CYP-450 Epoxygenase Derived Epoxyeicosatrienoic Acid Contribute To Reversal of Heart Failure in Obesity-Induced Diabetic Cardiomyopathy via PGC-1 α Activation.

We have previously shown that an Epoxyeicosatrienoic Acid (EET) -agonist has pleiotropic effects and reverses cardiomyopathy by decreasing inflammatory molecules and increasing antioxidant signaling. We hypothesized that administration of an EET agonist would increase Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α), which controls mitochondrial function and induction of HO-1 and negatively regulates the expression of the proinflammatory adipokines CCN3/NOV in cardiac and pericardial tissues. This pathway would be expected to further improve left ventricular (LV) systolic function as well as increase insulin receptor phosphorylation. Measurement of the effect of an EET agonist on oxygen consumption, fractional shortening, blood glucose levels, thermogenic and mitochondrial signaling proteins was performed. Control obese mice developed signs of metabolic syndrome including insulin resistance, hypertension, inflammation, LV dysfunction, and increased NOV expression in pericardial adipose tissue. EET agonist intervention decreased pericardial adipose tissue expression of NOV, while normalized FS, increased PGC-1α, HO-1 levels, insulin receptor phosphorylation and improved mitochondrial function, theses beneficial effect were reversed by deletion of PGC-1α. These studies demonstrate that an EET agonist increases insulin receptor phosphorylation, mitochondrial and thermogenic gene expression, decreased cardiac and pericardial tissue NOV levels, and ameliorates cardiomyopathy in an obese mouse model of the metabolic syndrome.

Supplementing an energy adequate, higher protein diet with protein does not enhance fat-free mass restoration after short-term severe negative energy balance.

Negative energy balance during military operations can be severe and result in significant reductions in fat-free mass (FFM). Consuming supplemental high-quality protein following such military operations may accelerate restoration of FFM. Body composition (dual-energy X-ray absorptiometry) and whole body protein turnover (single-pool [15N]alanine method) were determined before (PRE) and after 7 days (POST) of severe negative energy balance during military training in 63 male US Marines (means ± SD, 25 ± 3 yr, 84 ± 9 kg). After POST measures were collected, volunteers were randomized to receive higher protein (HIGH: 1,103 kcal/day, 133 g protein/day), moderate protein (MOD: 974 kcal/day, 84 g protein/day), or carbohydrate-based low protein control (CON: 1,042 kcal/day, 7 g protein/day) supplements, in addition to a self-selected, ad libitum diet, for the 27-day intervention (REFED). Measurements were repeated POST-REFED. POST total body mass (TBM; -5.8 ± 1.0 kg, -7.0%), FFM (-3.1 ± 1.6 kg, -4.7%), and net protein balance (-1.7 ± 1.1 g protein·kg-1·day-1) were lower and proteolysis (1.1 ± 1.9 g protein·kg-1·day-1) was higher compared with PRE (P < 0.05). Self-selected, ad libitum dietary intake during REFED was similar between groups (3,507 ± 730 kcal/day, 2.0 ± 0.5 g protein·kg-1·day-1). However, diets differed by protein intake due to supplementation (CON: 2.0 ± 0.4, MOD: 3.2 ± 0.7, and HIGH: 3.5 ± 0.7 g·kg-1·day-1; P < 0.05) but not total energy (4,498 ± 725 kcal/day). All volunteers, independent of group assignment, achieved positive net protein balance (0.4 ± 1.0 g protein·kg-1·day-1) and gained TBM (5.9 ± 1.7 kg, 7.8%) and FFM (3.6 ± 1.8 kg, 5.7%) POST-REFED compared with POST (P < 0.05). Supplementing ad libitum, energy-adequate, higher protein diets with additional protein may not be necessary to restore FFM after short-term severe negative energy balance.NEW & NOTEWORTHY This article demonstrates 1) the majority of physiological decrements incurred during military training (e.g., total and fat-free mass loss), with the exception of net protein balance, resolve and return to pretraining values after 27 days and 2) protein supplementation, in addition to an ad libitum, higher protein (~2.0 g·kg-1·day-1), energy adequate diet, is not necessary to restore fat-free mass following short-term severe negative energy balance.

Whole-body protein turnover response to short-term high-protein diets during weight loss: a randomized controlled trial.

To determine whole-body protein turnover responses to high-protein diets during weight loss, 39 adults (age, 21±1 years; VO2peak, 48±1 ml kg(-1) min(-1); body mass index, 25±1 kg m(2)) were randomized to diets providing protein at the recommend dietary allowance (RDA), 2 × -RDA or 3 × -RDA. A 10-day weight maintenance period preceded a 21-day, 40% energy deficit. Postabsorptive (FASTED) and postprandial (FED) whole-body protein turnover was determined during weight maintenance (day 10) and energy deficit (day 31) using [1-(13)C]leucine. FASTED flux, synthesis and breakdown were lower (P<0.05) for energy deficit than weight maintenance. Protein flux and synthesis were higher (P<0.05) for FED than FASTED. Feeding attenuated (P<0.05) breakdown during weight maintenance but not energy deficit. Oxidation increased (P<0.05) between dietary protein levels and feeding stimulated oxidation, although oxidative responses to feeding were higher (P<0.05) for energy deficit than weight maintenance. FASTED net balance decreased between dietary protein levels, but in the FED state, net balance was lower for 3 × -RDA as compared with RDA and 2 × -RDA (diet-by-state, P<0.05). Consuming dietary protein at levels above the RDA, particularly 3 × -RDA, during short-term weight loss increases protein oxidation with concomitant reductions in net protein balance.

A novel, noninvasive transdermal fluid sampling methodology: IGF-I measurement following exercise.

This study tested the hypothesis that transdermal fluid (TDF) provides a more sensitive and accurate measure of exercise-induced increases in insulin-like growth factor-I (IGF-I) than serum, and that these increases are detectable proximal, but not distal, to the exercising muscle. A novel, noninvasive methodology was used to collect TDF, followed by sampling of total IGF-I (tIGF-I) and free IGF-I (fIGF-I) in TDF and serum following an acute bout of exercise. Experiment 1: eight men (23 ± 3 yrs, 79 ± 7 kg) underwent two conditions (resting and 60 min of cycling exercise at 60% Vo(2)(peak)) in which serum and forearm TDF were collected for comparison. There were no significant changes in tIGF-I or fIGF-I in TDF obtained from the forearm or from serum following exercise (P > 0.05); however, the proportion of fIGF-I to tIGF-I in TDF was approximately fourfold greater than that of serum (P ≤ 0.05). These data suggest that changes in TDF IGF-I are not evident when TDF is sampled distal from the working tissue. To determine whether exercise-induced increases in local IGF-I could be detected when TDF was sampled directly over the active muscle group, we performed a second experiment. Experiment 2: fourteen subjects (22 ± 4 yr, 68 ± 11 kg) underwent an acute plyometric exercise condition consisting of 10 sets of 10 plyometric jumps with 2-min rest between sets. We observed a significant increase in TDF tIGF-I following exercise (P ≤ 0.05) but no change in serum tIGF-I (P > 0.05). Overall, these data suggest that TDF may provide a noninvasive means of monitoring acute exercise-induced changes in local IGF-I when sampled in proximity to exercising muscles. Moreover, our finding that the proportion of free to tIGF-I was greater in TDF than in serum suggests that changes in local IGF-I may be captured more readily using this system.

p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting.

Oxidative stress is a primary trigger of cachectic muscle wasting, but the signaling pathway(s) that links it to the muscle wasting processes remains to be defined. Here, we report that activation of p38 mitogen-activated protein kinase (MAPK) (phosphorylation) and increased oxidative stress (trans-4-hydroxy-2-nonenal protein modification) in skeletal muscle occur as early as 8 h after lipopolysaccharide (1 mg/kg) and 24 h after dexamethasone (25 mg/kg) injection (intraperitoneal) in mice, concurrent with upregulation of autophagy-related genes, Atg6, Atg7, and Atg12. Treating cultured C2C12 myotubes with oxidant hydrogen peroxide (4 h) resulted in increased p38 phosphorylation and reduced FoxO3 phosphorylation along with induced Atg7 mRNA expression without activation of NF-kappaB or FoxO3a transcriptional activities. Furthermore, inhibition of p38alpha/beta by SB202190 blocked hydrogen peroxide-induced atrophy with diminished upregulation of Atg7 and atrogenes [muscle atrophy F-box protein (MAFbx/Atrogin-1), muscle ring finger protein 1 (MuRF-1), and Nedd4]. These findings provide direct evidence for p38alpha/beta MAPK in mediating oxidative stress-induced autophagy-related genes, suggesting that p38alpha/beta MAPK regulates both the ubiquitin-proteasome and the autophagy-lysosome systems in muscle wasting.

Warfarin use and the risk of valvular calcification.

BACKGROUND: Warfarin affects the synthesis and function of the matrix Gla-protein, a vitamin K-dependent protein, which is a potent inhibitor of tissue calcification. OBJECTIVES: To investigate the incidence of mitral valve calcium (MVC), mitral annular calcium (MAC) and aortic valve calcium (AVC) in patients with non-valvular atrial fibrillation (AF) treated with warfarin vs. no warfarin. PATIENTS AND METHODS: Of 1155 patients, mean age 74 years, with AF, 725 (63%) were treated with warfarin and 430 (37%) without warfarin. The incidence of MVC, MAC and AVC was investigated in these 1155 patients with two-dimensional echocardiograms. Unadjusted logistic regression analysis was conducted to examine the association between the use of warfarin and the incidence of MVC, MAC or AVC. Logistic regression analyses were also conducted to investigate whether the relationship stands after adjustment for confounding risk factors such as age, sex, race, ejection fraction, smoking, hypertension, diabetes, dyslipidemia, coronary artery disease (CAD), glomerular filtration rate, calcium, phosphorus, calcium-phosphorus product, alkaline phosphatase, use of aspirin, beta blockers, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and statins. RESULTS: There was a significant association between the use of warfarin and the risk of calcification [unadjusted odds ratio = 1.71, 95% CI = (1.34-2.18)]. The association still stands after adjustment for confounding risk factors. MVC, MAC or AVC was present in 473 of 725 patients (65%) on warfarin vs. 225 of 430 patients (52%) not on warfarin (P < 0.0001). Whether this is a causal relationship remains unknown. CONCLUSIONS: Use of warfarin in patients with AF is associated with an increased prevalence of MVC, MAC or AVC.

Calpain-1 is required for hydrogen peroxide-induced myotube atrophy.

Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 microM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), alpha-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate alphaII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 microM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.

Redox regulation of diaphragm proteolysis during mechanical ventilation.

Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox-sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control, 2) 6 h of MV, 3) 6 h of MV with infusion of the antioxidant Trolox, 4) 18 h of MV, and 5) 18 h of MV with Trolox. Trolox did not attenuate MV-induced increases in diaphragmatic levels of ubiquitin-protein conjugation, polyubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome alpha-subunit 7, 14-kDa E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities in the diaphragm after 18 h of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (mug/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.

Heme oxygenase: a target gene for anti-diabetic and obesity.

Heme oxygenase-1 (HO-1) is central to the regulation of oxidative injury. The role of increased HO-1 expression and Heme oxygenase (HO) activity in mitigating the detrimental side effect of diabetes is examined. A review of the mechanism(s) of action is included. This may lead to the development of pharmacological and genetic approaches to mitigate the clinical complications associated with the progression of diabetes and obesity.

Antioxidant administration attenuates mechanical ventilation-induced rat diaphragm muscle atrophy independent of protein kinase B (PKB Akt) signalling.

Oxidative stress promotes controlled mechanical ventilation (MV)-induced diaphragmatic atrophy. Nonetheless, the signalling pathways responsible for oxidative stress-induced muscle atrophy remain unknown. We tested the hypothesis that oxidative stress down-regulates insulin-like growth factor-1-phosphotidylinositol 3-kinase-protein kinase B serine threonine kinase (IGF-1-PI3K-Akt) signalling and activates the forkhead box O (FoxO) class of transcription factors in diaphragm fibres during MV-induced diaphragm inactivity. Sprague-Dawley rats were randomly assigned to one of five experimental groups: (1) control (Con), (2) 6 h of MV, (3) 6 h of MV with infusion of the antioxidant Trolox, (4) 18 h of MV, (5) 18 h of MV with Trolox. Following 6 h and 18 h of MV, diaphragmatic Akt activation decreased in parallel with increased nuclear localization and transcriptional activation of FoxO1 and decreased nuclear localization of FoxO3 and FoxO4, culminating in increased expression of the muscle-specific ubiquitin ligases, muscle atrophy factor (MAFbx) and muscle ring finger-1 (MuRF-1). Interestingly, following 18 h of MV, antioxidant administration was associated with attenuation of MV-induced atrophy in type I, type IIa and type IIb/IIx myofibres. Collectively, these data reveal that the antioxidant Trolox attenuates MV-induced diaphragmatic atrophy independent of alterations in Akt regulation of FoxO transcription factors and expression of MAFbx or MuRF-1. Further, these results also indicate that differential regulation of diaphragmatic IGF-1-PI3K-Akt signalling exists during the early and late stages of MV.

Anatomic and physiological characteristics of the ferret lateral rectus muscle and abducens nucleus.

The ferret has become a popular model for physiological and neurodevelopmental research in the visual system. We believed it important, therefore, to study extraocular whole muscle as well as single motor unit physiology in the ferret. Using extracellular stimulation, 62 individual motor units in the ferret abducens nucleus were evaluated for their contractile characteristics. Of these motor units, 56 innervated the lateral rectus (LR) muscle alone, while 6 were split between the LR and retractor bulbi (RB) muscle slips. In addition to individual motor units, the whole LR muscle was evaluated for twitch, tetanic peak force, and fatigue. The abducens nucleus motor units showed a twitch contraction time of 15.4 ms, a mean twitch tension of 30.2 mg, and an average fusion frequency of 154 Hz. Single-unit fatigue index averaged 0.634. Whole muscle twitch contraction time was 16.7 ms with a mean twitch tension of 3.32 g. The average fatigue index of whole muscle was 0.408. The abducens nucleus was examined with horseradish peroxidase conjugated with the subunit B of cholera toxin histochemistry and found to contain an average of 183 motoneurons. Samples of LR were found to contain an average of 4,687 fibers, indicating an LR innervation ratio of 25.6:1. Compared with cat and squirrel monkeys, the ferret LR motor units contract more slowly yet more powerfully. The functional visual requirements of the ferret may explain these fundamental differences.

Ovarian hormone status and skeletal muscle inflammation during recovery from disuse in rats.

Resumption of normal muscle loading after a period of disuse initiates cellular processes related to mass accretion. The renewed loading also induces a significant amount of muscle damage and subsequent inflammation. Ovarian hormone depletion delays atrophied myofibre mass recovery. Ovarian hormones are also global regulators of immune system function. The purpose of this study was to determine whether ovarian hormone depletion-induced deficits in myofibre regrowth after disuse atrophy are related to the induction of muscle damage and the associated inflammatory response. We hypothesized that soleus muscle immune cell infiltration and inflammatory gene expression would be both accentuated and prolonged in ovarian hormone-depleted rats during the first week of recovery from disuse atrophy. Intact and ovariectomized (OVX) female rats were subjected to hindlimb suspension for 10 days and then returned to normal ambulation for a recovery period, the rats were killed and the soleus muscle removed for analysis. Although reloading increased both circulating creatine kinase and myofibre membrane disruption, there was no effect of ovarian hormones on these processes during recovery. Muscle neutrophil concentration was increased above baseline regardless of hormone status at days 1 and 3 of recovery; however, this increase was 43% greater at day 3 in the OVX group. Muscle ED1+ and ED2+ macrophage concentrations were increased during recovery in both groups. However, macropage concentrations remained elevated at day 7 of recovery in the OVX group, whereas they returned to control levels in the intact group. Cyclo-oxygenase-2, interleukin-6 and interleukin-1beta muscle mRNA expression increased similarly during recovery, regardless of ovarian hormone status. These results demonstrate that the initial myofibre damage and inflammatory gene expression induced during muscle recovery from disuse atrophy are independent of ovarian hormone status.

Estrogen status and skeletal muscle recovery from disuse atrophy.

Although estrogen loss can alter skeletal muscle recovery from disuse, the specific components of muscle regrowth that are estrogen sensitive have not been described. The primary purpose of this study was to determine the components of skeletal muscle mass recovery that are biological targets of estrogen. Intact, ovariectomized (OVX), and ovariectomized with 17beta-estradiol replacement (OVX+E2) female rats were subjected to hindlimb suspension for 10 days and then returned to normal cage ambulation for the duration of recovery. Soleus muscle mass returned to control levels by day 7 of recovery in the intact animals, whereas OVX soleus mass did not recover until day 14. Intact rats recovered soleus mean myofiber cross-sectional area (CSA) by day 14 of recovery, whereas the OVX soleus remained decreased (42%) at day 14. OVX mean fiber CSA did return to control levels by day 28 of recovery. The OVX+E2 treatment group recovered mean CSA at day 14, as in the intact animals. Myofibers demonstrating central nuclei were increased at day 14 in the OVX group, but not in intact or OVX+E2 animals. The percent noncontractile tissue was also increased 29% in OVX muscle at day 14, but not in either intact or OVX+E2 groups. In addition, collagen 1a mRNA was increased 45% in OVX muscle at day 14 of recovery. These results suggest that myofiber growth, myofiber regeneration, and extracellular matrix remodeling are estrogen-sensitive components of soleus muscle mass recovery from disuse atrophy.

Effects of 12 days of artificial rearing on morphology of hypoglossal motoneurons innervating tongue retrusors in rat.

The purpose of this study was to examine the influence of reduced tongue activity by artificial rearing on the morphology of motoneurons innervating the extrinsic tongue retrusors. Artificially reared rat pups were fed via gastric cannula from postnatal day 3 to postnatal day 14. Artificially reared animals and dam-reared controls had cholera toxin (subunit B) conjugate of horseradish peroxidase injected into the styloglossus to label motoneurons innervating hyoglossus and styloglossus on postnatal day 13 and postnatal day 59. Following perfusion on postnatal days 14 and 60, serial transverse sections treated with tetramethyl benzidine and counterstained neutral red were used to analyze motoneuron morphology. The shorter diameter of hyoglossus motoneurons increased with age for the dam-reared but not the artificially reared group. There was a tendency for a similar pattern for styloglossus motoneurons across the two rearing groups. The changes in form factor reflected the changes in shorter diameter for both motoneuron pools. Therefore, reducing suckling activity during normal postnatal development leads to diminished motoneuron somal growth in rats. This may also be the case in premature infants necessarily fed artificially.

Extraocular connective tissues: a role in human eye movements?

PURPOSE: This study presents a detailed anatomic analysis of the undisturbed connective tissues that surround the horizontal extraocular muscles (EOMs) of humans. Emphasis is placed on those EOM orbital side tissues that, in previous MRI studies, were assumed to couple the muscle to the pulley. METHODS: Serial 5-mum sections were prepared from paraffin-embedded blocks of the lateral and medial rectus muscles and their surrounding connective tissues. The sections were treated with Masson's trichrome stain for light microscopic examination of muscle fibers (red) and surrounding connective tissues (blue). RESULTS: Rectus muscle sections demonstrated the orbital connective tissues to be a collagenous bridge between the distal third of the muscle and the orbital periosteum (i.e., check ligament [CL]). The CL attaches to the muscle by investing itself around orbital muscle fibers whereas, at the point of attachment, those fibers remain aligned with the remainder of the muscle. The CL on the orbital side and the reflected bulbar fascia on the global side of the muscle constitute a tubelike sheath. The posterior border of the sheath insinuates into the muscle belly and its anterior aspect blends into the sides of the portal through Tenon's capsule. CONCLUSIONS: All rectus EOM fibers participate in eye rotation. The CL is the band of tissue present on the MRI images, but was previously described as the orbital layer insertion for the active pulley hypothesis (APH). The APH should now be questioned. Alternate theories incorporating accepted neurophysiological, anatomic, and ophthalmological principles of EOM movement are discussed.

Emergency department management of mild traumatic brain injury in the USA.

OBJECTIVE: To describe the emergency department (ED) management of isolated mild traumatic brain injury (TBI) in the USA and to examine variation in care across age and insurance types. METHODS: A secondary analysis of ED visits for isolated mild TBI in the National Hospital Ambulatory Medical Care Survey 1998-2000 was performed. Mild TBI was defined by International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9) codes for skull fracture, concussion, intracranial injury (unspecified), and head injury (unspecified). Available ED care variables were analysed by patient age and insurance categories using multivariate logistic regression. RESULTS: The incidence of isolated mild TBI cases attending ED was 153,296 per year, or 56.4/100,000 people. Of the patients with isolated mild TBI, 44.3% underwent computed tomography, 23.9% underwent other non-extremity, non-chest x rays, 17.1% received wound care and 14.1% received intravenous fluids. However, only 43.8% had an assessment of pain. Of those with documented pain, only 45.5% received analgesics in the ED. Nearly 38% were discharged without recommendations for specific follow up. Several aspects of ED care varied by age but not by insurance type. CONCLUSION: Substantial ED resources are devoted to the care of isolated mild TBI. The present study identified deficiencies in and variation around several important aspects of ED care. The development of guidelines specific for mild TBI could reduce variation and improve emergency care for this injury.

Postnatal development of hypoglossal motoneurons that innervate the hyoglossus and styloglossus muscles in rat.

Postnatal development of hyoglossus and styloglossus motoneurons was studied in this investigation of the hypoglossal nucleus. Our findings show separate and distinct locations for hyoglossus and styloglossus motoneurons within the retrusor (dorsal) subdivision of the hypoglossal nucleus for all age groups. Hyoglossus and styloglossus motoneuron cross-sectional area reached their adult size at different times (by weeks 2 and 3, respectively). Cell roundness, as measured by form factor (measure of cell perimeter relative to its area), decreased with advancing postnatal age for both populations of motoneurons. Differences in the direction of the dendritic projection between hyoglossus and styloglossus motoneurons were found. Hyoglossus and styloglossus motoneuron development was compared to genioglossus motoneuron postnatal development.

Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA(1)c.

AIMS/HYPOTHESIS: Patients with diabetes mellitus are well known to be at high risk for vascular disease. Circulating endothelial cells (CECs) have been reported to be an ex vivo indicator of vascular injury. We investigated the presence of CECs in the peripheral blood of 25 patients with diabetes mellitus and in nine non-diabetic control donors. METHODS: Endothelial cells were isolated from peripheral blood with anti-CD-146-coated immunomagnetic Dynabeads, and were stained with acridine orange dye and counted by fluorescence microscopy. The cells were also stained for von Willebrand factor and Ulex europaeus lectin 1. RESULTS: Patients with diabetes mellitus had an elevated number of CECs (mean 69+/-30 cells/ml, range 35-126) compared with healthy controls (mean 10+/-5 cells/ml, range 3-18) (p<0.001). The increase in CECs did not correlate with the levels of HbA(1)c. Circulating endothelial cell numbers were elevated regardless of glucose levels, suggesting that, even with control of glucose levels, there is increased endothelial cell sloughing. CONCLUSIONS: Our study suggests that the higher number of CECs in patients with type 2 diabetes may reflect ongoing vascular injury that is not directly dependent on glucose control.

Effects of electrode penetrations into the abducens nucleus of the monkey: eye movement recordings and histopathological evaluation of the nuclei and lateral rectus muscles.

Two adult rhesus monkeys that had undergone 2 years of electrode penetrations into their abducens and vestibular nuclei, for chronic eye movement studies, were examined histologically. An analysis of their VIth nucleus neurons and lateral rectus muscles revealed the following. Twenty-two percent of the large neurons (approximately 30 microm in diameter), on average, were missing and extensive neuropil disruption and gliosis was evident in the experimental side abducens nuclei as compared with the control side in each animal. While the lateral rectus muscles showed small, but inconsistent, changes in total fiber number, the muscle fiber diameters were altered, leading to a more homogenous muscle and making the typical orbital and global subdivisions of the muscle less distinct. Eye movement records from before and after the electrophysiological studies were comparable. We discuss how the complex architecture of the extraocular muscles as well as the possibility of polyneuronal innervation of single muscle fibers could explain our results.

RhoA expression during recovery from skeletal muscle disuse.

Functional overload and anabolic steroid administration induce signaling pathways that regulate skeletal muscle RhoA expression. The purpose of this study was to determine RhoA and associated protein expression at the onset of disuse and after a brief period of reloading. Male Sprague-Dawley rats were randomly assigned to cage control (Con), 3 days of hindlimb suspension (Sus), or 3 days of hindlimb suspension with 12 h of reloading (12-h Reload). The reloading stimuli consisted of 12 h of resumed normal locomotion after 3 days of hindlimb suspension. Plantaris muscle-to-body weight (mg/g) ratio decreased 17% from Con with Sus but returned to Con with 12-h Reload, increasing 13% from Sus. Sus decreased RhoA protein concentration 46%, whereas 12-h Reload induced a 24% increase compared with Sus. The ratio of cytosolic- to membrane-associated RhoA protein was not changed with either Sus or 12-h Reload. RhoA mRNA concentration was decreased 48% by Sus, and 12-h Reload induced a 170% increase from Sus. beta(1)-Integrin protein, a transmembrane protein associated with RhoA activation, was not altered by Sus but increased 155% with 12-h Reload. Although beta(1)-integrin mRNA was not altered by Sus, it increased 70% from Con with 12-h Reload. Rho family member Cdc42 protein associated with the muscle membrane was decreased 60% with Sus, and 12-h Reload induced a 172% increase compared with Sus. In conclusion, decreased RhoA protein expression and mRNA abundance are early adaptations to disuse but recover rapidly after normal locomotion is resumed.