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ATP‐sensitive K+ channels (KATP) have been implicated in the regulation of resting vascular smooth muscle membrane potential and tone. However, whether KATP channels modulate skeletal muscle microvascular hemodynamics at the capillary level (the primary site for blood‐myocyte O2 exchange) remains unknown. We tested the hypothesis that KATP channel inhibition would reduce the proportion of capillaries supporting continuous red blood cell (RBC) flow and impair RBC hemodynamics and distribution in perfused capillaries within resting skeletal muscle. RBC flux (fRBC), velocity (VRBC), and capillary tube hematocrit (Hctcap) were assessed via intravital microscopy of the rat spinotrapezius muscle (n = 6) under control (CON) and glibenclamide (GLI; KATP channel antagonist; 10 µM) superfusion conditions. There were no differences in mean arterial pressure (CON:120 ± 5, GLI:124 ± 5 mmHg; p > 0.05) or heart rate (CON:322 ± 32, GLI:337 ± 33 beats/min; p > 0.05) between conditions. The %RBC‐flowing capillaries were not altered between conditions (CON:87 ± 2, GLI:85 ± 1%; p > 0.05). In RBC‐perfused capillaries, GLI reduced fRBC (CON:20.1 ± 1.8, GLI:14.6 ± 1.3 cells/s; p 0.05). The absence of GLI effects on the %RBC‐flowing capillaries and Hctcap indicates preserved muscle O2 diffusing capacity (DO2m). In contrast, GLI lowered both fRBC and VRBC thus impairing perfusive microvascular O2 transport (Q̇m) and lengthening RBC capillary transit times, respectively. Given the interdependence between diffusive and perfusive O2 conductances (i.e., %O2 extraction∝DO2m/Q̇m), such GLI alterations are expected to elevate muscle %O2 extraction to sustain a given metabolic rate. These results support that KATP channels regulate capillary hemodynamics and, therefore, microvascular gas exchange in resting skeletal muscle.

Lower‐extremity peripheral artery disease (PAD) is associated with increased risk of cardiovascular events and impaired exercise tolerance. We have previously reported that leg heat therapy (HT) applied using liquid‐circulating trousers perfused with warm water increases leg blood flow and reduces blood pressure (BP) and the circulating levels of endothelin‐1 (ET‐1) in patients with symptomatic PAD. In this sham‐controlled, randomized, crossover study, sixteen patients with symptomatic PAD (age 65 ± 5.7 years and ankle‐brachial index: 0.69 ± 0.1) underwent a single 90‐min session of HT or a sham treatment prior to a symptom‐limited, graded cardiopulmonary exercise test on the treadmill. The primary outcome was the peak walking time (PWT) during the exercise test. Secondary outcomes included the claudication onset time (COT), resting and exercise BP, calf muscle oxygenation, pulmonary oxygen uptake (V̇O2), and plasma levels of ET‐1, interleukin‐6 (IL‐6) and tumor necrosis factor‐alpha (TNF‐α). Systolic, but not diastolic BP, was significantly lower (~7 mmHg, p, We show that a single session of leg heat therapy (HT) reduces systolic blood pressure and lowers post‐exercise circulating levels of the potent vasoconstrictor endothelin‐1. Leg HT also tended to improve peak walking time during a graded treadmill test. This practical and well‐tolerated therapy may be a useful adjunctive tool to improve cardiovascular health and restore functional capacity in PAD patients.

KEY POINTS Oxygen pressure gradients across the microvascular walls are essential for oxygen diffusion from blood to tissue cells. At any given flux, the magnitude of these transmural gradients is proportional to the local resistance. The greatest resistance to oxygen transport into skeletal muscle is considered to reside in the short distance between red blood cells and myocytes. Although crucial to oxygen transport, little is known about transmural pressure gradients within skeletal muscle during contractions. We evaluated oxygen pressures within both the skeletal muscle microvascular and interstitial spaces to determine transmural gradients during the rest-contraction transient in anaesthetized rats. The significant transmural gradient observed at rest was sustained during submaximal muscle contractions. Our findings support that the blood-myocyte interface provides substantial resistance to oxygen diffusion at rest and during contractions and suggest that modulations in microvascular haemodynamics and red blood cell distribution constitute primary mechanisms driving increased transmural oxygen flux with contractions. ABSTRACT Oxygen pressure (PO2) gradients across the blood-myocyte interface are required for diffusive O2 transport, thereby supporting oxidative metabolism. The greatest resistance to O2 flux into skeletal muscle is considered to reside between the erythrocyte surface and adjacent sarcolemma, although this has not been measured during contractions. We tested the hypothesis that O2 gradients between skeletal muscle microvascular (PO2 mv ) and interstitial (PO2 is ) spaces would be present at rest and maintained or increased during contractions. PO2 mv and PO2 is were determined via phosphorescence quenching (Oxyphor probes G2 and G4, respectively) in the exposed rat spinotrapezius during the rest-contraction transient (1 Hz, 6 V; n = 8). PO2 mv was higher than PO2 is in all instances from rest (34.9 ± 6.0 versus 15.7 ± 6.4) to contractions (28.4 ± 5.3 versus 10.6 ± 5.2 mmHg, respectively) such that the mean PO2 gradient throughout the transient was 16.9 ± 6.6 mmHg (P 0.05). However, the speed of the PO2 is fall during contractions was slower than that of PO2 mv (time constant: 12.8 ± 4.7 versus 9.0 ± 5.1 s, respectively; P

Key points • Neuronal nitric oxide (NO) synthase (nNOS) inhibition does not impact skeletal muscle blood flow or vascular conductance (VC) during low-speed (20 m min−1) treadmill running. • This may be due to the fact that low exercise intensities recruit primarily oxidative muscle and that nNOS-derived NO contributes to vascular control primarily within glycolytic muscle. • Rats ran in the severe-intensity domain at 15% above critical speed (an important glycolytic fast-twitch fibre recruitment boundary in the rat) before and after selective nNOS inhibition with S-methyl-l-thiocitrulline (SMTC). • SMTC reduced blood flow and VC during supra-critical speed treadmill running (52.5 ± 1.3 m min−1) with the greatest proportional reductions observed in glycolytic fast-twitch compared to oxidative slow- and fast-twitch muscle. There were no effects of SMTC on muscle blood flow or VC during low-speed running (20 m min−1). • The present data reveal important fibre-type- and exercise intensity-dependent peripheral vascular effects of nNOS-derived NO during whole-body exercise. Abstract We have recently shown that nitric oxide (NO) derived from neuronal NO synthase (nNOS) does not contribute to the hyperaemic response within rat hindlimb skeletal muscle during low-speed treadmill running. This may be attributed to low exercise intensities recruiting primarily oxidative muscle and that vascular effects of nNOS-derived NO are manifest principally within glycolytic muscle. We tested the hypothesis that selective nNOS inhibition via S-methyl-l-thiocitrulline (SMTC) would reduce rat hindlimb skeletal muscle blood flow and vascular conductance (VC) during high-speed treadmill running above critical speed (asymptote of the hyperbolic speed versus time-to-exhaustion relationship for high-speed running and an important glycolytic fast-twitch fibre recruitment boundary in the rat) principally within glycolytic fast-twitch muscle. Six rats performed three high-speed treadmill runs to exhaustion to determine critical speed. Subsequently, hindlimb skeletal muscle blood flow (radiolabelled microspheres) and VC (blood flow/mean arterial pressure) were determined during supra-critical speed treadmill running (critical speed + 15%, 52.5 ± 1.3 m min−1) before (control) and after selective nNOS inhibition with 0.56 mg kg−1 SMTC. SMTC reduced total hindlimb skeletal muscle blood flow (control: 241 ± 23, SMTC: 204 ± 13 ml min−1 (100 g)−1, P < 0.05) and VC (control: 1.88 ± 0.20, SMTC: 1.48 ± 0.13 ml min−1 (100 g)−1 mmHg−1, P < 0.05) during high-speed running. The relative reductions in blood flow and VC were greater in the highly glycolytic muscles and muscle parts consisting of 100% type IIb+d/x fibres compared to the highly oxidative muscles and muscle parts consisting of ≤35% type IIb+d/x muscle fibres (P < 0.05). These results extend our understanding of vascular control during exercise by identifying fibre-type-selective peripheral vascular effects of nNOS-derived NO during high-speed treadmill running.

Dietary nitrate (NO(3)(-)) supplementation, via its reduction to nitrite (NO(2)(-)) and subsequent conversion to nitric oxide (NO) and other reactive nitrogen intermediates, reduces blood pressure and the O(2) cost of submaximal exercise in humans. Despite these observations, the effects of dietary NO(3)(-) supplementation on skeletal muscle vascular control during locomotory exercise remain unknown. We tested the hypotheses that dietary NO(3)(-) supplementation via beetroot juice (BR) would reduce mean arterial pressure (MAP) and increase hindlimb muscle blood flow in the exercising rat. Male Sprague-Dawley rats (3-6 months) were administered either NO(3)(-) (via beetroot juice; 1 mmol kg(-1) day(-1), BR n = 8) or untreated (control, n = 11) tap water for 5 days. MAP and hindlimb skeletal muscle blood flow and vascular conductance (radiolabelled microsphere infusions) were measured during submaximal treadmill running (20 m min(-1), 5% grade). BR resulted in significantly lower exercising MAP (control: 137 ± 3, BR: 127 ± 4 mmHg, P < 0.05) and blood [lactate] (control: 2.6 ± 0.3, BR: 1.9 ± 0.2 mm, P < 0.05) compared to control. Total exercising hindlimb skeletal muscle blood flow (control: 108 ± 8, BR: 150 ± 11 ml min(-1) (100 g)(-1), P < 0.05) and vascular conductance (control: 0.78 ± 0.05, BR: 1.16 ± 0.10 ml min(-1) (100 g)(-1) mmHg(-1), P < 0.05) were greater in rats that received BR compared to control. The relative differences in blood flow and vascular conductance for the 28 individual hindlimb muscles and muscle parts correlated positively with their percentage type IIb + d/x muscle fibres (blood flow: r = 0.74, vascular conductance: r = 0.71, P < 0.01 for both). These data support the hypothesis that NO(3)(-) supplementation improves vascular control and elevates skeletal muscle O(2) delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO(3)(-) supplementation improves metabolic control.

Key points • Nitric oxide (NO) is an important vasodilatory signalling molecule that regulates O2 pressure within the skeletal muscle microvasculature (). In healthy subjects, NO is derived from two principal NO synthase (NOS) isoforms: neuronal NOS (nNOS) and endothelial NOS (eNOS). • Chronic heart failure (CHF) results in peripheral vascular dysfunction that is attributed, in part, to impaired NO function. This NO-mediated impairment is attributed generally to eNOS dysfunction. It is unknown if nNOS-mediated regulation of function is impaired in CHF. • Our present results demonstrate that skeletal muscle blood flow reductions and alterations during contractions observed following nNOS inhibition in healthy rats are markedly attenuated or absent in CHF rats, which is indicative of impaired nNOS function. • Identification of the mechanisms underlying impaired microvascular function in CHF is an important step in the development of treatments designed to improve CHF-induced skeletal muscle microvascular pathology. Abstract Chronic heart failure (CHF) impairs nitric oxide (NO)-mediated regulation of the skeletal muscle microvascular O2 delivery/ ratio (which sets the microvascular O2 pressure, ). Given the pervasiveness of endothelial dysfunction in CHF, this NO-mediated dysregulation is attributed generally to eNOS. It is unknown whether nNOS-mediated regulation is altered in CHF. We tested the hypothesis that CHF impairs nNOS-mediated control. In healthy and CHF (left ventricular end diastolic pressure (LVEDP): 6 ± 1 versus 14 ± 1 mmHg, respectively, P 0.05), at rest, or the MRT (Control: 22.8 ± 2.6, SMTC: 21.3 ± 3.0 s, P > 0.05). During the contracting steady-state, SMTC reduced blood flow (↓15%) and (↓15%) in healthy rats such that was unaltered (Control: 19.8 ± 1.7, SMTC: 20.7 ± 1.8 mmHg, P > 0.05). In marked contrast, in CHF rats SMTC did not change contracting steady-state blood flow, , or (Control: 17.0 ± 1.4, SMTC: 17.7 ± 1.8 mmHg, P > 0.05). nNOS-mediated control of skeletal muscle microvascular function is compromised in CHF versus healthy rats. Treatments designed to ameliorate microvascular dysfunction in CHF may benefit by targeting improvements in nNOS function.

Please cite this paper as: Copp, Hirai, Ferguson, Musch and Poole (2011). Role of Neuronal Nitric Oxide Synthase in Modulating Microvascular and Contractile Function in Rat Skeletal Muscle. Microcirculation 18(6), 501–511. Abstract Objective: This investigation tested the hypothesis that selective nNOS inhibition would lower the dynamic microvascular O2 delivery/utilization () balance (which sets the Po2mv) in rat skeletal muscle at rest and during contractions. Methods: Anesthetized male Sprague–Dawley rats had their spinotrapezius muscles exposed for blood flow (radiolabeled microspheres), (direct Fick calculation), Po2mv (phosphorescence quenching), or exteriorized for force production measurement during electrically induced contractions (1 Hz, 6–8 V, 180 seconds) pre- and post-nNOS inhibition with 2.1 μmol/kg of the selective nNOS inhibitor SMTC. Results: At rest, spinotrapezius blood flow was not different whereas SMTC reduced (27%) resulting in an elevated precontracting baseline Po2mv (control: 31.2 ± 1.6, SMTC: 37.1 ± 2.0 mmHg, p 0.05) which occurred despite an elevated (∼8%) muscle force production. Conclusions: These data demonstrate important physiological roles for nNOS-derived NO during contractions in healthy rat skeletal muscle and implicate maladaptations in nNOS function in pathological conditions associated with reduced NO bioavailability.

The O2 requirements of contracting skeletal muscle may increase 100-fold above rest. In 1919 August Krogh’s brilliant insights recognized the capillary as the principal site for this increased blood-myocyte O2 flux. Based on the premise that most capillaries did not sustain RBC flux at rest Krogh proposed that capillary recruitment (i.e., initiation of red blood cell (RBC) flux in previously non-flowing capillaries) increased the capillary surface area available for O2 flux and reduced mean capillary-to-mitochondrial diffusion distances. More modern experimental approaches reveal that most muscle capillaries may support RBC flux at rest. Thus, rather than contraction-induced capillary recruitment per se, increased RBC flux and hematocrit within already-flowing capillaries likely elevate perfusive and diffusive O2 conductances and hence blood-myocyte O2 flux. Additional surface area for O2 exchange is recruited but, crucially, this may occur along the length of already-flowing capillaries (i.e. longitudinal recruitment). Today, the capillary is still considered the principal site for O2 and substrate delivery to contracting skeletal muscle. Indeed, the presence of very low intramyocyte O2 partial pressures (PO2’s) and the absence of PO2 gradients, whilst refuting the relevance of diffusion distances, place an even greater importance on capillary hemodynamics. This emergent picture calls for a paradigm-shift in our understanding of the function of capillaries by de-emphasizing de novo ‘capillary recruitment.’ Diseases such as heart failure impair blood-myocyte O2 flux, in part, by decreasing the proportion of RBC-flowing capillaries. Knowledge of capillary function in healthy muscle is requisite for identification of pathology and efficient design of therapeutic treatments.

Critical speed (CS) constitutes an important metabolic and performance demarcator. However, active skeletal muscle blood flow distribution specifically surrounding CS remains unknown. We tested the hypotheses that CS could be accurately determined in the running rat and that measurement of hindlimb inter- and intramuscular blood flow below and above CS would support that the greatest muscle fibre recruitment above, relative to below, CS occurs in the predominantly glycolytic muscles. Seven male Sprague–Dawley rats performed five constant-speed tests to exhaustion at speeds between 95 and 115% of the speed that elicited to determine CS. Subsequent constant-speed tests were performed at speeds incrementally surrounding CS to determine time to exhaustion, , and hindlimb muscle blood flow distribution. Speed and time to exhaustion conformed to a hyperbolic relationship (r2= 0.92 ± 0.03) which corresponded to a linear 1/time function (r2= 0.93 ± 0.02) with a CS of 48.6 ± 1.0 m min−1. Time to exhaustion below CS was ∼5× greater (P 0.05 vs. above CS). The 11 individual muscles or muscle parts that evidenced the greatest blood flow increases above, relative to below, CS were composed of ≥69% Type IIb/d/x muscle fibres. Moreover, there was a significant correlation (P < 0.05, r= 0.42) between the increased blood flow above expressed relative to below CS and the percentage Type IIb/d/x fibres found in the individual muscles or muscle parts. These data validate the powerful CS construct in the rat and identify that running above CS, relative to below CS, incurs disproportionate blood flow increases (indicative of recruitment) in predominantly highly glycolytic muscle fibres.

Mass mortality of cultured yellowtail, Seriola quinqueradiata, has recently been reported from fish farms in western Japan. Previous studies revealed that diseased fish were characterized by encephalomyelitis and presporogonic stages of a myxosporean-like parasite in the spinal cord. However, the parasite has remained unidentified because of the lack of mature stages being present. Thus, in the present study, analysis of the small subunit ribosomal DNA (18S rDNA) of the parasite as well as in situ hybridization (ISH) studies using histological sections of the infected tissue was conducted. The 18S rDNA of the myxosporean had higher sequence similarities with those of bile-duct-infecting myxosporeans rather than those infecting nervous tissues and was identified as Myxobolus spirosulcatus. The ISH using specific probes demonstrated that the DNA amplified was derived from the multinuclear organisms found in histological sections. A highly sensitive and specific PCR-based assay for M. spirosulcatus was developed, which revealed a high prevalence of infection in cultured yellowtail that exhibited the clinical signs of encephalomyelitis.

The oxygen transport pathway from air to mitochondria involves a series of transfer steps within closely integrated systems (pulmonary, cardiovascular and tissue metabolic). Small and finite O(2) stores in most mammalian species require exquisitely controlled changes in O(2) flux rates to support elevated ATP turnover. This is especially true for the contracting skeletal muscle where O(2) requirements may increase two orders of magnitude above rest. This brief review focuses on the mechanistic bases for increased microvascular blood-myocyte O(2) flux ([Formula: see text] O(2)) from rest-to-contractions. Fick’s law dictates that [Formula: see text] O(2) elevations driven by muscle contractions are produced by commensurate changes in driving force (i.e., O(2) pressure gradients; ΔPO(2)) and/or effective diffusing capacity (DO(2)). While previous evidence indicates that increased DO(2) helps modulate contracting muscle O(2) flux, up until recently the role of the dynamic ΔPO(2) across the capillary wall was unknown. Recent phosphorescence quenching investigations of both microvascular and novel interstitial PO(2) kinetics in health have resolved an important step in the O(2) cascade between the capillary and myocyte. Specifically, the significant transmural ΔPO(2) at rest was sustained (but not increased) during submaximal contractions. This supports the contention that the blood-myocyte interface provides a substantial effective resistance to O(2) diffusion and underscores that modulations in erythrocyte hemodynamics and distribution (DO(2)) are crucial to preserve the driving force for O(2) flux across the capillary wall (ΔPO(2)) during contractions. Investigation of the O(2) transport pathway close to muscle mitochondria is key to identifying disease mechanisms and develop therapeutic approaches to ameliorate dysfunction and exercise intolerance.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is an integral mediator of vascular control during muscle contractions. However, it is not known whether neuronal NOS (nNOS)-derived NO regulates tissue hyperaemia in healthy subjects, particularly during exercise. We tested the hypothesis that selective nNOS inhibition would reduce blood flow and vascular conductance (VC) in rat hindlimb locomotor muscle(s), kidneys and splanchnic organs at rest and during dynamic treadmill exercise (20 m min−1, 10% grade). Nineteen male Sprague–Dawley rats (555 ± 23 g) were assigned to either rest (n= 9) or exercise (n= 10) groups. Blood flow and VC were determined via radiolabelled microspheres before and after the intra-arterial administration of the selective nNOS inhibitor S-methyl-l-thiocitrulline (SMTC, 2.1 ± 0.1 μmol kg−1). Total hindlimb muscle blood flow (control: 20 ± 2 ml min−1 100g−1, SMTC: 12 ± 2 ml min−1 100g−1, P 0.05) between control and SMTC conditions. SMTC reduced (P < 0.05) blood flow and VC at rest and during exercise in the kidneys, adrenals and liver. These results enhance our understanding of the role of NO-mediated circulatory control by demonstrating that nNOS does not appear to subserve an obligatory role in the exercising muscle hyperaemic response in the rat.

Aim: Lowered microvascular PO 2 (PO 2 mv) during the exercise off-transient likely impairs muscle metabolic recovery and limits the capacity to perform repetitive tasks. The current investigation explored the impact of altered nitric oxide (NO) bioavailability on PO 2 mv during recovery from contractions in healthy skeletal muscle. We hypothesized that increased NO bioavailability (sodium nitroprusside: SNP) would enhance PO 2 mv and speed its recovery kinetics while decreased NO bioavailability ( L -nitro arginine methyl ester: L -NAME) would reduce PO 2 mv and slow its recovery kinetics. Methods: PO 2 mv was measured by phosphorescence quenching during transitions (rest-1 Hz twitch-contractions for 3 min-recovery) in the spinotrapezius muscle of Sprague-Dawley rats under SNP (300 μM), Krebs-Henseleit (Control) and L -NAME (1.5 mM) superfusion conditions. Results: Relative to recovery in Control, SNP resulted in greater overall microvascular oxygenation as assessed by the area under the PO 2 mv curve (PO 2 AREA; Control: 3471 ± 292 mmHg s; SNP: 4307 ± 282 mmHg s; P < 0.05) and faster off-kinetics as evidenced by the mean response time (MRToff; Control: 60.2 ± 6.9 s; SNP: 34.8 ± 5.7 s; P < 0.05), whereas L -NAME produced lower PO 2 AREA (2339 ± 444 mmHg s; P < 0.05) and slower MRToff (86.6 ± 14.5 s; P < 0.05). Conclusion: NO bioavailability plays a key role in determining the matching of O 2 delivery-to-O 2 uptake and thus the upstream O 2 pressure driving capillary-myocyte O 2 flux (i.e. PO 2 mv) following cessation of contractions in healthy skeletal muscle. Additionally, these data support a mechanistic link between reduced NO bioavailability and prolonged muscle metabolic recovery commonly observed in ageing and diseased populations.

Considerable interindividual differences in the Q ˙ − V ˙ O 2 relationship during exercise have been documented but implications for submaximal exercise tolerance have not been considered. We tested the hypothesis that these interindividual differences were associated with differences in exercising muscle deoxygenation and ratings of perceived exertion (RPE) across a range of submaximal exercise intensities. A total of 31 (21 ± 3 years) healthy recreationally active males performed an incremental exercise test to exhaustion 24 h following a resting muscle biopsy. Cardiac output ( Q ˙ L/min; inert gas rebreathe), oxygen uptake ( V ˙ O 2 L/min; breath‐by‐breath pulmonary gas exchange), quadriceps saturation (near infrared spectroscopy) and exercise tolerance (6–20; Borg Scale RPE) were measured. The Q ˙ − V ˙ O 2 relationship from 40 to 160 W was used to partition individuals post hoc into higher ( n = 10; 6.3 ± 0.4) versus lower ( n = 10; 3.7 ± 0.4, P Q ˙ − V ˙ O 2 difference between responder types was not explained by arterial oxygen content differences ( P = 0.5) or peripheral skeletal muscle characteristics ( P from 0.1 to 0.8) but was strongly associated with stroke volume ( P Q ˙ − V ˙ O 2 difference between groups, no difference in quadriceps deoxygenation was observed during exercise (all P > 0.4). Lower cardiac responders had greater leg ( P = 0.027) and whole body ( P = 0.03) RPE only at 185 W, but this represented a higher %peak V ˙ O 2 in lower cardiac responders (87 ± 15% vs. 66 ± 12%, P = 0.005). Substantially lower Q ˙ − V ˙ O 2 in the lower responder group did not result in altered RPE or exercising muscle deoxygenation. This suggests substantial recruitment of blood flow redistribution in the lower responder group as part of protecting matching of exercising muscle oxygen delivery to demand.

Alterations of skeletal muscle redox state via antioxidant supplementation have the potential to impact contractile function and vascular smooth muscle tone. The effects of antioxidants on the regulation of muscle O(2) delivery-O(2) utilization (Q(O(2)m/V(O(2)m)) matching (which sets the microvascular partial pressure of O(2); P(O(2)mv)) in young healthy muscle are not known. Therefore, the purpose of this study was to test the effects of acute antioxidant supplementation on rat spinotrapezius muscle force production, blood flow, V(O(2)m) and P(O(2)mv) (phosphorescence quenching). Anaesthetized male Fischer 344 x Brown Norway rats (6-8 months old) had their right spinotrapezius muscles either exposed for measurement of blood flow and (n = 13) or exteriorized for measurement of muscle force production (n = 6). Electrically stimulated 1 Hz twitch contractions (approximately 7-9 V) were elicited for 180 s, and measurements were made before and after acute intra-arterial antioxidant supplementation (76 mg kg(-1) ascorbic acid, 52 mg kg(-1) tempol) dissolved in saline and infused over 30 min. The principal effects of antioxidants were a approximately 25% decrease (P < 0.05) in contracting spinotrapezius muscle force production concurrent with reductions in muscle blood flow and V(O(2)m) at rest and during contractions (P < 0.05 for both). Antioxidant supplementation reduced the resting baseline P(O(2)mv) (before, 29.9 +/- 1.2 mmHg; after, 25.6 +/- 1.3 mmHg; P < 0.05), and this magnitude of depression was sustained throughout the rest-to-exercise transition (steady-state value before, 16.4 +/- 0.7 mmHg; after, 13.6 +/- 0.9 mmHg; P < 0.05). In addition, the time constant of the P(O(2)mv) decrease was reduced after antioxidant supplementation (before, 23.4 +/- 4.3 s; after, 15.6 +/- 2.7 s; P < 0.05). These results demonstrate that antioxidant supplementation significantly impacts the control of (Q(O(2)m/V(O(2)m)) in young rats at rest and during contractions.

Helicobacter pylori infection is causally related to chronic type-B gastritis, and may also be associated with an increased risk of gastric carcinoma. Vacuolating cytotoxin, which is an 87-kDa protein secreted by H. pylori, induces eukaryotic cell vacuolation in vitro. To determine whether there is an association between H. pylori vacuolating cytotoxin and gastric carcinoma, we investigated several characteristics of H. pylori infection, i.e., isolation of H. pylori from gastric biopsies, antibodies specific for H. pylori, detection of neutralizing activity to vacuolating cytotoxin in serum and immunological detection of cytotoxin by serum. Out of 6 sera from gastric-carcinoma patients, all showed the neutralizing activity to vacuolating cytotoxin, in contrast to 3 of 5 sera from peptic-ulcer patients. Normal individuals showed no neutralizing activity. All sera possessing the neutralizing activity recognized an 87-kDa protein band by Western blot analysis. Our results confirmed that cytotoxin-neutralizing activity in human sera was associated with immunodetection of an 87-kDa protein. To further evaluate neutralizing activity in serum from gastric-carcinoma patients, we retrospectively analyzed frozen-stocked serum samples from 22 gastric-carcinoma patients. Sera from 21 of these 22 patients exhibited neutralizing activity. These sera were also checked for antibodies to H. pylori, using an ELISA; 16 sera showed positive results. Our results indicate that detection of cytotoxin-neutralizing activity in sera is strongly associated with H. pylori infection, and probably with gastric carcinoma, and is also of interest in the diagnosis of H. pylori infection. © 1994 Wiley-Liss, Inc.

Summary Screening a genomic library of Lolium temulentum (darnel) with synthetic (GT)15 and (GA)15 oligonucleotides, we identified microsatellite-containing clones. Based on the sequence data of the clones, specific primer pairs were synthesized and their effectiveness examined using 10 L. temulentum and two L. persicum accessions from Asia, Africa and Europe. Fifteen primer pairs amplified single fragments, and five of them showed polymorphism in polyacrylamide gel electrophoresis in the L. temulentum and L. persicum accessions. Fifteen primer pairs reported in L. perenne (perennial ryegrass) were also applied in this study. Nine of them amplified the microsatellite alleles, and two of the nine showed polymorphic alleles in L. temulentum and L. persicum. For the polymorphic markers, polymorphic information content values ranged from 0.60 to 0.86 with an average of 0.76 among 10 L. temulentum accessions. The microsatellite markers developed for L. temulentum and L. perenne should be informative and powerful in analysing the intraspecific genetic diversity and the phylogenetic relationships of L. temulentum and L. persicum.

Objective: We have assessed prognostic factors and the efficacy of adjuvant chemotherapy in stage I uterine endometrial carcinoma. Methods: 251 primary surgically treated stage I patients were studied. Prognostic factors were evaluated and 5-year and 10-year survival rates were compared in patients with lymph-vascular space invasion to investigate whether adjuvant chemotherapy improves survival. Results: The overall 5-year and 10-year survival rates were 94% and 93%. Multivariate analysis indicates that lymph-vascular space invasion is the most significant prognostic factor in both 5- and 10-year survival rates (P

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT-PCR using degenerate primers based on the N-terminal amino acid sequence of purified hJHBP and a conserved region near the C-terminus of other lepidopteran hJHBPs. 5'- and 3'-ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP-mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS-PAGE with a Mr of 32 kDa and showed a Kd of 4.5 x 10-7 M with JH III, both similar to those of native hJHBP.

Objective: Epidemiologic and clinicohistopathologic prognostic factors of uterine endometrioid carcinomas were analyzed. The association of estrogen related factors, focused on adenomyosis in the prognosis of endometrioid carcinomas was also examined. Methods: Risk factors of surgically treated 286 patients with endometrioid carcinoma (Stage I–III) were statistically analyzed. Results: Overall a recurrence-free 5-year survival rate was 81% (Stage I, 94%, Stage II, 71% and Stage III, 40%). Significant prognostic factors were lymph node metastases (P=0.0035) and serosal/parametrial invasion (P=0.014) by multivariate analysis. Endometrioid carcinomas with co-existing adenomyosis tend to be associated with endometrial hyperplasia (P=0.04, Fisher’s exact test), diagnosed in less invasive status (myometrial invasion, P=0.004 and serosal/parametrial invasion, P=0.006) and therefore have a favorable prognosis (P=0.01, log rank test). Conclusions: A favorable prognosis of endometrioid carcinomas with co-existing estrogen related factors (adenomyosis and endometrial hyperplasia) was suggested.

The E' center formed by γ-irradiation in crushed quartz has a distribution in the value of the hyperfine fine structure (hfs) constant of 29 Si (nuclear spin: I = 1/2) and of the g-factor. The widths of both distributions decreased after annealing. The central line due to the E'center associated with 28 Si (I = 0) shows a clear narrowing as to allow the numerical simulation of the distribution of g-factor based on the following assumptions. One is that the distribution is composed of Gaussians with various line widths. The other is that the broader component has a small thermal activation energy. The simulated spectrum shows good agreement with the experimental one. The distribution of the g-factor can be explained with an inhomogeneous eigenvector of a singly occupied molecular orbital (SOMO).

Background: Primary and acquired resistance to antibiotics is an important factor in determining the reason for treatment failure in Helicobacter pylori infection. We examined the relationship between the susceptibility of H. pylori isolates and the efficacy of chemotherapy. Methods: The minimal inhibitory concentrations (MICs) of metronidazole (MNZ), clarithromycin (CLAR) and amoxycillin (AMOX) of 320 H. pylori pre-treatment isolates were determined by the agar dilution method. In 290 patients with peptic ulcers, H. pylori infection was treated by dual or triple combination therapies for 2 weeks: one proton pump inhibitor (30 mg/day lansoprazole or 20 mg/day omeprazole) and one or two antibiotics (500 mg AMOX, 200 mg CLAR or 250 mg MNZ twice a day). MICs were also determined after the treatment failure. Results: Among the drugs tested, for MNZ and CLAR, 8.1% and 9.1% of the isolates, respectively, were resistant, while no isolate was resistant to AMOX. After unsuccessful treatment using MNZ and CLAR, 66.7% and 70.6% of the isolates changed from sensitive to resistant, respectively. All isolates were sensitive to AMOX after treatment failure. Conclusions: The failure of the H. pylori treatment results in the induction of resistance to CLAR and/or MNZ. Regimens with a high cure rate should be used in order to prevent the generation of acquired resistance to antibiotics.

Murine interferon-gamma (IFN-gamma) stimulates the murine macrophage tumour cell line RAW264-7 to produce nitric oxide (NO). IFN-gamma induces expression of inducible NO synthase (iNOS), manganese superoxide dismutase (Mn-SOD) and copper zinc SOD (CuZn-SOD) in these cells. To investigate the mechanism of induction of SOD expression, we added S-nitroso-N-acetyl penicillamine (SNAP) to RAW264-7 cells. SNAP enhanced the expression of Mn-SOD and CuZn-SOD. These results suggest that when producing NO, RAW264-7 cells express SOD that might protect them from NO toxicity.

Our objective was to evaluate whether intratumoral-blood flow analysis could differentiate uterine sarcoma from uterine leiomyoma. Color and pulsed Doppler findings obtained from 41 patients with histologically proven uterine leiomyoma and five with uterine sarcoma (four leiomyosarcoma, one mixed mesodermal tumor) were retrospectively assessed. Intratumoral blood flow velocity waveforms were recorded, and the resistance index (RI) and peak systolic velocity (PSV) were calculated. There was no significant difference between the RI (median 0.647; range 0.422-0.896) in the uterine leiomyomas and the RI (median 0.663; range 0.330-0.774) in the uterine sarcomas. The PSV (median 61.6 cm/s; range 40.0-124.0 cm/s) in the uterine sarcomas was significantly higher (median 21.6 cm/s, range 6.3-48.6 cm/s) than that in the uterine leiomyomas (p < 0.05). When a cut-off value for the PSV of 41.0 cm/s (mean PSV of the uterine leiomyomas plus 2 standard deviations) was considered, the detection rate for uterine sarcoma was 80.0%, and the false-positive rate was 2.4%. These results suggest that the PSV within the tumor detected by color and pulsed Doppler ultrasonography could be useful for the preoperative differential diagnosis of uterine sarcoma.

To determine whether hyperinsulinemia is related to gestational hypertension.We measured the arterial blood pressure and the level of immunoreactive insulin (IRI) during a 75 g oral glucose tolerance test in a total of 84 pregnant women. Hyperinsulinemia was defined as a fasting IRI levelor = 9 IU/l, while gestational hypertension was defined as arterial blood pressureor = 140/90 mmHg.The incidence of gestational hypertension was higher in the hyperinsulinemic group (n = 29) than in the control group (n = 55) (24.1% vs. 7.3%, respectively P0.05). After controlling for maternal age, parity, pre-pregnancy body mass index and the gestational age at the time of oral glucose tolerance test (OGTT), using a multiple regression model, the relative risk of developing gestational hypertension for a fasting insulin level was 1.19 (95% C.I., 1.03-1.38).Pregnant women with hyperinsulinemia are at increased risk of developing gestational hypertension.

The vacuolating cytotoxin and urease secreted by Helicobacter pylori are thought to be virulent factors. Because vacuolation is potentiated by the presence of ammonium ion, which is produced by urease in vitro, it is of interest to examine whether cytotoxin and urease work reciprocally in the development of atrophic gastritis or duodenal ulcer. In the present study, patients (all H. pylori-positive) were divided into four groups: mild atrophic gastritis (group 1; nine patients), severe atrophic gastritis (group 2; 36 patients), duodenal ulcer with mild atrophic gastritis (group 3; 19 patients) and duodenal ulcer with severe atrophic gastritis (group 4; 12 patients). Cytotoxin production and urease activity of H. pylori isolated from these patients were analysed. Cytotoxin production was observed in four of nine (44.4%), 28 of 36 (77.8%), 11 of 19 (57.9%) and eight of 12 (66.7%) isolates from groups 1, 2, 3 and 4, respectively. Cytotoxin-producing H. pylori isolates were found significantly more in patients with severe atrophy than in patients with mild atrophy (P = 0.048). The mean of relative activity of cytotoxin in H. pylori isolate was 1.6 +/- 2.3, 7.9 +/- 7.4, 5.8 +/- 6.0 and 9.0 +/- 9.1 in groups 1, 2, 3 and 4, respectively. Helicobacter pylori isolates from severe atrophy or duodenal ulcer patients in groups 2 or 4 possessed significantly higher activity than those from non-ulcer patients in group 1 (P = 0.017 and 0.030, respectively). The mean of urease activity was 8.6 +/- 4.6, 10.0 +/- 5.9, 10.0 +/- 8.5 and 11.2 +/- 7.7 IU/mg in groups 1, 2, 3 and 4, respectively. These differences indicated no statistical significance. In each H. pylori isolate, the production of cytotoxin and urease were independent, which indicated that there was no reciprocal effect between them in vivo. Thus, cytotoxin-producing H. pylori isolates were more prevalent in patients with severe atrophic gastritis and the cytotoxin activities of H. pylori isolates from the patients with severe atrophic gastritis or duodenal ulcer were much higher than those from the patients with mild atrophic gastritis, which suggested that vacuolating cytotoxin may be a disease-inducing factor.

Two types of magnesium oxide clusters, (MgO) and (MgO)nMg+ have been obtained by the bombardment of magnesium with a mixture of xenon and oxygen ions. Enhanced ion intensity in the cluster mass spectra was observed at certain n values. The correlation between the geometrical structures of the clusters and their abundances is discussed.

We assessed the possibility of transvaginal pulsed and color Doppler imaging as a screening method for malignant uterine tumors. Since the ultrasonic Doppler method was suggested as a preliminary screening method in the management of patients [l], we have evaluated a total of 51 cases of uterine tumors, including 22 of adenomyosis, 22 of myoma, three of uterine endometrial cancer, one of uterine cervical cancer and three of sarcoma. We used either a Hitachi EUB-555 or 565A unit equipped with a 6.5-MHz transvaginal probe. Intratumoral vessels were detected using Doppler imaging, and pulsed Doppler was then used to obtain signals from the intratumoral vessels. Efforts were made to detect the highest V,,, using our 2D velocity reconstruction technique, which has been described previously [2]. At least three optimal signals were obtained from each tumor. The

Intracoronary thrombolysis is a logical therapeutic method and one of the challenging new treatments of acute myocardial infarction. However, a wide dose range of urokinase has been reported, and the optimal dose has not yet been established. In this study the fibrinolytic activity in patients with recanalized coronary arteries was compared with that in those with nonrecanalized arteries. The mean doses of urokinase in the recanalized and non-recanalized groups were 910,700 +/- 161,730 international units (IU) and 1,008,000 +/- 151,800 IU, respectively. The fibrinolytic activity was measured with alpha 2-plasmin inhibitor, alpha 2-macroglobulin, fibrinogen, plasminogen, and fibrin degradation products. No significant difference was observed in the fibrinolytic activity between the recanalized and nonrecanalized groups. Because the fibrinolytic activity in the two groups was thought to be activated sufficiently and to a similar degree, it appears that 1,000,000 IU of urokinase is adequate for intracoronary thrombolysis and larger doses cannot be expected to result in a higher rate of recanalization.

The crystal structure of the γ-form of nylon 6 (polycaproamide) obtained by the iodine treatment of the α-from of nylon 6 has been determined by x-ray diffraction. The unit cell contains four repeating units and is monoclinic with a = 9.33 A., b = 16.88 A. (fiber axis), c = 4.78 A., β = 121°. The space group is P21/a. There is considerable deviation from the planar configuration in the amide group of the chain. The plane of the CH2 chain zigzag lies nearly parallel to the (001) plane. The crystal is composed of the pleated sheets of the parallel chains joined by hydrogen bond between the adjacent chains. The chain directions are opposite in alternating sheets. The hydrogen-bond length is 2.83 A., a reasonable figure for an NH·. O chains joined by a hydrogen bond between the adjacent chains. The direction of the hydrogen bond makes an angle of 12° with the (100) plane, and the CO and NH bonds are nearly colinear. The amide groups lie at the same level in the cell.