A symposium on the clinical aspects of metallothioneins (MTs) in neurodegenerative diseases was held at the 2003 Society of Metallothionein Meeting in Gifu, Japan. The objectives of the symposium were to review and speculate on the potential roles of MTs, especially MT-3 in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and spinocerebellar degeneration (SCD). Dr. Uchida discussed the controversial problem regarding the expression of MT-3 in AD brains. Dr. Asanuma addressed the function of MTs in the progression of PD and Dr. Hozumi described the therapeutic potential of MTs for ALS, while Dr. Yamada provided immunohistochemical findings of MT-3 in SCD for the first time. Although there are still controversial problems on MTs, this review provides proof that MTs are promising potential therapeutic targets for therapy for some neurodegenerative diseases.
The classical mammalian metallothioneins (MT-I/II) and the other subfamilies (MT-III/IV) both bind heavy metals by the well-conserved cysteine cluster structure, but their modes of expression differ from each other. MT-I/II is ubiquitously induced by heavy metals, but MT-III/IV is expressed in restricted tissues without obvious dependency on heavy metals. To understand the heavy metal dependent transcriptional activation of the MT-I/II genes, the mechanism of the heavy metal response of a pivotal transcription factor, MTF-1, has been extensively studied. On the other hand, in the case of MT-III, the mechanism of gene suppression has been investigated to clarify the basis of its tissue specific expression. In this review, recent progress in understanding the transcriptional regulation of these MT subfamilies is described.
Acupoints tend to show higher electric conductivity and have a higher concentration of neural and vascular elements as well as mast cells. According to one model, the meridian is an extravascular fluid (interstitial fluid) pathway and circulation of this fluid is driven by skeletal muscle contraction. Our result derived from laser Doppler flowmetry shows that Yanglao (SI6) and Xiaohai (SI8) have larger amplitude of heartbeat and vasomotion than non-acupuncture points. The amplitude of blood flow on the SI8 shows a more significant increase (4.3 ± 0.7 PU) than that of non-acupuncture points (1.1 ± 0.2 PU). Our results indicate that acupuncture increases blood flow when the De-Qi sensation takes place. In the experiment, the needle was only entered into the subject by 2 mm for about 15 sec during a SI6 acupuncture session, and the blood flow increased immediately. The blood flow of SI8 began to rise 5 sec after implementation and increased by 11.1 ± 0.9 PU. However, when the arm was tied up, the blood flow at the SI8 point slowed down quickly. Even though insertion with a twirling needle was followed after the drop in blood flow, the blood flow at the SI8 point did not accelerate again (0.7 ± 0.2 PU). The present study seems to show that acupuncture is effective in regulating the autonomic nervous system. Implementation of acupuncture causes the sphincter of microvessel to relax, and the tissue fluid to flow out, and the propagated sensation along the meridian is caused by this large amount of tissue fluid that flows along the loosen body stalk. The interstitial fluid increases during the needling and blood capillary expansion, which may be one of the mechanisms of acupuncture regulation.
Currently, erythropoietin (EPO) treatment in anemic patients is getting more interest, but in long term, development of hypertension is common problem in EPO treated patients. Since it isn't yet known whether EPO affects directly vascular and/or kidney functions, we aimed to study effects of acute EPO treatment on renal tissue nitrite level, and cardiovascular function and glomerular filtration rate (GFR). Experiments were done under 4 groups. Group I: Sham operated control, group II: 150 IU/kg EPO, group III: 50 mg/kg Nw-nitro-L-arginine methyl ester (L-NAME), group IV: L-NAME+EPO. Femoral artery, vein and bladder were catheterized under anesthesia. After stabilization and first 45 min basal control period, drugs were given i.v. bolus infusion according to the group protocols, then 2 × 45 min clearance periods were followed. EPO infusion increased renal tissue nitrite activity causing GFR increase without any influence on systemic blood pressure. L-NAME alone or together with EPO significantly raised the systemic blood pressure with a partial increase in GFR, but L-NAME treatment significantly reduced tissue nitrite level. The present study for the first time suggests that exogenous EPO treatment increases rat GFR and renal tissue nitrite level without affecting systemic blood pressure.
Cortical neuronal cultures were exposed to total ginkgolides (TG) in order to find out whether TG could rescue cultured cortical neurons from neurotoxicity damages. The cellular injuries were induced in the cells after 10 days in vitro by exposure to 1 mM L-glutamate (Glu) for 6-12 hr in serum-free medium, to 0.2 mM hydrogen peroxide (H2O2) for 6-12 hr, and then to free-glucose and hypoxia medium (an ischaemia model in vitro) for 4 hr. TG (0.01 to 100 ug/ml) was added to the growth medium 12 hr prior to or simultaneously the damage protected cortical neurons from toxic damages. Neuronal viability was confirmed by the assay of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium (MTT). The lactate dehydrogenase (LDH) release from the cultured medium was also detected. The results of present study demonstrated that TG protected cortical neurons from toxic damages induced by Glu, H2O2 or free-glucose and hypoxia.
Diabetic animal models are used extensively to dissect the mechanisms underlying diabetic cardiomyopathy. For such studies, detection of cardiac dysfunction is critical, and therefore, the present study comparatively examined cardiac function in mice 100 days after strepotozotocin (STZ)-induced diabetes using three complementary techniques: electrocardiography (ECG), echocardiography and left ventricular (LV) hemodynamic analysis. Histological changes were also assessed by periodic acid-Schiff (PAS)-positive materials and Masson's trichrome staining and immunohistochemical staining for type IV collagen. ECG monitoring for 2 hr in diabetic and control mice revealed no abnormality. By echocardiography, diabetic mice showed significant decreases in LV chamber diameter at end-diastole and end-systole but no other abnormalities, as compared to control mice. Hemodynamic evaluation in diabetic mice revealed that although basal parameters of cardiac function were similar to control, β-adrenergic responsiveness was significantly reduced in diabetic mice, indicating a loss of inotropic reserve and early myocardial dysfunction. Histological staining showed mild but significant increases in interstitial fibrosis in diabetic mice, confirming early diabetic cardiomyopathy. These results indicate that despite examination by ECG and resting hemodynamic may not sensitive approaches to reveal early diabetic cardiomyopathy, resting echocardiography and stressing hemodynamic analysis by evaluation of contractile provocation and inotropic reserve are able to uncover the evidence of early subtle mechanical dysfunction.
In this paper, we compared the bioavailability of penicillamine selenotrisulfide (PenSSeSPen) with the authentic selenium-source compound, selenite. When PenSSeSPen was orally administered to selenium-deficient mice for 7 days, selenium contents of blood, liver and heart significantly increased as well as selenite. In addition, when PenSSeSPen-supplemented diet was fed to 3-week old mice for 7 weeks, selenium contents of selected organs were almost the same as those of selenite, and hepatic cellular glutathione peroxidase (GPx) activity was also observed. These facts evidently indicate that PenSSeSPen, a totally foreign selenium compound, serves as a selenium-source compound.
Metallothionein (MT) is one of the stress proteins induced by various heavy metals and oxidative stress. Although induction by Fe has not been well established, its overload is documented to cause oxidative stress in experimental animals. To investigate alteration in tissue MT levels that were possibly caused by Fe overload, male Wistar rats were fed a diet containing 3.5% Fe(II) fumarate (1.2% as Fe) for up to 21 days. Tissue Fe levels in liver and kidney increased in time-dependent manners up to 7.2 and 2-fold, respectively, by the end of the experiment. Interestingly, plasma Fe levels showed a maximum on day 3, then decreased to the control level. The hepatic MT levels showed a transient decrease on day 3, then turned to increase throughout the experiment period, with the final level higher than that in control animals that fed on a normal diet. MT levels in the kidney decreased to nearly 1/3 of the control on day 3, with the values unchanged thereafter. Although Zn levels in liver, plasma and kidney showed a transient but significant reduction on day 3 of the Fe feeding, they recovered to the control values in the later period. Thus, a time-dependent change in the hepatic MT levels was quite similar to that of the tissue Zn levels. The renal Zn levels could not account for the change in the tissue MT levels, at least in the latter period of the experiment. Hybridization analysis of the MT mRNA in the kidney of Fe-fed rats did not differ from control-diet rats, suggesting Fe feeding would not alter synthetic rates of the renal MT. The turnover rates of the renal MT estimated from a time-dependent alteration after its induction by HgCl2 treatment seemed to be enhanced in Fe-fed rats. Thus, the Fe-induced decrease in the renal MT level could possibly be due to an enhanced turnover rate. Despite the reduced renal MT levels, the Fe-treated rats showed no sign of a renal failure indicated by a stable plasma creatinine levels and sustained increase of body weight, suggesting that the Fe-induced suppression of the renal MT levels would not be one of the toxic effects by this metal. Nevertheless, the HgCl2 injection experiment revealed Fe-fed rats had a slightly higher susceptibility against HgCl2 nephrotoxicity than the control-diet rats.
A new whole-cell bacterial sensor for the detection of low concentration of mercury in environment was constructed by gene fusion between a mercury resistance (mer) operon from pMR26 of Pseudomonas strain K-62 and a promoterless luxAB gene from Vibrio harveyi. The luminescence-based biosensor was evaluated for the selectivity and sensitivity of the detection of mercury. Cadmium, lead, chromium and zinc ions did not interfere with the assay even at same concentration compared to Hg2+. Methylmercury, phenylmercury and mercuric sulfide also did not affect the biosensor. These results reveal that the specificity of the construct is restricted to bioavailable Hg2+. The sensitivity of the biosensor was improved by decreasing the cell density in the bioassay in addition to genetically expressing an Hg2+ transport system which was expected to increase the amount of mer operon-inducing mercury in the cytoplasm. In optimized assay conditions, the lowest detectable concentration of Hg2+ was 2 pM with 1 ml sample. This detection limit is enough to detect this compound in many contaminated and some pristine environmental samples.
The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to phenobarbital (PB) in rodents. CYP2B forms are highly inducible in liver and intestine in male Wistar rats. In contrast, PB-dependent increases in CYP2B activity are rarely observed in female Wistar rats. The profiles of CAR mRNA expression measured by RT-PCR in various organs in male and female Wistar rats were consistent with those of PB-dependent expression of CYP2B genes. The sex-related dimorphic PB-responses of CYP2B genes in Wistar rats might be determined transcriptionally as are the organospecific PB-responses in both males and females.
There had been a smoking case in which dimethylamphetamine (DMA) and its metabolite DMA-N-oxide (DMAO) in addition to methamphetamine (MA) and amphetamine (AM), were detected in the abuser's urine. The analytical results of the confiscated leftover drug enabled us to consider that DMA detected in the abuser's urine would be the pyrolysis product formed by smoking MA. In this study, we carried out a smoking experiment based on this smoking case, and then gave the case full consideration. We heated MA hydrochloride in the range of 250°C to 350°C, in which demethylation and methylation reactions occurred mainly, with a smoke collection apparatus and a gas lighter, and trapped the generating vapor with an adsorption cartridge. AM and DMA were produced via the demethylation and methylation reactions of a methylamino group of MA. Allylbenzene, benzaldehyde, cis-β-methylstyrene, benzyl chloride and trans-β-methylstyrene were also formed as pyrolysis products. The sum of the formation ratio of DMA to the starting MA in the cartridge eluate and that in the residual materials inside the smoke collection apparatus was 4.98%. The adsorption of some pyrolyzates containing DMA on the cartridge means that these pyrolyzates can be taken into an abuser's body. The ratios of DMA to AM in the smoking experiments and the smoking case were 52.3% and 50.3%, respectively, and they were nearly equal. There will be no inconsistencies in considering that AM and DMA have been formed as pyrolysis products of MA in the smoking case, and DMA and its metabolite DMAO have been detected in the urine of this smoking abuser.
The aim of this study was to establish a preparation method needed to analyze the metabolites of an analytical procedure for antipsychotic phenothiazines and tertiary amino cyclic antidepressants by chemical reaction. These drugs were oxidized to their sulfoxide and N-oxide, respectively, with hydrogen peroxide using titanosilicate as the catalyst. An acidic medium (pH 3.0) containing 20% methanol was found to be optimal for the preparation of phenothiazine sulfoxide, and an alkaline medium (pH 10.5) containing 50% methanol was optimal for the preparation of the N-oxide of tertiary amino cyclic antidepressants. Each preparative scale reaction (2 mmol) was carried out and the crystallized product of each oxide was obtained. The amount of hydrogen peroxide needed to obtain the best yields of sulfoxide was 1.3-2.0 times the molar equivalent of the phenothiazines. The purities of the prepared crystallized product of the phenothiazine sulfoxides were good except for periciazine sulfoxide. The amounts of hydrogen peroxide needed to obtain the best yields of N-oxide were 4-5 times the molar equivalent of tertiary amino cyclic antidepressants. The purities of the crystals of each N-oxide were 99.5-100%. The characteristic mass fragment ions of each prepared oxide that were distinguishable as the N-oxides from parent drug could be confirmed when the collisional energy was decreased to 10 eV. 1H- and 13C-NMR spectral data confirmed the structure of the prepared mianserin N-oxide to be 2-oxide. In conclusion, a simple and rapid preparation method for oxide metabolites of phenothiazines and tertiary amino cyclic antidepressants available as analytical standards was established.
It is unknown which endocrine disruptors exert their effects on neuronal functions, particularly leading to behavioral alterations. To address this, we examined the effects of p-n-octylphenol, an endocrine disruptor, on rat behavior and cellular responses. Single intracisternal administration of p-n-octylphenol (87 nmol) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. The treated rats were about 1.5-fold more active in the nocturnal phase after administration of p-n-octylphenol than control rats. Immunohistochemical analyses revealed that p-n-octylphenol abolished immunoreactivity for tyrosine hydroxylase in the midbrain of 8 week-old rats, where terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)-positive cells were also seen. Thus, this is the first demonstration that p-n-octylphenol certainly affected the developing brain, resulting in hyperactivity in the rat, most likely due to degeneration of mesencephalic tyrosine hydroxylase.
To clarify the role of metallothionein (MT), an anti-stress protein, induced by psychological stress, we carried out an experiment to determine whether hepatic MT synthesis induced by restraint stress can be classically conditioned by an olfactory odor cue of camphor. Mice were conditioned by exposure to the odor of camphor 1 hr before restraint stress and partly during restraint stress. Although exposure to the odor of camphor did not elevate hepatic MT levels in itself, the odor stimuli significantly elevated hepatic MT levels in mice conditioned for 3 days. The results indicate that MT synthesis is induced by an association of the central nervous system with psychological stress.
Human T-cell leukemia virus type I U5 repressive element binding protein 1 (HTLV-I U5RE Binding protein 1; HUB1 and renamed ZNF282) — a member of the Krüppel type zinc finger family — represses HTLV-I long terminal repeat (LTR)-mediated transcription by binding to the TCCACCC motif of the U5RE. Sp1 and other Sp1 family proteins also recognize the CACCC box of the U5RE, as well as the GC box (GGGCGG). We would therefore expect HUB1 to compete with Sp1 for binding to the former. In the present study, we demonstrated that Sp1 activates transcription via the U5RE, and that HUB1 represses these Sp1-mediated effects. Electrophoretic mobility shift assays (EMSA) confirmed that HUB1 was bound to the CACCC box, but not the GC box. Consistent with that finding, overexpression of HUB1 repressed Sp1-mediated transcription reporter genes controlled via the CACCC box, but not via the GC box. These results suggest that, by binding to the CACCC box, HUB1 represses the Sp1-dependent transcriptional activation.
Zic family proteins are expressed in the cerebellum and play important roles in vertebrate development. However, very few observations have been made regarding the function of Zic proteins in transcription. We previously showed, using P19 cells as a model system, that the lamin A/C promoter has a retinoic acid responsive element (L-RARE), and that Sp1 and Sp3 bind the CACCC box of the L-RARE. Here, Zic1 is identified as a binding protein of the L-RARE by yeast one-hybrid screening. We show that Zic1 can bind to and repress transcription through the CACCC box of the L-RARE. Moreover, reporter assays using various fusion proteins of Zic1 with the GAL4 DNA binding domain reveal that Zic1 has an intrinsic transcriptional repressive and activation domain.
B-Lymphocytes are Elevated in Mouse Bone Marrow by Estrogen Deficiency, and Induce Receptor Activator of Nuclear Factor κB Ligand (RANKL) Expression in Osteoblasts via Cell Adhesion Chiho Matsumoto, Chisato Miyaura, and Akira Ito.....Vol. 50 No. 3 p. 312
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