Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7-biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels.

SCT1 mutants suppress the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I.

Camptothecin is a potent antineoplastic agent that interferes with the action of eukaryotic DNA topoisomerase I; the covalent enzyme-DNA intermediate is reversibly stabilized, leading to G2 arrest and cell death. We used a genetic screen to identify cellular factors, other than DNA topoisomerase I, that participate in the process of camptothecin-induced cell death. Following ethyl methanesulfonate mutagenesis of top1 delta yeast cells expressing plasmid-borne wild-type DNA topoisomerase I, six dominant suppressors of camptothecin toxicity were isolated that define a single genetic locus, sct1. Mutant SCT1 cells expressed DNA topoisomerase I protein of similar specific activity and camptothecin sensitivity to that of congenic, drug-sensitive sct1 cells, yet were resistant to camptothecin-mediated lethality. Moreover, camptothecin-treated SCT1 cells did not exhibit the G2-arrested, terminal phenotype characteristic of drug-treated wild-type cells. SCT1 cell sensitivity to other DNA-damaging agents suggests that alterations in SCT1 function suppress camptothecin-induced DNA damage produced in the presence of yeast DNA topoisomerase I.

Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes.

After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.

The weediness of wild plants: molecular analysis of genes influencing dispersal and persistence of johnsongrass, Sorghum halepense (L.) Pers.

Many major weeds rely upon vegetative dispersal by rhizomes and seed dispersal by "shattering" of the mature inflorescence. We report molecular analysis of these traits in a cross between cultivated and wild species of Sorghum that are the probable progenitors of the major weed "johnsongrass." By restriction fragment length polymorphism mapping, variation in the number of rhizomes producing above-ground shoots was associated with three quantitative trait loci (QTLs). Variation in regrowth (ratooning) after overwintering was associated with QTLs accounting for additional rhizomatous growth and with QTLs influencing tillering. Vegetative buds that become rhizomes are similar to those that become tillers--one QTL appears to influence the number of such vegetative buds available, and additional independent genes determine whether individual buds differentiate into tillers or rhizomes. DNA markers described herein facilitate cloning of genes associated with weediness, comparative study of rhizomatousness in other Poaceae, and assessment of gene flow between cultivated and weedy sorghums--a risk that constrains improvement of sorghum through biotechnology. Cloning of "weediness" genes may create opportunities for plant growth regulation, in suppressing propagation of weeds and enhancing productivity of major forage, turf, and "ratoon" crops.

Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors.

Neuroblastoma (NB), a tumor arising from the sympathetic nervous system, is one of the most common malignancies in childhood. Several recent reports on the p53 genotype found virtually exclusive wild-type status in primary tumors, and it was postulated that p53 plays no role in the development of NB. Here, however, we report that the vast majority of undifferentiated NBs exhibit abnormal cytoplasmic sequestration of wild-type p53. This inability of p53 to translocate to the nucleus presumably prevents the protein from functioning as a suppressor. Thirty of 31 cases (96%) of undifferentiated NB showed elevated levels of wild-type p53 in the cytoplasm of all tumor cells concomittant with a lack of nuclear staining. p53 immunoprecipitation from tumor tissues showed a 4.5- to 8-fold increase over normal protein levels. All of 10 tumors analyzed harbored wild-type p53 by direct sequencing of full-length cDNA and Southern blot. In addition, no MDM-2 gene amplification was seen in all 11 tumors analyzed. In contrast, no p53 abnormality was detected in 14 differentiated ganglioneuroblastomas and 1 benign ganglioneuroma. We conclude that loss of p53 function seems to play a major role in the tumorigenesis of undifferentiated NB. This tumor might abrogate the transactivating function of p53 by inhibiting its access to the nucleus, rather than by gene mutation. Importantly, our results suggest that (i) this could be a general mechanism for p53 inactivation not limited to breast cancer (where we first described it) and that (ii) it is found in a tumor previously not thought to be affected by p53 alteration.

Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.

Intermediate hosts of Toxoplasma gondii and food safety:epidemiology, genetics and parasite survival in meat-producing animals in Italy

Toxoplasma gondii is a coccidian parasite with a global distribution. The definitive host is the cat (and other felids). All warm-blooded animals can act as intermediate hosts, including humans. Sexual reproduction (gametogony) takes place in the final host and oocysts are released in the environment, where they then sporulate to become infective. In intermediate hosts the cycle is extra-intestinal and results in the formation of tachyzoites and bradyzoites. Tachyzoites represent the invasive and proliferative stage and on entering a cell it multiplies asexually by endodyogeny. Bradyzoites within tissue cysts are the latent form. T. gondii is a food-borne parasite causing toxoplasmosis, which can occur in both animals and humans. Infection in humans is asymptomatic in more than 80% of cases in Europe and North-America. In the remaining cases patients present fever, cervical lymphadenopathy and other non-specific clinical signs. Nevertheless, toxoplasmosis is life threatening if it occurs in immunocompromised subjects. The main organs involved are brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes, pancreas and parasite can be isolated from these tissues. Another aspect is congenital toxoplasmosis that may occur in pregnant women and the severity of the consequences depends on the stage of pregnancy when maternal infection occurs. Acute toxoplasmosis in developing foetuses may result in blindness, deformation, mental retardation or even death. The European Food Safety Authority (EFSA), in recent reports on zoonoses, highlighted that an increasing numbers of animals resulted infected with T. gondii in EU (reported by the European Member States for pigs, sheep, goats, hunted wild boar and hunted deer, in 2011 and 2012). In addition, high prevalence values have been detected in cats, cattle and dogs, as well as several other animal species, indicating the wide distribution of the parasite among different animal and wildlife species. The main route of transmission is consumption of food and water contaminated with sporulated oocysts. However, infection through the ingestion of meat contaminated with tissue cysts is frequent. Finally, although less frequent, other food products contaminated with tachyzoites such as milk, may also pose a risk. The importance of this parasite as a risk for human health was recently highlighted by EFSA’s opinion on modernization of meat inspection, where Toxoplasma gondii was identified as a relevant hazard to be addressed in revised meat inspection systems for pigs, sheep, goats, farmed wild boar and farmed deer (Call for proposals -GP/EFSA/BIOHAZ/2013/01). The risk of infection is more highly associated to animals reared outside, also in free-range or organic farms, where biohazard measure are less strict than in large scale, industrial farms. Here, animals are kept under strict biosecurity measures, including barriers, which inhibit access by cats, thus making soil contamination by oocysts nearly impossible. A growing demand by the consumer for organic products, coming from free-range livestock, in respect of animal-welfare, and the desire for the best quality of derived products, have all led to an increase in the farming of free-range animals. The risk of Toxoplasma gondii infection increases when animals have access to environment and the absence of data in Italy, together with need for in depth study of both the prevalence and genotypes of Toxoplasma gondii present in our country were the main reasons for the development of this thesis project. A total of 152 animals have been analyzed, including 21 free-range pigs (Suino Nero race), 24 transhumant Cornigliese sheep, 77 free-range chickens and 21 wild animals. Serology (on meat juice) and identification of T. gondii DNA through PCR was performed on all samples, except for wild animals (no serology). An in-vitro test was also applied with the aim to find an alternative and valid method to bioassay, actually the gold standard. Meat samples were digested and seeded onto Vero cells, checked every day and a RT-PCR protocol was used to determine an eventual increase in the amount of DNA, demonstrating the viability of the parasite. Several samples were alos genetically characterized using a PCR-RFLP protocol to define the major genotypes diffused in the geographical area studied. Within the context of a project promoted by Istituto Zooprofilattico of Pavia and Brescia (Italy), experimentally infected pigs were also analyzed. One of the aims was to verify if the production process of cured “Prosciutto di Parma” is able to kill the parasite. Our contribution included the digestion and seeding of homogenates on Vero cells and applying the Elisa test on meat juice. This thesis project has highlighted widespread diffusion of T. gondii in the geographical area taken into account. Pigs, sheep, chickens and wild animals showed high prevalence of infection. The data obtained with serology were 95.2%, 70.8%, 36.4%, respectively, indicating the spread of the parasite among numerous animal species. For wild animals, the average value of parasite infection determined through PCR was 44.8%. Meat juice serology appears to be a very useful, rapid and sensitive method for screening carcasses at slaughterhouse and for marketing “Toxo-free” meat. The results obtained on fresh pork meat (derived from experimentally infected pigs) before (on serum) and after (on meat juice) slaughter showed a good concordance. The free-range farming put in evidence a marked risk for meat-producing animals and as a consequence also for the consumer. Genotyping revealed the diffusion of Type-II and in a lower percentage of Type-III. In pigs is predominant the Type-II profile, while in wildlife is more diffused a Type-III and mixed profiles (mainly Type-II/III). The mixed genotypes (Type-II/III) could be explained by the presence of mixed infections. Free-range farming and the contact with wildlife could facilitate the spread of the parasite and the generation of new and atypical strains, with unknown consequences on human health. The curing process employed in this study appears to produce hams that do not pose a serious concern to human health and therefore could be marketed and consumed without significant health risk. Little is known about the diffusion and genotypes of T. gondii in wild animals; further studies on the way in which new and mixed genotypes may be introduced into the domestic cycle should be very interesting, also with the use of NGS techniques, more rapid and sensitive than PCR-RFLP. Furthermore wildlife can become a valuable indicator of environmental contamination with T. gondii oocysts. Other future perspectives regarding pigs include the expansion of the number of free-range animals and farms and for Cornigliese sheep the evaluation of other food products as raw milk and cheeses. It should be interesting to proceed with the validation of an ELISA test for infection in chickens, using both serum and meat juice on a larger number of animals and the same should be done also for wildlife (at the moment no ELISA tests are available and MAT is the reference method for them). Results related to Parma ham do not suggest a concerning risk for consumers. However, further studies are needed to complete the risk assessment and the analysis of other products cured using technological processes other than those investigated in the present study. For example, it could be interesting to analyze products such as salami, produced with pig meat all over the Italian country, with very different recipes, also in domestic and rural contexts, characterized by a very short period of curing (1 to 6 months). Toxoplasma gondii is one of the most diffuse food-borne parasites globally. Public health safety, improved animal production and protection of endangered livestock species are all important goals of research into reliable diagnostic tools for this infection. Future studies into the epidemiology, parasite survival and genotypes of T. gondii in meat producing animals should continue to be a research priority.

The Decline of Wild Pacific Salmon

As threatened and endangered species, wild Pacific salmon are in peril. This paper discusses the differences of the five species of wild Pacific salmon. As salmon go through several stages of their lifecycles, they face a myriad of threats to their existence. Threats from humans in the form of hydropower dams, habitat destruction, harvesting issues, and hatcheries are explained. A draft recovery plan for salmon in the Puget Sound area of Washington State is used as a case study. Strengths and weaknesses of this plan are discussed. The paper then discusses the need for growth management laws supporting salmon habitat and a change in individual behaviors if wild Pacific salmon sustainability is to become a reality.

Electroretinographical and histological study of mouse retina after optic nerve section: a comparison between wild-type and retinal degeneration 1 mice

Background: Retinal ganglion cell death underlies the pathophysiology of neurodegenerative disorders such as glaucoma or optic nerve trauma. To assess the potential influence of photoreceptor degeneration on retinal ganglion cell survival, and to evaluate functionality, we took advantage of the optic nerve section mouse model. Methods: Surviving retinal ganglion cells were double-stained by exposing both superior colliculi to fluorogold, and by applying dextran-tetramethylrhodamine to the injured optic nerve stump. To assess retinal function in wild-type animals, electroretinograms were recorded on the injured eyes and compared with the contralateral. Similar labelling experiments were carried out on retinal degeneration 1 mice. Surviving retinal ganglion cells were counted 21 days after axotomy and compared with wild-type mice. No functional experiments were performed on retinal degeneration 1 animals because they do not develop normal electroretinographical responses. Results: A significant decrease in retinal ganglion cell density was observed 6 days after axotomy in the wild type. Functional studies revealed that, in scotopic conditions, axotomy induced a significant amplitude decrease in the positive scotopic threshold response component of the electroretinogram. Such decrease paralleled cell loss, suggesting it may be an appropriate technique to evaluate functionality. When comparing retinal ganglion cell densities in wild-type and retinal degeneration 1 mice, a significant greater survival was observed on the latter. Conclusions: After optic nerve section, electroretinographical recordings exhibited a progressive decrease in the amplitude of the positive scotopic threshold response wave, reflecting ganglion cell loss. Interestingly, rod degeneration seemed, at least initially, to protect from axotomy-driven damage.

Main ecological gradients and landscape matrix affect wild rabbit Oryctolagus cuniculus (Linnaeus, 1758) (Mammalia: Leporidae) abundance in a coastal region (South-East Spain)

Landscape analysis with transects, in the Marina Baja area (province of Alicante, Spain), has contributed to establish the influence of different landscape matrices and some environmental gradients on wild rabbit Oryctolagus cuniculus (Linnaeus, 1758) (Mammalia: Leporidae) abundance (kilometric abundance index, KAI). Transects (n = 396) were developed to estimate the abundance of this species in the study area from 2006 to 2008.Our analysis shows that rabbits have preferences for a specific land use matrix (irrigated: KAI = 3.47 ± 1.14 rabbits/km). They prefer the coastal area (KAI = 3.82 ± 1.71 rabbits/km), which coincides with thermo-Mediterranean (a bioclimatic belt with a tempered winter and a hot and dry summer with high human density), as opposed to areas in the interior (continental climate with lower human occupation). Their preference for the southern area of the region was also noted (KAI = 8.22 ± 3.90 rabbits/km), which coincides with the upper semi-arid area, as opposed to the northern and intermediate areas (the north of the region coinciding with the upper dry and the intermediate area with the lower dry). On the other hand, we found that the number of rabbits increased during the 3-year study period, with the highest abundance (KAI = 2.71 ± 1.30 rabbits/km) inMay. Thus, this study will enable more precise knowledge of the ecological factors (habitat variables) that intervene in the distribution of wild rabbit populations in a poorly studied area.

Potential impact of Wild boar (Sus scrofa) on Pheasant (Phasianus colchicus) nesting success

In recent decades wild boar have greatly increased in Europe due a combination of environmental and human factors. Because of a high reproduction rate, wild boar cause conflicts with human activities and in particularly agriculture. However, there are concerns also about environmental impact and in particular on ground nesting birds. Our study aim to evaluate the potential impact of wild boar on pheasants nests. Predation rates were compared between two areas with different wild boar abundance and between nests placed in fenced plots with no wild boar access (but accessible to other predators) and open plots in which the wild boar gains access. Predation rate by wild boar in the area with higher wild boar abundance was 61.5% whereas in the area with lower abundance was 16.0%. In plots with free access, wild boar predation was 34.2% whereas in exclusion plots we did not registered predation by wild boar. Moreover, wild boar resulted the main nest predator (total loss 22.8%). Our findings suggest that wild boar could have e strong impact on pheasant reproductive success.