Structural and functional evolution of three cardiac natriuretic peptides

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.     

Vitamin D sensitizes breast cancer cells to the action of H2O2: mitochondria as a convergence point in the death pathway

Calcitriol, the hormonal form of vitamin D3, sensitizes breast cancer cells to reactive oxygen species (ROS)-dependent cytotoxicity induced by various anticancer modalities. This effect could be due to increased generation of ROS and/ or to increased sensitivity of the target cells to ROS. This work examined the effect of calcitriol on the damage inflicted on breast cancer cells by the direct action of ROS represented by H2O2. Treatment of MCF-7 cells with H2O2 resulted in activation of caspase 7 as well as induction of caspase-independent cell death. Both were enhanced by 48-72 h of pretreatment with calcitriol. This effect was not due to modulation of H2O2 degradation or to a specific effect on *OH-mediated cytotoxicity. The H2O2-induced drop in mitochondrial membrane potential and release of cytochrome c were enhanced by calcitriol. These findings indicate that calcitriol sensitizes breast cancer cells to ROS-induced death by affecting event(s) common to both caspase-dependent and -independent modes of cell death upstream to mitochondrial damage.     

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

Role of the p66Shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of actin cytoskeleton in rat myoblasts

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.     

Pravastatin up-regulates transforming growth factor-beta1 in THP-1 human macrophages: effect on scavenger receptor class A expression

Statins have been shown to interact with several monocyte/macrophage functions. We tested the effect of pravastatin on transforming growth factor-beta1 (TGF-beta1) production and its possible involvement in scavenger receptors class A (SRA) expression in human THP-1 cells. TGF-beta1s biological activity in THP-1 cell conditioned medium, evaluated by luciferase activity of transfected cell with a TGF-beta responsive promoter, was increased in a dose-dependent manner after incubation with pravastatin (1-20 microM). Pravastatin (1-20 microM) induced a dose-dependent increase in TGF-beta1 mRNA expression and protein production in THP-1 cells. PMA-induced SRA gene and protein expression was suppressed by pravastatin with a mean 3-fold decrease at 10 microM. This last effect was reversed by a mouse monoclonal anti-TGF-beta1 neutralizing antibody. PD98059, a specific inhibitor of MAP kinase cascade, completely reversed pravastatin-induced SRA down-regulation. p44 and p42 isoforms showed a dose-dependent phosphorylation after treatment with pravastatin (1-20 microM) which was inhibited by a mouse monoclonal anti-TGF-beta1 antibody. Our results demonstrate that pravastatin significantly up-regulates TGF-beta1 expression which may be in involved in down-regulation of SRA expression in THP-1 cell cultures. A new pathway for pravastatin effects in atherogenesis can be suggested.     

Cryptococcus neoformans population includes hybrid strains homozygous at mating-type locus

Recent attempts to characterise the hybrid strains of Cryptococcus neoformans have led to the identification of a cryptic population of hybrid strains ('H strains') with double DNA content but only a single mating-type allele. To verify a set of hypotheses concerning their origin, we investigated 14 previously isolated H strains and ten F1-progeny strains arising from H99 and JEC20 mating. The double DNA content was tested by flow cytometry; the presence of only one mating type was tested by amplifying 12 mating-type-specific genes and one gene unlinked with the mating-type locus (URA5). Analysis of the F1 progeny identified two H strains, and electrophoretic karyotyping confirmed the occurrence of genetic recombination. The simultaneous presence of the homozygous and heterozygous loci, and the fact that all of the F1-progeny strains presented a recombinant karyotype, suggest that the H strains originated from the post-meiotic random fusion of two of the four recombinant nuclei. Further studies are required to elucidate the role of the homozygous mating-type loci in the virulence of C. neoformans.     

Stringent requirement for HRD1, SEL1L,and OS-9/XTP3-B for disposal of ERAD-LS substrates

Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L_S substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L_M substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.