Methylation methods for the quantitative analysis of conjugated linoleic acid (CLA) isomers in various lipid samples

Precise methylation methods for various chemical forms of conjugated linoleic acid (CLA), which minimize the formation of t,t isomers and allylmethoxy derivatives (AMD) with the completion of methylation, were developed using a 50 mg lipid sample, 3 mL of 1.0 N H(2)SO(4)/methanol, and/or 3 mL of 20% tetramethylguanidine (TMG)/methanol solution(s). Free CLA (FCLA) was methylated with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). CLA esterified in safflower oil (CLA-SO) was methylated with 20% TMG/methanol (100 degrees C, 5 min), whereas CLA esterified in phospholipid (CLA-PL) was methylated with 20% TMG/methanol (100 degrees C, 10 min), followed by an additional reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). Similarly, CLA esterified in egg yolk lipid (CLA-EYL) was methylated by base hydrolysis, followed by reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). These results suggest that for the quantitative analysis of CLA in lipid samples by GC, proper methylation methods should be chosen on the basis of the chemical forms of CLA in samples.     

In vitro and ex vivo inhibition of canine cyclooxygenase isoforms by robenacoxib: A comparative study

In vitro whole blood canine assays were used to quantify the inhibitory actions of the novel non-steroidal anti-inflammatory drug (NSAID) robenacoxib on the cyclooxygenase (COX) isoenzymes, COX-1 and COX-2, in comparison with other drugs of the NSAID class. COX-1 activity was determined by measuring serum thromboxane (Tx)B₂ synthesis in blood samples allowed to clot at 37 °C for 1 h. COX-2 activity was determined by measuring prostaglandin (PG)E₂ synthesis in blood samples incubated at 37 °C for 24 h in the presence of lipopolysaccharide. The rank order of selectivity for inhibition of COX-2 versus COX-1 (IC₅₀ COX-1:IC₅₀ COX-2) for veterinary drugs was highest with robenacoxib (128.8) compared to deracoxib (48.5), nimesulide (29.2), S+ carprofen (17.6), meloxicam (7.3), etodolac (6.6), R? carprofen (5.8) and ketoprofen (0.88). Selectivity expressed as the clinically relevant ratio IC₂₀ COX-1:IC₈₀ COX-2 was highest for robenacoxib (19.8) compared to deracoxib (2.3), S+ carprofen (2.5), R? carprofen (2.1), nimesulide (1.8), etodolac (0.76), meloxicam (0.46) and ketoprofen (0.21).An in vivo pharmacokinetic ex vivo pharmacodynamic study in the dog established dosage and concentration–effect relationships for single oral doses of robenacoxib over the dosage range 0.5–8.0 mg/kg. Values of C_max and AUC were linearly related to dosage over the tested range. Robenacoxib did not inhibit serum TxB₂ synthesis (COX-1) ex vivo at dosages of 0.5–4.0 mg/kg and produced only transient inhibition (at the 1 h and 2 h sampling times) at the 8 mg/kg dosage. All dosages of robenacoxib (0.5–8 mg/kg) produced marked, significant and dose related inhibition of PGE₂ synthesis (COX-2) ex vivo.The data demonstrate that in the dog robenacoxib is a highly selective inhibitor of the COX-2 isoform of COX, and significantly inhibits COX-2 and spares COX-1 in vivo when administered orally over the dosage range 0.5–4.0 mg/kg.     

Sargachromenol Isolated from Sargassum horneri Attenuates Glutamate-Induced Neuronal Cell Death and Oxidative Stress through Inhibition of MAPK/NF-κB and Activation of Nrf2/HO-1 Signaling Pathway

Oxidative stress-induced neuronal cell loss is considered to be the major mechanism underlying the pathogenesis of neurodegenerative diseases, which could be induced by a high concentration of glutamate. In this study, sargachromenol (SC) was isolated from a marine brown seaweed Sargassum horneri (S. horneri) and its neuroprotective effects against glutamate-induced oxidative stress in HT22 cells were investigated. An MTT assay was applied to assess the cytotoxicity of the SC, and the efficacies of SC were determined by flow cytometry, an analysis of ROS production, quantitative Real-Time PCR, and the Western blot assay. Our results showed that the pretreatment of SC reduced glutamate-induced apoptosis in HT22 cells via inhibiting the sub-G₁ population, DNA fragmentation, and nuclear condensation, as well as up-regulating anti-apoptotic protein (Bcl-2) and down-regulating apoptotic proteins (Bax, p53, cleaved-PARP, caspase-3, caspase-9, and cytochrome c). Additionally, SC attenuated glutamate-induced oxidative stress by suppressing mitogen-activated protein kinases (MAPKs;ERK, JNK, and p38) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling (IκBα and NF-κB p65), while activating nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling (Nrf2; HO-1, and NQO-1). Our results suggest that SC could be used as a pharmacological candidate for the prevention and treatment of neurodegenerative diseases.     

Incorporation of alpha-tocopherol and linoleic acid in fresh lambs by feeding chemically treated dietary supplements containing dl-alpha-tocopheryl acetate and sunflower oil

The effects of feeding chemically treated dietary supplements (CTDS) containing sunflower oil and dl-alpha-tocopheryl acetate (TA) on alpha-tocopherol content and fatty acid profile in edible tissues of lambs were estimated. Compared with lambs fed control diet (CD), lambs fed CD plus 250 IU of either TA or CTDS increased serum alpha-tocopherol. The CTDS-fed lambs further increased serum alpha-tocopherol by 29% over those fed CD plus 250 IU of TA. Lambs supplemented with TA or CTDS increased alpha-tocopherol in muscle and adipose tissues as compared with lambs fed CD. The CTDS-fed lambs had higher levels of alpha-tocopherol in gluteus medius (7.55 vs 6.05 mug/g), psoas major (7.43 vs 6.02 mug/g), and subcutaneous fat (12.6 vs 9.98 mug/g) compared with the TA-fed lambs. Feeding lambs CTDS also substantially increased levels of linoleic acid in the adipose tissues while decreasing the content of palmitic and oleic acids.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Studien zur Bekämpfung von Phytophthora ramorum an Rhododendron im Rahmen der Entwicklung eines Behandlungskonzeptes für Park- und Gartenanlagen

In this study dealing with the development of management strategies for P. ramorum in Rhododendron stands several plant protection and fertilizer products were tested for their suitability to control this pathogen on Rhododendron. Tests were conducted on young potted plants and abscised leaves, using the following products: phosphite (Nutri-Phite^® PK), copper hydroxide (Cuprozin^® liquid), Bacillus subtilis (Serenade^®), a mycorrhizal preparation, and a product based on ‘effective microorganisms’ (EMa). Although transmission of the pathogen from infected to healthy plants was successfully inhibited by phosphite, Cu hydroxide and B. subtilis, subsequent tests showed that only phosphite and Cu hydroxide had a significant effect on disease development. All other preparations based on antagonistic or growth promoting microorganisms had no preventive or curative effect. While B. subtilis tended to increase disease symptoms, the remaining products caused a slight reduction of disease severity only. In contrast, phosphite and Cu hydroxide almost completely inhibited infection by P. ramorum. Therefore, in field situations, the use of the ecologically safe, systemic phosphite against P. ramorum in Rhododendron stands appears to be reasonable and advisable. Along with other supporting measures within the framework of a management strategy, this will allow to prevent or slow down further spread of the disease.     

Canine model of ischemic stroke with permanent middle cerebral artery occlusion: clinical and histopathological findings

The aim of the present study was to assess the clinical and histopathological findings in a canine model of ischemic stroke. Cerebral ischemic stroke was induced by middle cerebral artery occlusion in four healthy beagle dogs using silicone plugs. They showed neurological signs of forebrain dysfunction such as reduced responsiveness, head turning, circling, postural reaction deficits, perceptual deficits, and hemianopsia. These signs gradually regressed within 4 weeks without therapy. On magnetic resonance imaging, T2 hyperintensity and T1 hypointensity were found in the cerebral cortex and basal ganglia. These lesions were well-defined and sharply demarcated from adjacent brain parenchyma with a homogenous appearance. No abnormalities of the cerebrospinal fluid were observed. At necropsy, atrophic and necrotic lesions were observed in the cerebral cortex. The cerebral cortex, basal ganglia, and thalamus were partially unstained with triphenyl-tetrazolium chloride. Histopathologically, typical features of infarction were identified in cortical and thalamic lesions. This study demonstrates that our canine model resembles the conditions of real stroke patients.     

Anti-Photoaging Effect of Hydrolysates from Pacific Whiting Skin via MAPK/AP-1, NF-κB,TGF-β/Smad,and Nrf-2/HO-1 Signaling Pathway in UVB-Induced Human Dermal Fibroblasts

Chronic exposure to ultraviolet (UV) light promotes the breakdown of collagen in the skin and disrupts the extracellular matrix (ECM) structure, leading to skin wrinkling. Pacific whiting (Merluccius productus) is a fish abundant on the Pacific coast. In the current study, we investigated the anti-wrinkle effect of hydrolysate from Pacific whiting skin gelatin (PWG) in UVB-irradiated human dermal fibroblasts and the molecular mechanisms involved. PWG effectively restored type 1 procollagen synthesis reduced by UVB-irradiation. Also, we found that PWG inhibited collagen degradation by inhibiting MMP1 expression. Furthermore, PWG decreased cytokines TNF-α, IL-6, and IL-1β associated with inflammatory responses and increased antioxidant enzymes, HO-1, SOD, GPx, CAT, and GSH content, a defense system against oxidative stress. In terms of molecular mechanisms, PWG increased collagen synthesis through activating the transforming growth factor β (TGF-β)/Smad pathway and decreased collagen degradation through inhibiting the mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1) pathway. It also suppressed the inflammatory response through suppressing the nuclear factor-κB (NF-κB) pathway and increased antioxidant enzyme activity through activating the nuclear factor erythroid 2/heme oxygenase 1 (Nrf-2/HO-1) pathway. These multi-target mechanisms suggest that PWG may serve as an effective anti-photoaging material.