Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.

Evaluation of an endotoxin-specific limulus amebocyte lysate assay using leukocyte-rich plasma for the diagnosis of gram-negative bacterial infection.

We focused our attention on the endotoxin present within and on the surface of white blood cells and attempted to establish a new sample preparation method for endotoxin assays in leukocyte-rich plasma (LRP), taking advantage of the erythrocyte-aggregating property of hydroxyethyl starch. We used an endotoxin-specific turbidimetric kinetic assay, which is the conventional method used to assay endotoxin levels in platelet-rich plasma (PRP). Then, we comparatively assessed the assay results obtained with the endotoxin assay using PRP and LRP. It was found that the sensitivity of endotoxin assay in LRP was 88.5 %, which was superior to 73.1 % of the sensitivity in PRP in the diagnosis of infections caused by gram-negative bacteria. These results suggest that our newly developed LRP endotoxin assay may contribute to an improvement in the rate of sepsis diagnosis.

Experimental application of a synthetic luminescent substrate assay using endotoxin-specific limulus amebocyte lysate to human blood.

A synthetic luminescent substrate method, using a mutant-type luciferase whose luminescence intensity is more than ten times as intense as the wild type, was developed recently. We conducted the first basic studies on clinical application of the novel endotoxin measurement method. We assessed and established measurement conditions, including reagent concentrations and reaction time, so that it would be possible to apply the luminescent synthetic substrate method proposed by Noda et al. to measurements in human blood. When we added lipopolysaccharide (LPS) to water, it was possible to measure LPS at a concentration of 0.1 pg/ml, whereas it was possible to measure LPS in tenfold diluted and heated plasma at a concentration of 1 pg/ml. When plasma was further diluted, inhibiting activity decreased considerably. Thus, it will be necessary to completely eliminate the inhibitor present in plasma. However, the shortest time after collecting the specimen in which it was possible to make measurements was 30-40 min, suggesting that if an assay is established, it will be possible to use the method as a novel blood endotoxin assay.

[Study on the leukocyte-rich plasma as the specimen for the measurement of endotoxin].

PURPOSE: In the diagnosis of sepsis, the sensitivity of the endotoxin assay using platelet-rich plasma (PRP) is not as high as expected. In the endotoxin assay using PRP, endotoxin occurring within and on the surface of white blood cells cannot be measured. Thus, we devised a method of preparing leukocyte-rich plasma (LRP) using hydroxyethyl starch (HES) in order to improve the endotoxin assay. METHODS: The endotoxin concentration was measured by an endotoxin-specific Limulus amebocyte lysate assay method: turbidimetric kinetic assay. RESULTS: Only 1% of erythrocytes were recovered in LRP without the hemolysis of erythrocytes. Endotoxin contents in PRP obtained from blood incubated for 0, 30, 60, 120, and 180 min at 37 degrees C in the presence of lipopolysaccharide (50 pg/ml in a final concentration) were 1.18, 1.23, 1.23, 1.39, and 1.25 times higher (mean value from three bloods) than that in PRP, respectively. CONCLUSION: Thus, the superiority of the sensitivity of the LRP-based endotoxin assay over the PRP-based assay was identified. Our newly devised assay method may contribute to improvement of the diagnosis rate of endotoxemia in sepsis.

Retrospective study on the effect of tight glucose control in postoperative sepsis patients using an artificial pancreas.

Tight glucose control (TGC) using a sliding scale based on intermittent blood glucose measurements occasionally can have a fatal outcome as a result of insulin-induced hypoglycemia. The present study was undertaken to examine whether the use of an artificial pancreas to achieve TGC would be possible in postoperative patients with sepsis. The retrospective study was carried out as an exploratory study, focusing on the possibility of precise evaluation of the significance of TGC as a beneficial intervention by serological monitoring of various mediators. TGC was accomplished using an artificial pancreas (STG-22; (Nikkiso, Tokyo, Japan). The patients were divided into two groups: the TGC group (6 patients with sepsis in whom the target blood glucose level set at <150 mg/dl was attempted using the artificial pancreas), and the glucose control (GC) group (6 patients with sepsis in whom glucose control was attempted using a sliding scale; target blood glucose level was set at 200 mg/dl or lower). The mean blood glucose level was 129.7 ± 9.7 mg/dl in the TGC group and 200.9 ± 14.7 mg/dl in the GC group (P < 0.01, ANOVA). No hypoglycemia associated with the artificial pancreas was seen in any of the patients. The serum levels of S100A12 and HMGB-1 tended to decrease, and those of sRAGE tended to increase, in the TGC group. Further data collection from a larger number of cases would be expected to allow a precise assessment of TGC as a potentially beneficial intervention in sepsis patients.