Analysis of the Schwanniomyces occidentalis SWA2 gene promoter in Saccharomyces cerevisiae

The effect of different carbon sources on the expression in Saccharomyces cerevisiae of the SWA2 alpha-amylase gene from Schwanniomyces occidentalis was studied from constructs containing its 5' region (-223 to +15), which were fused in-frame to the lacZ gene coding sequence. Maximal expression was achieved with the non-fermentable substrates ethanol and/or glycerol, whereas lower levels were found with maltose or galactose. In contrast, glucose repressed it, even in the presence of any of these other carbon sources. Deletion analyses of the -233 to -85 SWA2 promoter region permitted the identification of two fragments involved in both glucose repression and ethanol activation. A possible region required for cAMP regulation was localised. The SWA2 promoter contains a MIG1-binding GC box whose deletion caused a five-fold increase in the glucose-repressed reporter expression. Despite this, expression of the SWA2 promoter was not MIG1-dependent.     

Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Embryonic stem (ES) cells have the ability to differentiate in vitro into a wide variety of cell types with potential applications for tissue regeneration. However, a large number of cells are required, thus strengthening the need to develop large-scale systems using chemically defined media for ES cell production and/or controlled differentiation. In the present studies, a stirred culture system (i.e. spinner flask) was used to scale-up mouse ES (mES) cell expansion in serum-containing (DMEM/FBS) or serum-free medium, both supplemented with leukemia inhibitory factor (LIF), using either Cytodex 3 or Cultispher S microcarriers. After 8 days, maximal cell densities achieved were (1.9+/-0.1), (2.6+/-0.7) and 3.5x10(6)cells/mL for Cytodex 3 in DMEM/FBS, Cultispher S in DMEM/FBS and Cultispher S in serum-free cultures, respectively, with fold increases of 38+/-2, 50+/-15 and 70. Both microcarriers were suitable to sustain mES cell expansion, though the macroporous Cultispher S seemed to be advantageous in providing a more protective environment against shear stress forces, which harmful effects are exacerbated in serum-free conditions. Importantly, mES cells expanded under stirred conditions using serum-free medium retained their pluripotency and the ability to commit to the neural lineage.     

Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?

We explore the relationship between general eggshell colour (background and spots) and proportion of surface covered by spots on the eggs of blue tits Cyanistes caeruleus and several variables reflecting female health and condition. Females laying more spotted eggs showed a poorer body condition, higher cellular concentration of the stress protein HSP70 and marginally lower total immunoglobulin levels in blood. In addition, these females were paired with males with higher levels of HSP70 and lower concentrations of immunoglobulins. We interpret these results based on the properties of the eggshell pigment protoporphyrin, which is known as an inductor of oxidative stress in females. Although observational, this study presents, for the first time, clear evidence of eggshell spottiness and general colour as indicators of general condition and stress in avian populations.     

Presence of cytosolic phosphoenolpyruvate carboxykinase activity in rat mammary gland

The cytosolic P-enolpyruvate carboxykinase activity was measured in mammary gland and liver of animals at all stages of the reproductive cycle. The specific activity of this enzyme was almost absent in the mammary gland of virgin rats. Different pattern of P-enolpyruvate carboxykinase activity was obtained in liver and mammary gland during lactation cycle. The specific activity of the enzyme increased more than 40-fold in mammary gland and 2-fold in liver during the transition from mid-pregnancy to mid-lactation. Weaning of mid-lactating rats for 48 h resulted in an abrupt change in the enzyme activity in mammary gland while there was a small change in liver. In all the experiments performed, the activity of P-enolpyruvate correlates inversely with the plasma insulin levels described for the lactogenic process.     

Involvement of diminution of glutathione, produced by deficiency of methionine in the diet, in the elevation of malic enzyme level in rat liver

Rats fed on a low protein diet show an increase in the specific activity of malic enzyme and a concomitant decrease of glutathione concentration. We have studied the effect on malic enzyme activity of supplementing of low protein diet with essential amino acids. Only when methionine was excluded from the diet did the specific activity of malic enzyme increase to the same extent as found in rats fed with low protein diet. Immunoprecipitation of malic enzyme indicated that specific activity changes are the result of changes in the amounts of enzyme. Under all dietary conditions studied, the increase in malic enzyme activity is associated with a decrease in the concentration of GSH. To evaluate the possible causative role of GSH in malic enzyme induction, the specific activity of malic enzyme was measured in rats treated with BSO, an inhibitor of GSH biosynthesis. The results show that in BSO-treated rats the decrease of GSH levels is also accompanied by an increase in the activity of malic enzyme.     

Microalgae-mediated chemicals production and wastes removal

Biotechnology of microalgae has gained importance in recent years due to the development of new production and environmental technologies. Because their growth requires unexpensive substrates such as solar light and CO₂, microalgae can be used as cheap and effective biocatalysts to obtain high added-value compounds, from simple metabolites to complex molecules, i.e., chemicals, vitamins, carotenoids, pigments, or polysaccharides. During productive processes, the algal biomass formed may be used as a food source like proteins. On the other hand, microalgae can also be employed in contaminant bioelimination processes especially for nitrogen, phosphorus, or sulfur compounds. Particularly relevant is their use for heavy metal removal from wastewaters; upon enriching the biomass in the metal, they can be recovered, thereby providing economic advantages.

The use of immobilized microalgae in these processes is very adequate and offers significant advantages in bioreactors.     

Cytogenetics of the South American Akodont rodents (Cricetidae) X. Akodon mollis: a species with XY females and B chromosomes

The morphology, G- and C-banding pattern of the Akodon mollis chromosome complement is analysed. Over a total of 14 males and 10 females studied, 8 males and 7 females had a modal chromosome number of 22, while 6 males and 3 females showed a modal number of 23 chromosomes. In the animals with 23 chromosomes the odd element was considered a B chromosome on the basis of: (a) its small size, (b) the lack of an homologous chromosome and the subsequent formation of univalents at diakinesis and metaphase I from testes, (c) the weak or null genetic action as evidenced by the lack of any obvious variation in the phenotype of carriers.Four females exhibited a sex-pair dimorphism indistinguishable from that observed in males. The G-banding analysis showed homology between the pattern found in the Y chromosome and that detected in the short arm of the X. The study of C-band distribution showed that several autosome pairs and the X chromosomes had small masses of centromeric heterochromatin. On the other hand, the Y and B chromosomes were C-band negative. The Y-like chromosome in females with dimorphism of the sex pair was also C-band negative. Accordingly these females were considered to be XY and not Xx (the x being an extensively deleted X chromosome).This work was supported by grants from UNESCO, OEA, CONICET and CIC. Requests for reprints should be addressed to N.O. Bianchi.     

Functional analysis of four CYP21 mutations from spanish patients with congenital adrenal hyperplasia.

Deleterious mutations in the CYP21 (steroid 21-hydroxylase) gene cause congenital adrenal hyperplasia (CAH). These mutations usually result from recombinations between CYP21 and an adjacent pseudogene, CYP21P, including deletions and transfers of deleterious mutations from CYP21P to CYP21 (gene conversions). Additional rare mutations that are not gene conversions account for 5-10% of 21-hydroxylase deficiency alleles. Recently, four novel CYP21 point mutations leading to amino acid changes were identified in a population of 57 Spanish families with CAH. A nonsense mutation, K74X, was also identified. The enzymatic activities of 21-hydroxylase mutants G90V, G178A, G291C, and R354H were examined in transiently transfected CHOP cells using progesterone and 17alpha-hydroxyprogesterone as substrates. The G90V, G291C, and R354H mutations effectively eliminated 21-hydroxylase activity. However, the G178A mutant retained significant activity when 17alpha-hydroxyprogesterone was the substrate. These results correlate well with the identification of G90V, G291C, and R354H in patients with severe "salt-wasting" disease and G178A in a patient with the milder simple virilizing form.     

Integration of lipid metabolism in the mammary gland and adipose tissue by prolactin during lactation

Prolactin deficiency, induced by bromocryptine treatment, brought about reciprocal changes in the ability of adipocytes and acini isolated from lactating rats to synthesize lipids. The capacity to synthesize fatty acids and phospholipids decreased in the mammary gland and increased in adipocytes by bromocryptine treatment. In the mammary gland, the maximum potential activity of the pentose shunt as well as the specific activities of the pathway dehydrogenases were significantly reduced by bromocryptine treatment. Simultaneously, adipose tissue increased its lipogenic capacity but neither the maximum potential of the shunt nor the specific activities of the pentose phosphate shunt dehydrogenases were significantly changed with respect to the control lactating rats. Thus, a differential regulatory mechanism(s) of the pentose phosphate shunt activity appears to operate in these two tissues. Adipocytes from lactating rats showed a poor responsiveness to insulin in terms of lipid synthesis from glucose. In contrast, in adipocytes from bromocryptine treated rats insulin was able to increase lipid synthesis (105%). Sheep prolactin administration in vivo partially reversed the effects of bromocryptine. These data suggest that prolactin mediates adipocytes resistance to insulin during lactation. Phospholipid synthesis, as occurred in fatty acid synthesis, is increased in adipose tissue and decreased in mammary gland by bromocryptine treatment. However, -adrenergic stimulation increases phosphatidylinositol turnover to about the same percentages in both mammary gland acini and adipocytes from lactating rats independently of bromocryptine treatment.     

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Background

Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods

A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 10⁹ copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results

Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion

Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.