(18)F-fluorodeoxyglucose positron-emission tomography-computed tomography to diagnose recurrent cancer.

BACKGROUND: Sometimes the diagnosis of recurrent cancer in patients with a previous malignancy can be challenging. This prospective cohort study assessed the clinical utility of (18)F-fluorodeoxyglucose positron-emission tomography-computed tomography ((18)F-FDG PET-CT) in the diagnosis of clinically suspected recurrence of cancer. METHODS: Patients were eligible if cancer recurrence (non-small-cell lung (NSCL), breast, head and neck, ovarian, oesophageal, Hodgkin's or non-Hodgkin's lymphoma) was suspected clinically, and if conventional imaging was non-diagnostic. Clinicians were asked to indicate their management plan before and after (18)F-FDG PET-CT scanning. The primary outcome was change in planned management after (18)F-FDG PET-CT. RESULTS: Between April 2009 and June 2011, 101 patients (age, median 65 years; 55% female) were enroled from four cancer centres in Ontario, Canada. Distribution by primary tumour type was: NSCL (55%), breast (19%), ovarian (10%), oesophageal (6%), lymphoma (6%), and head and neck (4%). Of the 99 subjects who underwent (18)F-FDG PET-CT, planned management changed after (18)F-FDG PET-CT in 52 subjects (53%, 95% confidence interval (CI), 42-63%); a major change in plan from no treatment to treatment was observed in 38 subjects (38%, 95% CI, 29-49%), and was typically associated with (18)F-FDG PET-CT findings that were positive for recurrent cancer (37 subjects). After 3 months, the stated post-(18)F-FDG PET-CT management plan was actually completed in 88 subjects (89%, 95% CI, 81-94%). CONCLUSION: In patients with suspected cancer recurrence and conventional imaging that is non-diagnostic, (18)F-FDG PET-CT often provides new information that leads to important changes in patient management.

A signal peptide that directs non-Sec transport in bacteria also directs efficient and exclusive transport on the thylakoid Delta pH pathway.

Signal peptides that specifically direct precursor proteins to the thylakoid Delta pH pathway possess an N domain RR motif. Signal peptides that direct transport of bacterial proteins across a non-Sec export pathway possess an N domain RRXFLK consensus motif. Recent genetic studies suggest an evolutionary link between these two protein translocation pathways. To further explore this relationship, we examined the thylakoid targeting capability of the signal peptide for Escherichia coli hydrogenase 1 small subunit (HyaA) by linking it to plastocyanin and assaying the chimeric protein in an in vitro thylakoid transport assay. The chimeric precursor was transported across thylakoids with high efficiency. Transport was characteristic of the Delta pH but not the Sec pathway, i.e. it was eliminated by ionophores that dissipate the DeltapH but occurred in the absence of stromal extract or ATP. This result was confirmed by competition with chemical quantities of a Delta pH pathway precursor. This indicates that the HyaA signal peptide has the necessary elements for efficient and exclusive targeting to the Delta pH pathway and further supports the notion that the alternate targeting pathways in prokaryotes and plant thylakoids are analogous.

Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway.

The thylakoid Delta pH pathway is a protein transport system with unprecedented characteristics. To investigate its mechanism, the topology of precursor insertion was determined. A fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17 (a Delta pH pathway precursor) was efficiently processed by thylakoid membranes. The amino terminus, including the targeting peptide, remained on the cis side of the membrane. Mature OE17 was transported to the lumen. These experiments demonstrate that Delta pH directed precursors enter the thylakoid membrane in a loop, implying that the Delta pH pathway has evolved from an export-type protein translocation system.

Complete sequence of the citrus tristeza virus RNA genome.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting.

The SecA protein is an essential, azide-sensitive component of the bacterial protein translocation machinery. A SecA protein homolog (CPSecA) now identified in pea chloroplasts was purified to homogeneity. CPSecA supported protein transport into thylakoids, the chloroplast internal membrane network, in an azide-sensitive fashion. Only one of three pathways for protein transport into thylakoids uses the CPSecA mechanism. The use of a bacteria-homologous mechanism in intrachloroplast protein transport provides evidence for conservative sorting of proteins within chloroplasts.

An Arabidopsis chloroplast RNA-binding protein gene encodes multiple mRNAs with different 5' ends.

An Arabidopsis cDNA (Atrbp33) encoding a nuclear-encoded chloroplast RNA-binding protein (RBP) has been isolated (A.J. DeLisle [1993] Plant Physiol 102: 313-314). ATRBP33 shares global structural homology with all known chloroplast RBPs: a chloroplast transit peptide in the amino terminus, followed by a unique acidic domain and a tandem pair of ribonucleoprotein consensus sequence-type RNA-binding domains in the carboxyl end. In vitro translation products of Atrbp33 were found to be imported into chloroplasts, suggesting that ATRBP33 is localized in chloroplasts. The expression of Atrbp33 was higher in chloroplast-containing organs than in nonchloroplast-containing organs. Furthermore, Atrbp33 was expressed in a light-dependent manner. These features are consistent with its postulated role in posttranscriptional control of chloroplast genes. Northern analyses and RNase protection assays showed that as many as nine messages are encoded by the single Atrbp33 gene. Sequence analysis of the cDNAs indicated that some of the transcripts have truncated 5' ends. Most interestingly, the multiple mRNAs potentially encode different polypeptides, one of which lacks a chloroplast transit peptide and acidic domain and contains only one intact RNA-binding domain. Unlike the chloroplast-localized ATRBP33, the truncated polypeptide may function in other cellular compartments.

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity.

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex (OE33) are two of several thylakoid lumen-located proteins that are made in the cytosol, imported into chloroplasts, and subsequently transported into thylakoids. Recently, competition studies showed that there are two pathways for protein transport into the thylakoid lumen and that plastocyanin and OE33 are on the same pathway (Cline, K., Henry, R., Li, C., and Yuan, J. (1993) EMBO J. 12, 4105-4114). Our expectation is that transport requirements reflect the steps of the process and that proteins on the same pathway share similar requirements. Unfortunately, the transport requirements for plastocyanin and OE33 are not well established. Here, we investigated transport in a reconstituted system with isolated thylakoids. Efficient transport of OE33 and plastocyanin was only obtained when stromal extract was included in the assay. Heat or protease treatment of stromal extract eliminated its ability to stimulate transport. Transport was abolished by treatments designed to deplete ATP or to prevent its formation and was greatly reduced in the presence of ionophores that dissipate the trans-thylakoidal proton gradient. These results show that transport of OE33 and plastocyanin requires ATP and is stimulated by stromal protein(s) and the trans-thylakoidal proton gradient. Taken together, these and previous results suggest that there are two mechanistically distinct pathways for protein transport into the thylakoid lumen.

Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport.

Nuclear encoded thylakoid lumen proteins are imported into the chloroplast storma and further directed across thylakoid membranes by lumen targeting domains. Recently, we showed that there are two protein-specific pathways for transport into the lumen. This was unexpected in that lumen targeting domains have similar properties, all containing bacterial signal peptide motifs. Nevertheless, sequence homology analysis suggests that pathway specificity is determined by elements in the lumen targeting domain. To test this, we constructed and analyzed chimeric proteins in which transit peptides from proteins transported by one pathway were fused to the mature domains of proteins directed by the other. We also investigated the transport characteristics of a previously unexamined protein whose pathway was predicted by sequence similarity analysis. Our results confirm that lumen targeting domains contain pathway sorting elements and further indicate that distinct energy and stroma requirements for transport are pathway characteristics, unrelated to the passenger protein. These findings suggest the operation of two mechanistically different translocators.

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.

Early events in the import/assembly pathway of an integral thylakoid protein.

The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.

An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited.

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates.

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.

Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts.

Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.

Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts.

Several proteases, i.e., pronase, a mixture of trypsin and chymotrypsin, and thermolysin were screened as potential surface probes of isolated intact pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Of these, only thermolysin met the criteria of a suitable probe. Thermolysin destroyed outer envelope polypeptides, but did not affect inner envelope polypeptides, envelope permeability properties or such chloroplast activities as metabolite transport and O(2) evolution.

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins.

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.

Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins.

Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides.

Separation and characterization of inner and outer envelope membranes of pea chloroplasts.

A procedure for purifying the chloroplast envelope subfractionates it into two membrane fractions of comparable quantities. This procedure differs from previous ones in that the chloroplasts are ruptured by freezing and thawing in hypertonic medium rather than by osmotic shock. The two membrane fractions have qualitatively similar polar lipid compositions but differ in their content of individual lipids, specifically monogalactosyldiacylglycerol and phosphatidylcholine. The two fractions also differ in their constituent polypeptides and in their appearance when examined by electron microscopy. The light (density = 1.08 g/ml) and heavy (density = 1.13 g/ml) membrane fractions have been tentatively identified as the outer and inner envelope membranes, respectively.