings of two pear (Pyrus communis L.) cultivars was investigated. Cultures of the easy to-root Conference and the difficult-to-root Doyenne d'Hiver were incubated for 21 days with or without indole-3-butyric acid (IBA) in the medium in darkness or under continuous far-red (8 µmol m^–2 s^–1), blue, white or red (15 or 36 µmol m^–2 s^–1) light. Conference rooted without IBA when exposed to red, blue or white light while no rooting was observed under far-red light and in darkness. The high rooting efficiency under red and, by contrast, the inhibition under far-red light and darkness suggest the involvement of the phytochrome system in rhizogenesis. The addition of IBA to the culture medium enhanced root production under all light regimes in both cultivars. Red light, especially at the lower photon fluence rate, had a positive effect by increasing root extension (number × length of roots) and stimulating secondary root formation.Abbreviations IBA Indole-3-butyric acid - R red light - B blue light - FR far-red light - W white light - D darkness - P_fr active (far-red light absorbing) form of phytochrome - P_tot total phytochrome - BA benzyl-adenine     

False occurrences of functional motifs in protein sequences highlight evolutionary constraints

Background

False occurrences of functional motifs in protein sequences can be considered as random events due solely to the sequence composition of a proteome. Here we use a numerical approach to investigate the random appearance of functional motifs with the aim of addressing biological questions such as: How are organisms protected from undesirable occurrences of motifs otherwise selected for their functionality? Has the random appearance of functional motifs in protein sequences been affected during evolution?

Results

Here we analyse the occurrence of functional motifs in random sequences and compare it to that observed in biological proteomes; the behaviour of random motifs is also studied. Most motifs exhibit a number of false positives significantly similar to the number of times they appear in randomized proteomes (=expected number of false positives). Interestingly, about 3% of the analysed motifs show a different kind of behaviour and appear in biological proteomes less than they do in random sequences. In some of these cases, a mechanism of evolutionary negative selection is apparent; this helps to prevent unwanted functionalities which could interfere with cellular mechanisms.

Conclusion

Our thorough statistical and biological analysis showed that there are several mechanisms and evolutionary constraints both of which affect the appearance of functional motifs in protein sequences.