Population data on benign and severe forms of X-linked muscular dystrophy

Summary Epidemiological data on Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) from a large sample of the Italian population are reported. For BMD the incidence rate was found to be 5.5x10^-5 liveborn males (lbm) and the prevalence rate, 13.1x10^-6; the mutation rate was estimated to be about 6.0x10^-6. For DMD the incidence and prevalence rates were found to be respectively 26x10^-5 lbm and 31.6x10^-6. The DMD mutation rate obtained by the Haldane formula was 86.6x10^-6 and by the semi-direct method, 65.6x10^-6. The results are discussed in the light of possible allelism of BMD and DMD.     

Characterization of maize polyamine oxidase.

Some structural and biochemical characteristics of polyamine oxidase (PAO) purified from maize shoots have been examined. The enzyme has only alanine as N-terminal amino acid and its N-terminal sequence shows a significant degree of homology with tryptophan 2-monooxygenase from Pseudomonas syringae pv. savastanoi. The pH optimum for the stability of the native enzyme is 5, similar to that of the barley leaf enzyme. Calorimetric analysis shows a single two-state transition at pH 6 with Tm 49.8 degrees. At pH 5 the thermal stability is increased by more than 14 degrees. Amine oxidation products, delta 1-pyrroline and diazabicyclononane, are competitive inhibitors of PAO activity (apparent Ki = 400 and 100 microM respectively). Moreover these compounds improve the thermal stability of the enzyme. N1-Acetylspermine, which is a good substrate for mammalian PAO, acts as a non-competitive inhibitor for the plant enzyme.     

Cyclic variation of chiasma frequency and distribution in Podarcis sicula (Reptilia: Lacertidae)

The seasonal variations of chiasma frequency and distribution have been studied in the lizard: Podareis sicula. In this species, as in Phyllodactylus (King & Hayman, Chromosoma 69: 131–154, 1978), chiasma frequencies vary following a definite annual cycle, and clearly different trends are shown by interstitial and terminal chiasmata.A comparison between these seasonal chiasma frequency variations and those of environmental temperature shows the existence of a clear correlation between these two parameters. However, this correlation is different in the two types of chiasmata, and may be different within the same type of chiasma depending on the period of the year.A more significant correlation is observed between chiasma cycles and annual variations of the haematic levels of sexual steroid hormones. In particular we observe a highly significant correlation between interstitial chiasma frequencies and testosterone concentration. A less precise correlation between terminal chiasma frequencies and estradiol concentration is also observed.In Podarcis, as in Phyllodactylus, the sperm that will be used for fertilization derive from the spermatocytes showing the highest rate of interstitial chiasmata. This supports the hypothesis that the cyclic variations in interstitial chiasma frequencies represent a mechanism to ensure an adequate level of variability in a given population. The above mentioned correlation between chiasma frequencies and steroia hormone concentrations suggests that the seasonal chiasma cycles are controlled by the same environmental and hormonal factors regulating the spermatogenetic cycle.     

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave-adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog-related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic 'hotspots' of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.     

Expression of estrogen receptor alpha switches off secretory activity in the epididymal channel of the lizard Podarcis sicula

The epididymis in the male reproductive tract allows the survival, viability, and storage of spermatozoa from the testis. In the lizard Podarcis sicula, the epididymis can be regionalized to an initial segment called the caput that comprises the efferent ductules, followed by the middle and terminal segments, respectively termed the corpus and cauda. By means of in situ hybridization and immunocytochemistry, we analyzed the expression of the estrogen receptors of the alpha and beta type (ERα and ERβ) in Podarcis to test the responsiveness of the epididymal regions to estrogen in the annual reproductive cycle of this seasonal breeder. The results show that the efferent ductules and the cauda always express both ERα and ERβ throughout the year. In the corpus, the expression of ERα takes place only at the end of the mating period and continues in the non-reproductive season whereas ERβ is expressed in all phases of the cycle. During the mating season, the cells of the corpus are engaged in massive secretory activity and do not express ERα. Experimental administration of E(2) during this season does not change the expression of ERβ, nor does it affect the efferent ductules and cauda; instead, it inhibits the secretory activity in the corpus and induces the expression of ERα. Taken together, our findings suggest that in the epididymis of Podarcis, the expression of ERα may act as a switch for the secretory activity of the epididymal corpus.     

The importance of myocardial amino acids during ischemia and reperfusion in dilated left ventricle of patients with degenerative mitral valve disease

Taurine, glutamine, glutamate, aspartate, and alanine are the most abundant intracellular free amino acids in human heart. The myocardial concentration of these amino acids changes during ischemia and reperfusion due to alterations in metabolic and ionic homeostasis. We hypothesized that dilated left ventricle secondary to mitral valve disease has different levels of amino acids compared to the right ventricle and that such differences determine the extent of amino acids' changes during ischemia and reperfusion. Myocardial concentration of amino acids was measured in biopsies collected from left and right ventricles before cardioplegic arrest (Custodiol HTK) and 10 min after reperfusion in patients undergoing mitral valve surgery. The dilated left ventricle had markedly higher (P < 0.05) concentrations (nmol/mg wet weight) of taurine (17.0 ± 1.5 vs. 10.9 ± 1.5), glutamine (20.5 ± 2.4 vs. 12.1 ± 1.2), and glutamate (18.3 ± 2.2 vs. 11.4 ± 1.5) when compared to right ventricle. There were no differences in the basal levels of alanine or aspartate. Upon reperfusion, a significant (P < 0.05) fall in taurine and glutamine was seen only in the left ventricle. These changes are likely to be due to transport (taurine) and/or metabolism (glutamine). There was a marked increase in the alanine to glutamate ratio in both ventricles indicative of ischemic stress which was confirmed by global release of lactate during reperfusion. This study shows that in contrast to the right ventricle, the dilated left ventricle had remodeled to accumulate amino acids which are used during ischemia and reperfusion. Whether these changes reflect differences in degree of cardioplegic protection between the two ventricles remain to be investigated.     

HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.Deregulation of the epidermal growth factor receptor signaling network contributes to initiate and/or maintain malignant growth (1). One of these alterations, aberrant cellular motility, is necessary for invasive growth, which eventually culminates with the establishment of distant metastases, the leading cause of death in patients with cancer.The epidermal growth factor receptor is the prototype of a family that also includes HER2 (ErbB2, Neu), HER3, and HER4 (ErbB3 and ErbB4). The analysis of cells expressing various HER receptors indicated that HER2 plays a critical role in the regulation of motility (2, 3). Upon activation through homo- or heterodimerization with other HER receptors, several tyrosines in the cytoplasmic tail of HER2 are phosphorylated and initiate intracellular signaling pathways, including the phospholipase C-γ1 and phosphatidylinositol 3-kinase pathways (4), which, in turn, promote cell migration through partially understood cascades. These cascades are largely regulated by phosphorylation/dephosphorylation of cellular components.A subgroup of HER2-positive patients expresses a series of carboxyl-terminal fragments (CTFs)⁵ of HER2. HER2 CTFs can be generated by two independent mechanisms: proteolytic processing and alternative initiation of translation. Metalloproteases with the so-called α-secretase activity shed the extracellular domain of HER2, leaving behind a fragment, known as P95, that starts around alanine 648 (5) (see also Fig. 1A). Alternative initiation of translation of the mRNA encoding HER2 from the methionine codons 611 and 687 generates two fragments: 611- and 687-CTF. These differ by a stretch of 76 amino acids, which includes the transmembrane domain and a cysteine-rich short extracellular domain (6) (see also Fig. 1A).Open in a separate windowFIGURE 1.Effect of HER2 CTFs on cell migration. A, schematic showing the different constructs used in these studies. The primary sequence of the extracellular and intracellular juxtamembrane domains of HER2 is shown. The transmembrane domain (TM) is represented by a hatched box. The positions of amino acids 611, 648, and 687 are indicated. The N at the beginning of the rectangle representing HER2 identifies the amino terminus. The vertical bold double line represents the plasma membrane, and the extracellular (out) and intracellular (in) regions are marked. B, MCF7 Tet-Off cells stably transfected with the empty vector (Vector) or with the vector containing the cDNAs encoding HER2 or 611-, 648-, or 687-CTFs under the control of a Tet/Dox-responsive element were kept with or without doxycycline for 24 h, lysed, and analyzed by Western blot with CB11, an antibody against the cytoplasmic domain of HER2. C, MCF7 Tet-Off cells transfected with empty vector or with 611-CTF were seeded in the absence of doxycycline, and motility was monitored by time lapse video microscopy. Representative fields of migrated cells at the indicated times are shown. Results indistinguishable from those corresponding to MCF7 Tet-Off cells transfected with vector and treated without doxycycline were observed when analyzing MCF7/611-CTF Tet-Off cells in the presence of doxycycline (data not shown). D, representative examples of the migratory behavior of MCF7 Tet-Off cells treated as in C. Digital images were taken every 30 min for 24 h (see supplemental videos), and the tracks were manually drawn. Results indistinguishable from those corresponding to MCF7 Tet-Off cells transfected with vector and treated without doxycycline were observed when analyzing MCF7/611-CTF Tet-Off cells in the presence of doxycycline (data not shown). E, tracks from cells analyzed as in D were measured in mm. Bars, average length ± S.D. of the tracks of five cells, each one on a different culture plate. F, MCF7 Tet-Off cells transfected with 611-CTF were seeded in the absence or the presence of doxycycline on transwell plates, as described under “Experimental Procedures.” After 24 h, cells were fixed and stained with DAPI. Representative fields are shown. G, MCF7 Tet-Off cells stably transfected with the empty vector (Vector) or with the vector containing the cDNAs encoding HER2 or 611-, 648-, or 687-CTFs were seeded on transwell plates with or without doxycycline as indicated. After 24 h, cells were fixed, stained with DAPI, and counted. Bars, average of the cells counted in three independent experiments, each one performed in triplicate, ± S.D. H, representative examples of the images obtained by time lapse microscopy of the closure of wounds made in a monolayer of control MCF7 Tet-Off cells stably transfected with 611-CTF and treated with or without doxycycline as indicated. The lines drawn in the images represent reference lines marking the width of the scratch when it was made. I, average values of migration distances in scratch wound assays as in H. Bars, means from three independent experiments, each one derived from evaluation of 10 fields ± S.D.We have recently shown that 687-CTF seems to be inactive (7). In contrast, the two CTFs containing the transmembrane domain, 648- and 611-CTFs, expressed at levels similar to those found in human breast tumors, can activate different intracellular signal transduction pathways (7). The level of activation of these pathways by HER2 CTFs is quite different. 611-CTF activates the mitogen-activated protein kinase and the Akt pathways to a greater extent because it constitutively forms homodimers maintained through disulfide bonds (7). In contrast, 648-CTF does not seem to form homodimers, and its activity is comparable with that of full-length HER2 (7). Therefore, cells expressing transmembrane CTFs, particularly 611-CTF, constitute a relevant model to study the consequences of the overactivation of HER2 signaling in tumors. Supporting this conclusion, it has been shown that breast cancer patients expressing CTFs have worse prognosis and are more likely to develop nodal metastasis compared with patients expressing predominantly full-length HER2 (8).Here we show that expression of 611-CTF enhances the migration of breast cancer cells as judged by monitoring single-cell migration, transwell migration, and wound healing assays. Since cell migration is frequently regulated by phosphorylation/dephosphorylation, we searched for phosphoproteins regulated by 611-CTF and probably contributing to cell migration using two independent proteomic approaches. The results of these analyses showed that difference gel electrophoresis (DIGE) is a particularly convenient methodology to analyze the regulation of multiphosphorylated proteins.Cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, was identified by DIGE as a phosphoprotein likely to be regulated by 611-CTF. Several assays showed that expression of 611-CTF leads to an increase in the phosphorylation of cortactin and to the generation of cell protrusions resembling lamellipodia or invadopodia. Confirming a role of cortactin on the increased cell migration induced by 611-CTF, down-modulation of the former with short hairpin RNAs leads to an impairment of the cell migration induced by the HER2 fragment. These results unveil a role of cortactin in the increased cell migration induced by hyperactive HER2 and strongly suggest that cortactin-dependent increased cell migration contributes to the tendency of breast tumors expressing CTFs to metastasize.     

Genomic aberrations in lung adenocarcinoma in never smokers

Background

Lung cancer in never smokers would rank as the seventh most common cause of cancer death worldwide.

Methods and Findings

We performed high-resolution array comparative genomic hybridization analysis of lung adenocarcinoma in sixty never smokers and identified fourteen new minimal common regions (MCR) of gain or loss, of which five contained a single gene (MOCS2, NSUN3, KHDRBS2, SNTG1 and ST18). One larger MCR of gain contained NSD1. One focal amplification and nine gains contained FUS. NSD1 and FUS are oncogenes hitherto not known to be associated with lung cancer. FISH showed that the amplicon containing FUS was joined to the next telomeric amplicon at 16p11.2. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. Other cancer genes present in aberrations included ARNT, BCL9, CDK4, CDKN2B, EGFR, ERBB2, MDM2, MDM4, MET, MYC and KRAS. Unsupervised hierarchical clustering with adjustment for false-discovery rate revealed clusters differing by the level and pattern of aberrations and displaying particular tumor characteristics. One cluster was strongly associated with gain of MYC. Another cluster was characterized by extensive losses containing tumor suppressor genes of which RB1 and WRN. Tumors in that cluster frequently harbored a central scar-like fibrosis. A third cluster was associated with gains on 7p and 7q, containing ETV1 and BRAF, and displayed the highest rate of EGFR mutations. SNP array analysis validated copy-number aberrations and revealed that RB1 and WRN were altered by recurrent copy-neutral loss of heterozygosity.

Conclusions

The present study has uncovered new aberrations containing cancer genes. The oncogene FUS is a candidate gene in the 16p region that is frequently gained in never smokers. Multiple genetic pathways defined by gains of MYC, deletions of RB1 and WRN or gains on 7p and 7q are involved in lung adenocarcinoma in never smokers.     

Antimicrobial activities of various essential oils against foodborne pathogenic or spoilage moulds

The use of essential oils in the food industry, as natural sanitizing agents, requires the definition of optimal conditions. The aim of the present work was to evaluate some antimicrobial activity parameters as mycelial growth inhibition, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of six essential oils against Aspergillus niger, Aspergillus terreus,Chaetomium globosum, Penicillium chrysogenum, Penicillium pinophilum, Trichoderma harzianum and Trichoderma viride. The antimicrobial activity of essential oils was monitored by the macrodiluition technique. The mycelial growth inhibition, fungistatic and fungicidal concentrations were recorded for each strain that showed sensitivity to the essential oils. The essential oils of catnip, cinnamon, tea tree and thyme essential oils exhibited a large spectrum antimicrobial activities; those of clary sage and laurel inhibited the mycelial growth in a few fungal strains. The essential oils of cinnamon and thyme had the lowest MIC and MFC values against all the fungi assayed, followed by catnip, tea tree, clary sage and laurel. The use of these natural products rather than, the currently used antifungal chemicals, may be of interest given that: i) essential oils are of natural origin which means they are safer for human health and the environment and ii) there is less chance that the pathogenic microorganisms will develop resistance.