Mossbauer spectroscopy of cells of cyanobacteria Synechocystis sp. PCC 6803 devoid of photosystem I and containing inactive phycobilisomes]

Mossbauer spectra of the psaAB mutant of Synechocystis sp. PPC 6803 devoid of photosystem I grown in a 57Fe-containing medium were measured. The spectrum is a broadened doublet whose size (about 20%) and parameters (isomeric shift delta = 0.3 mm/s and quadrupole splitting delta = 0.8 mm/s) suggest the presence of abundant nanoclusters of Fe3+ oxides in a superparamagnetic state tightly bound to the membrane. Treatment of cells with EDTA was accompanied by a substantial (tenfold) decrease in the amount of iron nonspecifically bound to the membrane and the appearance of Fe2+ localized, probably, inside cells and/or cell membranes. In addition, the spectrum of washed cells exhibited superfine magnetic splitting due to iron oxide clusters greater in size than nanoclusters present in the membrane prior to EDTA treatment.     

The role of visual afferentation in the development of early defense behavior of pied flycatcher nestlings

The development of defense behavior in normal and visually deprived pied flycatcher nestlings was studied in the wild. It was demonstrated that the young deprived of the visual afferentation did not develop the freezing posture. In the majority of cases, specific alarm call suppressed begging in visually deprived nestlings less effectively than in the control ones. Visually deprived nestlings could not discriminate between the alarm call and other rhythmically organized acoustic signals even though the latter effectively suppressed begging.     

Learning and memory: traditional and systemic approach

The same empiric event may appear as different facts for authors adhering to different theories. The present work was designed with analyze learning and memory from the viewpoint of systemic approach and to compare this view with the traditional one. Neuron's activity is considered not as a response to synaptic inflow that ensures the conduction of excitation but as means of changing the relation with environment, "action" that helps eliminate the discrepancy between cell's needs and its microenvironment. It is suggested that learning and memory consolidation is based not on a consistent increase in efficacy of synaptic transmission in neuronal chains but on systemogenesis--establishment of new systemic specializations of neurons not necessarily linked directly through synapses. The article discusses the role of systemogenetic processes taking place in normal as well as in pathological state: selection, reconsolidational modifications of previously formed memory store, genes activation, neurogenesis and apoptosis. The systemic understanding of the phenomenon of long-term potentiation is-substantiated. Finally, the scheme is suggested describing variants and stages of memory store formation.     

A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.     

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.     

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.     

Valid and reliable instruments for arm-hand assessment at ICF activity level in persons with hemiplegia: a systematic review

Background

Loss of arm-hand performance due to a hemiparesis as a result of stroke or cerebral palsy (CP), leads to large problems in daily life of these patients. Assessment of arm-hand performance is important in both clinical practice and research. To gain more insight in e.g. effectiveness of common therapies for different patient populations with similar clinical characteristics, consensus regarding the choice and use of outcome measures is paramount. To guide this choice, an overview of available instruments is necessary. The aim of this systematic review is to identify, evaluate and categorize instruments, reported to be valid and reliable, assessing arm-hand performance at the ICF activity level in patients with stroke or cerebral palsy.

Methods

A systematic literature search was performed to identify articles containing instruments assessing arm-hand skilled performance in patients with stroke or cerebral palsy. Instruments were identified and divided into the categories capacity, perceived performance and actual performance. A second search was performed to obtain information on their content and psychometrics.

Results

Regarding capacity, perceived performance and actual performance, 18, 9 and 3 instruments were included respectively. Only 3 of all included instruments were used and tested in both patient populations. The content of the instruments differed widely regarding the ICF levels measured, assessment of the amount of use versus the quality of use, the inclusion of unimanual and/or bimanual tasks and the inclusion of basic and/or extended tasks.

Conclusions

Although many instruments assess capacity and perceived performance, a dearth exists of instruments assessing actual performance. In addition, instruments appropriate for more than one patient population are sparse. For actual performance, new instruments have to be developed, with specific focus on the usability in different patient populations and the assessment of quality of use as well as amount of use. Also, consensus about the choice and use of instruments within and across populations is needed.