Phthalic Acid Induces Oxidative Stress and Alters the Activity of Some Antioxidant Enzymes in Roots of <Emphasis Type="Italic">Malus prunifolia</Emphasis>

Apple replant is a widespread agricultural problem documented in all of the major fruit-growing regions of the world. In order to better understand the phytotoxic mechanisms induced by allelochemicals involved with this problem, Malus prunifolia plants were grown hydroponically to the six-leaf-stage in the presence of phthalic acid (0 or 1 mM) for 5, 10, or 15 days. Apple plants were evaluated for: shoot and root length, fresh and dry weight, malondialdehyde (MDA) content, hydrogen peroxide (H₂O₂) content, superoxide radical (O₂ ^·−) generation rate, and antioxidant enzyme activities. Shoot and root lengths and fresh and dry weights of M. prunifolia decreased in plants exposed to phthalic acid. MDA and H₂O₂ content increased in phthalic acid-treated plants as did the generation rate of O₂ ^·− in M. prunifolia roots. The activities of superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1), and monodehydroascorbate reductase (EC 1.6.5.4) increased in phthalic acid-stressed roots compared with control roots. These results suggest that activation of the antioxidant system by phthalic acid led to the formation of reactive oxygen species that resulted in cellular damage and the decrease of M. prunifolia growth.     

Correlation of ATP/citrate lyase activity with lipid accumulation in developing seeds of Brassica napus L.

The temporal distribution of ATP/citrate lyase (ACL) activity in developing seeds of Brassica napus L. closely paralleled both that of acetyl-CoA carboxylase and the overall rate of lipid biosynthesis. Maximum ACL activities (250 nmol acetyl-CoA formed min⁻¹·g fresh seed) were recorded between 35 to 42 d after pollination and, if the in vitro data could be extrapolated to the situation in vivo, could account for half of the acetyl-CoA required for the measured rate of fatty acid biosynthesis during seed development. The enzyme appeared to be localized in a subcellular compartment, which was clearly separated from mitochondria on a sucrose gradient and by differential centrifugation, and which corresponded to the chloroplast organelle.     

Some new aspects of isoprenoid biosynthesis in plants—A review

Plants are capable of synthesizing a myriad of isoprenoids and prenyl lipids. Much attention has been focused on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the enzyme that synthesizes mevalonate and is generally considered responsible for the regulation of substrate flux to isoprenoids. In contrast to vertebrates, where there seems to exist only one HMGR gene, in plants a small family of isogenes appears differentially expressed in regard to location and time. Much less is known in plants about the preceding steps,viz. the conversion of acetyl-CoA to HMG-CoA. An enzyme system has been isolated from radish that can catalyze this transformation, and which shows some unusual propertiesin vitro. The intracellular localization of the early steps of isoprenoid biosynthesis in plant cells is still a matter of debate. The various observations and hypotheses derived from incorporation and inhibition studies are somewhat contradictory, and an attempt is being made to rationalize various findings that do not at first seem compatible. There are good arguments in favor of an exclusively cytoplasmic formation of isopentenyl pyrophosphate (IPP) via mevalonic acid, but other studies and observations suggest an independent formation in plastids. Other possibilities are being considered, such as the existence of independent (compartmentalized) biosynthetic pathways of IPP formationvia the socalled Rohmer pathway. Substrate channeling through the formation of end product-specific multienzyme complexes (metabolons) with no release of substrate intermediates will also be discussed. Based in part on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids,” Atlanta, Georgia, May 1994.     

Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver

Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (K_d=10⁻¹⁵ M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures—polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [¹⁴C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons ±3.3%. This protein was shown to bind avidin (M_r=16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [¹⁴C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein. The polyethylene glycol precipitation yielded 200 μg protein (4.4 μg/g liver), with a specific activity of 5 nmol malonyl-CoA/min/mg protein, whereas avidin affinity chromatography yielded 1.75±1.1 mg protein (9.0 μg/g liver), with a specific activity of 1.37±0.18 μmol malonyl-CoA/min/mg protein. The enzyme was citrate dependent showing maximum activity between 10 and 20 mM. Acetyl-CoA carboxylase-specific activity decreased by 50% in the presence of 0.2 M NaCl. These findings suggest that the major protein (M_r=230,000) purified from rainbow trout liver is acetyl-CoA carboxylase with enzyme characteristics comparable to mammalian acetyl-CoA carboxylase.     

Atorvastatin and simvastatin have distinct effects on hydroxy methylglutaryl-CoA reductase activity and mRNA abundance in the guinea pig

The effects of atorvastatin and simvastatin on hydroxy methylglutary (HMG)-CoA reductase activity and mRNA abundance were studied in guinea pigs randomized to three groups: untreated animals and those treated with 20 mg/kg of atorvastatin or simvastatin. Guinea pigs were fasted for 0, 6, 12, or 18 h in an attempt to remove the drug from their systems. Reductase activity and mRNA levels were analyzed after each time point. Reductase inhibitor treatment resulted in 50–60% lower cholesterol concentrations compared to untreated guinea pigs (P<0.0001), while plasma triacylglycerol (TAG) concentrations did not differ among groups. Plasma cholesterol and TAG were 50–70% lower after 18 h fasting in the three groups (P<0.001). In the nonfasting state, simvastatin and atorvastatin treatment did not affect HMG-CoA reductase activity compared with untreated animals. However, after 6 h of fasting, simvastatin-treated guinea pigs had higher HMG-CoA reductase activity than untreated animals (P<0.01), suggesting that the drug had been removed from the enzyme. In contrast, atorvastatin-treated guinea pigs maintained low enzyme activity even after 18 h of fasting. Further, HMG-CoA reductase mRNA abundance was increased by sevenfold after atorvastatin treatment and by twofold after simvastatin treatment (P<0.01). These results suggest that sinvastatin and atorvastatin have different half-lives, which may affect HMG-CoA reductase mRNA levels. The increase in reductase activity by simvastatin during fasting could be related to an effect of this statin in stabilizing the enzyme. In contrast, atorvastatin, possibly due to its longer half-life, prolonged inhibition of HMG-CoA reductase activity and resulted in a greater increase in mRNA synthesis.     

Abnormal suppression of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by hypertriglyceridemic very low density lipoprotein subclasses

Our previous studies showed that hypertriglyceridemic very low density lipoproteins (HTG VLDL) are functionally abnormal. HTG VLDL, but not normal VLDL, suppress HMG-CoA reductase in cultured normal human fibroblasts. To determine if the suppression by HTG VLDL resulted from a subpopulation of smaller suppressive particles, more homogeneous subclasses of VLDL-VLDL₁ (S_f 100–400), VLDL₂ (S_f 60–100), and VLDL₃ (S_f 20–60) were obtained from the d<1.006 (g°ml⁻¹) fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient and tested for suppression in normal human fibroblasts. VLDL₁ and VLDL₂ from each of the 12 normolipemic subjects tested failed to suppress HMG-CoA reductase activity in normal fibroblasts. Eleven out of 12 preparations of normal VLDL₃ suppressed HMG-CoA reductase, but only one-third as effectively as LDL. By contrast, the VLDL₁, VLDL₂ and VLDL₃ from 15 out of 17 hypertriglyceridemic patients (hyperlipoproteinemia Types IIb, III, IV and V) were highly effective in suppression, with half-maximal suppression at 0.1–2.0 μg VLDL protein/ml. The VLDL abnormality is apparently associated with hypertriglyceridemia and not hypercholesterolemia, since VLDL from a homozygous familial hypercholesterolemia patient with a Type IIa pattern did not suppress whereas each of the VLDL subclasses from a Type IIb patient suppressed. Suppression by HTG VLDL in normal cells is apparently a consequence of interaction of the protein portion of the VLDL with the specific LDL cell surface receptor since HTG VLDL₁ treated with 0.1 M 1,2-cyclohexanedione to block arginyl residues failed to suppress the enzyme. Moreover, hypertriglyceridemic S_f 60–400 VLDL failed to suppress HMG-CoA reductase activity in LDL receptor-negative fibroblasts. There were no consistent major compositional differences between comparable normal and hypertriglyceridemic VLDL subclasses which could account for differences in suppression. All VLDL subclasses from Type III subjects were enriched in cholesteryl esters and depleted in triglyceride, relative to the corresponding normal VLDL subclasses. However, Type IV and Type V VLDL subclasses were normal in this repect. We conclude from these studies that small particle diameter is not required for suppression, since HTG VLDL₁ and VLDL₂ which contained few, if any, small particles were effective in suppression. Presented as part of the symposium “Low Density and Very Low Density Lipoproteins” at the American Oil Chemists' Society meeting on May 2, 1979, in San Francisco.     

Enhancement of sterol synthesis by the monoterpene perillyl alcohol is unaffected by competitive 3-hydroxy-3-methylglutaryl-CoA reductase inhibition

Monoterpenes such as limonene and perillyl alcohol (PA) are currently under investigation for their chemotherapeutic properties which have been tied to their ability to affect protein isoprenylation. Because PA affects the synthesis of isoprenoids’ such as ubiquinone’ and cholesterol is the end product of the synthetic pathway from which this isoprenoid pathway branches’ we investigated the effects of this compound upon cholesterol metabolism in the colonic adenocarcinoma cell line SW480. PA (1 mM) inhibited incorporation of ¹⁴C-mevalonate into 21–26 kDa proteins by 25% in SW480 cells. Cholesterol (CH) biosynthesis was assessed by measuring the incorporation of ¹⁴C-acetate and ¹⁴C-mevalonate into 27-carbon-sterols. Cells treated with PA (1 mM) exhibited a fourfold increase in the incorporation of ¹⁴C-acetate but not ¹⁴C-mevalonate into cholesterol. Mevinolin (lovastatin)’ an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase’ at 2 μM concentration’ inhibited CH synthesis from ¹⁴C-acetate by 80%. Surprisingly’ concurrent addition of mevinolin and PA did not significantly alter the stimulatory effects of PA. As observed differences in ¹⁴C-acetate and ¹⁴C-mevalonate precursor labeling could indicate PA affects early pathway events’ the effects of this monoterpene on HMG-CoA reductase activity were evaluated. Unexpectedly’ 1 mM PA did not stimulate activity of this enzyme. Consistent with its action as a reversibly bound inhibitor’ in washed microsomes’ 2 μM mevinolin pretreatment increased reductase protein expression causing a 12.7 (±2.4)-fold compensatory HMG-CoA reductase activity increase; concurrent treatment with 1 mM PA attenuated this to a 5.3 (±0.03)-fold increase. Gas chromatographic analysis confirmed CH was the major lipid present in the measured thin-layer chromatography spot. Since ¹⁴C-acetate incorporation into free fatty acid and phospholipid pools was not significantly affected by PA treatment’ nonspecific changes in whole acetate pool sizes were not indicated. Because increases in endogenous CH synthesis should result in compensatory changes in exogenous sterol utilization’ the effects of PA upon low density lipoprotein (LDL) receptor activity were evaluated. Consistent with the observed increases in CH synthesis’ 1 mM PA decreased ¹²⁵I-LDL internalization to 50% of the fetal bovine serum control; concurrent addition of 2 μM mevinolin attenuated this effect to a reduction of 80% of the control value. Data suggest that in certain colonic tumor cells PA strongly affects cholesterol metabolism via a mechanism of action that is insensitive to the HMG-CoA reductase inhibitor mevinolin.     

Regulation of mevalonate synthesis in low density lipoprotein receptor knockout mice fed n-3 or n-6 polyunsaturated fatty acids

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, catalyzes the formation of mevalonate which is also required for cell proliferation. Changes in HMG-CoA reductase may mediate the differential effects of n-3 and n-6 polyunsaturated fatty acids (PUFA) on experimental mammary tumorigenesis, but the mechanisms by which these fatty acids regulate HMG-CoA reductase are unclear. To determine whether the low density lipoprotein receptor (LDL-R) is required for this regulation, groups of female LDL-R knockout (−/−) and wild-type (+/+) mice were fed 7% fat diets rich in either n-3 (menhaden oil) or n-6 (safflower oil) PUFA for 1 wk. Dietary PUFA and deletion of the LDL-R had independent effects on HMG-CoA reductase and serum lipids, and a significant diet-gene interaction was observed. The effects of PUFA on HMG-CoA reductase in the mammary gland, but not the liver, were mediated by the LDL-R. We also observed that differences in HMG-CoA reductase and serum LDL-cholesterol, high density lipoprotein cholesterol, and triglycerides between −/− and +/+ mice were dependent on whether the mice were fed n-3 or n-6 PUFA. Differences between −/− and +/+ mice were much greater when animals were fed n-6 PUFA rather than n-3 PUFA. These results show that the LDL-R mediates the effects of PUFA on HMG-CoA reductase in the mammary gland but not the liver. Furthermore, the composition of dietary PUFA profoundly influences the effects of deleting the LDL-R on HMG-CoA reductase and serum lipids and suggests that diet may influence the phenotype of other knockout or transgenic animals. This work was presented in part at the Third Congress of the International Society for the Study of Fatty Acids and Lipids, June 1–5, 1998, Lyon, France.     

Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava)

Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3′ and 5′ rapid amplification of cDNA ends. The sequence shows approximately 60–70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-β-d-thiogalactopyranoside and σ-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was≈2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.     

Spectrophotometric assay of 2,4-dienoyl coenzyme a reductase with 5-phenyl-2,4-pentadienoyl-coenzyme a as substrate

The spectrophotometric assay of 2,4-dienoyl coenzyme A (CoA) reductase (EC 1.1.1.34) was modified to improve the linearity and sensitivity of this method. 5-Phenyl-2,4-pentadienoyl-CoA, which has an absorbance maximum at 340 nm with an extinction coefficient of 44,300 M⁻¹ cm⁻¹, was synthesized and used as substrate. This compound is reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent 2,4-dienoyl-CoA reductase to 5-phenyl-3-pentenoyl-CoA. When a tissue homogenate serves as an enzyme source, the product is further metabolized by Δ³Δ²-enoyl-CoA isomerase (EC 5.3.3.8) to 5-phenyl-2-pentenoyl-CoA, which is hydrated to 5-phenyl-3-hydroxypentanoyl-CoA by enoyl-CoA hydratase (EC 4.2.1.17). The modified assay method, which measures the decrease in absorbance at 340 nm due to the reduction of 5-phenyl-2,4-pentadienoyl-CoA and the oxidation of NADPH, is linear for a longer period of time and is twice as sensitive as the conventional assay with 2,4-decadienoyl-CoA as substrate.     

Developmental regulation of sterol biosynthesis inZea mays

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods fromZea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-¹⁴C]mevalonic acid, [2-¹⁴C]acetate, and [2-³H]acetate), and stable isotopes (D₂O), we discovered that hydroxymethylglutaryl CoA reductase is not “the” rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24α-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an activede novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3β-ol. Examination of the mass spectrum and¹H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed. Dedicated to the distinguished insect steroid biochemist Dr. James A. Svoboda on occasion of his sixtieth birthday. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids”, Atlanta, Georgia, May 1994.     

Effects of cereals and culture filtrate ofTrichoderma viride on lipid metabolism of swine

Swine were fed corn-or barley-based diets with, or without, culture filtrate (CF) ofTrichoderma viride for 21 days. Weight gains were nonsignificantly but slightly increased by CF. The activities of β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase, acetyl-CoA carboxylase (ACX), fatty acid synthetase (FAS) and other lipogenic enzymes in several tissues were determined. Significant decreases in the activities of HMG-CoA reductase and cholesterol 7α-hydroxylase in all tissues of swine fed the CF-diets were observed. The major site for the regulation of cholesterol biosynthesis was adipose tissue followed by the intestine, liver, lung and muscle in order of activity The concentrations of cholesterol in serum and muscle were decreased 27% and 23%, respectively, by CF. ACX and FAS activities increased ca. 2-fold when CF was fed with either of the cereal-based diets. The major sites for fatty acid synthesis was the adipose tissue and, to a lesser extent, the liver. Very low rates of synthesis were detected in intestine, lung and muscle. Similar distributions of activities were found for related lipogenic enzymes. Cooperative investigation between the Science and Education Administration, US Department of Agriculture, and College of Agricultural and Life Sciences, University of Wisconsin, Madison. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol: A progress report

Our principal goal is the complete resolution and reconstitution of the microsomal enzymes of cholesterol biosynthesis. Elucidation of the enzymology has been achieved primarily through dissection of the membrane-bound, 19-step multienzymic process. This report describes the dissection approach through both interruption of specific steps and reconstitution of enzymes that catalyze oxidation of the 14α-methyl group. In earlier work, 4-demethylation was resolved into 3 component reactions catalyzed by: 4-methyl sterol oxidase (NAD[P] H- and O₂-dependnet); steroid 4α-carboxylic acid decarboxylase (NAD-dependent); and 3-ketosteroid reductase (NADPH-dependent). The 3-ketosteroid reductase and decarboxylase have been solubilized with Lubrol WX and deoxycholate, respectively, and characterized. The 4-methyl sterol oxidase (cytochrome b₅-dependent) recently has been solubilized with Renex 690. This study represents successful elucidation of a microsomal enzyme sequence by interruption of the central 10-step segment of the multienzymic formation of cholesterol from lanosterol. The initial C-32 oxidative reaction of 14α-methyl group elimination is catalyzed by a from of cytochrome P-450 that is induced by isosafrole. The induced cytochrome P-450 has been solubilized with Emulgen 913 and purified to homogeneity (17 nmol of cytochrome/mg protein). 24,25-Dihydrolanosterol is oxidized by combination of cytochrome P-450 reductase, hematin, NADPH, glutathione, and the purified, isosafrole-induced cytochrome in an artificial liposome. Oxidation product identification is underway. This study represents successful elucidation of a microsomal multienzymic sequence by solubilization and reconstitution of a segment of the pathway. The remaining enzymes under study are the Δ⁸→Δ⁷ isomerase and 3 NADPH-dependent double bond reductases that catalyze reduction of: Δ⁷, Δ¹⁴- Δ²⁴-sterol double bonds. Purification of these nonoxygenrequiring enzymes is in progress. Resolution of the enzymes has demonstrated unequivocally that cholesterol synthesis via this pathway could not have appeared biologically until membranes containedboth the cytochrome P-450- and cytochrome b₅-electron transport enzymes. Chemically, all enzymic attacks in the formation of cholesterol from lanosterol appear to be initiated on the α-face of the relatively planar steroids. Thus, considerable genetic pressure must have been needed for the stereospecific clearing of the steroidal α-face to form the mature membrane component, cholesterol.     

Simvastatin impairs mitogen-induced proliferation of malignant B-lymphocytes from humans—in vitro and in vivo studies

Chronic lymphocytic leukemia (CLL) cells express lower low density lipoprotein (LDL) receptor activity and higher 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity than normal mononuclear blood cells indicating that CLL cells may depend on cholesterol synthesis for their proliferation. We studied the effects of competitive inhibitors of HMG-CoA reductase on malignant lymphocyte proliferation in vitro and in vivo. Tumor B-cells from 13 patients with CLL, hairy cell leukemia, or immunoblastic B-cell lymphoma were cultured for 4 d in the presence of B-cell mitogens and cholesterol synthesis inhibitors. Simvastatin and lovastatin suppressed, in a concentration-dependent manner, the mitogen-induced cellular thymidin uptake in medium with 10% human AB-serum or lipoprotein-deficient serum. Pravastatin was active only in medium with lipoprotein-deficient serum. Ten previously untrated patients with CLL received simvastatin orally, 40 mg daily for 12 wk. Mean reductions in total plasma and LDL cholesterol were 30% (range 9–46%) and 37% (range 16–63%), respectively. Cells from four patients showed moderate to minor increases in the degradation rate of ¹²⁵I-LDL suggesting that the need for exogenous cholesterol had increased, three patients showed an increase in HMG-CoA reductase activity, and the cells from one patient showed both. There was no significant change in the clinical disease status during medication. However, four of the ten patients developed a therapy-demanding progressive disease during the subsequent year. Further clinical studies with cholesterol synthesis inhibitors in leukemia are warranted.     

Erythrocyte contamination of leukocyte populations following density-gradient centrifugation results in artificially high levels of human leukocyte HMG-CoA reductase activity

When measuring human leukocyte HMG-CoA reductase activity, special care must be taken to prevent erythrocyte contamination of the leukocyte layer during isopycnic centrifugation. Contamination during leukocyte isolation and subsequent erythrocyte lysis during NH₄Cl treatment results in increased leukocyte microsomal HMG-CoA reductase activity. Increased enzyme activity is not due to enzyme dephosphorylation, thiol-disulfide reduction or increased enzyme protein concentration. Leukocyte populations containing granulocytes appear to be most sensitive. Prevention of erythrocyte contamination during isopycnic centrifugation should aid in accurate measurement of human leukocyte HMG-CoA reductase activity.     

The effects of simvastatin and cholestyramine,alone and in combination,on hepatic cholesterol metabolism in the male rat

The influence of dietary simvastatin, cholestyramine, and the combination of simvastatin plus cholestyramine on hepatic cholesterol metabolism has been investigated in male rats. Recovery from the effects of the drugs was also investigated by refeeding normal chow for 24 h. Both drugs, alone and in combination, increased 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activityin vitro, but activity returned toward control values, after drug withdrawal. Acyl-CoA:cholesterol acyltransferase (ACAT) was significantly reduced (P<0.001) by simvastatin (−75%), cholestyramine (−71%), and by the drug combination (−81%), due both to a decrease in microsomal cholesterol and to nonsubstrate-dependent modulation of enzyme activity. Refeeding control diet increased ACAT activity but not to control levels. The enhanced activity arose partly from higher microsomal cholesterol and partly from increases in total enzyme activity. Cytosolic neutral cholesteryl ester hydrolase (CEH) activity was substantially elevated by simvastatin (3-fold) and by the drug combination (6-fold), whereas the effect of cholestyramine was smaller (1.5-fold). Normal chow for 24 h only partially returned cytosolic CEH activity to control values. Microsomal CEH activity was increased by simvastatin, alone and in combination with cholestyramine (1.4 to 1.7-fold), and was also enhanced, in the cholestyramine-treated animals, following drug withdrawal. Removal of simvastatin did not allow recovery of this enzyme activity, while withdrawal of the drug combination led to values 29% below controls. The results indicate that in the rat, simvastatin and cholestyramine alter both ACAT and CEH activity, as well as inhibiting HMG-CoA reductase activity.     

Properties of La1-xCexCoO3 system perovskite-type catalysts for diesel engine exhaust removal

A series of complex oxide La_1−x Ce _x CoO₃ catalysts was synthesized at a low temperature through a combustion process, in which x is among 0, 0.1, 0.2, 0.4 and 0.6 corresponding to the quantity of La³⁺ partial substitution by Ce⁴⁺. The catalysts were characterized for phase composition using chemical analysis and X-ray diffraction. The catalytic activity of the catalysts in removal of NO _x , total hydrocarbon (THC) and particulate matter (PM) from diesel exhaust gases were examined in detail using temperature-programmed reaction technique. The results show that after partial substitution of La³⁺ with Ce⁴⁺, the oxygen vacancy concentration increases significantly and a Co³⁺-Co²⁺ system is formed. Consequently, the catalytic activity in the removal of THC and NO _x is significantly improved. But for the PM, the effect is not so obvious. The possible catalytic mechanism for this was presented. It is also worth noting that the doped catalysts showed good stability. Translated from Journal of Fuel Chemistry and Technology, 2006, 34(1): 85–90 [译自: 燃料化学学报]     

Purification and characterization of thermophilic and alkalophilic tributyrin esterase fromBacillus strain A30-1 (ATCC 53841)

An extracellular esterase (EC 3.1.1.1) from a thermophilicBacillus A30-1 (ATCC 53841) was purified 139-fold to homogeneity by sodium chloride (6 M) treatment, ammonium sulfate fractionation (30–80%) and phenyl-Sepharose CL-6B column chromatography. The native enzyme was a single polypeptide chain with a molecular weight of about 65,000 and an isoelectric point at pH 4.8. The optimum pH for esterase activity was 9.0, and its pH stability range was 5.0–10.5. The optimum temperature for its activity was 60°C. The esterase had a half-life of 28 h at 50°C, 20 h at 60°C and 16 h at 65°C. It showed the highest activity on tributyrin, with little or no activity toward long-chain (12–20 carbon) fatty acid esters. The enzyme displayed K_m and K_cat values of 0.357 mM and 8365/min, respectively, for tributyrin hydrolysis at pH 9.0 and 60°C. Cyclodextrin (α, β, and γ), Ca²⁺, Co²⁺, Mg²⁺ and Mn²⁺ enhanced the esterase activity, and Zn²⁺ and Fe²⁺ acted as inhibitors of the enzyme activity. The enzyme activity was not affected by ethylenediaminetetraacetic acid, p-chloromercuribenzoate andN-bromosuccinimide. This paper was presented in part at the 82nd Annual Meeting and Exposition of the American Oil Chemists’ Society, held May 12–15, 1991, in Chicago, Illinois.     

A novel ∈-lysine acylase from <Emphasis Type="Italic">Streptomyces mobaraensis</Emphasis> for synthesis of <Emphasis Type="Italic">N∈</Emphasis>-acyl-<Emphasis Type="SmallCaps">l</Emphasis>-lysines

A novel ∈-lysine acylase (N ₆-acyl-l-lysine amidohydrolase; EC 3.5.1.17) was isolated from Streptomyces mobaraensis and purified to homogeneity by SDS-PAGE from the culture broth. The purified enzyme was monomeric, with a molecular mass of approximately 60 kDa. The enzyme was inactivated by the presence of 1,10-phenanthroline and activated in the presence of Co²⁺ and Zn²⁺. The enzyme showed a pH optimum of 8.0 and was stable at temperatures of up to 50°C for 1 h at pH 8.0. The enzyme specifically catalyzed the hydrolysis of the amide bond of various N∈-acyl-l-lysines. Furthermore, the enzyme efficiently catalyzed the synthesis of N∈-acyl-l-lysines with fatty and aromatic acyl groups in an aqueous buffer. In the syntheses of N∈-decanoyl-l-lysine, N∈-lauroyl-l-lysine, and N∈-myristoyl-l-lysine, the product precipitated and the yield was 90% or higher using 10 mM FA and 0.5 M l-lysine as the substrate.     

Effects of different fractions of the barley kernel on the hepatic lipid metabolism of chickens

Various physical fractions of the barley kernel were fed to one-day-old female and male chickens to determine their effect on hepatic β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase and the lipogenic enzymes, acetyl-CoA carboxylase (ACX), malic enzyme (ME), citrate-cleavage enzyme (CCE) and fatty acid synthetase (FAS) at the subcellular level. Significant inhibition (p<0.01) of cholesterol biosynthesis accompanied by significant decreases in plasma cholesterol concentrations and induction of fatty acid synthesis were found in diets based on pearled barley, barley pearlings and a high-protein barley flour (HPBF: aleurone and subaleurone layers of barley endosperm) separated from the pearlings when compared to corn. Lower weight gains in 1- to 4-week-old birds fed the high-protein barley flour were found to be the result of lower feed consumption; pair feeding of 12-week-old birds with diets based on corn and high-protein barley flour produced equal weight gains in both treatments and significant reductions in hepatic HMG-CoA reductase, plasma cholesterol and induction in several lipogenic enzymes in birds fed the high-protein barley flour. Substitutions of 5–20% high-protein barley flour for corn in a corn-based diet produced significant weight gains (p<0.01) of 10 to 20% in 2-week-old chickens, inhibited cholesterol biosynthesis by 45–65% and produced a 3-fold increase in a fatty acid synthetase. The results indicate that HPBF contains an inhibitor(s) of cholesterol biosynthesis and a growth factor(s) when compared to a corn-based diet. Cooperative investigation between the Science and Education Administration, US Department of Agriculture, and the College of Agricultural and Life Sciences, University of Wisconsin-Madison. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.