Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.     

A solar-powered transmitting video camera for monitoring cliff-nesting raptors

ABSTRACT.   We designed a system of solar-powered video cameras that transmitted images via telemetry to a monitor. This system allowed us to study the breeding behavior of the Bearded Vulture ( Gypaetus barbatus ) in the Pyrenees (northeastern Spain). From 2000 to 2004, 12 nests in seven territories were equipped with video cameras. To avoid disturbing the birds, equipment was installed 3–8 weeks before egg-laying. The acceptance rate was 75%. No decline in productivity was observed for nests monitored with video cameras compared to control nests. The cameras enabled us to document egg-laying, hatching asynchrony, the nestlings' diet, and the parents' breeding behavior from distances of 2–3 km, although technical problems temporarily interrupted the transmission of images. Video cameras can be used successfully to study the Bearded Vultures, and probably other cliff-nesting raptors, without reducing productivity.     

Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein

Adipose differentiation-related protein (ADFP; also known as ADRP or adipophilin), is a lipid droplet (LD) protein found in most cells and tissues. ADFP expression is strongly induced in cells with increased lipid load. We have inactivated the Adfp gene in mice to better understand its role in lipid accumulation. The Adfp-deficient mice have unaltered adipose differentiation or lipolysis in vitro or in vivo. Importantly, they display a 60% reduction in hepatic triglyceride (TG) and are resistant to diet-induced fatty liver. To determine the mechanism for the reduced hepatic TG content, we measured hepatic lipogenesis, very-low-density lipoprotein (VLDL) secretion, and lipid uptake and utilization, all of which parameters were shown to be similar between mutant and wild-type mice. The finding of similar VLDL output in the presence of a reduction in total TG in the Adfp-deficient liver is explained by the retention of TG in the microsomes where VLDL is assembled. Given that lipid droplets are thought to form from the outer leaflet of the microsomal membrane, the reduction of TG in the cytosol with concomitant accumulation of TG in the microsome of Adfp-/- cells suggests that ADFP may facilitate the formation of new LDs. In the absence of ADFP, impairment of LD formation is associated with the accumulation of microsomal TG but a reduction in TG in other subcellular compartments.     

Insulin and oxidative stress modulate proliferation of rat ovarian theca-interstitial cells through diverse signal transduction pathways

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.     

Iodination of proteins by IPy2BF4, a new tool in protein chemistry

Iodination is a very useful method for protein characterization and labeling. However, derivatization chemistries used in most conventional iodination procedures may cause substantial alterations in protein structure and function. The IPy(2)BF(4) reagent [bis(pyridine)iodonium (I) tetrafluoroborate] has been shown to be an effective iodinating reagent for peptides. Herein we report the first application of IPy(2)BF(4) in protein iodination in an aqueous medium using three representative substrates: insulin, lysozyme, and the enzyme 1,3-1,4-beta-d-4-glucanohydrolase. Our results show that IPy(2)BF(4) has clear advantages over existing methods in that the reaction is quantitative, fast, and selective for the most accessible Tyr residues of a protein, and it preserves the functional integrity of the protein when moderate Tyr labeling levels are pursued.     

Cytochrome oxidase in health and disease

Yeast and bovine cytochrome c oxidases (COX) are composed of 12 and 13 different polypeptides, respectively. In both cases, the three subunits constituting the catalytic core are encoded by mitochondrial DNA. The other subunits are all products of nuclear genes that are translated on cytoplasmic ribosomes and imported through different transport routes into mitochondria. Biogenesis of the functional complex depends on the expression of all the structural and more than two dozen COX-specific genes. The latter impinge on all aspects of the biogenesis process. Here we review the current state of information about the functions of the COX-specific gene products and of their relationship to human COX deficiencies.     

Influences of age and maxillary anterior teeth status on patient's satisfaction with dental appearance and tooth colour

doi: 10.1111/j.1741‐2358.2011.00543.x Influences of age and maxillary anterior teeth status on patient’s satisfaction with dental appearance and tooth colour Objectives: To study the impact of age, gender, tooth colour and maxillary anterior teeth status on patient’s satisfaction with their dental appearance. Material and methods: A total of 259 Caucasian subjects participated in the study (119 men, mean age 56 years; 140 women, mean age 61 years) divided into three age groups (young <35 age; middle aged 35–54 age; old ≥55 age). Their maxillary anterior teeth status was classified into three groups: (1) natural teeth (NTG) group; (2) composite filling group (CFG) and (3) porcelain‐fused‐to‐metal fixed prosthodontic restoration group (FPDG). The participants judged appearance and tooth colour using a scale with three categories: completely dissatisfied, moderately dissatisfied and completely satisfied. Results: Almost half of the participants were completely satisfied with their dental appearance and tooth colour. Half of the ‘young’ and ‘middle‐aged’ participants with natural maxillary anterior teeth were completely satisfied and half of the ‘old’ participants were moderately satisfied with their dental appearance and tooth colour. The majority of participants with composite restorations (45–51%) were moderately satisfied with their dental appearance, one‐third of ‘young’ and ‘middle‐aged’ participants were moderately satisfied or dissatisfied with their tooth colour and more than 70% of older participants were dissatisfied with their tooth colour (p > 0.05). Conclusions: Satisfaction with the appearance of the maxillary anterior teeth differed both between individuals of different age and different dental status.     

Random models of Menzerath-Altmann law in genomes

Recently, a random breakage model has been proposed to explain the negative correlation between mean chromosome length and chromosome number that is found in many groups of species and is consistent with Menzerath-Altmann law, a statistical law that defines the dependency between the mean size of the whole and the number of parts in quantitative linguistics. Here, the central assumption of the model, namely that genome size is independent from chromosome number is reviewed. This assumption is shown to be unrealistic from the perspective of chromosome structure and the statistical analysis of real genomes. A general class of random models, including that random breakage model, is analyzed. For any model within this class, a power law with an exponent of -1 is predicted for the expectation of the mean chromosome size as a function of chromosome length, a functional dependency that is not supported by real genomes. The random breakage and variants keeping genome size and chromosome number independent raise no serious objection to the relevance of correlations consistent with Menzerath-Altmann law across taxonomic groups and the possibility of a connection between human language and genomes through that law.     

Simvastatin decreases invasiveness of human endometrial stromal cells

Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.