Requirement of the Lec35 gene for all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals.

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-beta-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

The effects of circulating interleukin-8 and adhesion molecules on pulmonary dysfunction in pediatric orthotopic liver transplantation.

We investigated the effects of circulating inflammatory cytokines and adhesion molecules induced by ortho-topic liver transplantation (OLT) on pulmonary function. Although the plasma interleukin-8 (IL-8) levels increased gradually, peaking at the end of the operation, these increases were considered minimal. The baseline endothelial adhesion molecule (E-selectin) level was several times higher than the normal value, but after reperfusion of the new transplanted liver, the plasma E-selectin concentrations decreased to within the normal range and remained almost normal during the postoperative period. Similar changes were observed in the plasma levels of other types of adhesion molecules. Although PaO(2)/FIO(2) showed a significant inversed correlation with the peak IL-8 concentration, after the exclusion of two patients, one of whom died and one of whom rejected the transplanted liver, no correlation was able to be found between the PaO(2)/FIO(2) ratio and the maximum IL-8 concentration. Furthermore, there was no correlation between the adhesion moleclues and PaO(2)/FIO(2). These results suggest that IL-8 exerts only a slight effect on respiratory function following successful pediatric liver transplantation, and that circulating adhesion molecules do not affect perioperative lung function.

Expression cloning of a novel suppressor of the Lec15 and Lec35 glycosylation mutations of Chinese hamster ovary cells.

Lec15 and Lec35 are recessive Chinese hamster ovary (CHO) cell glycosylation mutations characterized by inefficient synthesis and utilization, respectively, of mannose-P-dolichol (MPD). Consequently, Lec15 and Lec35 cells accumulate Man5GlcNAc2-P-P-dolichol and glucosaminyl-acylphosphatidylinositol. This report describes the cloning of a suppressor (termed SL15) of the Lec15 and Lec35 mutations from a CHO cDNA library by functional expression in Lec15 cells, employing phytohemagglutinin/swainsonine selection. The SL15 protein has a predicted molecular weight of 26,693 with two potential membrane spanning regions and a likely C-terminal endoplasmic reticulum retention signal (Lys-Lys-Glu-Gln). Lec15 cells transfected with SL15 have normal levels of MPD synthase activity in vitro and convert Man5GlcNAc2-P-P-dolichol to Glc0-3Man9GlcNAc2-P-P-dolichol in vivo. Surprisingly, SL15 also corrects the defective mannosylation in Lec35 cells. The SL15 protein bears no apparent similarity to Saccharomyces cerevisiae MPD synthase (the DPM1 protein), but is highly similar to the hypothetical F38E1.9 protein encoded on Caenorhabditis elegans chromosome 5. These results indicate a novel function for the SL15 protein and suggest that MPD synthesis is more complex than previously suspected.

The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins.

Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Glc0-3Man9GlcNAc2 oligosaccharides resulted in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.

Orthotopic liver retransplantation in rats.

A surgical experience with a method of rate orthotopic liver retransplantation (OLRT), and a preliminary study of immunological responses after OLRT are reported. OLRT was performed on the same recipient after the fist orthotopic liver transplantation (1st-OLT) according to our original (Kamada's) cuff method. Replacement of the portal vein (PV) and infra-hepatic vena cava (IHVC) cuffs was not technically difficult. However, there were no survivors from the first 6 retransplanted rats, mainly due to complications from defective supra-hepatic vena cava (SHVC) anastomoses. Unlike the human intra-abdominal SHVC, the posterior wall of the intra-abdominal SHVC in rats is too short and fragile to perform an end-to-end anastomosis twice between donor and recipient SHVC. For a second group of seven retransplants, a modification of the SHVC anastomosis was made between donor and recipient SHVC in conjunction with the recipient's cuff diaphragm. This enabled reanastomosis to be secure, resulting in the improved 1-week survival after isogenic OLRT (85.7%). This OLRT model has been applied to the fully allogeneic combination for several immunological studies and led to novel findings. Thus, an experimental model of a rat orthotopic liver retransplant model has the potential to allow more valuable insights into the immunological study of chronic rejection, sensitization and chimerism following liver retransplantation.

Dynamic liver scanning in cirrhosis.

Dynamic hepatic scintigraphy was performed in 49 patients with established cirrhosis, using intravenous 99Tcm-pertechnetate and 99Tcm-sulphur colloid in a prospective study of its predictive value. There was a close correlation between the hepatic perfusion index (reflecting the ratio of arterial to total hepatic blood flow) obtained with pertechnetate (HPI-P) and with sulphur colloid (HPI-C) (r = 0.775; p less than 0.0001), and both indices correlated with disease severity (HPI-P p less than 0.0001; HPI-C p less than 0.01). HPI-P was significantly increased in patients who died, in patients with varices and in those with hepatic encephalopathy. HPI-C was significantly increased in patients with varices, in patients with hepatic encephalopathy and in those who had bled from varices. Neither HPI-P nor HPI-C was able accurately to predict the development of complications during the follow-up period. The trapping index (TI), reflecting a combination of hepatic extraction efficiency, degree of intrahepatic shunting and extrahepatic extraction of colloid, was significantly impaired in patients who died and in those with ascites, varices and/or variceal bleeding, but not in patients with hepatic encephalopathy. The trapping index correlated with disease severity, as did the computer-derived spleen-liver ratio (S-L ratio). Neither TI nor S-L ratio was able to predict the development of complications. The clearance rate constant of colloid from peripheral blood, the uptake rate constants for liver and spleen, and splenic volume were all found to be unhelpful as indicators of disease severity or as predictors of complications. While perfusion indices derived by dynamic hepatic scintigraphy reflect the severity of the underlying liver disease, their determination on a single occasion appears to offer no benefit in predicting the likelihood of major complications.

Reproducibility of oesophageal transit studies: several 'single swallows' must be performed.

The 'single swallow' technique for the isotopic investigation of oesophageal function has been widely accepted. In our experience the results obtained when several consecutive 'single swallow' tests are performed show considerable variation. Although one or two swallows may be enough to diagnose abnormality, the exclusion of a motility disorder requires more, preferably six consecutive swallows.

Use of a gamma camera for measuring limb blood flow in peripheral vascular disease.

A method for measuring limb blood flow during reactive hyperaemia is described. Pneumatic cuffs inflated to 300 mmHg are used to isolate the blood in the limbs from the rest of the circulation. The remaining blood is labelled with technetium. The increase in radioactivity in the limb following release of the cuffs is measured using a gamma camera. The mean rate of flow of blood to the limb (in ml (100 ml)-1 tissue min-1) is derived from the graph of radioactivity versus time and from the radioactivity in a sample of venous blood. The results of measurements carried out in patients with peripheral vascular disease (30 limbs) and normal controls (24 limbs) are presented. Repeated studies in 10 subjects (20 limbs) showed the method to be highly reproducible at high and low flow rates (r = 0.99). Case studies illustrating the use of the method as a screening test for peripheral vascular disease and to monitor the effects of treatment are presented.

The measurement of limb blood flow using technetium-labelled red blood cells.

A method for measuring blood flow below the knee during reactive hyperaemia induced by 3 min of arterial occlusion has been developed. Subjects are positioned with lower limbs within the field of view of a gamma camera and pneumatic cuffs are placed below the knees to isolate the blood and induce a hyperaemic response. The remaining blood pool is labelled with 99Tcm-labelled red cells. Blood flows have been derived from the initial gradients of time-activity curves and from equilibrium blood sampling. The technique has been validated using a tissue-equivalent leg phantom and peristaltic pump. The method has been applied to a small group of patients with peripheral vascular disease and to normal controls. The mean value (+/- SD) of limb perfusion for normal controls was found to be 16.4 +/- 3.0 ml/100 ml/min and for patients with intermittent claudication was 5.1 +/- 2.6 ml/100 ml/min. Flow measurements are found to correlate with clinical findings and with symptoms. Reproducibility (established by repeated measurements) is high. The method is well tolerated even by patients suffering from rest pain.

Monoclonal immunoscintigraphy for the detection of primary colorectal cancers: a prospective study.

A prospective study to investigate the value of monoclonal antibody (McAb) immunoscintigraphy for the detection of primary tumours of the colon and rectum has been undertaken. Twelve patients received 1 mg 131I-labelled McAb YPC 2/12.1, an antibody raised to a human colonic carcinoma membrane preparation. Imaging of the blood pool was achieved using 99mTc-labelled red cells. After surgery specimens were scanned and samples of normal and malignant tissue excised to obtain antibody binding ratios. In only one patient was the primary tumour visualized pre-operatively. Images of the resected specimens revealed the primary tumour in ten cases but this appeared to be a function of increased blood pool within the tumour rather than specific antibody localization. The mean tumour/normal tissue count ratio was 1.27:1 (range 0.63:1-1.93:1). It is concluded that the McAb YPC 2/12.1 is unlikely to be of use clinically. Furthermore when selecting McAbs for the purposes of immunoscintigraphy the results of in vitro binding to malignant cell lines, immunohistological studies and xenograft experiments may be misleading and careful clinical evaluation is necessary.