A phase II detoxification enzyme inducer from lemongrass: identification of citral and involvement of electrophilic reaction in the enzyme induction

We have developed a simple system for the sensitive detection and measurement of glutathione S-transferase (GST) activity that detoxifies polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, RL34 cells. Citral (3,7-dimethyl-2,6-octadienal) was isolated from the methanol extract of lemongrass (Cymbopogon citratus) and identified as a novel inducer of GST. Citral, a mixture of the two stereoisomers geranial and neral, dose- and time-dependently induced the total and pi-class-specific activities of GST. The structure-activity relationship study revealed that geranial, an E-isomer, was mainly responsible for the inducing activity of citral mixture and the aldehyde group conjugated with a trans-double bond is an essential structural factor. The data were consistent with the in vitro observation that both glutathione (GSH) and protein thiol quickly and specifically reacted with the active isomer geranial, but not neral. Pretreatment of the cells with diethyl maleate significantly enhanced not only the basal activity but also the citral-stimulated activity of GST, while pretreatment with N-acetyl-cysteine inhibited it. Moreover, the treatment of RL 34 cells with geranial for 30 min significantly attenuated the intracellular GSH level, while application for 18 h enhanced it. These results strongly suggested that the electrophilic property characterized by the reactivity with intracellular nucleophiles including protein thiol or glutathione (GSH) plays an important role in the induction of GST. The present study also implied the antioxidant role of GST induction by citral in mouse skin, providing a new insight into skin cancer prevention.     

A novel affinity chromatography method for the co-purification of deoxycytidine kinase and cytidine deaminase

By affinity chromatography with Sepharose coupled to 2'-deoxy-1-beta-D-ribofuranosyl-N4-dodecanoylcytosine, deoxycytidine kinase and cytidine deaminase were purified 1,950- and 2,240-fold, respectively, from Ehrlich carcinoma cells, and their enzyme activities for several deoxycytidine analogs were investigated.     

Isolation of water-soluble alginate from brown algae

Summary Water-soluble alginate was obtained from an aqueous extract of Kjellmaniella crassifolia by precipitation with HCl, calcium acetate or 20% ethanol in the presence of 0.05 M MgCl₂ Of these precipitation procedures, MgCl₂-ethanol gave the purest alginate preparation as judged by electrophoresis. The thin-layer and gas-liquid chromatography of its acid hydrolysate, and the IR spectra analysis of the whole alginate, suggested that the water-soluble alginate is similar to ordinary water-insoluble and alkali-soluble alginate such as Kelco alginate.However, the alginate obtained in the present work contained a great excess of mannuronic acid residues, giving an M:G ratio of about 13. Its molecular weight distribution was rather broad as with Kelco alginate, but the molecular weight of its major component was estimated to be 500 000 amu, whereas that Kelco alginate measured on the same column under the same condition was 1 700 000 amu. This suggests that water-soluble alginate was far smaller in average molecular size than Kelco alginate.     

Endogenous formation of protein adducts with carcinogenic aldehydes: implications for oxidative stress

In the present study, we characterize the covalent modification of a protein by crotonaldehyde, a representative carcinogenic aldehyde, and describe the endogenous production of this aldehyde in vivo. The crotonaldehyde preferentially reacted with the lysine and histidine residues of bovine serum albumin and generated a protein-linked carbonyl derivative. Upon incubation with the histidine and lysine derivatives, crotonaldehyde predominantly generated beta-substituted butanal adducts of histidine and lysine and N(epsilon)-(2,5-dimethyl-3-formyl-3,4-dehydropiperidino)lysine (dimethyl-FDP-lysine) as the putative carbonyl derivatives generated in the crotonaldehyde-modified protein. To verify the endogenous formation of crotonaldehyde in vivo, we raised the monoclonal antibody (mAb82D3) against the crotonaldehyde-modified protein and found that it cross-reacted with the protein-bound 2-alkenals, such as crotonaldehyde, 2-pentenal, and 2-hexenal. The anti-2-alkenal antibody recognized multiple crotonaldehyde-lysine adducts, including dimethyl-FDP-lysine and an unknown product, which showed the greatest immunoreactivity with the antibody. On the basis of the chemical and spectroscopic evidence, the major antigenic product was determined to be a novel Schiff base-derived crotonaldehyde-lysine adduct, N(epsilon)-(5-ethyl-2-methylpyridinium)lysine (EMP-lysine). It was found that the lysine residues that had disappeared in the protein treated with crotonaldehyde were partially recovered by EMP-lysine. The presence of immunoreactive materials with mAb82D3 in vivo was demonstrated in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate. In addition, the observations that the metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of proteins resulted in an increase in the antigenicity of the protein indicated that lipid peroxidation represents a potential pathway for the formation of crotonaldehyde/2-alkenals in vivo. These data suggest that the formation of carcinogenic aldehydes during lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.     

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.     

Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant

CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels. To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs. Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets. Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver. The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis. mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination. In contrast, mCLC-7 did not complement this gef1 phenotype. This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.     

Successful Survival of Grafted Transgenic Neural Plate Cells in Adult Central Nervous System Environment

1. Accumulating evidence indicates that damaged brain functions can be ameliorated in a variety of animal models by the grafting of fetal neuronal cell or tissue into damaged brain. Clinical trials are under way to determine whether human fetal mesencephalic tissue can ameliorate motor functions in patients with Parkinson's disease.2. Autopsy findings of parkinsonian patient implanted with human fetal mesencephalic tissue clearly revealed that the fetal neuronal graft can survive for an extended period of time in the human brain and densely reinnervate the surrounding host striatal tissue.3. It is, however, still important to obtain more practical, effective, and ethically justifiable donor material for the future clinical application of the procedures. Desirable properties for the donor cells include long-term survival in the brain, neuronal cell type for the reconstruction of damaged neural circuits, and susceptibility to genetic manipulation for the practical use.4. With the development of molecular biology techniques, genetic modification and transplantation of the donor neuronal cells might be a feasible way to cure many kinds of central nervous system diseases toward a graft-gene therapy.     

Intracellular mislocalization of mutant podocin and correction by chemical chaperones

The NPHS2 gene encoding the podocin protein was causally linked to the autosomal recessive type of steroid-resistant nephrotic syndrome. In this study, we investigated the consequence of the R138Q mutation of podocin, one of the most common missense mutations in the NPHS2 gene, by examining the expression of the wild-type and R138Q mutant podocins in mammalian cells. Either myc- or FLAG-tagged wild-type podocin was strongly stained in plasma membrane, particularly in the fine processes wherein the protein was colocalized with actin stress fibers. On the other hand, the R138Q mutant podocin was completely retained intracellularly and colocalized with the endoplasmic reticulum (ER) marker, calnexin. These results suggest that the R138Q mutation affected podocin protein folding, thereby interfering with the mutant protein's departure from the ER. To determine if the ER retention of R138Q mutant is correctable, cells were incubated with the chemical chaperones glycerol, trimethylamine-N-oxide, and DMSO. Using these two methods, namely, cell surface labeling with sulfo-NHS-S-S-biotin and Alexa 488-streptavidin, and immunostaining to detect the podocin protein close to the plasma membrane, we confirmed that these chemical chaperone treatments elicit a cellular redistribution of R138Q podocin. Our results reveal defective cellular processing of the mutant podocin, and provide evidence for pharmacological correction of the processing defect.