Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

Tubulin Tyrosine Ligase Like 12, a TTLL Family Member with SET- and TTL-Like Domains and Roles in Histone and Tubulin Modifications and Mitosis

hTTLL12 is a member of the tubulin tyrosine ligase (TTL) family that is highly conserved in phylogeny. It has both SET-like and TTL-like domains, suggesting that it could have histone methylation and tubulin tyrosine ligase activities. Altered expression of hTTLL12 in human cells leads to specific changes in H4K20 trimethylation, and tubulin detyrosination, hTTLL12 does not catalyse histone methylation or tubulin tyrosination in vitro, as might be expected from the lack of critical amino acids in its SET-like and TTLL-like domains. hTTLL12 misexpression increases mitotic duration and chromosome numbers. These results suggest that hTTLL12 has non-catalytic functions related to tubulin and histone modification, which could be linked to its effects on mitosis and chromosome number stability.     

An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae

Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO₄ system in the second.With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain.The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.     

Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

Rotavirus infection induces an increase in [Ca²⁺]_cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca²⁺ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca²⁺ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.     

Animal models of fibromyalgia

Animal models of disease states are valuable tools for developing new treatments and investigating underlying mechanisms. They should mimic the symptoms and pathology of the disease and importantly be predictive of effective treatments. Fibromyalgia is characterized by chronic widespread pain with associated co-morbid symptoms that include fatigue, depression, anxiety and sleep dysfunction. In this review, we present different animal models that mimic the signs and symptoms of fibromyalgia. These models are induced by a wide variety of methods that include repeated muscle insults, depletion of biogenic amines, and stress. All potential models produce widespread and long-lasting hyperalgesia without overt peripheral tissue damage and thus mimic the clinical presentation of fibromyalgia. We describe the methods for induction of the model, pathophysiological mechanisms for each model, and treatment profiles.     

Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

Background

Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line.

Methods and Principal Findings

We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis.

Conclusions

Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study were already known as TG2 substrates, we can also suppose that transamidating activity and differential phosphorylation of the same targets may represent a novel regulatory mechanism whose relevance in celiac disease pathogenesis is still unexplored.     

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L ‐lysine) (Ca‐alg‐PLL), and a novel calcium alginate‐poly(ethylene glycol) hybrid material (Ca‐alg‐PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca‐alg‐PLL capsules, while a resazurin‐based assay was used for comparison in case of the Ca‐alg‐PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter‐assay relative standard deviations below 10%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:986–993, 2013