The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro

The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-beta binding (IBB) domain and a COOH-terminal m3G-cap--binding region that bears no structural similarity to known import adaptors like importin-alpha (impalpha). Here, we show that recombinant snurportin1 and importin-beta (impbeta) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impalpha-dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1-impbeta, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impbeta and a snurportin1 mutant containing an impalpha IBB domain (IBBimpalpha), nuclear U1 snRNP import was Ran dependent. Furthermore, beta-galactosidase (betaGal) containing a snurportin1 IBB domain, but not IBBimpalpha-betaGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impbeta with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.     

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.     

SoFAR: software for fully automatic evaluation of real-time PCR data

Quantitative real-time PCR has proven to be an extremely useful technique in life sciences for many applications. Although a lot of attention has been paid to the optimization of the assay conditions, the analysis of the data acquired is often done with software tools that do not make optimum use of the information provided by the data. Particularly, this is the case for high-throughput analysis, which requires a careful characterization and interpretation of the complete data by suitable software. Here we present a software solution for the robust, reliable, accurate, and fast evaluation of real-time PCR data, called SoFAR. The software automatically evaluates the data acquired with the LightCycler system. It applies new algorithms for an adaptive background correction of signal trends, the calculation of the effective signal noise, the automated identification of the exponential phases, the adaptive smoothing of the raw data, and the correction of melting curve data. Finally, it provides information regarding the validity of the results obtained. The SoFAR software minimizes the time required for evaluation and increases the accuracy and reliability of the results. The software is available upon request.     

Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803

The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.     

Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Catabolite degradation of fructose-1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates the existence of two degradation pathways

Metabolic adaptation of Saccharomyces cerevisiae cells from a nonfermentable carbon source to glucose induces selective, rapid breakdown of the gluconeogenetic key enzyme fructose-1,6-bisphosphatase (FBPase), a process called catabolite degradation. Herein, we identify eight novel GID genes required for proteasome-dependent catabolite degradation of FBPase. Four yeast proteins contain the CTLH domain of unknown function. All of them are Gid proteins. The site of catabolite degradation has been controversial until now. Two FBPase degradation pathways have been described, one dependent on the cytosolic ubiquitin-proteasome machinery, and the other dependent on vacuolar proteolysis. Interestingly, three of the novel Gid proteins involved in ubiquitin-proteasome-dependent degradation have also been reported by others to affect the vacuolar degradation pathway. As shown herein, additional genes suggested to be essential for vacuolar degradation are unnecessary for proteasome-dependent degradation. These data raise the question as to whether two FBPase degradation pathways exist that share components. Detailed characterization of Gid2p demonstrates that it is part of a soluble, cytosolic protein complex of at least 600 kDa. Gid2p is necessary for FBPase ubiquitination. Our studies have not revealed any involvement of vesicular intermediates in proteasome-dependent FBPase degradation. The influence of Ubp14p, a deubiquitinating enzyme, on proteasome-dependent catabolite degradation was further uncovered.     

Neurofeedback treatment for attention-deficit/hyperactivity disorder in children: a comparison with methylphenidate

Clinical trials have suggested that neurofeedback may be efficient in treating attention-deficit/hyperactivity disorder (ADHD). We compared the effects of a 3-month electroencephalographic feedback program providing reinforcement contingent on the production of cortical sensorimotor rhythm (12–15 Hz) and beta1 activity (15–18 Hz) with stimulant medication. Participants were N = 34 children aged 8–12 years, 22 of which were assigned to the neurofeedback group and 12 to the methylphenidate group according to their parents' preference. Both neurofeedback and methylphenidate were associated with improvements on all subscales of the Test of Variables of Attention, and on the speed and accuracy measures of the d2 Attention Endurance Test. Furthermore, behaviors related to the disorder were rated as significantly reduced in both groups by both teachers and parents on the IOWA-Conners Behavior Rating Scale. These findings suggest that neurofeedback was efficient in improving some of the behavioral concomitants of ADHD in children whose parents favored a nonpharmacological treatment.     

Desmoid tumors of the abdominal wall: A case report

Background  

Desmoid tumors are slow growing deep fibromatoses with aggressive infiltration of adjacent tissue but without any metastatic potential.     

Structure-activity relationships of novel anti-malarial agents: part 5. N-(4-acylamino-3-benzoylphenyl)-[5-(4-nitrophenyl)-2-furyl]acrylic acid amides

We have developed the [5-(4-nitrophenyl)-2-furyl]acrylic acid substituted benzophenone 4g as a novel lead for anti-malarial agents. Here, we demonstrated that the acyl residue at the 2-amino group of the benzophenone core structure has to be a phenylacetic acid substructure substituted in its para-position with methyl or other substituents of similar size. The trifluoromethyl substituted derivative displayed an IC(50) of 47 nM against the multi-drug resistant Plasmodium falciparum strain Dd2.