Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Long-term effects of graduated compression stockings on cardiorespiratory performance

The use of graduated compression stockings (GCS) in sport has been increasing in the last years due to their potential positive effects for athletes. However, there is little evidence to support whether these types of garments actually improve cardiorespiratory performance. The aim of this study was to examine the cardiorespiratory responses of GCS during running after three weeks of regular use. Twenty recreational runners performed three tests on different days: test 1) – a 5-min maximal effort run in order to determine the participants’ maximal aerobic speed; and tests 2) and 3) – a fatigue running test of 30 minutes at 80% of their maximal aerobic speed with either GCS or PLACEBO stockings at random. Cardiorespiratory parameters (minute ventilation, heart rate, relative oxygen consumption, relative carbon dioxide production, ventilatory equivalents for oxygen and carbon dioxide, and oxygen pulse) were measured. Before each test in the laboratory, the participants trained with the randomly assigned stockings (GCS or PLACEBO) for three weeks. No significant differences between GCS and PLACEBO were found in any of the cardiorespiratory parameters. In conclusion, the present study provides evidence that running with GCS for three weeks does not influence cardiorespiratory parameters in recreational runners.     

Refining the structure of the Halobacterium salinarum flagellar filament using the iterative helical real space reconstruction method: insights into polymorphism

The eubacterial flagellar filament is an external, self-assembling, helical polymer approximately 220 A in diameter constructed from a highly conserved monomer, flagellin, which polymerizes externally at the distal end. The archaeal filament is only approximately 100 A in diameter, assembles at the proximal end and is constructed from different, glycosylated flagellins. Although the phenomenology of swimming is similar to that of eubacteria, the symmetry of the archebacterial filament is entirely different. Here, we extend our previous study on the flagellar coiled filament structure of strain R1M1 of Halobacterium salinarum. We use strain M175 of H.salinarum, which forms poly-flagellar bundles at high yield which, under conditions of relatively low ionic-strength (0.8 M versus 5 M) and low pH ( approximately 2.5 versus approximately 6.8), form straight filaments. We demonstrated previously that a single-particle approach to helical reconstruction has many advantages over conventional Fourier-Bessel methods when dealing with variable helical symmetry and heterogeneity. We show here that when this method is applied to the ordered helical structure of the archebacterial uncoiled flagellar filament, significant extensions in resolution can be obtained readily when compared to applying traditional helical techniques. The filament population can be separated into classes of different morphologies, which may represent polymorphic states. Using cryo-negatively stained images, a resolution of approximately 10-15 A has been achieved. Single alpha-helices can be fit into the reconstruction, supporting the proposed similarity of the structure to that of type IV bacterial pili.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly(244)-->Pro substitution into the enzyme. Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.     

Mechanisms of homogeneous nucleation of polymers of sickle cell anemia hemoglobin in deoxy state

The primary pathogenic event of sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within the red blood cells, occurring when HbS is in deoxy state in the venous circulation. Polymerization is known to start with nucleation of individual polymer fibers, followed by growth and branching via secondary nucleation, yet the mechanisms of nucleation of the primary fibers have never been subjected to dedicated tests. We implement a technique for direct determination of rates and induction times of primary nucleation of HbS fibers, based on detection of emerging HbS polymers using optical differential interference contrast microscopy after laser photolysis of CO-HbS. We show that: (i). nucleation throughout these determinations occurs homogeneously and not on foreign substrates; (ii). individual nucleation events are independent of each other; (iii). the nucleation rates are of the order of 10(6)-10(8)cm(-3)s(-1); (iv). nucleation induction times agree with an a priori prediction based on Zeldovich's theory; (v). in the probed parameter space, the nucleus contains 11 or 12 molecules. The nucleation rate values are comparable to those leading to erythrocyte sickling in vivo and suggest that the mechanisms deduced from in vitro experiments might provide physiologically relevant insights. While the statistics and dynamics of nucleation suggest mechanisms akin to those for small-molecule and protein crystals, the nucleation rate values are nine to ten orders of magnitude higher than those known for protein crystals. These high values cannot be rationalized within the current understanding of the nucleation processes.     

Yeast prions, mammalian amyloidoses, and the problem of proteomic networks

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are detected as the cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural-functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.