Gastric hibernoma: a novel location and presentation of a rare tumor.

We present a case of gastric hibernoma, an unusual tumor with a location novel to the literature. A 39-year-old female presented with one year of upper gastrointestinal bleeding and dysphagia. Gastroenterology performed an esophagogastroduodenoscopy with ultrasound and identified a 6 cm mass within the muscularis propria of the antrum. Computed tomography demonstrated a 9.7 × 7.8 × 4.8 cm3 heterogeneous antral mass with internal septa. A distal gastrectomy with Bilroth I gastroduodenostomy was performed with 4 cm proximal and 2 cm distal margins. Excision was appropriate to make the diagnosis, exclude malignancy, and remove a symptomatic mass. Hibernoma was confirmed by histopathology. These are rare tumors of brown fat named for their resemblance to the thermogenic tissue found in hibernating animals. They typically present as a slowly enlarging mass of the thigh or shoulder. To our knowledge, this is the first presentation of a hibernoma as a submucosal gastric mass.

Histopathology of locally advanced colorectal carcinoma, with emphasis on tumor invasion of adherent peritoneal membranes.

Locally advanced colorectal carcinomas are characterized by neoplastic cells that invade beyond the colon and directly into surrounding organs and structures that include the retroperitoneum and abdominopelvic sidewall. These aggressive tumors are prognostically adverse and are categorized with highest possible tumor stage in current cancer staging systems. Recognizing colorectal carcinoma with extensive locoregional invasion is typically straightforward, but some cases can be diagnostically challenging. These include tumors with limited invasion of extracolonic tissue such as the subserosa in which there are no cells or structures that are histologically or architecturally distinct from the colonic primary. Tumor-associated injury of the colonic peritoneum often precedes invasion by the neoplastic cells and can cause the peritoneal membrane of the colon to adhere and subsequently fuse to the peritoneal membrane of a neighboring organ or structure, thereby creating a trans-peritoneal bridge through which a tumor can directly invade the adherent extracolonic tissue. Hence, locally advanced colorectal carcinoma can be narrowly defined by neoplastic cells that completely invade through the fused peritoneal membrane and into the subserosa of the adherent extracolonic tissue. The evaluation of tumor invasion of the fused peritoneal membrane, which is enhanced by the combined use of an elastic stain and an immunostain for pan-keratin, is essential for the proper staging of locally advanced colorectal carcinoma and its clinical management.

Histopathology and enhanced detection of tumor invasion of peritoneal membranes.

Tumor invasion of the peritoneal membrane may have an adverse prognostic significance, but its histopathologic features can be diagnostically difficult to recognize. We observed that local peritoneal injury associated with tumor invasion is characterized by activation and proliferation of serosal stromal cells that express cytokeratin, a characteristic property of injured serosal membranes that may have diagnostic utility. To explore this, we examined 120 primary tumors of the gastrointestinal tract and pancreaticobiliary system using cytokeratin and elastic stains to assess for tumor invasion of peritoneal membranes. Peritoneal invasion by tumor was associated with retraction, splaying, and destruction of the elastic lamina and proliferation of keratin-expressing stromal cells of serosal membranes. All 82 peritoneal invasive tumors were characterized by neoplastic cells that invaded the elastic lamina and the serosal connective tissue with neoplastic cells that abutted or were surrounded by keratin-positive stromal cells, whereas all 38 tumors limited to the subserosa showed none of these features. The diagnosis of tumor invasion of peritoneal membranes is enhanced by the combined use of cytokeratin and elastic stains, which in turn would enable better histopathologic correlation with patient treatment and outcome.

Colorectal cancer in Crohn's colitis is comparable to sporadic colorectal cancer.

PURPOSE: It is now recognized that Crohn's disease (CD), similar to ulcerative colitis (UC), carries an up to 20-fold higher cancer risk, and the development of colorectal carcinoma (CRC) is a major long-term complication. Once CRC is present, molecular profiling is one of the components in selecting appropriate treatment strategies; however, in contrast to UC, genetic alterations in Crohn's colitis-associated CRC are poorly understood. METHODS: In a series of 227 patients with Crohn's colitis, we identified 33 cases of CRC (~14 %) and performed targeted mutational analysis of BRAF/KRAS/NRAS and determined microsatellite status as well as immunophenotype of the tumors. RESULTS: In the CRC cohort, the median age at time of cancer diagnosis was 58 (range 34-77 vs. 59.5 in sporadic; P = 0.81) and the median CD duration was 29 years (range 6-45). As a group, CRC complicating Crohn's colitis is BRAF (97 %) and NRAS (100 %) wild type and the vast majority is microsatellite stable (94 %); KRAS-mutations were found in six cases (18 %). Stage grouping, anatomic distribution, and overall survival were similar to sporadic CRC; however, long-standing CD (≥25 years) as well as gastric-immunophenotype (MUC5AC+) was associated with significantly shorter overall survival (P = 0.0029; P = 0.036, respectively). CONCLUSION: In summary, the clinicopathological and molecular profile of CD-associated CRC is similar to that observed in sporadic CRC.

Can endoscopic ultrasound predict pancreatic intraepithelial neoplasia lesions in chronic pancreatitis?: a retrospective study of pathologic correlation.

OBJECTIVE: This study aimed to evaluate associations between endoscopic ultrasound (EUS) criteria for chronic pancreatitis (CP) and coexisting pancreatic intraepithelial neoplasia (PanIN) lesions. METHODS: Patients with known or suspected CP who underwent pancreatic resection within a year of EUS were selected. Histology slides and EUS images were reviewed for evidence of pancreatic fibrosis. RESULTS: Ninety-seven (51 men; mean age, 53 [12] years) underwent EUS within a 1 year or less of EUS. Pancreatic intraepithelial neoplasia lesions were found in 84 (87%) patients. Pancreatic intraepithelial neoplasia 1, 2, and 3 lesions were seen in 71 (83%), 10 (14%), and 1 (2%), respectively. Two patients had more than 1 PanIN grade (one had PanIN 1 and 2 and the other had PanIN 1 and 3). The mean number of EUS criteria for PanIN 1, 2, and 3 lesions were 3.9, 4.5, and 5.5, respectively. The odds ratio for the association between PanIN 2 and hyperechoic foci without shadowing in the pancreas head was 8.5 (P = 0.05). The odds ratio for the association between PanIN 2 and lobularity with honeycombing was 2.7 (P = ns). Advanced PanIN lesions had greater than or equal to 4 EUS criteria for CP. CONCLUSIONS: Pancreatic intraepithelial neoplasia lesions were highly prevalent in CP resections. Increasing PanIN grade is observed with increasing fibrosis score and increasing number of EUS criteria for CP.

Endoscopic ultrasound and histology in chronic pancreatitis: how are they associated?

OBJECTIVES: This study aimed to correlate endoscopic ultrasound (EUS) criteria and pathology in patients with chronic pancreatitis (CP). METHODS: Endoscopic ultrasound reports and pathology specimens were reviewed from patients with known or suspected CP who underwent surgery within 1 year of EUS. The following information was abstracted: EUS criteria for CP, corresponding pathology results, and histologic features. The EUS and pathology results were correlated. RESULTS: One hundred patients (55 men; mean age, 54 years) underwent a pancreatic resection, median of 50 days (range, 1-363 days). The mean (SD) fibrosis scores in the head and body/tail specimens were 7.9 (3.0) and 6.4 (3.8), respectively (P = 0.02). The main pancreatic duct (MPD) dilation and irregularity were associated with moderate and severe fibrosis. Lobularity with honeycombing was associated with intralobular and interlobular fibrosis. Severe CP was associated with the following: lobularity with honeycombing, hyperechoic foci with shadowing, hyperechoic foci without shadowing, MPD dilation, MPD irregularity, and dilated side branches. CONCLUSIONS: Endoscopic ultrasound of the pancreas head may be considered in the evaluation of CP. The EUS criteria that were associated with severe CP included the following: lobularity with honeycombing, hyperechoic foci with shadowing, dilated MPD, irregular MPD, and dilated side branches. The importance of pancreatic ductal changes should not be minimized in the evaluation of CP.

Poorly differentiated neuroendocrine carcinomas of the pancreas: a clinicopathologic analysis of 44 cases.

BACKGROUND: In the pancreas, poorly differentiated neuroendocrine carcinomas include small cell carcinoma and large cell neuroendocrine carcinoma and are rare; data regarding their pathologic and clinical features are very limited. DESIGN: A total of 107 pancreatic resections originally diagnosed as poorly differentiated neuroendocrine carcinomas were reassessed using the classification and grading (mitotic rate/Ki67 index) criteria put forth by the World Health Organization in 2010 for the gastroenteropancreatic system. Immunohistochemical labeling for neuroendocrine and acinar differentiation markers was performed. Sixty-three cases were reclassified, mostly as well-differentiated neuroendocrine tumor (NET) or acinar cell carcinoma, and eliminated. The clinicopathologic features and survival of the remaining 44 poorly differentiated neuroendocrine carcinomas were further assessed. RESULTS: The mean patient age was 59 years (range, 21 to 82 y), and the male/female ratio was 1.4. Twenty-seven tumors were located in the head of the pancreas, 3 in the body, and 11 in the tail. The median tumor size was 4 cm (range, 2 to 18 cm). Twenty-seven tumors were large cell neuroendocrine carcinomas, and 17 were small cell carcinomas (mean mitotic rate, 37/10 and 51/10 HPF; mean Ki67 index, 66% and 75%, respectively). Eight tumors had combined components, mostly adenocarcinomas. In addition, 2 tumors had components of well-differentiated NET. Eighty-eight percent of the patients had nodal or distant metastatic disease at presentation, and an additional 7% developed metastases subsequently. Follow-up information was available for 43 patients; 33 died of disease, with a median survival of 11 months (range, 0 to 104 mo); 8 were alive with disease, with a median follow-up of 19.5 months (range, 0 to 71 mo). The 2- and 5-year survival rates were 22.5% and 16.1%, respectively. CONCLUSIONS: Poorly differentiated neuroendocrine carcinoma of the pancreas is a highly aggressive neoplasm, with frequent metastases and poor survival. Most patients die within less than a year. Most (61%) are large cell neuroendocrine carcinomas. Well-differentiated NET and acinar cell carcinoma are often misdiagnosed as poorly differentiated neuroendocrine carcinoma, emphasizing that diagnostic criteria need to be clearly followed to ensure accurate diagnosis.

The spectrum of Castleman's disease: mimics, radiologic pathologic correlation and role of imaging in patient management.

Castleman's disease (CD) is a rare benign lymphoid disorder with variable clinical course. The two principal histologic subtypes of CD are hyaline-vascular and plasma cell variants and the major clinicoradiological entities are unicentric and multicentric CD. Management of CD is tailored to clinicoradiologic subtype. In this review, we describe the CT, MR and PET/CT findings in Castleman's disease which can help suggest a diagnosis of CD as well as emphasize role of imaging in management of patients with CD.

EUS-FNA mutational analysis in differentiating autoimmune pancreatitis and pancreatic cancer.

BACKGROUND/AIMS: Autoimmune pancreatitis (AIP) may mimic pancreatic cancer (PC). The detection of DNA mutations in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) material may improve discrimination between AIP and PC and is the context for this study. METHODS: In a retrospective study, archived EUS-FNA material from patients with AIP and PC at two centers was analyzed for KRAS mutations and loss-of-heterozygosity analysis involving 18 microsatellite markers. KRAS status and the fractional allelic loss (number of affected microsatellites divided by informative ones) were compared for AIP and PC. RESULTS: Thirty-two patients with 33 samples were studied. There were 16 patients with AIP (17 samples) and 16 patients with PC. DNA amplification failed in 7 samples. Of 25 patients (26 samples), 14 had AIP (7 male, age 57 ± 17 years; mean ± SD) and 11 had PC (7 male, age 65 ± 14 years; mean ± SD). Cytology results for AIP were inflammatory = 3, inconclusive = 10, suspicious for malignancy = 2 and for PC were malignant = 5, suspicious for malignancy = 4 and inconclusive = 2, respectively. KRAS mutation was detected in none of the AIP cases and 10/11 PC cases (91%, Pearson χ(2) = 22.16, p < 0.001) or 10/16 PC cases (63%) accounting for PC cases with failed DNA amplification. Mean (±SD) fractional allelic loss for the AIP cases (0.16 ± 0.15) was not significantly different from the PC cases (0.26 ± 0.19). CONCLUSIONS: A KRAS mutation in EUS/FNA material from a pancreatic mass is associated with malignancy and may help discriminate from benign conditions such as AIP.

Survival after resection for invasive intraductal papillary mucinous neoplasm and for pancreatic adenocarcinoma: a multi-institutional comparison according to American Joint Committee on Cancer Stage.

BACKGROUND: Survival after resection for invasive intraductal papillary mucinous neoplasm (inv-IPMN) is superior to pancreatic ductal adenocarcinoma (PDAC). This difference may be explained by earlier presentation of inv-IPMN. We hypothesized that inv-IPMN has survival comparable with PDAC after resection when matched by stage. STUDY DESIGN: From 1999 to 2009, 113 patients underwent resection for inv-IPMN at 2 large academic institutions. These data were compared with 845 patients during the same period undergoing resection for PDAC. Demographics, pathology, and overall survival (OS) were compared according to current American Joint Committee on Cancer stage. RESULTS: Mean age with inv-IPMN and PDAC was 68 and 65 years, respectively. Follow-up was 33 and 24 months for inv-IPMN and PDAC, respectively. Median OS was 32 months for inv-IPMN and 17 months in PDAC (p < 0.001). Median OS in lymph node-negative inv-IPMN was 41 months and 24 months in PDAC (p = 0.003), with the greatest absolute difference in stage Ia patients with OS of 80 and 50 months in inv-IPMN and PDAC, respectively (p = 0.03). In node-positive patients, OS was 20 months in inv-IPMN and 15 months in PDAC (p = 0.06). Of inv-IPMN, 24% was colloid versus 75% of tubular subtype; 37(85%) of node-positive inv-IPMN were tubular subtype. Median OS was 23 and 127 months for tubular and colloid subtypes, respectively (p < 0.001). CONCLUSIONS: When matched by stage, inv-IPMN has superior survival after resection compared with PDAC. This disparity is greatest in node-negative and least in node-positive disease. These findings suggest the behaviors of inv-IPMN and PDAC, although different, converge with advancing American Joint Committee on Cancer stage because of a greater proportion of tubular subtype.

Clinicopathological and molecular characterization of nine cases of columnar cell variant of papillary thyroid carcinoma.

The majority of papillary thyroid carcinoma is indolent and associated with long-term survival. The columnar cell variant, however, is a rare subtype that is variable in biological behavior; some are clinically aggressive, whereas others are more clinically indolent. Tumor size, tumor circumscription, and encapsulation may influence the behavior of columnar cell carcinomas. Other variables including genetic changes and putative biomarkers associated with malignant growth have not been thoroughly examined in these neoplasms. In this study, nine cases of columnar cell variant of papillary thyroid carcinoma from three institutions were classified as clinically indolent or aggressive based on pathological features, clinical history, and outcome. Indolent tumors were typically small, circumscribed or encapsulated, and from younger female patients, whereas aggressive tumors were large, locally aggressive, associated with regional and distant metastasis, and from older male patients. The missense mutation, V600E in the BRAF oncogene (BRAF(V600E)), was detected in three of nine of cases, of which two were clinically aggressive. Immunohistochemical evaluation of neoplasia-associated markers showed increased nuclear cyclin D1 expression, elevated Ki-67 proliferation indices, and predominantly weak nuclear p53 staining in both indolent and aggressive tumors. Expression of ß-catenin was largely restricted to a membranous pattern in both tumor types. Cytoplasmic expression of bcl-2 was overall mildly reduced in indolent neoplasms. Nuclear expression of estrogen and progesterone receptors was increased in both indolent and aggressive neoplasms, but was without sex- or age-related differences; however, whereas progesterone receptor expression was diffuse and strong in clinically indolent carcinomas, its expression was diminished in aggressive neoplasms. Recognition of the clinicopathological characteristics and the molecular and immunophenotypic features of the columnar cell variant of papillary thyroid carcinoma may aid in characterizing neoplasms that behave indolently or aggressively.

Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers.

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.

Pioglitazone Attenuates Cystic Burden in the PCK Rodent Model of Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is a genetic disorder characterized by growth of fluid-filled cysts predominately in kidney tubules and liver bile ducts. Currently, the clinical management of PKD is limited to cyst aspiration, surgical resection or organ transplantation. Based on an observation that PPARγ agonists such as pioglitazone and rosiglitazone decrease mRNA levels of a Cl(-) transport protein, CFTR (cystic fibrosis transmembrane conductance regulator), and the Cl(-) secretory response to vasopressin in cultured renal cells, it is hypothesized that PPARγ agonists will inhibit cyst growth. The current studies show that a 7- or 14-week pioglitazone feeding regimen inhibits renal and hepatic bile duct cyst growth in the PCK rat, a rodent model orthologous to human PKD. These studies provide proof of concept for the mechanism of action of the PPARγ agonists and suggest that this class of drugs may be effective in controlling both renal and hepatic cyst growth and fibrosis in PKD.

A 61-year-old woman with osteomalacia and a thoracic spine lesion.

Phosphaturic mesenchymal tumor, mixed connective tissue variant (PMT-MCT) is a rare, largely benign, mesenchymal neoplasm almost invariably associated with oncogenic osteomalacia. It is generally found in the soft tissue and bone of the extremities. We report a case of a 61-year-old female with long-standing osteomalacia who was found to have PMT-MCT of the thoracic spine. There have been very few previously reported cases of PMT involving the spinal vertebrae and neuropathologists should be aware of this lesion. Recognition of PMT-MCT is critical for optimal patient care since complete surgical resection without additional therapy is curative.

Pathology of the liver in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis is a rare, rapidly progressive disorder characterized by an activation of the immune system resulting in a systemic proliferation of lymphocytes and histiocytes. The disease is genetically heterogeneous and maps to at least 4 loci including the gene encoding perforin, a protein critical for the cytotoxic and regulatory functions of T lymphocytes and natural killer (NK) cells. Hepatic dysfunction often occurs early in the clinical course, but the pathology of the liver is not well characterized. The clinical history, laboratory data, and pathologic material (25 hepatic specimens) from 19 children (11 boys, 7 girls, 1 unknown, 12 d to 11 mo of age, median 3 mo) with FHL were reviewed. Routine and immunohistochemical stains were carried out in all cases, and perforin gene sequencing in a subset. Common to all specimens was a portal and sinusoidal infiltrate of CD3, CD8, granzyme B+ lymphocytes admixed with CD68, CD1a- histiocytes that exhibited hemophagocytosis. There was endothelialitis of portal and central veins and lymphocyte-mediated bile duct injury. The degree of portal and sinusoidal lymphohistiocytic infiltrate and endothelialitis varied from mild to marked and correlated with clinical severity. In some specimens, histiocytic cells predominated and in others, there was extensive hepatocellular giant cell transformation. Accordingly, 4 histopathologic patterns were observed: (1) chronic hepatitis-like, (2) leukemia-like, (3) histiocytic storage disorder-like, and (4) neonatal giant cell hepatitis-like. Two siblings homozygous for a 50delT nucleotide deletion had no perforin immunoreactive cells, 1 compound heterozygote for a deletion and missense mutation had cells with markedly diminished perforin expression, and 1 infant hemizygous for a perforin missense mutation had intact expression. Recognizing the morphologic changes in the liver and the immunophenotypic features of the infiltrate are critical for a rapid diagnosis and a prompt institution of treatment.

Metastatic seminoma and grade 1 follicular lymphoma presenting concurrently in a supraclavicular lymph node: a case report.

An asymptomatic 67-year-old man presented with a left supraclavicular lymph node that enlarged over a 2-month period which was biopsied. Pathologic features were consistent with involvement by metastatic seminoma and follicular lymphoma, follicular pattern, grade 1 (of 3). Staging Positron Emission Tomography/Computed Tomography scans indicated several areas of enlarged lymph nodes. The patient completed chemotherapy with bleomycin, etoposide, and cisplatin chemotherapy. This is the first reported case of metastatic seminoma and follicular lymphoma occurring in the same lymph node. No obvious pathophysiologic link exists between these two malignancies and there are no shared common risk factors. Given the natural history of these two malignancies, if this patient develops recurrent lymphadenopathy, it will be difficult to identify whether the enlarged lymph nodes represent recurrent seminoma or follicular lymphoma without a biopsy of each pathologically enlarged node. Similarly, Fluorodeoxyglucose- Positron Emission Tomography is known to be active in both seminoma and follicular lymphoma, making this scan non-specific in this patient. Finally, this patient had no baseline elevation in any germ cell tumor marker. Thus, serum tumor markers cannot be relied upon as surrogates for response to chemotherapy or as identifiers of relapsed seminoma.

Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib.

BACKGROUND: Sunitinib, a multitargeted tyrosine-kinase inhibitor, which is approved by both US and European Commission regulatory agencies for clinical use, extends survival of patients with metastatic renal-cell carcinoma and gastrointestinal stromal tumours, but concerns have arisen about its cardiac safety. We therefore assessed the cardiovascular risk associated with sunitinib in patients with metastatic gastrointestinal stromal tumours. METHODS: We retrospectively reviewed all cardiovascular events in 75 patients with imatinib-resistant, metastatic, gastrointestinal stromal tumours who had been enrolled in a phase I/II trial investigating the efficacy of sunitinib. The composite cardiovascular endpoint was cardiac death, myocardial infarction, and congestive heart failure. We also examined sunitinib's effects on left ventricular ejection fraction (LVEF) and blood pressure. We investigated potential mechanisms of sunitinib-associated cardiac effects by studies in isolated rat cardiomyocytes and in mice. FINDINGS: Eight of 75 (11%) patients given repeating cycles of sunitinib in the phase I/II trial had a cardiovascular event, with congestive heart failure recorded in six of 75 (8%). Ten of 36 (28%) patients treated at the approved sunitinib dose had absolute LVEF reductions in ejection fraction (EF) of at least 10%, and seven of 36 (19%) had LVEF reductions of 15 EF% or more. Sunitinib induced increases in mean systolic and diastolic blood pressure, and 35 of 75 (47%) individuals developed hypertension (>150/100 mm Hg). Congestive heart failure and left ventricular dysfunction generally responded to sunitinib being withheld and institution of medical management. Sunitinib caused mitochondrial injury and cardiomyocyte apoptosis in mice and in cultured rat cardiomyocytes. INTERPRETATION: Left ventricular dysfunction might be due, in part, to direct cardiomyocyte toxicity, exacerbated by hypertension. Patients treated with sunitinib should be closely monitored for hypertension and LVEF reduction, especially those with a history of coronary artery disease or cardiac risk factors.

The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.

The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins.

ClC chloride channels are widely distributed in organisms across the evolutionary spectrum, and members of the mammalian family play crucial roles in cellular function and are mutated in several human diseases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Within the ClC-3, -4, -5 branch of the family that are intracellular channels, two alternatively spliced ClC-3 isoforms were recognized recently (Ogura, T., Furukawa, T., Toyozaki, T., Yamada, K., Zheng, Y. J., Katayama, Y., Nakaya, H., and Inagaki, N. (2002) FASEB J. 16, 863-865). ClC-3A resides in late endosomes where it serves as an anion shunt during acidification. We show here that the ClC-3B PDZ-binding isoform resides in the Golgi where it co-localizes with a small amount of the other known PDZ-binding chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator). Both channel proteins bind the Golgi PDZ protein, GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). Interestingly, however, when overexpressed, GOPC, which is thought to influence traffic in the endocytic/secretory pathway, causes a large reduction in the amounts of both channels, probably by leading them to the degradative end of this pathway. ClC-3B as well as CFTR also binds EBP50 (ERM-binding phosphoprotein 50) and PDZK1, which are concentrated at the plasma membrane. However, only PDZK1 was found to promote interaction between the two channels, perhaps because they were able to bind to two different PDZ domains in PDZK1. Thus while small portions of the populations of ClC-3B and CFTR may associate and co-localize, the bulk of the two populations reside in different organelles of cells where they are expressed heterologously or endogenously, and therefore their cellular functions are likely to be distinct and not primarily related.