Sustained QT prolongation induced by tacrolimus in guinea pigs.

Recently, clinical cases have been reported of QT prolongation and torsades de pointes associated with the use of tacrolimus (FK506). We examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs in order to evaluate the arrhythmogenicity of FK506 in comparison with quinidine (QND). FK506 (0.1 or 0.01 mg/hr/kg) or QND (30 mg/hr/kg) was intravenously infused to guinea pigs and time profiles of drug concentration in blood and QTc interval were examined during and after infusion. Both FK506 and QND evoked a significant QTc prolongation, and the dose-response relationship showed an anti-clockwise hysteresis, FK506-induced QTc prolongation persisted throughout the duration of the experiment despite a decline in the plasma FK506 concentration, whilst QND-induced QTc prolongation disappeared as plasma concentrations decreased. FK506 induced a sustained QTc prolongation in guinea pigs at drug concentrations in blood that correspond to its therapeutic range in human, suggesting that it might be of clinical significance to monitor the electrocardiogram, especially when patients have congenital or acquired QT-prolonging risk factors.     

Human RME-8 is involved in membrane trafficking through early endosomes

RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Cytotoxic 16-β-[(d-xylopyranosyl)oxy]oxohexadecanyl triterpene glycosides from a Malagasy plant, Physena sessiliflora

Brine shrimp lethality assay-guided separation of the MeOH extract of leaves of Physena sessiliflora, which is endemic to Madagascar, afforded eight triterpene glycosides, Physenoside S1–4 and 16-β-[(d-xylopyranosyl)oxy]oxohexadecanyl homologues, Physenoside S5–8. Structural elucidation of these compounds was based on both spectroscopic analyses and chemical properties. Physenoside S7 and S8 have significant cytotoxic activities in the brine shrimp lethality assay.     

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2μ2

Background

Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated.

Methods

We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out.

Results

We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules.

Conclusions

Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2.

General Significance

This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.