Gastroenteropancreatic neuroendocrine tumors in children and young adults We review the imaging findings of pediatric gastroenteropancreatic neuroendocrine tumors -LRB- GEP-NETs -RRB- using contemporary anatomic and molecular imaging techniques . A low index of suspicion can result in significant delays in diagnosis of pediatric GEP-NETs . A multimodality imaging approach , using both anatomic and functional imaging , is essential in the diagnosis , staging , and surveillance of these potentially malignant tumors . Introduction Neuroectodermal tumors arising from the neural crest , such as neuroblastomas , form a large proportion of childhood neoplasms . Gastroenteropancreatic neuroendocrine tumors -LRB- GEP-NETs -RRB- are a small subgroup of neural crest tumors whose imaging findings are not well described in children . These tumors arise from neuroendocrine cells found in the pancreas , the gut , and its derivatives including the bronchial tree . The term `` neuroendocrine tumors '' encompasses carcinoid tumors , islet cell tumors , and amine precursor uptake and decarboxylation -LRB- APUD -RRB- tumors . Although these tumors can produce distinct clinical syndromes because of their secretory capacity , they are underdiagnosed in children , resulting in delays in detection -LSB- 1 , 2 -RSB- . The small size of the primary tumor and its tendency to occur anywhere along the gastroenteropancreatic -LRB- GEP -RRB- system can make detection a challenge . Advances in anatomic imaging and incorporation of metabolic imaging techniques have led to improved detection of these tumors . Since 1990 , 13 patients 21 years of age or younger were seen at our hospital with a diagnosis of a GEP-NET . The primaries were located in the pancreas -LRB- n = 4 -RRB- , lung -LRB- n = 4 -RRB- , appendix -LRB- n = 1 -RRB- , thymus -LRB- n = 1 -RRB- and middle ear -LRB- n = 1 -RRB- , and in two patients the tumor was primary unknown . Six patients had functional tumors and were symptomatic for several months to 5 years -LRB- mean 2 years -RRB- with symptoms including diarrhea , peptic ulcer disease , abdominal pain , and weight loss . Metastases were present in 50 % of these patients by the time a GEP-NET was diagnosed . Two of the three patients with pulmonary NET tumors presented with recurrent pneumonia . One patient with an unsuspected NET developed carcinoid crisis after resection of the tumor and required intensive care management . One patient had a history of a familial disorder , von Hippel-Lindau -LRB- VHL -RRB- disease , predisposing to NET . Discussion Classification GEP-NETs have traditionally been classified into two groups : -LRB- 1 -RRB- carcinoids , which are subdivided by their origin into tumors of the foregut -LRB- lung , thymus , stomach , duodenum -RRB- , midgut -LRB- jejunum , ileum , appendix , right colon -RRB- , and hindgut -LRB- left colon , rectum -RRB- ; and -LRB- 2 -RRB- pancreatic tumors -LSB- 3 -RSB- . A more prognostically useful classification was proposed by the World Health Organization in 2000 : well-differentiated NETs , which show benign behavior -LRB- group 1a -RRB- ; well-differentiated neuroendocrine carcinomas -LRB- NEC -RRB- , which are characterized by low-grade malignancy -LRB- group 1b -RRB- ; and poorly differentiated NEC of high-grade malignancy -LRB- group 2 -RRB- -LSB- 4 -RSB- . Based on their clinical features , these tumors can be functional or nonfunctional . Functional tumors have clinical symptoms caused by hypersecretion of hormones such as gastrin , while nonfunctional tumors can produce clinically silent hormones such as pancreatic polypeptide . These tumors often produce more than one hormone , and the functional tumors are named according to the hormone responsible for the clinical syndrome , for example gastrinoma causing Zollinger-Ellison syndrome and insulinoma causing hypoglycemic syndrome . NETs can either be sporadic or occur as part of familial syndromes such as multiple endocrine neoplasia -LRB- MEN -RRB- I and II , VHL syndrome and neurofibromatosis type I -LRB- NF-I -RRB- . Imaging approach There is limited information in the pediatric literature regarding the imaging findings of NETs . Most of the information regarding imaging of these tumors is obtained from adult studies . The quoted diagnostic performance of different imaging modalities in various studies is highly variable depending on local expertise , different stages of technology development , and the imaging protocols used . A multimodality imaging approach using both anatomic and molecular imaging is essential for detection , staging , and surveillance of these tumors -LSB- 5 -RSB- . Anatomic imaging Multidetector CT Multidetector CT -LRB- MDCT -RRB- has been shown to have an accuracy of 92 % and 96 % for the detection of primary tumor and metastasis , respectively -LSB- 6 -RSB- . The advantages of MDCT include high temporal and spatial resolution , acquisition of isotropic data resulting in high-quality reformatted images , and bolus-tracking capability resulting in precise timing of the scan . However , it is imperative that the radiologist select appropriate imaging parameters -LRB- tube current , gantry rotation time , kilovoltage , table speed and detector configuration -RRB- to achieve a balance between image quality and radiation exposure -LSB- 7 -RSB- . Although in general multiphase examinations should be avoided in children to keep radiation exposure as low as is reasonably achievable -LRB- ALARA -RRB- , optimal evaluation of NETs requires dual-phase imaging . Images are obtained during arterial and portal venous phases of enhancement after administration of intravenous contrast agent at a rate of 3 -- 5 ml/s -LRB- Fig. 1 -RRB- . The appropriate delay after contrast agent injection and before scanning will vary with the size of the patient . In an average teenager , there will be a delay of approximately 25 s for the arterial phase and a delay of 60 to 70 s for visualization of the portal vein . This aids detection of NETs , which often demonstrate early enhancement with washout during the portal venous phase because of their hypervascular nature . Visualization of mural masses can be improved by using water as an oral contrast agent . MDCT can also be used to perform small-bowel enteroclysis to detect small mural and luminal lesions of gut NETs -LSB- 8 -RSB- . Fig. 1A 6-year-old girl with history of abdominal pain and diarrhea for several months . Contrast-enhanced axial CT image in the arterial phase -LRB- a -RRB- demonstrates multiple enhancing liver lesions -LRB- arrows -RRB- that are difficult to discern on the portal venous phase -LRB- b -RRB- consistent with hypervascular metastases . Also note the thickening of the gastric folds due to elevated gastrin levels . Fine-needle aspiration of a liver lesion confirmed metastatic gastrinoma MRI MRI has the highest sensitivity in detecting liver metastasis -LSB- 9 -RSB- . Arterial phase images after contrast agent administration and fat-suppressed fast spin echo T2-W images are the most sensitive in detecting liver lesions because of their hypervascular nature -LRB- Fig. 2 -RRB- . For the detection of pancreatic lesions , MRI has been shown to have a sensitivity of 94 % , with the tumor being most conspicuous on fat-saturated T1-W images -LSB- 10 -RSB- . Fig. 2A 14-year-old boy after resection of a bronchial NEC . Axial T1-W image -LRB- a -RRB- demonstrates a hypointense liver lesion that is moderately hyperintense on the T2-W image -LRB- b -RRB- . Arterial phase image after gadolinium administration -LRB- c -RRB- shows intense early enhancement with wash-out to isointensity on the portal venous phase images -LRB- d -RRB- , consistent with hypervascular metastasis Sonography Although traditional transabdominal US examinations have low sensitivity in detection of small tumors , contrast-enhanced , endoscopic , and intraoperative US examinations have been reported to have higher sensitivities in the detection of small lesions -LSB- 9 -RSB- . Endoscopic US with color Doppler imaging has been shown to have sensitivities in the range of 79 -- 100 % for the detection of small lesions in the pancreas head -LSB- 11 -RSB- . Intraoperative US examinations have also been shown to improve the accuracy of lesion detection to 97 % in the pancreatic head -LRB- Fig. 3 -RRB- -LSB- 12 -RSB- . Fig. 3A 21-year-old man with severe gastroesophageal reflux disease for 5 years and elevated gastrin levels of 5,258 pg/ml -LRB- normal range 0 -- 100 pg/ml -RRB- . Intraoperative US image shows a 3 × 3 cm hypoechoic mass in the pancreas -LRB- arrow -RRB- closely abutting the splenic vein -LRB- V -RRB- . Surgical resection confirmed pancreatic gastrinoma Molecular imaging The overexpression of somatostatin receptors and the presence of amine uptake and storage mechanisms allows targeted molecular imaging of NETs using somatostatin receptor scintigraphy -LRB- SRS -RRB- and metaiodobenzylguanidine -LRB- MIBG -RRB- , respectively . The sensitivity of octreotide and MIBG scintigraphy is known to range between 78 % and 100 % and 36 % and 85 % , respectively -LSB- 13 -RSB- . 111Indium diethylenetriaminepentaacetate -LRB- DTPA -RRB- octreotide is the most widely used radiopharmaceutical for SRS of NETs and is currently the modality of choice in the evaluation of these tumors -LRB- Fig. 4 -RRB- -LSB- 14 -RSB- . Octreotide has high affinity for somatostatin receptors , especially subtype 2 , resulting in good correlation between scintigraphic findings and the expression of somatostatin receptors on these tumors as determined by immunostaining . These receptors can also be targeted for radiotherapy with radiolabeled somatostatin analogues -LSB- 14 -RSB- . Fig. 4A 6-year-old girl with a history of abdominal pain and diarrhea for several months -LRB- same patient as in Fig. 2 -RRB- . SRS SPECT image -LRB- a -RRB- obtained 24 h after intravenous administration of indium 111 pentatreotide in the coronal plane shows multiple foci of increased uptake in the liver -LRB- arrowhead -RRB- with an intense focus inferior to the liver -LRB- arrow -RRB- , which corresponds to the exophytic lesion arising from the pancreatic neck -LRB- arrow -RRB- seen on the coronal reformatted CT image -LRB- b -RRB- . This is a surgically proven gastrinoma . c Photomicrograph of immunohistochemistry slide shows positive staining -LRB- brown -RRB- for somatostatin receptors Positron emission tomography -LRB- PET -RRB- has the advantage of high sensitivity , reasonable resolution , and the ability to perform whole-body scans -LRB- Fig. 5 -RRB- . 18Fluorodeoxyglucose -LRB- FDG -RRB- is the most widely used tracer but has limited sensitivity in detection of well-differentiated and slow-growing tumors -LSB- 15 -RSB- . PET using novel tracers such as 6 - -LSB- fluoride-18 -RSB- fluoro-levodopa -LRB- 18F-DOPA -RRB- , a catecholamine precursor , shows promise in the evaluation of NETs -LSB- 16 -RSB- . Fig. 5A 21-year-old male with history of metastatic neuroendocrine carcinoma with unknown primary . a , b 18-FDG PET images in the coronal plane demonstrate multifocal uptake in the left humerus , bilateral ribs , liver , iliac wings , and both femurs consistent with widespread metastatic disease Fusion imaging Coregistration of molecular and anatomic modalities with SPECT-CT or PET-CT can aid in accurate localization of disease and areas of physiologic uptake -LRB- Figs. 6 and 7 -RRB- . Fusion imaging with SPECT-CT has been shown to be more accurate than either SPECT or CT alone and resulted in management changes in 28 % of patients in one series -LSB- 17 -RSB- . Fig. 6A 19-year-old male after resection of bronchial NEC 7 years previously . a Surveillance CT demonstrates expansion of the left sacral neural foramen by a soft-tissue mass -LRB- arrow -RRB- . b FDG-PET demonstrates the lesion to be FDG avid raising the suspicion of tumor . Surgical resection confirmed metastasis to the left S1 nerve sheathFig . 7A 16-year-old boy after resection of pancreatic gastrinoma 7 years previously with recurrent symptoms of gastroesophageal reflux disease . a , b Coregistered SRS SPECT and contrast-enhanced CT images allow accurate localization of radionuclide uptake to a paracaval lymph node -LRB- arrow -RRB- and a perigastric -LRB- arrowhead -RRB- lymph node , both of which are less than 1 cm in short axis . Also note multiple hepatic metastases with intense octreotide uptake Common pediatric GEP-NETs by location Appendiceal NETs Appendiceal NETs are the most common pediatric GEP-NETs -LSB- 2 -RSB- . While appendiceal NETs in adults are generally incidentally detected , 78 -- 97 % of appendiceal NETs in children are associated with appendicitis -LRB- Fig. 8 -RRB- -LSB- 18 -- 20 -RSB- . In a series of 36 children with appendiceal carcinoids , the median age at diagnosis was 12.3 years , and three-fourths presented with acute appendicitis while the rest presented with chronic abdominal pain -LSB- 21 -RSB- . Although carcinoid syndrome has been reported in association with appendiceal NETs in adults , the largest series of these tumors in children failed to show any children with symptoms of carcinoid syndrome -LSB- 18 , 20 -RSB- . These tumors occur primarily at the appendix tip and are generally less than 2 cm in size . The long-term prognosis is good , with metastatic disease being unusual in children with appendiceal NETs , as shown in two large series with a median follow-up of 10 -- 26 years -LSB- 18 , 21 -RSB- . However , appendiceal carcinoids with synchronous and metachronous adenocarcinoma of the colon have been reported , even in children -LSB- 2 , 18 -RSB- . Fig. 8A 21-year-old female with a history of right lower quadrant pain for 6 days . a Contrast-enhanced axial CT image demonstrates a distended -LRB- 13 mm -RRB- non-opacified appendix -LRB- arrow -RRB- with minimal periappendiceal stranding . b Gross specimen of the appendix demonstrates obliteration of the lumen by the pale tumor that extends through the wall into the serosa near the tip -LRB- arrow -RRB- Gastrointestinal NETs GEP-NETs in other parts of the gastrointestinal tract are less common in children . Duodenal NETs arise from gastrin cells and tend to produce Zollinger-Ellison syndrome -LSB- 22 -RSB- . Duodenal carcinoids are most common in the proximal duodenum and present as early enhancing intraluminal polyps or mural masses -LSB- 23 -RSB- . Unlike duodenal NETs , jejunal and ileal NETs arise from the serotonin-producing enterochromaffin cells and can cause carcinoid syndrome . Serotonin production results in imaging findings of mesenteric desmoplasia , bowel kinking , elastic vascular sclerosis , and intestinal ischemia -LSB- 8 -RSB- . Ileal NETs might present with abdominal pain or diarrhea secondary to increased intestinal motility or obstruction secondary to intussusception . In the hindgut , the most common location of NETs is the rectum . Most of these tumors are nonfunctional , with rectal NETs having a better prognosis than NETs in other parts of the colon . Hepatic NETs The liver is a common site of extra-appendiceal NETs in children . In a series of 13 children with extra-appendiceal NETs , the liver was the second most common site of disease -LRB- 5 of 13 children -RRB- -LSB- 1 -RSB- . It is unclear whether these tumors represent true primary hepatic neoplasms or metastases from asymptomatic occult NETs . Like the primary NETs , liver lesions are hypointense on T1-W images , hyperintense on T2-W images , and demonstrate arterial phase enhancement . Pancreatic NETs Pancreatic NETs account for approximately 30 % of pancreatic tumors in patients younger than 20 years . While insulinomas are the most common in adults , gastrinomas are more common in children . Hypersecretion of gastrin by gastrinomas causes recurrent or ectopic peptic ulcers -LRB- Zollinger-Ellison syndrome -RRB- , or malabsorption diarrhea . Most gastrinomas are solitary lesions located in the gastrinoma triangle , formed by the junction of the pancreatic neck and body , the second and third parts of the duodenum , and the junction of the cystic and common bile ducts . Nongastrinoma neuroendocrine tumors can occur in any part of the pancreas -LSB- 24 -RSB- . Metastases are present in 60 -- 80 % of patients at initial diagnosis of gastrinoma , with the liver and lymph nodes being the most common sites of metastatic disease -LSB- 25 -RSB- . Most neuroendocrine tumors of the pancreas are hypointense on fat-suppressed T1-W images , moderately hyperintense to the pancreas on T2-W fat-suppressed images , and show moderately intense early enhancement -LSB- 24 -RSB- . Patients with familial NETs , such as MEN and VHL disease , might present with multiple tumors with the majority being nonfunctional -LRB- Fig. 9 -RRB- . Fig. 9A 21-year-old female with history of VHL disease . a , b Contrast-enhanced axial CT images during arterial phase demonstrate an enhancing mass in the body of the pancreas -LRB- arrow -RRB- . A second mass located in the uncinate process -LRB- arrowhead -RRB- adjacent to the portal vein was missed on initial reading . c , d FDG-PET axial images demonstrate two metabolically active foci in the pancreas , confirmed to be nonfunctional NETs at surgery Pulmonary NETs Pulmonary NETs are the most common extra-appendiceal NETs in children -LSB- 1 -RSB- . They are also the most common primary pulmonary neoplasm of childhood , accounting for between 16 % and 80 % of malignant lung tumors in this population -LSB- 1 , 26 -RSB- . Children with these tumors tend to present with wheezing and atelectasis , in addition to the classic adult triad of cough , hemoptysis , and obstructive pneumonitis -LSB- 27 -RSB- . Most present in late adolescence , though these tumors have been reported in children as young as 3 years -LSB- 28 -RSB- . Patients might be symptomatic for approximately a year prior to diagnosis -LSB- 27 -RSB- . Bronchial carcinoids in children tend to arise in the main , lobar , or segmental bronchi , in regions of bronchial bifurcation . They typically present as hilar or perihilar masses , with associated findings of endobronchial obstruction such as air-trapping , atelectasis , mucus-plugging , and obstructive pneumonitis -LRB- Fig. 10 -RRB- . The mass is usually a well-defined round or ovoid homogeneously enhancing lesion with slightly lobulated margins . The intense enhancement of the tumor is helpful for differentiating the mass from adjacent postobstructive parenchymal changes -LRB- Fig. 10 -RRB- . Calcification might be present in up to 30 % at histologic analysis . Carcinoid syndrome is unusual with bronchial NETs unless they have metastasized to the liver or are large in size with systemic drainage -LSB- 29 -RSB- . Intraoperatively , these tumors can release vasoactive mediators such as 5-hydroxyindoleacetic acid -LRB- 5-HIAA -RRB- , resulting in carcinoid crisis characterized by bronchospasm , hypotension , acidosis , and ventricular tachycardia . If the diagnosis of a NET is considered preoperatively , the life-threatening complication of carcinoid crisis can be prevented by pretreatment with octreotide -LSB- 30 -RSB- . Fig. 10A 20-year-old man with a history of `` asthma '' and recurrent pneumonia for 2 years . a Chest radiograph demonstrates right base consolidation and right pleural effusion with an in-dwelling chest tube . b Contrast-enhanced axial CT image demonstrates an enhancing right hilar mass -LRB- arrow -RRB- with right lower lobe obstructive pneumonitis . c Lung section at lobectomy demonstrates a fleshy circumscribed mass -LRB- arrow -RRB- with a small endobronchial and larger extraluminal component . The lung parenchyma is filled with cheesy purulent material -LRB- arrowhead -RRB- secondary to obstructive pneumonitis Thymic NETs Thymic NETs tend to be more aggressive than other foregut tumors and often have metastatic disease at initial presentation -LSB- 9 -RSB- . Not much is known about thymic carcinoids in children ; adult studies have shown that while bronchial carcinoids are malignant in about 26 % of patients , thymic carcinoids can be malignant in about 80 % of patients -LSB- 31 -RSB- . They can present with local symptoms -LRB- such as superior vena cava syndrome -RRB- , can be incidentally detected on radiographs , and can produce Cushing syndrome -LRB- Fig. 11 -RRB- . Fig. 11A 17-year-old female with an incidentally detected anterior mediastinal mass . a , b Axial contrast-enhanced CT images after biopsy demonstrate an infiltrating soft-tissue mass in the anterior mediastinum with enlarged pretracheal , subcarinal and hilar lymph nodes , and pleural studding -LRB- arrow -RRB- . c Bone window image demonstrates multiple sclerotic lesions in the vertebral body , sacrum , and iliac wings consistent with metastatic disease . d Photomicrograph of H&E - stained section shows round cells with regular nuclei and salt-and-pepper chromatin . e , f Immunostaining is positive for the neuroendocrine tumor markers chromogranin -LRB- e -RRB- and synaptophysin -LRB- f -RRB- Middle ear NETs NETs can also occur in the middle ear cavity , which is a derivative of the first pharyngeal pouch of the foregut -LRB- Fig. 12 -RRB- . Typical presenting symptoms of middle ear NETs include hearing loss , aural fullness , and tinnitus . Physical examination might reveal a mass behind the tympanic membrane . The imaging and histologic characteristics of these tumors can mimic those of paragangliomas . Although middle ear NETs are typically low-grade malignancies that have an indolent course , there is a 22 % risk of recurrence and 9 % risk of metastatic disease -LSB- 32 -RSB- . Fig. 12A 13-year-old boy with a history of a mass behind the left tympanic membrane . a , b Axial CT images through the middle ear cavity demonstrate a soft-tissue mass centered posterosuperior to the ossicles . Surgical resection led to a diagnosis of well-differentiated NET Conclusions A low index of suspicion for GEP-NETs in children combined with nonspecific clinical complaints can result in delay in the detection of these rare but treatable malignancies . A multimodality imaging approach using both anatomic and functional imaging is essential in the diagnosis , staging , and surveillance of these potentially malignant tumors in children .

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O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O

neuroendocrine tumors
carcinoid
islet cell
tumors
children


The inflammatory effects of UDP-glucose in N9 microglia are not mediated by P2Y14 receptor activation In this study we evaluated the functionality and inflammatory effects of P2Y14 receptors in murine N9 microglia . The selective P2Y14 receptor agonist UDP-glucose -LRB- UDPG -RRB- derived from microbial sources dose dependently stimulated expression of cyclooxygenase-2 and inducible nitric oxide synthase , and potentiated the effects of bacterial lipopolysaccharide on nitric oxide production . However , another selective P2Y14 receptor agonist , UDP-galactose , did not affect these endpoints either alone or in combination with lipopolysaccharide . Interestingly , synthetic UDPG also had no detectable pro-inflammatory effects , although P2Y14 receptors are both expressed and functional in N9 microglia . While synthetic UDPG decreased levels of phosphorylated cyclic AMP response element binding protein , an effect that was blocked by pertussis toxin , the pro-inflammatory effects of microbial-derived UDPG were insensitive to pertussis toxin . These data suggest that the pro-inflammatory effects of microbial-derived UDPG are independent of P2Y14 receptors and imply that microbial-derived contaminants in the UDPG preparation may be involved in the observed inflammatory effects .

O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O

cyclic amp response element binding protein (creb)


The PDE4 inhibitor rolipram reverses object memory impairment induced by acute tryptophan depletion in the rat Rationale The selective type IV phosphodiesterase inhibitor , rolipram , has been shown to improve long-term memory and can reverse the cholinergic deficit caused by scopolamine . However , the underlying mechanisms of action of rolipram remain obscure . Introduction Inhibition of phosphodiesterase type 4 -LRB- PDE4 -RRB- leads to an increase in intracellular cyclic adenosine monophosphate -LRB- cAMP -RRB- availability -LRB- Silvestre et al. 1999 -RRB- . cAMP is an important second messenger molecule in the process of intracellular signal transduction mechanisms -LRB- Bailey et al. 1996 -RRB- . Several studies have shown that rolipram , a selective PDE4 inhibitor , improves cognitive performance in young rats -LRB- Blokland et al. 2006 ; Imanishi et al. 1997 ; Rose et al. 2005 ; Zhang et al. 2000 , 2004 -RRB- and ameliorates scopolamine-induced memory deficits -LRB- Egawa et al. 1997 ; Imanishi et al. 1997 ; Rutten et al. 2006 ; Zhang and O'Donnell 2000 -RRB- . In a previous study , we have shown that rolipram reversed a scopolamine-induced deficit in an object recognition task -LRB- ORT -RRB- in male Wistar rats -LRB- Rutten et al. 2006 -RRB- . It was suggested that these effects may be related to the effects of elevated cAMP levels on neurotransmitter release . There is also evidence that there is a link between PDE4 inhibition and serotonergic neurotransmission -LRB- Schoffelmeer et al. 1985 ; West and Galloway 1996 -RRB- . The adenylate cyclase activator forskolin , as well as 8-bromo-cAMP , can enhance the electrically evoked release of 3H-5-hydroxytryptamine -LRB- Schoffelmeer et al. 1985 -RRB- . Thus , elevating cAMP levels by a selective PDE4 inhibitor could exert a general facilitatory effect on 5-HT release . Based on these findings , we argued that rolipram may also reverse memory deficits induced by a lowered 5-HT neurotransmission . Acute tryptophan depletion -LRB- ATD -RRB- is a well-established model to assess the role of serotonin in cognitive and affective functioning -LRB- Booij et al. 2003 -RRB- . The neurotransmitter serotonin is synthesized from its amino acid precursor tryptophan -LRB- TRP -RRB- , which is obtained from our food . Free TRP is transported into the brain across the blood -- brain barrier , but has to compete for entrance with five other large neutral amino acids -LRB- LNAAs : valine , leucine , isoleucine , phenylalanine , and tyrosine -RRB- . The ratio of TRP and these other LNAAs -LRB- TRP/ΣLNAA ratio -RRB- is thought to be a more sensitive index of brain TRP availability -LRB- Fernstrom 1981 ; Wurtman et al. 1980 -RRB- . As 5-HT is synthesized from TRP , through the rate-limiting enzyme TRP-hydroxylase , the availability of TRP in the brain determines the amount of central 5-HT -LRB- Wurtman et al. 1980 -RRB- . Due to its reversible and non-intrusive effects , the method of ATD can be repeatedly used in animals and humans . In humans , acute tryptophan depletion has a negative effect on memory consolidation -LRB- Park et al. 1994 ; Riedel et al. 1999 ; Schmitt et al. 2000 -RRB- . Furthermore , memory impairments in the ORT have been observed in rats after ATD -LRB- Lieben et al. 2004b , 2005 -RRB- . Therefore , we hypothesized that rolipram could reverse an ATD-induced memory deficit in rats by elevating 5-HT release in the brain . In addition to this aim of the study , we further examined the relation between peripheral TRP and memory performance . We investigated the effects of different pretreatment times of the TRP-depleted mixture on object recognition memory . These data should provide more experimental support for a relation between plasma TRP and , consequently , central 5-HT and memory performance . Materials and methods Animals All experimental procedures were approved by the local ethical committee of the Maastricht University for animal experiments according to governmental guidelines . A total of 42 4-month-old male Wistar rats -LRB- Charles River , The Netherlands -RRB- were used -LRB- 410 -- 450 g -RRB- . Rats were randomly assigned to either the biochemistry -LRB- n = 18 -RRB- or the behavior group -LRB- n = 24 -RRB- . In the biochemistry groups , the animals were randomly subdivided over three treatment groups , i.e. , saline , TRP + , or TRP − -LRB- n = 6/group -RRB- . The animals were housed individually in standard type 3 Makrolon cages on sawdust bedding in an air-conditioned room -LRB- about 20 °C -RRB- . They were kept under a reversed 12/12-h light/dark cycle -LRB- lights on from 18:00 to 6:00 hours -RRB- and had free access to water . Rats were housed in the same room as where they were tested . A radio , which played softly , provided background noise in all rooms . All testing were done between 9:00 and 17:00 hours . Drugs and chemicals The gelatin hydrolysate -LRB- Solugel C -RRB- was obtained from PB Gelatins -LRB- Tessenderlo , Belgium -RRB- . Glucodry 210 was obtained from the Amylumgroup -LRB- Koog aan de Zaan , The Netherlands -RRB- . l-Tryptophan and rolipram were obtained from Sigma-Aldrich -LRB- Zwijndrecht , The Netherlands -RRB- . Kaliumchloride -LRB- KCl -RRB- , calciumchloride-dihydrate -LRB- CaCl2 · 2H2O -RRB- , and 5-sulfosalicylic acid dihydrate were purchased from Merck -LRB- Darmstadt , Germany -RRB- . Treatment During a period of 2 weeks preceding the experiment , the rats were handled and habituated to oral injections with normal tap water -LRB- 10 ml/kg -RRB- . On experimental days , the rats were not fed 14 h before treatment until the testing period was completed . This was done to minimize the availability of TRP from food , which would counteract the effects of the ATD treatment . The rats were orally treated with a protein -- carbohydrate mixture containing TRP -LRB- TRP + group ; in which 0.28 % TRP of the total protein was added to the mixture -RRB- or one lacking TRP -LRB- TRP − group ; no TRP added to the mixture -RRB- , or with saline . The composition of the nutritional mixture is shown in Table 1 . Table 1Composition of mixture and determination of the amino acids in gelatin-based protein -LRB- mol -RRB- MixtureComposition Protein -LRB- Solugel -RRB- in 100 ml water100 g Aspartic acid4 .8 Glutamic acid10 .3 Hydroxyproline11 .4 Serine3 .4 Glycine23 .2 Histidine0 .8 Arginine10 .0 Threonine2 .0 Alanine10 .4 Proline12 .0 Tyrosine0 .4 Valine2 .2 Methionine0 .7 Isoleucine1 .0 Leucine2 .5 Hydroxylysine0 .9 Phenylalanine1 .1 Lysine2 .9 Carbohydrate -LRB- Glucodry 210 -RRB- in 80 ml water50KCl0 .094 CaCl2 · 2H2O2.32l-Tryptophan -LRB- TRP − group -RRB- 0l-Tryptophan -LRB- TRP + group -RRB- 0.28 The composition of the mixture -LRB- g -RRB- used in this experiment is described in italic . The amino acid spectrum -LRB- % -RRB- of the Solugel protein was obtained from PB Gelatins , Tessenderlo , Belgium . In every experiment , each dose contained Solugel C at 4.0 g/kg and Glucodry at 2.0 g/kg of the body weight and was given in a dose of 10 ml/kg between 8:30 and 12:30 hours . Blood samples were taken at baseline -LRB- 20 min before treatment -RRB- and 60 , 180 , and 360 min after the first treatment . For the behavioral studies in the object recognition task , the mixture was administered at different time points -LRB- see below -RRB- . Rolipram was freshly suspended in 5 % ethanol , 1 % tylose -LRB- methyl-cellulose -RRB- , and 94 % distilled water on every experimental day . The following doses of rolipram were tested : 0 , 0.01 , 0.03 , or 0.1 mg/kg always in combination with the TRP − mixture -LRB- 3 h before the first trial , T1 -RRB- . Rolipram was always administered 30 min before T1 -LRB- i.p. injection , dose 2 ml/kg -RRB- . Biochemistry For the determination of plasma amino acid levels , blood samples were taken at resting values and repeated at several points in time . Blood sampling was done via a tail-incision method -LRB- Fluttert et al. 2000 -RRB- . Promptly after collection of blood in sodium heparin tubes -LRB- Microvette ® CB 300 , Sarstedt , Germany -RRB- , the samples were kept on ice . After centrifugation of the blood samples -LRB- at 4 °C for 15 min at 3,000 × g in a Hettich EBA 12 centrifuge -RRB- , plasma was deproteinized with cups containing dry 5-sulfosalicylic acid -LRB- 6 mg/100 μl plasma -RRB- , and the protein was spun down . Samples were frozen in liquid nitrogen and stored at − 80 °C . Before analysis , samples were thawed at 4 °C , vortex-mixed vigorously , and centrifuged at 50,000 × g in a Hereaus Model Biogufe Stratos for 10 min at 4 °C . From the clear supernatant , 20 μl was mixed with 1,960 μl water and 20 μl norvaline and stored in the cooled -LRB- 7 °C -RRB- sample compartment until analysis . In addition to total plasma TRP , the concentrations of several other amino acids were determined with a fully automated high-performance liquid chromatography system after precolumn derivatization with ophthaldialdehyde -LRB- OPA ; Van Eijk et al. 1993 -RRB- . OPA amino acids derivates were quantified with fluorescence detection . The concentrations of the total plasma amino acids were expressed as micromole per liter . Object recognition memory The object recognition test was performed as described elsewhere -LRB- Prickaerts et al. 2002 -RRB- . The apparatus consisted of a circular arena , 83 cm in diameter . Half of the 40-cm high wall was made of gray polyvinyl chloride , the other half of transparent polyvinyl chloride . A light bulb was switched on during testing only and provided a light intensity -LRB- 20 lx -RRB- which was equal in the different parts of the apparatus . Two objects were placed in a symmetrical position about 10 cm away from the gray wall . We used four different sets of objects . The different objects were : -LRB- 1 -RRB- a cone consisting of a gray polyvinyl chloride base -LRB- maximal diameter 18 cm -RRB- with a collar on top made of brass -LRB- total height 16 cm -RRB- , -LRB- 2 -RRB- a standard 1-l transparent glass bottle -LRB- diameter 10 cm , height 22 cm -RRB- filled with sand , -LRB- 3 -RRB- a massive metal cube -LRB- 10.0 × 5.0 × 7.5 cm -RRB- with two holes -LRB- diameter 1.9 cm -RRB- , and -LRB- 4 -RRB- a massive aluminum cube with a tapering top -LRB- 13.0 × 8.0 × 8.0 cm -RRB- . The objects could not be displaced by a rat . In the first week , the animals were handled daily and were adapted to the procedure in 2 days ; that is , they were allowed to explore the apparatus -LRB- without any objects -RRB- twice for 3 min each day . In the two following weeks , the rats were adapted to the testing and i.p. administration procedure by a saline injection -LRB- 0.4 ml -RRB- 30 min before the first trial until they showed a stable discrimination performance , i.e. , good object discrimination -LRB- a d2 value of about 0.30 , see below -RRB- at a 1-h interval on two successive sessions . Subsequently , testing of the drugs began . A testing session comprised two trials . The duration of each trial was 3 min . During the first trial -LRB- T1 -RRB- the apparatus contained two identical objects -LRB- samples -RRB- . A rat was always placed in the apparatus facing the wall in the center of the transparent front segment . After the first exploration period , the rat was put back in its home cage . Subsequently , after a delay interval , the rat was put back in the apparatus for the second trial -LRB- T2 -RRB- , but now with two dissimilar objects , a familiar one -LRB- the sample -RRB- and a new one . The times spent exploring each object during T1 and T2 were recorded manually with a personal computer . Exploration was defined as follows : directing the nose to the object at a distance of no more than 2 cm and/or touching the object with the nose . Sitting on the object was not considered exploratory behavior . To avoid the presence of olfactory trails , the objects were always thoroughly cleaned with alcohol -LRB- 70 % -RRB- . Moreover , each object was available in triplicate so neither of the two objects from the first trial had to be used as the familiar object in the second trial . In addition , all combinations and locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations or objects . As we expected ATD to impair memory performance , we needed a delay interval at which normal rats discriminate between the novel and familiar object . Therefore , we selected a delay interval of 1 h , as Wistar rats show good discrimination between the two objects after this interval -LRB- Rutten et al. 2006 -RRB- . Furthermore , we hypothesized that rolipram may reverse deficits induced by ATD in a 1-h delay ORT . In 1 week , three testing sessions were given , with a 48-h washout period in between . Tests were always conducted on Mondays , Wednesdays , and Fridays . In the first behavioral experiment , we investigated the effects of administration with TRP + or TRP − mixture at different time points -LRB- i.e. , 30 min , 1 , 3 , or 6 h -RRB- before testing in the ORT . Each rat always participated in each condition , and the order of test conditions was decided at random . The second behavioral experiment examined the effect of rolipram -LRB- 30 min before T1 -RRB- on object recognition performance in combination with the TRP − mixture at the most effective time point -LRB- i.e. , 3 h before T1 -RRB- . Thus , five treatment conditions were tested , i.e. , saline , vehicle and TRP − , rolipram 0.01 mg/kg and TRP − , rolipram 0.03 mg/kg and TRP − , and rolipram 0.1 mg/kg and TRP − . Each rat was tested in each condition , and the order of treatment conditions was decided at random . The same animals -LRB- n = 24 -RRB- were used for both behavioral experiments ; thus , control conditions -LRB- TRP + -RRB- from experiment 1 and saline from experiment 2 can be compared to each other . Statistical analysis Biochemistry The mean concentrations of plasma amino acids were determined for each treatment and time condition separately . The extent of reduction -LRB- expressed in absolute values and in percentage decline from resting values -RRB- was calculated for total plasma TRP concentrations and for the TRP/ΣLNAA ratio . Extreme values , as determined by an extremity test -LRB- see Dixon 1959 -RRB- , were excluded from statistical analysis . Differences in plasma amino acid concentrations were analyzed with a general linear model , with main factor ` treatment ' and repeated measure factor ` time ' . Separate one-way analyses of variance -LRB- ANOVA -RRB- were performed to analyze differences between treatment conditions per time point . A post-hoc Bonferroni test was used to further characterize the differences between treatment conditions . Differences were regarded as statistically significant if P < 0.05 . Behavior The basic measures were the times spent by rats exploring an object during T1 and T2 . Table 2 shows the measures involved in the object recognition task -LRB- Prickaerts et al. 1997 -RRB- . e1 and e2 are measures of the total exploration time of both objects during T1 and T2 , respectively . d2 was considered as index measures of discrimination between the new and the familiar objects . d2 is a relative measure of discrimination which corrects the difference between exploring the old and the novel object for exploration activity -LRB- e2 -RRB- and appears to be independent of the total exploration times -LRB- see Şık et al. 2003 -RRB- . In the object recognition task , results are not reliable if an animal explores both objects in the second trial for less than 5 s -LRB- Şık et al. 2003 -RRB- . Therefore , in every session , animals that explored less than 5 s in the second trial -LRB- i.e. , e2 < 5 -RRB- were excluded from analysis . For all parameters , the effects of the different treatment , time , and interactions were analyzed with univariate ANOVA for the first experiment and one-way ANOVA for the second experiment . Significant effects were analyzed in more detail using the Bonferroni correction . To compare control conditions , i.e. , the TRP + -LRB- 3 h before the first trial -RRB- condition of the first experiment with the saline condition of the second experiment , a t test was performed . Differences were regarded as statistically significant if P < 0.05 . Table 2Measures involved in the object recognition testExplorationDiscriminatione1 is the measure of the time spent in exploring both identical objects -LRB- a1 and a2 -RRB- in T1 ; e2 is the measure of the time spent in exploring both the familiar -LRB- a -RRB- and new object -LRB- b -RRB- in T2 ; d2 is the measure of discrimination between the new and familiar objects . Results Plasma TRP values The plasma TRP/ΣLNAA ratio is shown in Fig. 1 . TRP/ΣLNAA levels changed over the 6 h -LSB- F -LRB- 3,60 -RRB- = 4.92 ; P < 0.01 -RSB- . There was a treatment effect for TRP/ΣLNAA ratio -LSB- F -LRB- 2,60 -RRB- = 36.72 , P < 0.01 -RSB- . Post hoc analysis showed that the TRP − condition was significantly different than the TRP + and the saline condition -LRB- Bonferroni , P < 0.05 -RRB- . In addition , the time × treatment interaction was also significant for the TRP/ΣLNAA ratio -LSB- F -LRB- 6,60 -RRB- = 7.80 ; P < 0.01 -RSB- . Separate one-way ANOVA analyses for each treatment time point showed differences between treatment conditions at the 1-h time point -LSB- F -LRB- 2,17 -RRB- = 31.44 ; P < 0.01 -RSB- and the 3-h time point -LSB- F -LRB- 2,16 -RRB- = 6.79 ; P < 0.01 -RSB- . However , no differences between treatment conditions were observed at the baseline time point -LRB- − 20 min -RRB- -LSB- F -LRB- 2,15 -RRB- = 2.03 ; n.s. -RSB- or the 6-h time point -LSB- F -LRB- 2.17 -RRB- = 3.33 ; n.s. -RSB- -LRB- see Fig. 1 -RRB- . Furthermore , post hoc analysis showed that the TRP/ΣLNAA ratio of the TRP-treated animals was lower than the TRP + - or saline-treated animals at the 1-h time point -LRB- Bonferroni ; P < 0.01 -RRB- and the 3-h time point -LRB- Bonferroni ; P < 0.05 -RRB- . Fig. 1The effects of treatment time on the ratio plasma TRP/ΣLNAA -LRB- mean values and SEM -RRB- . Percentages indicate the difference from baseline levels in the TRP − conditions . Asterisks indicate significant differences from baseline -LRB- * P < 0.05 ; ** P < 0.01 -RRB- Behavioral experiment 1 The effects of ATD on ORT performance are shown in Fig. 2 and Table 3 . There was a general treatment effect on ORT performance -LSB- F -LRB- 1,165 -RRB- = 36.44 ; P < 0.01 -RSB- . In addition , a time effect was observed on ORT performance -LSB- F -LRB- 3,165 -RRB- = 2.86 ; P < 0.05 -RSB- . The time × treatment interaction was also significant for the memory performance in the ORT -LSB- F -LRB- 3,165 -RRB- = 4.47 ; P < 0.01 -RSB- . Separate analysis per treatment condition showed that there was a significant time effect for the TRP − condition -LSB- F -LRB- 3,87 -RRB- = 5.41 ; P < 0.01 -RSB- in memory performance , but not for the TRP + condition -LSB- F -LRB- 3,84 -RRB- = 1.77 ; P = n.s. -RSB- . Post hoc analysis showed that object recognition performance was significantly impaired when the TRP − mixture was administered 3 h before the first trial . Fig. 2The effects of treatment time of TRP + or TRP − on the performance in object recognition task . Asterisks indicate a significant impairment in object recognition performance -LRB- * P < 0.05 -RRB- Table 3Exploration times in the object recognition task at baseline and after ATD T1 -- 30 minT1 -- 1 hT1 -- 3 hT1 -- 6 hTRP + e126 .62 -LRB- 2.09 -RRB- 32.30 -LRB- 1.37 -RRB- 27.82 -LRB- 2.06 -RRB- 29.00 -LRB- 2.50 -RRB- e233 .64 -LRB- 2.40 -RRB- 49.78 -LRB- 3.11 -RRB- * 28.61 -LRB- 2.39 -RRB- 28.37 -LRB- 2.15 -RRB- TRP − e126 .97 -LRB- 2.11 -RRB- 30.39 -LRB- 1.78 -RRB- 29.29 -LRB- 1.66 -RRB- 24.98 -LRB- 1.53 -RRB- e239 .26 -LRB- 4.17 -RRB- 37.32 -LRB- 2.40 -RRB- 30.06 -LRB- 1.77 -RRB- 28.91 -LRB- 2.69 -RRB- * P < 0.05 -LRB- indicates significant differences to the baseline group -RRB- Behavioral experiment 2 The effects of ATD in combination with rolipram on object recognition performance are depicted in Fig. 3 and Table 4 . A dose-dependent increase in discrimination performance was observed after treatment with rolipram -LSB- F -LRB- 4,104 -RRB- = 6.85 ; P < 0.01 -RSB- . Post hoc analysis showed that a dose of 0.1 mg/kg rolipram reversed the effects of TRP − treatment -LRB- P < 0.05 -RRB- . Rolipram treatment decreased exploration times in the first trial -LSB- F -LRB- 4,106 -RRB- = 16.24 ; P < 0.01 -RSB- , but had no effect on exploration times in the second trial -LSB- F -LRB- 4,106 -RRB- = 1.53 ; n.s. ; see Table 4 -RRB- . Post hoc analysis showed that for all of the rolipram doses the exploration in the first trial was lower than the saline control condition . Fig. 3The effects of rolipram treatment on ATD-induced deficits in the object recognition task . Asterisks indicate a significant difference in object recognition performance compared with the TRP − and vehicle condition -LRB- ** P < 0.01 -RRB- . The 0.1-mg / kg dose of rolipram reverses the ATD effectTable 4Exploration times in the object recognition task after treatment with rolipram and TRP − mixture SalineVehicle0 .01 mg/kg0 .03 mg/kg0 .1 mg/kgSalineTRP − TRP − TRP − TRP − e128 .72 -LRB- 2.52 -RRB- 26.18 -LRB- 1.72 -RRB- 19.15 -LRB- 1.66 -RRB- * 18.68 -LRB- 1.35 -RRB- * 12.02 -LRB- 1.53 -RRB- * e228 .73 -LRB- 1.42 -RRB- 27.43 -LRB- 1.80 -RRB- 21.68 -LRB- 1.96 -RRB- 27.89 -LRB- 2.63 -RRB- 27.29 -LRB- 2.70 -RRB- * P < 0.05 -LRB- indicates significant differences to the baseline group -RRB- A comparison between the d2 values of the control groups of the first and second experiment -LRB- TRP + 3 h vs saline -RRB- showed that there was no difference between these two control groups -LSB- t -LRB- 38 -RRB- = 0.906 ; n.s. -RSB- . Discussion The present study shows that acute tryptophan depletion results in reliable lowering of plasma TRP and the TRP/ΣLNAA ratio . When rats were administered one dose -LRB- 10 ml/kg -RRB- of the TRP − mixture , depletion was maximal -LRB- 48 % -RRB- 1 h after the treatment . The present data provide strong evidence that blood TRP levels predict object recognition performance and that ATD has only temporary effects on TRP levels and memory performance . When injected 3 h before T1 , object recognition was maximally impaired . This is in support of the results from the blood TRP values -LRB- see above -RRB- . Thus , TRP levels in the blood seem to be related with behavioral performance in rats . As previous studies have shown -LRB- Lieben et al. 2004a -RRB- a clear relation between plasma TRP and central 5-HT , these data provide further support that ATD can be used as a serotonergic-deficit model in the object recognition task . In the study by Lieben at al. -LRB- 2004a , b -RRB- , two doses of the TRP − mixture -LRB- 10 ml/kg -RRB- were administered , which resulted in a 70 % drop of the TRP/ΣLNAA ratio and in memory impairment in the ORT . In contrast , the present study showed that one dose of the TRP − mixture resulted in a 48 % drop in the TRP/ΣLNAA ratio , which was found to induce memory impairment in the ORT . These findings suggest that a 50 % reduction in plasma TRP levels is sufficient to impair object memory in rats . In the first behavioral experiment , no effects on exploration times in the ORT were observed , except from an increase in exploration time in the second trial of the TRP + 1 h before T1 group . However , in the second experiment , a dose-dependent decrease in the exploration time of T1 was observed after treatment with rolipram . Previous work from our group has shown that as long as exploration in trial 1 is higher than 10 s , a reliable discrimination index -LRB- d2 -RRB- can be calculated -LRB- Şık et al. 2003 -RRB- . Furthermore , as can be seen when exploration times are compared to d2 values , effects in exploration time are independent of effects in d2 values -LRB- Şık et al. 2003 -RRB- . Rolipram is known to have some sedative side effects -LRB- at higher dosages > 0.1 mg/kg -RRB- -LRB- Griebel et al. 1991 ; Silvestre et al. 1999 -RRB- ; nevertheless , the low dosages used in the present study improved memory performance . Rolipram has shown its pro-cognitive effects in several behavioral models . We have shown that rolipram treatment increases object memory in a time-dependent forgetting paradigm -LRB- Rutten et al. 2006 -RRB- . In addition , rolipram attenuated cholinergic deficits caused by scopolamine in several behavioral tasks -LRB- Imanishi et al. 1997 ; Rutten et al. 2006 ; Silvestre et al. 1999 ; Zhang and O'Donnell 2000 -RRB- . When rolipram -LRB- 0.1 mg/kg -RRB- was administered in combination with the TRP − mixture -LRB- i.e. , TRP − 3 h before T1 and rolipram 30 min before T1 -RRB- , we found that it reversed the effects of ATD . These are the first data suggesting that rolipram can reverse a serotonergic-induced memory deficit in rats . Our previous study indicated that rolipram can reverse the cholinergic deficit caused by scopolamine in the ORT -LRB- Rutten et al. 2006 -RRB- . Remarkably , a similar dose of 0.1 mg/kg rolipram was capable of reversing the serotonergic and cholinergic deficits in the object recognition task , which might be explained by a non-specific working mechanism of rolipram . Although the underlying mechanisms of action of the memory-enhancing effects of rolipram in these models remain to be determined , the present data could be explained via an enhanced cholinergic and serotonergic turnover by rolipram -LRB- Imanishi et al. 1997 ; Schoffelmeer et al. 1985 -RRB- . Alternatively , the effects of rolipram could also be explained in terms of only a cholinergic mechanism of action . Thus , previous studies have shown that the acetylcholinesterase inhibitor metrifonate ameliorates the effects of ATD as well and improves performance in scopolamine-deficit models of the ORT -LRB- Lieben et al. 2005 -RRB- . This suggests that the cholinergic and serotonergic systems act in a synergistic manner in memory performance -LRB- see Lieben et al. 2005 for a detailed discussion -RRB- . Clearly , further research is needed to scrutinize the mechanism of action rolipram in the different animal models of memory . Another mechanism of action of rolipram that could explain the present data is related to the intracellular signaling in long-term potentiation -LRB- LTP -RRB- . In the hippocampus , rolipram has shown to improve LTP through activation of the cAMP/PKA/CREB pathway -LRB- Frey et al. 1993 ; Impey et al. 1996 -RRB- . Moreover , these mechanisms have been linked to behavioral improvement in several behavioral tasks -LRB- Barad et al. 1998 ; Bernabeu et al. 1997 ; Blokland et al. 2006 ; Rutten et al. 2006 -RRB- . Consequently , it could be argued that the present data could be explained by the effects of rolipram on the cAMP/PKA/CREB pathway . However , a recent study by our group showed that the administration time of rolipram was of critical relevance . Rolipram was only effective in the 24-h interval ORT when administered 3 h after T1 -LRB- Rutten et al. 2006 -RRB- , and the mechanism of late LTP was proposed to explain these effects . However , in the present study , a 1-h interval in the ORT was examined , in which no gene transcription or protein synthesis is required . Therefore , we assume that the cAMP/PKA/CREB pathway can not explain these effects . A recent PET study used radioactive rolipram as a measure for PDE4 binding and as an indirect index of cAMP signaling -LRB- Lourenco et al. 2006 -RRB- . Acute elevation of synaptic neurotransmitter levels -LRB- noradrenaline , 5-HT , and histamine -RRB- resulted in elevated cAMP levels that in turn elevated -LRB- R -RRB- - -LSB- 11C -RSB- rolipram binding to PDE4 -LRB- Lourenco et al. 2006 -RRB- . According to this rationale , elevation of cAMP levels through inhibition of PDE4 can reverse cAMP-mediated deficits in behavior that occur due to a shortage of 5-HT , ie , reversing the ATD-induced object recognition impairment . As rolipram non-selectively facilitates several neurotransmitter systems , a broad mechanism of action can be expected . This can be favorable because influencing multiple systems increases the likelihood that several aspects of behavior could be enhanced . This may explain previous reports that rolipram was effective in depression models -LRB- Bobon et al. 1988 ; Norman et al. 1992 ; Weishaar et al. 1985 -RRB- . On the downside , the broad mechanism of action increases the risks for negative side effects , e.g. , emesis , sedation , and nausea . In summary , the method of ATD can be used as a reliable method for inducing object memory impairment in rats . The selective PDE4 inhibitor rolipram was able to reverse this serotonergic deficit , which could be explained by different mechanisms of action . These data provide further support that PDE4 inhibition could be considered as a potential target to improve memory performance .

O B O B O O B O O O O B I O O O O O O O O B O O B O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O 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O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O

phosphodiesterase
pde4
rolipram
tryptophan depletion
memory
object recognition
atd


Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode Light-harvesting antenna core -LRB- LH1-RC -RRB- complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane . Near infra-red -LRB- NIR -RRB- absorption , fluorescence , and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode . An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm . Introduction When light energy is absorbed in vivo by purple bacterial light-harvesting -LRB- LH -RRB- complexes it is rapidly transferred to the reaction centers -LRB- RC -RRB- where the light energy is efficiently used to drive chemical reactions -LRB- Ke 2001 -RRB- . In most types of purple bacteria there are two types of antenna complexes : peripheral LH2 complexes and the LH1 complexes -LRB- Ke 2001 -RRB- . The structure of the LH2 complex of Rhodopseudomonas acidophila strain 10050 has been resolved to a resolution of 2.0 Å -LRB- McDermott et al. 1995 -RRB- . This LH2 complex consists of a ring of nine heterodimeric subunits . However , such high-resolution structure has not been determined for the LH1 complex yet . There are , however , low-resolution projection structures produced by transmission electron microscopy -LRB- TEM -RRB- -LRB- Karrasch et al. 1995 -RRB- of two-dimensional -LRB- 2D -RRB- crystals of the LH1 complex and a 4.8 Å X-ray crystal structure of the LH1-RC core complex -LRB- Roszak et al. 2003 -RRB- . TEM analysis of the LH1 complexes revealed two types of complex , monomeric complexes from Rhodospirillum rubrum -LRB- R. rubrum -RRB- -LRB- Karrasch et al. 1995 -RRB- and dimeric complexes from Rhodobacter sphaeroides -LRB- R. sphaeroides -RRB- -LRB- Jungas et al. 1999 -RRB- . The recent crystal structure of the LH1-RC ` core ' complex from Rhodopseudomonas palustris -LRB- Rps . palustris -RRB- reveals that the LH1 complex surrounds the contours of the RC so that the ` core ' complex has an overall oval rather than a circular shape -LRB- Roszak et al. 2003 -RRB- . This structure showed the RC surrounded by the LH1 complex which consisted of 15 pairs of transmembrane helical α - and β-polypeptides and their coordinated BChls . The complete closure of the RC by the LH1 is prevented by a single transmembrane helix called W. Atomic force microscopy has also been used to observe antenna complexes in both natural and reconstituted membranes -LRB- Scheuring et al. 2001 , 2003 , 2004 ; Fotiadis et al. 2004 ; Bahatyrova et al. 2004a , b ; Stamouli et al. 2003 -RRB- . Scheuring et al. observed the LH1 complex as a minor component together with the major LH2 complexes from Rubrivivax geratinosus -LRB- Scheuring et al. 2001 -RRB- in reconstituted membranes and intact LH1-RC core complexes in native photosynthetic membranes -LRB- chromatophore -RRB- from Rhodopseudomonas viridis -LRB- Scheuring et al. 2003 -RRB- and Rhodospirillum photometricum -LRB- Scheuring et al. 2004 -RRB- . Fotiadis et al. observed the LH1-RC core complex from R. rubrum in membranes formed from Escherichia coli lipids -LRB- Fotiadis et al. 2004 -RRB- . Bahatyrova et al. showed that the LH1 complexes of a mutant lacking the RC from R. sphaeroides which form circular , elliptical , and even polygonal ring shapes as well as arcs and open rings -LRB- Bahatyrova et al. 2004b -RRB- and that the LH1 complexes are positioned to function as an energy collection hub from the LH2 complexes in native membranes -LRB- Bahatyrova et al. 2004a -RRB- . Our understanding of charge separation and energy transfer in these LH2 and LH1-RC core complexes has enabled the first steps to be taken towards generating artificial systems that convert light energy into usable electrical current . Previous attempts to produce an artificial , energy-converting electrode system used either the LH1 complexes -LRB- Ogawa et al. 2002 -RRB- or RC -LRB- Blankenship et al. 1995 -RRB- immobilized on the electrodes . Until now , there have only been a few attempts to immobilize intact ` core ' complexes , consisting of both the LH1 and the RC components together , onto an electrode -LRB- Ogawa et al. 2004 ; Das et al. 2004 -RRB- . We have recently developed a procedure to create a self-assembled monolayer -LRB- SAM -RRB- of reconstituted LH1 complexes on a transparent indium tin oxide -LRB- ITO -RRB- electrode modified with 3-aminopropyltriethoxysilane -LRB- APS -RRB- using electrostatic interactions or hydrogen bonding between the electrode surface and the anionic LH1 polypeptides at pH 8.0 -LRB- Ogawa et al. 2002 -RRB- . The near infra-red -LRB- NIR -RRB- absorption spectrum showed that the LH1 complex was stable when immobilized onto the electrode . Our current work extends this approach to the native LH1-RC core complexes . LH1-RC ` core ' complexes isolated from R. rubrum and Rps . palustris were successfully assembled on an ITO electrode modified with APS -LRB- APS-ITO -RRB- . Efficient energy transfer and photocurrent responses could be observed upon illumination at 880 nm . Materials and methods Growth of R. rubrum and Rps . palustris bacterium The photosynthetic bacteria , R. rubrum strain S1 and Rps . palustris strain 2.1.6 were grown anaerobically in the light in modified Hutner 's media as previously described -LRB- Roszak et al. 2003 ; Visschers et al. 1991 -RRB- . Purification of the LH1 complex of R. rubrum Chromatophores of R. rubrum were prepared as previously described -LRB- Visschers et al. 1991 -RRB- . Carotenoid was extracted from chromatophores using benzene . Usually approximately 30 mM OG -LRB- 1 mM Tris , pH 7.5 -RRB- was added to dissolve the chromatophores until the near IR absorption band shifted from 873 to 820 nm . Then the resultant aqueous solution was applied to a Sephadex G-100 column -LRB- 1.5 cm i.d. × 75 cm -RRB- to separate the RC and , LH1 complexes with carotenoids and subunit LH/BChl a complexes without carotenoid -LRB- B820 complexes -RRB- . The RC was eluted immediately after the void volume and then the LH1 complexes with carotenoid . Finally the B820 complexes were collected . Absorbance -LRB- λmax -RRB- of the LH1 complex in the absence of carotenoid -LRB- 15 mM OG at 25 °C -RRB- : 372 nm -LRB- 0.86 -RRB- , 586 -LRB- 0.23 -RRB- , 870 -LRB- 1.14 -RRB- . Absorbance -LRB- λmax -RRB- of the LH complex in the presence of carotenoid -LRB- 20 mM OG at 25 °C -RRB- : 372 nm -LRB- 0.86 -RRB- , 475 -LRB- 0.19 -RRB- , 512 -LRB- 0.22 -RRB- , 546 -LRB- 0.19 -RRB- , 586 -LRB- 0.235 -RRB- , 875 -LRB- 0.9 -RRB- . Isolation and purification of the core complex of R. rubrum and Rps . palustris The LH1-RC core complexes isolated from R. rubrum and Rps . palustris were purified essentially as described previously -LRB- Roszak et al. 2003 -RRB- . These LH1-RC core complexes from R. rubrum and Rps . palustris were initially solubilized by the addition of LDAO to 0.4 % or 1 % v/v in 20 mM Tris -- HCl pH 8.0 , respectively . The LH1-RC core complex of Rps . palustris was then separated from the LH2 complexes by sucrose density centrifugation , and was further purified by ion exchange chromatography by DE52 cellulose column . The OD880 of the isolated LH1-RC core complexes was adjusted to 0.3 . Preparation of the core complex assembled on APS-ITO electrodes The basic methods for this have been reported previously -LRB- Ogawa et al. 2002 -RRB- . Transparent indium tin oxide electrodes were cleaned by immersion in piranha solution -LRB- H2O2 : H2SO4 = 3:7 -RRB- . We obtained APS-ITO electrodes by reacting 3-aminopropyltriethoxysilane with the surface of ITO electrodes in dry benzene at 80 -- 90 °C for 4 h . The OD880 of the isolated core was adjusted to 0.3 . APS-ITO electrodes were immersed in the LH complex and the core complex solutions in Tris -- HCl pH 8.0 for 6 h at 4 °C , and then rinsed with Tris -- HCl pH 8.0 . The core complexes were immobilized , as a self-assembled monolayer on an APS-ITO electrode . Near IR , FT-IR , and fluorescence spectra Near IR spectra were recorded with Hitachi U-2000 and U-3500 . Fluorescence spectra were measured with a Nippon Roper fluorometer by using a halogen tungsten light bulb -LRB- TS-428 DC -RRB- , a single monochromator -LRB- SP-150M -RRB- for selection of the excitation wavelength , a monochromator -LRB- SP-306 -RRB- and a CCD detector -LRB- Spec 10-100 BR/LN -RRB- to detect the emitted fluorescence . The slits were set at 0.50 mm for the LH1 complexes in OG . Slits were set at 1.00 mm for the LH1 complex on the electrode . The samples were measured at 25 °C . FT-IR spectra were recorded with Perkin-Elmer Spectrum 2000 . Photocurrent measurements Photocurrents were measured at − 0.2 V -LRB- versus Ag/AgCl -RRB- in a home made cell that contained three electrodes ; an APS-ITO electrode incorporating the core complex as a working electrode , an Ag/AgCl -LRB- saturated KCl -RRB- as a reference electrode , and a platinum flake as a counter electrode . The working electrode was illuminated with a halogen lamp unit , AT-100HG , through a monochromator , SPG-120S -LRB- Shimadzu -RRB- . The solution consisted of 0.1 M phosphate buffer -LRB- pH 7.0 -RRB- , containing 0.1 M NaClO4 and 5 mM methyl viologen . Results and discussion Figure 1 shows the NIR absorption spectra of the isolated R. rubrum -LRB- a -RRB- and Rps . palustris -LRB- b -RRB- core complexes in 20 mM Tris -- HCl buffer pH 8.0 OG micelle -LRB- dotted line -RRB- and assembled onto an APS-ITO electrode -LRB- solid line -RRB- , respectively . These spectra show that these core complexes have the absorption maximum at 880 nm with two smaller peaks at 800 and 760 nm . The former peak is attributable to the overlap of bacteriochlorophyll a -LRB- BChla -RRB- in the LH1 complex -LRB- 880 nm -RRB- and the reaction center BChla dimer ` special pair ' -LRB- 870 nm -RRB- and the latter two peaks to the BChla called ` accessory ' -LRB- 800 nm -RRB- and bacteriopheophytin -LRB- 760 nm -RRB- in the RC , respectively -LRB- Blankenship et al. 1995 -RRB- . The NIR absorption spectra of these core complex on the electrode indicate that these complexes were not denatured when assembled onto an APS-ITO . In the previous study it was apparent that when the RC of R. rubrum was assembled , by itself , on the electrode it was relatively labile -LRB- Matsumoto et al. 1999 -RRB- . In present study , the complete core complex proved to be quite stable , when assembled onto the electrode . The enhanced stability of the RC surrounded by the LH1 complex probably results from supportive interactions between the two complexes . Fig. 1NIR absorption spectra of the isolated R. rubrum -LRB- a -RRB- and Rps . palustris -LRB- b -RRB- core complexes in 20 mM Tris -- HCl buffer pH 8.0 OG micelle -LRB- dotted line -RRB- and assembled onto an APS-ITO electrode -LRB- solid line -RRB- Table 1 shows the NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps . palustris in OG micelles and on an APS-ITO electrode . The fluorescence bands of these core complexes on the electrode are identical to those in the OG micelles , again indicating that these complexes are stable when assembled onto the APS-ITO electrode . Interestingly , when illuminating at 880 nm the fluorescence emission of BChla molecules in the LH complex of R. rubrum on the APS-ITO was strongly quenched , due to the presence of the RC of R. rubrum . This indicates that an efficient energy transfer from BChla in the LH1 complex to the RC in the core complex is still occurring on the electrode -LRB- data not shown -RRB- -LRB- Ogawa et al. 2004 -RRB- . FT-IR spectra of the LH complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps . palustris assembled on the APS-ITO show the absorptions at 1650 and 1550 cm − 1 . These bands can be assigned to the amide I -LRB- C =O stretching vibration -RRB- and amide II -LRB- N -- H deformation vibration -RRB- bands of α helical conformation , respectively -LRB- Miura et al. 1998 -RRB- . These results indicate that the LH polypeptides are in the same α helical configurations on the ITO electrode as in OG micelles -LRB- Parkes-Loach et al. 1988 ; Kashiwada et al. 2000 -RRB- . Table 1NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps . palustrisIn OG micelleOn APS-ITOAbsorbance -LRB- nm -RRB- Fluorescence -LRB- nm -RRB- Absorbance -LRB- nm -RRB- Fluorescence -LRB- nm -RRB- R. rubumLH1870890870890R . rubumLH1-RC880900880900Rps . palustrisLH1-RC878900878900 In summary , all of these results indicate that the LH1 and LH1-RC core complexes were not denatured by binding to the APS-ITO surface . Similar results were obtained using Langmuir -- Blodgett -LRB- LB -RRB- films to lay down LH1 complex membranes as a layer on a glass substrate -LRB- Iida et al. 2000 -RRB- . The LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps . palustris on the APS-ITO electrode were stable enough to handle at 4 °C in the dark condition least 24 h. Figure 2 shows the time course of the photocurrent generated from the LH1-RC core complex , LH1 complex or the RC of R. rubrum assembled onto an APS-ITO when the electrode was illuminated with a pulse of light at 880 nm . It is clear in Fig. 2 that an enhanced photocurrent was observed for the LH1-RC core complex . In contrast no photocurrent was observed for either LH1 complex or the RC . Under our experimental conditions a cathodic photocurrent was observed , implying that one-way electron transfer from pigments in the LH1-RC core complex to methyl viologen was occurring as shown in Fig. 3 -LRB- Nagata et al. 2003 ; Imahori et al. 2000 -RRB- . Fig. 2Time course of the photocurrent of the LH1-RC core complex , LH1 complex or the RC complex of R. rubrum on an APS-ITO electrode when the electrode is illuminated with pulsed light -LRB- 880 nm -RRB- firing continuously for 30 sFig . 3Schematic drawing of LH1-RC core complexes on an APS-ITO electrode generated cathodic photocurrent which shows the electron flow from the complex to methyl viologen -LRB- left -RRB- according to the cathodic photocurrent as shown in Fig. 2 . Energy diagram for cathodic photocurrent generation by the LH1-RC core complex -LRB- right -RRB- Figure 4 shows excitation spectrum of the photocurrent density -LRB- dots -RRB- and the NIR absorption spectra -LRB- solid line -RRB- from R. rubrum -LRB- a -RRB- and Rps . palustris -LRB- b -RRB- LH1-RC core complexes assembled onto an APS-ITO , respectively . These photocurrent responses showed a maximum at the wavelength corresponding to the absorption bands of the complex . Interestingly , an enhanced photocurrent was observed especially upon illumination at 880 nm for both R. rubrum -LRB- a -RRB- and Rps . palustris -LRB- b -RRB- LH1-RC core complexes . The quantum yield of the photocurrent was 0.05 % for both the R. rubrum and Rps . palustris complexes -LRB- Imahori et al. 2000 -RRB- . When the LH1 complex of R. rubrum alone , was immobilized on the electrode , the observed photocurrent was mainly generated by light absorbed at 770 nm , i.e. from monomeric BChla -LRB- Nagata et al. 2003 -RRB- . Furthermore , when the RC complex of R. rubrum only was immobilized on the electrode , an efficient photocurrent was not observed upon illumination at 880 nm as shown in Fig. 2 . Thus , the enhanced photocurrent observed at 880 nm in the assembled LH1-RC core complex can be ascribed to energy transfer from the LH1 to the RC and then electron transfer from the electrode to the RC as shown in Fig. 3 -LRB- Ogawa et al. 2004 -RRB- . This data indicates that the LH1-RC core complex was well organized on the ITO and the photocurrents were driven by light that was initially absorbed by the LH components . Fig. 4Photocurrent density -LRB- dots -RRB- and NIR absorption spectrum -LRB- solid line -RRB- of LH1-RC core complexes form : -LRB- a -RRB- R. rubrum and -LRB- b -RRB- Rps . palustris assembled on an APS-ITO electrode In conclusion , the SAM method is clearly successful in allowing assembly of functional LH1-RC core complexes on the electrode . This has been confirmed by NIR absorption spectroscopy , demonstrating that the photocurrent response , which is derived from electron transfer between the RC and the electrode , is enhanced by illumination at 880 nm .

O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O B I O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B 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light harvesting-reaction center complex