Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2011-31 March 2011

This article documents the addition of 111 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acipenser oxyrinchus desotoi, Anopheles nuneztovari sensu lato, Asellus aquaticus, Calopteryx splendens, Calopteryx virgo, Centaurea aspera, Centaurea seridis, Chilina dombeyana, Proctoeces cf. lintoni and Pyrenophora teres f. teres.     

Protective effect of prenatal water restriction on offspring cardiovascular homeostasis in response to hemorrhage

We determined the cardiovascular and AVP responses of prenatally dehydrated (PreDehy) neonates to intravascular hemorrhage. Ewes with singleton fetuses were subjected to water restriction from 110 days of gestation to full term to achieve hypernatremia of 8-10 meq/l. Water and food were provided ad libitum to control ewes. After delivery, water and food were provided ad libitum to ewes from both groups, and newborns were allowed to nurse ad libitum. At 15 +/- 2 days of age, PreDehy and control lambs were prepared with bladder and femoral catheters and studied at 25 +/- 2 days of age. After a 2-h basal period, lambs were hemorrhaged to 30% of blood volume over 1 h (0.5% of blood volume/min) and monitored 1 h after hemorrhage. Neonatal arterial blood pressure was measured, and blood samples were collected. Basal plasma sodium levels, plasma osmolality, hematocrit, and mean arterial pressure were increased in PreDehy lambs compared with controls. Both groups had similar basal AVP levels and heart rate. In response to hemorrhage, all parameters remained significantly elevated in PreDehy lambs. Blood pressure decreased less in PreDehy lambs than in controls. The hemorrhage-AVP threshold (percent blood volume withdrawal at which plasma AVP values significantly increased) was markedly elevated (20 vs. 15%) and peak hemorrhage-induced AVP plasma levels were lower (5.6 +/- 1.5 vs. 10.1 +/- 1.5 pg/ml, P < 0.01) in PreDehy lambs than in controls. Thus offspring of dehydrated ewes demonstrate enhanced AVP secretory responses to hypotension. Despite potential long-term adverse effects of systemic hypertension, these results suggest a protective effect of prenatal water restriction on offspring cardiovascular homeostasis during blood volume reduction.     

Microbiological control in stem cell banks: approaches to standardisation

The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a “feeder layer” for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.     

Relationship between expression of the PM H<Superscript>+</Superscript>-ATPase,growth and ion partitioning in the leaves of salt-treated <Emphasis Type="Italic">Medicago</Emphasis> species

The role of the plasma membrane (PM) H⁺-ATPase (E.C. 3.6.1.3) in the plants response to salt stress was studied in the perennial leguminosae forage Medicago arborea L. and its close relative Medicago citrina (Font-Quer) Greuter, a species exposed to saline conditions in its original habitat. Plants were solution cultured for 8 days in 1 or 100 mM NaCl. Leaf growth and CO₂ assimilation were more inhibited by salt in M. arborea than in M. citrina. Both species were able to osmoregulate, and salt-treated plants maintained turgor potentials, with no differences between species. Contrasting ion distribution patterns showed that M. citrina was able to exclude Na⁺ from the leaves more selectively, while M. arborea had a greater buildup of leaf blade Na⁺. Isolation of purified PM and quantification of H⁺-ATPase protein by Western blot analysis against the 46E5B11D5 or AHA3 antibodies showed an increase in response to salt stress in the expanding (92%) and expanded leaves (87%) of M. citrina, while no differences were found in the corresponding leaves of M. arborea. The assay of H⁺-ATPase specific activity of the two leaf types in salinized M. citrina confirmed this increase, as activities increased with 55% and 104% for the expanded and expanding leaves, respectively, while no significant differences were found for either leaf type of salinized M. arborea. A possible role of the increased expression of the PM H⁺-ATPase for leaf expansion and ion exclusion in salt-stressed plants is discussed.     

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConADelta9) was expressed in BY-2 cells. In contrast to proConA, proConADelta9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinant.     

Carbon coated magnetic nanoparticles for local drug delivery using magnetic implants

Bioferrofluids obtained from carbon coated iron nanoparticles are promising candidates for magnetic drug delivery. The carbon cages render the particles biocompatible, and provide a good support for drug adsorption. We propose a method in which gold plated permanent magnets are implanted directly in the affected organ, close to the tumour, by endoscopic techniques. The bioferrofluid charged with the chemotherapeutic agent is injected and the particles attracted to the magnet, then desorption of the drug takes place at the tumoral region. This method seems to be more promising, costless and effective than that based on the application of external magnetic fields. Preliminary results of drug adsorption and a preclinical experimental animal model are described.     

Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA

In Escherichia coli the codons CGG, AGG, UGG or GGG (NGG codons) but not GGN or GNG (where N is non-G) are associated with low expression of a reporter gene, if located at positions +2 to +5. Induction of a lacZ reporter gene with any one of the NGG codons at position +2 to +5 does not influence growth of a normal strain, but growth of a strain with a defective peptidyl-tRNA hydrolase (Pth) enzyme is inhibited. The same codons, if placed at position +7, did not give this effect. Other codons, such as CGU and AGA, at location +2 to +5, did not give any growth inhibition of either the wild-type or the mutant strain. The inhibitory effect on the pth mutant strain by NGG codons at location +5 was suppressed by overexpression of the Pth enzyme from a plasmid. However, the overexpression of cognate tRNAs for AGG or GGG did not rescue from the growth inhibition associated with these codons early in the induced model gene. The data suggest that the NGG codons trigger peptidyl-tRNA drop-off if located at early coding positions in mRNA, thereby strongly reducing gene expression. This does not happen if these codons are located further down in the mRNA at position +7, or later.     

Protein interactions within the TcZFP zinc finger family members of Trypanosoma cruzi: implications for their functions

The small zinc finger proteins tbZFP1 and tbZFP2 have been implicated in the control of Trypanosoma brucei differentiation to the procyclic form. Here, we report that the complete ZFP family in Trypanosoma cruzi is composed by four members, ZFP1A and B, and ZFP2A and B. ZFP1B is a paralog specific gene restricted to T. cruzi, while the ZFP2A and B paralogs diverged prior to the trypanosomatid lineage separation. Moreover, we demonstrate that TcZFP1 and TcZFP2 members interact with each other and that this interaction is mediated by a WW domain in TcZFP2. Also, TcZFP2B strongly homodimerizes by a glycine rich region absent in TcZFP2A. We propose a model to discuss the relevance of these protein-protein interactions in terms of the functions of these proteins.     

Ryanodine receptor channelopathies

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.