Serum release of hepatic calcium-binding protein regucalcin by liver injury with galactosamine administration in rats

The incorporation and 9 desaturation of exogenous [¹⁴C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0–160 M) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 Mca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [¹⁴C]stearate (9 desaturation) correlated well with the uptake curve between 0–80 M concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [¹⁴C]stearic acid (80 M) was also studied from 0–6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.Abbreviations FAME Fatty Acid Methyl esters - GLC gas-liquid chromatography - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanosulfonic acid - HTC Hepatoma Tissue Culture - IMEM-Zo Improved Minimal Essential Medium-zinc optional     

Effect of washing on the plasma membrane and on stress reactions of cultured rose cells

The effects of plant growth regulators, light intensity, and end-of-day (EOD) light quality treatments on node and microtuber induction (% of cultures with microtubers) and development (fresh weight of microtubers) in yam (Dioscorea alata L. cv. Oriental) cultures were investigated. Nodal segments were excised from plantlets cultured on tuberization medium containing growth regulators and exposed to various light treatments. Absciscic acid (1 M) stimulated and cytokinins (2.5 M) inhibited microtuber development from yam nodal segments cultured on Mantell's and Hugo's full-strength tuberization medium under 8-h photoperiods. EOD far-red (FR) light inhibited microtuber induction and development and enhanced node formation. EOD FR light effects were nullified by immediately following the FR treatment with red light. This suggested the involvement of phytochrome in these processes. The lowest light intensity evaluated (12 mol m^–2 s^–1) inhibited microtuber, root and shoot production as compared to light intensities of 42, 72 and 102 mol m^–2 s^–1. Kinetin (2.5 m) in half-strength tuberization medium inhibited microtuber induction and development but did not affect node production in the light intensity evaluation.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - 2iP 6-(c,c-dimethylallylamino)-purine - NAA napthaleneacetic acid - R light red light - FR light far-red light - EOD light end-of-day light     

Uptake and metabolism of eicosa-8,11,14-trienoic acid in normal hepatocytes and HTC cells

Summary The incorporation and conversion of eicosa-8, 11, 14-trienoic acid to arachidonic acid were studied in HTC cells (7288 c hepatoma cells) and isolated hepatocytes of rat. The cells were incubated at different concentrations of the acid during 0 to 6 hours. Both kinds of cells were able to take up the acid. However, the HTC cells have a greater avidity for the eicosatrienoic acid than normal liver cells. The incorporation of the acid modified the fatty acid composition of the cells and caused a decrease in the amount of saturated and monoenoic acid. In both cells eicosatrienoic acid was converted to arachidonic acid. However, in HTC cells arachidonic acid level was low and not correlated with the amount of the eicosatrienoic acid incorporated. This fact is apparently not due to an impairment in 5 desaturation activity since this enzyme is active in both cells. It would be possible that arachidonic acid production in malignant cells would be also interrupted in another metabolic pathway after 6 desaturation step. The strikingly low amount of arachidonic acid biosynthesized in HTC cells compared to normal hepatocytes could be interpreted as a consequence of a lower availability of eicosatrienoic acid for the microsomal desaturation system in malignant cells, in addition to the low A6 desaturase activity.     

Epizooic ciliates (Vorticella sp.) compete for food with their host Daphnia longispina in a small polyhumic lake

Summary Clearance rates of epizooic ciliates (Vorticella sp.) were measured together with their host, a planktonic cladoceran Daphnia longispina by using fluorescent latex beads as tracers of food. Vorticellans and their host graze on food of same size range (nanoplanktonic algae and bacteria). Individual clearance rates of Vorticella averaged 6.9 and 7.0 l ind^-1 h^-1 and those of Daphnia 463 and 708 l ind^-1 h^-1 for beads with diameter 2.00 and 3.92 m. On the average, epizooic vorticellans together on the carapace of Daphnia cleared particles with rates representing 25–33% of that the host cleared, the maximum rates being 50–80%. In a steeply stratified polyhumic lake vorticellans take advantage of following Daphnia to food patches and they can severely compete for food with their host.     

The regulation of total creatine content in a myoblast cell line

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 M) and largely dependent on extracellular [Na⁺]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na⁺,K⁺-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed - and -adrenergic agonist noradrenaline, the -adrenergic agonist isoproterenol, the ₂-agonist clenbuterol and the cAMP analogue N⁶,2-O-dibutyryladenosine 3,5-cyclic monophosphate, but was unaffected by the ₁ adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed - and -antagonist labetalol and by the -antagonist propranolol, but was unaffected by the ₂ antagonist phentolamine; greater inhibition was caused by the ₂ antagonist butoxamine than the ₁ antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3,5-triiodothyronine (at 70 M) and to 220 ± 35% of control by amylin (60 nM). As 3,3,5-triiodothyronine, amylin and isoproterenol all stimulate the Na⁺,K⁺-ATPase, we suggest that they stimulate Na⁺-creatine cotransport indirectly by increasing the transmembrane [Na⁺] concentration gradient and membrane potential.Abbreviations IGF-I insulin-like growth factor I - IGF-II insulin-like growth factor II - T₃ 3,3,5-triiodothyronine - CGRP calcitonin gene-related peptide     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

Tight-junction protein ZO-1 isoforms (α+ and α−) show differential extractability and epidermal-growth-factor-induced tyrosine phosphorylation in A431 cells

Summary ZO-1, a cytoplasmic plaque protein of tight junctions, exists in vivo as two major isoforms which are defined by the presence or absence of an 80 amino acid domain termed . The ZO-1 (+) isoform is expressed in most epithelial cells while ZO-1(–) isoform expression is restricted to endothelial cells and some highly specialized epithelial cells, suggesting that the isoforms serve different functions. We had previously demonstrated that both ZO-1 isoforms are expressed in A 431 cells and are tyrosine phosphorylated in response to epidermal-growth-factor treatment. In the present study, we found that the (-) isoform of ZO-1 was more tightly associated with the cytoskeleton than was the (+) isoform, based on extraction in nonionic detergent. In addition, the ZO-1(-) was preferentially tyrosine phosphorylated in response to epidermal-growth-factor treatment. However, both isoforms became more tightly associated with the cytoskeleton after A 431 cells were exposed to epidermal growth factor. Immunofluorescence analysis of A 431 cells with isoform-specific antibodies demonstrated that functional differences in ZO-1 isoform behavior were not due to differences in their subcellular locations. The coincident localization of these isoforms does not rule out different affinities for interacting proteins related to the presence or absence of the domain, and it is these interactions that are likely to explain functional differences between the isoforms.     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide     

Rhizogenesis and somatic embryogenesis in calli from wild olive (Olea europaea var. sylvestris (Miller) Lehr) mature zygotic embryos

In calli from distal and proximal cotyledon segments and from radicles of mature wild olive zygotic embryos, rhizogenesis prevailed during the first and somatic embryogenesis during the second 30 day subculture period. Rhizogenesis was enhanced by low IBA (0.5 and 2.5 M) and 5.0 M 2iP. By reducing by half the media salt concentrations and by inserting a 21 day dark period rhizogenesis was also enhanced. Somatic embryogenesis was inhibited by both 2iP and IBA at concentrations higher than 5.0 M, but was not influenced by lower salt concentrations or by inserting the dark period.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - 2iP N⁶-[2-isopentenyl]adenine - BA 6-benzylaminopurine - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA naphthaleneacetic acid - OM Olive medium [4] - OMc OM medium in which OM macroelements were replaced with the ones proposed by Bourgin & Nitsch [2] - OMe OM salts of quarter strength, with half strength vitamin mixture (except biotin which added in full strength), with 5 M 2iP and 20 g l^-1 sucrose [2] - OMr OM salts half strength containing 5 M IBA and 20 g l^-1 sucrose [2]     

Effects of corticosteroids on short-circuit current across the cecum of the domestic fowl,Gallus domesticus

Summary Both avian corticosteroid hormones, aldosterone and corticosterone, increased short-circuit current across the wall of the ceca of the domestic fowl (Gallus domesticus) in vitro. About 80% of this short-circuit current was inhibited by the Na-channel blocking drug amiloride. Corticosterone was about ten times less potent than aldosterone in increasing short-circuit current and it exerted a similar maximal effect. Cortisol (an endogenous corticosteroid hormone in mammals but not birds) was about ten times less potent than corticosterone and this difference appeared to reflect the presence of the 17-OH group in cortisol. Carbenoxolene, which inhibits 11-hydroxysteroid dehydrogenase, increased the effect of corticosterone. This effect is consistent with inhibition of the metabolism of corticosterone to 11-dehydrocorticosterone. The latter was found to be about 100 times less potent than corticosterone. The effects of both aldosterone and corticosterone (also dexamethasone) were abolished by the mineralocorticoid receptor antagonist spironolactone. The results suggest that corticosterone has an effect similar to aldosterone but in vivo its action may be depressed by the activity of 11-hydroxysteroid dehydrogenase. The sensitivity of the cecal preparations to corticosterone indicates that this hormone could contribute to the regulation of transcecal Na transport (absorption) in vivo.Abbreviations 11-HSD 11-Hydroxysteroid dehydrogenase - sc short-circuit current - KRB Krebs bicarbonate solution     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Antiproliferative effects of bacillus Calmette-Guérin and interferon α2b on human bladder cancer cells in vitro

Direct inhibitory effects of bacillus Calmette-Guérin (BCG) and interferon 2b (IFN2b) on six human bladder carcinoma cell lines, UCRU-BL-13, UCRU-BL-17, UCRU-BL-28, 5637, T24 and J82, were studied using an in vitro proliferation assay. Effects on proliferation following exposure to BCG or IFN2b were analysed by [³H]thymidine incorporation over 7 days. BCG had an antiproliferative effect on all bladder lines, while sensitivity to IFN2b varied greatly, being as remarkably low as 1 U/ml for some lines. The antiproliferative effect was greatest when cells were exposed continuously to either agent, but was still evident with a limited exposure. When clinical concentrations were simulated in vitro, BCG+IFN2b was more effective than BCG alone and as effective as a double BCG concentration. We conclude that, in addition to their immunomodulatory effects, BCG and IFN2b directly inhibit the proliferation of human bladder cancer cells, and often at extremely low concentrations.     

Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle inLocusta migratoria

Flight muscle and fat body extracts fromLocusta migratoria were incubated with D-[U-¹⁴C]-glucose or D-[3-³H]-3-deoxy-3-fluoroglucose and the products were analyzed. In the case of the latter compound, radio-chromatographic analysis yielded glycogen and trehalose fractions that were shown by¹⁹F nuclear magnetic resonance to contain fluorine. Acid hydrolysis of these fractions liberated tritium labelled 3-deoxy-3-fluoro-D-glucose. In addition to the formation of fluoroglycogen and fluorotrehalose in these tissue extracts, there was an accumulation of tritium labelled fructose.     

Formation of P1, P2-dinucleoside 5′-pyrophosphates under potentially prebiological conditions

Summary Organic pyrophosphates such as UppA and NAD are formed when a solution containing a nucleotide, a nucleoside 5-polyphosphate, Mg²⁺ and imidazole are allowed to dry out. We suggest that this synthesis may have occured concurrently with oligonucleotide formation.Abbreviations Im Imidazole - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - U uridine - p_nA adenosine 5-poly-phosphate containing n phosphate residues - pU uridine 5-phosphate - AppA P₁,P₂-diadenosine 5-pyrophosphate - UppA P₁-(uridine 5)-P₂-(adenosine 5)-pyrophosphate - ImpA adenosine 5-phosphorimidazolide - NMN nicotinamide mononucleotide - NAD nicotinamide-adenine dinucleotide     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Incorporation and metabolic conversion of saturated and unsaturated fatty acids in SK-Hep1 human hepatoma cells in culture

We report here a study of the incorporation and metabolism of various long chain fatty acids in SK-Hep-1 cultured hepatoma cells. Medium supplementation with radiolabelled palmitic, stearic, linoleic, -linolenic and eicosa-8, 11,14-trienoic acids (1 µM, 24 H) resulted in an active uptake of each of these precursors by the cultures. Subsequent analysis of the cellular lipids indicated that they exhibit almost all the enzymic activities of polyunsaturated fatty acid metabolism that are characteristic of normal hepatic cells. With respect to the desaturation capacities of this cell line, although -linolenic acid reacted more extensively than did linoleic acid and the conversion of 8,11,14-eicosatrienoic acid by the 5 specific enzyme was more avid than had been previously seen in normal rat or human liver: the saturated fatty acids constituted relatively poor substrates, being preferentially chain-elongated rather than (mono) desaturated at the 9 position. Analysis of the fatty acid profiles of total cellular lipids and of various lipid subclasses, however, revealed a relative paucity of essential fatty acids when compared with the abundance of endogenous monoenoic acids (particularly oleic). Of the total cellular fatty acids, 58% were present in the form of phospholipids; with 33% of the remaining 42% (i.e., the neutral lipids) being associated with triacylglycerol fraction. Within the total lipids, phosphatidyl-choline and phosphatidyl-ethanolamine were the major sites for the incorporation of all metabolic products derived from the incubated radiolabelled 16- and 18-carbon fatty acid precursors, whereas the phosphatidyl-inositol fraction was the predominat recipient of nascent arachidonic acid when the eicosatrienoate was the substrate. The express purpose of this investigation was to characterize the biochemical routes involved in the anabolism of various essential fatty acids in the human hepatocyte, through the use of cultured human hepatoma cells as an experimental model system. In view of the similarities between certain aspects of the polyunsaturated fatty acid metabolism of these cells and the corresponding properties of other mammalian hepatic or liver-derived tissues, the data presented here would thus constitute a significant beginning alone those lines. Moreover, considering the extreme difficulty in obtaining for such investigation relevant tissue samples from normal human sources, we regard these results — and the availability for use of this particular human hepatoma cell line — as important new developments in the effort to characterize a useful experimental model both for gaining immediate information and for designing future experiments.     

High affinity insulin binding in the human placenta insulin receptor requires αβ heterodimeric subunit interactions

Summary Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot.¹²⁵I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an heterodimeric insulin receptor complex from the ₂₂ heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional heterodimeric insulin receptor complex. (ii) the heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the ₂₂ association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the heterodimers.Abbreviations used are ATP adenosine 5-triphosphate - DTT dithiothreitol - SDS sodium dodecyl sulfate - DSS disuccinimidyl suberate - NEM N-ethylmaleimide - IGF-I insulin-like growth factor-I - EDTA ethylenediaminetetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

Somatic embryogenesis and regeneration of flowering plants in rose

Summary Somatic embryos of the cut rose cultivars Domingo and Vickey Brown were obtained from callus derived from leaf explants on half strength Murashige and Skoog medium with low concentrations of kinetin and 1-naphthyl acetic acid or 2-naphthyloxyacetic acid. Somatic embryos were first observed after 6 to 12 weeks of culture on callus formed at the basis or midrib of the leaf. Embryos could be grown to phenotypically true to type greenhouse plants.Abbreviations IAA 3-indole-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthylacetic acid - NOA 2-naphthyloxyacetic acid - BAP 6-benzylaminopurine - KIN 6-furfurylaminopurine - MS Murashige and Skoog     

Isolation of two protein factors from maize involved in poly U-dependent polyphenylalanine synthesis by maize ribosomes

Summary Two protein factors, isolated from S-100 of maize shoots, were found to be required not only for a Mg²⁺ shift for poly U-dependent polymerization of phenylalanine, but also for polyphenylalanine synthesis. They seem to be crude preparations of EF-1 and EF-2. The poly U-directed amino acid incorporation by washed ribosomes was completely dependent upon these factors.Abbreviation EF-1 Elongation factor 1 - EF-2 Elongation factor 2