Stearidonic acid-enriched flax oil reduces the growth of human breast cancer in vitro and in vivo.

The 20 and 22 carbon n-3 long-chain polyunsaturated fatty acids (LCPUFA) inhibit the growth of tumors in vitro and in animal models, but less is known about the 18 carbon n-3, stearidonic acid (SDA). This study aimed to establish and determine a mechanism for the anti-cancer activity of SDA-enriched oil (SO). SO (26 % of lipid) was produced by genetically engineering flax and used to treat human tumorigenic (MDA-MB-231, MCF-7) and non-tumorigenic (MCF-12A) breast cells. Nu/nu mice bearing MDA-MB-231 tumor were fed SO (SDA, 4 % of fat). Cell/tumor growth, phospholipid (PL) composition, apoptosis, CD95, and pro-apoptotic molecules were determined in SO-treated cells/tumors. Compared to a control lipid mixture, SO reduced (p < 0.05) the number of tumorigenic, but not MCF-12A cells, and resulted in higher concentration of most of the n-3 fatty acids in PL of all cells (p < 0.05). However, docosapentaenoic acid increased only in tumorigenic cells (p < 0.05). SO diet decreased tumor growth and resulted in more n-3 LCPUFA, including DPA and less arachidonic acid (AA) levels in major tumor PL (p < 0.05). Treatment of MDA-MB-231 cells/tumors with SO resulted in more apoptotic cells (in tumors) and in vivo and in vitro, more CD95+ positive cells and a higher expression of apoptotic molecules caspase-10, Bad, or Bid (p < 0.05). Supplementing SO alters total PL and PL classes by increasing membrane content of n-3 LCPUFA and lowering AA (in vivo), which is associated with increased CD95-mediated apoptosis, thereby suggesting a possible mechanism for reduce tumor survival.

Evaluating the conjugated linoleic acid and trans 18:1 isomers in milk fat of dairy cows fed increasing amounts of sunflower oil and a constant level of fish oil.

The objective was to evaluate different levels of sun-flower oil (SFO) in dairy rations to increase vaccenic (trans-11-18:1) and rumenic acids (cis-9,trans-11-18:2) in milk fat, and assess the content and composition of other trans-octadecenoic (trans-18:1) and conjugated linoleic acids (CLA) isomers. Eighty lactating Holstein cows were fed control diets for 4 wk and then placed on 4 diets for 38 d; milk fat was analyzed after 10 and 38 d. The treatments were: control, 1.5% SFO plus 0.5% fish oil (FO), 3% SFO plus 0.5% FO, and 4.5% SFO plus 0.5% FO. The forage-to-concentrate ratio was 50:50 and consisted of barley/alfalfa/hay silage and corn/barley grain concentrate. There were no differences in milk production. Supplementation of SFO/FO reduced milk fat compared with respective pretreatment periods, but milk protein and lactose levels were not affected. There was a linear decrease in all short- and medium-chain saturated fatty acids (SFA) in milk fat after 10 d (25.5, 24.1, 20.2, and 16.7%) and a corresponding linear increase in total trans-18:1 (5.2, 9.1, 14.1, and 21.3%) and total CLA (0.7, 1.9, 2.4, and 3.9%). The other FA in milk fat were not affected. Separation of trans-18:1 isomers was achieved by combination of gas chromatography (GC; 100-m highly polar capillary column) and prior separation of trans FA by silver ion-thin layer chromatography followed by GC. The CLA isomers were resolved by a combination of GC and silver ion-HPLC. The trans-11- and trans-10-18:1 isomers accounted for approximately 50% of the total trans-18:1 increase when SFO/FO diets were fed. On continued feeding to 38 d, trans-11-18:1 increased with 1.5% SFO/FO, stayed the same with 3%, and declined with 4.5% SFO/FO. Rumenic acid showed a similar pattern on continued feeding as trans-11-18:2; levels increased to 0.43, 1.5, 1.9, and 3.4% at 10 d and to 0.42, 2.15, 2.09, and 2.78% at 38 d. Rumenic acid was the major CLA isomer in all 4 diets: 66, 77, 78 and 85%. The CLA isomers trans-7,cis-9-, trans-9,cis-11-, trans-10,cis-12-, trans-11,trans-13-, and trans-9,trans-11-/trans-10,trans-12-18:2 also increased from 0.18 (control) to 0.52% (4.5% SFO/FO). Milk fat produced from 3% SFO/FO appeared most promising: trans-11-18:1 and cis-9,trans-11-18:2 increased 4.5-fold, total SFA reduced 18%, and moderate levels of trans-10-18:1 (3.2%), other trans-18:1 (6.6%) and CLA isomers (0.5%) were observed, and that composition remained unchanged to 38 d. The 4.5% SFO/FO diet produced higher levels of trans-11-18:1 and cis-9,trans-11-18:2, a 28% reduction in SFA, and similar levels of other trans-18:1 (9.2%) and CLA isomers (0.52%), but the higher levels of trans-11-18:1 and cis-9,trans-11-18:2 were not sustained. A stable milk fat quality was achieved by feeding moderate amounts of SFO (3% of DM) in the presence of 0.5% FO that had 4% vaccenic and 2% rumenic acids.

Developmental Profile of Diacylglycerol Acyltransferase in Maturing Seeds of Oilseed Rape and Safflower and Microspore-Derived Cultures of Oilseed Rape.

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was assayed during the maturation of seeds of oilseed rape (Brassica napus L.) and safflower (Carthamus tinctorius L.). Developmental studies were also conducted with microspore-derived embryos of oilseed rape (B. napus L. cv Topas) and an embryogenic microspore-derived cell-suspension culture of winter oilseed rape (B. napus L. cv Jet Neuf). In the maturing seeds, diacylglycerol acyltransferase activity increased to a maximum during rapid accumulation of lipid and declined, thereafter, with seed maturity. In microspore-derived embryos of oilseed rape (cv Topas), high levels of diacylglycerol acyltransferase activity were found throughout the early torpedo to late cotyledonary developmental stages with maximum enzyme specific activity associated with the mid-cotyledonary developmental stage. The cell-suspension culture of winter oilseed rape (cv Jet Neuf) contained 3 to 4% triacylglycerol on a dry weight basis and represented about half of the total lipid. The fatty acid profile of total lipid and triacylglycerol in the cell-suspension culture was similar in samples taken during a 1-year period. The Jet Neuf culture contained diacylglycerol acyltransferase with specific activity similar to that of Topas microspore-derived embryos. Jet Neuf diacylglycerol acyltransferase also displayed an enhanced specificity for erucoyl-CoA over oleoyl-CoA when assayed with 14 [mu]M acyl-coenzyme A in the reaction mixture. The specific activity of diacylglycerol acyltransferase in homogenates prepared from the Jet Neuf culture ranged from 5 to 15 pmol of triacylglycerol min-1 mg-1 of protein when assayed at intervals during a period of 1 year. Thus, the cell-suspension culture may represent an attractive tissue source for purification and characterization of triacyl-glycerol biosynthetic enzymes.

Human acylation stimulating protein enhances triacylglycerol biosynthesis in plant microsomes.

Diacylglycerol acyltransferase has a universal role in catalyzing the acyl-CoA-dependent formation of triacylglycerol in microorganisms, animals and plants. Acylation stimulating protein, from human blood, is known to enhance diacylglycerol acyltransferase activity and triacylglycerol biosynthesis in human adipocytes. In the current study, acylation stimulating protein was also shown to enhance diacylglycerol acyltransferase activity in microsomes from cell suspension cultures of oilseed rape. Enzyme stimulation occurred over the pH range of 6-9 but the degree of stimulation decreased with increasing ionic strength at pH 7.4. Varying acyl-CoA concentration did not affect the degree of stimulation. Membranes from triacylglycerol producing cells in plants and humans may have similar binding sites for acylation stimulating protein which have been preserved during molecular evolution. The results suggest that human acylation stimulating protein may be useful in modifying lipid biosynthesis in plants.

Isolation and identification of a new potent inhibitor of creatine kinase from human serum.

Human serum contains a very potent inhibitor of creatine kinase in addition to urate which was characterized as an inhibitor of this enzyme by Warren (Warren, W.A. (1975) Clin. Biochem. 8, 247). This new inhibitor was isolated from human serum by dialysis, negative DEAE-cellulose adsorption and a series of gel filtration chromatographies. The inhibitor so isolated was an organic compound. Kinetic investigation showed noncompetitive inhibition versus creatine, as well as MgATP. Infrared spectroscopy revealed amino and carboxyl groups. The isolated material was ninhydrin-positive. Thin-layer chromatography of the dansyl derivative showed several fluorescent spots. Quantitative amino acid analysis revealed the presence of several amino acids. Of these only cystine and cysteine were inhibitory in the standard inhibitor assay, using rabbit MM creatine kinase. Testing with human MM creatine kinase only cystine was found inhibitory. Cystine has a specific inhibitor activity of 600 anti-Units per mg, using rabbit or human MM creatine kinase, respectively. Urate by comparison has a specific inhibitor activity of 45 anti-Units per mg using human MM creatine kinase.

Effect of acute experimental influenza A virus pneumonia on concentration of alpha 1-acid glycoprotein in mouse serum.

In mice, the mean serum concentration of the acute-phase reactant alpha 1-acid glycoprotein increased 34-48% over 14 days following experimental induction of pneumonitis by intranasal inoculation of influenza A virus. Inoculation of undiluted (hemagglutination titer 640) and 10(-1) dilution of virus was followed by development of maximum concentrations of alpha 1-acid glycoprotein in serum at seven days, of 334 micrograms/ml, compared to a concentration in control mice inoculated with irradiated inactivated virus of 225 micrograms/ml (P = 0.002). Infection with 10(-2) virus yielded a peak serum alpha 1-acid glycoprotein of 301 micrograms/ml at four days, 34% higher than in control mice at four days (P = 0.04). There were no differences in alpha 1-acid glycoprotein concentrations among virus-infected mice. Influenza A virus pneumonitis was confirmed histologically, by virus isolation, and by serologic testing, but no inoculum-dependent differences were observed. On day 7, there was a direct relationship demonstrated between the severity of pneumonitis evaluated histologically and the serum alpha 1-acid glycoprotein concentration (r = 0.50; P less than 0.02). Influenza A pneumonia in mice is associated with increased concentrations of alpha 1-acid glycoprotein in serum; the increase may be directly related to the severity of the pulmonary inflammation.

Characterization of microsomal diacylglycerol acyltransferase activity from bovine adipose and muscle tissue.

The activity of the triacylglycerol bioassembly enzyme, diacylglycerol acyltransferase (DGAT), was characterized in microsomal fractions prepared from bovine subcutaneous (SC) adipose, intramuscular (IM) adipose, and muscle (pars costalis diaphragmatis) tissue. The activity of DGAT was generally higher from SC adipose tissue than from IM adipose or muscle tissue. The characteristics of DGAT activity from the three bovine tissues resembled the activity characteristics observed in previous studies from various other organisms and tissues; the pH optimum was near neutrality, the activity was almost completely inhibited by pre-incubation with N-ethylmaleimide (NEM), and the enzyme accepted a broad range of acyl-CoAs and sn-1,2-diacylglycerols. In some aspects, the SC adipose tissue DGAT activity was different from the DGAT activity from the other two tissues. The SC adipose tissue DGAT activity was not as susceptible to inhibition by NEM as the enzymes from the two other tissue sources, and it exhibited increased specificity for substrates containing oleoyl moieties. The differences in DGAT properties between the three bovine tissues may account to some extent for the differences in the relative fatty acid composition and the positional distribution of fatty acids in triacylglycerol between bovine tissues. The observed differences in enzymatic properties also support recent biochemical and molecular genetic observations that imply the existence of multiple DGAT genes and/or isoforms.

Phosphatidate phosphatases of mammals, yeast, and higher plants.

Phosphatidate phosphatase (EC 3.1.3.4) catalyzes the hydrolysis of phosphatidate to yield sn-1,2-diacylglycerol and inorganic phosphate. In mammalian systems, forms of phosphatidate phosphatase involved in glycerolipid synthesis and signal transduction have been identified. Forms of the enzyme involved in signal transduction have been purified and partially characterized. In yeast, phosphatidate phosphatases associated with the endoplasmic reticulum and mitochondria have also been purified and partially characterized. Information on phosphatidate phosphatases from mammals and yeast is useful in characterizing the enzyme from plant systems. This review examines progress on the characterization of phosphatidate phosphatases from mammals, yeast, and higher plants. The purification and characterization of the phosphatidate phosphatase involved in glycerolipid synthesis in developing oilseeds may lead to the identification of the encoding gene. Increasing our understanding of the enzymes of lipid synthesis in developing seeds will aid in the development of biotechnological strategies for seed oil modification.

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape.

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25 mM, MgSO4 and MgCl2 stimulated microsomal DGAT 25- and 10-fold, respectively. ATP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg2+ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.

Relationship of fatty acid composition to intramuscular fat content in beef from crossbred Wagyu cattle.

The deposition of i.m. fat, or marbling, in cattle is recognized as a desirable carcass trait in North American beef grading schemes. In order to investigate the relationship between degree of marbling and fatty acid composition of whole bovine muscle, we extracted the total lipid from pars costalis diaphragmatis (PCD) (n = 23) and longissimus (n = 36) muscles from Wagyu crossbred cattle that were assigned Canadian Grading Agency marbling scores ranging from 1 to 8 on an inverse 10-point scale (i.e., a score of 1 indicated "very abundant" marbling and a score of 10 would be assigned to a carcass "devoid" of marbling). Fatty acid methyl esters (FAME) of the total lipid and triacylglycerol fractions were resolved and quantified through GLC. Marbling scores were negatively associated with total lipid from both PCD (r = -.57, P < .01) and longissimus (r = -.80, P < .001). Differences between PCD and longissimus were found for almost all FAME studied from both lipid fractions, but no differences (P > .05) were seen when the monounsaturated:saturated fatty acid (MUFA/SFA) ratios were compared. Heifers had higher (P < .05) oleic acid content and lower (P < .05) palmitic acid content in lipid extracted from both muscles, resulting in higher (P < .05) MUFA/SFA ratios than those for steers. The relative amount of myristic acid increased as the lipid content (total lipid and triacylglycerol) increased in either longissimus (r values from .48 to .55; n = 36; P < .01) or PCD muscles (r from .67 to .76; n = 23; P < .001). The relative amount of linoleic acid (cis-9, cis-12 isomer) from total lipid was negatively associated with all chemical measurements of lipid from the longissimus (r from -.52 to -.64; n = 36; P < .001) and PCD muscles (r from -.75 to -.85; n = 23; P < .001). This association was not significant (P > .1) for either muscle when linoleic acid from the triacylglycerol fraction was examined, suggesting the negative association between this fatty acid and lipid content was due to a dilution of membrane phospholipids with increasing triacylglycerol. Indices of fatty acid elongase activity, calculated from FAME data, implicated the balance between this enzyme activity and fatty acid synthase as a source of variation between animals displaying various degrees of marbling and worthy of further investigation to better understand the process of marbling fat deposition in beef cattle.

A novel biological-based assay for the screening of neutralizing antibodies to ricin.

A novel approach to screen hybridomas producing antibodies directed against ricin was developed. Anti-ricin antibodies were produced and characterized based on protection of Sp2/mIL6 myeloma cells and in vivo studies demonstrating neutralization of the toxic effects of ricin in mice. During the production of hybridomas, cells were plated in media supplemented with hypoxanthine, aminopterin, and thymidine supplement (HAT), HAT + ricin, or ricin. Three hybridomas, designated HRF4, HHRD7 and HHRD9, were selected from the media supplemented with ricin and were shown to produce antibodies directed against ricin. Intraperitoneal injection of hybridoma supernatant containing anti-ricin antibodies combined with 0.5 microg ricin (a toxic dosage) protected Balb/C mice from the deleterious effects of the ricin. Hybridoma, HHRD9, did not contain high titre antibodies to ricin but appeared to neutralize the toxic effects of ricin in mice.

An endogenous alpha-amylase inhibitor in barley kernels.

Barley (Hordeum distichum cv Klages) kernels were shown to contain a factor that converted malted barley alpha-amylase II to the alpha-amylase III form. After purification by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel, and gel-filtration on Bio Gel P60, the factor gave a single band of protein on isoelectric focusing. The purified factor inhibited hydrolysis of soluble starch by alpha-amylase II from malted barley and germinated wheat (Triticum aestivum cv Neepawa). However, alpha-amylase I from these cereals was not affected. The inhibitor was not dialyzable and was retained by a PM 10 ultrafiltration membrane suggesting a molecular weight greater than 10,000 daltons. Heat treatment of the inhibitor at 70 degrees C for 15 minutes at pH 5.5 and 8.0 resulted in considerable loss of inhibitory activity.

A calorimetric study of human CuZn superoxide dismutase.

Structural alterations, as manifested by thermal transitions, caused by removal or binding of metal ions to human and bovine CuZn superoxide dismutases (SODs) were investigated by differential scanning calorimetry. Although holo forms of the two mammalian enzymes exhibited irreversible thermal transitions (delta Hcal. = 27.7 J/g and Td = 104 degrees C for bovine SOD; delta Hcal. = 23.6 J/g and Td = 101 degrees C for human SOD), only the bovine apoenzyme showed the presence of a less thermostable form (delta Hcal. = 10.7 J/g and Td = 63 degrees C). These observations suggested that human apo-SOD had considerably less conformational order than bovine apo-SOD. Reconstitution of human and bovine apoenzymes with Cu2+ and Zn2+ resulted in recovery of thermodynamic parameters and specific activity. Binding of Zn2+ alone to human apo-SOD resulted in the formation of two distinct structural units, detectable by differential scanning calorimetry, which underwent conformational disorder at 82 and 101 degrees C respectively. Saturation of binding sites with both Zn2+ and Cu2+ appeared to stabilize the enzyme structure further as shown by elimination of the low-temperature transition and the appearance of another thermal transition at a higher temperature.

Properties of Solubilized Microsomal Lipase from Germinating Brassica napus.

Lipase (triacylglycerol acylhydrolase [EC 3.1.1.3.]) was extracted from the microsomal fraction of cotyledons of dark grown seedlings of Canola (Brassica napus L. cv Westar) by treatment with Triton X-100. The enzyme was partially purified by chromatography on Sephacryl S-300 and DEAE Bio-Gel and was stable when stored at -20 degrees C in 50% (v/v) glycerol. The lipase aggregated readily but the distribution of species present in solution could be controlled by nonionic detergents. A species with an apparent M(r) of about 250,000 was obtained by gel filtration chromatography in the presence of 1% (v/v) Triton X-100. Lipase activity was optimal near neutral pH, and the reaction approached maximum velocity at a concentration of 0.5 to 1 millimolar emulsified triolein. The reaction rate responded linearly to temperature up to about 40 degrees C and the hydrolytic process had an activation energy of 18 kilocalories per mole. Microsomal lipase lost about 20% and 80% activity when heat-treated for 1 hour at 40 degrees C and 60 degrees C, respectively. At appropriate concentrations, the detergents Triton X-100, n-octyl-beta-d-glucopyranoside, (3-[(3-cholamidopropyl-O-dimethylammonio]-1-propanesulfonate, cetyl trimethylammonium bromide, and sodium dodecyl sulfate all inhibited lipase activity. n-Octyl-beta-d-glucopyranoside, however, was stimulatory in the 2 to 8 millimolar concentration range. The inhibitory effects of Triton X-100 were reversible.

Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography.

Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels.

An inhibitor of malted barley (Hordeum vulgare cv Conquest) alpha-amylase II was purified 125-fold from a crude extract of barley kernels by (NH(4))(2)SO(4) fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the alpha-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between alpha-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over alpha-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.

Partial characterization of alpha 1-acid glycoprotein isolated from mouse serum.

AGP was purified from mouse serum by perchloric acid treatment and CM-Sepharose chromatography. Induction of inflammation with turpentine resulted in a 10-fold increase in the serum level of mouse AGP, indicating mouse AGP is an acute phase reactant. Biochemical characterization of mouse AGP indicated similarity with human and rat AGP.