Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD). A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients. Studies in rats indicated that many grafted neurones die by apoptosis. CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis. We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats. CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress. Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats. Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth. Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.     

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.     

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.     

Transduction of terminally differentiated neurons by avian sarcoma virus

Recent studies have demonstrated that avian sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.     

Urinary 1-hydroxypyrene and mutagenicity in bus drivers and mail carriers exposed to urban air pollution in Denmark

BACKGROUND: Previous studies in Denmark have shown that bus drivers and tramway employees were at an increased risk for developing several types of cancer and that bus drives from central Copenhagen have high levels of biomarkers of DNA damage.AIMS: The present study evaluates 1-hydroxypyrene concentrations and mutagenic activity in urine as biomarkers of exposure in non-smoking bus drivers in city and rural areas on a work day and a day off and in non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). METHODS: Twenty-four hour urine samples were collected on a working day and a day off from 60 non-smoking bus drivers in city and rural areas and from 88 non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). The concentration of 1-hydroxypyrene was measured by means of HPLC and the mutagenic activity was assessed by the Ames assay with Salmonella tester strain YG1021 and S9 mix. The N-acetyltransferase (NAT2) phenotype was used as a biomarker for susceptibility to mutagenic/carcinogenic compounds. RESULTS: Bus drivers excreted more 1-hydroxypyrene in urine than did mail carriers. The differences were slightly smaller when NAT2 phenotype, cooking at home, exposure to vehicle exhaust, and performing physical exercise after work were included. The NAT2 slow acetylators had 29% (1.29 [CI: 1.15-1.98]) higher 1-hydroxypyrene concentrations in urine than the fast acetylators. Male bus drivers had 0.92 revertants/mol creatinine [CI: 0.37-1.47] and female bus drivers 1.90 revertants/mol creatinine [CI: 1.01-2.79] higher mutagenic activity in urine than mail carriers. CONCLUSION: The present study indicates that bus drivers are more exposed to polycyclic aromatic hydrocarbons (PAH) and mutagens than mail carriers. Mail carriers who worked outdoors had higher urinary concentration of 1-hydroxypyrene, a marker of exposure to PAH, than those working indoors. The individual levels of urinary mutagenic activity were not correlated to excretion of 1-hydroxypyrene. This might be due to the fact that the most potent mutagenic compounds in diesel exhaust are not PAH but dinitro-pyrenes. Among bus drivers, fast NAT2 acetylators had higher mutagenic activity in urine than slow NAT2 acetylators and female bus drivers had higher mutagenic activity than male bus drivers.     

New complexities in the genetics of stuttering: significant sex-specific linkage signals

Stuttering is a speech disorder long recognized to have a genetic component. Recent linkage studies mapped a susceptibility locus for stuttering to chromosome 12 in 46 highly inbred families ascertained in Pakistan. We report here on linkage studies in 100 families of European descent ascertained in the United States, Sweden, and Israel. These families included 252 individuals exhibiting persistent stuttering, 45 individuals classified as recovered from stuttering, and 19 individuals too young to classify. Primary analyses identified moderate evidence for linkage of the broader diagnosis of "ever stuttered" (including both persistent and recovered stuttering) on chromosome 9 (LOD = 2.3 at 60 cM) and of the narrower diagnosis of persistent stuttering on chromosome 15 (LOD = 1.95 at 23 cM). In contrast, sex-specific evidence for linkage on chromosome 7 at 153 cM in the male-only data subset (LOD = 2.99) and on chromosome 21 at 34 cM in the female-only data subset (LOD = 4.5) met genomewide criteria for significance. Secondary analyses revealed a significant increase in the evidence for linkage on chromosome 12, conditional on the evidence for linkage at chromosome 7, with the location of the increased signal congruent with the previously reported signal in families ascertained in Pakistan. In addition, a region on chromosome 2 (193 cM) showed a significant increase in the evidence for linkage conditional on either chromosome 9 (positive) or chromosome 7 (negative); this chromosome 2 region has been implicated elsewhere in studies on autism, with increased evidence for linkage observed when the sample is restricted to those with delayed onset of phrase speech. Our results support the hypothesis that the genetic component to stuttering has significant sex effects.     

Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection

We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3-10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5x10(2) to 1x10(3) with on-column UV detection at 280 nm.     

Comparative proteomic analysis of cisplatin sensitive IGROV1 ovarian carcinoma cell line and its resistant counterpart IGROV1-R10

Ovarian cancer is one of the leading causes of mortality due to gynaecological cancer. Despite a good response to surgery and initial chemotherapy essentially based on cisplatin (cis-diamino-dichloro-platinum(II) (CDDP)) compounds, late tumour detection and frequent recurrences with chemoresistance acquisition are responsible for poor prognosis. Several mechanisms have been implicated in CDDP resistance but they are not sufficient to exhaustively explain this resistance emergence. We applied a proteomic approach based on 2-DE coupled with MS to identify proteins associated with the chemoresistance process. We first established a proteomic pattern of the CDDP sensitive ovarian cell line IGROV1 using MALDI-TOF-MS and PMF. We then compared this 2-D pattern with that of the CDDP-resistant counterpart IGROV1-R10. Among the 40 proteins identified, cytokeratins 8 and 18 and aldehyde dehydrogenase 1 were overexpressed in IGROV1-R10, whereas annexin IV was down-regulated. These observations have been confirmed by Western blotting. The characterization of such variations could lead to the development of new protein markers or to the establishment of new therapeutic strategies. Moreover, the identification of proteins involved in CDDP resistance in ovarian tumours would be useful in completing our understanding on this complex mechanism.