Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco’s modified Eagle’s medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.     

Substrate specificity of strictosidine synthase

Strictosidine synthase catalyzes a Pictet-Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids to generate strictosidine. The substrate requirements for strictosidine synthase are systematically and quantitatively examined and the enzymatically generated compounds are processed by the second enzyme in this biosynthetic pathway.     

Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ∼5 m^-1 s^-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.The frequent abundance of arsenic in the environment has guided the evolution of enzymes for the reduction of arsenate (As(V))⁴ (1). Arsenate reductases (ArsCs) are unusual among well studied enzyme classes, because there is not a single family of evolutionarily related sequences. The structural folds and mechanisms that they are using are fundamentally different and arose independently during evolution (2). Arsenate reductases are small cytoplasmic redox enzymes that reduce arsenate to arsenite (As(III)) by the sequential involvement of three different thiolate nucleophiles that function as a redox cascade. As such, arsenate reductases from different organisms often work together with the thiol/disulfide mechanism in the cell.The major and most ubiquitous system for protection against oxidative stress and to maintain the intracellular thiol homeostasis is the thioredoxin system that is composed of Trx (thioredoxin) and TrxR (thioredoxin reductase) (3). In addition to the thioredoxin system, most living organisms contain low molecular weight thiol compounds that serve as a buffer to avert disulfide stress. In eukaryotes and Gram-negative bacteria, the redox level is maintained by redox cycling of glutathione (GSH) with Grx (glutaredoxin) and glutathione reductase (4). Gram-positive bacteria, like Staphylococcus aureus, produce no glutathione, but millimolar quantities of reduced coenzyme A is the predominant thiol, which is kept reduced with a NADPH-dependent flavoenzyme, coenzyme A disulfide reductase (5). Also actinobacteria, like Corynebacterium glutamicum, produce no GSH, but instead they contain millimolar concentrations of MSH (mycothiol; chemically 1D-myo-inosityl-2-[N-acetyl-l-cysteinyl] amido-2-deoxy-α-d-glucopyranoside), a pseudodisaccharide containing a cysteine moiety as a reactive thiol (6). Its oxidized form is mycothione (MSSM). In actinobacteria, MTR (mycothione reductase) is the NADPH-dependent flavoenzyme that reduces MSSM in order to maintain the intracellular redox homeostasis to allow the proper functioning of a variety of biological functions (7).Arsenate reductases are part of a defense mechanism of the cell against toxic arsenate. Their genes are most of the time found in an operon together with arsenite sensing and efflux genes (8). Based on the mechanism used to reduce arsenate to arsenite, two distinct ArsC classes can be defined. The first one is the thioredoxin-coupled ArsC class represented by S. aureus pI258 ArsC and Bacillus subtilis ArsC (9–11). Both enzymes use the structural fold of low molecular weight tyrosine phosphatase and need Trx to start a second catalytic cycle (12–14). The second class is the GSH/glutaredoxin-coupled class represented by Escherichia coli plasmid R773 ArsC (15, 16), the eukaryotic Acr2p reductase from Saccharomyces cerevisiae (17), and ArsC from Leishmania major (18). In this second class, two different structural folds are found; E. coli R773 ArsC partially resembles glutaredoxin (19), whereas the eukaryotic ArsCs have a rhodanese fold like the Cdc25a cell cycle control phosphatase (20). Notably, all arsenate reductases have a thiolate nucleophile at the N-terminal end of an α-helix. The active site of the ArsCs with a phosphatase-like scaffold is conserved (root mean square deviation of 0.54 Å) with a catalytically important Arg on position Cys⁺⁶.In C. glutamicum, there are four arsC genes located on different places in the chromosome (21): one orphan arsC gene (arsC4) and three arsC genes (arsC1-arsC1′ and arsC2) present in two ars operons. We show here that two of the encoded proteins, Cg_ArsC1 and Cg_ArsC2 (with 66% sequence identity) are members of a new third class, the mycothiol- and mycoredoxin-dependent arsenate reductases. Both the genes of arsC1 and arsC2, together with the genes for the enzymes of the mycothiol biosynthesis pathway are involved in arsenate resistance in C. glutamicum. We have reconstituted in vitro a novel electron transfer network containing, next to Cg_ArsC1 or Cg_ArsC2, mycothiol, mycoredoxin, and mycothione reductase. As such, the mechanism for the reduction of arsenate by C. glutamicum could be unraveled.     

Effects of iron deficiency upon the antibody response to influenza virus in rats

The effects of severe and moderate iron deficiency upon the antibody response to influenza virus were investigated in rats. Three groups of weanling male Wistar rats were fed one of two iron-deficient diets (5 mg and 15 mg iron/kg diet) or a normal iron-containing diet (35 mg iron/kg diet). A group of individually pair-fed rats was introduced with the low iron-consuming rats. The effects of the diets upon various iron status parameters were followed during the 4th, 5th, 6th, and 7th week of diet. After 4 weeks of feeding different diets, an intraperitoneal injection of inactivated influenza virus A/New Jersey/76 was performed and a recall injection was done at 5 weeks. Primary and secondary antibody responses were assayed. Rats were sacrificed at 7 weeks of diet. After 4 weeks of feeding different diets, the rats fed the 5 mg iron/kg diet were severely anemic and rats fed 15 mg iron/kg diet were moderately iron-deficient, as shown by their iron status parameters. Growth was delayed in anemic and matched pair-fed rats. A primary antibody response was almost nonexistent in all groups. Secondary antibody titers were significantly weaker in anemic rats than in ad libitum controls, but were not different from those of pair-fed rats. This response was similar in moderately iron-deficient, ad libitum, and pair-fed rats. These results show that antibody synthesis in response to the influenza virus vaccine is preserved in moderate iron deficiency but is reduced in severe anemia. The reduction in energy consumption associated with severe iron deficiency in the rat could play a part in the altered humoral response.     

Aeropalynological and phenological study in two different Mediterranean olive areas: Cordoba (Spain) and Perugia (Italy)

ABSTRACT

An aerobiological and phenological investigation on the olive tree was carried out during three years in two areas: Cordoba (Spain) and Perugia (Italy). In these countries, this species is economically important and those areas were chosen because of the long series of aeropalynological data (1982–1998) available, obtained by means of identical volumetric pollen traps. The aim of this study was to use phenological observations to prove the real contribution to the pollen curves in different cultivated areas. Results show that in Cordoba province (302.152 ha) the pollen curve is characterised by different peaks because of the pollination of different cultivated crops. In some cases, these crops are located far from the pollen trap (50 km) but pollen is transported thanks to favourable winds during the flowering period. In Perugia (750 ha) the pollen curve is characterised by only one peak; it is very concentrated because of the proximity of the investigated crops. The objective of this research was to obtain information on this species in order to elaborate statistical models aimed at forecasting the potential fruit production based on the amount of pollen released into the atmosphere.     

NMR Model of PrgI–SipD Interaction and Its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI–SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein—the N-terminal α-helical hairpin—has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.     

Glutathione oxidation correlates with one-lung ventilation time and PO2/FiO2 ratio during pulmonary lobectomy

Objectives: During lung lobectomy, the operated lung completely collapses with simultaneous hypoxic pulmonary vasoconstriction, followed by expansion and reperfusion. Here, we investigated glutathione oxidation and lipoperoxidation in patients undergoing lung lobectomy, during one-lung ventilation (OLV) and after resuming two-lung ventilation (TLV), and examined the relationship with OLV duration.

Methods: We performed a single-centre, observational, prospective study in 32 patients undergoing lung lobectomy. Blood samples were collected at five time-points: T0, pre-operatively; T1, during OLV, 5 minutes before resuming TLV; and T2, T3, and T4, respectively, 5, 60, and 180 minutes after resuming TLV. Samples were tested for reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione redox potential, and malondialdehyde (MDA).

Results: GSSG and MDA blood levels increased at T1, and increased further at T2. OLV duration directly correlated with marker levels at T1 and T2. Blood levels of GSH and glutathione redox potential decreased at T1?T3. GSSG, oxidized glutathione/total glutathione ratio, and MDA levels were inversely correlated with arterial blood PO₂/FiO₂ at T1 and T2.

Discussion: During lung lobectomy and OLV, glutathione oxidation, and lipoperoxidation marker blood levels increase, with further increases after resuming TLV. Oxidative stress degree was directly correlated with OLV duration, and inversely correlated with arterial blood PO₂/FiO₂.     

Corynebacterium glutamicum survives arsenic stress with arsenate reductases coupled to two distinct redox mechanisms

Arsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k(cat)/K(M) value which is 10(3) times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface.     

The design, synthesis, and biological evaluation of PIM kinase inhibitors

A series of substituted benzofuropyrimidinones with pan-PIM activities and excellent selectivity against a panel of diverse kinases is described. Initial exploration identified aryl benzofuropyrimidinones that were potent, but had cell permeability limitation. Using X-ray crystal structures of the bound PIM-1 complexes with 3, 5m, and 6d, we were able to guide the SAR and identify the alkyl benzofuropyrimidinone (6l) with good PIM potencies, permeability, and oral exposure.