Oleic acid synthesis in the fruit of Persea americana MILL

The tissue slices from the mesocarp of avocado could incorporateradioactive acetate into lipids. Oleic, palmitic and stearicacids were the most labeled fatty acids found to accumulatein triglycerides. The conclusions that oleic acid was formedby way of chain elongation of already unsaturated short-chainfatty acids, and that there was no evidence for the desaturationof stearic acid were based on the following observations: 1)Stearic acid-¹⁴C was incorporated into triglycerides by thetissue slices without formation of oleic acid. 2) The oleicacid synthesized from specifically labeled acetate was not randomlylabeled. The specific radioactivity of azelaic acid moiety ofoleic acid was rapidly increased while that of pelargonic acidmoiety was gradually increased. 3) An unexpected rise of stearicacid was observed among commonly occurring fatty acids in thetissue slices. This was accentuated by anaerobiosis which prevailedduring vacuum infiltration of labeled acetate. ¹Present address: Department of Botany, Faculty of Agriculture,Hokkaido University, Sapporo, Japan. (Received January 13, 1969; )     

3-Oxoacyl-(acyl-carrier protein) reductase from avocado (Persea americana) fruit mesocarp.

The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana). The enzyme is inactivated by low ionic strength and low temperature. On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa. Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric. The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH. Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate. It exhibits a broad pH optimum around neutrality. Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH. Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy. It is localized in plastids. N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f. Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp.     

Regulation of lipid synthesis in relation to keratinocyte differentiation capacity

Cultured keratinocytes and squamous carcinoma cells provide a useful model system for studying the processes involved in the regulation of differentiation, as the differentiation capacity of the cells can be modulated experimentally by changing the extracellular calcium concentration. Furthermore, the squamous carcinoma cell lines exhibit a defect in their differentiation capacity which they express to different extents. In this paper, the effect of external lipoproteins has been studied on lipid synthesis in normal keratinocytes and three squamous carcinoma cell (SCC) lines which showed a decreasing capacity to differentiate in the order of normal keratinocytes greater than SCC-12F2 greater than SCC-15 greater than SCC-4. The ability of the cells to form cornified envelopes was taken as a measure of differentiation capacity. The rate of total lipid synthesis as well as the phospholipid-neutral lipid ratio decreased in the order SCC-4 greater than SCC-15 greater than SCC-12F2 greater than or equal to normal keratinocytes, clearly correlating with the differentiation capacity of the cells. Because of the high rate of phospholipid synthesis and the low rate of ceramide synthesis, it is concluded that, under these in vitro conditions used, the maturation of keratinocytes proceeds to a lesser extent than that seen under in vivo conditions. In proliferating cells, in which the low-density lipoprotein (LDL) receptor is operative to a high extent, the rate of lipogenesis, especially that of neutral lipids, responded dramatically to changes of extracellular lipoprotein concentration. In the presence of lipoproteins a marked decrease of cholesterol and triacylglycerol synthesis and an increase of cholesterol ester synthesis has been observed. On the other hand, in differentiating cells lipogenesis appeared to be independent of extracellular lipoproteins, due to the absence of the LDL uptake mechanism, the only exception being the synthesis of triacylglycerols, the rate of which could be modulated to a certain extent by extracellular lipoproteins. The results presented here demonstrate a close inverse relationship between the regulation of lipogenesis by extracellular lipoproteins and the ability of the cells to differentiate.     

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.     

Modification of matrix polysaccharides during avocado (Persea americana) fruit ripening: an assessment of the role of Cx-cellulase

The role of C_x-cellulase (EC 3.2.1.4) in fruit ripening and softening is unknown. In the present study, avocado ( Persea americana ) fruit, a rich source of C_x-cellulase, were examined to determine if the enzyme plays a role in ripening-related hemicellulose metabolism. Hemicelluloses (4 M alkali-soluble) from avocado fruit exhibited a very broad distribution of polymer sizes and an overall decrease in M_r during ripening. Polymers affected were primarily those of large M_r (relative molecular mass). The characteristic total hemicellulose M_r distribution and changes with ripening were also evident for xyloglucan (XG), a putative substrate for avocado C_x-cellulase. Hydrolytic activity toward hemicelluloses from preripe fruit was detected in crude buffer-soluble protein extracts derived from ripe avocado mesocarp tissue. XG was also degraded, and in a pattern similar to that observed during ripening. Purified C_x-cellulase also exhibited activity against specific components of isolated hemicelluloses; however, in contrast to the crude protein. C_x-cellulase alone was without influence on the M_r distribution of avocado XG. Protein depleted of C_x-cellulase was capable of moderate XG depolymerization. We conclude from the present studies that the enzyme C_x-cellulase is not involved in the ripening-related depolymerization of XG in avocado fruit.     

Metabolism of free sugars in relation to starch synthesis in the developing Sorghum vulgare grain

Accumulation of starch at expense of its free-sugar precursors was studied in the developing grains of the ‘SL-44’variety of Sorghum vulgare Pers. The content of starch gradually increased with the maturation of the grain and this increase was relatively fast until 18 days after anthesis. The daily rate of starch accumulation was at a maximum 15 days after anthesis. The content of total free sugars, reducing sugars, non-reducing sugars other than sucrose, total and non-sucrosyl fructose, and glucose also increased, reaching maximum values at 18 days after anthesis. Sucrose content gradually increased with a concomitant decrease in the activity of invertase, and sucrose was the major non-reducing sugar in the matured grains. Detached heads incubated in labelled sugars indicated that, compared to sucrose and fructose. ¹⁴C was more efficiently incorporated from glucose into grain starch, which was maximally synthesized at the mid-milky stage of grain development. Exogenous supply of NAD⁺ plus ATP stimulated the in vivo incorporation of ¹⁴C from sucrose to starch. The decline in the rate of starch accumulation did not synchronise with that of protein synthesis.     

A new look at sporoderm ontogeny in Persea americana and the hidden side of development

Background and Aims

The phenomenon of self-assembly, widespread in both the living and the non-living world, is a key mechanism in sporoderm pattern formation. Observations in developmental palynology appear in a new light if they are regarded as aspects of a sequence of micellar colloidal mesophases at genomically controlled initial parameters. The exine of Persea is reduced to ornamentaion (spines and gemmae with underlying skin-like ectexine); there is no endexine. Development of Persea exine was analysed based on the idea that ornamentation of pollen occurs largely by self-assembly.

Methods

Flower buds were collected from trees grown in greenhouses over 11 years in order to examine all the main developmental stages, including the very short tetrad period. After fixing, sections were examined using transmission electron microscopy.

Key Results and Conclusions

The locations of future spines are determined by lipid droplets in invaginations of the microspore plasma membrane. The addition of new sporopollenin monomers into these invaginations leads to the appearance of chimeric polymersomes, which, after splitting into two individual assemblies, give rise to both liquid-crystal conical ‘skeletons’ of spines and spherical micelles. After autopolymerization of sporopollenin, spines emerge around their skeletons, nested into clusters of globules. These clusters and single globules between spines appear on a base of spherical micelles. The intine also develops on the base of micellar mesophases. Colloidal chemistry helps to provide a more general understanding of the processes and explains recurrent features of pollen walls from remote taxa.     

Metabolism of glucose 1,6-P2. I. Enzymes involved in the synthesis of glucose 1,6-P2 in pig tissues

Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose 1-P]----glucose 1,6-P2 + glucose; (B) glucose 1-P + ATP----glucose 1,6-P2 + ADP; (C) glucose 1-P + fructose 1,6-P2----glucose 1,6-P2 + fructose 6-P; (D) glucose 1-P + glycerate 1,3-P2----glucose 1,6-P2 + glycerate 3-P. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis. With the exception of skeletal muscle, activity "D" represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity "B" is the major activity. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction "D" is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography. There is an enzyme which specifically catalyzes reaction "C", not previously described. There is a glucose 1-P kinase not identical to phosphofructokinase.     

Expression of a Ripening-Related Avocado (Persea americana) Cytochrome P450 in Yeast

One of the mRNAs that accumulates during the ripening of avocado (Persea americana Mill. cv Hass) has been previously identified as a cytochrome P450 (P450) monooxygenase and the corresponding gene designated CYP71A1. In this report we demonstrate that during ripening the accumulation of antigenically detected CYP71A1 gene product (CYP71A1) correlates with increases in total P450 and two P450-dependent enzyme activities: para-chloro-N-methylaniline demethylase, and trans-cinnamic acid hydroxylase (tCAH). To determine whether both of these activities are derived from CYP71A1, we have expressed this protein in yeast (Saccharomyces cerevisiae) using a galactose-inducible yeast promoter. Following induction, the microsomal fraction of transformed yeast cells undergoes a large increase in P450 level, attributable almost exclusively to the plant CYP71A1 protein. These membranes exhibit NADPH-dependent para-chloro-N-methylaniline demethylase activity at a rate comparable to that in avocado microsomes but have no detectable tCAH. These results demonstrate both that the CYP71A1 protein is not a tCAH and that a plant P450 is fully functional upon heterologous expression in yeast. These findings also indicate that the heterologous P450 protein can interact with the yeast NADPH:P450 reductase to produce a functional complex.     

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (M_r), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the M_r of unbranched cell-wall polymers in the M_r range of 10⁶–10⁷ Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.Abbreviations CMC carboxymethylcellulose - DMAC dimethylacetamide - DS developmental stage - M molecular weight - XG xyloglucan     

N-demethylation of p-chloro-N-methylaniline catalyzed by subcellular fractions from the avocado pear (Persea americana)

Subcellular fractions from the avocado pear ( Persea americana) catalyzed formation of p-chloroaniline from p-chloro-N-methylaniline. Fractions prepared by centrifugation of avocado homogenates at 20, 000g for 20 min formed p-chloroaniline (2900 +/- 500 pmol min-1 mg protein-1) with an NADPH-generating system. p-Chloroaniline formation required reduced pyridine nucleotide (NADPH was 6-7 times more effective than NADH) and O2. N-Demethylation was inhibited by CO (55% inhibition at CO:O2 = 1) and was not inhibited by CN. Cytochrome P-450 was detected in the 20, 000g pellet at levels of 300-380 pmol/mg protein. This particulate preparation was also active in catalyzing the NADPH-dependent epoxidation of the chlorinated cyclodiene aldrin. Improvements to a colorimetric procedure for measuring p-chloroaniline increased the sensitivity of the procedure fourfold, and allowed use of samples containing high amounts of lipid. Avocado pear is suitable tissue for further studies on the oxidation of foreign compounds by higher plants.     

Some aspects of carnitine metabolism in avocado (Persea americana) (Short Communication)

The fact that palmitoyl-l-carnitine is oxidized by avocado mitochondria at a rate comparable with that of succinate oxidation suggests that there are at least two systems for β-oxidation in higher plants. The carnitine-associated system is located in a mitochondrial fraction, whereas the glyoxylate-cycle-associated system is located in the glyoxysomes.     

Dehydroepiandrosterone in relation to adiposity, glucose tolerance and lipid spectra in Czech non-diabetic population

This study aimed to examine relationships between DHEA(S), anthropometric parameters, oral glucose tolerance test derived data and lipid spectra in a Czech non-diabetic population. 380 healthy volunteers both with and without a family history of diabetes type 2 (DM2) were enrolled into the study (women: n=235, age 28.9+/-9.4 years, BMI 22.3+/-4.5 kg/m(2), men: n=145, age 32.3+/-10.0 years, BMI 24.7+/-3.6 kg/m(2)). Spearman's correlations (both without and with the adjustment for age, age and BMI), as well as ANCOVA were used. Non-adjusted data showed many "beneficial" correlations between DHEA(S) and both anthropometric and metabolic variables. Statistical analysis revealed that almost all correlations of DHEA(S) to adiposity and fat distribution in men as well as in women disappeared after the adjustment. There are, however, differences between men and women in the correlation of DHEA(S) to insulin sensitivity and lipid levels. The use of hormonal contraceptives (COC) is also an important factor in this relationship. In men and also in women using COC, DHEA-S after adjustment correlated positively with fasting and stimulated glucose, insulin and C-peptide, and negatively with insulin sensitivity. In this respect, the benefit of DHEA(S) supplementation seems -- at least in terms of its alleged antiobesity and antidiabetogenic effects -- to be more than controversial.     

On the Function of Seedcoats in Gibberellin-induced Hydrolysis of Starch Reserves during Avocado (Persea americana) Germination

Of all seed components investigated, the endogenous levels ofboth bound and free gibberellin (GA) are highest in embryo tissueof young immature avocado seeds. Experimentation with the gelatinaceousendosperm tissue of these seeds indicated that hydrolysis ofstarch reserves may be triggered by the embryo-contained GAacting on the ‘endosperm sheath’, a thin layer ofcells encompassing the endosperm. In the mature fruit when theendosperm and the encompassing layer are no longer present andin which starch reserves are present in the two stony cotyledons,the papery bi-integumental layer, commonly regarded as biologicallyinert, retains the ability to trigger starch hydrolysis uponcontact with GA. From these data and also based upon the findingsthat during germination levels of endogenous GA increase inembryo tissue and that exogenously applied GA₃ is effectivein induction of dormancy break it is proposed that in the avocadoseed an analogy, physiological but not anatomical, to the cerealendosperm system exists.     

Antioxidants in relation to lipid peroxidation

The role of antioxidants in lipid peroxidation is reviewed. Specifically, the rate and mechanism of inhibition of lipid peroxidation by water-soluble and lipid-soluble, chain-breaking antioxidants have been discussed.     

Primary Structure of a Cysteine Proteinase Inhibitor from the Fruit of Avocado (Persea americana Mill)

The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.     

Evidence for the presence of a native,non-auxinic rooting promoter in avocado (Persea americana Mill.)

The presence of a rooting promoter in paratially purified extracts of avocado (Persea amricana Mill.) organs has been demonstrated using the mung bean rooting bioassay. Extraction with 80% methanol was followed by partition into diethyl ether, paper chromatography (PC) and 3 steps of thin layer chromatography (TLC). The number of roots induced by the rooting promoter in the absence of exogenous auxin was 5 to 7 times higher than that of the water control and 50% higher than by 4-(indol-3-yl) butyric acid (IBA) at its reported optimum concentration. Rooting of tomato, Coleus and young avocado cuttings was also enhanced by the rooting promoter. The rooting promoter was inhibitory in the wheat coleoptile section elongation bioassay for auxins and had slight inhibitory activity in the split pea stem curvature test.The biological properties of the avocado rooting promoter may be comparable to those of -(p-chlorophenoxy) isobutyric acid (PCIB) which acts as an anti-auxin in certain bioassays and, nevertheless, promotes the rooting of mung bean cuttings.