Turnover of mitochondrial components of normal and essential fatty acid-deficient rats

1. Essential fatty acid (EFA)-deficient and control rats were injected intraperitoneally with [(32)P]phosphate, l-[(35)S]methionine and [2-(14)C]acetate. The animals were killed at various time-intervals after injection and their liver mitochondria fractionated into soluble protein, insoluble protein, and lipid. 2. The (35)S was assayed in the protein fractions and (32)P and (14)C were assayed in the lipid fraction. Curves of log (specific activity) plotted against time were prepared for the different fractions. 3. There was no significant difference between the insoluble protein results for control and EFA-deficient animals, both sets of results indicating the presence of a single component of half-life 9 days. 4. There was no significant difference between the soluble protein results for the two sets of animals and both sets of results indicated the presence of at least two components. 5. The [(32)P]-phospholipid results indicate that in the control animals the liver mitochondrial phospholipids contain components of half-life 1.6 and 10 days whereas the mitochondrial phospholipids of the EFA-deficient animals contain components of half-life 3 and 29 days. 6. The specific activity of mitochondrial [(14)C]phospholipid initially fell rapidly in both groups of animals, but after 17 days there was no further significant decrease. A fast component with maximum half-life 2-4 days was clearly demonstrated for both groups of animals. Whether or not these results also indicate the presence of a very long-lived mitochondrial phospholipid is discussed.     

Leukotriene B formation by neutrophils from essential fatty acid-deficient rats

Analysis of neutrophil phospholipids from rats fed an essential fatty acid-deficient diet revealed a 33% reduction in arachidonate and a 90% reduction in linoleate compared to neutrophil phospholipids of rats fed a normal diet. The neutrophil phospholipids from rats fed the essential fatty acid-deficient diet also contained significant amounts of 5,8,11-eicosatrienoate, a fatty acid not found in the neutrophils of rats fed a normal diet. Analysis of the production of leukotrienes of the B series by ionophore-stimulated neutrophils from rats fed an essential fatty acid-deficient diet revealed a 87% reduction in leukotriene B4 compared to neutrophils from rats fed a normal diet even though the arachidonate content was reduced by only 34%. Essential fatty acid-deficient neutrophils converted endogenous 5,8,11-eicosatrienoic acid to leukotriene A3 and its nonenzymatic degradation products, but little or no leukotriene B3 was formed. Neutrophils from rats fed a normal diet incubated with ionophore and exogenous 5,8,11-eicosatrienoate also produced leukotriene A3 and its nonenzymatic degradation products but little or no leukotriene B3. Exogenous 5,8,11-eicosatrienoate incubated with ionophore-stimulated normal neutrophils caused a dose-dependent inhibition of leukotriene A hydrolase resulting in diminished production of leukotriene B4 from endogenous arachidonate. Assays of leukotriene A hydrolase in the 10,000 X g supernatant fraction of a homogenate of RBL-1 cells revealed that a lipoxygenase metabolite of 5,8,11-eicosatrienoate rather than 5,8,11-eicosatrienoate itself is the inhibitor of leukotriene A hydrolase. Thus the finding that leukotriene B4 production by neutrophils from essential fatty acid-deficient rats is diminished out of proportion to the decrease in arachidonate content appears to be due to inhibition of leukotriene A hydrolase by a lipoxygenase metabolite.     

Visual acuity of essential fatty acid-deficient rats

1. Rats were maintained on a diet deficient in fat and on a normal diet of rat cubes. 2. Rats were trained to discriminate between vertical and horizontal striations. The minimal stripe width that could be used for discrimination was determined. 3. In bright illumination (0·7 or 4·5 ft.lamberts) both deficient and normal rats had the same ability to discriminate between black and white stripes. 4. With an illuminance of 0·002 ft.lambert, supplemented rats could discriminate as efficiently as at 0·7 ft.lambert, but deficient animals were unable to discriminate at 0·002 ft.lambert. 5. Control rats had 14% of docosahexaenoic acid in their retinal fats but the deficient rats had only 1%. 6. Deficient animals had no vitamin A stores in the liver whereas the control animals had about 190 i.u./g. 7. The visual acuity of the rats used was about 45′ of arc.     

Effects of infusion of some prostaglandins in essential fatty acid-deficient and normal rats

Infusion of 1 mg/kg per day of prostaglandin E(1) (PGE(1)) for 2 and 7 wk failed to correct the dermal signs of essential fatty acid (EFA) deficiency in rats despite the known conversion of EFA to certain prostaglandins. PGE(1) caused no significant changes in serum cholesterol, triglycerides, or phospholipids or in liver neutral lipids in EFA-deficient or normal rats. In normal rats epinephrine-induced lipolysis was greater in fat pads from infused than from untreated rats. The effect on epinephrine-induced lipolysis was greater after the 7 wk infusion than after the 2 wk infusion. The 7 wk infusion also lowered plasma free fatty acid (FFA) concentrations. Infusion of PGE(2) and PGF(2alpha) in combination for 4 wk had no significant effect on either dermal signs of EFA deficiency, lipolysis, or plasma FFA concentrations.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

In vitro drug permeation from chitosan pellets

The purpose of this study was to prepare and characterize coated pellets for controlled drug delivery. The influence of chitosan (CS) in pellets was evaluated by swelling, in vitro drug release and intestinal permeation assays. Pellets were coated with an enteric polymer, Kollicoat^® MAE 30 DP, in a fluidized-bed apparatus and the coating formulations were based on a factorial design. Metronidazole (MT) released from coated and uncoated pellets were assessed by dissolution method using Apparatus I. Intestinal permeation was evaluated by everted intestinal sac model in rats, used to study the absorption of MT from coated pellets containing CS or not through the intestinal tissue. Although the film coating avoided drug dissolution in gastric medium, the overall drug release and intestinal permeation were dependent on the presence of CS. Thus, pellets containing CS show potential as a system for controlled drug delivery.     

Synthesis of fatty acids, in vitro by choline-deficient and normal larvae of the housefly, Musca domestica

The synthesis of long chain fatty acids from acetate by a de novo pathway and by direct elongation of endogenous fatty acids has been demonstrated in homogenates of 4-day-old housefly larvae. The distribution of the synthesized fatty acids among the main classes of lipid has been studied. Addition of coenzyme-A to the medium inhibited the de novo synthesis pathway and made elongation the main synthetic route by which the radioactive acetate was incorporated into fatty acids. Direct elongation of palmitoleic to vaccenic acid has been demonstrated to occur in the homogenates. No consistent differences could be observed in the amount and distribution of the radioactivity incorporated into the fatty acids of homogenates prepared from larvae reared on a choline-deprived or a choline-sufficient diet. Addition of phosphatidylcholine to such homogenates also produced no changes in the labelling patterns. It was concluded that the changes seen, in vivo, in the fatty acids of the phospholipids, which accompany alteration of the amount of lipid-choline in the larvae, were unlikely to be due to any direct effect of the phosphatidylcholine on the enzymes involved in fatty acid synthesis.     

In vitro bulblet production of Brunsvigia undulata from twin-scales

Many South African medicinal plants are over-collected for use in traditional medicines. This necessitates developing methods for increasing production. Micropropagation can be used as an alternative to conventional propagation methods. Twin-scales, cut from large parent bulbs, were cultured on MS medium (Murashige and Skoog, 1962) supplemented with 25 plant growth regulator combinations. Bulblets formed on twin-scales in 24 of the treatments. All explants formed bulblets on plant growth regulator-free medium. The effect of plant growth regulators, activated charcoal, explant orientation, explant origin and photoperiod on bulblet production was investigated. Bulblet formation was greatest when twin-scales were excised from the middle of the parent bulb, placed adaxial side down on plant growth regulator-free medium and kept in a 16 h photoperiod.     

In vitro glucosylation of 1-naphthol by insects

Glucosylation in insects was investigated using tobacco hornworms (Manduca sexta) as the primary test insect and 1-naphthol-¹⁴C as the substrate. Of 6 common co-factors tested, only UDPG was utilized by the conjugating enzyme system. Neither the hornworm nor housefly enzymes could form the glucuronic acid derivative of 1-naphthol using UDPGA. Centrifugal fractionation of the hornworm homogenates showed that the glucosyltransferase activity was in the 105,000 g soluble fraction. In the housefly, the enzyme activity was associated with the 15,000 g pellet and to a lesser extent with the 105,000 g pellet. In vitro inhibition of the glucosyltransferase by sulphoxide, piperonyl butoxide, and other insecticide synergists was demonstrated.     

In vitro auxin production by Balansia epichloë

Balansia epichloë, a systemic plant pathogen isolated from Sporobolus poiretii, was shown to produce the plant growth regulators 3-indole acetic acid, 3-indole ethanol, 3-indole acetamide and methyl-3-indole carboxylate when grown on a medium containing tryptophan. When grown on a tryptophan deficient medium 3-substituted indole derivatives were not detected. However, extracts of the medium in lower doses increased and in higher doses inhibited the growth of wheat coleoptiles.     

Absorption of different amino acids by an intestinal preparation from normal rats and from rats exposed to supralethal X-Irradiation

Summary Absorption of different amino acids [lysine,-amino isobutyrate (AIB), methionine, alanine, glycine] was studied in an intestinal in vivo preparation from normal and X-irradiated rats (1 and 3 days after 2000 R). Absorption of amino acids under loading and in presence of other amino acids was also followed. Lysine and AIB absorption follow a saturation kinetics with a maximal velocity of 35 and 85.6 µmoles/min and aK _t of 10 and 67 µmoles for lysine and AIB respectively. Interference in absorption exists not only between amino acids of the same class, but also by lysine on neutral amino acids. One day after irradiation, absorption is normal or even enhanced, although active transport appears impaired as indicated by the increase inK _t. Three days after exposure, absorption, particularly the active one, is severely depressed. This is indicated by the low Vm and highK _t, as well as by the fact that loading has little influence on absorption.Supported by the Schutzkommission am Innenministerium der BRD. Publication No. 1389 of the Euratom Biology Division     

In vitro carotenogenesis by wild type and mutants of Phycomyces blakesleeanus

Cell extracts from shake cultures of the wild type and six mutant strains of Phycomyces converted [2-¹⁴C] MVA into carotenes, squalene and prenyl phosphates. Oxygen was required for the desaturation of phytoene. When compared with the wild type, cells extracts of carB and carR mutants are much less effective in phytoene dehydrogenation and lycopene cyclization, respectively. This confirms previous conclusions about the biochemical functions of the carB and carR genes, which were based on genetic and in vivo studies. CarA strain mutants accumulate, in vivo, much less β-carotene than the wild type. This correlates with a 10-fold decrease in carotenogenesis in vitro. The addition of retinol to incubations of cell extracts of the wild type and C2 strains stimulated β-carotene formation. Both carB and carR mutants show enhanced total carotenogenic activities in vitro and the carS mutant shows a higher β-carotene-synthesizing activity than the wild type. It is suggested that the feed-back regulatory mechanism known to control this pathway operates at the level of enzyme synthesis.     

In vitro regeneration of kidney from pluripotent stem cells

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.     

In vitro antioxidant and antitumor activities of polysaccharides extracted from Asparagus officinalis

Ultrasonic circulating extraction technology was applied for the polysaccharide extraction from Asparagus officinalis. The crude polysaccharides were deproteinized by Sevag method and three main polysaccharide fractions, AOP-4, AOP-6 and AOP-8 were obtained by fractional precipitation with gradient concentrations of ethanol (40%, 60% and 80%). The in vitro antitumor and antioxidant activities of the polysaccharide fractions were evaluated by MTT assay and free radical-scavenging assay, respectively. Deproteinized AOPs showed higher antioxidant and antitumor activities than crude AOP. AOP-4 with molecular weight 5.75 × 10⁴ Da showed significant function of scavenging hydroxyl radical. Three AOP fractions had significant antitumor activity against HeLa and BEL-7404 cells in a dose dependent manner. Furthermore, the inhibit activity of AOP-4 against HeLa cells was higher than those of other AOPs and the inhibition rate reached 83.96% at the concentration of 10 mg/mL. These results indicated that the AOP might be useful for developing natural safe antitumor drugs or health food.