The effects of creatine supplementation on high-intensity exercise performance in elite performers

The aim of this research was to determine whether creatine supplementation at a dose of 20 g · day⁻¹, given in 4 × 6-g doses (5 g creatine monohydrate and 1 g glucose) for 5 days, was effective in improving kayak ergometer performances of different durations. Sixteen male subjects with the following characteristics [mean (SEM)]: age 21 (1.2) years, height 170.2 (1.7) cm, weight 75.3 (2.3) kg, Σ8 skinfolds 59.3 (2.6) mm, and maximal oxygen consumption 67.1 ± (4.3) ml · kg · min⁻¹, undertook three maximal kayak ergometer tests of 90, 150 and 300 s duration on a wind-braked kayak ergometer (CON). Two groups were then randomly formed, with one group taking the supplement (SUP) while the other took a placebo (PLAC). No pre-test differences existed between the SUP and the PLAC groups in any of the variables measured. After supplementation each group then repeated the same kayak ergometer tests as performed previously and after a 4-week “washout period” the groups took either the PLAC or SUP for another 5 days and then completed the final tests. The SUP group completed significantly more work than either the CON or PLAC groups in all of the tests (90 s, P < 0.01; 150 s, P < 0.001; 300 s, P < 0.05). Body mass remained stable throughout the test period in both the CON and PLAC groups, but both were significantly less than the SUP body mass of 77.3 (1.0) kg (P < 0.01). The results of this work indicate that creatine supplementation can significantly increase the amount of work accomplished during kayak ergometer performance at durations ranging from 90 to 300 s. Accepted: 8 January 1998     

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM₂ were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM₄ was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02–0.1 m hexafluoroacetone/NH₃, pH 7.2 for highly acidic peptides; and (2) 6 m formic acid/0.13 m trimethylamine, pH 1.5, vs 4 m formic acid/0.09 m trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.     

Dramatic Potentiation of the Antiviral Activity of HIV Antibodies by Cholesterol Conjugation

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10–100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.     

Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis

A lot of studies have been carried out on Saccharomyces cerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyveromyces lactis as a good alternative model to analyse variants in the oxidative stress response, since the respiratory metabolism in this yeast is predominant under aerobic conditions and it shows other important differences with S. cerevisiae in catabolic repression and carbohydrate utilization. The knowledge of oxidative stress response in K. lactis is still a developing field. In this article, we summarize the state of the art derived from experimental approaches and we provide a global vision on the characteristics of the putative K. lactis components of the oxidative stress response pathway, inferred from their sequence homology with the S. cerevisiae counterparts. Since K. lactis is also a well-established alternative host for industrial production of native enzymes and heterologous proteins, relevant differences in the oxidative stress response pathway and their potential in biotechnological uses of this yeast are also reviewed.     

Reoxidation of cytosolic NADPH in Kluyveromyces lactis

Saccharomyces cerevisiae and Kluyveromyces lactis are considered to be the prototypes of two distinct metabolic models of facultatively-aerobic yeasts: Crabtree-positive/fermentative and Crabtree-negative/respiratory, respectively. Our group had previously proposed that one of the molecular keys supporting this difference lies in the mechanisms involved in the reoxidation of the NADPH produced as a consequence of the activity of the pentose phosphate pathway. It has been demonstrated that a significant part of this reoxidation is carried out in K. lactis by mitochondrial external alternative dehydrogenases which use NADPH, the enzymes of S. cerevisiae being NADH-specific. Moreover, the NADPH-dependent pathways of response to oxidative stress appear as a feasible alternative that might co-exist with direct mitochondrial reoxidation.     

ATP-binding cassette transporter G1 and lipid homeostasis

PURPOSE OF REVIEW: This review briefly discusses the ATP-binding cassette transporter G (ABCG) family members and emphasizes recent studies that identify ABCG1 as a key regulator of cellular lipid homeostasis. RECENT FINDINGS: The in-vivo importance of ABCG1 has recently been demonstrated with both loss-of-function and gain-of-function studies in mice. Administration of a diet high in both fat and cholesterol to ABCG1 mice results in massive cholesterol accumulation in both the liver and lungs. In contrast, lipid accumulation is greatly attenuated in transgenic mice that express both the murine and human ABCG1 genes. Despite the observed tissue lipid accumulation, plasma lipid levels and lipoprotein cholesterol distribution are not significantly different between wild-type, ABCG1, and hABCG1 transgenic mice. Other studies show that ABCG1 expression is induced following activation of the nuclear receptor LXR and that over expression of ABCG1 results in increased efflux of cellular cholesterol to HDL or phospholipid vesicles. SUMMARY: The ABCG1 transporter plays a key role in regulating cellular cholesterol and lipid homeostasis. Elucidation of the molecular mechanism by which ABCG1 controls sterol flux should provide critical information that may link ABCG1 to the reverse cholesterol transport pathway or diseases such as atherosclerosis.     

Bacterial Macroscopic Rope-like Fibers with Cytopathic and Adhesive Properties

We present a body of ultrastructural, biochemical, and genetic evidence that demonstrates the oligomerization of virulence-associated autotransporter proteins EspC or EspP produced by deadly human pathogens enterohemorrhagic and enteropathogenic Escherichia coli into novel macroscopic rope-like structures (>1 cm long). The rope-like structures showed high aggregation and insolubility, stability to anionic detergents and high temperature, and binding to Congo Red and thioflavin T dyes. These are properties also exhibited by human amyloidogenic proteins. These macroscopic ropes were not observed in cultures of nonpathogenic Escherichia coli or isogenic espP or espC deletion mutants of enterohemorrhagic or enteropathogenic Escherichia coli but were produced by an Escherichia coli K-12 strain carrying a plasmid expressing espP. Purified recombinant EspP monomers were able to self-assemble into macroscopic ropes upon incubation, suggesting that no other protein was required for assembly. The ropes bound to and showed cytopathic effects on cultured epithelial cells, served as a substratum for bacterial adherence and biofilm formation, and protected bacteria from antimicrobial compounds. We hypothesize that these ropes play a biologically significant role in the survival and pathogenic scheme of these organisms.     

A Mechanism of Release of Calreticulin from Cells During Apoptosis

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca²⁺ homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca²⁺-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.