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The diaphragm is a unique skeletal muscle; it is active throughout the lifetime and, therefore, differs from locomotor muscles in the properties of muscle fibers and the mechanisms of blood supply control. The aim of this review was to survey the structural characteristics of diaphragm muscle tissue, which provide its integral contractile properties; to compare the activity of vascular tone control mechanisms in the diaphragm and locomotor muscles; and to explore their relationship with the regulation of contractile function. The diaphragm differs from the majority of skeletal muscles by a high content of both slow fatigue-resistant muscle fibers of type I and fast fibers of type IIb, which provides endurance and high force-velocity characteristics of the diaphragm. The muscle fibers in the diaphragm are smaller, and the density of capillarization is much higher than in locomotor muscles. Arteries and arterioles that regulate blood supply to the diaphragm capillary bed combine the properties of arteries from muscles composed mainly of oxidative or mainly of glycolytic fibers. Such variety provides blood flow in the diaphragm adequate to its functional load with various patterns of activity. The mechanisms of vasoregulation in the diaphragm can qualitatively differ in the proximal and distal parts of the vascular bed. The functional properties of the proximal arteries can in part be explained by their proximity to the aorta and their small length. The contractile characteristics and blood supply of the diaphragm in various conditions should be considered when conducting respiratory muscle training in sports and rehabilitation medicine.

Lysenko, EA, Popov, DV, Vepkhvadze, TF, Sharova, AP, and Vinogradova, OL. Moderate-intensity strength exercise to exhaustion results in more pronounced signaling changes in skeletal muscles of strength-trained compared with untrained individuals. J Strength Cond Res 34(4): 1103-1112, 2020-The aim of our investigation was to compare the response pattern of signaling proteins and genes regulating protein synthesis and degradation in skeletal muscle after strength exercise sessions performed to volitional fatigue in strength-trained and untrained males. Eight healthy recreationally active males and 8 power-lifting athletes performed 4 sets of unilateral leg presses to exhaustion (65% 1 repetition maximum). Biopsy samples of m. vastus lateralis were obtained before, 1 and 5 hours after cessation of exercise. Phosphorylation of p70S6k, 4EBP1, and ACC increased, whereas phosphorylation of eEF2 and FOXO1 decreased only in the trained group after exercise. Expression of DDIT4, MURF1, and FOXO1 mRNAs increased and expression of MSTN mRNA decreased also only in the trained group after exercise. In conclusion, moderate-intensity strength exercise performed to volitional fatigue changed the phosphorylation status of mTORC1 downstream signaling molecules and markers of ubiquitin-proteasome system activation in trained individuals, suggesting activation of protein synthesis and degradation. In contrast to the trained group, signaling responses in the untrained group were considerably less pronounced. It can be assumed that the slowdown in muscle mass gain as the athletes increase in qualification cannot be associated with a decrease in the sensitivity of systems regulating protein metabolism, but possibly with inadequate intake or assimilation of nutrients necessary for anabolism. Perhaps, the intake of highly digestible protein or protein-carbohydrate dietary supplements could contribute to the increase in muscle mass in strength athletes.

The goal of this study was to describe and validate a muscle specific method to evaluate the anaerobic threshold in a working muscle based on the simultaneous measurement of EMG activity and the deoxyhemoglobin content (ATHHb-EMG). The study involved males with different fitness levels. During the cycling (n = 40) and ski double poling (n = 9) incremental ramp tests, blood lactate concentration, and muscle deoxyhemoglobin content and EMG activity were measured. Some participants were involved in the cycling test-retest study (n = 11). In cycling and double poling tests, close and significant correlations (r = 0.89 – 0.92, P < 0.002) were found between lactate threshold (a marker of the anaerobic threshold at the organism level) and the ATHHb-EMG (a marker of the AT at the working muscle level). The coefficient of variation of the ATHHb-EMG in the cycling test-retest was low (~3%). The muscle specific ATHHb-EMG demonstrates low variability and is appropriate to detect the fitness level and training-induced increase in aerobic performance in a working muscle. The emergence on the market of miniature EMG amplifiers and near-infrared spectrometers opens wide possibilities for using our method in laboratory studies and in field tests.

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.

The design and fabrication of lattice-strained platinum catalysts achieved by removing a soluble core from a platinum shell synthesized via atomic layer deposition, is reported. The remarkable catalytic performance for the oxygen reduction reaction (ORR), measured in both half-cell and full-cell configurations, is attributed to the observed lattice strain. By further optimizing the nanoparticle geometry and ionomer/carbon interactions, mass activity close to 0.8 A mgPt-1 @0.9 V iR-free is achievable in the membrane electrode assembly. Nevertheless, active catalysts with high ORR activity do not necessarily lead to high performance in the high-current-density (HCD) region. More attention shall be directed toward HCD performance for enabling high-power-density hydrogen fuel cells.

The well-defined roles and specific protein-protein interactions of many integral membrane proteins (IMPs), such as those functioning as receptors for extracellular matrix proteins and soluble growth factors, easily align with considering IMP structure as a classical "lock-and-key" concept. Nevertheless, continued advances in understanding protein conformation, such as those which established the widespread existence of intrinsically disordered proteins (IDPs) and especially intrinsically disordered regions (IDRs) in otherwise three-dimensionally organized proteins, call for ongoing reevaluation of transmembrane proteins. Here, we present basic traits of IDPs and IDRs, and, for some select single-span IMPs, consider the potential functional advantages intrinsic disorder might provide and the possible conformational impact of disease-associated mutations. For transmembrane proteins in general, we highlight several investigational approaches, such as biophysical and computational methods, stressing the importance of integrating them to produce a more-complete mechanistic model of disorder-containing IMPs. These procedures, when synergized with in-cell assessments, will likely be key in translating in silico and in vitro results to improved understanding of IMP conformational flexibility in normal cell physiology as well as disease, and will help to extend their potential as therapeutic targets.

Objective Insulin-like growth factor I (IGF1) is an important regulator of collagen and extracellular matrix protein expression. We aimed to evaluate the effect of amino acids (AAs) on expression of IGF1 and IGF1-dependent genes in human myotubes and skeletal muscle and supposed that AAs administration increases IGF1 levels in blood and expression of IGF1 and IGF1-dependent genes in trained skeletal muscle, thereby reducing training-induced muscle damage. Design Human myotubes were incubated with Arg and Leu for 24 h. Then, the effects of long-term branched chain AAs administration (10 weeks, 0.1 g/kg body mass/day) to volunteers (six subjects per AAs and placebo groups) performing large training volumes regularly (cross country skiers, training twice a day) were examined. Results Incubating the myotubes with AAs increases expression of IGF1 mRNA isoforms and IGF1 secretion by 2–3 times. In athletes, long-term AAs administration increased basal blood levels of IGF1 (~50%) and expression of IGF1Ea mRNA slightly in skeletal muscle. There is no marked increase in expression of COL1A1, COL3A1, COL5A1, and LOX genes in skeletal muscle after AAs administration. However, expression of these genes in the combined group (placebo + AAs; n = 12) significantly correlated with the expression of IGF1Ea mRNA in muscle and did not correlate with IGF1 levels in the blood. Conclusions AAs administration increases IGF1 expression in vitro and in vivo. To obtain more pronounced changes in expression of IGF1 and IGF1-dependent genes in skeletal muscle, it may be necessary to increase the dose and/or duration of AAs administration.

Improved activity and stability Pt-based catalysts for the oxygen reduction reaction (ORR) are needed to perpetuate the deployment of polymer electrolyte fuel cells (PEFCs) in the transportation sector. Here, we use atomic layer deposition of TiO2 and Pt coupled with thermal reductive annealing to prepare Pt3Ti electrocatalysts. The atomic level synthetic control resulted in Pt3Ti nanoparticles with high ORR performance, including a mass activity of 1.84 A/mgPt and excellent electrochemical stability. The Pt3Ti nanoparticles show excellent specific activity — 5.3-fold higher than commercial Pt/C and 3-fold higher than polycrystalline Pt, exceeding the performance of any PtTi catalysts reported to date. Combined experimental and computational efforts indicate that Pt enrichment on the Pt3Ti enhances the activity, and the intrinsic stability of the Pt3Ti phase provides durability. This knowledge, along with the facile fabrication of alloys by atomic layer deposition, can be leveraged to designed improved performance catalysts.

Herein we present the effect of artificially imposed topological constraint on calmodulin (CaM) backbone dynamics and its molecular recognition behavior. While backbone dynamics of CaM remain largely unperturbed, the thermodynamic profile of CaM binding to the smooth-muscle myosin light-chain kinase (smMLCK) peptide is modulated significantly.

The goal of treatment of relapsed/refractory multiple myeloma (RRMM) is to control the disease, prolong survival, reduce disease-related symptoms, and improve quality of life (QoL). Comprehensive evaluation of new treatment regimens in RRMM pts is worthwhile. We aimed to evaluate QoL, treatment satisfaction, response to treatment and safety during Ixazomib-Lenalidomide-Dexamethasone (IRd) treatment as ≥ 2nd line in RRMM pts in a real world setting. Adult pts with RRMM who have been assigned IRd as ≥2nd line treatment were enrolled in multicenter observational prospective study. Treatment response was evaluated by IMWG 2011, adverse events (AEs) - by CTCAE v.4.0. Pts filled out RAND SF-36 and ESAS-R at baseline and at 1 and 3 mos, and thereafter every 3 mos till 18 mos after IRd treatment start; Patient Treatment Satisfaction Cheklist (PTSC) - at each time-point after IRd treatment start. For analysis of meaningful QoL changes during IRd treatment the proportion of pts with baseline significant QoL impairment who experienced meaningful QoL improvement during IRd treatment was evaluated as well as the number of pts without baseline QoL impairment who maintained it during IRd treatment. For statistical analysis GLM paired test and GEE were employed with adjustment to age, gender and baseline QoL. In total, 40 pts with RRMM were enrolled into the pilot study: median age - 64 years (range, 33-80), 29% males, Durie-Salmon stage at study entry: I/II/III - 3/41/56%, ECOG status 0/1 - 68.7%, 2/3 - 31.6%. Median time since initial MM diagnosis - 51.4 mos (range, 2.9-100.7), disease status at study entry: relapsed - 26%, refractory - 21%, relapsed and refractory - 53%. Median number of lines of prior therapy is 3 (range, 1-3). At the time of analysis the median number of IRd cycles administered is 5, median follow-up - 4.6 (0.4-13.7) mos. Treatment response was not evaluated in 11 pts: 1 - death (at 3 months), 1- refusal, 9 - too early for evaluation. Out from 29 pts one patient achieved complete response (CR), seven pts achieved partial response (PR) and one patient - very good partial response (VGPR), 12 - minor response (MR). Thus, a clinical benefit rate was 70%. Also, seven pts achieved stable disease (23%), one patient was primary refractory to IRd (3.5%), one patient died (3.5%). At the time of analysis all the pts with CR/PR/VGPR (30%) maintained their response to treatment, 4 pts (13%) maintained stable disease and 9 pts (30%) maintained minor response; 7 pts (23%) experienced disease progression. AEs were revealed in 47% pts: grades 1-2 AEs - 12 pts; grades 3-4 AEs - 4 pts; SAEs - 3 pts (neurological toxicity, gastric bleeding, hypotension), all pts with SAEs discontinued IRd treatment. Baseline QoL was dramatically impaired by the majority of SF-36 scales with significant QoL impairment in 42% pts. The most worsening was revealed for physical functioning, role functioning, general health and vitality (Mean scores varied from 24.6 to 47.0 out from 100 scores). At baseline 88% pts had moderate-to severe symptoms (≥4 scores on the scale from 0 to 10); moderate-to severe worse wellbeing, tiredness, pain and shortness of breath had 72,5%, 67,5%, 61,5% and 45% pts, respectively. At 1 month of IRd treatment QoL meaningfully improved or was stable without significant impairment in 61% pts, at 3 months - in 50% pts. In 1 mos of IRd treatment significant improvement was revealed for physical functioning (GLM, 44.9 vs 54.7, p=.01) and general health (GLM, 47.8 vs 56.3, p.05). At 1 month of treatment, meaningful decrease of shortness of breath (in 42% pts), tiredness (36%), and pain (28%) was revealed; at 3 months of IRd treatment this proportion was 33%, 27%, and 13%, accordingly. Regarding treatment satisfaction pts reported the following: at 1 month after treatment start 94% of pts were satisfied with symptoms decrease due to IRd, 89% pts confirmed that IRd was convenient and 97% pts reported global satisfaction with IRd; at 3 months of treatment all the pts confirmed the convenience of IRd regimen, 84% pts were satisfied with IRd treatment. The results obtained in a real-world setting demonstrate clinical benefits of IRd regimen in RRMM pts, which were achieved without negatively affecting QoL and with satisfactory symptom control in these heavily pretreated patients. IISR funded by Takeda Disclosures Ionova: BMS: Research Funding; Takeda: Research Funding. Vinogradova:Novartis: Honoraria, Research Funding.

8020 Background: MM patients (pts) often receive several lines of therapy with multiple drug combinations and, as lenalidomide (R)-containing regimens are commonly used as first-line therapy, R-free options for subsequent lines are necessary. Additionally, as pts age and become less tolerant to treatment, more convenient regimens, such as all-oral options, with less toxicity are needed. Dexamethasone (dex)-based doublets are effective and tolerable in this setting. Methods: Proteasome inhibitor (PI)-exposed and/or intolerant and R-refractory pts who had ≥2 prior therapies (N = 122) were randomized 3:2 to receive: ixazomib (ixa) 4 mg (5.5 mg from cycle 2 if tolerated) on day (d) 1, 8, 15, and dex 20 mg (≥75 years [yrs], 10 mg) on d 1, 2, 8, 9, 15, 16, 22, 23; or pomalidomide (pom) 4 mg on d 1–21, and dex 40 mg (≥75 yrs, 20 mg) on d 1, 8, 15, 22, in 28-d cycles until progressive disease (PD) or unacceptable toxicity. Pts were stratified by age ( < 65 vs ≥65 yrs), International Staging System (ISS) disease stage at study entry (I/II vs III), and prior lines of therapy (2 vs ≥3). The study was powered to test the primary endpoint of progression-free survival (PFS). Results: In the ixa-dex (n = 73) vs pom-dex (n = 49) arms, median age was 72 vs 68 yrs (36% vs 18% ≥75 yrs), 25% vs 22% of pts had ISS stage III MM, and 52% vs 53% had received ≥3 prior therapies (per stratification). At data cutoff (5/31/2020), 19% vs 20% of pts were ongoing on treatment with ixa-dex vs pom-dex; primary reasons for discontinuation were PD (47% vs 57%) and adverse events (AEs; 23% vs 12%). With median follow-up of 15.3 vs 17.3 months (mos), median PFS (mPFS) was 7.1 vs 4.8 mos with ixa-dex vs pom-dex (hazard ratio [HR] 0.847; 95% confidence interval [CI] 0.535–1.341; p = 0.477); the Table shows mPFS by prior lines, and secondary endpoints. Pts received a median of 6 cycles with both ixa-dex (range 1–25) and pom-dex (range 1–27); 64% of ixa-dex pts were able to escalate to a 5.5 mg dose of ixa. 69% vs 81% of ixa-dex vs pom-dex pts had grade (G) ≥3 AEs, 51% vs 53% had serious AEs, 39% vs 36% had an AE leading to drug discontinuation, 44% vs 32% had an AE leading to dose reduction, and 13% vs 13% died on study. Health-related quality of life (HRQoL; EORTC QLQ-C30/MY20, and EQ-5D-5L) was maintained, and similar between arms. Conclusions: Ixa-dex prolonged PFS vs pom-dex in these heavily pretreated, PI-exposed and/or intolerant, R-refractory pts, but the difference was not statistically significant. Ixa-dex was well tolerated, with lower G≥3 AE rates vs pom-dex, and comparable HRQoL. Clinical trial information: NCT03170882.

Small isoprenoid diphosphates, such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST296AA or T304A) investigated, confirm that the backbone amide of at least one Thr (Thr304), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr296/297) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM2-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.—Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

We report here a comparative study of activity in signal pathways and gene expression in the red (RGM) and white (WGM) parts of the gastrocnemius muscle in rats after series of short (1 sec) tetanic contractions evoked by stimulation of the motor nerve at a frequency of 100 Hz and an amplitude sufficient to activate all the motor units of the muscle. At 2 h after stimulation, WGM showed more marked increases in the level of ERK1/2 phosphorylation than RGM, though increases in AMPK phosphorylation were no different. Furthermore, the increases in MyoD and myogenin mRNA in WGM were significantly greater than those in RGM, while the effects of stimulation on expression of the IGF-1, MaFbx, and MuRF genes were weak and similar in WGM and RGM. There was also an increase in the content of myostatin mRNA in RGM. Thus, glycolytic muscle fibers in WGM display more marked regulatory hypertrophic-type shifts than the oxidative muscle fibers making up RGM.

Butyrophilin 3A1 (BTN3A1) binds small phosphorous-containing molecules, which initiates transmembrane signaling and activates butyrophilin-responsive cells. We synthesized several phosphinophosphonates and their corresponding tris-pivaloyloxymethyl prodrugs and examined their effects on BTN3A1. An analog of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) bound to BTN3A1 with intermediate affinity, which was enthalpy-driven. Docking studies revealed binding to the basic surface pocket and interactions between the allylic hydroxyl group and the BTN3A1 backbone. The phosphinophosphonate stimulated proliferation of Vγ9Vδ2 T cells with moderate activity (EC50 = 26 µM). Cellular potency was enhanced >600-fold in the tris-POM prodrug (EC50 = 0.041 µM). The novel prodrug also induced T cell mediated leukemia cell lysis. Analysis of dose response data reveals HMBPP-induced Hill coefficients of 0.69 for target cell lysis and 0.68 in interferon secretion. Together, tris-POM prodrugs enhance the cellular activity of phosphinophosphonates, reveal structure-activity relationships of butyrophilin ligands, and support a negatively cooperative model of cellular butyrophilin activation.

New Findings What is the central question of this study? This study was designed to investigate the role of AMPK in the regulation of PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. What is the main finding and its importance? Low-intensity exercise markedly increased the expression of PGC-1α mRNA via the alternative promoter, without increases in ACCSer79/222 (a marker of AMPK activation) and AMPKThr172 phosphorylation. A single dose of the AMPK activator metformin indicated that AMPK was not involved in regulating PGC-1α mRNA expression via the alternative promoter in endurance-trained human skeletal muscle. In human skeletal muscle, PGC-1α is constitutively expressed via the canonical promoter. In contrast, the expression of PGC-1α mRNA via the alternative promoter was found to be highly dependent on the intensity of exercise and to contribute largely to the postexercise increase of total PGC-1α mRNA. This study investigated the role of AMPK in regulating PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. AMPK activation and PGC-1α gene expression were assayed in skeletal muscle of nine endurance-trained men before and after low-intensity exercise (38% of maximal oxygen uptake) and with or without administration of a single dose (2 g) of the AMPK activator metformin. Low-intensity exercise markedly and significantly increased (∼100-fold, P

A third of the genes in prokaryotic and eukaryotic genomes encode membrane proteins that are either essential for signal transduction and solute transport or function as scaffold structures. Unlike many of their soluble counterparts, the overall structural and functional organization of membrane proteins is sparingly understood. Recent advances in X-ray crystallography, cryo-EM, and nuclear magnetic resonance (NMR) are closing this gap by enabling an in-depth view of these ever-elusive proteins at atomic resolution. Despite substantial technological advancements, however, the overall proportion of membrane protein entries in the Protein Data Bank (PDB) remains

OBJECTIVE Phase synchronization of arterial pressure (AP) and pulse interval (PI) oscillations in the low-frequency band (around 0.4 Hz in rats) is governed by baroreflex activity. In long-term stationary data recordings, such synchronization can be estimated by the coherence. The phase synchronization index (PSI) can be used as well. The aim of this study was to correlate PSI and the coherence of AP and PI under stationary conditions and to estimate the informativity of PSI as a measure of baroreflex activity during transient processes. APPROACH AP and PI were recorded in conscious Wistar rats using femoral artery catheters. To study the hemodynamics during hemorrhage, blood was gradually withdrawn (20 ml × kg-1 over 30 min) through a catheter in the carotid artery. MAIN RESULTS PSI and coherence spectra calculated from 30-minute AP and PI recordings demonstrated distinct peaks at the frequency of 0.4 Hz; these indicators correlate well with each other (Pearson r = 0.920, p

TNFAIP3 interacting protein 1 (TNIP1) interacts with numerous non-related cellular, viral, and bacterial proteins. TNIP1 is also linked with multiple chronic inflammatory disorders on the gene and protein levels, through numerous single-nucleotide polymorphisms and reduced protein amounts. Despite the importance of TNIP1 function, there is limited investigation as to how its conformation may impact its apparent multiple roles. Hub proteins like TNIP1 are often intrinsically disordered proteins. Our initial in silico assessments suggested TNIP1 is natively unstructured, featuring numerous potentials intrinsically disordered regions, including the ABIN homology domain 1-ubiquitin binding domain in ABIN proteins and NEMO (AHD1-UBAN) domain associated with its anti-inflammatory function. Using multiple biophysical approaches, we demonstrate the structural flexibility of full-length TNIP1 and the AHD1-UBAN domain. We present evidence the AHD1-UBAN domain exists primarily as a pre-molten globule with limited secondary structure in solution. Data presented here suggest the previously described coiled-coil conformation of the crystallized UBAN-only region may represent just one of possibly multiple states for the AHD1-UBAN domain in solution. These data also characterize the AHD1-UBAN domain in solution as mostly monomeric with potential to undergo oligomerization under specific environmental conditions (e.g., binding partner availability, pH-dependence). This proposed intrinsic disorder across TNIP1 and within the AHD1-UBAN region is likely to impact TNIP1 function and interaction with its multiple partners.

Cardiovascular system is adapted to gravity, and reactions to its vanishing in space are presumably dependent on body size. Dependency of hematological parameters and body fluids reaction to simulated microgravity have never been studied as an allometric function before. Thus we estimated RBC, blood and extracellular fluid volumes in hindlimb-unloaded (HLU) or control (ATT) mice, rats and rabbits.RBC decrease was found to be size-independent, and the allometric dependency for red blood loss in HLU and ATT animals shared a common power (−0.054±0.008) but differrent Y0 (8.66±0.40 and 10.73±0.49 correspondingly, pOur data underscore the importance of size-independent mechanisms of cardiovascular adaptation to weightlessness. Despite use of mice hampers application of a straightforward translational approach, this species is useful for gravitational biology as a tool to investigate size-independent mechanisms of mammalian adaptation to microgravity.

A method to evaluate aerobic-anaerobic transition (AAT) during exercise is suggested. The subjects performed two tests with incremental increase of load: bicycle exercise and one leg knee extensions. In both tests the relation of deoxygenated hemoglobin (HHb) to EMG-activity of m. vastus lateralis during test has characteristic peak corresponding to AAT. The statistically significant correlation (r = 0.78, p < 0.05) between the load corresponding to AAT and anaerobic threshold (blood [La] = 4 mmol) was found during bicycle test. The method is applicable to determination of AAT during exercise of small muscle group.

The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro. Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data.

The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSPN and MOSPC. Biophysical analysis of constructs refolded in vitro demonstrated that MOSPC, previously shown to possess porin activity, forms amphiphilic trimers, while MOSPN forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSPC is OM-embedded and surface-exposed, while MOSPN resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the β-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.

The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes built onto a common protein scaffold to facilitate the breakdown of the polymeric substrate, cellulose. One strategy for mimicking these highly effective nanomachines may involve the use of synthetic scaffolds that can react to and organize multiple engineered enzymes to promote synergistic action between the enzymes on the scaffold. As an example of the earlier strategy, we describe here an approach for the expression of cellulolytic enzymes with a serine esterase tag, and the rapid reaction between the tag and the end-functionalized polymers to form enzyme–polymer–enzyme multienzyme conjugates. In principle, this general and versatile supramolecular approach may be used to organize specific cellulolytic enzymes onto synthetic scaffolds to form multienzyme complexes to potentially work in synergy for enhanced biological activities. Best reaction conditions, good activities of the armored cellulolytic enzymes and the design of optimal protein linker in the fusion protein are discussed in detail. If other reactive tags are included on the enzyme in future, multiple types of synergistic enzymes may be positioned at specific sites on a designed polymer scaffold that mimics the complex structure and enhanced function of natural cellulosomes. This type of nanoarmoring of multiple enzymes on a nanoscale might also enhance enzyme stability, when compared to the unprotected enzymes.

SummaryPhosphoantigen-sensitive Vγ9Vδ2 T cells are important responders to infections and malignancy. However, the mechanisms by which phosphoantigens stimulate Vγ9Vδ2 T cells are unclear. Here, we synthesized phosphoantigen prodrugs and used them to demonstrate that intracellular delivery of phosphoantigens is required for their activity. The pivaloyloxymethyl prodrug is the most potent phosphoantigen described to date, with stronger stimulation of Vγ9Vδ2 T cells from human peripheral blood and greater ability to induce lysis of Daudi lymphoma cells relative to the previously most potent compound, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). We demonstrate high binding affinity between phosphoantigens and the intracellular region of butyrophilin 3A1 (BTN3A1), localized to the PRY/SPRY (B30.2) domain, but also affecting the membrane proximal region. Our findings promote a phosphoantigen prodrug approach for cancer immunotherapy and unravel fundamental aspects of the mechanisms of Vγ9Vδ2 T cell activation.

Background: PF-114 mesylate is a 4th-generation oral tyrosine kinase-inhibitor (TKI) active against wild-type and mutated BCRABL1 isoforms including those with a BCRABL1T315I. We present data from a phase-1 study in subjects with chronic or accelerated phase chronic myeloid leukaemia (CML) failing ≥2 TKIs or who have BCRABL1T315I (NCT02885766). Methods: 3+3 dose-escalation design to determine maximum tolerated dose (MTD) followed by expanded cohorts for doses ≤MTD. The primary objective was to determine the MTD and identify dose-limiting toxicities (DLTs) during cycle 1 (28 days). Secondary objectives included safety and anti-CML activity based on hematological, cytogenetic, and molecular criteria. Adverse events (AEs) were assessed and graded using NCI-CTCAE v4.03. Results: 51 subjects were enrolled as of June 26, 2018. Daily doses were 50 mg (n=3), 100 mg (n=3), 200 mg (n=9), 300 mg (n=11), 400 mg (n=12), 500 mg (n=3), 600 mg (n=6), 750 mg (n=4) given on a continuous QD schedule. Median age was 50 years (range, 29-82 years). Median interval from diagnosis to study-entry was 10 years (range, 0-23 years). Subjects had baseline ECOG performance scores Conclusion: MTD of PF-114 is 600 mg with skin toxicity as the DLT. The best safety/efficacy ratio was seen at doses of 200 and 300 mg which are being studied in expanded cohorts and soon in a phase-2 study. Disclosures Turkina: Novartis: Other: provided consultations; Bristol Myers Squibb: Other: provided consultations; Phizer: Other: provided consultations; Fusion Pharma: Other: provided consultations. Shukhov:Novartis: Other: provided consultations and performed lectures ; Bristol Myers Squibb: Other: provided consultations and performed lectures . Chelysheva:Bristol Myers Squibb: Other: provided consultations and performed lectures; Fusion Pharma: Other: provided consultations ; Novartis: Other: provided consultations and performed lectures. Cortes:Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Arog: Research Funding. Ottmann:Novartis: Consultancy; Pfizer: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Fusion Pharma: Consultancy, Research Funding. Mikhailov:Fusion Pharma: Employment. Novikov:Fusion Pharma: Employment. Shulgina:Fusion Pharma: Employment. Chilov:Fusion Pharma: Employment.

Despite arduous efforts and recent technological developments structural investigation of integral membrane proteins remains a challenge. The primary deterrents include difficulties with their expression, low inherent solubility, and problems associated with existing membrane mimicking systems. A relatively new class of membrane mimetics, nanodiscs, is emerging as a promising alternative. Although nanodiscs have been proven successful for several biophysical applications, they yet remain to become the system of preferred choice for structure determination. We have hereby made nanodiscs more suitable for solution NMR applications by reducing the diameter of the self-assembly complex to its potential limit. We achieved a noticeable improvement in the quality of NMR spectra obtained for the transmembrane and cytoplasmic domains of integrin IIb incorporated into these smaller discs rendering them susceptible for a thorough structural investigation. In addition, we also present an on-column method for a rapid, efficient, single-step preparation of protein incorporated nanodiscs at high concentrations. These discs have been fully characterized by transmission electron microscopy, dynamic light scattering, and differential scanning calorimetry. Proteins 2013; 81:1222-1231. (c) 2013 Wiley Periodicals, Inc.

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, αIIbβ3 integrin. We have analyzed phosphorylated (Tyr747 and Tyr759) β3 integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr747 phosphorylation prevents β3CT from binding to its heterodimer partner αIIbCT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of β3CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.

We report the NMR solution structure of a synthetic 40-mer (T(377)-E(416)) that encompasses human cannabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30% trifluoroethanol/H(2)O. Structural features include, from the peptide's amino terminus, a hydrophobic alpha-helix (TMH7); a loop-like, 11 residue segment featuring a pronounced Pro-kink within the conserved NPxxY motif; a short amphipathic alpha-helix (H8) orthogonal to TMH7 with cationic and hydrophobic amino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structure suggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and a hydrogen-bond network involving the peptide's loop-like region. Potential cation-pi and cation-phenolic OH interactions between Y(397) in the TMH7 NPxxY motif and R(405) in H8 are identified as candidate structural forces promoting interhelical microdomain formation. This microdomain may function as a flexible molecular hinge during ligand-induced hCB1 conformer transitions.

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide's NMR structural biology is characterized by two alpha-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide's flexible region, W(258) may hydrogen-bond with L(255) to help stabilize the Pro-kinked hCB2 TMH6 structure and position C(257) advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.

There is a significant need for new therapeutics to treat infections caused by the biodefense agent Bacillus anthracis. In pursuit of drug discovery against this organism, we have developed novel propargyl-linked inhibitors that target the essential enzyme dihydrofolate reductase (DHFR) from B. anthracis. Previously, we reported an initial series of these inhibitors and a high-resolution crystal structure of the ternary complex of the enzyme bound to its cofactor and one of the most potent inhibitors, UCP120B [Beierlein, J., Frey, K., Bolstad, D., Pelphrey, P., Joska, T., Smith, A., Priestley, N., Wright, D., and Anderson, A. (2008) J. Med. Chem. 51, 7532-7540]. Herein, we describe a three-dimensional solution structure of the ternary complex as determined by NMR. A comparison of this solution structure to the crystal structure reveals a general conservation of the DHFR fold and cofactor interactions as well as differences in the location of an active site helix and specific ligand interactions. In addition to data for the fully assigned ternary complex, data for the binary (enzyme-cofactor) complex were collected, providing chemical shift comparisons and revealing perturbations in residues that accommodate ligand binding. Dynamics of the protein, measured using (15)N T(1) and T(2) relaxation times and {(1)H}-(15)N heteronuclear NOEs, reveal residue flexibility at the active site that explains enzyme inhibition and structure-activity relationships for two different series of these propargyl-linked inhibitors. The information obtained from the solution structure regarding active site flexibility will be especially valuable in the design of inhibitors with increased potency.

Characteristics of the small arteries (with a diameter of 200-250 μm) feeding the medial gastrocnemius muscle and diaphragm were studied. Recording of the mechanical activity of ring segments under isometric conditions demonstrated that, similar to other arteries feeding the muscles with a high content of slow fibers, the diaphragm arteries are highly sensitive to adrenoceptor agonists and acetylcholine. The differences in the endothelium-dependent relaxation in response to acetylcholine were retained in the presence of L-NAME and diclofenac. The diaphragm and gastrocnemius arteries similarly responded to serotonin. On the other hand, a high innervation density was characteristic of the diaphragm arteries unlike the arteries of other slow muscles. The density of adrenergic nerve plexus in the diaphragm arteries was considerably higher than in the gastrocnemius arteries. The results suggest that the characteristics of small diaphragm arteries are determined not only by the oxidative capacity of diaphragm muscle fibers, but also by the fact that this is a respiratory muscle.

Effects of low-frequency electrical stimulation, which is currently considered to be a possible countermeasure for long-duration spaceflights, with and without stretch were evaluated. Twelve young male volunteers were randomly distributed into two groups. In one group anterior thigh muscles—knee extensors of both legs were stimulated with frequency of 15 Hz for 4.5 wks, six times a week; each session was 6-h long. In the other group, electrical stimulation with the same parameters was applied to stretched knee extensors. Following stimulation the subjects exhibited an increase in fatigue resistance, and in the succinate dehydrogenase activity and a 10% gain in the percentage of muscle fibers with slow myosin heavy chain isoforms. In a stimulated group the peak voluntary strength went down significantly, the CSA of fast muscle fibers in m. quadriceps femoris became slightly less in size (10%). Electrical stimulation of the stretched muscles induced an insignificant decline in their strength and an increase of cross-sectional area of muscle fibers of both types. Thus chronic low-frequency electrical stimulation may be proposed as a candidate countermeasure against muscle strength and mass loss if it is combined with stretch.

The study investigated the effects of chronic low-frequency electrical stimulation (CLFES) of the stretched knee extensor muscles versus CLFES without a resistance load. A total of 19 male volunteers were randomized into two groups with similar aerobic and force-velocity properties. In group 1, anterior thigh muscles (the knee extensors) of both legs were exposed to CLFES (15 Hz). In group 2, the CLFES of the knee extensors was combined with stretching. The stimulated subjects demonstrated a marked tendency towards increasing endurance and a significant increase (by 10%) in both succinate dehydrogenase activity and the percentage of muscle fibers containing slow myosin heavy chain (MHC) isoforms. At the same time, the proportion of fibers reacting with the antibody against fast MHC isoforms decreased by 12%. In group 1, the maximum voluntary force (MVF) significantly decreased, the volume of m. quadriceps femoris (as measured by magnetic resonance imaging) remained unchanged, and the size of the fast fibers slightly decreased (by 11%). In group 2, no significant decrease in the MVF was observed, while there was a significant increase in muscle volume and the cross-sectional area of muscle fibers.

The aim of the present study was to investigate the effect of acute aerobic exercise on the expression of PGC-1α transcript variants in human skeletal muscle. Seven endurance-trained athletes performed a 90-min cycling test (62% of VO2max). At resting state, the levels of N-truncated (NT)-PGC-1α and PGC-1α exon 1a-derived transcripts were significantly higher (20-fold; P0.05) than those of PGC-1α exon 1b- and 1c-derived transcripts. Acute exercise did not change the PGC-1α exon 1a-derived expression level, but it did increase the expression level of NT-PGC-1α mRNAs 6-fold, and the expression levels of PGC-1α exon 1b- and 1c-derived mRNAs200-fold (P0.05). We conclude that NT-PGC-1α transcript expression in resting muscle and after acute moderate-intensity exercise constituted a significant share of total PGC-1α expression. The exercise led to a higher level of PGC-1α expression from alternative promoters (exon 1b- and 1c-derived mRNA) than from the canonical proximal promoter (exon 1a-derived mRNA).

The adenovirus E3-13.7 protein interferes with endosomal protein sorting to down-regulate the epidermal growth factor receptor and related tyrosine kinase receptors. The cytoplasmic C terminus of this protein contains three protein sorting motifs which are related to the function of E3-13.7. In this study, the structure of a 23-residue polypeptide corresponding to this domain was examined using solution NMR and CD spectroscopic methods. The peptide was observed to exist in a mostly random structural state in aqueous solution but underwent high affinity association with dodecylphosphocholine micelles, where it adopted an ordered structure. The affinity of this peptide for the micellar surface and the structure of the bound peptide were independent of pH variation, surface charge, or attachment of a myristoyl anchor to the N-terminal. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. On the micellar surface all three sorting motifs are closely associated with the water/apolar interface: 72-YLRH and 87-LL lie within interfacial amphipathic helices, while 76-HPQY is non-helical and dimples just above the surface. These results contribute to the development of an understanding of the basis for specificity in recognition of sorting motifs by components of the cellular protein trafficking machinery.

Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (× 10-7cm2/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggesting that this well-known protein denaturant should be used with care in studies of complex membrane proteins.

Several model systems are currently available for studying membrane proteins. One such relatively new system called nanodiscs has been emerging as a promising alternative. Nanodiscs are discoidal nanolipoproteins, having a nanoscale phospholipid bilayer held together by an amphipathic helical membrane scaffold protein (MSP). Due to their soluble nature, ease of concentration, monodispersity, temperature stability, and compatibility with cell free expression, nanodiscs present a competitive edge over other available systems for studying membrane proteins. With our aim of carrying out structural investigation of membrane proteins within nanodiscs using solution NMR we reduced the diameter of the self assembly complex to its potential limit there by achieving shorter rotational correlation times. This is shown by the noticeable improvement in the spectrum quality of the transmembrane and cytoplasmic domains of integrin αIIb incorporated into these newly developed minimalistic nanodiscs. With the vested interest in generating large “NMR” quantities of homogenous samples in the shortest possible time we developed an on-column method for preparing protein incorporated nanodiscs. Not only did we achieve a rapid, single step, homogenous preparation at one third the time, but we did so with the concurrent separation of protein incorporated discs from the empty discs. This is beneficial as it improves the yield by reducing the number of purification steps, providing a more pure homogeneous sample typically required for structural studies. We have exemplified our new method by reconstituting both bacteriorhodopsin and integrin αIIb using two different strategies within nanodiscs. The nanodiscs prepared by our method have been characterized by Transmission Electron Microscopy, Dynamic Light Scattering and Differential Scanning Calorimetry.

Protein-protein interactions are crucial for a wide variety of biological processes. These interactions range from high affinity (K (d)nM) to very low affinity (K (d)mM). While much is known about the nature of high affinity protein complexes, our knowledge about structural characteristics of weak protein-protein interactions (wPPIs) remains limited: in addition to the technical difficulties associated with their investigation, historically wPPIs used to be considered physiologically irrelevant. However, emerging evidence suggests that wPPIs, either in the form of intact protein complexes or as part of large molecular machineries, are fundamentally important for promoting rapid on/off switches of signal transduction, reversible cell-cell contacts, transient assembly/disassembly of signaling complexes, and enzyme-substrate recognition. Therefore an atomic-level elucidation of wPPIs is vital to understanding a cornucopia of diverse cellular events. Nuclear magnetic resonance (NMR) is famous for its unique abilities to study wPPIs and, by utilization of the new technical developments combined with sparse data based computational analysis, it now allows rapid identification and structural characterization of wPPIs. Here we present our perspective on the NMR methods employed.

Calpains are ubiquitous intracellular calcium- and thiol-dependent proteases. Their over activation, resulting in the degradation of various substrates, has been implicated in a number of cardiovascular and neurological disorders. Here, we present the first structural characterization of LSEAL penta-peptide, a potent calpain inhibitor, bound to the calmodulin-like domain of calpain. Our in vitro binding data supports the idea that domains other than calpain’s active site may be suitable targets for future development of therapeutic agents to be used to treat heart attack, traumatic brain injuries or a variety of neurodegenerative conditions, such as ischemic stroke.

The subject of this study is the frequency distribution of alleles of the vascular endothelial growth factor gene (VEGF; the G-634C polymorphism) in athletes (n = 670) and in a control group (n = 1073) and the relationships of genotypes with aerobic performance in rowers (n = 90). Genetic typing was performed using the analysis of restriction fragment length polymorphism. The frequency of the VEGF C allele in the group of endurance athletes (n = 294) was significantly higher than in the control group and increased together with increasing sports qualification. In addition, a correlation of the VEGF C allele with a high aerobic performance of athletes (according to data on the maximal power and maximal oxygen consumption) and with a substantial contribution to the energy supply of aerobic metabolism (according to the values of maximal lactate content) has been found. It is inferred that the G-634C polymorphism of the VEGF gene is associated with physical performance of athletes and plays a key role in sports selection.

A series of 6-mono-, di-, and trifluoro analogs of S-phenyl 2,3,4-tri-O-benzyl-D- or L-thiorhamnopyranoside has been synthesized and used as donors in glycosylation reactions, with activation by the 1-benzenesulfinyl piperidine/triflic anhydride system. The stereochemical outcome of the glycosylation reactions was found to depend on the electron-withdrawing capability of the disarming substituent at the 6-position, i.e., on the number of fluorine atoms present. The results are explained with regard to the increased stability of the glycosyl triflates, shown to be intermediates in the reaction by low-temperature 1H NMR experiments, with increased fluorine content.

A synthesis of the pentenyl glycoside of the tetrasaccharide side chain from the major glycoprotein of Bacillus anthracis by a [3 + 1] approach is described. The construction of the 1,2-trans-glycosidic linkage in the terminal anthrose moiety was achieved through the application of known alpha-nitrilium ion-mediated beta-selective glycosylation methodology. An iterative glycosylation strategy was used for the assembly of the trirhamnan building block. A new route to the anthrose saccharide was developed from D-galactose.

A series of 6-mono-, di-, and trifluoro analogs of S-phenyl 2,3,4-tri-O-benzyl-D- or L-thiorhamnopyranoside has been synthesized and used as donors in glycosylation reactions, with activation by the 1-benzenesulfinyl piperidine/triflic anhydride system. The stereochemical outcome of the glycosylation reactions was found to depend on the electron-withdrawing capability of the disarming substituent at the 6-position, i.e., on the number of fluorine atoms present. The results are explained with regard to the increased stability of the glycosyl triflates, shown to be intermediates in the reaction by low-temperature 1H NMR experiments, with increased fluorine content.

On exposure to dichlorodicyanoquinone in wet dichloromethane at room temperature, equatorial 4-O-benzyl ethers are removed with moderate selectivity in the presence of other benzyl ethers in glycopyranosides and glycothiopyranosides.

Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates genes responsible for skeletal and heart muscle fatty acid oxidation. Previous studies have shown that the PPARalpha intron 7 G/C polymorphism was associated with left ventricular growth in response to exercise. We speculated that GG homozygotes should be more prevalent within a group of endurance-oriented athletes, have normal fatty acid metabolism, and increased percentages of slow-twitch fibers. We have tested this hypothesis in the study of a mixed cohort of 786 Russian athletes in 13 different sporting disciplines prospectively stratified by performance (endurance-oriented athletes, power-oriented athletes and athletes with mixed endurance/power activity). PPARalpha intron 7 genotype and allele frequencies were compared to 1,242 controls. We found an increasing linear trend of C allele with increasing anaerobic component of physical performance (P=0.029). GG genotype frequencies in endurance-oriented and power-oriented athletes were 80.3 and 50.6%, respectively, and were significantly (P0.0001) different compared to controls (70.0%). To examine the association between PPARalpha gene variant and fiber type composition, muscle biopsies from m. vastus lateralis were obtained and analyzed in 40 young men. GG homozygotes (n=25) had significantly (P=0.003) higher percentages of slow-twitch fibers (55.5+/-2.0 vs 38.5+/-2.3%) than CC homozygotes (n=4). In conclusion, PPARalpha intron 7 G/C polymorphism was associated with physical performance in Russian athletes, and this may be explained, in part, by the association between PPARalpha genotype and muscle fiber type composition.