Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study. After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats

We examined the effects of intracerebroventricular (i.c.v.) administration of adrenomedullin 2 (AM2) on plasma oxytocin (OXT) and arginine vasopressin (AVP) levels in conscious rats. Plasma OXT levels were markedly increased 5 min after i.c.v. administration of AM2 (1 nmol/rat) compared with vehicle and remained elevated in samples taken at 10, 15, 30, and 60 min. By contrast, plasma AVP levels were not significantly elevated in samples taken between 5 and 180 min after i.c.v. administration of AM2 except at the 30-min time point. Fos-like immunoreactivity (Fos-LI) was observed in various brain areas, including the paraventricular (PVN) and the supraoptic nuclei (SON) after i.c.v. administration of AM2 (2 nmol/rat) in conscious rats (measured at 90 min post-AM2 infusion). Dual immunostaining for OXT/Fos and AVP/Fos showed that OXT-LI neurons predominantly exhibited nuclear Fos-LI compared with AVP-LI neurons in the PVN and the SON. In situ hybridization histochemistry showed that i.c.v. administration of AM2 (0.2, 1, and 2 nmol/rat) caused marked induction of the expression of the c-fos gene in the PVN and the SON. This induction was significantly reduced by pretreatment with both the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) (3 nmol/rat) and the AM receptor antagonist AM-(22-52) (27 nmol/rat). These results suggest that centrally administered AM2 mainly activates OXT-secreting neurons in the PVN and the SON, at least in part through the CGRP and/or AM receptors with marked elevation of plasma OXT levels in conscious rats.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Differential expression pattern of C4 bundle sheath expression genes in rice, a C3 plant

NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase(PCK) are specifically expressed in bundle sheath cells (BSCs)in NADP-ME-type and PCK-type C₄ plants, respectively. Unlikethe high activities of these enzymes in the green leaves ofC₄ plants, their low activities have been detected in the leavesof C₃ plants. In order to elucidate the differences in the geneexpression system between C₃ and C₄ plants, we have producedchimeric constructs with the ß-glucuronidase (GUS)reporter gene under the control of the maize NADP-Me (ZmMe)or Zoysia japonica Pck (ZjPck) promoter and introduced theseconstructs into rice. In leaves of transgenic rice, the ZmMepromoter directed GUS expression not only in mesophyll cells(MCs) but also in BSCs and vascular cells, whereas the ZjPckpromoter directed GUS expression only in BSCs and vascular cells.Neither the ZjPck nor ZmMe promoters induced GUS expressiondue to light. In rice leaves, the endogenous NADP-Me (OsMe1)was expressed in MCs, BSCs and vascular cells, whereas the ricePck (OsPck1) was expressed only in BSCs and vascular cells.Taken together, the results obtained from transgenic rice demonstratethat the expression pattern of ZmMe or ZjPck in transgenic ricewas reflected by that of its counterpart gene in rice. (Received August 8, 2004; Accepted February 20, 2005 )     

A single bout of exercise influences natural killer cells in elderly women, especially those who are habitually active

The purpose of our study was to examine the effects of a single exercise bout on the natural killer (NK) cell count and activity in physically active elderly people, sedentary elderly people, and sedentary young people. Eight elderly women who trained by walking (age, 64 +/- 1 years; Vo(2peak), 32.2 +/- 1.1 ml.kg(-1).min(-1)), 8 age-matched untrained women (63 +/- 1 years, 28.8 +/- 1.0 ml.kg(-1).min(-1)), and 8 young untrained women (25 +/- 1 years, 37.6 +/- 1.6 ml.kg(-1).min(-1)) were studied. Blood samples were taken before, immediately after, and 2 hours after a 30-minute bout of exercise at an intensity equivalent to 70-75% of Vo(2peak). Peripheral blood mononuclear cells were isolated and the NK cell count and activity were analyzed. The NK cell count of the elderly sedentary group immediately after exercise was significantly higher than those of the elderly women who walked and young sedentary women, whereas no significant interaction was detected in NK cell activity and NK cell activity per cell number among the 3 groups. Consequently, an intrinsic defect in the cytotoxic ability of NK cells appeared in sedentary elderly people but not in active elderly people who perform habitual exercise.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.     

Decolorization of Alcohol Distillery Wastewater by Thermotolerant White Rot Fungi

To determine a thermotolerant fungus strain for decolorization of alcohol distillery wastewater (ADW), 38 fungus strains were studied. Capacity for ligninolytic enzyme production was examined at 35 and ⁴³C on agar media containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and MnCl₂. At 43°C, four Pycnoporus coccineus strains showed a higher potential for ADW decolorization both on agar media and in liquid media. Immobilized mycelia on polyurethane foam removed about threefold more total phenol than did free mycelia under conditions of shaking at 43°C. Moreover, immobilized mycelia removed nearly 50% more color than did free mycelia.