Utilising magnetic resonance imaging as the gold-standard in management of suspected scaphoid fractures in the emergency department setting.

Scaphoid fractures are the most common carpal bone fracture. Up to 40% of scaphoid fractures can be missed at initial presentation and investigation. Follow-up plain film radiograph has overall poor sensitivity and reliability. MRI has been shown to have an almost 100% sensitivity and specificity and so is the gold standard in scaphoid fracture diagnosis. Additionally, early specialist involvement is recommended. We proposed that following a designated pathway, there would be no significant increase in MRI requests. Following implementation of a pathway for the management of suspected scaphoid fractures in St James's Hospital in 2012 re-auditing demonstrated that management changed to either MRI directly after initial x-ray (16/145, 11%), MRI after second x-ray (9/28, 32%) or orthopaedic follow-up (19/28, 68%). The number of MRIs requested was consistent with our predictors of demand. Thus, our new protocol maximises diagnostics, cost effectiveness and quality of patient care.

Disease models provide insights into post-golgi protein trafficking, localization and processing.

Over the past two years, the use of in vitro systems and the identification of autoantibodies to Golgi proteins have provided important new tools for analyzing vesicle and cargo trafficking in the distal secretory pathway. In addition, the phenotypic characterization of mice with knockouts of various prohormone convertases has led to significant progress in understanding the biological relevance of prohormone processing in post-Golgi compartments.

Günter Blobel--still passionate after all these years.

UNLABELLED: What one word best describes Günter Blobel? ANSWER: passion. Almost all of the nearly 200 investigators who have worked in the Blobel laboratory in the past quarter century would describe him as highly enthusiastic, intense and, above all, passionate. Whether it was in the early days of the signal hypothesis, the initial characterization of nuclear pores and lamina or his more recent foray into rebuilding the historic German City of Dresden, Günter attacks every project with unbridled passion, intensity, boundless energy and determination.

Retrovirus-mediated expression of preprosomatostatin: posttranslational processing, intracellular storage, and secretion in GH3 pituitary cells.

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, preproSRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone, which is located at the carboxyl terminus of the precursor, is preceded by a single pair of basic amino acids. We are studying preproSRIF to investigate intracellular sorting, proteolytic processing, and storage of peptide hormone precursors in the secretory pathway. We used a retroviral expression vector to achieve the high levels of precursor synthesis which are necessary for detailed characterization of processing intermediates and mature somatostatin. Recombinant retroviruses containing RNA transcripts encoding anglerfish preproSRIF I were used to infect rat pituitary GH3 cells which secrete growth hormone and prolactin, neither of which are substrates for endoproteolytic cleavage. In these cells preproSRIF was accurately processed to the mature hormone with an efficiency of approximately 75%. Of the newly synthesized mature SRIF, 55% was sorted into the regulated secretory pathway and released in response to the secretagogue 8-Br-cAMP. The remaining 45% of mature SRIF and residual unprocessed precursor was rapidly secreted. In contrast to SRIF, only 5% of newly synthesized endogenous growth hormone was stored intracellularly, whereas 95% was sorted to the constitutive pathway and secreted rapidly with kinetics identical to proSRIF. Our results show that proSRIF processing is not necessarily dependent on a specific protease found only in SRIF-producing cells and suggest that proteolytic cleavage is not restricted to cells that process endogenous hormones. Moreover, these results demonstrate that GH3 cells have the capacity to discriminate between endogenous and foreign hormones and target the foreign molecule significantly more efficiently to the regulated secretory pathway.

The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes.

To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells.

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP-binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.

The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin. Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP.

Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

Somatostatin discriminates between the intracellular pathways of secretory and membrane proteins.

Somatostatin is a14-amino acid peptide hormone that inhibits the secretion of a variety of other polypeptide hormones, including growth hormone. Here we describe an experimental system used to determine whether somatostatin can discriminate in its inhibition between secretory and plasma membrane proteins. Growth hormone-secreting cells (GH3) were infected with vesicular stomatitis virus and pulse-chased with [35S]methionine to follow the simultaneous intracellular transit of growth hormone and the viral membrane glycoprotein, G protein. Secretion of growth hormone was monitored by immunoprecipitation of chase media, while appearance of G protein on the plasma membrane was detected by cell surface labeling and virus purification. In the presence of somatostatin (10 micrograms/ml), the secretion of growth hormone was inhibited by 80%. In contrast, G protein appeared on the plasma membrane with slightly enhanced kinetics. When cells were treated with the ionophore monensin (0.2 microM), there was a dramatic inhibition of both the secretion of growth hormone and the incorporation of G protein into plasma membranes. Our results on the differential effect of somatostatin provide evidence for sorting of secretory and membrane proteins into distinct compartments in the secretory pathway. The data further suggest that this sorting event occurs late in the Golgi complex or after proteins exit from that organelle.

Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence.

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.

Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly.

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.

A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase.

Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.

Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Chromosomal location and evolutionary rate variation in enterobacterial genes.

The basal rate of DNA sequence evolution in enterobacteria, as seen in the extent of divergence between Escherichia coli and Salmonella typhimurium, varies greatly among genes, even when only "silent" sites are considered. The degree of divergence is clearly related to the level of gene expression, reflecting constraints on synonymous codon choice. However, where this constraint is weak, among genes not expressed at high levels, divergence is also related to the chromosomal location of the gene; it appears that genes furthest away from oriC, the origin of replication, have a mutation rate approximately two times that of genes near oriC.

Genetic polymorphisms in platelet-related proteins and coronary artery disease: investigation of candidate genes, including N-acetylgalactosaminyltransferase 4 (GALNT4) and sulphotransferase 1A1/2 (SULT1A1/2).

BACKGROUND: Both platelet function and heart disease show strong genetic components, many of which remain to be elucidated. MATERIALS AND METHODS: The roles of candidate polymorphisms in ten platelet-associated genes were compared between 1,237 Acute Coronary Syndrome (ACS) cases (with myocardial infarction and unstable angina) and 386 controls, from an Irish Caucasian population. Additionally, 361 stable angina patients were investigated. Two genes of interest were followed up in a separate Irish study of 1,484 individuals (577 with IHD and 907 unaffected). RESULTS: The GALNT4 (N-acetyl galactosaminyl transferase 4) 506I allele was significantly underrepresented in ACS (OR = 0.66, CI = 0.52-0.84; P = 0.001; P = 0.01 after correction for multiple testing), while the SULT1A1 (Sulphotransferase 1A1) 213H allele was associated with risk of ACS (OR = 1.37, CI = 1.08-1.74; P = 0.01; P = 0.1 after correction for multiple testing). Subsequent genotyping of further SNPs in GALNT4 in the family-based (IHD) group revealed that the 506I allele showed the same trend towards protecting against ACS but the haplotypic test over the four commonest haplotypes was not significant (P = 0.55). In contrast, the SULT1A1/SULT1A2 gene complex showed suggestive haplotypic association in the family-based study (P = 0.07), with the greatest increase in risk conferred by the SULT1A2 235T allele (P = 0.025). CONCLUSION: We have identified two risk genes for cardiovascular disease, one of whose (GALNT4) effects may be on either platelet or endothelial function through modifications of PSGL1 or other important glycosylated proteins. The role of sulphotransferases (SULT1A1/2) in cardiovascular disease requires further exploration. Further validation of cardiovascular risks conferred by both genes in other populations (including gene copy number variation) is warranted.

Sources of nitrogen dioxide (NO2) in New Zealand homes: findings from a community randomized controlled trial of heater substitutions.

UNLABELLED: Houses in New Zealand have inadequate space heating and a third of households use unflued gas heaters. As part of a large community intervention trial to improve space heating, we replaced ineffective heaters with more effective, non-polluting heaters. This paper assesses the contribution of heating and household factors to indoor NO2 in almost 350 homes and reports on the reduction in NO2 levels due to heater replacement. Homes using unflued gas heaters had more than three times the level of NO2 in living rooms [geometric mean ratio (GMR) = 3.35, 95% CI: 2.83-3.96, P < 0.001] than homes without unflued gas heaters, whereas homes using gas stove-tops had significantly elevated living room NO2 levels (GMR = 1.42, 95% CI: 1.05-1.93, P = 0.02). Homes with heat pumps, flued gas heating, or enclosed wood burners had significantly lower levels of NO2 in living areas and bedrooms. In homes that used unflued gas heaters as their main form of heating at baseline, the intervention was associated with a two-third (67%) reduction in NO2 levels in living rooms, when compared with homes that continued to use unflued gas heaters. Reducing the use of unflued gas heating would substantially lower NO2 exposure in New Zealand homes. PRACTICAL IMPLICATIONS: Understanding the factors influencing indoor NO2 levels is critical for the assessment and control of indoor air pollution. This study found that homes that used unflued gas combustion appliances for heating and cooking had higher NO2 levels compared with homes where other fuels were used. These findings require institutional incentives to increase the use of more effective, less polluting fuels, particularly in the home environment.

Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis.

BACKGROUND/INTRODUCTION: Sarcoidosis is a multi-systemic disorder of unknown etiology, characterized by the presence of non-caseating granulomas in target organs. In 90% of cases, there is thoracic involvement. Fifty to seventy percent of pulmonary sarcoidosis patients will experience acute, self-limiting disease. For the subgroup of patients who develop persistent disease, no targeted therapy is currently available. AIM: To investigate the potential of the single nucleotide polymorphism (SNP), Toll-like receptor 3 Leu412Phe (TLR3 L412F; rs3775291), as a causative factor in the development of and in disease persistence in pulmonary sarcoidosis. To investigate the functionality of TLR3 L412F in vitro in primary human lung fibroblasts from pulmonary sarcoidosis patients. DESIGN: SNP-genotyping and cellular assays, respectively, were used to investigate the role of TLR3 L412F in the development of persistent pulmonary sarcoidosis. METHODS: Cohorts of Irish sarcoidosis patients (n = 228), healthy Irish controls (n = 263) and a secondary cohort of American sarcoidosis patients (n = 123) were genotyped for TLR3 L412F. Additionally, the effect of TLR3 L412F in primary lung fibroblasts from pulmonary sarcoidosis patients was quantitated following TLR3 activation in the context of cytokine and type I interferon production, TLR3 expression and apoptotic- and fibroproliferative-responses. RESULTS: We report a significant association between TLR3 L412F and persistent clinical disease in two cohorts of Irish and American Caucasians with pulmonary sarcoidosis. Furthermore, activation of TLR3 in primary lung fibroblasts from 412 F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-ß and TLR3 expression, reduced apoptosis- and dysregulated fibroproliferative-responses compared with TLR3 wild-type patients. DISCUSSION/CONCLUSION: This study identifies defective TLR3 function as a previously unidentified factor in persistent clinical disease in pulmonary sarcoidosis and reveals TLR3 L412F as a candidate biomarker.

Stereotactic cortical resection in non-lesional extra-temporal partial epilepsy.

The presentation and treatment of a patient with extra-temporal non-lesional partial epilepsy is discussed herein. His clinical semiology was consistent with supplementary motor area seizures; however, MR imaging did not demonstrate a lesion. A region of stable cortical glucose hypermetabolism in the left frontal region was noted with 2-fluoro-2-deoxy-D-glucose (FDG)-PET. This was consistent with the frequent interictal discharges evident over the left fronto-temporal region and the stereotypic high amplitude ictal discharges arising with highest amplitude from the left frontal region. Epileptiform activity evident on an intracranial 64-point subdural recording grid placed over the left dorsolateral frontal cortex confirmed a distribution concordant with FDG-PET findings. The subsequent resection was guided by the PET and EEG findings rather than structural MR imaging, and a limited cortical resection led to an immediate and substantial reduction in seizure frequency.

Intracellular localization of phospholipase D1 in mammalian cells.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.