Endurance treadmill training in rats alters CRH activity in the hypothalamic paraventricular nucleus at rest and during acute running according to its period

Running training on the treadmill increases the resting hypothalamic corticotropin-releasing hormone (CRH) content in rats, though is still unknown whether and how it occurs in the parvocellular region of the hypothalamic paraventricular nucleus (PVN) where is a predominant region of pituitary-adrenal activity and where CRH and arginine vasopressin (AVP) are colocalized. We thus aimed at examining whether treadmill training would alter the CRH and AVP mRNA levels in the PVN at rest and during acute running with different lengths of a training regime. Male Wistar rats were subjected to treadmill running (approximately 25 m/min, 60 minutes/day, 5 times/week) for training regimes of 0, 1, 2 or 4 weeks. All training regimes induced an adrenal hypertrophy. Plasma corticosterone levels before acute running increased with lengthening the training period. Four weeks of training produced a significant increase in the resting CRH, but not AVP, mRNA levels in the PVN though relatively shorter training regimes did not. Acute responses of lactate and ACTH release were reduced after 2 and 4 weeks of training, respectively. The responsive PVN CRH mRNA level to acute running decreased with 4 weeks of training but increased with relatively shorter training regimes. These results indicate that running training changes the PVN CRH biosynthetic activity with the regime lasting for 4 weeks, which follows adaptive changes in adrenal functions. Thus, running training-induced changes in hypothalamic CRH activity would originate from the PVN and be induced according to the training period.     

Phosphorus foraging root development of Brassica rapa nothovar. grown in a phosphorus-deficient nonallophanic Andisol

Lateral root of Brassica crops firmly aggregated around Ca-alginate gel beads containing dicalcium phosphate dihydrate and -cyclodextrin (DCPD gel bead) in a phosphate (P)-deficient soil (Nanzyo et al., 2002, Soil Sci. Plant Nutr. 48, 847–853). The first aim of the present study was to identify the component in the DCPD gel beads that accounts for the special root proliferation. This P-foraging root growth was observed in plots applied with either polyolefin-coated NH₄H₂PO₄ (POC-MAP) or DCPD powder instead of the DCPD gel beads. The POC-MAP neither contains Ca, alginate nor -cyclodextrin. The DCPD powder was applied in a similar number of spots with the number of DCPD gel beads. Thus, the essential component in the DCPD gel beads for the P-foraging root growth around them was P. The second aim was to examine the effect of various inorganic P sources on the P uptake of B. rapa nothovar. While significant P uptake was obtained in the plot applied with apatite from Florida, USA, sediment origin (F-Ap), almost no P uptake was obtained in that with apatite from Quebec, Canada, igneous origin in the P-deficient nonallophanic Andisol. Hence, a P-release level from F-Ap was near the lower limit for the P uptake by the B. rapa nothovar. under the present experimental conditions. These results indicate the P foraging characteristics of the Brassica roots contribute to improve the P recovery rate in the agricultural fields with localized application of moderately-soluble P fertilizers.     

Stable, stoichiometric delivery of diverse protein functions

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.     

Superoxide generation mediated by 8-nitroguanosine, a highly redox-active nucleic acid derivative

Formation of 8-nitroguanosine may be characterized as nucleic acid modification induced by nitric oxide (NO). We show here that 8-nitroguanosine is a highly redox-active nucleic acid derivative that strongly stimulated superoxide generation from various NADPH-dependent reductases, including cytochrome P450 reductase and all isoforms of NO synthase. This reaction involves these reductases in a redox cycling reaction via single-electron reduction of 8-nitroguanosine to form 8-nitroguanosine anion radical. One electron is then transferred from this radical to molecular oxygen. 8-Nitroguanosine formed in vivo may function as a potent redox cofactor that intensifies oxyradical generation by various NADPH/reductase-like enzymes and thus participates in diverse physiological and pathological events.     

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.     

Effects of soy protein isolate on LEC rats,a model of Wilson disease: mechanisms underlying enhancement of liver cell damage

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.     

Disturbed secretion of mutant adiponectin associated with the metabolic syndrome

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.     

Muscle type difference in the regulation of UCP3 under cold conditions

We found opposite regulation of uncoupling protein 3 (UCP3) in slow-twitch soleus and fast-twitch gastrocnemius muscles of rats during cold exposure. Namely, the UCP3 mRNA level was downregulated in the soleus muscles, but upregulated in the gastrocnemius muscles after a 24-h cold exposure. In the analysis of UCP3 protein, we first succeeded in detecting UCP3 short-form as well as the long-form in vivo, which levels were decreased markedly in the cold-exposed soleus muscles. However, the levels of UCP3 and cytochrome oxidase subunit IV were well maintained in the cold-exposed gastrocnemius muscles with a rise in the total mitochondrial protein level, suggesting an increase of total oxidative ability. The fast-twitch muscle rather than the slow-twitch one may play an important role in adaptive responses, including thermogenesis under acute cold exposure.