Methylation and downregulated expression of mac25/insulin-like growth factor binding protein-7 is associated with liver tumorigenesis in SV40T/t antigen transgenic mice, screened by restriction landmark genomic scanning for methylation (RLGS-M)

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis. An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5' flanking region and 5' portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5' to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene.     

Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.     

Construction of new ribozymes requiring short regulator oligonucleotides as a cofactor

A hairpin loop and an oligonucleotide bound to the loop form one-half of the pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short complementary oligonucleotide as a cofactor. Stem II of the hammerhead ribozyme was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage activity of the target RNA. However, it exhibited the cleavage activity in the presence of a cofactor oligoribonucleotide, which is complementary to loop II of the ribozyme. The activity is assumed to be derived from the formation of a pseudo-stem structure between the cofactor oligonucleotide and loop II. The structure including the loop may be similar to the pseudo-half-knot structure. The activation efficiencies of the cofactor oligonucleotides were decreased as the lengths of the oligonucleotides increased, and the ribozyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3'-dangling purine bases served as efficient cofactors of the ribozyme, and a 2'-O-methyloligonucleotide enhanced the cleavage activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligonucleotides with a cytidine base at the 3'-end also activated a ribozyme with the G10.1.G11.1 mutation, which eliminates the cleavage activity in the wild-type. The binding sites of the oligonucleotide were identified by photo-crosslinking experiments and were found to be the predicted sites in the loop. This is the first report of a design aimed at positively controlling the activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin loops.     

Excretion of copper complexed with thiomolybdate into the bile and blood in LEC rats

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was removed with ammonium tetrathiomolybdate (TTM), and the fate of the Cu complexed with TTM and mobilized from the liver was determined. TTM was injected intravenously as a single dose of 2, 10 or 50 mg TTM/kg body weight into LEC and Wistar (normal Cu metabolism) rats, and then the concentrations of Cu and molybdenum (Mo) in the bile and plasma were monitored with time after the injection. In Wistar rats, most of the Mo was excreted into the urine, only a small quantity being excreted into the bile, while Cu excreted into the urine decreased. However, in LEC rats, Cu and Mo were excreted into the bile and blood, and the bile is recognized for the first time as the major route of excretion. The Cu excreted into both the bile and plasma was accompanied by an equimolar amount of Mo. The relative ratio of the amounts of Cu excreted into the bile and plasma was 40/60 for the low and high dose groups, and 70/30 for the medium dose group. The systemic dispositions of the Cu mobilized from the liver and the Mo complexed with the Cu were also determined for the kidneys, spleen and brain together with their urinal excretion. Although Mo in the three organs and Cu in the kidneys and spleen were increased or showed a tendency to increase, Cu in the brain was not increased at all doses of TTM.     

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.     

Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis

The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.     

GABAergic contribution to rat bladder hyperactivity after middle cerebral artery occlusion

To evaluate the influences of gamma-aminobutyric acid (GABA) mechanisms on bladder hyperactivity after left middle cerebral artery occlusion, cystometric recordings were obtained from unanesthetized female rats. Intracerebroventricular administration of both muscimol (GABA(A) receptor agonist; 0.1-10 nmol) and baclofen (GABA(B) receptor agonist; 0.1-3 nmol) produced dose-dependent inhibitions of micturition with increases in bladder capacity (BC). The effects of high doses (1-10 nmol) were similar in sham-operated (SO) and cerebral-infarcted (CI) rats. However, lower doses of muscimol (0.1 or 0.3 nmol) and baclofen (0.1 nmol) reduced BC in CI rats. After bicuculline (GABA(A) receptor antagonist; 1 or 3 nmol) administration, BC in both SO and CI rats first decreased and subsequently increased. An increase in urethral pressure was observed after administration of bicuculline (3 nmol) but not with either muscimol or baclofen. Infarct volumes in muscimol-, bicuculline-, or baclofen-treated rats were not significantly different from those of vehicle-treated rats. These results suggest that GABAergic mechanisms inhibit the micturition reflex at the supraspinal level but that this can change as a result of CI.     

Changes in trace element concentrations in hair of growing children

Head hair concentrations of zinc, copper, manganese, and iron from a total of 418 subjects (154 male and 264 female) aged between 6 mo and 20 yr were measured mainly with flameless atomic absorption spectrophotometry. Only zinc analysis of a part of the female samples (n=140) were analyzed with inductively coupled plasma-atomic emission spectrometry. The two analytical methods showed close agreement. The mean concentration of copper and manganese were significantly higher in male subjects than in female subjects. The trace element concentrations in hair varied with the subject’s age. Zinc concentration in hair decreased from 6 mo to 14 yr in the male subjects and decreased from 6 mo to 12 yr in the female subjects. Then, the concentrations increased gradually to 20 yr in the both sexes. Age-dependent variations of copper and manganese concentrations in hair showed similar trends to those of zinc. The results of this study suggest that a higher concentration in the diet of these trace elements may be required for growing children, especially in the period of adolescence.     

Stress-induced factor involved in flower formation of Lemna is an alpha-ketol derivative of linolenic acid

A stress-induced substance(s) (factor C) incubated with norepinephrine (NE) has strong flower-inducing activity in Lemna paucicostata. We isolated an essential component (FIF) of factor C, and clarified its chemical structure as 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid, an alpha-ketol derivative of linolenic acid, which is formed via 9-hydroperoxy linolenic acid. Synthesized FIF showed flower-inducing activity after incubation with NE (factor C activity) equivalent to that formed in the stressed Lemna. Jasmonic acid and 13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid (12,13-alpha-ketol linolenic acid), both of which are formed via 13-hydroperoxide of linolenic acid and all other derivatives of FIF synthesized by chemical and enzymatic processes failed to show the factor C activity. These results suggest that the molecular structure of FIF is very specific for the factor C activity.