Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Making sense of lung-cancer gene-expression profiles

A report on the Critical Assessment of Microarray Data Analysis (CAMDA'03) meeting and competition, Durham, USA, 12-14 November 2003.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Molecular characterization of the <Emphasis Type="Italic">singed wings</Emphasis> locus of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

Hormones frequently guide animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone. singed wings (swi) is the only gene known in Drosophila melanogaster for which mutations specifically interrupt the transmission of the regulatory signal from early to late ecdysone inducible genes.     

Average protein density is a molecular-weight-dependent function

The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.     

Reliability and factorial validity of squat and countermovement jump tests

The primary aim of this study was to determine reliability and factorial validity of squat (SJ) and countermovement jump (CMJ) tests. The secondary aim was to compare 3 popular methods for the estimation of vertical jumping height. Physical education students (n = 93) performed 7 explosive power tests: 5 different vertical jumps (Sargent jump, Abalakow's jump with arm swing and without arm swing, SJ, and CMJ) and 2 horizontal jumps (standing long jump and standing triple jump). The greatest reliability among all jumping tests (Cronbach's alpha = 0.97 and 0.98) had SJ and CMJ. The reliability alpha coefficients for other jumps were also high and varied between 0.93 and 0.96. Within-subject variation (CV) in jumping tests ranged between 2.4 and 4.6%, the values being lowest in both horizontal jumps and CMJ. Factor analysis resulted in the extraction of only 1 significant principal component, which explained 66.43% of the variance of all 7 jumping tests. Since all jumping tests had high correlation coefficients with the principal component (r = 0.76-0.87), it was interpreted as the explosive power factor. The CMJ test showed the highest relationship with the explosive power factor (r = 0.87), that is, the greatest factorial validity. Other jumping tests had lower but relatively homogeneous correlation with the explosive power factor extracted. Based on the results of this study, it can be concluded that CMJ and SJ, measured by means of contact mat and digital timer, are the most reliable and valid field tests for the estimation of explosive power of the lower limbs in physically active men.     

The mechanism [correction of mehanism] of spatial [correction of spetial] orientation in conditions of G-stress

Six test subjects were subjected to lateral (+Gy) and longitudinal-lateral (+Gz/+Gy) accelerations in a centrifuge with a rotation radius of 6.55 m. During rotation, test subjects were instructed to indicate the position of subjective visual vertical. Results of this study demonstrated that during exposure to +Gy and +Gz/+Gy accelerations, the direction of the indicated subjective vertical approached the direction of the resultant acceleration vector when the lateral component increased. This observed effect decreases with an increase of the longitudinal component of the acceleration. It was suggested that exposure to (i.e. "pulling") high lateral acceleration (up to 2-3 Gy) in highly maneuverable aircraft can hinder spatial orientation of a pilot due to this persistent illusory spatial position as reported above. Our analysis showed that the process of spatial orientation under the conditions of G-load influence becomes more difficult and it is depending on the compromise between visual and vestibular-proprioceptive inputs. On account of this finding, it may be proposed that under conditions of G-load influence, pilots that rely primarily on visual perception may be exposed to higher risk of spatial disorientation.     

Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities

DNA polymerase (pol) λ is homologous to pol β and has intrinsic polymerase and terminal transferase activities. However, nothing is known about the amino acid residues involved in these activites. In order to precisely define the nucleotide-binding site of human pol λ, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol β, which are involved in nucleotide binding. Our analysis demonstrated that pol λ Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol λ, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.