Contrast-enhanced intra-operative ultrasound as a clinical decision making tool during surgery for colorectal liver metastases: The ULIIS study.

BACKGROUND: Detecting more colorectal liver metastases (CRLMs) during surgery may help optimise strategy and improve outcomes. Our objective was to determine clinical utility (CU) of contrast-enhanced intra-operative ultrasound (CE-IOUS) using sulphur hexafluoride microbubbles during CRLM surgery. METHOD: A prospective phase II trial performed at two comprehensive cancer research centres. Patients operated for CRLMs were eligible and assessable if intra-operative ultrasound (IOUS) and CE-IOUS had been performed and pathological results were available and/or 3-month imaging. CU was defined as the justified change in planned surgical strategy or procedure using CE-IOUS. RESULTS: Out of the 68 patients enrolled, 54 were eligible and assessable. 43 patients underwent pre-operative chemotherapy. The median number of CRLMs was 2 (range, 1-11). Pre-operative staging was performed using MRI. IOUS allowed identification of 45 new CRLMs in 13 (24.7%) patients. Compared to IOUS, CE-IOUS allowed identification of 10 additional CRLMs in 9 (16.7%) patients. Surgery was altered and justified in 4 patients only, leading to a CU rate of 7.70% (95 CI, [3.2, 18.6]). No missing CRLMs were identified by CE-IOUS. CONCLUSIONS: Although the primary endpoint was not met for one protocol violation, secondary endpoints indicate that CE-IOUS has an intermediate added-value for surgeons treating CRLMs. TRIAL REGISTRATION: NCT01880554 (https://clinicaltrials.gov/).

Incidentally-discovered gallbladder cancer: When, why and which reoperation?

Cancer of the gallbladder, a rare entity with a poor prognosis, is often discovered incidentally during or after cholecystectomy. It tends to disseminate early via lymphatic, peritoneal, endobiliary, and hematogenous pathways. Diagnosis is made intra-operatively in only a quarter of cases, by examination of the opened cholecystectomy specimen in the operating room by the surgeon; this procedure should be routine. For incidentally-discovered cancers, survival was 28% at five years. Prognostic factors include age, TNM stage, gallbladder perforation during cholecystectomy and less-than-optimal resection at re-operation. Whether the laparoscopic route for the initial cholecystectomy has an impact on survival remains a subject of debate. R0 surgery is the only potentially curative treatment: simple cholecystectomy with clear margins is adequate resection for stage T1a tumors; extended cholecystectomy with lymphadenectomy and possibly resection of the bile duct is required for more advanced stages. After curative resection, neo-adjuvant or adjuvant chemotherapy and radiotherapy have not, so far, proven effective. Improvement of surgical practices (systematic review of cholecystectomy specimens in the OR, prevention of gallbladder perforation with bile spillage during surgery, early re-intervention for optimal resection) could improve the prognosis of these cancers.

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system.

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.

Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons.

The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses.

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases.

The unc-47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc-47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS-7 and PC12 cells induced an intracellular, glycine-sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.

The photoactivatable inhibitor 7-azido-8-iodoketanserin labels the N terminus of the vesicular monoamine transporter from bovine chromaffin granules.

In monoaminergic cells, the hormone or neurotransmitter is concentrated into secretory vesicles by a tetrabenazine- and reserpine-sensitive vesicular monoamine transporter (VMAT), catalyzing a H+/monoamine antiport. Ketanserin is another powerful inhibitor of VMAT that binds to the tetrabenazine binding site. A photoactivatable derivative, 7-azido-8-iodoketanserin (AZIK), labels covalently the transporter from bovine chromaffin granules, VMAT-2. Digestion with endoproteinases V8 or Lys-C, which cleave peptide bonds at acidic or lysine residues, respectively, revealed that the AZIK label is located in a 7 kDa segment of the VMAT-2 polypeptide. The photolabeled chromaffin granule transporter was purified by DEAE and WGA chromatography followed by selective aggregation and size-exclusion HPLC. After treatment by V8 or Lys-C, digestion products were separated by electrophoresis in SDS and sequenced. For both enzymes, the material comigrating with the labeled peptide produced a sequence matching the N terminus of VMAT-2. A K55E mutant of the bovine VMAT-2 cDNA was constructed and expressed in COS-7 cells. The mutant protein exhibited a full VMAT activity and could be labeled by AZIK. However, the formation of the 7 kDa labeled peptide upon Lys-C proteolysis was prevented in the mutant, with a redistribution of the label in higher-molecular mass digestion products. The localization of the label upstream of lysine 55 was confirmed by an immunological and enzymatic analysis. We conclude that the segment 2-55 of bovine VMAT-2, which encompasses the cytosolic N terminus and the first transmembrane segment in the current topological model of the transporter, contains residues involved in the binding of ketanserin and, possibly, tetrabenazine.

SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.

Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules.

Prior to secretion, monoamines (catecholamines, serotonin, histamine) are concentrated from the cytoplasm into vesicles by vesicular monoamine transporters (VMAT). These transporters also carry non-physiological compounds, e.g. the neurotoxin methyl-4-phenylpyridinium. VMAT acts as an electrogenic antiporter (exchanger) of protons and monoamines, using a proton electrochemical gradient. Vesicular transport is inhibited by specific ligands, including tetrabenazine, ketanserin and reserpine. The mechanism of transport and the biochemistry of VMAT have been analyzed with the help of these tools, using mainly the chromaffin granules from bovine adrenal glands as a source of transporter. Although biochemical studies did not suggest a multiplicity of VMATs, two homologous but distinct VMAT genes have recently been cloned from rat, bovine and human adrenal glands. The VMAT proteins are predicted to possess 12 transmembrane segments, with both extremities lying on the cytoplasmic side. They possess N-glycosylation sites in a putative luminal loop and phosphorylation sites in cytoplasmic domains. In rat, VMAT1 is expressed in the adrenal gland whereas VMAT2 is expressed in the brain. In contrast, we found that the bovine adrenal gland expressed both VMAT1 and VMAT2. VMAT2 corresponds to the major transporter of chromaffin granules, as shown by partial peptidic sequences of the purified protein and by a pharmacological analysis of the transport obtained in transfected COS cells (COS cells are monkey kidney cells possessing the ability to replicate SV-40-origin-containing plasmids). We discuss the possibility that VMAT1 may be specifically addressed to large secretory granules vesicles, whereas VMAT2 may also be addressed to small synaptic vesicles; species differences would then reflect the distinct physiological roles of the small synaptic vesicles in the adrenal gland.

Expression and regulation of the bovine vesicular monoamine transporter gene.

In monoaminergic cells, the neurotransmitter is accumulated into secretory or synaptic vesicles by a tetrabenazine- and reserpine-sensitive transporter, catalyzing an H+/monoamine antiport. The major vesicular monoamine transporter from bovine chromaffin cells was cloned, using sequences common to adrenal medulla and brain rat vesicular monoamine transporters. Its identity was confirmed by peptide sequences, determined from the purified protein. Surprisingly, the bovine adrenal medulla sequence, bVMAT2, is more related to the transporter from human and rat brain than to that from rat adrenal medulla. PCR amplification showed that bVMAT2 is expressed in both adrenal medulla and brain, in contrast with the situation reported in rats, where distinct genes appear to be expressed in brain (SVAT or MAT, now renamed rVMAT2) and in the adrenal medulla (CGAT, now renamed rVMAT1). In bovine chromaffin cells, long-term depolarization by KCl resulted in an increase in the level of bVMAT2 mRNA, in agreement with the previously observed increase in the transporter binding sites, suggesting that a coupling between stimulation, secretion and synthesis changes the composition of the secretory granule membrane.

Characterization and purification of the monoamine transporter of bovine chromaffin granules.

The monoamine transporter of the chromaffin granule membranes can be specifically labeled by the photoaffinity reagent 7-azido-8-[125I]iodoketanserin. The characteristics of the labeled protein have been investigated. Two-dimensional gel electrophoresis of the labeled membranes indicated a MW of about 70,000 and an isoelectric point ranging from 3.8 to 4.6. No clear protein spot was associated with the radioactive material, which migrated between glycoproteins GPII and GPIV. The diffuse aspect of the radioactive material indicated a heterogeneity, which was not modified after a second electrophoresis. This heterogeneity was, at least partially, due to glycosylation of the transporter; neuraminidase treatment increased the protein pI up to 6.3, whereas digestion with N-glycopeptidase markedly decreased the apparent MW, from 70,000 to 50,000. SDS-polyacrylamide gel electrophoresis showed that, at low acrylamide concentrations, the labeled material migrated more rapidly than predicted from the mobility of the markers of molecular weight, a behavior which indicated a marked hydrophobicity of the transporter. The labeled protein was purified to homogeneity by a combination of chromatography on DEAE-cellulose at pH 4.5, on immobilized wheat germ agglutinin, and on hydroxylapatite in the presence of SDS. During this purification, the specific radioactivity was increased by a factor of 300-500, with a yield of 10-20%.

Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-[125I]iodoketanserin.

An iodinated azido derivative of ketanserin, 7-azido-8-[125I]iodoketanserin ( [125I]AZIK), has been used to label the monoamine transporter of bovine chromaffin granule membranes by the technique of photoaffinity labeling. In the dark, this derivative was found to bind reversibly to the membranes, with an equilibrium dissociation constant estimated to be 6 nM at 0 degrees C. As for ketanserin, binding occurred at the tetrabenazine site: (i) [125I]AZIK was displaced efficiently from its binding site by tetrabenazine, ketanserin, and 7-azidoketanserin, whereas serotonin, which is a substrate for the transporter but has a low affinity for tetrabenazine binding site, was a poor displacer; pipamperone and pyrilamine, two antagonists of respectively serotonin S2 and histamine H1 receptors, were inactive. (ii) 7-Azidoketanserin was a competitive inhibitor of [3H]dihydrotetrabenazine binding, and it inhibited the ATP-dependent uptake of serotonin by chromaffin granule ghosts. Irradiation of [125I]AZIK with long-wavelength UV light, followed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and autoradiography, revealed irreversible labeling of a membrane component with an apparent molecular weight of 73,000. Tetrabenazine inhibited the labeling of this 73-kDa band in a manner parallel to the binding of [125I]AZIK in the dark. Such a labeling is totally compatible with previous results obtained through photolabeling with a tetrabenazine derivative or by target size analysis. Moreover, preliminary experiments showed that [125I]AZIK can label the tetrabenazine binding sites of various sources including rat striatum, rabbit platelets, human pheochromocytoma, and human adrenal medulla. Therefore, this molecule appears to be an excellent probe to label the monoamine transporter of different amine storage vesicles even without purification.

[Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla]. / Pharmacologie moléculaire du transporteur des catécholamines des granules chromaffines de la médullo-surrénale bovine.

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of the tetrabenazine binding sites of bovine chromaffin granule membranes.

An azido derivative of tetrabenazine, a specific inhibitor of the monoamine carrier of chromaffin granule membranes, has been synthesized. In the dark, this compound, 3H-labeled N-(3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo [a]quinolizin-2-yl)-4-[(4-azido-2-nitrophenyl)amino]butanamide+ ++ ([3H]TBA), bound reversibly to purified chromaffin granule membranes. Centrifugation through SP-Sephadex columns was used to separate bound and free [3H]TBA. This technique gave low levels of nonspecific binding and allowed recovery of [3H]TBA-membrane complexes. Scatchard analysis of the data indicated one class of sites with an equilibrium dissociation constant KD of 50 nM and a density of sites of 40-50 pmol/mg of protein, consistent with reported densities of reserpine and dihydrotetrabenazine binding sites. Competition experiments showed that TBA and tetrabenazine bound to the same site. Irradiation at 435 nm of [3H]TBA-membrane mixtures induced some irreversible binding of the probe to membranes. After irreversible binding of TBA, the number of dihydrotetrabenazine binding sites was decreased, indicating that the probe was covalently bound to the monoamine carrier. [3H]TBA-membrane complexes isolated by centrifugation through SP-Sephadex columns were irradiated, and their radioactivity was analyzed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. A polypeptide with a molecular weight of 70 000 was labeled. This polypeptide was different from dopamine beta-hydroxylase, and it was not adsorbed on concanavalin A-Sepharose. It is proposed that the monoamine carrier of chromaffin granule membrane has an oligomeric structure, involving a 45K subunit [Gabizon, R., Yetinson, T., & Schuldiner, S. (1982) J. Biol. Chem. 257, 15145] and a 70K subunit.

Characterization of the monoamine uptake system in catecholamine storage vesicles isolated from a pheochromocytoma taken from a child.

The catecholamine storage vesicles of a pheochromocytoma taken from a child have been isolated and characterized. The tumor contained almost exclusively noradrenaline and a large proportion of this amine was vesicle-bound. The noradrenaline-containing vesicles showed great resemblance to bovine chromaffin granules. Their catecholamine and dopamine beta-hydroxylase contents were that of chromaffin granules; their morphology and density were similar to those of the subpopulation of these granules that contain noradrenaline. The pheochromocytoma vesicles contained in their membranes an abundant polypeptide of mol. wt 110,000, which was not apparent in bovine adrenal medulla vesicle membranes. Monoamine uptake by pheochromocytoma noradrenaline vesicles did not differ significantly from that observed in bovine chromaffin granules. The time-course, plateau level and KM for noradrenaline were similar for both types of organelles. Both had an oligomycin-resistant ATPase with similar properties. Investigations using the tetrabenazine derivative [2-3H]dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,2,3,4,6, 7-hexahydro-11b-H-benzo[a]quinolizine), which binds specially to the bovine chromaffin granule monoamine carrier indicated that granule membranes from the tumor have a 10-fold increased number of [2-3H]dihydrotetrabenazine binding sites, with no change in dissociation constant. As in the case of bovine chromaffin granules, [2-3H]dihydrotetrabenazine can be totally displaced by noradrenaline and serotonin. To account for the discrepancy observed between the uptake data (which indicated no difference with bovine chromaffin granules) and the [2-3H]dihydrotetrabenazine binding studies (which showed a large excess of binding sites in the tumor membranes), we propose that granules in the investigated tumor contained a large amount of inactive monoamine carrier.

Solubilization and reconstitution of the adenosine 5'-triphosphate dependent noradrenaline uptake system of bovine chromaffin granule membrane.

The ATP-dependent catecholamine uptake system of chromaffin granule membrane has been solubilized and reconstituted in phospholipidic vesicles. The activity of the vesicles implies that both the ATP-dependent H+-translocase and the noradrenaline carrier have been successfully reconstituted. The membrane was solubilized by sodium cholate in presence of asolectin and the asolectin to cholate ratio appeared to be critical. Omission of asolectin resulted in reconstitution of vesicles with an active H+-pump and an inactive transport system. The detergent was removed from the solubilized membranes by filtration on Sephadex G-50 and it has been verified that the residual detergent of the reconstituted preparation was below the concentration inhibitory to the ghost H+-pump. The pH-dependence, Km for ATP and Km for noradrenaline of the reconstituted vesicles were similar to those of the ghosts, but their specific activity and reserpine-resistance were somehow variable. Vesicle activity was limited by transporter reconstitution, thus suggesting that reconstitution of the complete system might be used as an assay for the transporter. The noradrenaline carrier is not physically linked to dopamine beta-hydroxylase and bears no wheat germ agglutinin binding sites.