What best animal science teachers do.

Great teachers have the extraordinary ability to inspire and motivate even those students who resist learning. The top educators are knowledgeable not only about the content of the course they are teaching but also of the information, literature, and practice of instructional delivery to their audience. Many exemplary educators have been profiled and studied; however, there is a paucity of information pertaining to how the top animal science teachers teach. The objective of this study was to identify and describe characteristics of award-winning animal science teachers. The inclusion criterion for selecting faculty was being bestowed an excellence in teaching award through their professional organization. Each teacher answered a series of questions about themselves, their students, and the class being taught. Lecture was captured using a digital all-inclusive camera and later analyzed for pedagogical trends and instructor-student interactions. Despite a variety of topics being taught by award-winning teachers, there were multiple trends emerging from their classrooms. Common events included reviewing highlights of previous lectures, distributing something to students, posing questions during class, and calling on students by name. Each teacher taught differently, but they all understood their audience; they grasped the subject matter and most importantly, they valued students learning. Collectively, these findings can be utilized and applied by animal science teachers in their own environments in an attempt to foster improved student learning through excellent teaching.

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes.

High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48h and 144h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (PPPP<0.05) of cleavage and blastocyst formation by 48 and 144h after IVF compared with all other groups. These results indicate that supplementing with 0.005mM glucuronic acid and 0.005mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.

The effects of N-acetyl-L-cysteine supplementation on in vitro porcine oocyte maturation and subsequent fertilisation and embryonic development.

The effects of supplementation with 1.5 mM n-acetyl-l-cysteine (NAC) during in vitro oocyte maturation were studied. Oocytes were supplemented with 1.5 mM NAC during maturation for 0 to 24 h, 24 to 48 h, or 0 to 48 h then subjected to IVF and embryo development. Oocytes were evaluated after maturation for intracellular glutathione concentration, superoxide dismutase and glutathione peroxidase activities and DNA fragmentation. Fertilisation and embryonic development success were also evaluated. There was no effect of treatment on intracellular glutathione concentrations, enzyme activities or fertilisation success rates. Supplementing NAC during maturation significantly decreased (P < 0.05) the percentage of oocytes with fragmented DNA compared with no NAC supplementation. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of oocytes with male pronuclei than for oocytes from the other treatment groups. There was no difference in the percentage of embryos cleaved by 48 h after IVF between treatment groups. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of embryos reaching the blastocyst stage by 144 h after IVF compared with the other treatment groups. These results indicate that supplementation of the oocyte maturation medium with 1.5 mM NAC, specifically during the last 24 h, improves male pronucleus formation and blastocyst development in pigs.

Effects of dietary supplementation with an organic source of selenium on characteristics of semen quality and in vitro fertility in boars.

Semen characteristics in boars fed organic or inorganic sources of Se were assessed in 3 experiments. Crossbred boars were randomly assigned at weaning to 1 of 3 dietary treatments: I) basal diets with no supplemental Se (control), II) basal diets with 0.3 mg/kg of supplemental Se from an organic source (Sel-Plex, Alltech Inc., Nicholasville, KY), and III) basal diets supplemented with 0.3 mg/kg of supplemental Se from sodium selenite (Premium Selenium 270, North American Nutrition Co. Inc., Lewisburg, OH). For Exp. 1, semen was collected from boars (n = 10/dietary treatment) on 5 consecutive days at 15 mo of age. Effects of treatment × day were detected for the proportions of progressively motile (P = 0.02) and rapidly moving (P = 0.03) spermatozoa, and measures of sperm velocity, including path velocity of the smoothed cell path (P = 0.05) and average velocity measured in a straight line from the beginning to the end of the track (P = 0.05). Negative effects of day of semen collection on sperm motility were least pronounced in boars fed Sel-Plex. Experiment 2 was conducted when boars were 17 mo of age, and semen was collected (n = 10 boars/dietary treatment), diluted in commercially available extenders, and stored at 18°C for 9 d. Effects of treatment × day were detected for percentages of motile (P = 0.01) and static (P = 0.01) spermatozoa, amplitude of lateral head displacement (P = 0.02), frequency with which the sperm track crossed the sperm path (P = 0.04), straightness (P = 0.01), and average size of all sperm heads (P = 0.03). In general, sperm cells from boars fed Sel-Plex were better able to maintain motility during liquid storage compared with boars fed sodium selenite. For Exp. 3, semen was collected from boars (n = 6/dietary treatment) at 23 mo of age, and spermatozoa were evaluated at d 1 and 8 after semen collection using in vitro fertilization procedures. There was a tendency for an effect (P = 0.11) of dietary treatment on fertilization rate with Sel-Plex-fed boars having the greatest value (70.7%). The results of this study suggest that there are positive effects of dietary supplementation with Sel-Plex on boar semen characteristics and that organic Se supplementation may help ameliorate the negative effects of semen storage on characteristics of sperm motility.

N-acetyl-l-cysteine supplementation improves boar spermatozoa characteristics and subsequent fertilization and embryonic development.

The effects of 1.0 mmN-acetyl-l-cysteine (NAC) supplementation during the incubation of frozen-thawed and preserved boar sperm were studied in addition to subsequent oocyte IVF. Frozen-thawed and preserved boar sperm were supplemented with 1.0 mm NAC and incubated for 60 min to allow capacitation to occur followed by the addition of calcium ionophore 23187 to induce the acrosome reaction. The number of sperm having undergone the acrosome reaction was determined using the Wells-Awa staining technique. DNA damage was detected using single-cell gel electrophoresis. Membrane lipid peroxidation was estimated by the end point generation of malondialdehyde (MDA). Frozen-thawed sperm was not different in the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) more DNA damage (59.8 ± 1.0) compared to preserved sperm (32.0 ± 1.0%). Supplementing 1.0 mm NAC did not have an effect on the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) less DNA (39.2 ± 1.0%) damage compared to no antioxidant supplementation (52.7 ± 1.0%). Frozen-thawed sperm produced a significantly higher (p < 0.05) concentration of MDA (2.08 ± 0.05 µm MDA/10(7) cells) compared to preserved sperm (1.82 ± 0.05 µm MDA/10(7) cells), and non-supplemented sperm produced a significantly higher (p < 0.05) concentration of MDA (3.62 ± 0.05 µm MDA/10(7) cells) compared to the 1.0 mm NAC-supplemented sperm (0.28 ± 0.05 µm MDA/10(7) cells. Supplementation or semen storage method had no effect on IVF or embryonic development. These results indicate that supplementation with 1.0 mm NAC improved the ability to use frozen-thawed boar sperm during IVF as it reduces the DNA fragmentation and lipid peroxidation of the sperm.

Effects of N-acetyl-cysteine and N-acetyl-cysteine-amide supplementation on in vitro matured porcine oocytes.

This study was conducted to evaluate the effects of different concentrations of the antioxidant N-acetyl-cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC-amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post-fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development.

Mechanisms of oxidative stress in porcine oocytes and the role of anti-oxidants.

The mechanisms of oxidative stress in in vitro maturing porcine oocytes and the effects of anti-oxidant supplementation of the medium in ameliorating these effects were investigated in the present study. In addition to intracellular reduced glutathione (GSH) concentrations and DNA fragmentation, the present study focused on superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity. The anti-oxidants used were N-acetylcysteine (NAC) and its derivative NAC-amide (NACA). The results indicate that when SOD is inhibited, supplementation of the maturarion medium with 1.5 mm NAC or NACA compensates for the decrease in SOD activity by reducing the degree of DNA fragmentation (P < 0.05). When GPx is inhibited, supplementation of the maturarion medium with 1.5 mm NAC alleviates the effects of no GPx activity, as indicated by a decrease in the degree of DNA fragmentation (P < 0.05). When the maturarion medium was supplemented with 1.5 mm NACA, intracellular GSH concentrations decreased (P < 0.05) and SOD and catalase activities increased (P < 0.05) along with the degree of DNA fragmentation. These results indicate that the mechanisms of alleviating oxidative stress in porcine oocytes are very complex and supplementing maturing oocytes with anti-oxidants may enhance enzyme activities and eliminate free radicals.

Novel fatty acid esters of p-coumaryl alcohol in epicuticular wax of apple fruit.

Hexane extracts of epicuticular wax from cv. Gala apples were noted to have an unusual, broad absorbance maximum at approximately 258 nm, which led us to isolate and identify the primary UV-absorbing compounds. Column and thin-layer chromatography yielded a fraction that gave a series of paired, 260-nm-absorbing peaks on C(18) HPLC. These were shown to be a family of phenolic fatty acid esters, for which retention times increased with increasing fatty acid chain length, and paired peaks were esters of two related phenolics with the same fatty acid moiety. Alkaline hydrolysis of the esters released two water-soluble phenolics separable by C(18) HPLC. Electrospray ionization mass spectrometry gave a molecular mass of 150 for both, and (1)H NMR plus UV absorbance spectra identified them as E and Z isomers of p-coumaryl alcohol. Alkaline cleavage of the fatty acid esters in the presence of methanol or ethanol resulted in partial derivatization of E-p-coumaryl alcohol to the corresponding gamma-O-methyl or O-ethyl ether. Gradient HMQC NMR of the HPLC-purified stearate ester of E-p-coumaryl alcohol indicated that fatty acid esterification occurs at the gamma-OH rather than at the 4-OH on the phenyl ring. This is the first report of fatty acid esters of monolignols as a natural plant product.

Temperature-dependent autoxidation of conjugated trienols from apple peel yields 6-methyl-5-hepten-2-one, a volatile implicated in induction of scald.

Conjugated triene (CT) oxidation products of alpha-farnesene have long been thought to be involved in development of superficial scald in apple fruit. Early studies found that CT hydroperoxides and the volatile 6-methyl-5-hepten-2-one (MHO) are major in vitro autoxidation products of alpha-farnesene. However, it was recently shown that > or =99% of the oxidation products of alpha-farnesene that accumulate in apple peel are conjugated trienols (CTols), isomers of 2,6,10-trimethyldodeca-2,7,9,11-tetraene-6-ol. HPLC-purified CTols from fruit of two scald-susceptible cultivars, Granny Smith (GS) and Red Delicious (RD), were used to study autoxidation of these compounds in vitro. Incubation of CTols in sealed glass vials under air resulted in accumulation of MHO. Oxygen enrichment did not increase the amount of MHO produced. Regardless of which cultivar CTols were derived from, at 0 degrees C autoxidation yielding MHO was quite slow and linear, whereas at 20 degrees C MHO production was much more rapid, and after several hours the rate increased abruptly. However, CTols isolated from GS and RD fruit differed in the duration of the initial lag phase and the overall level of MHO generated at 20 degrees C. The sharp increase in MHO production occurred after 3 h with GS CTols and at about 12 h with RD CTols. Also, the yield of MHO from GS CTols after 6 h at 20 degrees C was nearly 6-fold greater than that from RD CTols after 20 h at 20 degrees C. The antioxidants butylated hydroxytoluene and diphenyamine reduced the yield of MHO by about 97%. Recent work has shown that MHO can induce scald-like symptoms in apple peel and that tissue sensitivity increases with time in storage. This may explain the correlation between high CTol levels and scald development, and why symptoms rapidly intensify when fruits are removed from cold storage.

Characterization of a phospholipase Dalpha cDNA from tomato fruit.

Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid increases in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLDalpha cDNA as a probe, a PLDalpha cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLDalpha shares >75% sequence identity with PLDalphas from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel-blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded active enzyme, and a FLAG-PLDalpha fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS/PAGE.

Steryl esters in the elaioplasts of the tapetum in developing Brassica anthers and their recovery on the pollen surface.

The tapetum cells in the developing anthers of Brassica napus contained abundant elaioplasts, which had few thylakoid membranes but were packed with globuli of neutral esters. Of the neutral esters, the major ester group possessed mainly 24-methylenecholesterol, 31-norcycloartenol, 24-dehydropollinastanol, and pollinastanol esterified to 18:3 and other unsaturated and saturated fatty-acyl moieties. The minor ester group had a dominant component tentatively identified as 12-dehydrolupeol esterified to mostly 18:0, 16:0, and 20:0 fatty-acyl moieties. The elaioplasts also contained a high proportion (16% w/w of total lipids) of monogalactosyldiacylglycerols (MGDG). This is the first report of plastids having steryl esters as the predominant lipids. We propose that the globuli contain steryl esters and are stabilized by surface MGDG and structural proteins. The tapetosomes, the other abundant lipid-containing organelles in the tapetum, possessed triacylglycerols (TAG) as the predominant lipids. At a late stage of anther development, the minor group of neutral esters and MGDG of the elaioplasts, as well as the TAG of the tapetosomes, were degraded. Steryl esters similar to those of the elaioplasts were recovered from the pollen surface and were the major lipids of the pollen coat. The pollen coat steryl esters and proteins could be extracted with moderately polar or nonpolar solvents. These proteins, which were mostly fragments of oleosins derived from the tapetosomes, had a high proportion of lysine (13 mol %). The possible functions of the steryl esters and the proteins on the pollen surface are discussed.

The effect of ethanol and oxygen on the growth of Zymomonas mobilis and the levels of hopanoids and other membrane lipids.

Zymomonas mobilis (ATCC 29191) was grown either aerobically or anaerobically in the presence of 2% (wt/vol) glucose and 0, 3, or 6% (vol/vol) ethanol. The rates of growth and the composition of hopanoids, cellular fatty acids, and other lipids in the bacterial membranes were quantitatively analyzed. The bacterium grew in the presence of 3% and 6% ethanol and was more ethanol tolerant when grown anaerobically. In the absence of ethanol, hopanoids comprised about 30% (by mass) of the total cellular lipids. Addition of ethanol to the media caused complex changes in the levels of hopanoids and other lipids. However, there was not a significant increase in any of the hopanoid lipid classes as ethanol concentration was increased. As previously reported, vaccenic acid was the most abundant fatty acid in the lipids of Z. mobilis, and its high constitutive levels were unaffected by the variations in ethanol and oxygen concentrations. A cyclopropane fatty acid accounted for 2.6-6.4 wt % of the total fatty acids in all treatments.

Quantitative high-performance liquid chromatography analysis of plant phospholipids and glycolipids using light-scattering detection.

Application of the evaporative light-scattering principle to quantitative high-performance liquid chromatography (HPLC) analyses of plant membrane lipids has received little study. Light-scattering detection response curves were generated for nine classes of plant membrane phospholipid and glycolipids. Quantitative results obtained by HPLC/light-scattering detection and conventional lipid analytical methods (thin-layer chromatography and lipid-P assay) were in close agreement, confirming the reliability of HPLC/evaporative light-scattering detection (ELSD) analyses. Only three of the nine plant lipid classes gave linear detector response functions above 10 micrograms injected lipid mass. This finding contradicts earlier precepts involving light-scattering detection of lipids. At a given mass, appreciable variation in ELSD signal intensity and detection limit was found to exist among the various plant membrane lipid classes. The variation in detector response among plant lipid classes is an important consideration in achieving accurate quantitative results in plant lipid analyses.

Analysis of intact hopanoids and other lipids from the bacterium Zymomonas mobilis by high-performance liquid chromatography.

Hopanoids and other lipids were extracted from Zymomonas mobilis and quantitatively analyzed by high-performance liquid chromatography. Previous methods for hopanoid analysis required derivatization of the hopanoids via periodate oxidation or acetylation. The current method employs a normal-phase silica gel column, a ternary gradient of hexane-isopropanol-water-triethylamine, and detection with a flame ionization detector. Three major hopanoid classes were separated and quantified by this new method, and together they comprised 30 to 40% of the total lipid in these cells. Mass spectrometry confirmed that chemical structures of these hopanoids were consistent with those previously proposed. In addition, several other common lipid classes (free fatty acids and six classes of common phospholipids), comprising the remaining 60 to 70% of the total lipids, were also separated and quantified. This new chromatographic method represents the first technique for the analysis and purification of intact hopanoids. This method should be useful for the analysis and purification of hopanoids from other bacterial species.

Plasma Membrane Lipids Associated with Genetic Variability in Freezing Tolerance and Cold Acclimation of Solanum Species.

Simultaneous comparisons were made between a freezing-tolerant, cold-acclimating (CA) wild potato species (Solanum commersonii) and a freezing-sensitive, nonacclimating (NA) cultivated species (Solanum tuberosum). Comparative studies allowed differentiation of plasma membrane lipid changes associated with increased freezing tolerance following CA from lipid changes that can result from metabolic adjustment to reduced temperature during CA. Following CA treatment lipid changes found in both the NA and CA species included a decrease in palmitic acid, an increase in unsaturated to saturated fatty acid ratio, an increase in free sterols, an increase in sitosterol, and a slight decrease in cerebrosides. Lipid changes detected only in the acclimating species included an increase in phosphatidylethanolamine, a decrease in sterol to phospholipid ratio, an increase in linoleic acid, a decrease in linolenic acid, and an increase in acylated steryl glycoside to steryl glycoside ratio. These changes were either absent or opposite in the NA species, suggesting an association of these lipid changes with CA. Furthermore, the lipid changes associated with increased freezing tolerance during CA were distinct from lipid differences between the two species in the NA state.

Glycerolipid-fatty-acid desaturase deficiencies in chloroplasts from fruits of Capsicum annuum L.

Chloroplasts from fruits and leaves of Capsicum annuum cv. 'Bell Tower' were purified on sucrose gradients, and the lipids were separated by column and thin-layer chromatography. The glycerolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) were quantified, and the fatty-acid composition at the 1 and 2 positions of the glycerol moiety (sn-1 and sn-2) was determined after hydrolysis with position-specific lipases. In fruit chloroplasts, Δ (3)-trans hexadecenoate (trans-3-16∶1) was absent and replaced by palmitate (16∶0) at sn-2 of PG, and Δ (7,10,13)-hexadecatrienoate (16∶3) at sn-2 of MGDG was greatly reduced and largely replaced by linoleate (18∶2). The ratio of 18∶2 to linolenate (18∶3) was consistently greater in glycerolipids from fruit compared with leaf chloroplasts. The lower percentage of C-16 fatty acids at sn-2 indicated that "prokaryotic" molecular species were reduced by ≤15% in DGDG, ∼40% in SQDG, and ∼90% in MGDG, in fruit compared with leaf chloroplasts. The MGDG∶DGDG ratios in fruit and leaf chloroplasts were 1.2∶1 and 2.2∶1, respectively. Taken together, the data indicate that chloroplasts in Capsicum fruit are deficient in three desaturases: those that convert 1) 16∶0 to Δ (3)-trans-16∶1 at sn-2 of PG, 2) 16∶0 to Δ (7)-cis-16∶1 at sn-2 of MGDG, and 3) 18∶2 to 18∶3 at both sn-1 and sn-2 of various chloroplast glycerolipids.

Sterol synergism in Paramecium tetraurelia.

Paramecium tetraurelia is a naturally occurring sterol auxotroph with an absolute nutritional requirement for one of a small group of structurally related phytosterols. We report here a quantitative study demonstrating that a low, otherwise sub-supportive, concentration (approximately 0.020-0.050 micrograms ml-1) of an essential phytosterol (stigmasterol) is adequate for growth of this ciliate, provided that a second, relatively non-specific sterol is available at a higher concentration (1.0 micrograms ml-1) to allow for membrane biosynthesis. This phenomenon, referred to as sterol synergism, has been observed in a broad taxonomic range of organisms, with the conclusion that small amounts of specific sterols are required to perform some previously unknown, vital metabolic or regulatory function. Paramecium promises to be an excellent model organism for the elucidation of essential sterol function.

Growth support and metabolism of phytosterols inParamecium tetraurelia.

The basis of the growth requirement ofParamecium for one of several structurally similar phytosterols is not known. Previous research has indicated that selective esterification of only growth-promoting sterols may be a key. In this study, it was found that under certain conditions sterols that fail to support growth (e.g., cholesterol) can be esterified in large amounts inParamecium. We found no compelling evidence to support the hypothesis that steryl esters serve a specialized role in the fatty acid metabolism of the cell. Octadecenoic acid, essential for cell growth, was the major fatty acid in both steryl esters and triglycerides. It was also shown thatP. tetraurelia can dehydrogenate Δ(0) and Δ(7), as well as Δ(5)-3ß-hydroxy sterols, to yield the conjugated 5,7-diene derivative. These results indicate the presence of a Δ(5), in addition to a Δ(7), desaturase of the sterol nucleus in this ciliate. Two C24 α-ethyl sterols, Δ(22)-stigmasterol (Δ(22)) and stigmastanol (Δ(0)), were shown for the first time to promote growth. Finally, we found that non-growth-promoting sterols may compose a high percentage of the free sterols of the surface membrane without adversely affecting cell growth or viability. These data support the conclusion that the growth requirement for select phytosterols inParamecium does not involve the structural or functional role of "bulk" sterols in cell membranes.

Fatty-acid composition of polar lipids in fruit and leaf chloroplasts of "16:3"- and "18:3"-plant species.

The fatty-acid composition of polar lipids from fruit and leaf chloroplasts was compared in five Solanaceous and two cucurbit species. The acylated fatty acids in monogalactosyl diglycerides (MGDG) from leaf chloroplasts of all five Solanaceous species included substantial amounts of Δ (7,10,13)-hexadecatrienoic acid (16:3). In contrast, the MGDG from fruit chloroplasts of the Solanaceae contained very little of this plastid-specific polyunsaturate, and instead included a proportionately greater percentage of linoleic acid (18:2). In MGDG from leaf chloroplasts of two cucurbits, α-linolenic acid (18:3) constituted 94-95% of the acylated fatty acids. Fruit-chloroplast galactolipids of the cucurbits had a greater abundance of 18:2, and hence a higher 18:2/18:3 ratio, than found in the corresponding leaf lipids. Among the phosphoglycerides, the unusual fatty acid Δ3-trans-hexadecenoate (trans-16:1) constituted from 15 to 24% of the acylated fatty acids in phosphatidyl glycerol (PG) from leaf chloroplasts (all species). In sharp contrast, trans-16:1 was virtually absent in PG from fruit chloroplasts of both Solanaceous and cucurbit species, and was replaced by a proportionate increase in the content of palmitate (16:0). The observed differences in the polar lipid fatty-acid composition of fruit and leaf chloroplasts are discussed in terms of the relative activity of several intrachloroplastic enzymes involved in lipid synthesis and fatty-acyl desaturation.