Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

Differential synthesis of glyceraldehyde-3-phosphate dehydrogenase polypeptides in stressed yeast cells

Abstract Three unlinked genes, TDH1, TDH2 and TDH3 , encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (triose-phosphate dehydrogenase; TDK) in the yeast Saccharomyces cerevisiae . We demonstrate that the synthesis of the three encoded TDK polypeptides (TDHa, TDHb and TDHc, respectively) is not co-ordinately regulated and that TDHa is only synthesised as cells enter stationary phase, due to glucose starvation, or in heat-shocked cells. Furthermore, the synthesis of TDHb, but not TDHc, is strongly repressed by a heat shock. Hence, the TDHa enzyme may play a cellular role, distinct from glycolysis, that is required by stressed cells.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

Assessing “Dangerous Climate Change”: Required Reduction of Carbon Emissions to Protect Young People,Future Generations and Nature

We assess climate impacts of global warming using ongoing observations and paleoclimate data. We use Earth’s measured energy imbalance, paleoclimate data, and simple representations of the global carbon cycle and temperature to define emission reductions needed to stabilize climate and avoid potentially disastrous impacts on today’s young people, future generations, and nature. A cumulative industrial-era limit of ∼500 GtC fossil fuel emissions and 100 GtC storage in the biosphere and soil would keep climate close to the Holocene range to which humanity and other species are adapted. Cumulative emissions of ∼1000 GtC, sometimes associated with 2°C global warming, would spur “slow” feedbacks and eventual warming of 3–4°C with disastrous consequences. Rapid emissions reduction is required to restore Earth’s energy balance and avoid ocean heat uptake that would practically guarantee irreversible effects. Continuation of high fossil fuel emissions, given current knowledge of the consequences, would be an act of extraordinary witting intergenerational injustice. Responsible policymaking requires a rising price on carbon emissions that would preclude emissions from most remaining coal and unconventional fossil fuels and phase down emissions from conventional fossil fuels.     

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.     

Differences in daily stem size variation and growth in two hybrid eucalypt clones

Understanding daily stem size variation is important as the net increment of a forest stand is ultimately determined by the accumulation of daily increment events. In this study, measurements of stem size at high spatial and temporal resolution were made using two commercial hybrid Eucalyptus clones [E. grandis × urophylla (GU) and E. grandis × camaldulensis (GC)] over a period of more than 3.5 years in order to better understand how daily stem growth is effected by variations in environmental conditions. It was evident that GU had fewer days on which net growth occurred than GC. However, when growth did occur, GU grew for longer each day and at a higher rate than GC. Thus, it still had an overall larger net stem increment during the study period. The GU clone had a markedly intermittent pattern of growth, such that growth essentially ceased under drought conditions, but responded rapidly when water became available. This confirms other findings that E. grandis × urophylla is more susceptible to drought stress than E. grandis × camaldulensis, but emphasizes that a strategy of “rapid response” when environmental conditions become temporarily non-limiting is a good one in terms of net increment at sites such as in this study.

Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III-I-IV for PpcB, as opposed to I-IV-III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.     

Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.     

Pitipeptolides C-F, antimycobacterial cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula from Guam

Pitipeptolides A (1) and B (2) are cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula from Piti Bomb Holes, Guam. Additional analogues have now been isolated by revisiting larger collections of the same cyanobacterium. The four identified analogues, pitipeptolides C–F (3–6), are the tetrahydro analogue (3), an analogue with a lower degree of methylation (4) as well as two homologues (5 and 6) of pitipeptolide A. Their structures were elucidated using 2D NMR experiments, chiral HPLC analysis and comparison with pitipeptolide A. The identified analogues showed weaker cytotoxic activities compared to the two major parent compounds, pitipeptolides A (1) and B (2), against HT-29 colon adenocarcinoma and MCF7 breast cancer cells. On the other hand, pitipeptolide F (6) was the most potent pitipeptolide in a disc diffusion assay against Mycobacterium tuberculosis. The latter finding suggests that the structure of pitipeptolides could be optimized for selective antibacterial activity.     

Zinc transporter expression profiles in the rat prostate following alterations in dietary zinc

Zinc plays important roles in numerous cellular activities and physiological functions. Intracellular zinc levels are strictly maintained by zinc homeostatic mechanisms. Zinc concentrations in the prostate are the highest of all soft tissues and could be important for prostate health. However, the mechanisms by which the prostate maintains high zinc levels are still unclear. In addition, the response of the prostate to alterations in dietary zinc is unknown. The current study explored cellular zinc levels and zinc transporter expression profiles in the lobes of the prostate during dietary marginal zinc depletion. Rats were given either zinc-adequate (ZA, 30 mg Zn/kg) or marginal zinc-deficient (MZD, 5 mg Zn/kg) diet for 9 weeks. In addition, a subgroup of the MZD rats was supplemented with phytase (1,500 unit/kg diet) to improve zinc bioavailability. We found that both zinc concentrations and ZnT2 expression in the prostate dorsolateral lobes were substantially higher than in the ventral lobes (P < 0.05). Marginal zinc depletion significantly decreased ZnT2 expression in the dorsolateral lobes (P < 0.05), and phytase supplementation had a trend to increase ZnT2 expression. In addition, of all measured zinc transporters, only ZnT2 mRNA abundance was significantly correlated to the zinc concentrations in the dorsolateral lobe. No correlations were found between zinc transporter expression and zinc concentrations in the ventral lobes. These results indicate that ZnT2 may play a significant role in the maintenance of zinc homeostasis in the prostate.