Phosphatidylinositol 3-kinase binding to polyoma virus middle tumor antigen mediates elevation of glucose transport by increasing translocation of the GLUT1 transporter.

Elevation in the rate of glucose transport in polyoma virus-infected mouse fibroblasts was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137) binding to complexes of middle tumor antigen (middle T) and pp60c-src. Wild-type polyoma virus infection led to a 3-fold increase in the rate of 2-deoxyglucose (2DG) uptake, whereas a weakly transforming polyoma virus mutant that encodes a middle T capable of activating pp60c-src but unable to promote binding of PI 3-kinase induced little or no change in the rate of 2DG transport. Another transformation-defective mutant encoding a middle T that retains functional binding of both pp60c-src and PI 3-kinase but is incapable of binding Shc (a protein involved in activation of Ras) induced 2DG transport to wild-type levels. Wortmannin (< or = 100 nM), a known inhibitor of PI 3-kinase, blocked elevation of glucose transport in wild-type virus-infected cells. In contrast to serum stimulation, which led to increased levels of glucose transporter 1 (GLUT1) RNA and protein, wild-type virus infection induced no significant change in levels of either GLUT1 RNA or protein. Nevertheless, virus-infected cells did show increases in GLUT1 protein in plasma membranes. These results point to a posttranslational mechanism in the elevation of glucose transport by polyoma virus middle T involving activation of PI 3-kinase and translocation of GLUT1.

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum.

Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum (ER), Sec63p and BiP (Kar2p), are required for posttranslational translocation of yeast alpha-factor precursor in vitro. To investigate the role of these proteins in cotranslational translocation, we examined the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes. We found that Sec63p and Kar2p are necessary for both co- and posttranslational translocation in yeast. Several kar2 mutants, one of which had normal ATPase activity, were defective in cotranslational translocation of invertase. We conclude that the requirement for BiP/Kar2p, which is not seen in a reaction reconstituted with pure mammalian membrane proteins [Görlich, D. & Rapoport, T.A. (1993) Cell 75, 615-630], is not due to a distinction between cotranslational translocation in mammalian cells and posttranslational translocation in yeast cells.

Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines.

Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA)n.(dT)n mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

Cre-mediated site-specific translocation between nonhomologous mouse chromosomes.

Chromosome rearrangements, such as large deletions, inversions, or translocations, mediate migration of large DNA segments within or between chromosomes, which can have major effects on cellular genetic control. A method for chromosome manipulation would be very useful for studying the consequences of large-scale DNA rearrangements in mammalian cells or animals. With the use of the Cre-loxP recombination system of bacteriophage P1, we induced a site-specific translocation between the Dek gene on chromosome 13 and the Can gene on chromosome 2 in mouse embryonic stem cells. The estimated frequency of Cre-mediated translocation between the nonhomologous mouse chromosomes is approximately 1 in 1200-2400 embryonic stem cells expressing Cre recombinase. These results demonstrate the feasibility of site-specific recombination systems for chromosome manipulation in mammalian cells in vivo, breaking ground for chromosome engineering.

Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

The acute effects of contraction and insulin on the glucose transport and GLUT4 glucose transporter translocation were investigated in rat soleus muscles by using a 3-O-methylglucose transport assay and the sensitive exofacial labeling technique with the impermeant photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-y loxy)-2- propylamine (ATB-BMPA), respectively. Addition of wortmannin, which inhibits phosphatidylinositol 3-kinase, reduced insulin-stimulated glucose transport (8.8 +/- 0.5 mumol per ml per h vs. 1.4 +/- 0.1 mumol per ml per h) and GLUT4 translocation [2.79 +/- 0.20 pmol/g (wet muscle weight) vs. 0.49 +/- 0.05 pmol/g (wet muscle weight)]. In contrast, even at a high concentration (1 microM), wortmannin had no effect on contraction-mediated glucose uptake (4.4 +/- 0.1 mumol per ml per h vs. 4.1 +/- 0.2 mumol per ml per h) and GLUT4 cell surface content [1.75 +/- 0.16 pmol/g (wet muscle weight) vs. 1.52 +/- 0.16 pmol/g (wet muscle weight)]. Contraction-mediated translocation of the GLUT4 transporters to the cell surface was closely correlated with the glucose transport activity and could account fully for the increment in glucose uptake after contraction. The combined effects of contraction and maximal insulin stimulation were greater than either stimulation alone on glucose transport activity (11.5 +/- 0.4 mumol per ml per h vs. 5.6 +/- 0.2 mumol per ml per h and 9.0 +/- 0.2 mumol per ml per h) and on GLUT4 translocation [4.10 +/- 0.20 pmol/g (wet muscle weight) vs. 1.75 +/- 0.25 pmol/g (wet muscle weight) and 3.15 +/- 0.18 pmol/g (wet muscle weight)]. The results provide evidence that contraction stimulates translocation of GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

Genetics and the origin of European languages.

A new set of European genetic data has been analyzed to dissect independent patterns of geographic variation. The most important cause of European genetic variation has been confirmed to correspond to the migration of Neolithic farmers from the area of origin of agriculture in the Middle East. The next most important component of genetic variation is apparently associated with a north-south gradient possibly due to adaptation to cold climates but also to the differentiation of the Uralic and the Indo-European language-speaking people; however, the relevant correlations are not significantly different from zero after elimination of the spatial autocorrelation. The third component is highly correlated with the infiltration of the Yamna ("Kurgan") people, nomadic pastoralists who domesticated the horse and who have been claimed to have spread Indo-European languages to Europe; this association, which is statistically significant even when taking spatial autocorrelations into account, does not completely exclude the hypothesis of Indo-European as the language of Neolithic farmers. It is possible that both expansions were responsible for the spread of different subfamilies of Indo-European languages, but our genetic data cannot resolve their relative importance.

Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation.

Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.

We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.

Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver.

Intraperitoneal injection of epidermal growth factor into mice results in the appearance of multiple tyrosine-phosphorylated proteins in liver nuclei within minutes after administration. We have previously identified three of these proteins as Stat 1 alpha, Stat 1 beta (p91, p84), and Stat 3 (p89). In the present report we demonstrate that Stat 5 (p92), the recently described prolactin inducible transcription factor detected in mammary glands, is the major tyrosine-phosphorylated protein translocated to the nucleus in mouse liver in response to epidermal growth factor. Furthermore, gel-shift analysis and affinity purification revealed that Stat 5, Stat 1 alpha, and Stat 1 beta specifically bind to the prolactin inducible element upstream of the beta-casein promoter.

Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent. Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional in promoting the active nuclear transport of MyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells. Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor. In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.

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.

Advanced Genetics (2015-2016). Units 1 to 4

Ppoint presentations of Units 1 to 4 corresponding to Advanced Genetics course.

Molecular Techniques (2015-2016). Protocol for the Genetics Laboratory practices

Protocol for the Genetics Laboratory practices.