Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.     

The Positive Correlations Between the Activity of Superoxide Dismutase and Dehydration Tolerance in Wheat Seedlings

Damage to crops by drought is still a serious problem in large areas of the world. Considerable research has been undertaken to discover the mechanisms of drought injury and drought resistance of plants. However, the critical features of drought injury have not yet been identified. In the past ten years a free radical hypothesis has been suggested to account for subcellular damage caused by severe environments. Superoxide (oxygen radical) is normally produced in hydrated tissues. It is controlled by free radical scavenging reactions. One such scavenger is the enzyme superoxide dismutase (SOD). Under water stress, production of excess free radicals may occur in dehydrated plant tissues and this probably damages the membranes by causing peroxidation of the lipid components. So far few studies have been done to determine if drought injury is correlated with the free radical mechanism. In the present study, the SOD activities in wheat seedlings under water stress have been investigated by measuring the photoreduction of nitro blue tetrazolium using a spectrometric method. Meanwhile, the viabilities of wheat seedlings during drying were followed by tetrazolium test. These results provided information on the relationship between SOD activity and the dehydration tolerance of the plant. Results indicated that SOD activity changed with the time after germination. The activity of SOD of 24 h seedlings was 1.9 times higher than those of 72 h seedlings based on fresh weight. SOD activity in shoot was also higher than in root. These results were consistent with the results obtained from rating of the viabilities of seedlings during drying. The 24h seedlings were more tolerant of dehydration than 72 h seedlings and root were more sensitive of drought than shoot. In addition, shoot and root tips showed the higher SOD activities than non-tip region and they also showed a higher survival ability upon dehydration. In dehydration and subsequent rehydration, SOD activity, different from many other enzymes in plants, increased rather than declined during drying. After rehydration SOD activity returned to nearly the original level. Therefore, the positive correlations were found to exist between SOD activity and dehydration tolerance. It is reasonable to suggest that SOD enzyme may play a protective role against damage caused by free radicals which may be produced excessively during dehydration in wheat seedling.     

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate, P < 0.001) at a concentration of 3 μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P < 0.001 at 3 μM) but the peptide was cytotoxic at this concentration.     

Comparative study between the free radicals and tumor markers in patients with gastrointestinal tumors

The tumorous processes, increased level of tumor markers and the change of free radical status are associated in patents with gastrointestinal tumors. The aim of this study was to examine free radical status and tumor markers in patients with gastrointestinal tumors. Two hundered and thirteen patients with gastrointestinal tumor were examined. In the control group 44 non-tumorous patients were examined. The tumor markers (CEA, CA 19-9, CA 72-4, AFP, TPA, AGP) and free radical status (total scavenger capacity) were diagnosed using venal blood (obtained by LIA-kits and chemiluminescent methods, LIA-mAT and the Lumat Berthold instrument). It has been found that: (1) The results showed that the tumor markers, TPA and AGP are the best indicators for the tumorous process; (2) The AGP serum level was in the operable case 91.56+/-38.29 mg/dl meanwhile its value was, 128.46+/-47.62 mg/dl (P<0.001) in the inoperable case; and (3) The TPA value was 118.37+/-155.47 mg/dl in the operable case, (P<0.001) while its value was 227.32+/-244.39 mg/dl in inoperable cases. The significantly high levels of the plasma Chemiluminescent Light Intensity (CLI)=28.12+/-25.96; was obtained in patients with rectal tumors vs. in the control cases CLI= 4.27+/-5.12 RLU% (Relative Light Unit; mean+S.D.; P<0.005). In six of these cases, the free radical status examination indicated the presence of the tumor, even though the level of tumor markers was normal. It has been concluded that the testing of both regular tumor markers and free radical status has an important role in the diagnosis and monitoring of the patients with gastrointestinal tumors.     

Redesign of LAOBP to bind novel l‐amino acid ligands

Computational protein design is still a challenge for advancing structure‐function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

The inclusion complex of 4-hydroxynonenal with a polymeric derivative of β-cyclodextrin enhances the antitumoral efficacy of the aldehyde in several tumor cell lines and in a three-dimensional human melanoma model

4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, β-cyclodextrin (βCD) polymers are playing a very important role in drug formulation by virtue of the ability of βCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several βCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of βCD, the βCD–poly(4-acryloylmorpholine) conjugate (PACM-βCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-βCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.     

Combination of Ascorbate and α-Tocopherol as a Preventive Therapy against Structural and Functional Defects of Erythrocytes in Visceral Leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of α-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of α-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with α-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with α-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

雌牛生殖道内游离氨基酸种类及含量分析

根据黄体的大小和形态，判断屠宰母牛的发情时期，分别收集发情第3天(D3)和第7天(D7)的输卵管液(OF)和子宫液(UF)，通过HPLC分析游离氨基酸种类及含量。总共检测到23种氨基酸，包括除Cys外的19种必需和非必需氨基酸，以及另外四种(βAla,Tau,Orm和Cit)非蛋白质氨基酸。OFD3、UFD3、OFD7和UFD7的游离氨基酸总量分别为31．6、46．9、26．3和17．2mmol/L，其中，Gly含量最高，分别为14．7、14．4、11．1和4．4，其次为Glu、Gln和A1a，其他氨基酸的含量均接近或低于lmmol/L。结果表明：氨基酸总量及个别氨基酸含量在不同体液及不同发育时期均存在一定程度的差异.