Effect of Docosahexaenoic Acid on the Synthesis of Phosphatidylserine in Rat Brain Microsomes and C6 Glioma Cells

Abstract: Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 ± 0.1%) when compared with control animals (15.0 ± 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [³H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10–40 µ M ) showed a significant increase in the synthesis of [³H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.     

Characterization of human serum albumin's interactions with safranal and crocin using multi-spectroscopic and molecular docking techniques

Interaction mechanisms of human serum albumin (HSA) with safranal and crocin were studied using UV–Vis absorption, fluorescence quenching and circular dichroism (CD) spectroscopies as well as molecular docking techniques. Changes in absorbance and fluorescence of HSA upon interactions with both compounds were attributed to their binding to amino acid chromophores located in subdomains IIA and IIIA. Fluorescence secondary inner filter effect was excluded using 278 nm and 340 nm as the wavelengths of HSA's excitation and fluorescence while safranal and crocin absorbed at 320 nm and 445 nm, respectively. Stern-Volmer model revealed a static quenching mechanism involve the formation of non-fluorescent ground state complexes. Stern-Volmer, Hill, Benesi-Hilbrand and Scatchard models gave apparent binding constants ranged in 4.25 × 10³ - 2.15 × 10⁵ for safranal and 7.67 × 10³ - 4.23 × 10⁵ L mol^?1 for crocin. CD measurements indicated that 13 folds of safranal and crocin unfolded the α-helix structure of HSA by 7.47–21.20%. In-silico molecular docking revealed selective exothermic binding of safranal on eight binding sites with binding energies ranged in ?3.969 to ?6.6.913 kcal/mol. Crocin exothermally bound to a new large pocket located on subdomain IIA (sudlow 1) with binding energy of ?12.922 kcal/mol.These results confirmed the formation of HSA stable complexes with safranal and crocin and contributed to our understanding for their binding characteristics (affinities, sites, modes, forces … etc.) and structural changes upon interactions. They also proved that HSA can solubilize and transport both compounds in blood to target tissues. The results are of high importance in determining the pharmacological properties of the two phytochemical compounds and for their future developments as anticancer, antispasmodic, antidepressant or aphrodisiac therapeutic agents.     

Brain Micro vessel 12-Hydroxyeicosatetraenoic Acid Is the (S) Enantiomer and Is Lipoxygenase Derived

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.     

Single-dose murine monoclonal antibody ricin A chain immunotoxin in the treatment of metastatic melanoma: a phase I trial.

To determine the maximally tolerated dose of a ricin A chain-conjugated antimelanoma antibody (XomaZyme-Mel), 20 patients with metastatic melanoma were treated with escalating doses of the murine immunotoxin given as single intravenous infusion over 30 minutes. The starting dose was 0.6 mg/kg and was escalated in five groups to a maximum of 1.6 mg/kg. The maximally tolerated dose was 1.25 mg/kg as three of six patients treated at 1.6 mg/kg developed unacceptable toxicity. The dose-limiting toxicity consisted of profound fatigue, myalgias, and arthralgias. These occurred within 4 days and resolved in 7 to 10 days. Other non-dose-limiting toxicities encountered consisted of hypoalbuminemia, weight gain, peripheral edema, mild hypotension, and flu-like syndrome; the severity of these was also dose related. In addition, two allergic reactions occurred, one severe. There was one durable complete response of 12+ months' duration and one brief mixed response lasting 3 months. We conclude that the maximum tolerated single dose of XomaZyme-Mel is 1.25 mg/kg. Phase I studies evaluating 1.25 mg/kg given in multiple doses at 2- to 4-week intervals and phase II studies to determine the response rate of a single 1.25 mg/kg dose are warranted.     

Physiological compartmental analysis of alpha-linolenic acid metabolism in adult humans

A physiological compartmental model of alpha-linolenic acid metabolism was derived from the plasma concentration-time curves for d5-18:3n-3, d5-20:5n-3, d5-22:5n-3, and d5-22:6n-3 in eight healthy subjects. Subjects received a 1-g oral dose of an isotope tracer of alpha-linolenate (d5-18:3n-3 ethyl ester) while subsisting on a rigorously controlled beef-based diet. By utilizing the Windows Simulation and Analysis Modeling program, kinetic parameters were determined for each subject. Half-lives and mean transit times of the n-3 fatty acids in the plasma were also determined. The model predicted plasma values for the n-3 fatty acids in good accordance with the measured steady state concentrations and also predicted dietary linolenic acid intake for each subject in accordance with values determined by lipid analysis of the diet. Only about 0.2% of the plasma 18:3n-3 was destined for synthesis of 20:5n-3, approximately 63% of the plasma 20:5n-3 was accessible for production of 22:5n-3, and 37% of 22:5n-3 was available for synthesis of 22:6n-3. The inefficiency of the conversion of 18:3n-3 to 20:5n-3 indicates that the biosynthesis of long-chain n-3 PUFA from alpha-linolenic acid is limited in healthy individuals. In contrast, the much greater rate of transfer of mass from the plasma 20:5n-3 compartment to 22:5n-3 suggests that dietary eicosapentaenoic acid may be well utilized in the biosynthesis of 22:6n-3 in humans.     

Inflammatory markers in chronic kidney disease and end stage renal disease patients

Molecular Biology Reports - Chronic kidney disease (CKD) is condition characterized by a gradual loss of kidney function, patient with CKD suffering from a variety of immune system defects. This...     

Effect of dietary sesame oil as antioxidant on brain hippocampus of rat in focal cerebral ischemia

Oxidative stress may be regarded as an imbalance between free radical production and opposing antioxidant defenses. Free radical oxidative stress is implicated in rat cerebral ischemia and naturaceutical antioxidants are dietary supplements that have been reported to have neuroprotective activity. Many studies have reported dietary sesame oil (SO) as an effective antioxidant. In the present study the neuroprotective effect of dietary SO was evaluated against middle cerebral artery occlusion (MCAO)-induced cerebral ischemia injury in rats. Rats were fed on diet (20% SO) for 15 days. The middle cerebral artery of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. The antioxidant properties of brain were measured as levels of reduced glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxide (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and thiobarbituric acid reactive substance (TBARS). A decrease in the activity of all the enzymatic and non-enzymatic antioxidants was observed along with an increase in lipid peroxidation (LPO) in MCAO group. The neurobehavioral activity of rats was also observed by using videopath analyzer. Dietary SO improved the antioxidant status in MCAO+SO group when compared with MCAO group. The results of neurobehavioral activity also support our biochemical data. The results obtained suggest protective effect of SO against cerebral ischemia in rat brain through their antioxidant properties.     

Advantages of intramedullar fixation in treatment of congenital tibial pseudoarthrosis--a case report

This report describes a case of a 29-year old patient with congenital pseudoarthrosis of the distal tibia previously treated unsuccessfully by a conventional surgical method. Tibial congenital pseudoarthrosis is a rare disease characterized by segmental osseous weakness resulting in deformation of the bone and spontaneous fractures which progresses to a tibial nonunion. In our case we used intramedullary stabilization with bone grafting and six month after operation congenital pseudarthrosis of the tibia healed.     

Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation

Muscle growth is determined primarily by the balance between protein synthesis and degradation. When rates of protein synthesis are similar between individuals, protein degradation is critical in explaining differences in growth efficiency. Studies in mammals showed that muscle atrophy results from increased protein breakdown, and is associated with activation of the ubiquitin proteasome pathway, including induction of the muscle-specific ubiquitin protein ligase, MuRF1. Animals lacking MuRF1 are resistant to muscle atrophy. In fish, little is known about the role of the proteasome/MuRF pathway in muscle degradation. The objectives of this study were to: 1) clone and characterize MuRF genes in rainbow trout; and 2) determine expression of MuRF genes in association with starvation- and vitellogenesis-induced muscle atrophy in rainbow trout. We have identified full-length cDNA sequences for three MuRF genes (MuRF1, MuRF2, and MuRF3). These genes encode proteins with typical MuRF structural domains, including a RING-finger, a B-box and a Leucine-rich coiled-coil domain. RT-PCR analysis showed that MuRF genes are predominantly expressed in muscle and heart tissues. Real time PCR analysis revealed that expression of all MuRF genes is up-regulated during starvation and MuRF3 is up-regulated in vitellogenesis-associated muscle degradation. These results suggest that MuRF genes have an important role in fish muscle protein degradation. Further studies are warranted to assess the potential use of MuRF genes as tools to monitor fish muscle growth and degradation.     

Current status and future possibilities of molecular genetics techniques in <Emphasis Type="Italic">Brassica napus</Emphasis>

As PCR methods have improved over the last 15 years, there has been an upsurge in the number of new DNA marker tools, which has allowed the generation of high-density molecular maps for all the key Brassica crop types. Biotechnology and molecular plant breeding have emerged as a significant tool for molecular understanding that led to a significant crop improvement in the Brassica napus species. Brassica napus possess a very complicated polyploidy-based genomics. The quantitative trait locus (QTL) is not sufficient to develop effective markers for trait introgression. In the coming years, the molecular marker techniques will be more effective to determine the whole genome impairing desired traits. Available genetic markers using the single-nucleotide sequence (SNP) technique and high-throughput sequencing are effective in determining the maps and genome polymorphisms amongst candidate genes and allele interactions. High-throughput sequencing and gene mapping techniques are involved in discovering new alleles and gene pairs, serving as a bridge between the gene map and genome evaluation. The decreasing cost for DNA sequencing will help in discovering full genome sequences with less resources and time. This review describes (1) the current use of integrated approaches, such as molecular marker technologies, to determine genome arrangements and interspecific outcomes combined with cost-effective genomes to increase the efficiency in prognostic breeding efforts. (2) It also focused on functional genomics, proteomics and field-based breeding practices to achieve insight into the genetics underlying both simple and complex traits in canola.