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Purpose: Obesity is a chronic metabolic disease with global distribution among adults and children which affects daily functioning and ultimately quality of life. Primary care physicians (PCPs) provide an important role for the treatment of severe obesity. Better understanding of obesity and its treatment options may increase patients' referral rates to the various treatment modalities, including metabolic/bariatric surgery (MBS)., Materials and Methods: A quantitative cross-sectional study used a self-reported questionnaire among PCPs of Clalit Health Services (CHS) in Northern Israel. The quantitative questionnaire examined the PCP's knowledge, opinions, attitude, and approaches to managing severe obesity., Results: A total of 246 PCPs from Northern Israel filled the questionnaire (42.9%), the majority were Muslim Arabs (54.5%), who gained their medical degree outside of Israel (73.8%) and practicing for over 10 years (58.8%). 64.3% of PCPs had a high workload (over 100 appointments per week), 77.1% did not know the definition of severe obesity, and 69.17% did not attend educational meetings regarding obesity during the previous year. The referral rate for MBS was 50.4% ± 23.3. Two prognostic factors that had a statistically significant effect on the referral rate for bariatric surgery were the total appointments per week, and the number of practice years. Both had a negative association., Conclusion: The knowledge and referral rates for bariatric surgery are higher among PCPs with lower workload and relatively fewer practice years. Workshops and annual training courses may fortify knowledge and awareness for the treatment of obesity, which in turn could increase the referral rate for MBS., (© 2024. The Author(s).)

Background: Acute cholecystitis is one of the most common acute surgical diseases. Diabetic patients have been shown to have an increased risk for gallbladder disease, but the correlation between the severity of gallstone disease and diabetes is still debated. The aim of this study is to examine the possible difference in the disease process between patients with diabetes mellitus (DM) and those without., Patients and Methods: A retrospective study was conducted of all patients who underwent percutaneous cholecystostomy between 2005 and 2015 at Emek Medical Center, Afula, Israel. Demographic and medical history including data on bile and blood culture results, antimicrobial susceptibility, and clinical outcomes were retrieved from patient files., Results: The cohort included 272 patients. Mean age was 68 years old, 50.74% were male and 43.75% had diabetes mellitus. Bile cultures were obtained from 252 (92.64%) patients and were positive in 134 (53.2%) patients. In 11 patients (4%) two pathogens were isolated. Blood cultures obtained from 231 patients and were positive in 35 (15.2%). Escherichia coli was the most common isolate, and was seen in 22.3% of positive bile cultures and 40% of blood cultures. Although diabetic patients had significantly more positive bile cultures, the severity of the disease, according to the Tokyo guidelines, was not higher., Conclusions: Acute cholecystitis was neither more severe nor had significant difference in bacteriological properties when comparing diabetic patients to non-diabetic ones.

We previously reported that in the adult animal extinction in pairs resulted in enhanced extinction, showing that social presence can reduce previously acquired fear responses. Based on our findings that juvenile and adult animals differ in the mechanisms of extinction, here we address whether the social presence of a conspecific affects extinction in juvenile animals similarly to adults. We further address whether such presence has a different impact on juvenile males and females. To that end, we examined in our established experimental setting whether conditioned male and female animals extinguish contextual fear memory better while in pairs. Taking advantage of the role of oxytocin (OT) in the mediation of extinction memory and social interaction, we also study the effect of antagonizing the OT receptors (OTR) either systemically or in the prefrontal cortex on social interaction-induced effects of fear extinction. The results show that social presence accelerates extinction in males and females as compared to the single condition. Yet, we show differential and opposing effects of an OTR antagonist in both sexes. Whereas in females, the systemic application of an OTR antagonist is associated with impaired extinction, it is associated with enhanced extinction in males. In contrast, prefrontal OT is not engaged in extinction in juvenile males, while is it is critical in females. Previously reported differences in the levels of prefrontal OT between males and females might explain the differences in OT action. These results suggest that even during the juvenile period, critical mechanisms are differently involved in the regulation of fear in males and females.

Introduction: Primary lymphoma of the colon is exceedingly rare and comprises 0.2-1% of all colon tumors. The most common subtype of lymphoma in the colon is non-Hodgkin lymphoma. Symptoms are often nonspecific, and treatment varies between chemotherapy alone and a combination of surgery and chemotherapy., Case Presentation: We describe a case of a Ashkenazi Jew patient who presented in the typical way that carcinoma of the colon might present but turned out to have a very rare type of tumor in both its histology and its location., Conclusion: There was apparent discordance between the relative bulkiness and gross appearance of the tumor with the unrevealing result of the biopsies, demanding a high level of suspicion as to the actual presence and possible type of such a tumor in the future.

Accumulating evidence suggests that the cyclooxygenase-2 (COX-2) enzyme has additional catalytic-independent functions. Here we show that COX-2 appears to be cleaved in mouse and human tumors, which led us to hypothesize that COX-2 proteolysis may play a role in cell proliferation. The data presented herein show that a K598R point mutation at the carboxyl-terminus of COX-2 causes the appearance of several COX-2 immunoreactive fragments in nuclear compartments, and significantly enhances cell proliferation. In contrast, insertion of additional mutations at the border of the membrane-binding and catalytic domains of K598R COX-2 blocks fragment formation and prevents the increase in proliferation. Transcriptomic analyses show that K598R COX-2 significantly affects the expression of genes involved in RNA metabolism, and subsequent proteomics suggest that it is associated with proteins that regulate mRNA processing. We observe a similar increase in proliferation by expressing just that catalytic domain of COX-2 (ΔNT- COX-2), which is completely devoid of catalytic activity in the absence of its other domains. Moreover, we show that the ΔNT- COX-2 protein also interacts in the nucleus with β-catenin, a central regulator of gene transcription. Together these data suggest that the cleavage products of COX-2 can affect cell proliferation by mechanisms that are independent of prostaglandin synthesis., Competing Interests: The authors declare no conflict of interest

Background: The effect of 1-3 months of preoperative exclusive total parental nutrition (TPN) in active Crohn's disease (CD) patients is not well established. We investigated the efficacy of exclusive TPN in active CD patients., Methods: In a retrospective multi-visit study with data according to our standard care therapy, we assessed clinical and laboratory remission to refractory CD with exclusive preoperative TPN. Inclusion required exclusive preoperative home TPN without additional oral intake for 1-3 months prior to planning surgery., Results: Twenty preoperative CD patients (65% male; 35% female) were on exclusive TPN. The mean age of the cohort was 30.8 ± 11.6 years. Mean duration of preoperative TPN treatment was 73 days (range: 24-142 days). Most patients had terminal ileal (35%) or ileocolonic CD (30%), and with stricturing (B2) phenotype. All 20 patients had significant clinical improvement in all disease activity indices at the end of preoperative TPN (baseline vs. post TPN): HBI 14.5 vs. 4.0 ( p = 0.001); BMI 19.2 vs. 19.7 kg/m 2 ( p = 0.017); CRP 57.2 vs. 10.3 mg/L ( p = 0.001); Fecal calprotectin (FC) 672 vs. 200 (μg/g); albumin 2.7 vs. 3.6 g/dL ( p = 0.001). Two patients (10%) no longer required surgery after completion of exclusive TPN., Conclusion: Exclusive preoperative TPN was found to provide significant improvement in nutritional status, and clinical and laboratory remission in severe active Crohn's patients.

The limbic circuit is still undergoing maturation during juvenility and adolescence, explaining why environmental and metabolic challenges during these developmental periods can have specific adverse effects on cognitive functions. We have previously shown that long-term exposure (8-12 weeks) to high-fat diet (HFD) during adolescence (from weaning to adulthood), but not at adulthood, was associated with altered amygdala and hippocampal functions. Moreover, these HFD effects were normalized by treatment with glucocorticoid receptor (GR) antagonists. Here, we examined in male rats whether acute exposure (7-9 days) to HFD during juvenility [from postnatal day (PND) 21 to PND 28-30] or adulthood (from PND 60 to PND 67-69) is sufficient to affect hippocampal functions and whether it is also dependent on GRs activation. Juvenile HFD abolished both hippocampal synaptic plasticity, assessed through in vivo long-term potentiation (LTP) in CA1, and long-term hippocampal-dependent memory, using object location memory (OLM). No effect of HFD was observed in short-term OLM suggesting a specific effect on consolidation process. In contrast, adult HFD enhanced in vivo LTP and OLM. Systemic application of GR antagonist alleviated HFD-induced LTP and OLM impairments in juveniles. These results suggest that acute exposure to HFD during juvenility is sufficient to impair hippocampal functions in a GR-dependent manner. Interestingly, this effect depends on the developmental period studied as acute exposure to HFD at adulthood did not impair, but rather enhanced, hippocampal functions.

Juvenility represents a critical developmental phase during which exposure to a high fat diet (HFD) can severely modify cognitive and emotional functioning. The purpose of this study was to address how short and acute exposure to a HFD during juvenility affects social memory recognition and prefrontal long-term potentiation (LTP). As LTP and social memory depend on the neuromodulator oxytocin (OXY) and due to its role in metabolism, we also examined the effects of OXY in mediating HFD-induced alterations in social memory and LTP. Our results show that short exposure to a HFD during juvenility impairs social preference memory and prefrontal LTP. Interestingly, whereas systemic injections of OXY reversed the impairments in HFD-fed animals and impaired LTP and memory in control animals; prefrontal injections of the OXY agonist TGOT reversed the effects in HFD animals without affecting control animals. Exposure to HFD was associated with a reduction in the levels of OXY in the prefrontal compared to control animals. Interestingly, the restoration of social memory by TGOT in HFD animals was also associated with normalization of OXY in the prefrontal. These results point to a role that prefrontal OXY has in mediating the effects of HFD on memory and plasticity., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Metaplasticity is the dynamic regulation of the ability to induce activity-dependent synaptic plasticity and is governed by the prior history of the synapses. Previous reports by others and us have shown that behavioral stress induces a form of emotional metaplasticity that affects the ability to induce LTP in the subiculum-medial prefrontal cortex pathway, which depends on NMDA receptors (NMDAr). However, studies addressing the effects of stress on LTP and metaplasticity have mainly focused on the adult animal. Here we compared the effects of exposure to stress on the induction of LTP in adult and juvenile animals and examined whether a low dose of NMDAr antagonist (MK801) that does not affect LTP per se would differentially affect stress-induced metaplasticity in adult and juvenile animals. Our findings show that exposure to the elevated platform differentially affects the induction of LTP in adult and juvenile animals. Specifically, whereas exposure to stress resulted in impaired LTP in adult animals, it resulted in enhanced LTP in juvenile animals. Similarly, while MK801 failed to inhibit the induction of LTP in both age groups, it resulted in inhibition of stress-induced enhanced LTP in juvenile animals, but did not affect stress-induced impaired LTP in adult animals. Taken together, these findings demonstrate that emotional metaplasticity is differently dependent on NMDAr in adult and juvenile animals that may stem from developmental differences in the NMDA receptor representation. These results further confirm that the mechanisms of plasticity following stress are distinctive in the two groups of age., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Impaired microvascular function leads to a poor outcome in a variety of medical conditions. Our aim was to determine whether vasodilator responses to acetylcholine (Ach) are impaired in human omental arterioles from patients with severe trauma., Materials and Methods: Patients with massive blood loss and severe shock requiring damage control procedures were included. Tissues were collected at the first (FEL) and the second explorative laparotomy (SEL). Control tissues were collected from nontrauma patients. Freshly isolated 50-200-μm-diameter omental arterioles were analysed using videomicroscopy. Dihydroethidine and DCF-DA fluorescence were used to assess reactive oxygen species (ROS) production. MnTBAP was used to determine the contribution of excess vascular superoxide contribution to endothelial dysfunction., Results: After constriction (30-50%) with endothelin-1, dilation to graded doses of Ach (10 -9 -10 -4 M) was greater in control vessels compared to FEL and SEL (max dilation at 10 -4 M (MD) = 25 ± 3%, n = 8; and 59 ± 8%, n = 8, respectively, and controls MD = 93 ± 10%, n = 6, P < 0·05). Fluorescence imaging of ROS production showed significant increases in superoxide (225·46 ± 12·86; 215·77 ± 10·75 vs. 133·75 ± 7·26, arbitrary units; P < 0·05) and peroxide-related ROS (240·8 ± 20·42; 234·59 ± 28·86, vs. 150·78 ± 15·65, arbitrary units; P < 0·05), in FEL and SEL microvessels compared to control, respectively. FEL pretreated with MnTBAP demonstrated significant improvement in Ach-induced vasodilation (25·5 ± 3·0% vs. 79·5 ± 8·2%; P < 0·05)., Conclusions: Severe shock associated with microvascular endothelial dysfunction enhances production of ROS in human omental tissues. The altered flow regulation may contribute to a mismatch between local blood supply and demand, exacerbating abnormal tissue perfusion and function., (© 2016 Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Chronic groin pain appears in athletes with a diverse etiology. In a select few, it can be defined as a sportsman's hernia, that may be related, among other pathologies, to weakness of the posterior inguinal wall and may successfully respond to surgery., Hypothesis: Surgical repair of the sportsman's hernia is associated with good functional outcomes, if the diagnosis is based on meticulous examination and follows a simple selection flowchart., Study Design: Prospective case cohort study., Methods: The study assessed patients recruited from 2006 until the present assessed by a dedicated team with clinical and radiographic features of a sportsman's hernia who had failed a specified period of conservative therapies. Surgery was performed using a tension-free mesh open inguinal hernia repair., Results: Of 246 male patients with chronic groin pain, 51 underwent surgery (mean age 20.7 years, range 14-36 years) with 58 inguinal procedures performed. Of the operated group, seven underwent bilateral surgery with a direct hernia found in 9/58 operated sides (15.5%), an indirect hernial sac in 8/58 (14%) and a direct and indirect hernia being found in 3/58 (5%) of operated sides. There was no post-operative morbidity (median follow-up 36.1 months; range 1-74 months), with two failures (3.45 % of operated sides). All other patients were asymptomatic, returned to full sports activity within 4.3 weeks (range 3-8 weeks) after surgery, and required no analgesics or further treatment., Conclusion: Selective surgical hernia repair, based on meticulous anamnesis and physical examination is effective in the management of chronic groin pain in athletes.

Laser tissue soldering is a method of repairing incisions. It involves the application of a biological solder to the approximated edges of the incision and heating it with a laser beam. A pilot clinical study was carried out on 10 patients who underwent laparoscopic cholecystectomy. Of the four abdominal incisions in each patient, two were sutured and two were laser soldered. Cicatrization, esthetical appearance, degree of pain, and pruritus in the incisions were examined on postoperative days 1, 7, and 30. The soldered wounds were watertight and healed well, with no discharge from these wounds or infection. The total closure time was equal in both methods, but the net soldering time was much shorter than suturing. There was no difference between the two types of wound closure with respect to the pain and pruritus on a follow-up of one month. Esthetically, the soldered incisions were estimated as good as the sutured ones. The present study confirmed that temperature-controlled laser soldering of human skin incisions is clinically feasible, and the results obtained were at least equivalent to those of standard suturing.

Most cases of blunt hepatic trauma are treated nowadays non-operatively. This type of conservative treatment has resulted in increased complication rate. Delayed complications occur in cases that didn't require surgical intervention during the first 24 hours. The most common late complication is hemorrhage. We report a case of two weeks delayed hemorrhage after blunt hepatic trauma in an adult. We describe the diagnostic procedures, the surgical treatment and review the relevant literature.

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

The creation of anastomoses between various parts of the GI tract is a major task in the daily practice of oncological, reconstructive and transplant surgery. The most widely used anastomosing techniques today involve the use of sutures or metal titanium staples. Both techniques involve foreign material penetrating the tissue and evoking localized inflammatory response, tissue injury and breaking of mucosal barriers that may facilitate bacterial growth within the anastomotic line, increasing the propensity to anastomotic-related morbidity. Different types of compression devices were successfully used clinically in the past. The history and evolving characteristics of this technology is reviewed. Nitinol-based solutions for the creation of compression anastomosis are evaluated as a possible potential for revolutionary impact on the current surgical methods and anastomosing technology in the alimentary tract and beyond.

We tested the capacity of a newly developed portable gamma camera to precisely locate sentinel nodes by injecting a radiotracer. Two sets of experiments were performed on eight pigs under general anesthesia. 99mTc-Nanocolloid and dye complex was injected in the submuscular layer of the small bowel in the first set and subcutaneously in the knee region in the second set of experiments. Image acquisition of the sentinel nodes was performed with the Camera placed at various angles. A mosaic of images was obtained encompassing the injection sites, lymphatic pathways, and sentinel lymph nodes. Three-dimensional visualizations were obtained, allowing the precise location and complete excision of these nodes. The use of the portable gamma camera allowed the rapid visualization of the lymphatic pathways leading from the injection sites to the sentinel nodes and precise location of these nodes. The Camera was also useful to verify the complete removal of the labeled target tissues.

Background: Generations of investigators have attempted to achieve compression bowel anastomosis by a sutureless device, providing temporary support to the tissue and facilitating the natural healing process. The biocompatibility of nickel-titanium alloy has made it attractive for use in medical implants and devices, and several studies have described the creation of a side-to-side compression anastomosis in colon surgery with a nickel-titanium clip. We evaluated the feasibility and safety of a newly designed gun for applying a nickel-titanium compression anastomosis ring (CAR) to create an end-to-end colorectal anastomosis in a porcine model., Materials and Methods: A segment of the proximal rectum was resected in 25 pigs. The bowel ends were anastomosed transanally by an end-to-end CAR device. The animals' follow-up continued for up to 8 weeks, and included general health status, weight gain, blood tests, and abdominal X-ray. They were then sacrificed. The anastomoses were studied for burst pressure, anastomotic index, and histopathology., Results: One pig died due to iatrogenic bowel injury unrelated to the CAR device. There was no other morbidity/mortality. The other animals recovered and gained weight. Burst pressure studies demonstrated a minimum pressure of 160 mmHg at time point 0 that escalated quickly to >300 mmHg. The mean anastomotic index after 8 weeks was 0.81. Histologic evaluation revealed minimal inflammation and minimal fibrosis at the anastomosis site., Conclusion: The principles of compression anastomosis are better executed with the use of memory shape alloys. The promising results of this novel technique should encourage further studies of this technology.

Flow-induced dilation (FID) is an important physiological stimulus that regulates tissue blood flow and is mediated by endothelium-derived factors that play a role in vascular integrity and the development of atherosclerosis. In coronary artery disease (CAD), conduit artery FID is impaired. The purpose of this study was to determine the mechanism of FID in human visceral adipose and examine whether the presence of conduit coronary atherosclerosis is associated with altered endothelial function in visceral fat. FID was determined in isolated visceral fat arterioles from patients with and without CAD. After constriction with endothelin-1, increases in flow produced an endothelium-dependent vasodilation that was sensitive to N(omega)-nitro-l-arginine methyl ester (l-NAME) in visceral fat arterioles from patients without CAD. In contrast, l-NAME alone or in combination with indomethacin had no effect on FID in similarly located arterioles from patients with CAD. Flow increased dichlorofluorescein (DCF) and dihydroethidium fluorescence accumulation in arterioles from patients with CAD versus without, indicative of the production of oxidative metabolites and superoxide, respectively. Both the dilation and DCF fluorescence to flow were reduced in the presence of the H(2)O(2) scavenger polyethylene glycol-catalase. Exogenous H(2)O(2) elicited similar relaxations of arterioles from patients in both groups. These data indicate that FID in visceral fat arterioles is nitric oxide dependent in the absence of known CAD. However, in the presence of CAD, H(2)O(2) replaces nitric oxide as the mediator of endothelium-dependent FID. This study provides evidence that adverse microvascular changes during CAD are evident in human visceral adipose, a tissue associated with CAD.

Acute lung injury (ALI) carries a high mortality in critically ill patients. Recent reports correlate elevated concentrations of endothelium-derived microparticles (EMPs) with diseases of endothelial dysfunction. Many of these diseases have ALI sequelae. We hypothesize that EMPs contribute to endothelial cell (EC) dysfunction and development of ALI. To test this hypothesis, we treated isolated vessels with EMPs and examined changes in vasodilation. Endothelial cell cultures were incubated with EMPs and examined for changes in stimulated nitric oxide (*NO) production and nitric oxide synthase (eNOS) activation. Finally, EMPs were injected into rats and mice and lungs examined for ALI. In both mouse and human ex vivo vessel preparations, we found a marked attenuation of endothelium-mediated vasodilation after EMP treatment (4 x 10(6)/mL). This dysfunction was not corrected by pretreatment of EMPs with free radical scavengers. Coincubation of EMPs with EC cultures yielded a three-fold reduction in A23187-stimulated *NO release. Western analysis of these cells showed a corresponding decrease in eNOS phosphorylation at Ser1179 and a decrease in hsp90 association. Measurements of lung permeability, myeloperoxidase activity, and histology of EMPs-treated Brown Norway rats demonstrated pulmonary edema, neutrophil recruitment, and compromise of the endothelial-alveolar barrier as a second hit phenomenon. In C57BL/6 mice, exogenous EMPs caused a significant rise in pulmonary capillary permeability both as a primary and secondary injury. These findings demonstrate EMPs are capable of inducing significant lung injury at pathophysiologically relevant concentrations. Endothelium-derived microparticles inhibit endothelium-mediated vasodilation and *NO generation from eNOS. Once elucidated, EMP mechanisms of inducing ALI and endothelial dysfunction may present new therapeutic targets.

The Purpose of This Study: To evaluate the safety and efficacy of an enhanced magnetic resonance-guided focused ultrasound (MRgFUS) emission protocol that results in more extensive treatment by increasing the volume of each focal ablation using the same energy., Materials and Methods: Six pigs were treated with an MRgFUS system combined with real-time MR, for imaging and temperature mapping, with 102 "enhanced" and 97 "regular" focal ablations performed on both buttock muscles. Real-time imaging, temperature mapping, and acoustic reflected spectrum data enabled immediate evaluation of the results. MR contrast-enhanced images and pathology examinations were used for confirmation., Results: The location of the ablated volume by "enhanced" sonication is predictable, with a maximum possible shift of 6 mm toward, and 3 mm away, from the transducer. The ablated volume after enhanced sonication was, on average, 1.8 times larger than after a regular sonication of the same energy. Pathology results showed the same thermally induced damage patterns in the enhanced sonications and the regular sonications., Conclusion: Accelerated MRgFUS with enhanced sonication is a safe, controllable, and more effective tissue ablative modality than standard sonication. This new technology may significantly reduce the length of tumor ablation procedures. (Isn't the new technology you're talking about MRgFUS? If so, you don't need to repeat it at the end of this sentence.).

Chronic inflammation is a complex biologic process which involves immune as well as non-immune cells including the microvasculature and its endothelial lining. Growing evidence suggests that the microvasculature plays an integral role in the pathophysiology of inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis). The microvasculature contributes to chronic inflammation through altered leukocyte recruitment, impaired perfusion, and angiogenesis leading to tissue remodeling. These diverse areas of IBD microvascular biology represent therapeutic targets that are currently undergoing investigation.

The importance of endothelium-derived nitric oxide in coronary vascular regulation is well-established and the loss of this vasodilator compound is associated with endothelial dysfunction, tissue hypoperfusion and atherosclerosis. Numerous studies indicate that the endothelium produces another class of compounds, the epoxyeicosatrienoic acids (EETs), which may partially compensate for the loss of nitric oxide in cardiovascular disease. The EETs are endogenous lipids which are derived through the metabolism of arachidonic acid by cytochrome P450 epoxygenase enzymes. Also, EETs hyperpolarize vascular smooth muscle and induce dilation of coronary arteries and arterioles, and therefore may be endogenous mediators of coronary vasomotor tone and myocardial perfusion. In addition, EETs have been shown to inhibit vascular smooth muscle migration, decrease inflammation, inhibit platelet aggregation and decrease adhesion molecule expression, therefore representing an endogenous protective mechanism against atherosclerosis. Endogenous EETs are degraded to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. Pharmacological inhibition of soluble epoxide hydrolase has received considerable attention as a potential approach to enhance EET-mediated vascular protection, and several compounds have appeared promising in recent animal studies. The present review discusses the emerging role of EETs in coronary vascular function, as well as recent advancements in the development of pharmacological agents to enhance EET bioavailability.

Objective: Endothelial dysfunction and vascular dysregulation contribute to the pathological effects of radiation on tissues. The objectives of this study were to assess the acute effect of irradiation on acetylcholine (Ach)-induced dilation of gut submucosal microvessels., Methods and Results: Rats were exposed in vivo to 1 to 9 cGy in 3 fractions per week on alternate days for 3 successive weeks for a total dose of up to 2250 cGy. Submucosal microvessels were isolated after varying levels of irradiation. Diameters of isolated vessels were measured using videomicroscopy, and the dose-response relationship to Ach was determined. Dihydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate fluorescent probes were used to assess reactive oxygen species (ROS) production. After constriction (30% to 50%) with endothelin, dilation to graded doses of Ach (10(-9)-10(-4) M) was observed in control vessels (maximal dilation [MD] 87+/-3%; n=7). However, Ach-induced dilation was reduced in vessels from irradiated rats (MD=3+/-9%; n=7; P= or <0.05 versus controls). Significant increases in superoxide and peroxides were observed in irradiated microvessels. Irradiated microvessels pretreated with superoxide dismutase-mimetic demonstrated significant improvement in Ach-induced vasodilation compared with irradiation alone, suggesting that superoxide contributes to impaired dilation to Ach after irradiation., Conclusions: Radiation induces acute microvascular dysfunction in the resistance arterioles of the intestine. Enhanced ROS contribute to this dysfunction and therefore may represent a novel therapeutic target to minimize radiation toxicity in the gut.

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.

Recent evidence suggests that the adipose tissue-derived cytokine leptin (LEP) is involved in modulation of growth and differentiation of normal small intestine. The purpose of the present study was to evaluate the effects of parenteral LEP on structural intestinal adaptation, cell proliferation and apoptosis in a rat model of short bowel syndrome (SBS). Male Sprague-Dawley rats were divided into three experimental groups: Sham rats underwent bowel transection and re-anastomosis, SBS-rats underwent a 75% small bowel resection, and SBS-LEP-rats underwent bowel resection and were treated with LEP given subcutaneously at a dose of 20 mug/kg, once daily, from day 3 through 14. Parameters of intestinal adaptation (bowel and mucosal weights, mucosal DNA and protein, villus height and crypt depth in jejunum and ileum), enterocyte proliferation and enterocyte apoptosis were determined on day 15 following operation. Ileal tissue samples were taken for detection of bax and bcl-2 gene expression using RT-PCR technique. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA test, with P<0.05 considered statistically significant. Treatment with subcutaneous LEP resulted in a significant increase in jejunal (17%, P<0.05) and ileal (13%, P<0.05) bowel weight, jejunal (10%, P<0.05) and ileal (25%, P<0.05) mucosal weight, jejunal (26%, P<0.05) and ileal (38%, P<0.05) mucosal DNA, ileal (25%, P<0.05) mucosal protein, jejunal (41%, P<0.05) and ileal (21%, P<0.05) villus height, jejunal (37%, P<0.05) crypt depth, and jejunal (24%, P<0.05) and ileal (21%, P<0.05) enterocyte proliferation compared to SBS-animals. Enterocyte apoptosis increased significantly after bowel resection in jejunum and ileum compared to sham animals and was accompanied by an increased bax gene expression and a decreased bcl-2 gene expression in ileal samples. SBS-LEP rats showed a trend toward a decrease in enterocyte apoptosis in ileum and a mild decrease in bax gene expression compared to SBS-untreated animals. In conclusion, in a rat model of SBS parenteral LEP stimulates structural intestinal adaptation. Increased cell proliferation and decreased cell death via apoptosis may be responsible for this increased cell mass.

Background and Aims: Radiation therapy of abdominal and pelvic solid tumours results in late intestinal toxicity of a severe nature in approximately 5% of cases. These manifestations may include ischaemia and stricture formation, which may present as "webs". These webs are likely to play a role in the pathogenesis of recurrent bowel obstruction. The mechanisms of microvascular injury to the bowel in the setting of radiation have not been defined. We hypothesised that microvascular dysfunction with impaired vasodilation to acetylcholine (Ach) would be an acquired pathophysiological abnormality in radiation and "web" formation., Methods: A 40 year old patient treated with radiation, two years previously, for an anal squamous cell cancer presented with recurrent small bowel obstruction. "Webs" in the distal ileum were detected using wireless capsule endoscopy, after small bowel barium radiographs failed to demonstrate a lesion. Following resection, freshly isolated 50-150 mum diameter arterioles from the "web" and adjacent normal calibre bowel were analysed with histology and microvessel physiological studies., Results: After constriction (30-50%) with endothelin, dilation to graded doses of Ach (10(-9)-10(-4) M) was observed in vessels dissected from the stricture and the adjacent normal calibre area. Ach dilation was reduced in vessels from "web" (mean diameter 7 (2)%; n = 3, p < 0.01) compared with the adjacent unaffected bowel (mean diameter 85 (5)%). Dihydroethidine and dichlorofluorescein diacetate intravital staining demonstrated increased reactive oxygen species production in microvessels from "web" compared with adjacent normal calibre bowel. Histology from the strictured bowel demonstrated narrowing of the arterial lumen due to intimal and muscularis propria fibrosis, with endothelial preservation., Conclusions: External radiation is associated with acquired microvascular endothelial dysfunction and "web" formation in the small bowel.

Objective: Endothelium-dependent dilation to acetylcholine (Ach) is reduced in mucosal arterioles from patients with inflammatory bowel disease (IBD). The contributions of both nitric oxide (NO) and endothelial-derived hyperpolarizing factor (EDHF) are decreased. We hypothesized that the remaining dilation results from products of cyclooxygenase., Methods and Results: High-performance liquid chromatography (HPLC) was used to isolate eicosanoid vasodilator products and videomicroscopy was used to examine vasomotor responses in human mucosal arterioles from subjects with or without IBD undergoing bowel resection surgeries. In subjects without IBD, Ach constricted (-52%+/-10%) arterioles devoid of endothelium. Indomethacin (INDO) (cyclooxygenase inhibitor) had no effect. In contrast, Ach dose-dependently dilated both intact and endothelial denuded arterioles from patients with IBD. The dilation was converted to constriction by INDO (-54%+/-9%; P<0.05 versus non-IBD) or by BWA868C (PGD2 receptor antagonist). Only in arterioles from subjects with IBD did Ach produce an arachidonic acid metabolite that comigrated on HPLC with PG D2 (PGD2). Exogenous PGD2 dilated (max=66%+/-4%) IBD arterioles., Conclusions: In arterioles from IBD patients, Ach-mediated dilation shifts from endothelial production of NO and EDHF to nonendothelial generation of a PG, likely PGD2. This is a novel dilator mechanism arising from nonendothelial vascular tissue that compensates for loss of endothelium-dependent dilation. PGD2 appears to be important in regulating mucosal blood flow in patients with IBD, implicating potentially detrimental effects from nonsteroidal antiinflammatory drugs.

This review has focused on evidence regarding intestinal perfusion of inflammatory bowel disease (IBD). Basic investigation has defined an altered microvascular anatomy in the affected IBD bowel, which corresponds with diminished mucosal perfusion in the setting of chronic, long-standing inflammation. Diminished perfusion is linked to impaired wound healing, and may contribute to the continued refractory mucosal damage, which characterizes IBD. Alterations in vascular anatomy and physiology in IBD suggests additional possible mechanisms by which micro-vessels may contribute to the initiation and perpetuation of IBD. This begs the following questions: will angiogenesis within the gut lead to sustained inflammation, does the growing vasculature generate factors that transform the surrounding tissue and does angiogenesis generate vascular anastomosis within the gut, with shunting of blood away from the mucosal surface, impairment of metabolism and potentiation of gut damage? Further studies are required to define the mechanisms that underlie the vascular dysfunction and its role in pathophysiology of IBD.

Adenosine is a key myocardial metabolite that elicits coronary vasodilation in a variety of pathophysiological conditions. We examined the mechanism of adenosine-induced vasodilation in coronary arterioles from patients with heart disease. Human coronary arterioles (HCAs) were dissected from pieces of the atrial appendage obtained at the time of cardiac surgery and cannulated for the measurement of internal diameter with videomicroscopy. Adenosine-induced vasodilation was not inhibited by endothelial denudation, but A(2) receptor antagonism with 3,7-dimethyl-1-propargylxanthine and adenylate cyclase (AC) inhibition with SQ22536 significantly attenuated the dilation. In contrast, A(1) receptor antagonism with 8-cyclopentyl-1,3-dipropylxanthine significantly augmented the sensitivity to adenosine. Moreover, dilation to A(2a) receptor activation with 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine hydrochloride was reduced by the A(1) receptor agonist (2S)-N(6)-(2-endo-norbornyl)adenosine. The nonspecific calcium-activated potassium (K(Ca)) channel blocker tetrabutylammonium attenuated adenosine-induced dilation, as did the intermediate-conductance K(Ca) blocker clotrimazole. Neither the large-conductance K(Ca) blocker iberiotoxin nor small-conductance K(Ca) blocker apamin altered the dilation. In conclusion, adenosine endothelium independently dilates HCAs from patients with heart disease through a receptor-mediated mechanism that involves the activation of intermediate-conductance K(Ca) channels via an AC signaling pathway. The roles of A(1) and A(2) receptor subtypes are opposing, with the former being inhibitory to AC-mediated dilator actions of the latter. These observations identify unique fundamental physiological characteristics of the human coronary circulation and may help to target the use of novel adenosine analogs for vasodilation in perfusion imaging or suggest new strategies for myocardial preconditioning.

Mesenteric venous thrombosis (MVT) is a rare but potentially catastrophic clinical complication, which may lead to ischemia or infarction of the intestine and/or the emergence of portal hypertension. An association between inflammatory bowel disease (IBD) and MVT has previously been described, but clinical factors that may contribute to this complication in the setting of IBD are not well characterized. Diagnosis of MVT in IBD is difficult, as patients frequently present with nonspecific abdominal discomfort, which may delay diagnosis and initiation of treatment. We report 6 of 545 IBD patients at our center (1.1%) that developed MVT, and describe presentation, diagnostic approaches, treatment options, underlying contributing factors, and outcome. The diagnosis was determined with abdominal computed tomography (CT) in 5 of 6 cases. Clinical factors, which were thought to contribute to MVT, included underlying hypercoagulability, low-flow state, uncontrolled inflammation, perioperative time period, and prior surgical manipulation of the portal vein following orthotopic liver transplantation. There were no deaths as a result of MVT, although 1 patient developed severe portal hypertension and another experienced intestinal infarction requiring extensive resection. We conclude that MVT is an important clinical consideration in IBD patients, specifically during the perioperative setting, and diagnosis is facilitated with the use of CT scan.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several mediators of vasodilation, which include prostacyclin (PGI(2)), nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently defined the role of nitric oxide and PGI(2) in the dilation of submucosal intestinal arterioles from patients with normal bowel function. However, significant endothelium-dependent dilator capacity to ACh remained after inhibiting both these mediators. The current study was designed to examine the potential role of EDHF in human intestinal submucosal arterioles. ACh elicited endothelium-dependent relaxation in the presence of inhibitors of nitric oxide synthase and cyclooxygenase (23 +/- 10%, n = 6). This ACh-induced relaxation was inhibited and converted to constriction by catalase (-53 +/- 10%, n = 6) or KCl (-30 +/- 3%, n = 7), whereas 17-octadecynoic acid and 6-(2-propargylloxyphenyl) hexanoic acid, two inhibitors of cytochrome P450 monooxygenase, had no significant effect (3 +/- 1% and 20 +/- 8%, n = 5, respectively). Exogenous H(2)O(2) elicited dose-dependent relaxation of intact microvessels (52 +/- 10%, n = 7) but caused frank vasoconstriction in arterioles denuded of endothelium (-73 +/- 8%, n = 7). ACh markedly increased the dichlorofluorescein fluorescence in intact arterioles in the presence of nitric oxide synthase and cyclooxygenase inhibitors compared with control and compared with catalase-treated microvessels (363.6 +/- 49, 218.8 +/- 10.6, 221.9 +/- 27.9, respectively, P < 0.05 ANOVA, n = 5 arbitrary units). No changes in the dichlorofluorescein fluorescence were recorded in vessels treated with ACh alone. These results indicate that endothelial production of H(2)O(2) occurs in response to ACh in human gut mucosal arterioles but that H(2)O(2) is not an EDHF in this tissue. Rather, we speculate that it stimulates the release of a chemically distinct EDHF.

Background: The purpose of this study was to determine the accuracy of barium radiography compared with intraoperative evaluation with passage of a balloon catheter for assessment of stricturing Crohn's disease (CD)., Methods: After institutional review board approval, we retrospectively reviewed a tertiary inflammatory bowel disease center's consecutive records of surgical patients between 1998 and 2003 with small intestinal CD to compare the number of strictures found at surgery with those identified preoperatively by barium imaging. Age, gender, prior surgical procedures, and steroid usage were recorded. By decision of the surgeons, all patients were treated with an identical approach that utilized intraluminal sizing with passage of a balloon-tipped catheter., Results: In 118 patients, 230 strictures were identified by barium examination; 365 strictures were identified using the balloon catheter technique. Barium examination overestimated or underestimated the number of strictures in 43 of 118 patients (36%). Overall, barium radiography was least accurate in patients with strictures amenable to strictureplasty. Prior surgery and multiple strictures identified preoperatively by barium studies were found to decrease the accuracy of the barium examination, but the decrease did not reach statistical significance. After successful surgery for stricturing small intestinal CD, more than 90% of patients can successfully be weaned from their steroids within 3 months. Failure to be able to wean from steroids may suggest a missed stricture., Conclusions: Our data suggest that careful exploration and intraoperative, intraluminal testing of intestinal patency identify additional strictures compared with barium radiographs in a significant number of patients with CD undergoing small bowel surgical intervention.

Background & Aims: Inflammatory bowel disease (IBD; i.e., Crohn's disease, ulcerative colitis) is characterized by refractory inflammatory ulceration and damage to the intestine. Mechanisms underlying impaired healing are not defined. Because microvascular dysfunction resulting in diminished vasodilatory capacity and tissue hypoperfusion is associated with impaired wound healing, we hypothesized that microvascular dysfunction may also occur in chronic IBD., Methods: Intact submucosal arterioles from control, involved, and uninvolved IBD specimens were assessed using in vitro videomicroscopy to assess endothelium-dependent vasodilation in response to acetylcholine (Ach) and fluorescence microscopy to detect oxyradicals., Results: Normal microvessels dilated in a dose-dependent and endothelium-dependent manner to Ach (maximum, 82% +/- 2%; n = 34). Inhibition of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester (L-NAME) reduced maximal dilation to 54% +/- 6% (P < 0.05, n = 7), and further reduction was observed after inhibiting cyclooxygenase (indomethacin; 23% +/- 10%, n = 6). Chronically inflamed IBD microvessels showed significantly reduced Ach-induced vasodilation (maximum, 15% +/- 2%; n = 33), with no effect of L-NAME. Indomethacin eliminated the remaining Ach-induced vasodilation, resulting in frank vasoconstriction (-54% +/- 9%, n = 6). Uninvolved IBD gut vessels and non-IBD inflammatory controls responded in a fashion similar to normal vessels. IBD-involved microvessels generated significantly higher levels of reactive oxygen species compared with control and uninvolved IBD vessels (P < 0.01)., Conclusions: Human intestinal microvessels from chronically inflamed IBD show microvascular endothelial dysfunction, characterized by loss of NO-dependent dilation that may contribute to reduced perfusion, poor wound healing, and maintenance of chronic inflammation.

Background: Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)-dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O2*-) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor-null (Ldlr-/-) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O2*- generation., Methods and Results: Arterioles were isolated from hypercholesterolemic Ldlr-/- mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O2*- generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase-inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr-/- mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2*- in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice., Conclusions: These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD. Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases.

Thrombin (Thromb), activated as part of the clotting cascade, dilates conduit arteries through an endothelial pertussis toxin (PTX)-sensitive G-protein receptor and releases nitric oxide (NO). Thromb also acts on downstream microvessels. Therefore, we examined whether Thromb dilates human coronary arterioles (HCA). HCA from right atrial appendages were constricted by 30-50% with endothelin-1. Dilation to Thromb (10(-4)-1 U/ml) was assessed before and after inhibitors with videomicroscopy. There was no tachyphylaxis to Thromb dilation (maximum dilation = 87.0%, ED(50) = 1.49 x 10(-2)). Dilation to Thromb was abolished with either hirudin or denudation but was not affected by PTX. Neither N(omega)-nitro-l-arginine methyl ester (n = 7), indomethacin (n = 9), (1)H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (n = 6), tetraethylammonium chloride (n = 5), nor iberiotoxin (n = 4) reduced dilation to Thromb. However, KCl (maximum dilation = 89 +/- 5 vs. 20 +/- 10%; P < 0.05; n = 7), tetrabutylammonium chloride (maximum dilation = 79 +/- 7 vs. 21 +/- 4%; P < 0.05; n = 5), and charybdotoxin (maximum dilation = 89 +/- 4 vs. 10 +/- 2%; P < 0.05; n = 4) attenuated dilation to Thromb. In contrast to animal models, Thromb-induced dilation in human arterioles is independent of G(i)-protein activation and NO release. However, Thromb dilation is endothelium dependent, is maintained on consecutive applications, and involves activation of K(+) channels. We speculate that an endothelium-derived hyperpolarizing factor contributes to Thromb-induced dilation in HCA.

We have previously observed that bolus fluid resuscitation in uncontrolled hemorrhagic shock induced by solid organ injury leads to increased blood loss and mortality. In the present investigation, we studied the effect of continuous fluid resuscitation on the hemodynamic response and survival following massive splenic injury (MSI) in rats. The animals were randomized into 11 groups: group 1, sham-operated; group 2, MSI untreated; group 3, MSI treated with 17.5 mL/kg/h of Ringers lactate (RL) solution (RL-17.5); group 4, MSI treated with 35 mL/kg/h RL (RL-35); group 5, MSI treated with 70 mL/kg/h RL (RL-70); group 6, MSI treated with 7.5 mL/kg/h of 7.5% NaCl (HTS-7.5); group 7, MSI treated with 15 mL/kg/h of 7.5% NaCl (HTS-15); group S, MSI treated with 30 mL/kg/h of 7.5% NaCl (HTS-30); group 9, MSI treated with 7.5 mL/kg/h 6% hydroxyethyl starch (HES-7.5); group 10, MSI treated with 15 mL/kg/h 6% hydroxyethyl starch (HES-15); and group 11, MSI treated with 30 mL/kg/h 6% hydroxyethyl starch (HES-30). MSI in untreated group 2 was followed by a fall of mean arterial pressure (MAP) to 50.1 +/- 6.7 mmHg (P < 0.001) in 15 min. Mean survival time (MST) was 99.5 +/- 16.6 min, and total blood loss (TBL) was 37.8% +/- 2.6% of blood volume. Fluid treatment with increasing volumes of RL in groups 3, 4, and 5 was followed by a gradual increase in TBL compared with untreated animals, and MST remained unchanged. Increasing volumes of HTS infusion in groups 6, 7, and 8 was also followed by incease in TBL, but MST remained unchanged except for an increase to 123.0 +/- 20.5 min (P < 0.05) in group 6. Increasing volumes of HES in groups 9, 10, and 11 was also followed by increase in TBL, but MST remained unchanged. In conclusion, continuous infusion of LR, HTS, and HES following massive splenic injury resulted in a significant increase in intra-abdominal bleeding, but survival time in the first hour following injury remained unchanged in contrast to bolus fluid infusion, which increases early mortality.

MyoD is a tissue-specific transcriptional activator involvd in skeletal muscle differentiation. It is induced during transition from proliferating, non-differentiated myoblasts to the resting and well differentiated myotubes. Like many other transcriptional regulators, it is short-lived, however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved have remained obscure. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In cells, degradation is inhibited by lactacystin, a specific inhibitor of the 20S proteasome. Inhibition is accompanied by accumulation of MyoD-ubiquitin conjugates. In a cell free system, the proteolytic process requires both ATP and ubiquitin and is preceded by formation of MyoD-ubiquitin adducts. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds. Analysis of the ubiquitination site has revealed that the N-terminal residue of MyoD is sufficient and essential to promote conjugation and subsequent degradation of the protein: conjugation to internal Lys residues is not necessary. Substitution of all Lys residues did not affect significantly its degradation either in intact cells or in a reconstituted cell free system. Degradation was inhibited by specific proteasome inhibitors and was accompanied by accumulation of ubiquitinated species of the protein. We concluded that the first ubiquitin moiety is attached via its C-terminal Gly to the N-terminal residue of MyoD, and the polyubiquitin chain is then synthesized on Lys48 of this moiety.