The impact of dnaKJ overexpression on recombinant protein solubility results from antagonistic effects on the control of protein quality

We have produced increasing levels of DnaK and its co-chaperone DnaJ along with the model VP1LAC misfolding-prone protein, to explore the role of DnaK on the management of Escherichia coli inclusion bodies. While relative solubility of VP1LAC is progressively enhanced, the heat-shock response is down-regulated as revealed by decreasing levels of GroEL. This is accompanied by an increasing yield of VP1LAC and a non-regular evolution of its insoluble fraction, at moderate levels of DnaK resulting in more abundant inclusion bodies. Also, the impact of chaperone co-expression is much more pronounced in wild type cells than in a DnaK- mutant, probably due to the different background of heat shock proteins in these cells. The involvement of DnaK in the supervision of misfolding proteins is then pictured as a dynamic balance between its immediate holding and folding activities, and the side-effect downregulation of the heat shock response though the limitation of other chaperone and proteases activities.     

Disruption of the serine/threonine kinase 9 gene causes severe X-linked infantile spasms and mental retardation

X-linked West syndrome, also called "X-linked infantile spasms" (ISSX), is characterized by early-onset generalized seizures, hypsarrhythmia, and mental retardation. Recently, we have shown that the majority of the X-linked families with infantile spasms carry mutations in the aristaless-related homeobox gene (ARX), which maps to the Xp21.3-p22.1 interval, and that the clinical picture in these patients can vary from mild mental retardation to severe ISSX with additional neurological abnormalities. Here, we report a study of two severely affected female patients with apparently de novo balanced X;autosome translocations, both disrupting the serine-threonine kinase 9 (STK9) gene, which maps distal to ARX in the Xp22.3 region. We show that STK9 is subject to X-inactivation in normal female somatic cells and is functionally absent in the two patients, because of preferential inactivation of the normal X. Disruption of the same gene in two unrelated patients who have identical phenotypes (consisting of early-onset severe infantile spasms, profound global developmental arrest, hypsarrhythmia, and severe mental retardation) strongly suggests that lack of functional STK9 protein causes severe ISSX and that STK9 is a second X-chromosomal locus for this disorder.     

The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates--nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I(50) (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.     

Evidence that high telomerase activity may induce a senescent-like growth arrest in human fibroblasts

Expression of the catalytic subunit of human telomerase (hTERT), in normal human fibroblasts allows them to escape replicative senescence. However, we have observed that populations of hTERT-immortalized human fibroblasts contain 3-20% cells with a senescent morphology. To determine what causes the appearance of these senescent-like cells, we used flow cytometry to select them from the population and analyzed them for various senescence markers, telomere length, and telomerase activity. This subpopulation of cells had elevated levels of p21 and hypophosphorylated Rb, but telomere length was similar to that of the immortal cells in the culture that was sorted. Surprisingly, telomerase activity in the senescent-like cells was significantly elevated compared with immortal cells from the same population, suggesting that high telomerase activity may induce the senescent phenotype. Furthermore, transfection of normal fibroblasts with a hTERT-expressing plasmid that confers high telomerase activity led to the induction of p21, a higher percentage of SA-beta-galactosidase-positive cells, and a greater number of cells entering growth arrest compared with controls. These results suggest that excessive telomerase activity may act as a hyperproliferative signal in cells and induce a senescent phenotype in a manner similar to that seen following overexpression of oncogenic Ras, Raf, and E2F1. Thus, there must be a critical threshold of telomerase activity that permits cell proliferation.     

Towards proteome database of Francisella tularensis

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.     

Simultaneous liquid chromatography-tandem mass spectrometric determination of albendazole sulfoxide and albendazole sulfone in plasma

This paper describes a simple, fast, sensitive and reliable method for the simultaneous determination of albendazole sulfoxide (ASOX) and albendazole sulfone (ASON), the two most important metabolites of the drug albendazole (ABZ), in plasma samples using liquid chromatography and tandem mass spectrometry. After liquid-liquid extraction with dichloromethane, the two albendazole metabolites and the internal standard phenacetin were resolved in a CN column using the mobile phase methanol-water (4:6, v/v) acidified with 1% acetic acid. Detection by electrospray mass spectrometry was carried out in the positive ion mode. The method was linear up to 2500 and 250 ng/ml for ASOX and ASON, respectively, with mean recoveries of more than 85%. The precision and accuracy data, based on within- and between-day variations over 5 days, were lower than 15%. The quantitation limits of 0.5 and 5.0 ng/ml for ASON and ASOX are low enough for the method to be suitable for pharmacokinetic studies. Pharmacokinetic data obtained with the proposed method following oral administration of ABZ to a patient with neurocysticercosis are also reported.     

Molecular evolution of rDNA external transcribed spacer and phylogeny of sect. Petota (genus Solanum)

The 5(') external transcribed spacer (ETS) region of ribosomal DNA of 30 species of Solanum sect. Petota and the European Solanum dulcamara were compared. Two structural elements can be distinguished in the ETS: (i). a variable region (VR), demonstrating significant structural rearrangements and (ii). a conservative region (CR), evolving mainly by base substitutions. In VR, a conservative element (CE) with similarity to the ETS of distantly related Nicotiana is present. The ancestral organization of ETS (variant A) was found for non-tuber-bearing species of ser. Etuberosa, tuber-bearing wild potatoes of Central American ser. Bulbocastana, Pinnatisecta, and Polyadenia and S. dulcamara. Duplication of CE took place in the ETS of species from ser. Commersoniana and Circaeifolia (variant B). South American diploids and Mexican polyploids from superser. Rotata also possess two CE, and additionally two duplications around CE1 are present in VR (variant C). Three major lineages could be distinguished: non-tuber-bearing species of ser. Etuberosa, tuber-bearing Central American diploids and all South American species radiated from a common ancestor at early stages of evolution, indicating a South American origin of the tuber-bearing species. Later, Central and South American diploids evolved further as independent lineages. South American species form a monophyletic group composed of series with both stellata and rotata flower morphology. Solanum commersonii represents a sister taxon for all rotata species, whereas ser. Circaeifolia diverged earlier. Two main groups, C1 and C2, may be distinguished for species possessing ETS variant C. C1 contains ser. Megistacroloba, Conicibaccata, Maglia, and Acaulia, whereas all diploids of ser. Tuberosa are combined into C2. A closer relationship of Solanum chacoense (ser. Yungasensa) to the C2 group was found. The origin of polyploid species Solanum maglia, Solanum acaule, Solanum tuberosum, Solanum iopetalum, and Solanum demissum is discussed.     

Mitochondrial K(ATP) channels and sarcoplasmic reticulum influence cardiac force development under anoxia in the Amazonian armored catfish Liposarcus pardalis

The contribution of alterations in mitochondrial K(ATP) channel activity and the sarcoplasmic reticulum (SR) to anaerobic cardiac function in the anoxia tolerant armored catfish Liposarcus pardalis were assessed. K(ATP) channels contribute to hypoxic cardioprotection in mammals, but little is known of their action in more hypoxia tolerant animals. Anoxia resulted in a decrease in force in isometrically contracting ventricle strips to approximately 40% of the pre-anoxic level. This was maintained for at least 2 h. Upon reoxygenation, hearts recovered to the same level as control preparations. Treatment with 5-hydroxydecanoic acid (5HD), a specific mitochondrial K(ATP) blocker significantly increased force in preparations during anoxia and caused hypercontracture at reoxygenation. Ryanodine, a specific inhibitor of SR function, significantly increased force loss in ventricle preparations under anoxia. Results show that mitochondrial K(ATP) channel activity and SR function are important in anaerobic and post-anaerobic contractility in armored catfish heart.