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Michael W Mulholland

Publications and source records attributed to Michael W Mulholland.

At least 19 recordsLinked to original sources
PubMed2006

Melanocortin-3 receptor activates MAP kinase via PI3 kinase.

HEK 293 cells stably expressing human melanocortin-3 receptor (MC3R) were exposed to melanocortin receptor agonist, NDP-MSH (10(-)(10)-10(-)(6) M). ERK1/2 was phosphorylated in a dose-dependent manner with an EC(50) of 3.3+/-1.5 x 10(-)(9) M, similar to the IC(50) of NDP-MSH binding to the MC3R. ERK1/2 phosphorylation was blocked by the melanocortin receptor antagonists SHU9119. NDP-MSH-induced ERK1/2 phosphorylation was sensitive to pertussis toxin and the PI3K inhibitor, wortmannin. Rp-cAMPS, BAPTA-AM and Myr-PKC did not inhibit the NDP-MSH-induced ERK1/2 phosphorylation. NDP-MSH stimulated cellular proliferation in a dose-dependent manner with a similar EC(50) to ERK1/2 phosphorylation, 2.1+/-0.6 x 10(-)(9) M. Cellular proliferation was blocked by AGRP (86-132) and by the MEK inhibitor, PD98059. The NDP-MSH did not inhibit serum deprivation-induced apoptosis. MC3R activation induces ERK1/2 phosphorylation via PI3K and this pathway is involved in cellular proliferation in HEK cells expressing MC3R.

Agouti-Related Protein↗
PubMed2006

Melanocortin-4 receptor-mediated inhibition of apoptosis in immortalized hypothalamic neurons via mitogen-activated protein kinase.

The melanocortin-4 receptor (MC4R) is a seven transmembrane member of the melanocortin receptor family. The GT1-1 cell line exhibits endogenous expression of MC4R. In this study, GT1-1 cells were used to study MC4R signaling pathways and to examine the effects of melanocortin receptor agonist NDP-MSH on apoptosis. MC4R mRNA expression was demonstrated by RT-PCR. Functional melanocortin receptor expression was implied by specific binding of NDP-MSH and cAMP production. NDP-MSH-stimulated GnRH release in a dose-dependent manner. Serum deprivation-induced apoptosis in GT1-1 cells, and the NDP-MSH inhibited this effect. The melanocortin receptor antagonist SHU9119 blocked the antiapoptotic actions of NDP-MSH, and the MAP kinase inhibitor PD98059 significantly attenuated the antiapoptotic effect. NDP-MSH-stimulated ERK1/2 phosphorylation in a dose-dependent manner. ERK1/2 phosphorylation could be abolished by SHU9119. In GT1-1 cells, melanocortin receptor activation causes ERK1/2 phosphorylation. In these cells, MC4R activation is also associated with antiapoptotic effects.

Animals↗
PubMed2006

Activation of the melanocortin-4 receptor mobilizes intracellular free calcium in immortalized hypothalamic neurons.

BACKGROUND: At least 4% of childhood obesity is due to mutations in the hypothalamic melanocortin-4 receptor. The melanocortin-4 receptor, a seven transmembrane G-protein-coupled receptor, is important in the regulation of feeding behavior and body weight. The specific pathways of intracellular signaling remain in investigative stages. To further understand its function, we hypothesized that the melanocortin-4 receptor activates the Galphaq/phospholipase C signaling pathway, resulting in alterations of cytoplasmic calcium in immortalized hypothalamic (GT1-1) neurons. MATERIALS AND METHODS: Changes in intracellular calcium were measured after loading GT1-1 cells with fura-2-AM. Cells were treated with NDP-alphaMSH, an alpha-melanocortin stimulating hormone analogue, and intracellular calcium changes were recorded. Cells treated with NDP-alpha-MSH were also treated with the melanocortin-4 receptor antagonist, SHU-9119. To assess the specific G-protein subunit involved, GT1-1 neurons were treated with the phospholipase C inhibitor U73122 and its inactive analogue, U73433. Experiments were also performed after inhibition of IP3 receptors with 2-aminoethoxydiphenylborate (2APB). Additional experiments were conducted in a calcium-depleted buffer environment. Data were analyzed by ANOVA with statistical significance of P < 0.05. RESULTS: Agonist treatment (0.01-1000 nm) of GT1-1 neurons resulted in dose-dependent increases in intracellular calcium. SHU-9119 (0.01-1000 nm) abolished the calcium response. Treatment with U73122 (10 microm) attenuated the calcium response, while U73433 (10 microm) had minimal effect. 2APB (200 microm) inhibited the calcium transient, and the use of calcium-free buffer did not affect the amplitude of the calcium spike. CONCLUSIONS: Our study demonstrates that, upon agonist binding, the melanocortin-4 receptor mediates increases in intracellular calcium through the Galphaq-protein/phospholipase C dependent signaling pathway. Understanding the physiological importance of calcium signaling by the melanocortin-4 receptor may be important for future development of therapeutic targets.

Animals↗
PubMed2006

Adjuvant treatment strategies for pancreatic cancer.

Pancreatic cancer is a difficult and unsolved surgical problem. It remains one of the top five causes of cancer-related deaths and has the lowest 5-year survival of any cancer, largely due to late diagnosis, low resection rates, and local recurrence. Clinical trials examining the optimal timing and delivery of adjuvant therapies for pancreatic cancer have yielded controversial results. Although most experts agree that the addition of chemotherapy has survival benefit in patients with resectable pancreatic cancer, there is no consensus regarding the optimal therapeutic agents, timing (neoadjuvant versus adjuvant), and the addition of radiation therapy to the treatment regimen. Multiple phase III trials are in progress in efforts to examine these issues. Additionally, exciting progress has been made with novel chemotherapeutic combinations, and alternative treatment modalities including interferon-alpha, immunotherapy, and pancreatic cancer stem cells. Given the high failure pattern after surgical resection, with more than half of patients developing locoregional recurrence, all patients undergoing pancreaticoduodenectomy are candidates for adjuvant therapy.

Adenocarcinoma↗
PubMed2006

Prophylactic gastrectomy for hereditary diffuse gastric cancer syndrome.

BACKGROUND: Germline mutations in the E-cadherin gene, CDH1, were recently identified in families with hereditary diffuse gastric cancer. The efficacy of endoscopic surveillance by current methods is largely ineffective because most tumors spread diffusely. There has been little evidence that prophylactic gastrectomy should be performed in patients with the genetic mutation, and few data are available on the outcomes of operations in patients with the CDH1 mutation undergoing prophylactic gastrectomy. We report the outcomes of patients with the CDH1 mutation who have undergone prophylactic gastrectomy in the year 2003 to 2004. STUDY DESIGN: After genetic counseling and informed consent, two patients underwent total gastrectomy with Roux-en-Y reconstruction. Demographic information, pedigree analysis, preoperative screening results, operative course, and postoperative data, including complications, were collected and reviewed for each patient. RESULTS: Pathologic examination of the stomach revealed no evidence of in situ or invasive carcinoma. There were no operative complications. Both patients reported diarrhea and moderate weight loss. There were no other postoperative complications. CONCLUSIONS: Prophylactic gastrectomy can be performed safely, without mortality or severe morbidity, in patients with CDH1 mutations, and should be curative in this population of patients.

Adolescent↗
PubMed2005

Interactions of human melanocortin 4 receptor with nonpeptide and peptide agonists.

Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane alpha-helices (TM) 2, 3, 6, and 7 (residues Glu(100), Asp(122), Asp(126), Phe(261), His(264), Leu(265), and Leu(288)). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor-ligand complexes with their agonists were generated by distance-geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys(40)-Cys(279), Cys(271)-Cys(277)) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His(6), d-Phe(7), Arg(8), and Trp(9)) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His(6) and Arg(6) of NDP-MSH; their substituted piperidines mimic Trp(9) of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu(133), Trp(258), and Phe(261) residues.

Amino Acid Sequence↗
PubMed2005

Serum-free culture of rat postnatal neurons derived from the dorsal motor nucleus of the vagus.

Previous studies on dorsal motor nucleus of the vagus (DMNV) neurons have mainly used in vivo animal models and in vitro brainstem slices. Primary culture of postnatal DMNV neurons in defined serum free medium has not been reported. We report a method for culture of postnatal rat DMNV neurons using serum free medium. Cultured DMNV neurons contain both Hu positive precursor cells and mature cells staining positively for microtubule associated protein 2 (MAP2) and choline acetyltransferase. Exposure of cultured DMNV neurons to glutamate (10(-7) to 10(-3)M) induced an increase in intracellular calcium concentration ([Ca(2+)](i)) in a dose-dependent manner, indicating the functional presence of glutamate receptors. Voltage-dependent calcium currents were present in cultured DMNV neurons. Active cell proliferation was demonstrated by BrdU incorporation. Upon removal of beta FGF, the percentage of MAP2 positive mature neurons was significantly increased from 36+/-3 to 73+/-3%. Our study demonstrates that postnatal rat DMNV neurons cultured in serum free medium retain morphological and physiological characteristics of DMNV neurons in situ.

Animals↗
PubMed2005

Stimulation of neurogenesis in rat nucleus of the solitary tract by ghrelin.

Ghrelin, a gastric hormone, regulates growth hormone secretion and energy homeostasis. The present study shows that ghrelin promotes neural proliferation in vivo and in vitro in the rat nucleus of the solitary tract (NTS). Systemic administration of ghrelin significantly increased 5-bromo-2'-deoxyuridine (BrdU) incorporation in the NTS in adult rats with cervical vagotomy. Cultured NTS neurons contain immature precursor cells as shown by expression of Hu protein. Exposure of cultured NTS neurons to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose- and time-dependent manners. Co-localization of Hu immunoreactivity with BrdU labeling was demonstrated by double fluorescent staining, suggesting that cells labeled with BrdU are neuronal cells. Ghrelin receptor mRNA was detected in tissues from the NTS. The mitotic effect of ghrelin was abolished by treatment of cultured NTS neurons with ghrelin receptor antagonists: D-Lys-3-GHRP-6 and [D-Arg1, D-Phe-5, D-Trp-7, 9, Leu-11] substance P. Diltiazem, a L-type calcium channel blocker, significantly attenuated ghrelin-mediated increments in BrdU incorporation. Ghrelin acts directly on NTS neurons to stimulate neurogenesis.

Animals↗
PubMed2005

Sphingosine-1-phosphate induces early response gene expression in C6 glioma cells.

Sphingosine-1-phosphate (S1P) caused dose-dependent and time-dependent increases in c-fos mRNA. Pretreatment with pertussis toxin (PTX; 100 ng/mlx24 h) reduced c-fos activation by S1P (100 microM-187+/-6% vs. 411+/-27%) and lysophosphatidic acid (LPA; 100 microM-90+/-34% vs. 188+/-41%), but not by sphingosylphosphorylcholine (SPC; 100 microM-390+/-47% vs. 420+/-44%). RT-PCR analysis and sequencing demonstrated the presence of previously unidentified LPA-responsive Endothelial Differentiation Gene (EDG) receptor mRNAs in C6 cells: EDG-2 and EDG-4.

Animals↗
PubMed2005

Linear programming to optimize performance in a department of surgery.

BACKGROUND: Linear programming is an analytic method that can be used to develop models for health care that optimize distribution of resources through mathematical means. STUDY DESIGN: The linear programming model contained objective, decision, and constraint elements. The objective was to optimize financial outcomes for both the hospital and physicians in the Department of Surgery. The decision concerns procedure mix or the number of each type of surgical procedure. Constraints apply to resources that are consumed during the course of the patient's surgical encounter. RESULTS: The optimal solution produced an increase in professional payments of 3.6% and an increase in hospital total margin of 16.1%. This solution favored surgical procedures that require inpatient care; these patients had greater comorbidity, reflected in a higher case-mix index of 3.74 compared to 2.97. Substantial differences were noted in use of general care and ICU days, and in consumption of preoperative, intraoperative, and recovery room time. CONCLUSIONS: Aligning quality surgical care with optimal financial performance may be assisted by mathematical models such as linear programming.

Comorbidity↗
PubMed2004

Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus.

Ghrelin, a gastric peptide hormone, has been reported to regulate growth hormone secretion and energy homeostasis. Here we show that ghrelin promotes neural proliferation in vivo and in vitro in the rat dorsal motor nucleus of the vagus (DMNV). Ghrelin receptor mRNA and immunoreactivity were detected in tissues from DMNV. Systemic administration of ghrelin (130 nmol kg(-1)) significantly increased 5-bromo-2'-deoxyuridine (BrdU) incorporation in the DMNV in adult rats with cervical vagotomy (BrdU positive cells; from 27 +/- 4 to 69 +/- 14 n = 5, P < 0.05). In vitro, exposure of cultured DMNV neurones to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose-dependent (10(-9) -10(-6)m), and time-dependent (6 h to 48 h) manners. Ghrelin significantly increased voltage-activated calcium currents in isolated single DMNV neurones from a mean maximal change of 141 +/- 26 pA to 227 +/- 37 pA. Upon removal of ghrelin, calcium currents slowly returned to baseline. Blocking L-type calcium channels by diltiazem (10 microm) significantly attenuated ghrelin-mediated increments in BrdU incorporation (n = 5, P < 0.05). Ghrelin acts directly on DMNV neurones to stimulate neurogenesis.

Animals↗
PubMed2004

Inhibition of adipogenesis by ghrelin.

Ghrelin, a novel gastric hormone, regulates food intake and energy metabolism via central mechanisms. The peripheral effect of ghrelin on adiposity is poorly understood. We established a stable 3T3-L1 cell line expressing ghrelin to study the direct effect of ghrelin on adipogenesis. Cells overexpressing ghrelin demonstrate significantly attenuated differentiation of preadipocytes into adipocytes. Expression of peroxisome proliferator-activator receptor-gamma is significantly inhibited as demonstrated by decrease of peroxisome proliferator-activator receptor-gamma mRNA and protein. Both ghrelin overexpression and exogenous ghrelin stimulate cell proliferation. Phosphorylation of mitogen-activated protein kinase is increased after treatment of cells with ghrelin. Ghrelin binding activity is demonstrated in both native and ghrelin-overexpressing 3T3-L1 cells by radiolabeled ghrelin, although reverse transcription-polymerase chain reaction with the primer sequence of the previously identified ghrelin receptor subtypes detected no signal. Our results demonstrate that ghrelin inhibits adipogenesis by stimulation of cell proliferation via the mediation of a ghrelin receptor, likely a novel unidentified subtype.

Adipocytes↗
PubMed2003

Activation of c-fos expression in the rat inferior olivary nucleus by ghrelin.

Ghrelin, a novel 28-amino-acid hormone secreted by gastric oxyntic glands, stimulates food intake and induces adiposity. We examined whether ghrelin activates the inferior olivary nucleus. Systemic administration of ghrelin (37 nmol/kg) induced the expression of c-fos immunoreactivity in inferior olive neurons (n=6 rats). The number of neurons containing c-fos staining was significantly increased in the ghrelin-treated rats (65+/-14 vs.11+/-6 positive neurons, n=5). No significant difference in c-fos-positive neurons was observed between left (32+/-5) and right (33+/-6) inferior olivary nuclei. The number of c-fos-positive neurons in rats with bilateral vagotomy was not significantly different from those with intact vagal nerves. The present study demonstrates that ghrelin induces c-fos expression in inferior olivary nucleus via a central mechanism.

Animals↗
PubMed2003

Intercellular calcium waves in cultured enteric glia from neonatal guinea pig.

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.

Adenosine Triphosphate↗
PubMed2003

Epidemiology of surgically treated gastric cancer in the United States, 1988-2000.

The incidence of gastric cancer and the need for subsequent surgery has been decreasing in the United States. However, very few population-based studies on the magnitude of these changes are available. The objective of the present study was to characterize temporal trends in the use of gastric resection in the treatment of gastric cancer. Patients with a primary diagnosis code for gastric cancer (N=105,887) and a procedure code for gastric resection (N=23,690) in the Nationwide Inpatient Sample for 1988-2000 were included. The Nationwide Inpatient Sample represents a 20% stratified random sample representative of all United States hospitals. Outcome variables included the overall incidence, in-hospital mortality rate, and length of stay. Rates of surgery are shown as the number of cases per 100,000 hospital discharges. Hospital volume was defined as follows: low volume (1 to 4 cases per year), medium volume (5 to 8 cases per year), and high volume (9 or more cases per year). Rates of gastric resection have shown a 20% decline from 30 cases per 100,000 (1988-1989) to 24 cases per 100,000 (1999-2000) (P=0.001). In-hospital mortality has not changed over the 13-year period and remains at 7.4%. There was significant variation in mortality across hospitals, with very low-volume centers having an 8.9% mortality rate, whereas very high-volume centers had a 6.4% mortality rate (P<0.001). The market share of gastric resections performed at high-volume centers increased a small amount from 43% (1988-1989) to 48% (1999-2000) (P=0.023). Over the 13-year period, length of stay decreased from 15 days (interquartile range [IQR] 11-23) in 1988 to 11 days (interquartile range [IQR] 8-16) in 2000 (P<0.001). Rates of gastric resection for cancer have shown a modest decline over the past 13 years in the United States. Although the length of stay for these patients has decreased, no significant changes to in-hospital mortality have occurred. Given the declining rates of gastric cancer surgery, and the superior outcomes at high-volume centers, regionalization of care may improve mortality rates for this high-risk surgical procedure.

Gastrectomy↗
PubMed2003

101 laparoscopic splenectomies for the treatment of benign and malignant hematologic disorders.

BACKGROUND: Laparoscopic splenectomy (LS) is the surgical approach of choice for patients with hematologic disorders requiring splenectomy. Patients with idiopathic thrombocytopenic purpura (ITP) have normal to slightly enlarged spleens and benefit the most from LS. METHODS: We reviewed the perioperative outcomes in 101 patients who underwent LS between May 1996 and December 2002. Patients were divided into three groups--ITP, other benign, and malignant hematologic disorders--and compared. RESULTS: The ITP patients (n = 48) had significantly smaller spleens and operative times compared with the other groups. Splenomegaly in the other benign (n = 23) and malignant hematologic disorders (n = 30) groups was responsible for higher open conversion rates and greater need for hand-assisted laparoscopic splenectomy (HALS). CONCLUSIONS: Laparoscopic splenectomy and HALS can be performed with good results for benign and malignant hematologic disorders. The benefits of HALS are similar to LS, so there should be a low threshold for HALS in patients with large spleens.

Case-Control Studies↗
PubMed2003

Stimulation of rat pancreatic exocrine secretion by urocortin and corticotropin releasing factor.

Neural and hormonal mechanisms control pancreatic secretion. The effects of the corticotropin releasing factor (CRF) related neuropeptide urocortin (UCN) on pancreatic exocrine secretion were examined. In anesthetized male rats, pancreatic secretion volume and total protein were assayed. UCN increased pancreatic secretory volume and protein secretion and potentiated cholecytokinin-stimulated protein secretion. Astressin, a non-specific CRF receptor antagonist, inhibited UCN-stimulated protein output while CRF(2) receptor antagonist, antisauvagine-30, was without effect. Atropine, but not subdiaphragmatic vagotomy, inhibited UCN-mediated secretion. In acinar cells, UCN did not stimulate release of amylase nor intracellular cAMP. UCN is a pancreatic exocrine secreatagogue with effects mediated through cholinergic intrapancreatic neurons.

Animals↗