Esophagojejunal reconstruction after total gastrectomy for gastric cancer using a transorally inserted anvil delivery system.

INTRODUCTION: Total gastrectomy (TG) is commonly performed for the treatment of patients with gastric cancer. However, reconstruction of the esophagojejunal (EJ) anastomosis can be technically demanding, with reported anastomotic leak rates in the Western world still approaching 10-15%. We report our experience using the transoral anvil delivery system (OrVil™) for creation of the EJ anastomosis after TG. METHODS: From 2007 to 2011, 48 consecutive patients with gastric cancer underwent open (n=31) or laparoscopic (n=17) TG. EJ reconstruction was performed with the transoral anvil deliver system (OrVil™) in an end-to-side fashion. Demographic, clinic, and perioperative data were obtained from a prospectively maintained database. RESULTS: Of the 48 patients, 83% were male. Median age at resection was 64 years. Median body mass index was 27.1 kg/m2. Seventy-nine percent (n=38) of patients had at least one comorbidity. Fifteen patients (31%) had at least one perioperative complication. There was one perioperative death (2%) following a duodenal stump leak. There were four EJ leaks (8%) and two EJ stenoses (independent of leak; 4%). There was one EJ leak (6%) and one EJ stenosis (6%) following a case that was first attempted laparoscopically. There were no deaths as a consequence of an EJ leak. CONCLUSIONS: The use of the transoral anvil delivery system during EJ reconstruction is a safe and effective option for reconstruction after open or laparoscopic TG with acceptable mortality and morbidity. The anastomotic leak rate appears to be comparable to that of other techniques.

Essential role of the disulfide-bonded loop of chromogranin B for sorting to secretory granules is revealed by expression of a deletion mutant in the absence of endogenous granin synthesis.

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide- bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Deltacys-hCgB) lacking the 22-amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Deltacys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Deltacys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus-mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.

Spontaneous isolated dissection of the celiac trunk with rupture of the proximal splenic artery: a case report.

INTRODUCTION: Spontaneous visceral artery dissection is an uncommon cause of acute abdominal pain. Complications are ischemia, aneurysm formation and rupture. We present a case with synchronous rupture of the splenic artery causing massive bleeding and demanding urgent surgery. To our knowledge, only 24 previous cases are reported in the literature. REPORT: The patient was a 56-year-old male smoker with no previous medical history who was treated surgically with exposure of the suprarenal aorta through left-sided medial visceral rotation and isolation of the celiac artery. The origin of the bleeding was identified as a longitudinal rupture of the splenic artery just distal to the hepatic artery. The artery was ligated and splenectomy was performed because of splenic infarction. The hepatic artery was patent and no reconstruction was needed. The postoperative course was uneventful, treatment with antiplatelets and antihypertensive drugs was initiated. The patient was discharged after ten days and at monthly follow-up the patient was in good condition. CT angiography was performed six months postoperative and the celiac trunk was patent but a small aneurysm had developed. DISCUSSION: Dissection of the celiac artery is uncommon and is rarely considered in the diagnosis of acute abdominal pain. The condition could be mistaken for a ruptured AAA. The condition may be underdiagnosed and it seems likely that more cases will be identified in the future as a result of the rapidly evolving vascular imaging modalities.

The granin (chromogranin/secretogranin) family.

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy.

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.

Protein secretion: puzzling receptors.

All known sorting receptors for soluble cargo in the secretory pathway are transmembrane proteins. For sorting to the regulated pathway, however, a subpopulation of secretory proteins, associated with the membrane but not membrane-spanning, appears to link cargo and membrane in storage granule biogenesis.

Dynamics of immature secretory granules: role of cytoskeletal elements during transport, cortical restriction, and F-actin-dependent tethering.

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Expansion of the epithelial cell proliferative compartment and frequency of adenomatous polyps in the colon correlate with the strength of family history of colorectal cancer.

Expansion of the proliferative compartment of epithelial cells in colonic crypts and colonic adenomas have been described as phenotypic precursors to colon cancer in individuals affected with hereditary or sporadic colon cancer. This study measured the size of the proliferative compartment in colonic crypts and the frequency of adenomas in asymptomatic members of families having sporadic colorectal cancer. The subjects were divided into 2 groups according to the frequency of colorectal cancer in their families. A shift of the compartment of proliferating epithelial cells toward the lumenal surface of colonic crypts was seen in the group of subjects with a stronger family history of colorectal cancer, with significant differences in the numbers of proliferative cells in the upper and the lower crypt compartments (P < 0.05) and in the fraction of proliferative cells at the highest compartment at the lumenal surface of the crypts (P < 0.05). Cell proliferation patterns in normal-appearing mucosa of the 2 groups revealed no difference in whole crypt [3H]thymidine labeling index. Colonoscopic examination of the 56 subjects revealed an overall prevalence of adenomas of 21%; when stratified by frequency of colorectal cancer in their families, 3 of 22 subjects (14%) with a weaker family history had adenomas, while 9 of 34 (26%) with a stronger family history had adenomas. Thus, parallel abnormalities of colonic epithelial cell proliferation and neoplasia were seen in individuals with a family history of colorectal cancer, both of which were more pronounced with increasing strength of family history. This observation provides further evidence of relationships among these factors in the etiology of "sporadic" colorectal cancer.

Gene response upon illumination in forming mRNA encoding peroxisomal glycollate oxidase.

Glycollate oxidase is a constituent of leaf peroxisomes. Its biosynthesis is, like the biosynthesis of many chloroplastic proteins, controlled by light, via phytochrome. The level of mRNA coding for glycollate oxidase was determined at different stages of greening of etiolated plant cells. The appearance of glycollate oxidase mRNA in the cytoplasm was measured by hybridization with cDNA containing part of the coding sequence for glycollate oxidase. cDNA was prepared from enriched mRNA, inserted into the Pst I site of pBR 322, and cloned in Escherichia coli DH-1. By differential colony hybridization and hybrid selection, a clone containing a 670 bp sequence complementary to mRNA encoding glycollate oxidase was selected and identified. Northern blot hybridization was used to investigate mRNA levels induced by light. It was found that continuous light affected the formation of glycollate oxidase mRNA. When a large population of microbodies was present in the cells being induced, the immediate mRNA increase was very pronounced, and was detectable as little as 20 min after the beginning of the light treatment. In contrast, a lag period in the mRNA increase was observed when the induction was performed with etiolated leaves which are characterized by the occurrence of a rather small population of microbodies. For comparison, we measured the time-course of formation of mRNA coding for a light-induced chloroplastic protein, i.e., a protein of the light-harvesting complex. The time-courses of levels of the two mRNAs indicate that the program of gene expression differs between the two particular proteins destined either for chloroplasts or for peroxisomes. The formation of glycollate oxidase mRNA could also be stimulated by a short pulse of light, a treatment of 15 s being a sufficient trigger.

Endoscopic ultrasound in the evaluation of gastric small lymphocytic mucosa-associated lymphoid tumors.

PURPOSE: Low-grade, small lymphocytic lymphomas of the mucosa-associated lymphoid tissue (MALT) have recently been shown to be associated with Helicobacter pylori infections. Regression of these tumors has been reported with antibiotic therapy. Here we evaluate endoscopic ultrasound (EUS) as on objective method to evaluate pretreatment disease and posttherapy response. MATERIALS AND METHODS: We retrospectively reviewed 20 patients initially diagnosed elsewhere with MALT lymphoma. All patients had their initial endoscopic biopsies (EGDs) reviewed at Memorial Sloan-Kettering Cancer Center (MSKCC). All patients had EUS performed at the time of consultation and on completion of therapy if treated at our center. Antral biopsies were stained with a modified Steiner preparation to determine infection by H pylori. RESULTS: Gastric low-grade lymphoma was confirmed in 16 of 20 patients; 11 of 16 had previously received antibiotic therapy for biopsy-positive H pylori infection. All gastric lymphomas had an abnormal EUS: eight with discrete tumor masses and eight with gastric wall infiltration (submucosa, n = 4; muscularis propria, n = 3; serosa, n = 1). On completion of lymphoma treatment with chemotherapy, radiotherapy, or surgery, 11 of 16 patients underwent follow-up EUS. Five patients received care elsewhere and did not return for posttreatment EUS. The gastric wall was normal with no evidence of disease on EUS-guided biopsy in eight of 11 patients. The remaining three patients had abnormal gastric walls. One was biopsy-negative and two had residual lymphoma. Four patients were found to have benign lymphoid aggregates in association with H pylori on initial EGD and EUS biopsies. All four patients were previously untreated with antibiotics. EUS showed prominent mucosa, but no significant findings within the gastric wall. CONCLUSION: EUS appears useful to stage objectively and evaluate therapeutic outcome in the management of gastric, low-grade MALT lymphomas. It also helps to distinguish benign lymphoid aggregates from lymphoma associated with H pylori infection. EUS findings may have a significant impact on assessment and therapeutic recommendations.

Preoperative endoscopic ultrasound can predict the risk of recurrence after operation for gastric carcinoma.

PURPOSE: Endoscopic ultrasonography (EUS) has been shown to determine accurately the depth of invasion of the stomach wall in gastric carcinoma. We undertook this study to determine if T (tumor) stage as determined by EUS correlated with recurrence after resection and could be used to identify patients preoperatively at high risk for recurrence. MATERIALS AND METHODS: We reviewed the surgical pathology and obtained follow-up data from the first 50 patients who underwent preoperative EUS for staging of gastric carcinoma. Rotating sector-scan ultrasound endoscopes were used with switchable frequencies of 7.5 MHz and 12 MHz. RESULTS: Of 50 patients, 43 underwent resection with curative intent and were available for follow-up. The concordance of EUS T stage with pathologic T stage in these patients was 86%. At a median follow-up duration of 25 months, only two of 13 patients with preoperative EUS stage T1 or T2 disease were found to have recurrence, while 23 of 30 patients with EUS stage T3 or T4 disease had recurrence (P = .0002) and 22 died. CONCLUSION: We conclude that patients with a preoperative EUS stage T1 or T2 are at low risk for postoperative recurrence, while patients with EUS stage T3 or T4 are at high risk for early postoperative recurrence. The latter patients are reasonable candidates for controlled trials of alternative preoperative management programs, such as chemotherapy, in an effort to improve their poor prognosis.

Neoadjuvant therapy of high-risk gastric cancer: a phase II trial of preoperative FAMTX and postoperative intraperitoneal fluorouracil-cisplatin plus intravenous fluorouracil.

PURPOSE AND METHODS: We identified patients with gastric cancer at high risk for recurrence before therapy using endoscopic ultrasonography (EUS). Neoadjuvant therapy using the fluorouracil, doxorubicin, and metrotrexate (FAMTX) regimen was given for three courses before planned laparotomy with the intention to perform curative resection. Postoperatively, intraperitoneal (IP) cisplatin and fluorouracil (FU) and intravenous (i.v.) FU were administered to patients undergoing resection. RESULTS: Fifty-six assessable patients were treated. Preoperative FAMTX therapy was tolerable, with the major toxicity being neutropenic fever. One treatment-related death was seen. Eighty-nine percent of patients underwent surgical exploration and 61% had potentially curative resections. There were two postoperative deaths. Comparison of pathologic tumor (pT) stage with EUS-predicted tumor stage showed apparent downstaging in 51% of patients. Postoperative IP chemotherapy was delivered to 75% of eligible patients. Toxicity was acceptable. There was no increase in operative morbidity or mortality compared with concurrent nonstudy patients undergoing a similar operative procedure and not receiving preoperative therapy. With a median follow-up time of 29 months, the median survival duration was 15.3 months. For patients who underwent potentially curative resections, the median survival duration was 31 months. Peritoneal failure was seen in 16% of patients. CONCLUSION: Chemotherapy with the FAMTX regimen is tolerable in patients with locally advanced gastric cancer, without an increase in operative morbidity or mortality. IP therapy can be successfully delivered to most resected patients. The intraabdominal failure pattern appears to be decreased compared with expected. This approach is an appropriate investigational arm to pursue in future studies.

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells.

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.

Targeting of green fluorescent protein to neuroendocrine secretory granules: a new tool for real time studies of regulated protein secretion.

Human chromogranin B (hCgB), a soluble marker protein of neuroendocrine secretory granules, was fused to green fluorescent protein (GFP). Two GFP-mutants with different folding properties, S65T and EGFP, were used to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB-EGFP elicited green fluorescence in the neuroendocrine cell line PC12. Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG). Confocal microscopy revealed that fluorescent hCgB-EGFP colocalized largely with synaptotagmin, a membrane marker of secretory granules and synaptic-like microvesicles, and significantly with endogenous rat chromogranin B (rCgB), a soluble marker of secretory granules. Upon stimulation of transfected cells with 5 mM Ba2+ or by depolarization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma membrane (PM) of living PC12 cells were visualized by confocal microscopy. The majority of these vesicles did not move within 8.5 sec as if they were docked. In contrast, in NGF-induced cells most of the secretory granules beneath the somatic PM moved within the same time period whereas only little movement was observed in the neurites. These findings indicate that in differentiated PC12 cells the majority of the docking zones are not in the soma but are distributed along the neurites. In conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of protein secretion.

Neuroendocrine cell type-specific and inducible expression of the secretogranin II gene: crucial role of cyclic adenosine monophosphate and serum response elements.

Secretogranin II, an acidic protein in the chromogranin/secretogranin family, is widely distributed in neuroendocrine secretory granules. What factors govern such widespread, yet selective, expression? The 5' deletions localized neuroendocrine cell type-specific expression to the proximal mouse secretogranin II promoter: such expression was abolished after deletion past the cAMP response element (CRE; [-67 bp]TGACGTCA[-60 bp]), and transfer of the CRE to a neutral promoter conferred 3.4- to 5.3-fold neuroendocrine selectivity. Thus, the CRE is, at least partly, sufficient to confer tissue-specific expression. Substantial (48-59%) loss of cell type-specific expression also occurred upon deletion past the serum response element (SRE; [-302 bp]GATGTCC[-296 bp]), and transfer of the SRE to a neutral promoter also conferred neuroendocrine selectivity. Expression of both the endogenous gene and the transfected secretogranin II promoter was up-regulated after secretagogues, and the degree of trans-activation of the transfected promoter (2.2- to 5.4-fold) paralleled activation of the endogenous gene (1.8- to 3.2-fold). The 5' promoter deletions revealed complete loss of secretagogue responses after deletion past the CRE. Transfer of the CRE to a neutral promoter conferred secretagogue responses (by 2.2- to 18.6-fold). Substantial (59-74%) falls in secretagogue responses also occurred after deletion past the promoter's SRE. Transfer of the SRE to a neutral promoter conferred secretagogue responses (by 2.7- to 8.3-fold). We conclude that the CRE is a crucial determinant of cell type-specific constitutive and secretagogue-inducible expression of the secretogranin II gene and that the SRE also plays a substantial role in both processes.

Neuroendocrine cell type-specific and inducible expression of the chromogranin B gene: crucial role of the proximal promoter.

Chromogranin B, a soluble acidic secretory protein, is widely distributed in neuroendocrine and neuronal cells, although not in other cell types. To identify the elements governing such widespread, yet selective, expression of the gene, we characterized the isolated mouse chromogranin B promoter. 5'-Promoter deletions localized neuroendocrine cell type-specific expression to the proximal chromogranin B promoter (from -216 to -91 bp); this region contains an E box (at [-206 bp]CACCTG[-201 bp]), four G/C-rich regions (at [-196 bp]CCCCGC[-191 bp], [-134 bp]CCGCCCGC[-127 bp], [-125 bp]GGCGCCGCC[-117 bp], and [-115 bp]CGGGGC[-110 bp]), and a cAMP response element (CRE; at [-102 bp]TGACGTCA[-95 bp]). A 60-bp core promoter region, defined by an internal deletion from - 134 to -74 bp upstream of the cap site and spanning the CRE and three G/C-rich regions, directed tissue-specific expression of the gene. The CRE motif directed cell type-specific expression of the chromogranin B gene in neurons, whereas three of the G/C-rich regions played a crucial role in neuroendocrine cells. Both the endogenous chromogranin B gene and the transfected chromogranin B promoter were induced by preganglionic secretory stimuli (pituitary adenylyl cyclase-activating polypeptide, vasoactive intestinal peptide, or a nicotinic cholinergic agonist), establishing stimulus-transcription coupling for this promoter. The adenylyl cyclase activator forskolin, nerve growth factor, and retinoic acid also activated the chromogranin B gene. Secretagogue-inducible expression of chromogranin B also mapped onto the proximal promoter; inducible expression was entirely lost upon internal deletion of the 60-bp core (from 134 to -74 bp). We conclude that CRE and G/C-rich domains are crucial determinants of both cell type-specific and secretagogue-inducible expression of the chromogranin B gene.

Green fluorescent protein: applications in cell biology.

The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.

Visualization of protein transport along the secretory pathway using green fluorescent protein.

We have expressed green fluorescent protein (GFP) from A. victoria in the secretory pathway of HeLa cells by fusing it to the C-terminus of a secretory protein, chromogranin B. Under normal culture conditions at 37 degrees C maturation of GFP to the fluorescent form was not detectable. However, fluorescent GFP was observed when biosynthetic protein transport was arrested at the intermediate compartment or the trans-Golgi network by temperature blocks (15 degrees C and 20 degrees C, respectively). Reversal of the temperature blocks allowed the visualization of secretion of fluorescent GFP and offers the possibility to analyse transport in the secretory pathway in living cells.

The organisation of the mouse chromogranin B (secretogranin I) gene.

A cosmid clone containing the gene for mouse chromogranin B (secretogranin I), a secretory protein found in secretory granules of most endocrine cells and neurons, was isolated and sequenced. The chromogranin B protein was found to be encoded by 5 exons which correspond to the cleaved signal peptide, the short N-terminal sequence preceding the disulfide-bonded loop structure, the disulfide-bonded loop structure itself, the large, variable region comprising approximately 90% of the protein, and the conserved C-terminal sequence. The promoter region of the chromogranin B gene is very GC-rich and contains a CATAA motif, a cAMP-responsive element and an Sp1 binding site.

The organisation of the mouse secretogranin II gene.

We have characterized the gene which encodes mouse secretogranin II (previously also referred to as chromogranin C), a tyrosine-sulfated secretory protein belonging to the granin (chromogranin/secretogranin) family which is found in secretory granules of most endocrine cells and neurons. The secretogranin II gene was found to contain 2 exons. In contrast to chromogranin A and chromogranin B, the two previously characterized granin genes, the entire secretogranin II protein is encoded by a single exon, exon 2, with exon 1 containing only a 5'-untranslated sequence. Consistent with previous data on the expression of secretogranin II, the putative promoter region was found to contain a cAMP-responsive element and a potential AP-1 binding site.