Search

Heliothine eggs are commonly found on agricultural commodities and ornamental plants transported through domestic and international commerce. Tobacco budworm [Chloridea (Heliothis) virescens (F.)], Chloridea subflexa (Guenée), and the corn earworm [Helicoverpa zea (Boddie)] are indigenous pests of the American continent. Interceptions of the Old World bollworm [Helicoverpa armigera (Hübner)] at various ports of entry into the United States have been detected due to the invasion of this pest in South America, adding to the complexity and importance of decision making at plant inspection stations. The ability to distinguish the eggs of C. virescens from C. subflexa and Helicoverpa species is a critical component for conducting risk assessments by quarantine authorities, taxonomists, and crop consultants. We developed a simple, rapid (~60 min), inexpensive ($0.06 per sample), and accurate (100% reliability) technique to distinguish C. virescens eggs from the possibility of being H. armigera based on the presence, number, and/or size of aeropyle holes on the primary ribs of eggs, near the micropylar rosette. In this location, aeropyles were easily visible at 40× magnification in 213 fresh and ethanol-preserved C. virescens eggs once treated with Hoyer’s solution. A small number of C. subflexa had one or two aeropyles on a few of the primary ribs, whereas no aeropyles were found on 411 H. zea and 269 H. armigera eggs analyzed using optical, electron, and confocal microscopy. We conclude that in most cases multiple aeropyle holes positively identify H. virescens eggs from H. zea or H. armigera, and C. subflexa. This technique potentially will reduce the number of specimens that need to be sent for molecular identification thereby saving program time and resources.

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. 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. 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. 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.

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.

Improved reproductive management allowed dairy cow pregnancies to be optimized for beef production. The objective of this sire-controlled study was to characterize effects of maternal genetics and the dairy management system on calf growth. Pregnancies were created with a 2 × 2 factorial arrangement of dam breed (Holstein or Jersey) and mating type (artificial insemination or implantation of an in vitro produced embryo from a commercial beef cow oocyte). Resulting calves were reared in a calf ranch. Additionally, commercial beef cows were inseminated and reared resulting calves on range. Therefore, the five treatments were Angus × Holstein (A×H; n = 19), Angus × Jersey (A×J; n = 22), Angus × Beef gestated by Holstein (H-ET; n = 18), Angus × Beef gestated by Jersey (J-ET; n = 8), and Angus × Beef raised by beef (A×B; n = 20). Beginning at birth, calf body weight, cannon circumference, forearm circumference, top width, hip width, and hip height were measured approximately every 28 days until approximately 196 days of age. At birth, A×J calves weighed the least (P < 0.01). At 150 days of age, body weight was greatest (P < 0.05) among A×B calves, intermediate among H-ET and A×H calves, and least among J-ET and A×J calves (P < 0.05). Morphometric differences were detected between treatments (MANOVA P < 0.01). Primary discriminant function scores identified A×B calves having lesser values than A×J or A×H calves (ANOVA P < 0.01); A×B calves had greater cannon circumference, greater top width, and less hip height (standardized loadings of -0.47, -0.48, and 0.63, respectively). The dairy management system limited growth rate of beef genetics compared to the beef management system. In addition, Holstein dams transmitted greater growth potential than Jersey dams. Replacing maternal dairy genetics with beef genetics increased top width and decreased hip height.

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.

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.

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 ...

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.

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.

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.

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.

Roller massage (RM) has been reported to reduce pain associated with exercise-induced muscle soreness and increase range of motion without force or activation impairments. The objective was to examine RM effects on evoked pain and contractile properties. Twelve men received three sets of 30-s RM at a perceived discomfort level of 7/10 on a visual analogue scale on the ipsilateral (IPSI-R) stimulated plantar flexors (PF), contralateral PF (CONTRA-R), Sham (light rolling on stimulated PF), or Control. At pre-test, post-test, and 5-min post-test, they received evoked maximal twitch, tetanus, and 70% maximal tetanic stimulation, and performed a maximal voluntary isometric contraction (MVIC). Data analysis included perceived pain and contractile properties. The 70% tetanus illustrated significant 9–10% increases in pain perception with Sham and Control at post- and 5-min post-test, respectively (p

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.

The following chapter provides photographs with explanations of a variety of stretching exercises that can be employed within a standard athletic warm-up or dedicated flexibility training sessions. A standardized warm-up is presented in Table 11.1, which outlines in general the allocation of flexibility, mobility, and activation activities.

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.

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

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.

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.

Grabow, L, Young, JD, Alcock, LR, Quigley, PJ, Byrne, JM, Granacher, U, Škarabot, 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.

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.

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.

Nucleoside transport in humans is mediated by members of two unrelated protein families, the SLC28 family of cation-linked concentrative nucleoside transporters (CNTs) and the SLC29 family of energy-independent, equilibrative nucleoside transporters (ENTs). These families contain three and four members, respectively, which differ both in the stoichiometry of cation coupling and in permeant selectivity. Together, they play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis. Moreover, they facilitate cellular uptake of several nucleoside and nucleobase drugs used in cancer chemotherapy and treatment of viral infections. Thus, the transporter content of target cells can represent a key determinant of the response to treatment. In addition, by regulating the concentration of adenosine available to cell surface receptors, nucleoside transporters modulate many physiological processes ranging from neurotransmission to cardiovascular activity. This review describes the molecular and functional properties of the two transporter families, with a particular focus on their physiological roles in humans and relevance to disease treatment.

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K (i) values, 3-26 μM), hENT1/2 and hCNT2 weakly (K (i) values, 0.5-3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [(3)H]gemcitabine, [(14)C]azacitidine, and [(3)H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC (50) values, μM) conferred by hNTs were hCNT1 (0.1)hENT1 (0.3) ≫ hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3)hCNT1 (0.8) ⋙ hENT2, hCNT2 (100) for decitabine. Protection against cytotoxicity was observed for both drugs in the presence of inhibitors of nucleoside transport, thus suggesting the importance of hNTs in manifestation of toxicity. In summary, all seven hNTs transported azacitidine, with hCNT3 showing the highest rates, whereas hENT1 and hENT2 showed modest transport and hCNT1 and hCNT3 poor transport of decitabine. Our results show for the first time that azacitidine and decitabine exhibit different human nucleoside transportability profiles and their cytotoxicities are dependent on the presence of hNTs, which could serve as potential biomarkers of clinical response.

Adenosine concentrations are regulated by purinergic enzymes and nucleoside transporters. Transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1) have been generated (Parkinson et al., 2009 [7] ). The present study tested the hypothesis that mice homozygous and heterozygous for the transgene exhibit differences in hENT1 mRNA and protein expression, and in behavioral responses to caffeine and ethanol, two drugs with adenosine-dependent actions. Real time polymerase chain reaction (PCR) was used to identify mice heterozygous and homozygous for the transgene. Gene expression, determined by real time PCR of cDNA reverse transcribed from cerebral cortex RNA, was 3.8-fold greater in homozygous mice. Protein abundance, determined by radioligand binding assays using 0.14 nM [ 3 H]S-(4-nitrobenzyl)-6-thioinosine ([ 3 H]NBTI), was up to 84% greater in cortex synaptosome membranes from homozygous than from heterozygous mice. In western blots with an antibody specific for hENT1, a protein of approximately 40 kDa was strongly labelled in cortex samples from homozygous mice, weakly labelled in samples from heterozygous mice and absent from samples from wild type mice. In behavioral assays, transgenic mice showed a greater response to ethanol and a reduced response to caffeine than wild type littermates; however, no significant differences between heterozygous and homozygous mice were detected. These data indicate that the difference in ENT1 function between wild type and heterozygous mice was greater than that between heterozygous and homozygous mice. Therefore, either heterozygous or homozygous hENT1 transgenic mice can be used in studies of ENT1 regulation of adenosine levels and adenosine dependent behaviors.

Owing to the overlapping and redundant roles of the seven mammalian nucleoside transporters (NTs), which belong to two protein families (ENTs and CNTs), the physiological importance of individual NTs has been difficult to assess. Mice that have NT genes knocked out can be a valuable tool in gaining an understanding of the NT proteins. We have generated a strain of mice that is homozygous for a disruption mutation between exons 2 and 3 of the mouse equilibrative nucleoside transporter, mENT1. We have undertaken a quantitative survey of NT gene expression in 10 tissues, as well as microarray analysis of heart and kidney, from wild-type and mENT1 knockout mice. Rather than a consistent change in expression of NT genes in all tissues of mENT1 knockout mice, a complex pattern of changes was found. Some genes, such as those encoding mCNT1 and mCNT3 in colon tissue, exhibited increased expression, whereas other genes, such as those encoding mCNT2 and mENT4 in lung tissue, exhibited decreased expression. Although mCNT3 has been shown to be important in human and rat kidney tissue, we were unable to detect mCNT3 transcripts in the kidney of either the wild-type or mENT1 knockout mice, suggesting differences in renal nucleoside resorption between species.

Human concentrative nucleoside transporters (hCNTs) mediate electrogenic secondary active transport of physiological nucleosides and nucleoside drugs into cells. Six fused-pyrimidine ribonucleosides and one 2'-deoxynucleoside were assessed for their abilities to inhibit [(3)H]uridine transport in the yeast Saccharomyces cerevisiae producing recombinant hCNT1, hCNT2 or hCNT3. Six of the analogs inhibited hCNT1 with K(i) values1μM whereas only two analogs inhibited hCNT3 with K(i) values1μM and none inhibited hCNT2. To assess if the inhibitory analogs were also permeants, currents evoked were measured in oocytes of Xenopus laevis producing recombinant hCNT1, hCNT2 or hCNT3. Significant inward currents, indicating permeant activity, were generated with (i) three of the analogs in hCNT1-producing oocytes, (ii) none of the analogs in hCNT2-producing oocytes and (iii) all of the analogs in hCNT3-producing oocytes. Four were not, or were only very weakly, transported by hCNT1. The thienopyrimidine 2'-deoxynucleoside (dMeThPmR, 3) and ribonucleoside (MeThPmR, 4) were the most active inhibitors of uridine transport in hCNT1-producing oocytes and were an order of magnitude more effective than adenosine, a known low-capacity transport inhibitor of hCNT1. Neither was toxic to cultured human leukemic CEM cells, and both protected CEM cell lines with hCNT1 but not with hENT1 against gemcitabine cytotoxicity. In summary, dMeThPmR (3) and MeThPmR (4) were potent inhibitors of hCNT1 with negligible transportability and little apparent cytotoxicity, suggesting that pending further evaluation for toxicity against normal cells, they may have utility in protecting normal hCNT1-producing tissues from toxicities resulting from anti-cancer nucleoside drugs that enter via hCNT1.

Nucleosides with an aromatic five-membered ring heterocycle (N, O, or S) fused at C4–C5 of pyrimidin-2-one were prepared by ring closures with 5-(alkyn-1-yl)pyrimidin-2-one intermediates, heterocyclic atom replacements, and ring closure with a 5-aminocytidine derivative. Ultraviolet absorption and emission properties of the autofluorescent products enabled studies on permeation and inhibition of the trans-cellular trafficking effected by human equilibrative nucleoside transporters (hENTs). Some of the autofluorescent nucleosides were shown to be potent and selective inhibitors of human concentrative nucleoside transporters (hCNTs) in a companion study reported elsewhere.

(18)F-3'-Deoxy-3'-fluorothymidine ((18)F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for (18)F-FLT uptake in normal tissues and tumors.ENT1-knockout (ENT1(-/-)) mice were generated and compared with wild-type (ENT1(+/+)) mice using small-animal (18)F-FLT PET. In addition, ENT1(+/+) mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by (18)F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and (18)F-FLT uptake using real-time polymerase chain reaction and (3)H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by (18)F-FLT small-animal PET.Compared with noninjected ENT1(+/+) mice, ENT1(+/+) mice injected with NBMPR-P and ENT1(-/-) mice displayed a reduced percentage injected dose per gram (%ID/g) for (18)F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for (18)F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1(-/-) mice displayed 1.65-fold greater plasma thymidine levels than did ENT1(+/+) mice. Spleen tissue from ENT1(+/+) and ENT1(-/-) mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold (3)H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection.Loss of ENT1 activity significantly affected (18)F-FLT biodistribution in mice and (18)F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of (18)F-FLT uptake in normal and transformed cells.

The abundance of human equilibrative nucleoside transporter 1 (hENT1) has recently been shown to be a predictive marker of benefit from gemcitabine therapy in patients with pancreatic cancer. Since hENT1 is also important for the uptake of positron emission tomography (PET) tracer 3′-deoxy-3′-fluorothymidine (FLT) in various cultured human cell lines, this study was undertaken to determine if FLT uptake predicts gemcitabine uptake and/or toxicity in a panel of human pancreatic cancer cell lines (Capan-2, AsPC-1, BxPC-3, PL45, MIA PaCa-2, and PANC-1). Capan-2 cells displayed the lowest levels of (1) extracellular nitrobenzylmercaptopurine ribonucleoside (NBMPR) binding, which represents cell-surface hENT1, (2) FLT and gemcitabine uptake during short (1–45 s) and prolonged (1 h) periods, and (3) gemcitabine sensitivity. Exposure to NBMPR (inhibits only hENT1) or dilazep (inhibits hENT1 and hENT2) reduced FLT and gemcitabine uptake and gemcitabine sensitivity, with dilazep having greater effects than NBMPR. Gemcitabine permeation was almost completely mediated, primarily by hENT1 and to a lesser extent by hENT2, whereas FLT permeation included a substantial component of passive diffusion. In five of six cell lines, correlations were observed between (1) FLT and gemcitabine initial rates of uptake, (2) gemcitabine uptake and gemcitabine toxicity, (3) FLT uptake and gemcitabine toxicity, and (4) ribonucleotide reductase subunit M1 expression and gemcitabine toxicity. FLT and gemcitabine uptake were comparable for predicting gemcitabine toxicity in the tested pancreatic cancer cell lines suggesting that FLT PET may provide clinically useful information about tumor gemcitabine transport capacity and sensitivity.

This review describes recent advances in developing human nucleoside transporters (hNTs) as biomarkers to predict response to nucleoside analog drugs with clinical activity. Understanding processes that contribute to drug response or lack thereof will provide strategies to potentiate efficacy or avoid toxicities of nucleoside analog drugs. hNT abundance, evaluated by immunohistochemical methods, has shown promise as a predictive marker to assess clinical drug response that could be used to identify patients who would most likely benefit from nucleoside analog drug treatment.

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na(+) and H(+) to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na(+)/nucleoside and H(+)/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H(+) dependence (all mutants), shift in Na(+):nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na(+)-specific hCNT1, uncoupled Na(+) currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na+-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H+/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C-exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na+ and not H+. Site-directed mutagenesis experiments in wild-type and hCNT3C-backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na+-binding phenotype. Alanine at this position restored Na+ binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H+-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H+-induced conformational state of the protein (Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem.283 ,8496 -850718199742). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na+- as well as H+-coupled modes of hCNT3 nucleoside transport.

1. The human (h) SLC29 family of integral membrane proteins is represented by four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterized family member, hENT1. They belong to the widely distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporter proteins. 2. A predicted topology of eleven transmembrane helices has been experimentally confirmed for hENT1. The best-characterized members of the family, hENT1 and hENT2, possess similar broad permeant selectivities for purine and pyrimidine nucleosides, but hENT2 also efficiently transports nucleobases. hENT3 has a similar broad permeant selectivity for nucleosides and nucleobases and appears to function in intracellular membranes, including lysosomes. 3. hENT4 is uniquely selective for adenosine, and also transports a variety of organic cations. hENT3 and hENT4 are pH sensitive, and optimally active under acidic conditions. ENTs, including those in parasitic protozoa, function in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and, in humans, are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. 4. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission.

The Nantucket pine tip moth, Rhyacionia frustrana (Scudder in Comstock) is a common pest of several southern pine species. Some tip moth research requires the sexing of pupae in order to collect emerging adults of known ages or unmated females. Characters for determining the sex of tip moth pupae were originally described in 1969. Here, we revise one of the published characters and describe seven new characters that may be used for gender determination. The new sexually dimorphic characters are: articulation of the seventh abdominal segment, dorsal spines of the second abdominal segment, length of the metathoracic tarsi, distribution of cremasteral setae, shape of the mandibular sclerite, antenna length and antenna shape.

Interpatient variability in renal elimination of fludarabine (9-beta-D-arabinosyl-2-fluoroadenine) by renal human nucleoside transporters (hNTs) may contribute to unpredictable toxicities including rare nephrotoxicities. This study assessed relationships between hNT levels and fludarabine uptake and cytotoxicity in cultures of human renal proximal tubule cells (hRPTCs) that produce multiple transporter types.hRPTC cultures were established from ten different individuals and their hNT characteristics were assessed by measuring RNA expression by TaqMantrade mark reverse transcriptase polymerase chain reaction, protein abundance by quantitative immunoblotting of cell surface protein preparations, and uptake by radiolabeled nucleoside uptake assays. Fludarabine cytotoxicity against hRPTC cultures was quantified using methoxyphenyl tetrazolium inner salt (MTS) assays.RNA, protein and activities for human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) were identified in cultures of hRPTCs from ten different individuals. Significant differences in hCNT3 activities were exhibited among hRPTC cultures and correlated positively with cell surface levels of hCNT3 protein, but did not correlate with hCNT3 mRNA levels.Observed differences in hCNT3-mediated uptake activities, hNT-mediated fludarabine uptake activities, and fludarabine cytotoxicities correlated positively with each other, suggesting that hCNT3 is a primary determinant of fludarabine uptake and cytotoxicity in hRPTC cultures. Variations in hCNT3 abundance in renal proximal tubules, and hence nucleoside reabsorption, may explain interpatient variability in fludarabine's pharmacokinetics and toxicities.

Nucleoside analogue chemotherapy has assumed increasing importance in the therapy of many hematological malignancies. Although fludarabine is a front-line therapy for low-grade lymphomas such as sm...

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50Na+ and/or Kmuridine values and diminished VmaxNa+ and Vmaxuridine values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays. hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [3H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [3H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs.

The Na + /nudeoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser 319 -Gln 320 and Ser 353 -Leu 354 ) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T-and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNTI mutation S353T produced a profound decrease in cytidine transport efficiency (V max /K m ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNTI for Na + and Li + . Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore.

Renal handling of physiological and pharmacological nucleosides is a major determinant of their plasma levels and tissue availabilities. Additionally, the pharmacokinetics and normal tissue toxicities of nucleoside drugs are influenced by their handling in the kidney. Renal reabsorption or secretion of nucleosides is selective and dependent on integral membrane proteins, termed nucleoside transporters (NTs) present in renal epithelia. The 7 known human NTs (hNTs) exhibit varying permeant selectivities and are divided into 2 protein families: the solute carrier (SLC) 29 (SLC29A1, SLC29A2, SLC29A3, SLC29A4) and SLC28 (SLC28A1, SLC28A2, SLC28A3) proteins, otherwise known, respectively, as the human equilibrative NTs (hENTs, hENT1, hENT2, hENT3, hENT4) and human concentrative NTs (hCNTs, hCNT1, hCNT2, hCNT3). The well characterized hENTs (hENT1 and hENT2) are bidirectional facilitative diffusion transporters in plasma membranes; hENT3 and hENT4 are much less well known, although hENT3, found in lysosomal membranes, transports nucleosides and is pH dependent, whereas hENT4–PMAT is a H+-adenosine cotransporter as well as a monoamine–organic cation transporter. The 3 hCNTs are unidirectional secondary active Na+-nucleoside cotransporters. In renal epithelial cells, hCNT1, hCNT2, and hCNT3 at apical membranes, and hENT1 and hENT2 at basolateral membranes, apparently work in concert to mediate reabsorption of nucleosides from lumen to blood, driven by Na+gradients. Secretion of some physiological nucleosides, therapeutic nucleoside analog drugs, and nucleotide metabolites of therapeutic nucleoside and nucleobase drugs likely occurs through various xenobiotic transporters in renal epithelia, including organic cation transporters, organic anion transporters, multidrug resistance related proteins, and multidrug resistance proteins. Mounting evidence suggests that hENT1 may have a presence at both apical and basolateral membranes of renal epithelia, and thus may participate in both selective secretory and reabsorptive fluxes of nucleosides. In this review, the renal handling of nucleosides is examined with respect to physiological and clinical implications for the regulation of human kidney NTs and adenosine signaling, intracellular nucleoside transport, and nephrotoxicities associated with some nucleoside drugs.

Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K m values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia.

Hydrophilic purine and pyrimidine nucleosides rely on specialized carrier proteins for their membrane translocation. The recent identification of two gene families encoding equilibrative and concentrative nucleoside transporters in mammals and other organisms has provided the essential breakthrough to a more complete understanding of the biological significance of nucleoside transport. Although nucleoside salvage is a primary function of these proteins, recent data indicate functions beyond metabolic recycling. In brain and spinal cord, for example, nucleoside transporters have the potential to regulate synaptic levels of neuroactive purines such as adenosine and, thereby, indirectly modulate physiological processes through G-protein-coupled purine P1 receptors. As described in this review, recent research indicates novel putative functions for CNS nucleoside transporters in sleep, arousal, drug and alcohol addiction, nociception and analgesia. The therapeutic use of nucleoside analogue drugs and nucleoside transporter inhibitors in viral, neoplastic, cardiovascular and infectious disease is also described.

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.