Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

The Discovery of Stereoselectivity at Biological Receptors: Arnaldo Piutti and the Taste of the Asparagine Enantiomers—History and Analysis on the 125th Anniversary

In 1886, Italian chemist Arnaldo Piutti isolated, for the first time, d ‐asparagine, the enantiomer of the known l ‐asparagine. He obtained 100 g of d ‐asparagine from 6500 kg of vetches. Using an ingenious synthetic scheme, Piutti established the chemical structure of asparagine and demonstrated that his isolation of d ‐asparagine from plants was not the result of the racemization of l ‐asparagine during the extraction procedure. He found a striking difference in the taste of asparagine: l ‐asparagine was without taste, while d ‐asparagine was intensely sweet. This was the first example of enantioselectivity in a receptor‐mediated biological activity. Receptors constitute one of the most important and most intensively studied phenomena in biology, and enantioselectivity in receptor‐mediated activity, including at the sweetness receptor, is today an important and commonly seen aspect of receptor function. Therefore, Piutti's discovery, although made ca. 15 years before the emergence of the receptor concept, was a milestone. The publication of Piutti's asparagine work prompted several eminent scientists, including Louis Pasteur and Arthur Cushny, a leading pharmacologist of the time, to remark on the importance of the discovery. Piutti also carried out investigations in many other fields, e.g., other organic compounds and reactions, pharmaceuticals, alimentary products, radioactivity, noble gases, and spectroscopy. Considerable progress has been made in recent decades concerning the biology and chemistry of sweet taste, but the details of the interactions of chiral molecules with the sweetness receptor remain poorly understood. Piutti and his discovery are largely forgotten today; they deserve the attention of the chirality and receptor “communities.” Chirality 24:959–976, 2012. © 2012 Wiley Periodicals, Inc.     

Residue cysteine 232 is important for substrate transport of neutral amino acid transporter,SNAT4

SNAT4 is a system A type amino acid transporter that primarily expresses in liver and mediates the transport of L-alanine. To determine the critical amino acid residue(s) involved in substrate transport function of SNAT4, we used hydrosulfate cross-linking MTS reagents - MMTS and MTSEA. These two reagents caused inhibition of L-alanine transport by wild-type SNAT4. There are 5 cysteine residues in SNAT4 and among them; residues Cys-232 and Cys-345 are located in the transmembrane domains. Mutation of Cys-232, but not Cys-345, inhibited transport function of SNAT4 and also rendered SNAT4 less sensitive to the cross-linking by MMTS and MTSEA. The results suggested that TMD located Cys-232 is an aqueous accessible residue, likely to be located close to the core of substrate binding site. Mutation of Cys-232 to serine similarly attenuated the transport of L-alanine substrate. Biotinylation analysis showed that C232A mutant of SNAT4 was equally capable as wild-type SNAT4 of expressing on the cell surface. Moreover, single site mutant, C232A was also found to be more resistant to MTS inhibition than double mutant C18A,C345A, further confirming the aqueous accessibility of Cys-232 residue. We also showed that mutation of Cys-232 to alanine reduced the maximal velocity (Vmax), but had minimal effect on binding affinity (Km). Together, these data suggest that residue Cys-232 at 4^th transmembrane domain of SNAT4 has a major influence on substrate transport capacity, but not on substrate binding affinity.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Plasma glucocorticoid concentrations in free-ranging platypuses (Ornithorhynchus anatinus): response to capture and patterns in relation to reproduction

Energy demands in the platypus are likely to increase in the breeding season, which occurs from winter to early spring. Glucocorticoids, which play a major role in energy mobilisation, were measured in consecutive blood samples from free-ranging adults at approximately monthly intervals throughout the year. Glucose and free fatty acids were also measured in some samples. Plasma concentrations of glucocorticoids rose significantly during the first 30 min after capture, accompanied by a rise in free fatty acids, but no corresponding increase in glucose concentrations. We observed a strong pattern in plasma glucocorticoids in samples collected within 15 min of capture (indicative of pre-disturbance concentrations) in different phases of the annual reproductive cycle, with significantly higher levels in both males and females in the breeding season compared to the non-breeding season. These data, and the decline in tail fat stores that occur towards the end of the mating period (around October), suggest that platypus experience high-energy demands during this phase of reproduction. Plasma glucocorticoid concentrations in females sampled during the lactation period (October-February) were relatively low, and similar to those in females sampled in the non-breeding, non-lactation period (March-June). The latter requires further investigation as results may have been influenced by sampling limitations.     

Environmental factors affecting the lipid metabolism in Rosmarinus officinalis L

The effect of some environmental factors on the lipid metabolism was studied in two chemotypes of Rosmarinus officinalis L. Epicuticular hydrocarbons (EH), epicuticular fatty acids (EFA), whole leaf fatty acids (WLFA) and essential oils (EO) were extracted and analysed by GC-MS during winter 1991–1992 and related to temperature and moisture variations. Leaf fresh and dry wts were determined along with some morphophysiological parameters such as specific leaf weight (SLW) and specific leaf area (SLA). Leaf areas were calculated by image analysis and statistically processed as for chemical data. The results indicated that in R. officinalis the response to some environmental factors, with particular reference to temperature and moisture, was an increase in epicuticular hydrocarbons and a decrease in epicuticular fatty acids, leaf fatty acids and essential oils. Qualitative changes in the chemical composition of the above lipid classes were found to be correlated with temperature changes. From a chemosystematic viewpoint, a clear separation between the two chemotypes was achieved only when epicuticular hydrocarbons and essential oils were considered as chemosystematic characters.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

Paradoxical effect of l-arginine: Acceleration of endothelial cell senescence

We have recently shown that inhibition of nitric oxide (NO) synthesis by asymmetrical dimethylarginine (ADMA) accelerated endothelial cell (EC) senescence which was prevented by coincubation with l-arginine; however the effect of long-term treatment of l-arginine alone on senescence of ECs have not been investigated. Human ECs were cultured in medium containing different concentrations of l-arginine until senescence. l-Arginine paradoxically accelerated senescence indicated by inhibiting telomerase activity. Moreover, l-arginine decreased NO metabolites, increased peroxynitrite, and 8-iso-prostaglandin F_2α formation. In old cells, the mRNA expression of human amino acid transporter (hCAT)2B, the activity and protein expression of arginase II were upregulated indicated by enhanced urea, l-ornithine, and l-arginine consumption. Inhibition of arginase activity, or transfection with arginase II siRNA prevented l-arginine-accelerated senescence. The most possible explanation for the paradoxical acceleration of senescence by l-arginine so far may be the translational and posttranslational activation of arginase II.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

花脸香蘑菌丝体氨基酸分析

以花脸香蘑菌丝体为实验材料,用美国戴安AAA型氨基酸分析仪测定了18种氨基酸的组成及含量,得出花脸香蘑菌丝体18种氨基酸质量分数为259.7 g.kg-1干样,其中100 g干样中谷氨酸2.84 g,天冬氨酸2.56 g,亮氨酸2.24 g,赖氨酸1.79 g,苯丙氨酸1.76 g,蛋氨酸0.47 g,人体必需氨基酸质量分数为133.7 g.kg-1干样,占所测氨基酸的51.49%,因此花脸香蘑菌丝体具有很高的营养价值。