The incidence of ΔF508 CF mutation,and associated haplotypes,in a sample of English CF families

Summary Data are presented for ΔF508 screening and KM19/XV2c haplotype analysis of 195 cystic fibrosis (CF) chromosomes from the British Caucasian population. We report the frequency of ΔF508 in this group to be 80% and find pronounced disequilibrium between the deletion and the KM 2, XV 1 haplotype. Haplotype analysis of 71 normal chromosomes is also presented. We report one individual who had meconium ileus and who does not have the ΔF508 mutation on either chromosome.     

Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method

A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.     

A new photoprobe for studying biological activities of secreted phospholipases A2

Ammodytoxin (Atx) is a snake venom phospholipase A2 (sPLA2s) with presynaptic toxicity, anticoagulant activity and the ability to influence cell cycle progression. These multiple physiological activities make this molecule a promising tool for studying processes influenced by the highly homologous mammalian sPLA2s-for example cell proliferation and apoptosis. Secreted PLA2s can act on cells as enzymes or as ligands for cellular receptors. To further characterize the sPLA2-binding molecules in cells we have developed a new method based on AtxC and a biotin-containing cross-linking reagent sulfo-SBED which possesses both an amine-reactive and a photo-reactive site, together with a biotin moiety that enables specific detection and affinity-based concentration. The biological activity of the AtxC derivatized by sulfo-SBED was demonstrated by biotin-tagging of calmodulin and R25, both known AtxC targets, but not of other proteins. In addition, using the new protocol we specifically labelled 14-3-3 proteins, protein disulfide isomerase and two unknown proteins of 45 and 46kDa in the mitochondrial-synaptosomal fraction of porcine cerebral cortex, none of which could be tagged by the previously used methods. The new methodology, which can be used for any sPLA2, constitutes a novel approach to discovering and purifying sPLA2-binding proteins, to studying the topology of their respective complexes and to following sPLA2s in different biological systems.     

Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity

FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3′ flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity.     

Biomotor development in 1992 and 2002 samples of seven-year-old children

The aim of the study was to determine quantitative and qualitative differences in the morphological-motor status between elementary school first-graders of both sexes in 1992 and 2002. A standard set of 11 variables currently used in the Croatian school system to evaluate the morphological, motor and functional status of school children was employed at the beginning of academic years in a sample of 635 children (325 male and 310 female) in 1992 and a sample of 850 children (430 male and 420 female) in 2002. The mean age of study children was 7 years (+/- 2 months). Results of canonic discriminative analysis revealed the male children enrolled in elementary school first grade in 2002 to show better performance on the tests of aerobic endurance, static strength and explosive strength, and to have greater mass of muscle tissue and less adipose tissue, while achieving poorer results on the test of movement frequency than their 1992 counterparts. Female children tested in 2002 showed better results on the test of aerobic endurance and all tests of strength factors, with greater mass of muscle tissue and less adipose tissue, while yielding poorer results on the tests of flexibility, coordination and movement frequency as compared with their 1992 counterparts. Factor analysis in the morphological-motor system isolated three varimax factors each in children of both sexes tested in 1992 and 2002. First factor showed a pattern of a general morphological factor predominantly defined by body weight and volume in the children of both sexes from both study years. Second factor showed a pattern of a general motoricity factor predominantly defined by explosive strength, coordination and speed in children tested in 1992, whereas in their 2002 counterparts the general motoricity factor was predominantly defined by the factors of strength and endurance. Third factor was defined by flexibility in both 1992 and 2002 children. In female children tested in 1992, second factor mostly defined energy regulation with predominance of explosive and static strength, followed by coordination, whereas third factor was defined by movement frequency and aerobic endurance. In female children tested in 2002, second factor mostly defined energy regulation with predominance of explosive and repetitive strength, followed by aerobic endurance and coordination, whereas third factor was defined by movement frequency followed by muscle tone regulation.     

Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior

Hallucinogens, including mescaline, psilocybin, and lysergic acid diethylamide (LSD), profoundly affect perception, cognition, and mood. All known drugs of this class are 5-HT(2A) receptor (2AR) agonists, yet closely related 2AR agonists such as lisuride lack comparable psychoactive properties. Why only certain 2AR agonists are hallucinogens and which neural circuits mediate their effects are poorly understood. By genetically expressing 2AR only in cortex, we show that 2AR-regulated pathways on cortical neurons are sufficient to mediate the signaling pattern and behavioral response to hallucinogens. Hallucinogenic and nonhallucinogenic 2AR agonists both regulate signaling in the same 2AR-expressing cortical neurons. However, the signaling and behavioral responses to the hallucinogens are distinct. While lisuride and LSD both act at 2AR expressed by cortex neurons to regulate phospholipase C, LSD responses also involve pertussis toxin-sensitive heterotrimeric G(i/o) proteins and Src. These studies identify the long-elusive neural and signaling mechanisms responsible for the unique effects of hallucinogens.     

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l⁻¹ without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg⁻¹) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.     

Values of vanillylmandelic acid and homovanillic acid in the urine as potential prognostic biomarkers in ischaemic stroke patients

Background: Suitable biomarkers that have prognostic values are one of the key points of interest in ischaemic stroke. Increased sympathetic nervous system activity in ischaemic stroke causes multiple local and systemic effects that can be detrimental to the outcome. The mechanism of action is increased secretion and activity of catecholamines, whose end metabolic products are vanillylmandelic acid and homovanilic acid. Aim of our study was to determine whether these compounds can be used as potential prognostic biomarkers in ischaemic stroke, as a unique insight into the activity of the sympathetic nervous system.

Methods: Urine samples of 96 patients with ischaemic stroke and transitory ischaemic attacks were analysed. Values of vanillylmandelic and homovanillic acids in urine were tested using liquid chromatography on the first and third day post-stroke. Severity of stroke was determined using the NIHSS scale, while functional outcome was determined using the Modified Rankin Scale.

Results: Values of vanillylmandelic and homovanillic acids positively correlated with functional outcome of ischaemic stroke. Favorable outcomes correlated with decreased values, on contrary to increased values, which were associated with unfavourable outcomes.

Conclusion: Determining the values of these compounds in the urine is an easily available prognostic tool for the ischaemic stroke outcome, while also influencing potential therapeutic changes.     

Rheological investigation on dynamic and structure of separated phases in polymer mixture–ionic surfactant ternary system

Ternary systems composed of 0.30% sodium carboxymethyl cellulose (NaCMC), 0.70% hydroxypropylmethyl cellulose (HPMC), and sodium dodecylsulfate (SDS) of various concentrations (0.00–2.00%), were investigated by oscillatory and flow shear rheometry. The investigation on binary mixtures of HPMC/SDS (0.7% HPMC, 0.00–2.00% SDS) and HPMC/NaCMC (1.00% polymer mixtures with HPMC/NaCMC mass ratio ranging from 1/0 to 0/1) was also carried out. In the examined systems, various interactions between HPMC–NaCMC, HPMC–SDS and NaCMC–(HPMC–SDS) take place. The interactions may bring about phase separation in the ternary system into HPMC–SDS complex rich phase (coacervate), and NaCMC rich phase (supernatant). The possibility of employing the interactions in the ternary system to vary structural properties of the coacervate, in terms of viscoelasticity and shear rate influence, was investigated. Rheological properties of the ternary system, as well as of the separated coacervate and the supernatant were tested. Influence of shear rate on the interactions taking place in both the binary and the ternary mixtures was examined. It was found that the applied shear rate can influence the interactions. Molecular mechanisms determining the rheological properties of the coacervate were suggested. It was found that complex intermolecular interactions in the ternary system can be employed to attain control over the structural properties of the coacervate phase.