The Effect of Low-Volume Preseason Plyometric Training on Force-Velocity Profiles in Semiprofessional Rugby Union Players.

ABSTRACT: Watkins, CM, Gill, ND, Maunder, E, Downes, P, Young, JD, McGuigan, MR, and Storey, AG. The effect of low-volume preseason plyometric training on force-velocity profiles in semiprofessional rugby union players. J Strength Cond Res 35(3): 604-615, 2021-Rugby union is a physically demanding and complex team sport requiring athletes across all positions to express speed and acceleration. Plyometrics can effectively improve speed profiles by enhancing both force- and velocity-(FV) characteristics; however, the optimal dose and exercise direction for trained athletes is still relatively unknown. Therefore, the aim of this investigation was to determine the efficacy of a low-dose, directionally specific plyometric training program for improving speed profiles in semiprofessional rugby players. Players were randomly allocated to one of 2 plyometric training groups that performed low-volume (40-60 ground contacts per session) plyometrics twice weekly, or a control group that did not participate in any plyometric training. The 2 training groups underwent reverse back-to-back three-week vertically and horizontally focused plyometric training programs, with a 12-day washout. Body composition, aerobic capacity, and sprint performance (10-, 20-, 30-m split time, horizontal FV profile) were measured. During the intervention, HV-1 (horizontal/vertical training group 1) improved sprint performance (n = 12; ∆30 m = -0.020 seconds; p = 0.038), VH-2 (vertical/horizontal training group 2) maintained sprint performance (n = 8; ∆30 m = +0.049 seconds; p = 0.377), and the control group progressively declined in sprint performance (n = 12; ∆30 m = +0.071; p = 0.019). In addition, vertical plyometrics may preferentially benefit secondary acceleration (∆10-20 m split time: -0.01 seconds; p = 0.03) and many force-oriented FV profile characteristics. Correlational analyses (r2 = -0.568 to 0.515) showed sprint improvements were hindered in athletes with lower initial aerobic fitness, suggesting accumulated fatigue may have limited the magnitude of adaptation. Therefore, including low-volume plyometric training may be beneficial for improving sprint profiles or attenuating decrements realized during periods of high-volume sport-specific training.

HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport.

Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported [3H] or [14C] pyrimidine and purine nucleosides once inside cells. With the exception of adenosine, transported nucleosides were generally subject to minimal intracellular metabolism. We also used radiochemical HPLC analysis to study the mechanism by which adenosine functions as a low Km, low Vmax permeant of hCNT1. hCNT1-producing oocytes were pre-loaded with [3H] uridine, after which efflux of accumulated radioactivity was measured in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. hCNT1-mediated [3H]-efflux was stimulated by extracellular uridine, but inhibited by extracellular adenosine, with >95% of the radioactivity exiting cells being unmetabolized uridine, consistent with a low transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC 29) family, hENT1-4. Of these, hENT1 and hENT2 transport both nucleosides and nucleobases into and out of cells, but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. We therefore used HPLC to determine plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice, and to compare the findings with knockout of mCNT3. Results demonstrated that ENT1 was more important than ENT2 or CNT3 in determining plasma adenosine concentrations, indicated modest roles of ENT1 in the homeostasis of other nucleosides, and suggested that none of the transporters is a major participant in handling of nucleobases.

The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.

Unilateral hamstrings static stretching can impair the affected and contralateral knee extension force but improve unilateral drop jump height.

PURPOSE: Prolonged static stretching (SS) in isolation (no dynamic warm-up) can impair muscle performance. There are conflicting reports whether impairments are present in antagonist and contralateral muscles. The objective of this study was to assess the effect of unilateral hamstrings SS on ipsilateral stretched and contralateral limbs' strength and jump power. METHODS: The SS (four repetitions of 30-s) and control sessions involved unilateral testing of the stretched leg and contralateral leg for knee extension (KE) maximum voluntary isometric contraction (MVIC) force and electromyography (EMG), drop jump (DJ) height and contact time at 1-min post-stretching. RESULTS: There were significant KE MVIC force impairments for both the SS (p = 0.006, d = 0.3, - 8.1%) and contralateral (p = 0.02, d = 0.20, - 4.2%) leg. With normalized data, there was a near-significant (p = 0.1), small magnitude (d = 0.29), greater force impairment with the ipsilateral (93.0 ± 12.8% of pre-test) versus the contralateral (96.2 ± 9.1% of pre-test) KE MVIC force. DJ height significantly improved for the stretched leg (p = 0.03, d = 0.18, + 9.2%) with near-significant, improvements for the contralateral leg (p = 0.06, d = 0.22, + 12.1%). For the stretched leg, DJ contact time was significantly (p = 0.04, d = 0.18, + 3.4%) prolonged, but there was no significant change with the contralateral leg. CONCLUSIONS: Unilateral hamstrings SS induced strength deficits in the ipsilateral and contralateral knee extension MVIC and a prolongation of the stretched leg DJ contact period. In anticipation of maximal force outputs, prolonged SS in isolation (no dynamic warm-up included) can have negative consequences on antagonist and contralateral muscle performance.

Unilateral Knee and Ankle Joint Fatigue Induce Similar Impairment to Bipedal Balance in Judo Athletes.

The purpose of this study was to compare the effects of unilateral ankle fatigue versus the knee muscles with and without vision on bipedal postural control. Elite judo athletes who competed at the national level with at least 10 years of training experience, were randomised into KNEE (n = 10; 20 ± 2 years) and ANKLE (n = 9; 20 ± 3 years) groups, who performed dynamic isokinetic fatiguing contractions (force decreased to 50% of initial peak torque for three consecutive movements) of the knee flexors and extensors or ankle dorsiflexors and plantar flexors, respectively. Static bipedal postural control (French Posturology Association normative standards) with eyes open and eyes closed was examined before and immediately after the fatiguing task. Postural variables examined were the centre of pressure (CoP) sway in the medio-lateral and antero-posterior directions, total CoP area sway and CoP sway velocity. Although unilateral ankle and knee fatigue adversely affected all bipedal postural measures, with greater disturbances with eyes closed, there were no significant main group or interaction effects between KNEE and ANKLE groups. Unilateral lower limb fatigue adversely affected bipedal balance, with knee extension/flexion fatigue affecting bipedal postural control to a similar extent as unilateral ankle dorsiflexion/plantar flexion fatigue. Hence unilateral fatigue can affect subsequent bilateral performance or also have implications for rehabilitation exercise techniques. Our findings may be limited to judo athletes as other populations were not tested.

Global Training Effects of Trained and Untrained Muscles With Youth Can be Maintained During 4 Weeks of Detraining.

Chaouachi, A, Ben Othman, A, Makhlouf, I, Young, JD, Granacher, U, and Behm, DG. Global training effects of trained and untrained muscles with youth can be maintained during 4 weeks of detraining. J Strength Cond Res 33(10): 2788-2800, 2019-Global (whole-body) effects of resistance training (i.e., cross-education) may be pervasive with children. Detraining induces less substantial deficits with children than adults. It was the objective of this study to investigate the global responses to 4 weeks of detraining after 8 weeks of unilateral leg press (LP) training in 10-13-year-old, pre-peak-height-velocity stage boys. Subjects were randomly separated into 2 unilateral resistance training groups (high load/low repetitions [HL-LR] and low load/high repetitions [LL-HR], and control group). Assessments at pre-training, post-training, and detraining included dominant and nondominant limbs, unilateral, 1 repetition maximum (1RM) and 60% 1RM LP, knee extension, knee flexion, elbow flexion, and handgrip maximal voluntary isometric contraction (MVIC), and countermovement jump (CMJ). All measures significantly increased from pre-test to detraining for both training programs, except for elbow flexion MVIC with increases only with HL-LR. All measures except CMJ and handgrip MVIC significantly decreased from post-test to detraining, except for elbow flexion MVIC with decreases only with HL-LR. The dominant trained limb experienced significantly greater LP improvements (pre- to detraining) and decrements (post- to detraining) with LP 1RM and 60% 1RM LP. In conclusion, youth HL-LR and LL-HR global training effects of trained and untrained limbs demonstrate similar benefits (pre- to detraining) and decrements (post- to detraining) with detraining. The findings emphasize that training any muscle group in a child can have positive global implications for improved strength and power that can persist over baseline measures for at least a month.

In vitro inhibition of human nucleoside transporters and uptake of azacitidine by an isocitrate dehydrogenase-2 inhibitor enasidenib and its metabolite AGI-16903.

1. The present study investigated inhibitory effects of enasidenib and its metabolite AGI-16903 on (a) recombinant human nucleoside transporters (hNTs) in hNT-producing Xenopus laevis oocytes, and (b) azacitidine uptake in a normal B-lymphoblast peripheral blood cell line (PBC) and acute myeloid leukemia (AML) cell lines. 2. Enasidenib inhibited hENT1, hENT2, hENT3, and hENT4 in oocytes with IC50 values of 7, 63, 27, and 76 µM, respectively, but exhibited little inhibition of hCNT1-3. AGI-16903 exhibited little inhibition of any hNT produced in oocytes. 3. Azacitidine uptake was more than 2-fold higher in AML cells than in PBC. Enasidenib inhibited azacitidine uptake into OCI-AML2, TF-1 and PBC cells in a concentration-dependent manner with IC50 values of 0.27, 1.7, and 1.0 µM in sodium-containing transport medium, respectively. 4. IC50 values shifted approximately 100-fold higher when human plasma was used as the incubation medium (27 µM in OCI-AML2, 162 µM in TF-1, and 129 µM in PBC), likely due to high human plasma protein binding of enasidenib (98.5% bound). 5. Although enasidenib inhibits hENTs and azacitidine uptake in vitro, plasma proteins attenuate this inhibitory effect, likely resulting in no meaningful in vivo effects in humans.

The Addition of Transcutaneous Electrical Nerve Stimulation with Roller Massage Alone or in Combination Did Not Increase Pain Tolerance or Range of Motion.

Roller massage (RM) can be painful and induce muscle activity during application. Acute increases in pain pressure threshold (PPT) and range of motion (ROM) have been previously reported following RM. It is unclear whether the RM-induced increases in PPT and ROM can be attributed to changes in neural or muscle responses. To help determine if neural pain pathways are affected by roller massage, transcutaneous electrical nerve stimulation (TENS) was utilized as a form of electroanalgesia during RM with PPT and ROM tested on the affected and contralateral quadriceps. The purpose of this study was to evaluate in both quadriceps, the effect of brief intense TENS on PPT and ROM following unilateral RM of the quadriceps. A randomized within subjects' design was used to examine local and non-local effects of TENS and roller massage versus a control condition (rolling without TENS application). Four 30s bouts of roller massage of the dominant quadriceps were implemented with 30s of rest. The researcher applied the RM using a constant pressure device with approximately 70% of the maximum tolerable load. Perceived pain was monitored using a visual analog scale (VAS) during RM. Ipsilateral and contralateral quadriceps ROM and PPT were measured immediately following RM. Significant main effects for time showed increased PPT and ROM in both the treated and contralateral quadriceps, with no significant main effects for intervention or interactions for intervention and time. Moderate to large effect sizes and minimal clinically important differences (MCID) were detected when comparing baseline to pre- and post-tests respectively. VAS scores were significantly (main effect for intervention) and near significantly (interactions) reduced with MCID when TENS was applied during rolling. The addition of TENS to rolling did not increase PPT or ROM in the affected or contralateral quadriceps, likely due to a repeated testing effect.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

Inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hCNT3) predicts an elevator-type transport mechanism.

The human SLC28 family of concentrative (Na+-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNTNW to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter's outward-facing conformation. In addition, both the Na+ and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.

Higher Quadriceps Roller Massage Forces Do Not Amplify Range-of-Motion Increases nor Impair Strength and Jump Performance.

Grabow, L, Young, JD, Alcock, LR, Quigley, PJ, Byrne, JM, Granacher, U, Skarabot, J, and Behm, DG. Higher quadriceps roller massage forces do not amplify range-of-motion increases nor impair strength and jump performance. J Strength Cond Res 32(11): 3059-3069, 2018-Roller massage (RM) has been reported to increase range of motion (ROM) without subsequent performance decrements. However, the effects of different rolling forces have not been examined. The purpose of this study was to compare the effects of sham (RMsham), moderate (RMmod), and high (RMhigh) RM forces, calculated relative to the individuals' pain perception, on ROM, strength, and jump parameters. Sixteen healthy individuals (27 ± 4 years) participated in this study. The intervention involved three 60-second quadriceps RM bouts with RMlow (3.9/10 ± 0.64 rating of perceived pain [RPP]), RMmod (6.2/10 ± 0.64 RPP), and RMhigh (8.2/10 ± 0.44 RPP) pain conditions, respectively. A within-subject design was used to assess dependent variables (active and passive knee flexion ROM, single-leg drop jump [DJ] height, DJ contact time, DJ performance index, maximum voluntary isometric contraction [MVIC] force, and force produced in the first 200 milliseconds [F200] of the knee extensors and flexors). A 2-way repeated measures analysis of variance showed a main effect of testing time in active (p < 0.001, d = 2.54) and passive (p < 0.001, d = 3.22) ROM. Independent of the RM forces, active and passive ROM increased by 7.0% (p = 0.03, d = 2.25) and 15.4% (p < 0.001, d = 3.73) from premeasure to postmeasure, respectively. Drop jump and MVIC parameters were unaffected from pretest to posttest (p > 0.05, d = 0.33-0.84). Roller massage can be efficiently used to increase ROM without substantial pain and without subsequent performance impairments.

The effects of different durations of static stretching within a comprehensive warm-up on voluntary and evoked contractile properties.

Evidence for performance decrements following prolonged static stretching (SS) has led to a paradigm shift in stretching routines within a warm-up. Rather than SS, dynamic stretching (DS) and dynamic activity (DA) have replaced SS within warm-up routines. The objective of the present study was to compare the effect of differing lower limb SS durations (30 [SS30s], 60 [SS60s] or 120 s [SS120s] of SS per muscle group or no-stretch control) within a comprehensive warm-up protocol consisting of aerobic activity, DS and DA. Sixteen male participants completed the four stretching conditions in a randomized order, after a 5-min low-intensity (cycle) warm-up and before a DS/DA component on separate days. Tests included passive hip and knee ranges of motion (ROM), maximum voluntary knee extensor/flexor force, force produced at 100 ms (F100), vertical jump height and evoked knee extensor contractile properties. For hip flexion (hamstrings) ROM, SS120s provided the largest increase (5.6-11.7%) followed by SS60s (4.3-11.4%), control (4.4-10.6%) and SS30s (3.6-11.1%). For knee flexion (quadriceps) ROM, SS30s provided the largest increase (9.3-18.2%) followed by SS120s (6.5-16.3%), SS60s (7.2-15.2%) and control (6.3-15.2%). There were decreases in quadriceps F100 following SS in SS120s (29.6%) only. There were increases in vertical jump performance in the control (6.2%), SS60s (4.6%) and SS30s (3.3%). While 120 s SS per muscle increased ROM, even within a comprehensive warm-up routine, it also elicited notable performance decrements. However, moderate durations of SS were observed to improve ROM whilst either having negligible or beneficial (but not detrimental) effects on specific aspects of athletic performance.

Roller massage decreases spinal excitability to the soleus.

Roller massage (RM) interventions have shown acute increases in range of motion (ROM) and pain pressure threshold (PPT). It is unclear whether the RM-induced increases can be attributed to changes in neural or muscle responses. The purpose of this study was to evaluate the effect of altered afferent input via application of RM on spinal excitability, as measured with the Hoffmann (H-) reflex. A randomized within-subjects design was used. Three 30-s bouts of RM were implemented on a rested, nonexercised, injury-free muscle with 30 s of rest between bouts. The researcher applied RM to the plantar flexors at three intensities of pain: high, moderate, and sham. Measures included normalized M-wave and H-reflex peak-to-peak amplitudes before, during, and up to 3 min postintervention. M-wave and H-reflex measures were highly reliable. RM resulted in significant decreases in soleus H-reflex amplitudes. High-intensity, moderate-intensity, and sham conditions decreased soleus H-reflex amplitudes by 58%, 43%, and 19%, respectively. H-reflexes induced with high-intensity rolling discomfort or pain were significantly lower than moderate and sham conditions. The effects were transient in nature, with an immediate return to baseline following RM. This is the first evidence of RM-induced modulation of spinal excitability. The intensity-dependent response observed indicates that rolling pressure or pain perception may play a role in modulation of the inhibition. Roller massage-induced neural modulation of spinal excitability may explain previously reported increases in ROM and PPT. NEW & NOTEWORTHY Recent evidence indicates that the benefits of foam rolling and roller massage are primarily accrued through neural mechanisms. The present study attempts to determine the neuromuscular response to roller massage interventions. We provide strong evidence of roller massage-induced neural modulation of spinal excitability to the soleus. It is plausible that reflex inhibition may explain subsequent increases in pain pressure threshold.

Effectiveness of Traditional Strength vs. Power Training on Muscle Strength, Power and Speed with Youth: A Systematic Review and Meta-Analysis.

Numerous national associations and multiple reviews have documented the safety and efficacy of strength training for children and adolescents. The literature highlights the significant training-induced increases in strength associated with youth strength training. However, the effectiveness of youth strength training programs to improve power measures is not as clear. This discrepancy may be related to training and testing specificity. Most prior youth strength training programs emphasized lower intensity resistance with relatively slow movements. Since power activities typically involve higher intensity, explosive-like contractions with higher angular velocities (e.g., plyometrics), there is a conflict between the training medium and testing measures. This meta-analysis compared strength (e.g., training with resistance or body mass) and power training programs (e.g., plyometric training) on proxies of muscle strength, power, and speed. A systematic literature search using a Boolean Search Strategy was conducted in the electronic databases PubMed, SPORT Discus, Web of Science, and Google Scholar and revealed 652 hits. After perusal of title, abstract, and full text, 107 studies were eligible for inclusion in this systematic review and meta-analysis. The meta-analysis showed small to moderate magnitude changes for training specificity with jump measures. In other words, power training was more effective than strength training for improving youth jump height. For sprint measures, strength training was more effective than power training with youth. Furthermore, strength training exhibited consistently large magnitude changes to lower body strength measures, which contrasted with the generally trivial, small and moderate magnitude training improvements of power training upon lower body strength, sprint and jump measures, respectively. Maturity related inadequacies in eccentric strength and balance might influence the lack of training specificity with the unilateral landings and propulsions associated with sprinting. Based on this meta-analysis, strength training should be incorporated prior to power training in order to establish an adequate foundation of strength for power training activities.

Unilateral Rolling of the Foot did not Affect Non-Local Range of Motion or Balance.

Non-local or crossover (contralateral and non-stretched muscles) increases in range-of-motion (ROM) and balance have been reported following rolling of quadriceps, hamstrings and plantar flexors. Since there is limited information regarding plantar sole (foot) rolling effects, the objectives of this study were to determine if unilateral foot rolling would affect ipsilateral and contralateral measures of ROM and balance in young healthy adults. A randomized within-subject design was used to examine non-local effects of unilateral foot rolling on ipsilateral and contralateral limb ankle dorsiflexion ROM and a modified sit-and-reach-test (SRT). Static balance was also tested during a 30 s single leg stance test. Twelve participants performed three bouts of 60 s unilateral plantar sole rolling using a roller on the dominant foot with 60 s rest intervals between sets. ROM and balance measures were assessed in separate sessions at pre-intervention, immediately and 10 minutes post-intervention. To evaluate repeated measures effects, two SRT pre-tests were implemented. Results demonstrated that the second pre-test SRT was 6.6% higher than the first pre-test (p = 0.009, d = 1.91). There were no statistically significant effects of foot rolling on any measures immediately or 10 min post-test. To conclude, unilateral foot rolling did not produce statistically significant increases in ipsilateral or contralateral dorsiflexion or SRT ROM nor did it affect postural sway. Our statistically non-significant findings might be attributed to a lower degree of roller-induced afferent stimulation due to the smaller volume of myofascia and muscle compared to prior studies. Furthermore, ROM results from studies utilizing a single pre-test without a sufficient warm-up should be viewed critically.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance.

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C-), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C-) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C-) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.

The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey.

Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na(+)-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

Synthesis of purine and 7-deazapurine nucleoside analogues of 6-N-(4-Nitrobenzyl)adenosine; inhibition of nucleoside transport and proliferation of cancer cells.

Human equilibrative nucleoside transporter 1 (hENT1) is a prototypical nucleoside transporter protein ubiquitously expressed on the cell surface of almost all human tissue. Given the role of hENT1 in the transport of nucleoside drugs, an important class of therapeutics in the treatment of various cancers and viral infections, efforts have been made to better understand the mechanisms by which hENT1 modulates nucleoside transport. To that end, we report here the design and synthesis of novel tool compounds for the further study of hENT1. The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by a Dimroth rearrangement to the 4-N-isomer or by fluoro-diazotization followed by SN Ar displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at micromolar concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2',3',5'-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1.

Transmembrane water-flux through SLC4A11: a route defective in genetic corneal diseases.

Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11(-/-) mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.