Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants.

The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays) floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.

Calcium-regulated proteolysis of eEF1A.

Eukaryotic elongation factor 1alpha (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [(14)C]Ethanolamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incorporated detectable quantities of [(14)C]ethanolamine (Ransom et al., 1998). When 1 mM CaCl(2) was added to microsomes containing [(14)C]ethanolamine-labeled eEF1A ([(14)C]et-eEF1A), there was a 60% decrease in the amount of [(14)C]et-eEF1A recovered after 10 min. The loss of endogenous [(14)C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca(2+)-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [(14)C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A(2), resulted in a decrease in [(14)C]et-eEF1A from carrot microsomes. The fact that there was no loss of [(14)C]et-eEF1A with phospholipase A(2) treatment even in the presence of 1 mM Ca(2+) suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca(2+) concentrations, proteolysis of eEF1A was pH sensitive. When 1 microM CaCl(2) was added at pH 7.2, 35% of [(14)C]et-eEF1A was lost; while at pH 6.8, 10 microM CaCl(2) was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca(2+) and lipid-mediated signal transduction cascades.

Inositol phospholipids as plant second messengers.

Two plasma membrane lipids, phosphatidylinositol monophosphate (PtdIns4P) and phosphatidylinositol bisphosphate (PtdIns(4,5)P2), have been shown to be key intermediates in stimulus response pathways in many animal cells. PtdIns4P and PtdIns(4,5)P2 act as sources of second messengers and they directly alter the activity of membrane enzymes. These lipids and the enzymes involved in their metabolism are found in the plasma membranes of plant cells. A clear role for the inositol phospholipids in plant signal transduction, however, has not emerged. In this chapter we present some of the questions raised by current work in the area and propose an alternative focus for phosphoinositide metabolism in plants: a role for PtdIns4P and PtdIns(4,5)P2 as direct effectors of membrane structure and function.

Colonization of human genital tract by Streptococcus agalactiae.

This bacteriological study involved 92 young married couples who required medical help because of infertility. Colonization of male urethra or the presence of Streptococcus agalactiae in the ejaculate was detected in 9.78% of men. Vagina or vagina and cervix were colonized in 4.35% of women. Oral application of phenoxymethylpenicillin temporarily eliminated S. agalactiae from the genital tract of all colonized partners. Repeated colonization occurred 3-7 months later, in three cases by different serotypes compared with pretreatment period, in one case by the same serotype.

Phagetypes of human and bovine Streptococcus agalactiae isolates in Slovenia.

In two rural areas 34 human (colonization) and 2 bovine (mastitis) Str. agalactiae strains were isolated. Identical serotypes and phagetypes in persons and cows were established: serotype R with phagetype 14/27/30/31 in two persons and one cow from two neighbouring villages, and serotype Ic with phagetype 4/12/16/18/20 in a child and a cow from the same household. Identical serotypes and phagetypes were also found in two married couples and in members of other families. Serotypes with phagetypes confirm the transmission of Str. agalactiae from person to person, but also show, that the transmission from cows to humans might be possible.

Factors influencing the uptake of cholesterol by isolated brush border membranes from rabbit small intestine.

The uptake of [3H] cholesterol by isolated brush border membranes from rabbit small intestine was studied under different conditions. In initial experiments the uptake from taurocholate-micellized suspensions was found to be greatly enhanced by Ca2+. Maximum stimulation was obtained with 10 mM Ca2+ and this condition was adopted for some of the subsequent experiments. In the presence of this cation, the uptake was too rapid to allow kinetic studies and only equilibrium values could be measured. Addition of lecithins to the micelles had an inhibitory effect on the equilibrium uptake in the presence of Ca2+ and on the rate of uptake in the absence of this cation which increased with acyl chain length. Admixture of the sterol with dipalmitoylphosphatidylethanolamine stimulated the uptake whereas admixture with dipalmitoylphosphatidylglycerol as well as with 1-monoolein alone or together with oleic acid had little or no effect on uptake in the presence of Ca2+. Again with this cation present, preincubation of membranes with the dipalmitoyl analogue of lecithin increased the equilibrium uptake of the sterol and abolished the inhibitory effect of the choline lipid. This inhibitory effect could be seen only with untreated membranes when lecithin and cholesterol were micellized together. In the absence of Ca2+, preincubation with dipalmitoylphosphatidylcholine had no marked effect on the rate of cholesterol uptake and the suppressive effect of lecithin on this rate could be seen even with lipid-treated membranes provided the lecithin and sterol were micellized together.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine.

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

A 17 beta-hydroxysteroid dehydrogenase of female rabbit liver cytosol. Purification and characterization of multiple forms of the enzyme.

Multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver were identified. NAD-dependent and NADP-dependent enzyme activities were separated by affinity chromatography on agarose-immobilized Procion Red HE3B, and three forms of the NADP-dependent enzyme activity were purified by chromatofocusing. These three enzyme forms are charge isomers and have no quaternary structure. The enzymes catalysed the C-17 oxidoreduction of oestrogens and androgens; with all enzyme forms the activity towards androgens was higher than that toward oestrogens. The enzymes also exhibited 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. Comparison of the relative activities of the enzymes towards a number of oestrogen and androgen substrates revealed differences among the enzyme forms for both the oxidative and the reductive reactions. In particular, one enzyme form had a significantly lower Km for the 3 alpha-hydroxysteroid substrate and a higher 3 alpha-/17 beta-hydroxysteroid dehydrogenase activity ratio than the other two enzyme forms.

Studies on the mechanism of cholesterol uptake and on the effects of bile salts on this uptake by brush-border membranes isolated from rabbit small intestine.

The effect of bile salts and other surfactants on the rate of incorporation of cholesterol into isolated brush-border membranes was tested. At constant cholesterol concentration, a stimulatory effect of taurocholate was noticed which increased as the bile salt concentration was raised to 20 mM. Taurodeoxycholate was as effective as taurocholate at concentrations of up to 5 mM and inhibited at higher concentrations. Glycocholate was only moderately stimulatory whereas cholate was nearly as effective as taurocholate at concentrations above 5 mM. Other surfactants such as sodium lauryl sulfate and Triton X-100 were very inhibitory at all concentrations tried whereas cetyltrimethyl ammonium chloride was stimulatory only at a very low range of concentrations. These micellizing agents all caused some disruption of the membranes and the greater effectiveness of taurocholate in stimulating sterol uptake was partly relatable to the weaker membrane solubilizing action of this bile salt. Preincubation of membranes with 20 mM taurocholate followed by washing and exposure to cholesterol-containing lipid suspensions lacking bile salt, did not enhance the incorporation of the sterol. In the absence of bile salt the incorporation of cholesterol was unaffected by stirring of the incubation mixtures. Increasing the cholesterol concentration in the mixed micelle while keeping the concentration of bile salt constant caused an increase in rate of sterol incorporation. This increased rate was seen whether the cholesterol suspension was turbid, i.e., contained non-micellized cholesterol, or whether it was optically-clear and contained only monomers and micelles. When the concentration of taurocholate and cholesterol were increased simultaneously such that the concentration ratio of these two components was kept constant, there resulted a corresponding increase in rate of cholesterol uptake. The initial rates of cholesterol incorporation from suspensions containing micellar and monomer forms of cholesterol were much larger than from solutions containing only monomers of the same concentration. The rates of incorporation of cholesterol and phosphatidylethanolamine from mixed micelles containing these lipids in equimolar concentrations were very different. The results as a whole suggest at least for those experimental conditions specified in this study, that uptake of cholesterol by isolated brush-border membranes involves both the monomer and micellar phases of the bulk lipid and that the interaction of the micelles with membrane does not likely involve a fusion process.

The effect of phosphoglycerides on the incorporation of cholesterol into isolated brush-border membranes from rabbit small intestine.

The incorporation of cholesterol from bile salt micelles into brush-border membranes was found to be similar whether these originated from jejunum, ileum or whole small intestine. This incorporation, however, was appreciably lower in membranes obtained from duodenum. Studies pursued with membranes from whole small intestine revealed that dipalmitoyl or dioleoylphosphatidylcholine, when micellized together with the sterol and taurocholate markedly inhibited the incorporation. The didecanoyl and dilauroyl analogues of this lipid class were without significant effect. Preincubation of the membranes for 30 min at 37 degrees C with or without dipalmitoylphosphatidylcholine had no effect on cholesterol incorporation. Again in this case, suppression of sterol uptake could be seen only when phosphatidylcholine was admixed with the sterol. In contrast, dipalmitoyl and dilauroylphosphatidylethanolamines were found to be stimulatory rather than inhibitory. Addition of palmitic acid to the sterol-bile salt micelles had no effect on the uptake of cholesterol; however, this fatty acid could completely reverse the inhibition of cholesterol uptake by dipalmitoylphosphatidylcholine. The present study supports the conclusion that cholesterol incorporation into isolated brush-border membranes is governed largely by factors which affect the partitioning of the sterol out of the bile salt micelle.

Pigment production in human and bovine streptococcus agalactiae strains.

Most of human isolates (66%) and more than a half of bovine isolates from privately-owned herds (57.1%) produced pigment at least on one of two media (DMS-agar, Todd-Hewitt broth). None of bovine isolates from socially-owned farms produced pigment on DMS and TH broth. The results are in accord with previous investigations, in which some biochemical properties and serotypes of human and bovine isolates in Slovenia were studied.

Interaction of lipids with intestinal brush border membrane preparations.

Conditions for uptake of lipids by rabbit intestinal brush border membrane preparations were investigated. A variety of lipids were found to be incorporated, including choline and ethanolamine phosphatides as well as cholesterol, diglyceride, and fatty acid. The incorporation of those lipids tested was enhanced by Ca2+ and other divalent cations but not by monovalent cations. The optimal Ca2+ concentration was approximately 10 mM. The uptake varied with lipid and membrane protein concentration and proceeded at rates which were too rapid to measure under several assay conditions tried. Incorporations were decreased substantially outside the pH range of 6.5-8.0. The effect of one lipid, phosphatidylcholine, on the structural appearance of the membrane fraction was examined by electron microscopy. No free or surface-bound lipid structures could be detected and the membrane fractions appeared to be unchanged after uptake.

Comparative studies of some biochemical properties of human and bovine Streptococcus agalactiae strains.

Both, bovine and human strains of Str. agalactiae grew well in sterile cow-milk, the majority of bovine strains curdled the milk. The majority of human strains grew well in human urine. Bovine strains, isolated from the milk of cows from large socially-owned farms, had biochemical properties characteristical for bovine strains. Bovine strains, isolated from the milk of cows of private raisers and human strains, isolated from the raisers of the cows, deviated in their characteristical biochemical properties. It seems, that with these strains it came to reciprocal influences. This confirms our previous presumption, that infection due to Str. agalactiae in humans and cows may take a reciprocal course, if the circumstances are adequate. The tracing of infection sources is possible only by serotyping.

A contribution to the research of infection of cows and humans with Streptococcus agalactiae.

In the small area of eight alpine villages in Slovenia 159 cows and 113 persons from 34 family-farms on Streptococcus agalactiae (Str. agal.) were examined. From the milk of 35 cows (22%) from 21 farms and from the throat and urine of 17 persons (15%) from 14 farms Str. agal. was isolated. Identical serotypes of Str. agal. were established on the farms, where infected cows and infected persons were detected: in cows serotypes II and III and in persons serotypes II, III and R. In persons from the farms, where the cows were negative, other serotypes were found: Ib, Ic and II. In 12 persons Str. agal. was isolated from urine, in 3 persons from the throat and in 2 persons from the urine and throat. All positive persons were without visible clinical symptoms. We suggest, that one of the ways of infection with Str. agal. in humans is probably a direct route with infected milk or from the infected cows.

Findings of serotypes of group B streptococci in human and bovine sources.

Five hundred and fifty-five strains of S. agalactiae of human or bovine origin were serologically typed. In human strains, serotype Ia was the most frequent irrespective of the source and kind of cultivation material, but serotype R was very frequent in urine. In bovine strains, one serotype was found as a rule in one stable both in small private and large socialist farms. The reason for such uniformity of serotypes is not known. Monocolonisation is one of the alternatives, but it seems more reasonable to assume that, the most resistant or more invasive strain will predominate in the herd in the course of time.