Spatial distribution and developmental appearance of postjunctional P2X(1) receptors on smooth muscle cells of the mouse vas deferens

P2X(1)-type purinoceptors, have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage clamp techniques were used to estimate the amplitudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta -MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X(1) receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X(1) staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X(1) mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X(1) were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X(1) receptors, distributed diffusely across the smooth muscle cell surface. Synapse 42:1-11, 2001. (C) 2001 Wiley-Liss, Inc.

Effect of propionyl-L-carnitine on exercise performance in peripheral arterial disease

Background: Supplementation with propionyl-L-carnitine (PLC) may be of use in improving the exercise capacity of people with peripheral arterial disease. Methods: After a 2-wk exercise familiarization phase, seven subjects displaying intermittent claudication were studied over a 12-wk period consisting of three 4-wk phases, baseline (B), supplementation (S), and placebo (P). PLC was supplemented at 2 g(.)d(-1), and subjects were blinded to the order of supplementation. Unilateral calf strength and endurance were assessed weekly. Walking performance was assessed at the end of each phase using an incremental protocol, during which respiratory gases were collected. Results: Although there was not a significant increase in maximal walking time (similar to 14%) in the whole group, walking time improved to a greater extent than the individual baseline coefficient of variation in four of the seven subjects. The changes in walking performance were correlated with changes in the respiratory exchange ratio both at steady state (r = 0.59) and maximal exercise (r = 0.79). Muscle strength increased significantly from 695 +/- 198 N to 812 +/- 249 N by the end of S. Changes in calf strength from B to S were modestly related to changes in walking performance (r = 0.56). No improvements in calf endurance were detected throughout the study. Conclusions: These preliminary data suggest that, in addition to walking performance, muscle strength can be increased in PAD patients after 4 wk of supplementation with propionyl-L-carnitine.

Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ER alpha gene but not ERP by reverse transcriptase-polymerase chain reaction, possessed ER alpha protein on Western blotting, and displayed specific 17 beta -estradiol (E-2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ER alpha complement for males or females. There were no gender differences in ER alpha complement for subcutaneous or visceral samples. We conclude that ER alpha but not ERP is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ER alpha.

Post-antibiotic growth suppression of linezolid against Gram-positive bacteria

The in vitro post-antibiotic effects (PAEs) of eight different concentrations of linezolid against Gram-positive cocci were investigated and the results analysed using the sigmoid E-max model for mathematically modelling the PAE. Mean maximal linezolid PAEs against strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae were 2.2, 1.8, 2.8, 2.0 and 3.0 h, respectively. Resistance to methicillin (for the staphylococci), vancomycin (for the enterococci) and penicillin (for the pneumococci) had no effect on the duration of the PAE. Results of PAE testing support twice-daily dosing of linezolid in humans.

Dexamethasone enhances SOX9 expression in chondrocytes

SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared From rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox-consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes ch differentiation through enhancement of SOX9.

Interferon-gamma enhances cytotoxic T lymphocyte recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide

Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV-mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL-sensitive E7-expressing cell lines but are not susceptible to CTL-mediated lysis in vitro . E7 transgenic and non-transgenic KC are susceptible to conventional mechanisms of CTL-mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL-4), despite large differences in H-2D(b) expression at the cell surface. Furthermore, exposure of KC to IFN-gamma increased H-2D(b) expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN-gamma. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.

The number of rational points of a class of Artin-Schreier curves

We determine the number of F-q-rational points of a class of Artin-Schreier curves by using recent results concerning evaluations of some exponential sums. In particular, we determine infinitely many new examples of maximal and minimal plane curves in the context of the Hasse-Weil bound. (C) 2002 Elsevier Science (USA).

Impact of malocclusion and orthognathic reconstruction surgery on resonance and articulatory function: an examination of variability in five cases

Articulatory patterns and nasal resonance were assessed before and 6 months after orthognathic reconstruction surgery in five patients with dentofacial deformities. Perceptual and physiological assessments showed disorders of nasality and articulatory function preoperatively, two patients being hyponasal, and one hypernasal. Four patients had mild articulatory deficits, and four had reduced maximal lip or tongue pressures. Operation resulted in different patterns of change. Nasality deteriorated in three patients and articulatory precision and intelligibility improved in only one patient and showed no change in the other four. Operation improved interlabial pressures in three patients, while its impact on tongue pressures varied, being improved in one case, deteriorating in one, and remaining unchanged in the other three. The variability in the results highlights the need for routine assessment of speech and resonance before and after orthognathic reconstruction. (C) 2002 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Science Ltd. All rights reserved.

Physiological and symptomatic responses to cycling and walking in intermittent claudication

To shed light on the potential efficacy of cycling as a resting modality in the treatment of intermittent claudication (IC), this study compared physiological and symptomatic responses to graded walking and cycling tests in claudicants. Sixteen subjects with peripheral arterial disease (resting ankle:brachial index (ABI) < 0.9) and IC completed a maximal graded treadmill walking (T) and cycle (C) Lest after three familiarization tests on each mode. During cacti test, symptoms, oxygen uptake (VO2), minute ventilation (V-E), (respiratory exchange ratio) (RER) and heart rate (HR) were measured, and for 10 min after each Lest the brachial and ankle systolic pressures were recorded, All but One subject experienced calf pain as the primary limiting symptom during T whereas the symptoms were more varied during C and included thigh pain, calf pain and dyspnoea, Although maximal exercise time was significantly longer on C than T (690 +/- 67 vs, 495 +/- 57 s), peak VO2, peak, V-E and peak heart rate during C and T were not different; whereas peak RER was higher during C. These responses during C and T were also positively 1, (P < 0.05) with each other, with the exception of RER. The postexercise systolic pressures were also not different between C and T. However, the peak decline ill ankle pressures from resting values after C and T were not correlated with each other. Thew data demonstrate that cycling and walking induce a similar level of metabolic and cardiovascular strain, but that the primary limiting symptoms and haemodynamic response in an individual's extremity, measured after exercise, can differ substantially between these two modes.

Skeletal muscle mitochondrial ATP production rate and walking performance in peripheral arterial disease

This study tested the hypotheses that skeletal muscle mitochondrial ATP production rate (MAPR) is impaired in patients with peripheral arterial disease (PAD) and that it relates positively to their walking performances. Seven untrained patients, eight exercise-trained patients and 11 healthy controls completed a maximal walking test and had muscle sampled from the gastrocnemius medialis muscle. Muscle was analysed for its MAPR in the presence of pyruvate, palmitoyl-L-carnitine or both, as well as citrate synthase (CS) activity. MAPRs were not different between untrained PAD and controls. In contrast, MAPRs (pyruvate) were significantly higher in trained PAD vs. controls. MAPR (pyruvate combinations) was also significantly higher in trained than untrained PAD muscle. MAPR and CS activity were highly correlated with walking performance in patients, but not in controls. These data do not support the hypothesis that isolated mitochondria are functionally impaired in PAD and demonstrate that the muscle mitochondrial capacity to oxidize carbohydrate is positively related to walking performance in these patients.

Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [H-3]MK-801 binding site were delineated along with the enhancement of [H-3]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.

Repeatability of surface EMG variables in the sternocleidomastoid and anterior scalene muscles

In this study we examined the repeatability and reliability of the surface electromyographic (sEMG) signal mean frequency (MNF), average rectified value (ARV) and conduction velocity (CV) measured for the sternocleidomastoid (SCM) and the anterior scalene (AS) muscles in nine healthy volunteers during 15-s isometric cervical flexion contractions at 50% of the maximal voluntary contraction level over 3 non-consecutive days. Repeatability and reliability estimates were obtained for the initial values and rates of change of each sEMG variable by using both the Intraclass Correlation Coefficient (ICC) and the normalised standard error of the mean (nSEM). Results from SCM indicated good levels of repeatability for the initial value and slope of ARV (ICC > 65%). For the AS, high levels of repeatability were identified for the initial value of MNF (ICC > 70%) and the slope of ARV (ICC > 75%). Values of nSEM in the range 2.8-7.2% were obtained for the initial values of MNF and CV for both SCM and AS, indicating clinically acceptable measurement precision. The low value obtained for the nSEM of the initial value of MNF for the AS, in combination with the high ICC, indicates that of all of the variables examined, this variable could offer the best normative index to distinguish between subjects with and without neck pain, and represents the sEMG variable of choice for future evaluation purposes.

Thiazolidinediones and type 2 diabetes: new drugs for an old disease

Thiazolidinediones are a new class of drugs for the treatment of type 2 diabetes, and act by improving insulin sensitivity in adipose tissue, liver and skeletal muscle. Rosiglitazone and pioglitazone are registered for use in monotherapy, and in combination with sulfonylureas and metformin. Pioglitazone is also licensed for use in combination with insulin. There is level II evidence that in patients with inadequate glycaemic control both drugs reduce the level of HbA(1c) and fasting plasma glucose (FPG) when used as monotherapy and in combination with sulfonylurea or metformin or insulin; and both drugs increase levels of HDL and LDL and lower free fatty acid levels, but only pioglitazone significantly lowers triglyceride levels. Both drugs lower fasting insulin and C-peptide levels. In monotherapy, they may be slightly less potent at reducing the level of HbA(1c) than sulfonylureas or metformin. The maximal effect of these agents may not be seen for 6-14 weeks after commencement. Both drugs are well tolerated but liver function must be checked at baseline every second month for the first year, and periodically thereafter. The drugs are currently contraindicated in patients with moderate to severe liver dysfunction and alanine aminotransferase levels more than 2.5 times normal, New York Heart Association III-IV cardiac status, pregnancy, lactation and in children. The main side effects include weight gain, oedema, and mild dilutional anaemia.

Intrinsic regional differences in androgen receptors and dihydrotestosterone metabolism in human preadipocytes

Androgens play an important role in regulating the central obesity that is a strong risk factor for cardiovascular disease and insulin resistance. This study confirms that androgen receptors are present in subcultured human preadipocytes, with androgen receptor gene expression and saturable specific dihydrotestosterone binding, dissociation constant 1.02 - 2.56 nM and maximal binding capacity 30.8 - 55.7 fmol/mg protein. There was an intrinsic regional difference in androgen receptor complement, with more androgen receptors in visceral than in subcutaneous preadipocytes. Dihydrotestosterone was metabolised by human preadipocytes, with more androstanediol produced by subcutaneous than visceral preadipocytes. While dihydrotestosterone metabolism was insufficient to explain the regional variation in androgen binding, both of these differences would reduce the androgen responsiveness of the subcutaneous preadipocytes compared with visceral preadipocytes. There were no gender differences in androgen binding or metabolism. While the direct effects of androgens on human PAS remain uncertain, these regional differences suggest that AR-mediated regulation of certain PA functions influences adipose tissue distribution.

Physiological responses to the menstrual cycle - Implications for the development of heat illness in female athletes

Fluctuations in estrogen and progesterone during the menstrual cycle can cause changes in body systems other than the reproductive system. For example, progesterone is involved in the regulation of fluid balance in the renal tubules and innervation of the diaphragm via the phrenic nerve. However, few significant changes in the responses of the cardiovascular and respiratory systems, blood lactate, bodyweight, performance and ratings of perceived exertion are evident across the cycle. Nevertheless, substantial evidence exists to suggest that increased progesterone levels during the luteal phase cause increases in both core and skin temperatures and alter the temperature at which sweating begins during exposure to both ambient and hot environments. As heat illness is characterised by a significant increase in body temperature, it is feasible that an additional increase in core temperature during the luteal phase could place females at an increased risk of developing heat illness during this time. In addition, it is often argued that physiological gender differences such as oxygen consumption, percentage body fat and surface area-to-mass ratio place females at a higher risk of heat illness than males. This review examines various physiological responses to heat exposure during the menstrual cycle at rest and during exercise, and considers whether such changes increase the risk of heat illness in female athletes during a particular phase of the menstrual cycle.