Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase

In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions. In this study, we report that substitution of the divalent metal ion Mg²⁺ with Mn²⁺ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus. The ability of Mn²⁺ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or δ polymerases. However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3′) guanine of the d(GpG) intrastrand cisplatin lesion. Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3′ guanine of the lesion. Furthermore, substitution of MgCl₂ with MnCl₂ greatly inhibited the 3′ to 5′ exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase. Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate. Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro.     

Genetic polymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by a PCR‐based assay of the invasive Mediterranean strain

Abstract An invasive, cold‐tolerant strain of the tropical green alga Caulerpa taxifolia was introduced recently in the Mediterranean Sea and along the Californian coast. We screened 50 aquarium and open‐sea C. taxifolia specimens for the presence/absence of an intron located in the rbcL gene of chloroplast DNA. We also reanalysed a total of 229 sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA, combining previously published sequences from different studies with 68 new sequences to complement rbcL data. The introduced Mediterranean strain was found to be characterized by the absence of the rbcL intron and by the occurrence of a particular monomorphic ITS type. A PCR assay based on rbcL gene was developed to detect new introductions of the invasive strain of C. taxifolia. This rapid and inexpensive test could be useful to assist environment managers in the preservation of coastal marine ecosystems.     

Turning regenerative technologies into treatment to repair myocardial injuries

Regenerative therapies including stem cell treatments hold promise to allow curing patients affected by severe cardiac muscle diseases. However, the clinical efficacy of stem cell therapy remains elusive, so far. The two key roadblocks that still need to be overcome are the poor cell engraftment into the injured myocardium and the limited knowledge of the ideal mixture of bioactive factors to be locally delivered for restoring heart function. Thus, therapeutic strategies for cardiac repair are directed to increase the retention and functional integration of transplanted cells in the damaged myocardium or to enhance the endogenous repair mechanisms through cell-free therapies. In this context, biomaterial-based technologies and tissue engineering approaches have the potential to dramatically impact cardiac translational medicine. This review intends to offer some consideration on the cell-based and cell-free cardiac therapies, their limitations and the possible future developments.     

Cell Survival and Polarity of Drosophila Follicle Cells Require the Activity of Ecdysone Receptor B1 Isoform

Proper assembly and maintenance of epithelia are critical for normal development and homeostasis. Here, using the Drosophila ovary as a model, we identify a role for the B1 isoform of the ecdysone receptor (EcR-B1) in this process. We performed a reverse genetic analysis of EcR-B1 function during oogenesis and demonstrate that silencing of this receptor isoform causes loss of integrity and multilayering of the follicular epithelium. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and basolateral cell polarity markers including atypical-protein kinase C (aPKC), Discs-large (Dlg), and Scribble (Scrib) and aberrant accumulation of adherens junctions and F-actin cytoskeleton. We find that the EcR-B1 isoform is required for proper follicle cell polarity both during early stages of oogenesis, when follicle cells undergo the mitotic cell cycle, and at midoogenesis when these cells stop dividing and undergo several endocycles. In addition, we show that the EcR-B1 isoform is required during early oogenesis for follicle cell survival and that disruption of its function causes apoptotic cell death induced by caspase.     

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.     

Use of an inhibitor to identify members of the hormone-sensitive lipase family

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.     

4-hydroxynonenal and TGF-beta1 concur in inducing antiproliferative effects on the CaCo-2 human colon adenocarcinoma cell line

4-Hydroxynonenal (HNE) has been demonstrated to exert its antiproliferative effect by up-regulating the c-Jun-N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family (MAPKs). Transforming growth factor-beta1 (TGF-beta1) is the major negative regulatory factor in controlling cell proliferation, and Smads are its intracellular transducers. Recent data on human colon adenocarcinoma has shown a low HNE content paralleled by a marked alteration of TGF-beta1 levels within the tumor mass. The two events appear related because of the demonstrated marked ability of HNE to up-regulate expression and synthesis of TGF-beta1; the combined decreases of HNE and TGF-beta1 found in cancer cells provide a favorable condition for neoplastic progression. Furthermore, HNE is likely able to interact with the cytokine to enhance apoptosis and increase intracellular reactive oxygen species (ROS) formation in the CaCo-2 colon carcinoma cell line. The probable mechanism whereby HNE and TGF-beta1 interact to induce apoptosis is through cross-talk between the main signaling pathways of the two molecules (JNK and Smads), and the observed ROS production might only contribute to amplifying the apoptotic pathways. The network between the two signaling pathways here involved is now under investigation.     

Protein multiple sequence alignment by hybrid bio-inspired algorithms

This article presents an immune inspired algorithm to tackle the Multiple Sequence Alignment (MSA) problem. MSA is one of the most important tasks in biological sequence analysis. Although this paper focuses on protein alignments, most of the discussion and methodology may also be applied to DNA alignments. The problem of finding the multiple alignment was investigated in the study by Bonizzoni and Vedova and Wang and Jiang, and proved to be a NP-hard (non-deterministic polynomial-time hard) problem. The presented algorithm, called Immunological Multiple Sequence Alignment Algorithm (IMSA), incorporates two new strategies to create the initial population and specific ad hoc mutation operators. It is based on the 'weighted sum of pairs' as objective function, to evaluate a given candidate alignment. IMSA was tested using both classical benchmarks of BAliBASE (versions 1.0, 2.0 and 3.0), and experimental results indicate that it is comparable with state-of-the-art multiple alignment algorithms, in terms of quality of alignments, weighted Sums-of-Pairs (SP) and Column Score (CS) values. The main novelty of IMSA is its ability to generate more than a single suboptimal alignment, for every MSA instance; this behaviour is due to the stochastic nature of the algorithm and of the populations evolved during the convergence process. This feature will help the decision maker to assess and select a biologically relevant multiple sequence alignment. Finally, the designed algorithm can be used as a local search procedure to properly explore promising alignments of the search space.     

Role of adrenergic innervation in experimental renal hypertension

Renal hypertension was induced by ligation of the aorta between renal arteries in rats sympathectomized with 6-hydroxydopamine. In the early phase, equally severe hypertension developed in the denervated group as compared to innervated controls. Later, blood pressure was lower in the denervated rats. Initially, increases in plasma renin were seen in both groups; the levels, however, were markedly lower in the denervated rats. Later, the renin levels were similar and not different from baseline. It is concluded that adrenergic neural activity is not essential in the development of renal hypertension; the maintenance of the chronic state, however, depends in part on adrenergic innervation.