Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles.

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

Case-controlled identification of colorectal cancer based on proteomic profiles and the potential for screening.

AIM: Colorectal cancer (CRC) screening programmes detect early cancers but unfortunately have limited sensitivity and specificity. Mass spectrometry-based determination of serum peptide and protein profiles provides a new approach for improved screening. METHOD: Serum samples were obtained from 126 CRC patients before treatment and 277 control individuals. An additional group of samples from 50 CRC patients and 82 controls was used for validation. Peptide and protein enrichments were carried out using reverse-phase C18 and weak-cation exchange magnetic beads in an automated solid-phase extraction and spotting procedure. Profiles were acquired on a matrix-assisted laser desorption/ionization time-of-flight system. Discriminant rules using logistic regression were calibrated for the peptide and protein signatures separately, followed by combining the classifications to obtain double cross-validated predicted class probabilities. Results were validated on an identical patient set. RESULTS: A discriminative power was found for patients with CRC representative for all histopathological stages compared with controls with an area under the curve of 0.95 in the test set (0.93 for the validation set) and with a high specificity (94-95%). CONCLUSION: The study has shown that a serum peptide and protein biomarker signature can be used to distinguish CRC patients from healthy controls with high discriminative power. This relatively simple and cheap test is promising for CRC screening.

Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion.

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.

Minimally invasive vein harvest and wound healing using the SaphLITE Retractor System.

BACKGROUND: In response to reported wound complication rates of 19% to 43% for traditional saphenous vein harvest, several minimally invasive vein harvest (MIVH) techniques have been developed. The purpose of this investigation is to determine the effectiveness of one such MIVH technology, the Genzyme SaphLITE Retractor System (Genzyme Biosurgery, Cambridge, MA). METHODS: Since May 2000, saphenectomy was undertaken in 305 coronary artery bypass graft (CABG) patients using SaphLITE in a prospective, nonrandomized trial at three centers. Patients were assessed for wound healing (ASEPSIS tool) and incisional pain (numeric scale) through the postoperative visit. Harvest times, incision lengths, and vein lengths were recorded. RESULTS: ASEPSIS indicated satisfactory healing in 96.0%. Infection rate was 1.3% with four patients requiring antibiotics and debridement of one incision. Of hospitalized patients, 85.4% had no or minimal affected leg pain. Additional mean data include: harvest time 43.4 +/- 17.6 minutes, incision number 3.0 +/- 1.2, incision length 2.9 +/- 1.4 cm, and vein length 46.0 +/- 15.2 cm. CONCLUSIONS: SaphLITE provides an effective alternative to traditional saphenous vein harvest, with improved wound healing, decreased pain, and acceptable harvest times.

Genes and signal molecules involved in the rhizobia-leguminoseae symbiosis.

The symbiosis between Rhizobium bacteria and their host plants is dependent on the specific recognition of signal molecules produced by each partner. Many players in the signal exchange have been identified. Among them are signal molecules such as flavonoids, LCOs, auxin, cytokinin, ethylene and uridine and genes such as Enod40, Enod2 and Enod12. Their interconnection, however, is only starting to be understood. The most recent insights into their interconnection include: advances in the use of transgenic leguminous plants containing reporter gene constructs for studying the effect of the signal molecules; novel methods for delivery of signal molecules using ballistic microtargeting; and the discovery of the role of chitin oligosaccharides in animal embryogenesis.

Role of lipid peroxidation in electroporation-induced cell permeability.

Erythroleukemia K562 cells and lentil (Lens culinaris) root protoplasts have been subjected to pore-forming electric fields suitable for transfection experiments. Evidence is presented that the amount of hydroperoxides formed in cell membranes of both cell-types is a function of field strength applied. On the other hand, the electroporation-induced lipid peroxidation paralleled the enhancement of membrane permeability and was associated with greater membrane fluidity. The membrane hydroperoxides formed upon electric shock enhanced cell luminescence, and lipoxygenase activity appeared to be involved in the process. Electroporation of prokaryotic cells of Escherichia coli also enhanced light emission, which was higher in lipoxygenase-expressing clones.

Accelerated transmural gradients of energy compound metabolism resulting from left ventricular hypertrophy.

Eighteen dogs underwent transmural left ventricular biopsies for adenosine triphosphate and suturing of the noncoronary cusp, creating valvular aortic stenosis. Three months after aortic stenosis and the subsequent development of left ventricular hypertrophy, animals underwent repeat transmural left ventricular biopsies followed by total myocardial ischemia at 37 degrees C. Left ventricular tissue samples for adenosine triphosphate and lactate levels were determined at 15-minute intervals and compared with 15 control animals. No significant difference between subendocardial and subepicardial adenosine triphosphate levels was found between left ventricular samples taken before left ventricular hypertrophy and 3 months after left ventricular hypertrophy. Significant differences in adenosine triphosphate utilization occurred between subendocardial and subepicardial layers in control and left ventricular hypertrophy myocardium, however. The gradient between the subendocardium and the subepicardium was significantly increased by left ventricular hypertrophy (p less than 0.05). Significant differences also occurred within the same layer when left ventricular hypertrophy and control groups were compared. During total ischemia, lactate concentration was significantly greater within the subendocardium than within the subepicardium in left ventricular hypertrophy. The onset of ischemic contracture was 48.2 +/- 2.1 minutes in left ventricular hypertrophy versus 62.3 +/- 1.8 minutes in control hearts (p less than 0.01). Subendocardial intramyocardial pressure increased significantly earlier than subepicardial in both left ventricular hypertrophy and control hearts. Adenosine triphosphate was used, and lactate accumulated more rapidly in animals with a more pronounced hemodynamic gradient. These data show that after left ventricular hypertrophy, adenosine triphosphate stores in the subendocardium and the subepicardium are unchanged from control values, yet the rates of adenosine triphosphate utilization and lactate accumulation during total ischemia are significantly increased. Furthermore, the subendocardial to subepicardial gradient of adenosine triphosphate utilization during ischemia found in normal hearts is markedly increased by left ventricular hypertrophy.

Diagnosis and recommended management of esophageal perforation and rupture.

Over the past 47 years (1937 to 1984), a total of 127 patients with esophageal perforation or rupture were evaluated at Duke Medical Center or the Durham Veterans Administration Medical Center. In 13 patients, the diagnosis was established at the time of autopsy and in the remaining 114, the diagnosis was established clinically. The etiology, radiological findings, underlying esophageal disease, time interval between onset of symptoms and therapy, and eventual outcome were evaluated. Patients with anastomotic leaks and those in whom carcinoma resulted in perforation or fistula were excluded. Iatrogenic causes were responsible for 55% of perforations, followed by spontaneous rupture in 15%, foreign body perforation in 14%, and traumatic perforation in 10%. Of the 127 patients, 114 underwent treatment involving primary closure (43%), drainage alone (28%), resection (9%), or nonoperative therapy (20%). The overall mortality among these 114 patients was 21%. Fourteen patients sustained a major complication requiring additional operative intervention. The overall mortality among patients requiring reoperation was 57%. Survival was significantly influenced by a delay in treatment of greater than 24 hours. With the exception of nonoperative therapy, survival was improved for all forms of treatment instituted within 24 hours. Primary closure within 24 hours resulted in the most favorable outcome (92% survival). In addition to early treatment, other factors associated with a favorable outcome included traumatic perforation (100% survival), foreign-body perforations (94% survival), and iatrogenic causes (80% survival). Spontaneous rupture resulted in the lowest survival (37%). The incidence of esophageal perforation has increased dramatically since 1967.(ABSTRACT TRUNCATED AT 250 WORDS)

Prehospital cardiopulmonary resuscitation of the critically injured patient.

Prehospital cardiopulmonary resuscitation combined with endotracheal intubation, vigorous fluid resuscitation, and rapid transport can be effective in resuscitating trauma patients in cardiopulmonary arrest. Survival does not correlate with the injury severity score or transport time once the patient has arrested but does correlate with the mechanism of injury, endotracheal intubation, and placement of intravenous lines.