Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C₁₈ precursors.     

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca²⁺ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca²⁺ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.     

Isolation of an anorexigenic protein from membranes

Satiety means an internal state that leads to termination of eating. We have isolated an anorexigenic glycoprotein (MW 50,000 dalton) from human and rat erythrocyte membrane and from rat liver plasma membrane. The substance isolated from all these membrane sources has almost same onset and offset anorectic effect in rats deprived of food for 96 h as well as in normally fed rats without any rebound. Similar properties of membrane anorectic substance and plasma satietin indicated that it has membrane origin. The results also suggest that the loss of appetite in chronic diseases involving damage or turn-over of cell membranes could be due to release of a glycopeptide from cellular membranes into the circulation.     

Formation of strand breaks and interstrand cross-links in DNA by methylglyoxal

Methylglyoxal (MG), a dietary mutagen, is present in various frequently consumed beverages and foods and in cigarette smoke. A combination of S1 nuclease hydrolysis and alkaline unwinding assay was used to demonstrate the formation of single-strand breaks and interstrand cross-links in DNA upon treatment with MG. Calf thymus DNA, when treated with increasing concentrations of MG, showed an increasing degree of S1 nuclease hydrolysis. It also showed the formation of an increasing number of strand breaks per molecule as determined by an alkaline unwinding assay. Incubation of DNA with relatively higher concentrations of methylglyoxal or prolonged treatment gave increased thermal melting temperatures and an enhanced rate of reannealing after thermal denaturation. These results indicated the formation of interstrand cross-links. Upon treatment with MG, A-T base pair depleted DNA showed a reduced number of single-strand break formation. It also showed a significantly lower decrease in Tm as compared with MG-treated normal DNA. These results showed that under the conditions used, MG primarily reacts with A-T base pairs in duplex DNA.     

Liposome uptake into human colon adenocarcinoma cells in monolayer, spinner, and trypsinized cultures

The purpose of this study was to begin investigating the nature of liposome interactions with colon tumor cells. Thus, experiments were performed to study the uptake and incorporation of multilamellar and of reverse-phase evaporation liposomes of neutral charge into monolayers, suspended spinner cultures, and trypsinized cells of a human colon adenocarcinoma cell line, LS174T. The results showed that the same tumor cells cultured under each condition exhibited a distinct pattern of vesicle uptake as determined at 0, 15, 30, 60, and 120 min. In monolayer cultures of LS174T cells, the uptake of liposomes bearing [3H]actinomycin D in the lipid bilayers was linear throughout the incubation period. In contrast, in trypsinized and spinner suspension cultures, uptake of liposomes was biphasic. There was a proportional uptake of both liposome (labeled with [3H]phosphatidylcholine or [14C]cholesterol) and of actinomycin D (trace labeled with 3H) into the cells under all culture conditions, indicating quantitative delivery of the drug with the intact lipid vesicle. Although the amount of actinomycin D presented to tumor cells by the two liposomes was equivalent, reverse-phase evaporation liposomes were more effective than multilamellar vesicles in inhibiting uridine uptake. In the presence of excess liposomes (10 times the uptake studies), saturation of the tumor cell surface occurred by 120 min. However, the liposomes remained accessible to enzymatic removal for 60 min. Liposome-saturated tumor cells remained refractory to further binding of liposomes for at least 2 hr. The results thus revealed that differences in cell uptake were due to the state of the target cells and not the liposome types, or their differential leakage of labels.     

Utilization of Glucose in Heterotrophic Media by Thiobacillus intermedius

The growth yield of Thiobacillus intermedius is greater in glucose-yeast extract or glucose-casein hydrolysate broth than in comparable media without glucose. The quantity of glucose utilized in the glucose-supplemented media is much greater than the increase in cell yield observed relative to the unsupplemented media. Addition of glucose to cell-free extracts of glucose-yeast extract or glucose-casein hydrolysate grown cells results in the reduction of endogenous cytochrome c. Thus, in these media, glucose serves as a source of energy. This is in contrast to thiosulfate-glucose broth in which glucose provides only cell carbon. The presence of thiosulfate in glucose-casein hydrolysate broth results in a marked decrease in glucose consumption. Cytochrome c in extracts of cells grown in this medium is not reduced by glucose addition. The data suggest that thiosulfate prevents the utilization of glucose for energy generation. The final growth yield in glucose-casein hydrolysate broth is directly proportional to the initial glucose concentration, although not all the glucose was utilized even at the lowest concentration tested. This effect may be due to an inefficient glucose transport in this organism.     

Regulation of Glucose Metabolism in Thiobacillus intermedius

Glucose-yeast extract or glucose-casein hydrolysate-grown Thiobacillus intermedius cells, which use glucose for energy generation, possess high specific activities of the Entner-Doudoroff pathway and related enzymes, 6-phosphogluconate dehydrase, 2-keto-3-deoxy-6-phosphogluconate aldolase, glucokinase, and glucose-6-phosphate dehydrogenase, but low activities of enzymes unique to the pentose shunt and Embden-Meyerhof pathways. Although the synthesis of the latter enzymes remains largely unaffected by the growth environment, that of the former is stimulated by glucose. Radiorespirometric measurements demonstrate an early and parallel respiration of glucose carbon atoms one and four in glucose-casein hydrolysate broth. It is concluded that the Entner-Doudoroff pathway performs an energetic role in glucose metabolism by T. intermedius with the pentose shunt and Embden-Meyerhof pathways functioning mainly in biosynthesis. The presence of thiosulfate in the growth medium inhibits the synthesis of the Entner-Doudoroff pathway and related enzymes. In addition, both thiosulfate and glucose inhibit the synthesis of the Krebs cycle enzymes, nicotinamide adenine dinucleotide phosphate-linked isocitrate and alpha-ketoglutarate dehydrogenases. Thus, repression of enzymes is of significance in the adaptation of T. intermedius to its nutritional environment. The activity of glucose-6-phosphate dehydrogenase of T. intermedius is inhibited by adenosine triphosphate. Such a control could afford the organism a mechanism to regulate the flow of glucose into major energetic and biosynthetic routes.     

A long-term follow-up study on the efficacy of further attenuated live measles vaccine, Biken CAM vaccine

Antibody persistence was measured in 39 children in an open community 12-13 years after immunization against measles with further attenuated live vaccine, Biken CAM. Serum samples of the children taken every two or three years after vaccination had higher, lower, or the same HI antibody titers as those in samples taken 6 weeks after vaccination. These differences reflected a decrease in the titer in some children and subclinical natural reinfection in others. However, all the children still retained detectable antibody in 12 or 13 years after vaccination, indicating long-term persistence of immunity after immunization with Biken CAM vaccine. For evaluation of the protective efficacy of the vaccine, matched controls were studied during the same period. Serological examination revealed that 97.5% of the controls were infected with measles and contracted the disease. In contrast, none of the vaccinees developed clinical infection after close contact with measles patients.     

The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.