A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors.

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10(4) U·mg(-1), corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.

Use of chemometrics in the selection of a Saccharomyces cerevisiae expression system for recombinant cyprosin B production.

Two multivariate statistical methods, factor analysis (FA) and hierarchical cluster analysis (HCA), were applied to experimental data set to evaluate their usefulness in selecting the adequate expression system and optimal growth parameters for recombinant cyprosin B production. Using FA, the large data set was reduced to two factors representing 73.4% of variability. Factor 1, with 53.5% of variability, corresponds to recombinant cyprosin B expression and efficient secretion, while factor 2, accounting for 19.9% of variability, represents cell growth and physiological characteristics. FA and HCA allowed the establishment of correlations among different variables and the clusters obtained providing clear identification of the experimental parameters related to cyprosin B production, which results on more accurate scientific output and time saving when selection of an adequate expression system is concerned.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots / PtSRR1, um possível receptor simbiose-regulado de Pisolithus tinctorius é expresso nas primeiras horas de interação ectomicorrízica com raízes de Castanea sativa

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1 foi isolado preliminarmente de P. tinctorius nas primeiras horas da interação com raízes de C. sativa. Análises de QRT-PCR confirmaram sua expressão positiva (12 h) e seu peptídeo putativo indicou forte possibilidade para proteína transmembranar. Estes dados estimulam o estudo do PtSRR1 durante a formação de ectomicorrizas.

Outcome of pregnancy in renal allograft recipients.

OBJECTIVE: To evaluate risk factors affecting pregnancy, perinatal outcomes and graft condition in women who underwent renal transplantation. METHODS: Retrospective study of 34 pregnancies in 28 renal recipients followed in a single tertiary center from January 1989 to January 2007. MAIN OUTCOME MEASURES: Pregnancy outcome, kidney allograft function, maternal complications and perinatal outcomes were evaluated in these patients. RESULTS: Mean maternal age at time of pregnancy was 27+/-5.1 years (18-37) and the interval between transplant and pregnancy varied between 1 and 134 months (mean 51.3+/-34.2). Most pregnant women (25/28) were submitted to triple immunosuppression during the entire pregnancy. The fetal outcome included 27 live births (79.4%), 2 stillbirths (5.9%), 3 spontaneous abortions (8.8%) and 2 therapeutic abortions (5.9%). The most frequent maternal complications were hypertension in 18 pregnancies, 2 of which ended in pre-eclampsia; urinary tract infections in 10 pregnancies; gestational diabetes mellitus in 3, anemia in 3 and 2 acute graft rejections. The major fetal complications observed consisted of four (13. 8%) intrauterine growth restrictions and two (6.9%) stillbirths. Vaginal delivery occurred in 10 women (34.5%); in the other 19 (65.5%), a cesarean section was performed. Of the 27 successful pregnancies, 11 (40.7%) resulted in term deliveries and 16 (59.3%) in preterm deliveries (range 31-39 weeks). The mean birth weight of the offspring was 2,465 g (range 1,300-3,530). There were no major perinatal complications, but two allograft rejections occurred after pregnancy. CONCLUSIONS: This series results are in agreement with those in other studies. Even though pregnancy does not seem to adversely affect short-term renal allograft function, risks of obstetric and perinatal complications seem to be increased. Further studies of long term graft function and pediatric follow-up are needed.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots.

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

Molecular phylogeny of wild hops, Humulus lupulus L.

We have analysed wild hops collected widely from the Northern Hemisphere, assessing the genetic diversity and the geographical distribution of haplotypes, to investigate the evolution and phylogeny of hops, Humulus lupulus. The haplotypes were characterized by the nuclear ribosomal DNA spacer region (length and DNA sequence) and chloroplast DNA noncoding regions (DNA sequences). The results indicated that primary divergence into European (including Caucasus and Altai hops), and Asian-North American types, was 1.05+/-0.28 to 1.27+/-0.30 million years ago. Although an Eastern boundary for European nuclear haplotype distribution was unclear due to the ambiguous origin of Northern Chinese samples, the European hop group showed a wide geographical distribution across Eurasia from the Altai region to Portugal. The low genetic variation in this group suggested rapid and recent expansion. The North American hop group showed high diversity, and is considered to include hops that have migrated from Asia. Japanese and Chinese hops were identified as genetically distinct. This study has shown that wild hops in each growing region are genetically differentiated with considerable genetic diversity. It gives insights into the evolution and domestication of hops that are discussed.

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture.

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P

Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine sodium caseinates.

The rates and extents of hydrolysis of alpha(S)- and beta-caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficus-indica, (L.) Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo-first-order enzymatic reaction, in the presence of first-order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet exhibited higher enzymatic efficiency than the fruit extract, irrespective of the source (i.e., bovine, caprine, or ovine) and the type (i.e., alpha(S)- or beta-casein) of substrate. The enzymatic efficiency (k(cat)/K(m)) for alpha(S)-casein ranged from 72 to 220 and from 43 to 65 L g(-1) h(-1), and for beta-casein from 242 to 742 and from 55 to 164 L g(-1) h(-1), for the animal rennet and the enzymatic extract of O. ficus-indica, respectively. Finally, it was observed that beta-casein from caprine and ovine caseinates was degraded by O. ficus-indica faster than its alpha(S) counterpart, but the reverse was observed for bovine caseinate.

An electron probe X-ray microanalysis study during organogenesis from internode-derived nodules of Humulus lupulus var. Nugget.

Elemental changes during the induction of organogenesis from internode-derived nodules of Humulus lupulus var. Nugget were studied by electron probe X-ray microanalysis (EPMA) of specimens submitted to physical fixation procedures. X-ray spectra were collected from cambial and cortical cells. Four days after explants inoculation an increase of K and Ca was detected in cells of both regions. Four to twelve days after explants inoculation an increase of Cu, Zn, Fe, S and Mn was therein detected. Values of Cu, Zn, Fe, K and S were lower in control explants than in induced explants 12 days after induction. Although S presented fluctuations it increased throughout the induction period. X-ray spectra collected from organogenic nodules revealed higher levels of Ca, K, Fe, P and S on peripheral regions where regeneration was occurring. Ca was mobilized in several directions, from inner regions of nodules towards their periphery at the onset of plantlet regeneration. Levels of Mg and Na were low or absent. Control explants neither formed nodules nor regenerated plantlets. The results suggest that EPMA can be used to study relative elemental changes during plant morphogenesis induction and enables the early establishment of organogenic regions in nodules.

Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa.

Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full-length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresponding enzyme was purified in its precursor form from fresh flowers using ammonium-sulfate precipitation followed by ion-exchange and hydrophobic-interaction chromatography. The processing of the precursor into its mature form was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16- and 30-kDa chains were obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content.

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.

Production and characterization of a specific rubisco monoclonal antibody, and its use in rubisco quantification during Zantedeschia aethiopica spathe development.

Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified from leaves of Zantedeschia aethiopica and used to immunize female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A random selected IgG2a subclass MAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose CL-4B, with a recovery of 84.3% and it was apparently homogeneous on native PAGE. The monoclonality of the purified MAb was determined by IEF. The MAb was highly specific for Rubisco from leaves of Z. aethiopica as determined by Western blotting and was used to determine the concentration of Rubisco protein by enzyme-linked immunoadsorbent assay (ELISA), at three distinct stages of Z. aethiopica spathe development and in the leaf. The results suggest de novo synthesis of Rubisco during the spathe regreening, which could explain, at least in part, the increase of photosynthetic activity observed during regreening.