Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines

The oxidative modification of low-density lipoprotein (LDL) is involved in atherogenesis. Among a variety of modified LDLs mentioned in the literature, so-called minimally modified LDL (MM-LDL) was reported to have pro-atherogenic properties despite minimal changes in its oxidative measures. After treatment of LDL with 1 micro M FeSO(4) at 4 degrees C for 96 h, the resulting MM-LDL showed a slight increase in thiobarbituric acid-reactive substances (TBARS) and little association with macrophages. On the other hand, heavily oxidized LDL, which was prepared by copper-induced oxidation of LDL at 37 degrees C, showed a sharp increase in TBARS and strong association with macrophages. By introducing a fluorometric procedure to detect aldehyde-containing phosphatidylcholines (aldehyde-PCs), we examined the amounts of aldehyde-PCs in modified LDL preparations. Aldehyde-PCs increased to 23.4 pmol/ microg protein in MM-LDL, which was more than four-fold higher than in the heavily oxidized LDL. We conclude that MM-LDL is a unique type of oxidized LDL enriched with aldehyde-PCs.     

Synthesis of amyloid β-peptides in solution employing chloroform-phenol mixed solvent for facile segment condensation of sparingly soluble protected peptides

Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl₃-phenol mixed solvent without danger of epimerization or of significant ester formationwith the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employedin the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine(HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl₃-phenol were determined and successfully applied to the synthesis of amyloid -peptide (1-42), (1-43) and [Pyr³]-(3-42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra andfluorimetric assay.     

LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation

A recently identified lectin-like oxidized low-density lipoprotein receptor (LOX-1) mediates endothelial cell injury and facilitates inflammatory cell adhesion. We studied the role of LOX-1 in myocardial ischemia-reperfusion (I/R) injury. Anesthetized Sprague-Dawley rats were subjected to 60 min of left coronary artery (LCA) ligation, followed by 60 min of reperfusion. Rats were treated with saline, LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat IgG (10 mg/kg) before I/R. Ten other rats underwent surgery without LCA ligation and served as a sham control group. LOX-1 expression was markedly increased during I/R (P < 0.01 vs. sham control group). Simultaneously, the expression of matrix metalloproteinase-1 (MMP-1) and adhesion molecules (P-selectin, VCAM-1, and ICAM-1) was also increased in the I/R area (P < 0.01 vs. sham control group). There was intense leukocyte accumulation in the I/R area in the saline-treated group. Treatment of rats with the LOX-1 antibody prevented I/R-induced upregulation of LOX-1 and reduced MMP-1 and adhesion molecule expression as well as leukocyte recruitment. LOX-1 antibody, but not nonspecific IgG, also reduced myocardial infarct size (P < 0.01 vs. saline-treated I/R group). To explore the link between LOX-1 and adhesion molecule expression, we measured expression of oxidative stress-sensitive p38 mitogen-activated protein kinase (p38 MAPK). The activity of p38 MAPK was increased during I/R (P < 0.01 vs. sham control), and use of LOX-1 antibody inhibited p38 MAPK activation (P < 0.01). These findings indicate that myocardial I/R upregulates LOX-1 expression, which through p38 MAPK activation increases the expression of MMP-1 and adhesion molecules. Inhibition of LOX-1 exerts an important protective effect against myocardial I/R injury.     

Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase.

HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the "palm and fingers" substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and fingers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutions in vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivo and in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity.     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.     

Hinge-mediated dimerization of SMC protein is essential for its dynamic interaction with DNA

Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans. As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled-coil arms with a flexible hinge. Site-directed cross-linking experiments show here that dimerization of BsSMC is mediated by a hinge-hinge interaction between self-folded monomers. This architecture is conserved in the eukaryotic SMC2-SMC4 heterodimer. Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA-binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer. Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA. Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross-link double-stranded DNA in a nucleotide-dependent manner. We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC-DNA interactions.