Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C₃ and C₄ plant species, we analyzed kinetic parameters of purified recombinant C₃ (Arabidopsis thaliana) and C₄ (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The ¹²C/¹³C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the ¹³C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C₃ and C₄ enzymes.     

Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors

Segregation of homologs at the first meiotic division (MI) is facilitated by crossovers and by a physical constraint imposed on sister kinetochores that facilitates monopolar attachment to the MI spindle. Recombination failure or premature separation of homologs results in univalent chromosomes at MI, and univalents constrained to form monopolar attachments should be inherently unstable and trigger the spindle assembly checkpoint (SAC). Although univalents trigger cell-cycle arrest in the male, this is not the case in mammalian oocytes. Because the spindle assembly portion of the SAC appears to function normally, two hypotheses have been proposed to explain the lack of response to univalents: (1) reduced stringency of the oocyte SAC to aberrant chromosome behavior, and (2) the ability of univalents to satisfy the SAC by forming bipolar attachments. The present study of Mlh1 mutant mice demonstrates that metaphase alignment is not a prerequisite for anaphase onset and provides strong evidence that MI spindle stabilization and anaphase onset require stable bipolar attachment of a critical mass--but not all--of chromosomes. We postulate that subtle differences in SAC-mediated control make the human oocyte inherently error prone and contribute to the age-related increase in aneuploidy.     

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylethanolamine Bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and members of the homologous series of zwitterionic n-saturated diacyl phosphatidylethanolamines (PEs). Analogs of L24, in which the lysine residues were replaced by 2,3-diaminopropionic acid (acetyl-DAP2-Leu24-DAP2-amide (L24DAP)) or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (Ac-K2-W-L22-W-K2-amide (WL22W)), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the PE bilayers is altered by these peptides in a hydrophobic mismatch-independent manner, in contrast to the hydrophobic mismatch-dependent manner observed previously with zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) bilayers. Moreover, all three peptides reduce the phase transition temperature to a greater extent in PE bilayers than in PC and PG bilayers, indicating a greater disruption of PE gel-phase bilayer organization. Moreover, the lysine-anchored L24 reduces the phase transition temperature, enthalpy, and the cooperativity of PE bilayers to a much greater extent than DAP-anchored L24DAP, whereas replacement of the terminal leucines by tryptophan residues (Ac-K2-W-L22-W-K2-amide) only slightly attenuates the effects of this peptide on the chain-melting phase transition of the host PE bilayers. All three peptides form very stable alpha-helices in PE bilayers, but small conformational changes occur in response to mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. These results suggest that the lysine snorkeling plays a significant role in the peptide-PE interactions and that cation-pi-interactions between lysine and tryptophan residues may modulate these interactions. Altogether, these results suggest that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer but also by the electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the polar headgroups of PE bilayers.