Rat hypocretin/orexin neurons are maintained in a depolarized state by TRPC channels

In a previous study we proposed that the depolarized state of the wake-promoting hypocretin/orexin (hcrt/orx) neurons was independent of synaptic inputs as it persisted in tetrodotoxin and low calcium/high magnesium solutions. Here we show first that these cells are hyperpolarized when external sodium is lowered, suggesting that non-selective cation channels (NSCCs) could be involved. As canonical transient receptor channels (TRPCs) are known to form NSCCs, we looked for TRPCs subunits using single-cell RT-PCR and found that TRPC6 mRNA was detectable in a small minority, TRPC1, TRPC3 and TRPC7 in a majority and TRPC4 and 5 in the vast majority (～90%) of hcrt/orx neurons. Using intracellular applications of TRPC antibodies against subunits known to form NSCCs, we then found that only TRPC5 antibodies elicited an outward current, together with hyperpolarization and inhibition of the cells. These effects were blocked by co-application of a TRPC5 antigen peptide. Voltage-clamp ramps in the presence or absence of TRPC5 antibodies indicated the presence of a current with a reversal potential close to -15 mV. Application of the non-selective TRPC channel blocker, flufenamic acid, had a similar effect, which could be occluded in cells pre-loaded with TRPC5 antibodies. Finally, using the same TRPC5 antibodies we found that most hcrt/orx cells show immunostaining for the TRPC5 subunit. These results suggest that hcrt/orx neurons are endowed with a constitutively active non-selective cation current which depends on TRPC channels containing the TRPC5 subunit and which is responsible for the depolarized and active state of these cells.     

The radiolysis of aqueous ammonium cyanide: Polymers of interest to chemical evolution studies

Some of the polymers isolated from the mixture of products obtained upon irradiation of dilute oxygen-free solutions of NH₄CN with gamma rays from a⁶⁰Co source were examined. Their molecular weights were estimated to be in the range 4000–20000 daltons; elements of their structure were inferred from chemical and spectroscopic studies; and their interactions with nucleic acid components were examined. Significance of these results for chemical evolution studies is summarized.     

Properties of glutamate dehydrogenase from developing maize endosperm

Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o₂) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca²⁺. Saturation curves for (NH₄)₂SO₄ and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca²⁺, but substrate inhibition occurred without Ca²⁺. The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca²⁺ and Mn²⁺, but not by Cu₂₊ and Mg²⁺.     

Activity and distribution of nitrogen-metabolism enzymes in the developing maize kernel

The activities of glutamine synthetase (EC 6.3.1.2), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and soluble protein content in the developing endosperm and embryo of normal (Oh-43) and mutant (Oh-430₂) maize were investigated. Maize inbred lines were grown under field conditions and all plants were self-pollinated. Ears for experiments were harvested over the period of 15 lo 45 days after pollination. After pollination kernel capacity for soluble protein synthesis is located mainly in the endosperm. This progressively decreases and about 40 days after pollination soluble protein synthesis is taken over by the embryo. Comparative data on the activity of the investigated enzymes in the embryo and endosperm indicate that the capacity for synthesis of glutamine and glutamate predominates in the embryo tissue, whereas transamination processes at the initial stages of the embryo development are less intensive than their counterparts in the endosperm. The roles of embryo and endosperm subsequently interchange. Biosynthetic processes of soluble precursors for protein synthesis in the embryo and endosperm of the developing kernel are mutually coordinated.     

Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development⋆

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1 mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.     

Post-transcriptional control of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression: formation of a nuclear HuR-C/EBPbeta mRNA complex determines the amount of message reaching the cytosol

In 3T3-L1 cells, HuR is constitutively expressed and prior to induction of differentiation localized predominantly to the nucleus. Within minutes of induction of differentiation, nuclear HuR binds to its target ligand mRNAs, and the complexes appear to move to the cytosol. One ligand mRNA is the CCAAT/enhancer-binding protein beta (C/EBPbeta) message. To examine the function and importance of the HuR-C/EBPbeta interaction, retroviral expression constructs were created in which the HuR binding site was altered by deletion (betadel) or deletion and substitution (betad/s). Expression of these constructs in murine embryonic fibroblasts resulted in significant adipose conversion relative to those cells expressing wild type C/EBPbeta. C/EBPbeta protein content was increased markedly in both betadel and betad/s, which correlated with the acquisition of the adipocyte phenotype. Analysis of the betad/s cell line demonstrated a robust expression of C/EBPalpha coincident with peroxisome proliferator-activated receptor gamma expression. Total C/EBPbeta mRNA accumulation indicated no difference between cells harboring either the wild type C/EBPbeta cDNA or betad/s construct. However, cytosolic C/EBPbeta mRNA in the cells expressing the betad/s construct was maintained at levels between 2- and 7-fold greater than in the cells expressing the wild type construct. Alteration in mRNA half-life was not responsible for the increased accumulation. Mechanistically, these data suggest that HuR binding results in nuclear retention of the C/EBPbeta mRNA and is consistent with HuR control, at least in part, of mRNA processing.     

Zinc or Magnesium Supplementation Modulates Cd Intoxication in Blood,Kidney, Spleen,and Bone of Rabbits

The objective of this study was to examine the influence of oral supplementation with Zn or Mg on Cd content in the blood and organs of rabbits exposed to prolonged Cd intoxication. Rabbits were divided into the following groups: Cd group-received orally every day for 4 weeks 10 mg Cd/kg body weight (b.w.), Cd+Zn group and Cd+Mg group-exposed to Cd and supplemented with 20 mg Zn/kg b.w. or 40 mg Mg/kg b.w. 1 h after Cd treatment. Cd content in biological material was determined by atomic absorption spectrophotometry. Blood Cd concentration was determined in all investigated groups at time 0 and after 10, 14, 18, 22, 25, and 28 days, whereas Cd content in the brain, heart, lungs, liver, kidney, spleen, pancreas, skeletal muscle, and bone was determined after 28 days. Blood Cd concentration was significantly increased in all groups from the 14th day of Cd intoxication and lasted till the end of the experiment. Zn or Mg supplementation significantly reduced blood Cd content on the 18th and 25th days. Supplementation with Zn or Mg significantly decreased Cd concentration in the kidney, spleen, and bone and, in addition, Zn reduced Cd content in the brain. Supplementation with Zn or Mg in Cd-intoxicated rabbits caused similar reduction of blood Cd concentration; however, reduction of tissue Cd content was more pronounced in Zn- than in Mg-supplemented group.     

Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages

Oxidative stress is considered to be implicated in the pathophysiology of breast cancers. In this study we investigated the level of oxidative stress and antioxidant (AO) status in the blood of breast cancer patients of different ages. The level of lipid hydroperoxides (LP) was measured in blood plasma and the activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) enzymes, as well as the level of total glutathione (GSH) and CuZnSOD protein were measured in blood cells of breast cancer patients and age-matched healthy subjects. Our results showed that breast carcinoma is related to increase of lipid peroxidation in plasma with concomitant decrease of AO defense capacity in blood cells, which becomes more pronounced during aging of the patients. Suppression of CuZnSOD activity related to breast cancer is most likely caused by decreased de novo synthesis of this enzyme. Similar patterns of suppression in CuZnSOD and CAT activities related to aging were recorded both in controls and patients. Age-related decrease in CuZnSOD activity seems not to be caused by altered protein levels of this enzyme. Suppression of AO enzymes associated with breast cancer and aging is most likely the cause of increased levels of reactive oxygen species (ROS). Our results indicate significant role of oxidative-induced injury in the breast carcinogenesis, particularly during the later stages of aging. Overall, our data support the importance of endogenous AOs in the etiology of breast cancer across all levels of predicted risk.