3-isobutylmethylxanthine inhibits hepatic urea synthesis: protection by agmatine

We previously showed that agmatine stimulated hepatic ureagenesis. In this study, we sought to determine whether the action of agmatine is mediated via cAMP signaling. A pilot experiment demonstrated that the phosphodiesterase inhibitor, 3-isobutylmethylxanthine (IBMX), inhibited urea synthesis albeit increased [cAMP]. Thus, we hypothesized that IBMX inhibits hepatic urea synthesis independent of [cAMP]. We further theorized that agmatine would negate the IBMX action and improve ureagenesis. Experiments were carried out with isolated mitochondria and (15)NH(4)Cl to trace [(15)N]citrulline production or [5-(15)N]glutamine and a rat liver perfusion system to trace ureagenesis. The results demonstrate that IBMX induced the following: (i) inhibition of the mitochondrial respiratory chain and diminished O(2) consumption during liver perfusion; (ii) depletion of the phosphorylation potential and overall hepatic energetic capacity; (iii) inhibition of [(15)N]citrulline synthesis; and (iv) inhibition of urea output in liver perfusion with little effect on [N-acetylglutamate]. The results indicate that IBMX directly and specifically inhibited complex I of the respiratory chain and carbamoyl-phosphate synthase-I (CPS-I), with an EC(50) about 0.6 mm despite a significant elevation of hepatic [cAMP]. Perfusion of agmatine with IBMX stimulated O(2) consumption, restored hepatic phosphorylation potential, and significantly stimulated ureagenesis. The action of agmatine may signify a cascade effect initiated by increased oxidative phosphorylation and greater ATP synthesis. In addition, agmatine may prevent IBMX from binding to one or more active site(s) of CPS-I and thus protect against inhibition of CPS-I. Together, the data may suggest a new experimental application of IBMX in studies of CPS-I malfunction and the use of agmatine as intervention therapy.     

Molecular ecology of nifH genes and transcripts in the eastern Mediterranean Sea

The eastern Mediterranean Sea is one of the most extreme oligotrophic oceanic regions on earth in terms of nutrient concentrations and primary productivity. Nitrogen fixation has been suggested to contribute to the high N : P molar ratios of approximately 28:1 found in this region. Surprisingly, no molecular biological work has been performed in situ to assess whether N(2) fixation genes actually occur in the eastern Mediterranean Sea, or to determine which organisms are responsible for this process. In this study, we examined the presence and expression of nitrogenase genes (nifH) in the upper water layer of the eastern Mediterranean. Clone libraries constructed from both DNA and reverse-transcribed PCR-amplified mRNA were examined and compared. We observed different nifH genes from diverse microbial groups, such as Cyanobacteria, Proteobacteria and methanogenic Archaea. Interestingly, numerous phylotypes were observed in coastal stations at the DNA level but none were active. However, in far offshore stations, the phylotypes observed at the DNA level were the ones that were actually active. Our preliminary study revealed diverse diazotrophs that possess and express nifH genes, which may support N(2) fixation in the eastern Mediterranean Sea.     

Ethylene action blockade and cold storage affect ripening of ‘Golden’ papaya fruit

The purpose of this work was to evaluate the effects of ethylene action blockade and cold storage on the ripening of ‘Golden’ papaya fruit. Papayas harvested at maturity stage 1 (up to 15% yellow skin) were evaluated. Half of the fruits, whether treated or not treated with 100 nL L⁻¹ of 1-methylcyclopropene (1-MCP), were stored at 23°C, while the other half were stored at 11°C for 20 days prior to being stored at 23°C. Non-refrigerated fruits receiving 1-MCP application presented a reduction in respiratory activity, ethylene production, skin color development and pectinmethylesterase activity. Even with a gradual increase in ethylene production at 23°C, fruits treated with 1-MCP maintained a high firmness, but presented a loss of green skin color. Cold storage caused a decrease in ethylene production when fruits were transferred to 23°C. The results suggest that pulp softening is more dependent on ethylene than skin color development, and that some processes responsible for loss of firmness do not depend on ethylene.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erratum

Costal and auricular cartilage obtained from mutant rabbits exhibiting lysozyme deficiency has been found to be identical to similar tissue from control animals in a variety of biochemical parameters. These data seriously question the putative role of lysozyme as a structural component of cartilage.     

Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

Never Resting Brain: Simultaneous Representation of Two Alpha Related Processes in Humans

Brain activity is continuously modulated, even at “rest”. The alpha rhythm (8–12 Hz) has been known as the hallmark of the brain''s idle-state. However, it is still debated if the alpha rhythm reflects synchronization in a distributed network or focal generator and whether it occurs spontaneously or is driven by a stimulus. This EEG/fMRI study aimed to explore the source of alpha modulations and their distribution in the resting brain. By serendipity, while computing the individually defined power modulations of the alpha-band, two simultaneously occurring components of these modulations were found. An ‘induced alpha’ that was correlated with the paradigm (eyes open/ eyes closed), and a ‘spontaneous alpha’ that was on-going and unrelated to the paradigm. These alpha components when used as regressors for BOLD activation revealed two segregated activation maps: the ‘induced map’ included left lateral temporal cortical regions and the hippocampus; the ‘spontaneous map’ included prefrontal cortical regions and the thalamus. Our combined fMRI/EEG approach allowed to computationally untangle two parallel patterns of alpha modulations and underpin their anatomical basis in the human brain. These findings suggest that the human alpha rhythm represents at least two simultaneously occurring processes which characterize the ‘resting brain’; one is related to expected change in sensory information, while the other is endogenous and independent of stimulus change.