Combined effects of oleic, linoleic and linolenic acids on lactation performance and the milk fatty acid profile in lactating dairy cows.

The potential combined effects of oleic, linoleic and linolenic acids supplementation on lactation performance and the milk fatty acid (FA) profile in dairy cows have not been well investigated. Our objective was to examine the effects of supplementation with a combination of these FA as well as the effects of removing each from the combination on lactation performance and the milk FA profile in dairy cows. Eight Holstein cows (101±11 days in milk) received four intravenously infused treatments in a 4×4 Latin square design, and each period lasted for 12 days which consisted of 5 days of infusion and 7 days of recovery. The control treatment (CTL) contained 58.30, 58.17 and 39.96 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively. The other three treatments were designated --C18 : 1 (20.68, 61.17 and 41.72 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively), -C18 : 2 (61.49, 19.55 and 42.13 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively) and -C18 : 3 (60.89, 60.16 and 1.53 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively). Dry matter intake and lactose content were not affected by the treatments, but the milk protein content was lower in cows treated with -C18 : 2 than that in CTL-treated cows. Milk yield as well as milk fat, protein and lactose yields were higher in cows treated with -C18 : 3 than the yields in CTL-treated cows, and these yields increased linearly as the unsaturation degree of the supplemental FA decreased. Compared with the CTL treatment, the -C18 : 2 treatment decreased milk C18 : 2 cis-9 content (by 2.80%) and yield (by 22.12 g/day), and the -C18 : 3 treatment decreased milk C18 : 3 cis-9, cis-12, cis-15 content (by 2.72%) and yield (by 22.33 g/day). In contrast, removing C18 : 1 cis-9 did not affect the milk content or yield of C18 : 1 cis-9. The -C18 : 2-treated cows had a higher C18 : 1 cis-9 content and tended to have a higher C18 : 1 cis-9 yield than CTL-treated cows. The yields of C8 : 0, C14 : 0 and C16 : 0 as well as

Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol.

A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (-61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2-2 phenotype.

Paradigm to test a drug-induced aversion to ethanol.

The screening of new agents for aversive therapy of alcoholism requires a simple animal model. Animals trained to ingest ethanol solutions and subsequently administered a drug known to produce an aversion to ethanol in humans, do not readily make the association between the malaise induced by the aversive drug-ethanol reaction and the consumption of the same ethanol-containing solution that has been consumed previously without ill effects. An experimental paradigm is reported in which the malaise of the drug-ethanol reaction is quickly recognized by rats as derived from ethanol. Disulfiram was used as the model drug. Lewis rats were deprived of water for 18 h after which 6% (v/v) ethanol was offered as the only fluid. During the first hour of ethanol access, both controls (vehicle) and disulfiram (100 mg/kg)-treated animals consumed intoxicating amounts of ethanol (0.7-0.9 g ethanol/kg). Plasma acetaldehyde levels developed were 3-5 microM and 40-50 microM in the two groups respectively. After this time, disulfiram-treated animals virtually ceased consuming alcohol (90% inhibition), indicating that the disulfiram-ethanol reaction is associated with alcohol ingestion. Control animals continued consuming the alcohol solution for the additional 4-5 h tested. This model should be of value in the testing of new agents that reduce aldehyde dehydrogenase levels for prolonged periods for their potential as an aversive treatment in alcoholism.

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Tetranucleotide GGGA motif in primary RNA transcripts. Novel target site for antisense design.

Selecting effective antisense target sites on a given mRNA molecule constitutes a major problem in antisense therapeutics. By trial-and-error, only 1 in 18 (6%) of antisense oligonucleotides designed to target the primary RNA transcript of tumor necrosis factor-alpha (TNF-alpha) strongly inhibited TNF-alpha synthesis. Subsequent studies showed that the area in RNA targeted by antisense oligonucleotides could be moved effectively 10-15 bases in either direction from the original area. We observed that only molecules that incorporated a tetranucleotide motif TCCC (complementary to GGGA on RNA) yielded potent antisense oligonucleotides against TNF-alpha. A comprehensive literature survey showed that this motif is unwittingly present in 48% of the most potent antisense oligonucleotides reported in the literature. This finding was prospectively used to predict the sequences of additional antisense oligonucleotides for the rat TNF-alpha primary RNA transcript. Over 50% of antisense constructs (13 of 22) containing the TCCC motif were found to effectively inhibit TNF-alpha synthesis. Marked reductions in mRNA were also observed. This motif was found to be most effective when targeting introns in the primary RNA transcript, suggesting a nuclear localization for the antisense action. Predicting target sites based on the presence of this motif in primary RNA transcripts should be of value in the development on new antisense pharmacotherapy.

Inhibition of gene expression by triple helix formation in hepatoma cells.

The aim of this study was to selectively inhibit human mitochondrial aldehyde dehydrogenase (ALDH2) gene expression by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to two purine-rich sequences in the 5'-flanking region of the human ALDH2 gene. Gel mobility shift assays showed that triplex formation is sequence-specific for the target duplex and the third strand oligonucleotide. In the presence of Mg2+, but absence of K+, triplex-forming oligonucleotides bind to their target sites with apparent dissociation constants (Kd) in the 10(-7) to 10(-9) M range. Potassium cation virtually suppressed the triplex formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent triplex formation with phosphorothioate-modified oligonucleotides. Phosphorothioate-modified oligonucleotides were delivered into human hepatoma Hep G2 cells by cationic liposomes. The reduction in ALDH2 mRNA levels in the cells was determined by the competitive reverse transcription-polymerase chain reaction. One of the phosphorothioate-modified oligonucleotides designed to forma an antiparallel triplex with a target in the 5'-flanking region of human ALDH2 gene (-105 to -125 from the translation initiation codon ATG) reduced by 80-90% the ALDH2 mRNA levels without affecting albumin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH2 gene expression for therapeutic use.

Presence of cytosolic aldehyde dehydrogenase isozymes in adult and fetal rat liver.

A colony of Wistar-strain rats bred at Purdue University was composed of animals with two different isozyme patterns of liver cytosolic aldehyde dehydrogenase (EC 1.2.1.3, ALDH) as determined by isoelectric focusing. One cytosolic isozyme pattern had a major activity band with a pI = 5.8 and a minor activity band at pI = 6.2. The other pattern contained three major isozymes with pI values of 5.3, 5.4 and 5.6 along with the pI 6.2 isozyme and a trace of the 5.8 one. The 5.8 and 6.2 isozymes were recognized by antibodies produced against horse and beef liver cytosolic ALDH, whereas the set of three (5.3-5.6) were not. The cytosolic isozymes were inhibited by low levels of disulfiram and had Km values for acetaldehyde in the 100 microM range, properties typical for cytosolic ALDHs. All animals contained the same isozymes of liver mitochondrial ALDH. These were a major activity with a pI = 5.2 and minor activities associated with isozymes of pI = 6.4 and 6.6. These isozymes were recognized by antibodies produced against pure horse and beef liver mitochondrial ALDHs. Both cytosolic and mitochondrial ALDHs were found in fetal liver as early as day 15 of gestation. The total activity for mitochondrial ALDH increased between day 15 and day 21 whereas that for cytosolic ALDHs remained relatively constant during development. It appeared that both cytosolic and mitochondrial ALDH were present by at least the third trimester and could afford the fetus some protection against the toxic action of endogenous or exogenous aldehydes.

Purification and characterization of beef and pig liver aldehyde dehydrogenases.

Beef liver cytosolic, mitochondrial, and pig liver mitochondrial aldehyde dehydrogenases (ALDH) had been purified to homogeneity. The two mitochondrial enzymes as with other mammalian mitochondrial enzymes had properties very similar to that of the corresponding human enzyme. These include immunological as well as basic kinetic properties such as low Km for aldehyde, activation by Mg2+ ions, and lack of inhibition by disulfiram. A major difference between these two enzymes and the human mitochondrial enzyme was that they contained an N-terminal-blocked amino acid. Cytosolic ALDHs from human and horse liver have been shown to possess an N-acetyl serine as the N-terminal residue; beef cytosolic ALDH was also found to be blocked. Tissue preparations and subcellular fractions from beef or pig liver could be used to study acetaldehyde oxidation. This is the subject of the accompanying paper (Cao Q-N, Tu G-C, Weiner H, Alcohol Clin Exp Res 12:xxx-xxx, 1988).

Mitochondria as the primary site of acetaldehyde metabolism in beef and pig liver slices.

Aldehyde dehydrogenase (ALDH) is the major enzyme involved in the oxidation of acetaldehyde. It has been shown that the liver enzyme is located in both cytosol and mitochondria. It has not been established where the subcellular oxidation of acetaldehyde occurs in species other than rat. Using slices isolated from beef and pig livers and selectively inhibiting the mitochondria enzyme with cyanamide or the cytosolic enzyme with disulfiram, it was possible to address this question. It was found that with both beef and pig liver slices 60% of the oxidation was catalyzed by the mitochondrial ALDH and 20% by the higher Km cytosolic enzyme. The remainder of the metabolism was the result of non-ALDH involvement. Furthermore, any decrease in the level of the low Km mitochondrial aldehyde dehydrogenase activity resulted in a decreased rate of acetaldehyde oxidation showing that its activity governed the rate of acetaldehyde oxidation. These were the same conclusions previously reached using rat liver tissue slices. Thus, it appears that for all mammalian tissue, mitochondria is the primary location of acetaldehyde oxidation.