THE INCORPORATION OF [1-14C]LINOLENATE INTO LIPIDS OF DEVELOPING RAT BRAIN DURING ESSENTIAL FATTY ACID DEPRIVATION

Abstract— Twenty-one-day old essential fatty acid (EFA) deprived rats incorporated about twice the radioactivity from [1-¹⁴C]linolenate into brain lipid fractions as did controls. At 5 min after injection, 2/3 of the radioactivity was associated with the less polar lipid fraction of both control and EFA deprived animals. By 30 min after injection, 70% of the radioactivity was in the phospholipid fraction. This value increased to 90% at later time points.
The specific activity of brain phospholipids from EFA deprived rats was always greater than that of controls. This held true for the individual phosphatide fractions also. In general, phosphatidylcholine (PC) was labeled most rapidly. With increasing time intervals, radioactivity was transferred to phospha-tidylethanolamine (PE) and phosphatidylserine + phosphatidylinositol (PS + PI).
The transfer of fatty acid radioactivity into phospholipid and the distribution of radioactivity among individual phosphatides did not appear to be affected by the dietary state. However, the total amount of radioactivity incorporated was related to the amount initially retained by brain after injection. Our data suggest that one or more components of the less polar lipid fraction may act as a 'trap' or reservoir for fatty acids which are required for phospholipid synthesis.     

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of Ultrafiltration To Isolate Viruses from Seawater Which Are Pathogens of Marine Phytoplankton

Viruses may be major structuring elements of phytoplankton communities and hence important regulators of nutrient and energy fluxes in aquatic environments. In order to ascertain whether viruses are potentially important in dictating phytoplankton community structure, it is essential to determine the extent to which representative phytoplankton taxa are susceptible to viral infection. We used a spiral ultrafiltration cartridge (30,000-molecular-weight cutoff) to concentrate viruses from seawater at efficiencies approaching 100%. Natural virus communities were concentrated from stations in the Gulf of Mexico, a barrier island pass, and a hypersaline lagoon (Laguna Madre) and added to cultures of potential phytoplankton hosts. By following changes in in vivo fluorescence over time, it was possible to isolate several viruses that were pathogens to a variety of marine phytoplankton, including a prasinophyte (Micromonas pusilla), a pennate diatom (likely a Navicula sp.), a centric diatom (of unknown taxa), and a chroococcoid cyanobacterium (a Synechococcus sp.). As well, we observed changes in fluorescence in cultures of a cryptophyte (a Rhodomonas sp.) and a chlorophyte (Nannochloropsis oculata) which were consistent with the presence of viral pathogens. Although pathogens were isolated from all stations, all the pathogens were not isolated from every station. Filterability studies on the viruses infecting M. pusilla and the Navicula sp. showed that the viruses were consistently infective after filtration through polycarbonate and glass-fiber filters but were affected by most other filter types. Establishment of phytoplankton-pathogen systems will be important in elucidating the effect that viruses have on primary producers in aquatic systems.     

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [¹⁴C]methylamine. The pH of unfractionated blood cells is 7.39±0.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.     

A quantitative autoradiographic method for the measurement of local rates of brain protein synthesis

We have developed a new method for measuring local rates of brain protein synthesis in vivo. It combines the intraperitoneal injection of a large dose of low specific activity amino acid with quantitative autoradiography. This method has several advantages: 1) It is ideally suited for young or small animals or where immobilizing an animal is undesirable. 2) The amino acid injection floods amino acid pools so that errors in estimating precursor specific activity, which is especially important in pathological conditions, are minimized. 3) The method provides for the use of a radioautographic internal standard in which valine incorporation is measured directly. Internal standards from experimental animals correct for tissue protein content and self-absorption of radiation in tissue sections which could vary under experimental conditions.A preliminary report of this work was presented at the 11th annual meeting of the Society for Neuroscience, Los Angeles, California, October 18–23, 1981.     

Prostaglandin E2 on the electroencephalogram

Present study was prompted by the report from another center on the occasional occurrence of convulsions and abnormal electroencephalogram (E.E.G.) patterns in women aborted with intraamniotic prostaglandin F2α (PGF2α). Fifty four subjects were investigated by means of an E.E.G. taken before and after initiation of PGE2 administration. They included pregnant and non-pregnant patients, nearly half (23) of whom were known epileptics. One seizure was observed during PG administration in a man with daily psychomotor attacks who had not taken his anticonvulsants on the day the test was performed. PGE2 caused no alteration of the E.E.G. in subjects with a normal control tracing; in those with an abnormal E.E.G., a deterioration was seen in one and an improvement in three. It is concluded that PGE2 is not epileptogenic at doses required for termination of pregnancy.     

Simultaneous tissue profiling of eicosanoid and endocannabinoid lipid families in a rat model of osteoarthritis

We describe a novel LC method for the simultaneous and quantitative profiling of 43 oxylipins including eicosanoids, endocannabinoids, and structurally related bioactive lipids with modified acyl groups. The LC-MS/MS method uses switching at a defined time between negative and positive electrospray ionization modes to achieve optimal detection sensitivity for all the lipids. The validated method is linear over a range of 0.01–5 nmol/g (0.1–50 nmol/g for 2-arachidonoyl glycerol) with intra- and interday precision and accuracy between 1.38 and 26.76% and 85.22 and 114.3%, respectively. The method successfully quantified bioactive lipids in different tissue types in the rat, including spinal cord, dorsal root ganglia (DRGs), knee joint, brain, and plasma. Distinct regional differences in the pattern of lipid measured between tissue types were observed using principle component analysis. The method was applied to analyze tissue samples from an established preclinical rat model of osteoarthritis (OA) pain and showed that levels of 12-hydroxyeicosatetraenoic acid were significantly increased in the OA rat knee joint compared with controls, and that 15-hydroxyeicosatetraenoic acid was significantly increased in the DRGs in the model of OA compared with controls. The developed LC-MS/MS method has the potential to provide detailed pathway profiling in tissues and biofluids where the disruption of bioactive oxylipins may be involved in disease states.