Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

 The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed. Received: 25 November 1996 / Accepted: 2 June 1997     

Cartographic study: Breakpoints in 1574 families carrying human reciprocal translocations

Reciprocal translocations (rcp) are among the most common constitutional chromosomal aberrations in man. Using a European database of 1574 families carrying autosomal rcp, a cartographic study was done on the breakpoints involved. The breakpoints are non-randomly distributed along the different chromosomes, indicating “hot spots”. Breakpoints of rcp that result in descendants that are unbalanced chromosomally at birth are more frequent in a distal position on chromosomal arms, and 65% of them are localised in R-bands. Among the R-bands, bands rich in GC islands and poor in Alu repetitive sequences are more frequently the site of breakpoints, as well as bands that include a fragile site. This result suggests that the variation in degree of methylation in GC islands could be involved in chromosomal breakage and hence in chromosomal rearrangements. Received: 10 April 1995 / Revised: 1 July 1995     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca(2+) entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca(2+) entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca(2+) stores via a luminal N-terminal Ca(2+)-binding EF-hand domain. Dissociation of Ca(2+) from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca(2+) channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca(2+)-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca(2+) entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca(2+) entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca(2+)-selective (ARC) channels-a widely expressed mode of agonist-activated Ca(2+) entry whose activation is completely independent of Ca(2+) store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca(2+)-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca(2+) entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca(2+)entry remains an intriguing, yet currently unresolved, question.     

Effect of two different long-sprint training regimens on sprint performance and associated metabolic responses

The purpose of this study was to analyze 2 different long-sprint training programs (TPs) of equal total work load, completed either with short recovery (SR) or long recovery (LR) between sets and to compare the effects of 6 long-sprint training sessions (TSs) conducted over a 2-week period on a 300-m performance. Fourteen trained subjects performed 3 pretraining maximal sprints (50-, 100-, and 300-m), were paired according to their 300-m performance, and randomly allocated to an LR or SR group, which performed 6 TSs consisting of sets of 150, 200, or 250 m. The recovery in the LR group was double that of the SR group. During the third TS and the 300-m pretest and posttest, blood pH, bicarbonate concentration ([HCO??]), excess-base (EB), and lactate concentration were recorded. Compared with a similar TS performed with SR, the LR training tends to induce a greater alteration of the acid-base balance: pH: 7.09 ± 0.08 (LR) and 7.14 ± 0.05 (SR) (p = 0.10), [HCO??]: 7.8 ± 1.9 (LR) and 9.6 ± 2.7 (SR) (p = 0.04), and EB: -21.1 ± 3.8 (LR) and -17.7 ± 2.8 (SR) (p = 0.11). A significant improvement in the 300-m performance between pre-TP and post-TP (42.45 ± 2.64 vs. 41.52 ± 2.45, p = 0.01) and significant decreases in pH (p < 0.01), EB (p < 0.001) and increase in [La] (p < 0.001) have been observed post-TP compared with those pre-TP. Although sprint training with longer recovery induces higher metabolic disturbances, both sprint training regimens allow a similar 300-m performance improvement with no concomitant significant progress in the 50- and 100-m performance.     

8p22 MTUS1 Gene Product ATIP3 Is a Novel Anti-Mitotic Protein Underexpressed in Invasive Breast Carcinoma of Poor Prognosis

Background

Breast cancer is a heterogeneous disease that is not totally eradicated by current therapies. The classification of breast tumors into distinct molecular subtypes by gene profiling and immunodetection of surrogate markers has proven useful for tumor prognosis and prediction of effective targeted treatments. The challenge now is to identify molecular biomarkers that may be of functional relevance for personalized therapy of breast tumors with poor outcome that do not respond to available treatments. The Mitochondrial Tumor Suppressor (MTUS1) gene is an interesting candidate whose expression is reduced in colon, pancreas, ovary and oral cancers. The present study investigates the expression and functional effects of MTUS1 gene products in breast cancer.

Methods and Findings

By means of gene array analysis, real-time RT-PCR and immunohistochemistry, we show here that MTUS1/ATIP3 is significantly down-regulated in a series of 151 infiltrating breast cancer carcinomas as compared to normal breast tissue. Low levels of ATIP3 correlate with high grade of the tumor and the occurrence of distant metastasis. ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy. Functional studies indicate that silencing ATIP3 expression by siRNA increases breast cancer cell proliferation. Conversely, restoring endogenous levels of ATIP3 expression leads to reduced cancer cell proliferation, clonogenicity, anchorage-independent growth, and reduces the incidence and size of xenografts grown in vivo. We provide evidence that ATIP3 associates with the microtubule cytoskeleton and localizes at the centrosomes, mitotic spindle and intercellular bridge during cell division. Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis.

Conclusions

Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome. ATIP3 re-expression limits tumor cell proliferation in vitro and in vivo, suggesting that this protein may represent a novel useful biomarker and an interesting candidate for future targeted therapies of aggressive breast cancer.