An endogenous metabolite of dopamine, 3,4-dihydroxyphenylethanol, acts as a unique cytoprotective agent against oxidative stress-induced injury

A natural compound contained in olive oil, 3,4-dihydroxyphenylethanol (DOPE), is also known as an endogenous metabolite of dopamine. The role of DOPE in oxidative stress-induced cell damage was investigated using differentiated PC12 cells. Superoxide (O(2)(-)) and H(2)O(2) induced a dose-dependent leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by MTT assay. While O(2)(-) -induced cell damage was not affected by DOPE, pretreatment of the cells with DOPE dose-dependently prevented the leakage of LDH induced by H(2)O(2). In these cells, augmented activity of catalase was demonstrated, while the levels of glutathione and glutathione peroxidase activity remained unchanged. The effect of DOPE was abolished when an inhibitor of catalase 3-amino-l, 2,4-triazole, was included in the medium. DOPE also protected against cell damage induced by H(2)O(2), and Fe(2+). In the hydroxyl radical ((.-)OH) assay using p-nitroso-N, N-dimethylaniline (PNDA), oxidation of PNDA by (.-)OH generated by the Fenton reaction was significantly attenuated in the presence of DOPE. By an electron spin resonance spin trapping study that represents the direct activity of DOPE to scavenge (.-)OH, however, limited scavenging activity was demonstrated for DOPE. Taken together, DOPE may act as a unique cytoprotective compound in nerve tissue subjected to oxidative stress.     

Loss of function mechanism in aprataxin-related early-onset ataxia

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is an autosomal recessive form of cerebellar ataxia that occurs most commonly in Japan but is also frequently seen in Europe. This disease is caused by mutations in the aprataxin gene, but the functions of the gene product and the pathogenic mechanism remain unclear. The present study provides experimental evidence that the histidine triad (HIT) domain in aprataxin has enzymatic activity that is negatively regulated by the intramolecular interaction of the N-terminal domain. Furthermore, the reduction in HIT activity seen in all the disease-causing mutants tested, and the correlation between the reduced activity and the severe phenotype, support that aprataxin's physiological function is associated with its catalytic activity. Our findings suggest that the clinical phenotypes are caused by a loss of aprataxin function, attributable largely to diminished HIT activity but partially to a reduction in the levels of gene products.     

Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases

Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.     

Metals accelerate production of the aberrant splicing isoform of the presenilin-2

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.     

Rapid replacement of somatic linker histones with the oocyte-specific linker histone H1foo in nuclear transfer

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.     

A novel kinesin-like protein, KIF1Bbeta3 is involved in the movement of lysosomes to the cell periphery in non-neuronal cells

The kinesin superfamily protein, KIF1Bβ, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non-neuronal tissues. To elucidate the functions of KIF1Bβ in non-neuronal cells, we analyzed the intracellular localization of KIF1Bβ and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bβ, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bβ3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bβ-specific tail region. Overexpression of KIF1Bβ3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bβ3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bβ3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.     

Isolation and characterization of simple repeat sequences from the yellow fin sea bream Acanthopagrus latus (Sparidae)

We isolated DNA fragments containing various repetitive elements from the genome of a sea bream Acanthopagrus latus. Sequence analysis indicated that two fragments have particularly interesting features. Fragment AL87 contained a tetranucleotide repeat and a quasipalindromic sequence. Sequence comparison suggested that AL87 may be a part of a gene encoding a serine/threonine protein kinase, and that the quasipalindrome is situated at the junction of an intron and an exon. Moreover, the quasipalindrome is conserved in several other fishes, even though it has the potential to form a stem-loop structure at the splicing site. Fragment AL79 contained a minisatellite sequence made up of six 30-bp units in tandem. DNase I sensitivity assays and statistical analyses showed the repeat region to be flexible when subjected to bending stress. In addition, atomic force microscopic imaging of AL79 showed the presence of highly curved (kinked) segments flanking the repeat region. The structural features of these repetitive elements may be key factors facilitating the amplification of the repeats.     

Role of premature leptin surge in obesity resulting from intrauterine undernutrition

Intrauterine undernutrition is closely associated with obesity related to detrimental metabolic sequelae in adulthood. We report a mouse model in which offspring with fetal undernutrition (UN offspring), when fed a high-fat diet (HFD), develop pronounced weight gain and adiposity. In the neonatal period, UN offspring exhibited a premature onset of neonatal leptin surge compared to offspring with intrauterine normal nutrition (NN offspring). Unexpectedly, premature leptin surge generated in NN offspring by exogenous leptin administration led to accelerated weight gain with an HFD. Both UN offspring and neonatally leptin-treated NN offspring exhibited an impaired response to acute peripheral leptin administration on a regular chow diet (RCD) with impaired leptin transport to the brain as well as an increased density of hypothalamic nerve terminals. The present study suggests that the premature leptin surge alters energy regulation by the hypothalamus and contributes to “developmental origins of health and disease.”     

Ca2+-dependent cessation of cytoplasmic streaming induced by hypertonic treatment in Vallisneria mesophyll cells: possible role of cell wall-plasma membrane adhesion

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, a rapid and transient inhibition of cytoplasmic streaming was induced by hypertonic treatment with sorbitol. Higher concentrations of sorbitol induced the response more rapidly and in more cells. The response to hypertonic treatment was strictly dependent on the presence of extracellular Ca2+ and was sensitive to Ca2+-channel blockers, including the stretch-activated Ca2+-channel blocker Gd3+. Deplasmolyzed cells never responded to a second hypertonic treatment administered immediately after plasmolysis and subsequent deplasmolysis. Responsiveness was gradually recovered during 24 h of incubation; however, cycloheximide, cordycepin, and trypsin completely suppressed the recovery. Although an Arg-Gly-Asp (RGD) hexapeptide markedly disturbed the pattern of cytoplasmic streaming, it exhibited no specific effects on the response to hypertonic treatment or on the recovery of responsiveness. Taken together, our results demonstrate that leaf mesophyll cells in a multicellular plant can respond to mechanical stimuli and that a Ca2+ influx through stretch-activated Ca2+ channels plays an indispensable role in the response. Furthermore, the possible involvement of RGD-insensitive but trypsin-sensitive protein factor(s), whose function is impaired by detachment of the plasma membrane from the cell wall, is suggested.