In vitro expansion of human cardiac progenitor cells: exploring 'omics tools for characterization of cell-based allogeneic products.

Human cardiac stem/progenitor cells (hCPCs) have been shown to be capable to regenerate contractile myocardium. However, because of their relative low abundance in the heart, in vitro expansion of hCPC is mandatory to achieve necessary quantities for allogeneic or autologous cardiac regeneration therapy applications (10(6)-10(9) cells/patient). Up to now, cell number requirements of ongoing phase I/IIa trials have been fulfilled with production in static monolayer cultures. However, this manufacturing process poses critical limitations when moving to the following clinical phases where hundreds of patients will be enrolled. For this, increased process yield is required, while guaranteeing the quality of the cell-based products. In this work, we developed and validated a robust, scalable, and good manufacturing practice (GMP)-compatible bioprocess for the expansion of high-quality hCPC. We applied platforms extensively used by the biopharmaceutical industry, such as microcarrier technology and stirred systems, and assessed culture conditions' impact on hCPC's quality and potency, as required by regulatory agencies. Complementary analytical assays including gene expression microarrays and mass spectrometry-based approaches were explored to compare transcriptome, proteome, surface markers, and secretion profiles of hCPC cultured in static monolayers and in stirred microcarrier-based systems. Our results show that stirred microcarrier-based culture systems enabled achieving more than 3-fold increase in hCPC expansion, when compared with traditional static monolayers, while retaining cell's phenotype and similar "omics" profiles. These findings demonstrate that this change in the production process does not affect cell's identity and quality, with potential to be translated into a transversal production platform for clinical development of stem-cell therapies.

The analysis of Lupinus albus root proteome revealed cytoskeleton altered features due to long-term boron deficiency.

Boron (B) deficiency greatly limits plants' growth and development. Since the root is the organ that first senses the deficiency, we have analyzed the adaptive responses of Lupinus albus roots to long-term B deficiency. Large morphological differences were observed between plants grown with or without B, and 265 polypeptides were found to be responsive to B deficiency out of a total of 406 polypeptides detected by two-dimensional electrophoresis in the L. albus root proteome. By using mass spectrometry techniques we were able to securely identify 128 of the responsive polypeptides that are related to cell wall metabolism, cell structure, defense, energy pathways and protein metabolism. The detection of multiple peptide isoforms is striking, suggesting that protein modification may have an important contribution during the plant response to long-term B deficiency. Furthermore, detected changes in cytoskeletal associated proteins indicate altered cytoskeletal biosynthesis and suggest that B may have an important contribution in this process.

Effect of water stress on lupin stem protein analysed by two-dimensional gel electrophoresis.

Lupinus albus plants can withstand severe drought stress and show signs of recovery 24 h after rewatering (RW). Two-dimensional gel electrophoresis was used to evaluate the effect of water deficit (WD) on the protein composition of the two components of the lupin stem (stele and cortex). This was performed at three distinct stress levels: an early stage, a severe WD, and early recovery. Protein characterisation was performed through mass spectrometric partial sequencing. Modifications in the protein expression were first noticed at 3 days of withholding water, when the plant water status was still unaffected but some decrease in the relative soil water content had already occurred. An increase in serine proteases, possibly associated with WD sensing, was an early alteration induced by WD. When the stress severity increased, a larger number of stem proteins were affected. Immunophilin, serine protease and cysteine protease (well-known components of animal sensing pathways) were some of these proteins. The simultaneous expression of proteases and protease inhibitors that reacted differently to the stress level and to RW was found. Although the level of protease inhibitors was significantly raised, RW did not cause de novo expression of proteins. Many amino acid sequences did not match known sequences of either protein or expressed sequence tag databases. This emphasises the largely unknown nature of stem proteins. Nevertheless, some important clues regarding the way the lupin plant copes with WD were revealed.

How plants cope with water stress in the field. Photosynthesis and growth.

Plants are often subjected to periods of soil and atmospheric water deficit during their life cycle. The frequency of such phenomena is likely to increase in the future even outside today's arid/semi-arid regions. Plant responses to water scarcity are complex, involving deleterious and/or adaptive changes, and under field conditions these responses can be synergistically or antagonistically modified by the superimposition of other stresses. This complexity is illustrated using examples of woody and herbaceous species mostly from Mediterranean-type ecosystems, with strategies ranging from drought-avoidance, as in winter/spring annuals or in deep-rooted perennials, to the stress resistance of sclerophylls. Differences among species that can be traced to different capacities for water acquisition, rather than to differences in metabolism at a given water status, are described. Changes in the root : shoot ratio or the temporary accumulation of reserves in the stem are accompanied by alterations in nitrogen and carbon metabolism, the fine regulation of which is still largely unknown. At the leaf level, the dissipation of excitation energy through processes other than photosynthetic C-metabolism is an important defence mechanism under conditions of water stress and is accompanied by down-regulation of photochemistry and, in the longer term, of carbon metabolism.

Rapid deposition of extensin during the elicitation of grapevine callus cultures is specifically catalyzed by a 40-kilodalton peroxidase.

Elicitation or peroxide stimulation of grape (Vitis vinifera L. cv Touriga) vine callus cultures results in the rapid and selective in situ insolubilization of an abundant and ionically bound cell wall protein-denominated GvP1. Surface-enhanced laser desorption/ionization/time of flight-mass spectrometry analysis, the amino acid composition, and the N-terminal sequence of purified GvP1 identified it as an 89.9-kD extensin. Analysis of cell walls following the in situ insolubilization of GvP1 indicates large and specific increases in the major amino acids of GvP1 as compared with the amino acids present in salt-eluted cell walls. We calculate that following deposition, covalently bound GvP1 contributes up to 4% to 5% of the cell wall dry weight. The deposition of GvP1 in situ requires peroxide and endogenous peroxidase activity. Isoelectric focusing of saline eluates of callus revealed only a few basic peroxidases that were all isolated or purified to electrophoretic homogeneity. In vitro and in situ assays of extensin cross-linking activity using GvP1 and peroxidases showed that a 40-kD peroxidase cross-linked GvP1 within minutes, whereas other grapevine peroxidases had no significant activity with GvP1. Internal peptide sequences indicated this extensin peroxidase (EP) is a member of the class III peroxidases. We conclude that we have identified and purified an EP from grapevine callus that is responsible for the catalysis of GvP1 deposition in situ during elicitation. Our results suggest that GvP1 and this EP play an important combined role in grapevine cell wall defense.

Alterations in carbon and nitrogen metabolism induced by water deficit in the stems and leaves of Lupinus albus L.

Water deficit (WD) in Lupinus albus L. brings about tissue-specific responses that are dependent on stress intensity. Carbohydrate metabolism is very sensitive to changes in plant water status. Six days from withholding water (DAW), sucrose, glucose and fructose levels of the leaf blade had already increased over 5-fold, and the activities of SS and INV(A) had increased c. 1.5-2 times. From 9 DAW on, when stress intensity was more pronounced, these effects were reversed with fructose and glucose concentrations as well as INV(A) activity dropping in parallel. The stem (specifically the stele) responded to the stress intensification with striking increases in the concentration of sugars, N and S, and in the induction of thaumatin-like-protein and an increase in chitinase and peroxidase. At 13 DAW, the plants lost most of the leaves but on rewatering they fully recovered. Thus, the observed changes appear to contribute to a general mechanism of survival under drought, the stem playing a key role in that process.

Differential expression of four genes encoding 1-aminocyclopropane-1-caroboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding.

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a "silent" twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings.

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds.

A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein.

Doença hemorrágica do recém-nascido na forma tardia: descriçäo de casos / Late hemorragic disease of the newborn: case report

Objetivo: Chamar atençäo para a doença hemorrágica do recém-nascido em sua forma tardia, que poderá ter graves consequências, incluindo óbito e seqüelas neurológicas. Métodos: Säo apresentados e descritos quatro casos de recém-nascidos, com idaade variando de 12 a 21 dias de vida, diagnosticados como doença hemorrágica tardia por dificiência de vitamina k. Resultados: Nos casos apresentados as manifestaçöes hemorrágicas foram múltiplas. Os locais acometidos em nossos casos foram o aparelho digestivo, trato urinário, côto umbilical, aparelho respiratório e SNC. Conclusäo: O estudo mostra a importância da forma tardia da doença hemorrágica do recém-nascido, destacando-se sua possível gravidade, risco de morte e de seqüelas neurológicas. A administraçäo profilática de vitamina k após o nascimento, preconizada pela OMS, continua sendo a melhor forma de previnir a doença...

Study of the intercellular fluid of healthy Lupinus albus organs. Presence of a chitinase and a thaumatin-like protein.

Proteins in the intercellular fluid (IF) of healthy Lupinus albus leaves were characterized. Silver staining of the proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed more than 30 polypeptides, with the major ones having a molecular mass lower than 36 kD. After amino-terminal amino acid sequence analysis, one of the major polypeptides, IF4, was shown to have no identity with any of the proteins present in the data bases. Two others, IF1 and IF3, showed identity with previously reported pathogenesis-related proteins, IF1 with an antifungal protein from Hordeum vulgare that belongs to the thaumatin family (PR-5 family), and IF3 with class III chitinase-lysozymes. IF3 was also present in the IF of stem and root and it represents the major polypeptide in the medium of L. albus cell-suspension cultures. The ubiquitous presence of this enzyme in healthy, nonstressed tissues of L. albus cannot be explained.

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: 1KG(R)V5KGTGD10(T)PXXV15XLY(N)R20T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man alpha 2Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4 GlcNAc (13%); +/- Man alpha 2Man alpha 6(Man alpha 3)Man alpha 6(+/- Man alpha 2 Man alpha 2 Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (29%); Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (13%); Man alpha 6(Man alpha 3)Man alpha 6(Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (16%); Man alpha 6(Man alpha 3)(Xyl beta 2)Man beta 4GlcNAc beta 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.

Lupinus albus L. pathogenesis-related proteins that show similarity to PR-10 proteins.

We describe a group of three acidic proteins, pathogenesis-related (PR)-p16.5a, PR-p16.5b, and PR-p16.5c, that accumulate in the leaves of Lupinus albus L. cv Rio Maior plants when infected with the fungus Colletotrichum gloeosporioides Penz. These proteins co-migrate in sodium dodecyl sulfate-polyacrylamide gels as a single band of 16.5 kD, behaving as charge isomers, and are related to several members of the defense-related PR-10 protein family. Localization of the proteins was investigated by techniques of tissue printing and immunogold electron microscopy; they are predominantly associated with the vascular system and are localized extracellularly. The accumulation of PR-p16.5a, PR-p16.5b, and PR-p16.5c also seems to be induced by cucumber mosaic virus and by two forms of abiotic stress, salicylic acid and ultraviolet, suggesting a general defense role for these proteins.

Cloning and characterization of two ubiquitin::79-amino-acid extension protein-encoding fusion genes from Lupinus albus.

Two different ubiquitin::79-amino-acid (aa) extension protein-encoding fusion genes were isolated from a Lupinus albus nuclear DNA library and sequenced. Both genes have 465-nucleotide open reading frames encoding a single ubiquitin (Ub) monomer fused in frame to a 79-aa extension (Ext) protein. The deduced aa sequences of the encoded Ub are identical to the aa sequences of Ub from other plants. The encoded Ext proteins are putative ribosomal proteins, highly basic, differing by 2 aa from each other, and have a high degree of similarity to Ext proteins from other plants.

Molecular cloning and sequencing analysis of a beta-tubulin gene from Lupinus albus.

Genomic lambda-Dash library constructed from Lupinus albus nuclear DNA was screened using a fragment of the beta-tubulin cDNA (beta 8-31) clone of Chlamydomonas reinhardtii as probe. One of the positive recombinant phages was isolated, subcloned and analysed by sequencing. We present here nucleotide and derived amino acid sequences of the beta-tubulin gene, designated as L beta 1 and identified by similarity with other beta-tubulins. The L beta 1-encoded protein reveals a very high degree of similarity with other plant tubulins and contains consensus sequences for binding guanine base, phosphate and Mg2+. Northern analysis of total RNA isolated from roots, leaves, flowers and pools revealed that Lupinus albus beta-tubulin genes are constitutively expressed in all studied plant tissues.

Cytochrome c aided resolution of Lupinus albus isoperoxidases in a cathodal polyacrylamide gel electrophoresis system.

In a cathodal polyacrylamide gel electrophoresis system, three distinct groups of isoperoxidases from Lupinus albus were found to achieve retention factors (rf) dependent on the quantity of sample applied onto the gel. The possibility of extract-derived substances weakly associating with peroxidase samples was investigated. Association of the putative agents survived dialysis against electrophoresis buffer with and without 2 M CaCl2 and freeze-thaw treatments. The addition of polyvinylpolypyrrolidone and polyethylene glycol to the homogenization buffer also proved ineffective in eliminating the variation in isoperoxidase rf although differences in the zymogram profiles of these samples were evident. The addition of spermine and cytochrome c to samples was found to increase the rf of some peroxidase bands. Electrophoresis of samples with cytochrome c resulted in the resolution of peroxidase groups to distinct bands at rf independent of the quantity of peroxidase applied. Control experiments indicate that this treatment did not introduce any detectable artifacts.

Development of tuberous roots and sugar accumulation as related to invertase activity and mineral nutrition.

Sucrose storage in tuberous roots was not observed when the tissues had very high activities of acid invertase. High activities of the enzyme were always present in the roots at early stages of their development. In species where the activity of the enzyme decreased during root development, sucrose was stored. Thus, acid invertase was undetectable in mature roots of carrots (Daucus carota L.) where sucrose formed almost 80% of the dry matter. Conversely, radish (Raphanus sativus L.) and turnip (Brassica rapa L.) roots, in which the activity of the enzyme remained high until maturity, did not store appreciable amounts of sucrose (2% and 9%, respectively, of the dry matter in the mature roots), reducing sugars being the main reserve (more than 80% of the dry matter in mature turnips). The correlation between sucrose content and acid invertase activity was furthermore evident in both sucrose- and hexose-storing roots when the activity of this enzyme was affected by changes in the mineral nutrition. Deficiencies of nitrogen and sulphur reduced the activity of acid and alkaline invertases and led to increase in sucrose content and decrease in reducing sugars. However, the decline of alkaline invertase activity in tissues low in acid invertase had no clear effect on sugar content. Sodium chloride (10(-1)M) affected acid invertase and sugars in a manner similar to that of the two deficiencies, but had practically no effect on alkaline invertase. The changes in sugar content produced by the variations in mineral nutrition were small in hexose-storing roots in relation to those of sucrose-storing roots. It is possible that this result is related to the different levels of acid invertase in the two types of roots.

Alkaline ß-fructofuranosidases of tuberous roots: Possible physiological function.

Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K i value being ca. 4×10(-2) M and 3×10(-1)M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.