An atypical proprotein convertase in Giardia lamblia differentiation.

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles.

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.

Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway.

To survive in the environment and infect a new host, Giardia lamblia secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid (t1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

A signal recognition particle receptor gene from the early-diverging eukaryote, Giardia lamblia.

The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes. To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor. SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER. Sequence and phylogenetic analyses showed that SRalpha from G. lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs. Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane. While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions. Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop. The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon. RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation. Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G. lamlia. Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote. Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.

Giardia lamblia: evidence for carrier-mediated uptake and release of conjugated bile acids.

Giardia lamblia trophozoites colonize the human small intestine, where they are exposed to high concentrations of conjugated bile acids. Previous work has shown that bile acids enhance trophozoite survival, multiplication, and differentiation into the cyst stage. Therefore, experiments were performed to test whether carrier-mediated uptake of conjugated bile acids is present in this primitive parasite. Uptake of both cholyltaurine (C-tau) and cholylglycine (C-gly) was increased manyfold after culturing trophozoites in medium lacking bile acids. Absence of uptake at 4 degrees C and inhibition by other conjugated bile acids provided additional evidence for carrier-mediated uptake. Uptake of C-tau was greater than that of C-gly under all experimental conditions and appeared to be mediated by a different carrier. The major evidence for different carriers is that C-tau uptake was Na(+)-dependent, while C-gly uptake was not. In addition, C-tau uptake was more strongly inhibited by DTNB and several organic anions than C-gly uptake. Radiolabeled C-tau and C-gly were each released rapidly from trophozoites at 37 degrees C but not at 4 degrees C, suggesting that release of conjugated bile acids was also carrier-mediated. These findings are consistent with the notion that multiple transporters for conjugated bile acids are present in a lower eukaryote. We speculate that intracellular bile acids may facilitate lipid trafficking and membrane biosynthesis.

Cell biology of the primitive eukaryote Giardia lamblia.

Giardia lamblia is an extremely primitive or early-diverging eukaryote that has been considered to have no typical ER or Golgi apparatus, although it is a complex and highly developed cell. Both the trophozoite and cyst have unusual surface proteins that enable these stages to survive in very different and hostile environments. We found that G. lamblia forms novel encystation-specific secretory vesicles and can sort cyst wall proteins to a regulated secretory pathway distinct from the constitutive pathway used to transport the variable cysteine-rich protein to the trophozoite surface. Our studies, utilizing novel ultrastructural methods that preserve the endomembranes, as well as IEM, support the idea that G. lamblia has many of the endomembrane protein transport elements and sorting functions of higher cells and that these appeared very early in the evolution of eukaryotic cells.

A lipoprotein-cholesterol-albumin serum substitute stimulates Giardia lamblia encystation vesicle formation.

We found previously that the A6 clone of Giardia lamblia strain WB that did not encyst in vitro was blocked at an early stage in differentiation, as it did not form encystation secretory vesicles (ESV) efficiently or express cyst antigens, in comparison with the related clone C6. We now report that A6 formed ESV normally in the suckling mouse model. Therefore, we asked whether our serum-containing encystation media might lack a stimulus or component or contain an inhibitor of ESV formation to which A6 was especially sensitive. We found that replacing bovine serum with a lipoprotein-cholesterol solution and bovine serum albumin (LPC) in pre-encystation and encystation media increased ESV formation by both A6 and C6. The % of A6 cells with ESV increased from 8% in BS medium to 48% in LPC medium, compared with 64% and 98% for C6. Similarly, the average number of ESV/positive cell increased from 1.5 in BS medium to 7.7 in LPC medium for A6, and from 13.3 to 19.7 for C6. Moreover, in LPC encystation media, A6 expressed the cyst wall epitope recognized by monoclonal GCSA-1. Although formation of water-resistant cysts by A6 was increased > 60 fold in LPC media, the numbers of cysts remained only approximately 3-15% that of C6. This suggests that LPC may primarily affect early events in encystation and that A6 may require additional factors later in encystation.

Giardia lamblia: absence of cyst antigens and reduced secretory vesicle formation and bile salt uptake in an encystation-deficient subline.

Encystation of Giardia lamblia entails the appearance of a number of new antigens, as well as formation of a novel class of large encystation-specific secretory vesicles (ESV) that transport stage-specific proteins to the nascent cyst wall. The monoclonal antibody GCSA-1, which was raised against purified cyst walls, recognizes protein species of approximately 26-46 kDa that are regulated by exposure to bile (plus lactic acid) and alkaline pH, the factors that induce encystation. The GCSA-1 epitope is maximally expressed after approximately 14 hr of encystation and localizes to the interior, but not the membrane of the ESV as shown by frozen section immunoelectron microscopy. To further understand the process of encystation, we compared two sublines of strain WB that differ in their ability to encyst in vitro. Water-resistant cysts were not detected in subline A6 under conditions in which subline C6 formed approximately 2 x 10(5) cysts/ml. Moreover, subline A6 did not form ESV efficiently or detectably express antigens recognized by mAb GCSA-1 or by polyclonal anti-cyst sera. Finally, uptake of the bile salt taurocholate by A6 was reduced 4- to 20-fold, compared with that of C6, although transport by both strains was sodium-dependent and regulated by bile salt starvation. The decrease in bile salt uptake by A6 may be related to its defect in encystation.

The evaluation and management of known or suspected stones of the common bile duct in the era of minimal access surgery.

Laparoscopic cholecystectomy (LC) has rapidly become the procedure of choice for symptomatic cholelithiasis. The perioperative diagnosis and modern treatment of suspected stones of the common bile duct (CBD) remains controversial. A database of 573 patients undergoing LC was compiled during an 11 month period. Sixty-seven patients (47 females, 20 males) (13 percent) were suspected of having CBD stones based upon clinical, laboratory and roentgenographic evidence. Fifty-two patients underwent endoscopic retrograde cholangiopancreatography (ER-CP) (45 preoperative and seven postoperative). Seventeen patients were studied with intraoperative cholangiogram (IOC). The indications for cholangiography (either ERCP or IOC) included pancreatitis (group 1), clinical history suggestive of CBD stones, but normal preoperative liver function tests (LFT) (group 2), cholangitis (group 3), grossly abnormal LFT (group 4) and dilated CBD (greater than 7 millimeters on sonogram) (group 5). The incidence of CBD stones was three of 14 (21 percent) in group 1, six of 20 (30 percent) in group 2, two of three (67 percent) in group 3, 16 of 26 (62 percent) in group 4 and two of four (50 percent) in group 5. Overall, 29 patients (23 females and six males) had stones retrieved from the CBD. Of the 52 ERCP, 20 endoscopic sphincterotomies were performed for documented CBD stones. Of the group that had pre-LC ERCP, three (6 percent) ultimately required an open procedure. There were three instances of post-ERCP pancreatitis (6 percent) and ERCP was not able to opacify or clear the CBD in four instances. Seven patients had postoperative ERCP with successful retrieval of retained CBD stones (100 percent). Of the 17 IOC, eight were positive--two patients underwent laparoscopic clearance of the CBD and six required conversion to an open procedure. There were no deaths or extensive complications. Total and direct bilirubin, alkaline phosphatase and serum glutamic pyruvic transaminase were independently related to the presence of a CBD stone, while demographic data, past medical history, preoperative symptoms, leukocyte count, vital signs, amylase, serum glutamic-oxalacetic transaminase nuclear scintigraphic visualization of the duodenum or size of CBD on sonography, were not. No patient with biliary pancreatitis had CBD stones without abnormalities in the LFT or the preoperative sonogram. ERCP is a useful technique to clear the CBD pre-LC. However, ERCP in patients with biliary pancreatitis, but otherwise normal preoperative tests, has a low yield. In this group of patients, IOC is an appropriate alternative to pre-LC ERCP.

Encystation of Giardia lamblia leads to expression of antigens recognized by antibodies against conserved heat shock proteins.

During in vitro encystation, Giardia lamblia expresses several stage-specific proteins which are recognized in immunoblots by antisera raised against antigens from three different pathogens. The antigens belong to two different families of conserved stress proteins: (i) HSP60 purified from Legionella pneumophila and recombinant HSP60 from Mycobacterium bovis BCG and (ii) recombinant HSP70 from Plasmodium falciparum.

Human secretory and serum antibodies recognize environmentally induced antigens of Giardia lamblia.

The variability in duration and severity of infection with Giardia lamblia is likely to be due to trophozoite interactions with immune and nonimmune components of the small intestinal milieu. Despite its potential importance, nothing is known of the isotype or the specificity of the secretory antibody response to G. lamblia. In the present study, we show that serum and secretory antibodies recognize many Giardia antigens whose expression is induced by exposure to selected intestinal conditions. Isotype-specific immunoblots of antigens from trophozoites grown at pH 7.0 without bile or at the intestinal pH of 7.8 with bile were reacted with milk or serum antibodies from subjects with or without histories of giardiasis. While the results were complex, several key observations emerged. Serum and secretory immunoglobulin A (IgA), IgM, and IgG antibodies reacted with many regulated antigens. Antigen recognition patterns varied with isotype and between milk and serum antibodies of the same isotype. Antigen recognition also differed among subjects. Antibodies from virtually every patient recognized some G. lamblia antigens. Furthermore, milk and/or serum samples from putative controls without histories of giardiasis were positive more frequently than would be predicted from published prevalence studies, suggesting either that these antibodies may be cross-reactive or that undiagnosed infections with G. lamblia may be more common than previously thought. Thus, recognition of neoantigens induced by host conditions may be due to conserved or cross-reactive epitopes which could constitute a form of immune evasion by G. lamblia.

Postoperative peritoneal blood salvage with autotransfusion after hepatic trauma.

Postoperative peritoneal blood salvage with autotransfusion is technically feasible and may serve as an adjunctive measure in the management of severely injured patients with hepatic trauma and nonmechanical hemorrhage. Its effectiveness and ultimate place in the care of patients with trauma will be determined only after continued study.

A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor.

Since Giardia lamblia trophozoites are exposed to high concentrations of fatty acids in their human small intestinal milieu, we determined the pattern of incorporation of [3H]palmitic acid and myristic acid into G. lamblia proteins. The pattern of fatty acylation was unusually simple since greater than 90% of the Giardia protein biosynthetically labeled with either [3H]palmitate or myristate migrated at approximately 49 kDa (GP49) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis during both growth and differentiation. GP49, which partitions into the Triton X-114 detergent phase, is localized on the cell surface since it is 125I-surface-labeled. GP49 was also biosynthetically labeled with [14C]ethanolamine and [3H]myoinositol, suggesting that it has a glycosylphosphatidylinositol (GPI) anchor. Moreover, phospholipase A2 (PLA2) or mild alkaline treatment released free fatty acids, indicating a diacylglycerol moiety with ester linkages. Finally, a 3H- and 14C-labeled species was released by nitrous acid deamination from [14C]palmitate- and [3H]myoinositol-labeled GP49. The GPI anchor of GP49 is unusual, however, because purified GP49 was cleaved by Bacillus cereus phosphatidylinositol (PI)-specific PLC, but not by Staphylococcus aureus PI-PLC, or plasma PLD, and did not react with antibody against the variant surface glycoprotein cross-reactive determinant. Moreover, the double-labeled deaminated GP49 anchor migrated faster than authentic PI in TLC and produced [3H]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich G. lamblia surface antigens described previously, GP49 was identified in Western blots of every isolate tested, as well as in subclones of a single isolate which differ in expression of a major cysteine-rich 85/66-kDa surface antigen, which does not appear to be GPI-anchored. These observations suggest that GP49, the first common surface antigen to be described in G. lamblia, may play an important role in the interaction of this parasite with its environment.

Laparoscopic Stamm gastrostomy with gastropexy.

Recent technologic advances in video equipment and instruments have enabled therapeutic gastrointestinal operations to be performed laparoscopically. Using these advances and laparoscopic techniques, we have performed a Stamm gastrostomy with gastropexy by using the laparoscope in a single patient. The technique, indication, and considerations are discussed herein.

Giardia lamblia: regulation of secretory vesicle formation and loss of ability to reattach during encystation in vitro.

Encystation of Giardia lamblia is required for survival outside the host, as well as for initiation of new infections. Previously, we induced cultured G. lamblia trophozoites to encyst in vitro for the first time. During encystation, we observed the appearance of a new class of large secretory vesicle (encystation-specific vesicle; ESV) within which cyst antigens are concentrated and transported to the nascent wall. The present kinetic and physiologic studies now show that ESV are the earliest morphologic change observed in encystation. Expression of ESV, as well as subsequent encystation, are regulated by exposure to bile at the slightly alkaline pH which is typical of the human intestinal tract. ESV formation appears to be less stringently regulated than formation of water-resistant cysts because omission of either encystation stimuli or alkaline pH preferentially inhibits encystation. Since cysts do not attach, we asked when in encystation this physiologic transition occurs. We found that most encysting trophozoites remain attached until they begin to round up (greater than 24 hr). However, if they are made to detach, as early as 12 hr in encystation, well before they round up, they are defective in the ability to reattach. If trophozoites also become less able to reattach to the intestinal epithelium early in encystation in vivo, this would increase their exposure to lumenal encystation stimuli and promote encystation. These studies have provided new insights into the complex sequence of morphologic and physiologic alterations which occur during encystation of G. lamblia in vitro and their regulation by host intestinal factors.

Simple technique for long term central venous access in the patient with thrombocytopenic carcinoma.

A technique for the insertion of a central venous access device in the patient with thrombocytopenia is described. Using the Seldinger technique, a wire is placed into the internal jugular vein. A catheter tunneled from the anterior part of the chest is inserted through a peel-away sheath into the central venous system. The incision is then closed.

Organelles of protein transport in Giardia lamblia.

Giardia occupies a unique evolutionary position since it is considered to belong to the earliest known lineage to diverge from the eukaryotic line of descent. Although organelles of protein transport are thought to have evolved with the nuclear membrane, G. lamblia is reported to have no Golgi apparatus. Therefore, Frances Gillin, David Reiner and Michael McCaffery have investigated how it exports glycoproteins to the cyst wall during encystation and whether a Golgi might become evident during an active secretory phase. They have found both functional and morphological evidence of a Golgi in Giardia and have shown that trophozoites are capable of sophisticated protein recognition, sorting and trafficking. These studies suggest that membranous organelles of protein transport appeared early in the evolution of the eukaryotic cell.