GLycol-EThanol-WAter (3:3:4); A SOlvent for SUdan BLack B STaining

The previously used supersaturated 60% isopropanol method (Lillie 1954, p. 303) protected the more readily soluble lipids from the solvent action of 70% alcohol but precipitation of dye particles within and around sections is sometimes a disturbing complication (cf. Vlachos 1959). The propylene glycol method of Chiffelle and Putt (Lillie, p. 304) has been found to remove much of the birefringent lipid from the sections. Glacial acetic acid dissolves most tissue lipids except those of the solvent-resistant ceroid or lipofuscin type. While solutions in 60-70% acetic acid conserve ordinary fats well (Sills and Marsh 1959) their effect on the more easily soluble lipids should be further explored.     

A Cytochemical Technique for Demonstration of Lipids,Polysaccharides and Protein Bodies in Thick Resin Sections

An effective cytochemical technique for the simultaneous demonstration of lipids, polysaccharides and protein bodies in the same section from the tissue embedded in Epon 812 is described. Thick sections of peanut cotyledon are used for a typical sample according to the following procelures. Firstly, PAS reaction: (1) Oxidize sections in 0.5% periodic acid in 0.3% nitric acid for 10 min, (2) Wash in running water for 1–2 min and then pass through distilled water, (3) Stain in Schiff's reagent for 30 min, (4) Wash in sodium metabisulfite 3 times, 2 min for each time, (5) Wash in running water for 5 min and then pass through distilled water. Secondly, Sudan black B staining: (1) Rinse section in 70% ethanol for 1-2 min, (2) Stain in fresh 1% Sudan black B in 70% ethanol for 30–60 min at 40–60℃, (3)Rinse in 70% ethanol for 1 min and then in distilled water. Thirdly, Coomassie brilliant blue R staining: (1) Rinse sections in 7% acetic acid for 1–2 min, (2) Stain in I% Coomassie brilliant blue R in 7% acetic acid for 20 min at 60℃, (3) Differentiate in 0.1% acetic acid for I min, (4) Rinse in lunning water for 5 min and then pass through distilled water, (5) Dry at room temperature or in oven, 40℃. The dry sections mount in glycerin-gelatin. After the above three step staining, the three main compounds of the cell can be stained simultaneously. Starch grains and cellulose cell wall take cherry red colour, lipids appear in black, protein bodies are blue. The sealed slides can be kept permanently.     

A new method for the preparation of band 3, the main integral protein of the human erythrocyte membrane

Band-3 protein from human erythrocyte membranes was isolated, without using detergents, by a two-step procedure: (1) The peripheral proteins were removed from the membrane by treatment with 10% acetic acid. (2) The remaining lipoprotein complex was solubilized in approximately 92% (v/v) acetic acid and then separated into its components by preparative zonal electrophoresis in a gradient made up of acetic acid, water and sucrose. Band 3 was recovered from the gradient at a yield of 60 - 70% and purity of about 95%. Approximately 25 mg of band 3 could be prepared in one run. The protein is soluble in aqueous solutions, even in the absence of organic solvents or detergents. In addition to band 3, the proteins stained by periodic acid/Schiff's reagent (the sialoglycoproteins) are also separated from the other proteins.     

Direct somatic embryogenesis fromBegonia gracilis explants

Direct somatic embryogenesis ofBegonia gracilis was achieved from microcultured laminar segments and petioles on Murashige and Skoog medium with 0.5 mg 1^–1 kinetin and 2% coconut water. Somatic embryos were obtained with greater frequency from petiole explants than from leaf blade sections. Under red light (45 mol m^–2 s^–1), approximately 80% of the petiole explants successfully produced somatic embryos but only 30% of the leaf blade sections responded. However, somatic embryos were significantly more abundant on responding lamina explants (60–70 embryos/leaf section) than on petioles (40–50 embryos/petiole). These trends were similar for explants kept in the dark, but overall production was lower. Somatic embryos were produced more quickly (5 weeks) from petioles than from lamina explants (8 weeks). The somatic embryos germinated to produce plantlets and subsequently shoot cultures with the same appearance as the parental clone.Abbreviations BA benzyladenine - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - SE somatic embryo     

In vitro regeneration and Agrobacterium-mediated genetic transformation of Euonymus alatus

An in vitro plant regeneration method and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Euonymus alatus. More than 60% of cotyledon and 70% of hypocotyl sections from 10-day-old seedlings of E. alatus produced 2–4 shoots on woody plant medium (WPM) supplemented with 5.0 mg/l 6-benzylaminopurine (BA) plus 0.2 mg/l α-naphthalene acetic acid (NAA), and 77% of shoots produced roots on WPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tumefaciens strain EHA105 harboring a gusplus gene that contained a plant recognizable intron from the castor bean catalase gene to ensure plant-specific β-glucuronidase (GUS) expression, 16% of cotyledon and 15% of hypocotyl explants produced transgenic shoots using kanamycin as a selection agent, and 67% of these shoots rooted. Stable insertion of T-DNA into the host genome was determined with organ- and tissue-specific expression of the gusplus gene and further confirmed with a PCR-based molecular analysis.     

Nile Blue Histochemical Method for Phospholipids

The technic recommended is: Fix 6-12 hr. in 10% formalin containing 1% CaCl₂. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile blue sulfate, 500 ml. plus 50 ml. of 0.5% H₂SO₄, boiled 2 hr. before use. Rinse in distilled water, and place in acetone heated to 50°C. Remove the acetone from the source of heat and allow the sections to remain 30 min. Differentiate in 5% acetic acid 30 min., rinse in distilled water, and refine the differentiation in 0.5% HCl for 3 min. Wash in several changes of distilled water and mount in glycerol jelly. Results: phospholipids - blue; everything else - unstained. Counterstaining nuclei with safranin is optional, but if done, it preferably precedes the Nile blue and is then differentiated by the acetic acid. The histochemical principles on which the method is based are as follows: (1) The calcium compounds of phospholipids combine with the oxazine form of Nile blue sulfate and survive subsequent treatment; (2) neutral lipids are dissolved out by acetone; (3) proteins and other interfering substances are destained by the acetic acid and hydrochloric acid baths.     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Factors influencing extract of Hibiscus sabdariffa staining of rat testes

Abstract

Some plant extracts can be used in biology and medicine to reveal or identify cellular components and tissues. We investigated the effects of time and concentration on staining of histological sections of rat testes by an acidified extract of Hibiscus sabdariffa. An ethanolic extract of H. sabdariffa was diluted using 1% acetic acid in 70% ethanol to stain histological sections of testes at concentrations of 0.2, 0.1 and 0.05 g/ml for 5, 10, 15, 30, 45 and 60 min. The sections of testes were stained deep red. The staining efficiency of H. sabdariffa was greater at a high concentration and required less time to achieve optimal staining. H. sabdariffa is a strongly basic dye that can be used for various diagnostic purposes. Staining time and concentration must be considered to achieve optimal results.     

Trans-3-Methylthioacrylamide,a New Metabolic Product from Streptomyces sioyaensis

Rhodosporidium toruloides is a lipid-producing yeast, the growth of which is severely suppressed when hydrolysates of lignocellulosic biomass are used as carbon source. This is probably due to the toxic substances, such as organic acids, furans, and phenolic compounds produced during the preparation of the hydrolysates. In order to solve this problem, R. toruloides cultures were subjected to atmospheric room-temperature plasma mutagenesis, resulting in the isolation of mutants showing tolerance to sugarcane bagasse hydrolysate (SBH). Three mutant strains, M11, M13, and M18, were found to grow with producing lipids with SBH as carbon source. M11 in particular appeared to accumulate higher levels (up to 60% of dry cell weight) of intracellular lipids. Further, all three mutant strains showed tolerance of vanillin, furfural, and acetic acid, with different spectra, suggesting that different genetic determinants are involved in SBH tolerance.     

Lipase-catalyzed synthesis of two new antioxidants: 4-O- and 3-O-palmitoyl chlorogenic acids

Chlorogenic acid (5-caffeoyl quinic acid (CQA)) extracted from Hydrangea macrophylla (44%, w/w) with 98% purity, was acylated with palmitic acid by Novozym 435 to yield mono-acylated CQA. Acylation of CQA was achieved in 2-methyl-2-butanol at 60°C, and yielded two mono-acylated products: a major product acylated at the C-4 of the quinic moiety (4-O-palmitoyl chlorogenic acid) and a minor product acylated at the C-3 (3-O-palmitoyl chlorogenic acid). The bioconversions obtained in 7 days ranged from 14 to 60% and were influenced by the molar ratio of palmitic acid/CQA, which ranged from 10 to 80. The regioselectivity (4-O-palmitoyl/3-O-palmitoyl ratio) of the reaction was also affected by the molar ratio, and ranged from 90 to 70%. The scavenging activities against 1,1-diphenyl-2-picryl-hydrazyl radicals demonstrated that these palmitoyl CQA derivatives are associated with antioxidant activity (70% vs CQA).     

The use of tetrahydrofuran for delipidation and water solubilization of brain proteolipid proteins.

Tetrahydrofuran, an efficient total lipid solvent, was found to precipitate chloroform-methanol soluble proteins from CM (chloroform-methanol, 2:1) lipid extracts of bovine and rat white and grey matter. To total lipid extracts containing proteolipid proteins 4 volumes of tetrahydrofuran are added; the precipitate is centrifuged and washed once with the same solvent. The product contains all the protein and about 1 to 2% of the lipids of the original lipid extract. It is insoluble in CM; solubility in CM is restored by addition of acetic acid or of the lipids recovered from the tetrahydrofuran-soluble fraction It is also soluble in glacial acetic acid and this solution can be diluted either with CM or with distilled water.     

Production of gamma-linolenic acid by Mucor circinelloides and Mucor rouxii with acetic acid as carbon substrate

Summary The production of gamma-linolenic acid (GLA) by Mucor circinelloides CBS 203.28 and M. rouxii CBS 416.77 in fed-batch cultures operated in pH-stat mode with acetic acid as carbon substrate and titrant compared favourably with the performance of M. circinelloides in batch culture on glucose. On acetic acid M. circinelloides accumulated up to 39.8 mg GLA/g biomass, with a crude oil content of 28% containing 91% neutral lipids. The GLA content of the neutral lipid fraction was 15.6%.     

Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [14C]acetic and [14C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [14C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [14C]acetic acid. Similarly, [14C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [14C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of (3H)-arachidonic acid into cattle retina lipids: high uptake in triacylglycerols, diacylglycerols, phosphatidylcholine and phosphatidylinositol.

The incubation of (³H)-arachidonic acid-prelabeled cattle retinas for 20 min in the presence of glucose under a gas phase of 5% carbon dioxide in oxygen showed uneven labeling in lipid classes. Total phospholipids, acylglycerides and free fatty acids contained 35, 37 and 31 per cent of the total radioactivity. In phosphatidylinositol and phosphatidylcholine almost 70% of the polar lipid (³H)-arachidonate was recovered. About 70% of the total fatty acid esterified in retina lipids was found in diacylglycerols, triacylglycerols, phosphatidylinositol and phosphatidylcholine. It is concluded that the cattle retina “in vitro” takes up free arachidonic acid and that this fatty acid is further unevenly acylated into lipids.The apolar fatty acyl residues of lipids display an independent turnover and their composition may be modified by acylation-deacylation reactions. In several cellular lipids, a differential turnover of the fatty acids as compared with other lipid moieties has been indicated, such as the case of phosphatidylinositol (1–3) and cardiolipin (4). The latter is enriched in the inner mitochondrial membrane where energy conservation processes take place and the former has been implicated in synaptic transmission (5) and related with a protein identified as the acetylcholine receptor (6). In brain phosphoinositides tetraenoic molecular species are by far the largest (2) and an active acylation-deacylation cycle of arachidonic acid occurs (7). However data regarding retina phosphoinositides composition and metabolism is limited to: fatty acid distribution (8), to some studies on the phosphodiester metabolism by ³²p (9) and to a study reporting that in frog rod outer segments and retina, polyphosphoinositides are undetectable (10). The purpose of the present investigation was to observe the (³H)-arachidonic acid labeling of acylglycerides and of phosphoglyceride classes of cattle retina.     

Some novel N-fatty acyl derivatives of a microbial galactosaminan

Novel seven N-fatty acyl derivatives (degree of substitution 0.78–0.96) of a microbial galactosaminan were prepared in 59–79% yields by its reaction with fatty acid anhydrides in aqueous acetic acid-methanol. N-Acetyl and N-propionyl derivatives were soluble in water, aqueous 2% sodium hydroxide, and aqueous 2% acetic acid, but N-higher fatty acyl (>C6) derivatives were insoluble. Gel was not formed in these react ions     

Effects of acetic and formic acid on ABE production by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> and <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

The effect of acetic acid and formic acid on acetone-butanol-ethanol (ABE) production by solventogenic Clostridia was investigated. The ABE concentration in Clostridium acetobutylicum was found to have increased slightly on addition of 3.7 ∼ 9.7 g/L acetic acid, but was found to have drastically reduced in the presence of 11.7 g/L acetic acid. However, the solvent production of C. beijerinckii was not affected by addition of acetic acid in the range of 3.7 ∼ 11.7 g/L. C. acetobutylicum was more vulnerable to formic acid than C. beijerinckii. In C. acetobutylicum, the total ABE production decreased to 77% on addition of 0.4 g/L formic acid and 25% with 1.0 g/L formic acid. The total ABE production by C. acetobutylicum was also noted to have decreased from 15.1 to 8.6 g/L when 8.7 g/L acetic acid and 0.4 g/L formic acid co-existed. The solvent production by C. beijerinckii was not affected at all under the tested concentration range of formic acid (0.0 ∼ 1.0 g/L) and co-presence of acetic acid and formic acid. Therefore, C. beijerinckii is more favorable than C. acetobutylicum when the ABE is produced using lignocellulosic hydrolysate containing acetic and formic acid.     

High-throughput liquid chromatography for drug analysis in biological fluids: investigation of extraction column life

A novel method was developed and assessed to extend the lifetime of extraction columns of high-throughput liquid chromatography (HTLC) for bioanalysis of human plasma samples. In this method, a 15% acetic acid solution and 90% THF were respectively used as mobile phases to clean up the proteins in human plasma samples and residual lipids from the extraction and analytical columns. The 15% acetic acid solution weakens the interactions between proteins and the stationary phase of the extraction column and increases the protein solubility in the mobile phase. The 90% THF mobile phase prevents the accumulation of lipids and thus reduces the potential damage on the columns. Using this novel method, the extraction column lifetime has been extended to about 2000 direct plasma injections, and this is the first time that high concentration acetic acid and THF are used in HTLC for on-line cleanup and extraction column lifetime extension.     

An Orcein-Oil Red O Stain for Concomitant Demonstration of Elastic Tissue and Lipid

Orcein, 0.5% in 50% isopropanol, 0.5-1 hr, followed by saturated oil red O in isopropanol diluted 3:2 with distilled water, 10-15 min, was used to demonstrate lipids and elastic tissue simultaneously in 10 μ frozen sections of formalin-fixed aortas of the wild African buffalo, showing atherosclerotic lesions. A comparison was made with the oil red O-aldehyde fuchsin (AF) method of Kwaan and Hopkins (Stain Techn., 39: 123-5, 1964) and the resorcin fuchsin (RF)-oil red O method of Lillie (Histopathologic Technic and Practical Histochemistry, McGraw-Hill, 1954), but both gave marked background staining by AF or RF that obscured the smaller deposits of lipid. Sudan IV could be substituted for oil red but did not demonstrate many of the finest deposits of lipids. Sudan black, in combination with orcein, AF or RF, was very satisfactory for demonstrating lipids but obscured many elastic fibres. Sudan dyes I, II, III, brown, blue, and green, with orcein, AF or RF, showed less contrast between lipids and elastic tissue or failed to stain the lipids adequately.     

Lipid metabolism in green leaves of developing monocotyledons

Lipid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivum L.) leaves. The endogenous levels of acyl lipids and their constituent fatty acids from the same leaf sections were also analysed. The principle chloroplast acyl lipids showed a relative increase in amount with the age of the leaf section. Their content of -linolenic acid also increased whereas there was little change in the amount of this acid in phosphatidylcholine and phosphatidylethanolamine, which are primarily non-chloroplastic. The content of trans-3-hexadecenoic acid in phosphatidylglycerol increased approximately 20-fold between the youngest (basal) and oldest (distal) leaf sections.The incorporation of [¹⁴C]acetate was always high into monogalactosyldiacylglycerol, phosphatidylcholine and the neutral lipid (mainly pigments) fractions. With increasing age, the neutral lipids were less well labelled. In three of the plant species but not in barley, phosphatidylglycerol was heavily labelled. Monogalactosyldiacylglycerol usually contained the highest amount of radioactivity in the middle leaf sections. Apart from these generalisations, each plant type had its own specific pattern of radiolabelling.     

Synthesis of Poly(3‐hydroxybutyrate‐co‐4‐hydrobutyrate) with a Target Mole Fraction of 4‐Hydroxybutyric Acid Units by Two‐Stage Continuous Cultivation of Delftia acidovorans P4a

Delftia acidovorans P4a (DSMZ 10474) was grown in mineral medium on acetic acid at pH 8.0 without an additional supply of nutrients like yeast extract or polypeptone. Using acetic acid and γ‐butyrolactone (GBL), copolymers with a 4HB content from 2–90 mol % were detected in batch experiments, depending on the ratio of the both carbon substrates. Due to the different consumption rates of the individual carbon substrates a multitude of different target mole fractions were difficult to produce by fed‐batch fermentation. Therefore, the two‐stage continuous cultivation technique was applied with two fermenters connected in series. At stage 2, the optimum PHA productivity of the bioreactor and a target 4HB content of the polymer could be precisely adjusted by the composition of the two substrates. This cultivation strategy was especially convenient when toxic substrates like acetic acid and GBL were employed. Using mixtures of acetic acid and GBL (3.5–23.5 mol % GBL), copolymers with a target mole fraction of 2.7–19 % 4HB could be produced. The PHA content was in the range of 52–60 %. The dilution rates (D) of the first and second fermenter were 0.2 h^–1 and 0.06 h^–1, respectively.