Effect of varying levels of fatty acids from palm oil on feed intake and milk production in Holstein cows.

To determine the optimum feeding level of fatty acids of palm oil (PALM; Energizer RP10; 86.6% palmitic acid) on milk production, lactating cows (n = 18) were randomly assigned to a treatment sequence in replicated 4 x 4 Latin squares. Animals were assigned to squares by parity (3 multiparous and 1 primiparous squares with primiparous in the incomplete square). The 4 diets were designed to provide 0, 500, 1,000, and 1,500 g of PALM per day. Cows were fed individually with feed intake measured daily. Each period lasted 16 d with milk production and composition determined the final 2 d. Milk production, milk composition and feed intake data were analyzed using the MIXED procedure of SAS. Milk yields were 30.9, 34.0, 34.2, and 34.2 kg/ d (SEM = 1.9) for the 0, 500, 1,000, and 1,500 g levels, respectively. Milk yield was increased by the addition of PALM; however, there were no differences among the levels of PALM. Milk fat percentage was also increased from 3.44% for 0 g to 3.95% (SEM = 0.17) across all levels of PALM but there were no differences among the PALM treatments. Dry matter intakes were 23.3, 26.4, 24.7, and 23.8 kg/d (SEM = 1.4) for the 0, 500, 1,000 and 1,500 g levels, respectively. The addition of PALM increased milk yield and milk fat percentage, and no adverse effects on dry matter intake were observed.

What five clinical pastoral education residents discovered from a JCAHO review.

Discusses the process one pastoral care department and its five residents used to prepare for a JCAHO review and reports the experience, detailing questions asked and chaplains' responses when they sat in on twenty unit reviews.

CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context.

The yeast CCR4 protein is required for the expression of a number of genes involved in nonfermentative growth, including glucose-repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance. We show here that although CCR4 did not bind DNA under the conditions tested, it was able to activate transcription when fused to a heterologous DNA-binding domain. The transcriptional activation ability of CCR4, in contrast to that of many other activators, was glucose regulated. Two activation domains one of which was glucose responsive and encompassed a glutamine-proline-rich region similar to that found in other eukaryotic transcriptional factors were identified. The two transactivation regions, when separated from the leucine-rich repeat and the C terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C terminus make contacts that link the activation regions to the proper gene context. Native immunoprecipitation of CCR4 revealed that CCR4 was complexed with at least four other proteins. The leucine-rich repeat of CCR4 was both necessary and sufficient for interaction with at least two of these factors. We propose that the leucine-rich repeat links CCR4 through its associated factors to its promoter context at ADH2 and other loci where it is required for proper transcriptional regulation.

Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation.

The expression of the yeast ADH2 gene is controlled by the transcriptional activator ADR1, a zinc-finger protein that binds to an upstream activating sequence (UAS1) in the ADH2 promoter. We report here the isolation of seven mutations in the ADR1-5c allele, defining five different amino acid changes, that suppress the enhanced ADH2 expression caused by the ADR1-5c allele. Each of the mutations was shown to reduce the activation of ADH2 by a wild-type ADR1 gene, suggesting the mutations disrupt a domain important to the function of both the ADR1 and ADR1-5c proteins. All five amino acid changes occurred within the DNA-binding domain of ADR1 and were shown to severely inhibit the ability of ADR1 to bind UAS1 in vitro. These mutations were found, however, to also affect the ability of ADR1 to activate transcription independent of its ability to bind DNA. These results indicate that the DNA-binding region of ADR1 is involved in both transactivation and DNA binding.

Evaluation of three techniques for differential diagnosis of prostatic needle biopsy specimens.

AIMS: To determine whether acidic mucin staining, lectin histochemistry using Wisteria floribunda agglutinin, and immunohistochemistry using the monoclonal antibody EAB 903 are of benefit in distinguishing between hyperplastic and carcinomatous prostatic glandular tissue in needle biopsy specimens. METHODS: Formalin fixed, paraffin wax embedded prostatic needle biopsy specimens of benign and malignant tissue were examined. Alcian blue-periodic acid Schiff staining was performed on 33 benign and 34 malignant cases. Wisteria floribunda agglutinin (WFA) binding sites were demonstrated by the avidin-biotin peroxidase (ABC) technique with and without neuraminidase pretreatment on 34 benign cases and 32 malignant cases. EAB903 anticytokeratin antibody binding sites were demonstrated using both an indirect immunoperoxidase (IIP) technique and an avidin-biotin peroxidase complex method on seven benign and 31 malignant cases. RESULTS: Acidic mucin staining was found in 17 of 34 malignant glands and was weakly positive in five of 33 benign glands. WFA positivity before neuraminidase pretreatment was present in 29 of 32 malignant glands and in 19 of 34 benign glands. After neuraminidase all benign and malignant cases showed positivity. EAB 903 positivity was seen in 11 of 31 malignant glands using the IIP technique and in two of 31 malignant glands using the ABC technique. In seven benign cases there was positivity in all glands using the IIP method with predominant basal cell staining in three and superficial cell staining in four. In benign cases using the ABC method two cases were negative. CONCLUSIONS: None of the three methods studied showed sufficient sensitivity and specificity to allow their recommendation for routine diagnostic use.

Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria.

Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase. To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced. The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450. The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family. Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene. No major rearrangements were detected in the mevalonic aciduria subject. The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts. Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue. The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother. Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism. Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity. Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.

Inhibitory effects of terpene alcohols and aldehydes on growth of green algaChlorella pyrenoidosa.

The growth of the green algaChlorella pyrenoidosa was inhibited by terpene alcohols and the terpene aldehyde citral. The strongest activity was shown by citral. Nerol, geraniol, and citronellol also showed pronounced activity. Strong inhibition was linked to acyclic terpenes containing a primary alcohol or aldehyde function. Inhibition appeared to be taking place through the vapor phase rather than by diffusion through the agar medium from the terpene-treated paper disks used in the system. Inhibition through agar diffusion was shown by certain aged samples of terpene hydrocarbons but not by recently purchased samples.

Effects of long-term administration of HMG-CoA reductase inhibitors on cholesterol synthesis in lens.

The effects of long-term dosing with inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase on the rate of cholesterol biosynthesis were examined in the lens and liver of rats and hamsters. While both pravastatin and lovastatin inhibited incorporation of [14C]acetate into cholesterol in liver slices 2-4 hr after an oral dose, lovastatin, but not pravastatin, inhibited sterol synthesis in lens as well. At 24 hr after a single oral dose, cholesterol synthesis in livers from drug-treated animals was increased compared to controls. This induction of the cholesterol synthetic pathway was observed for both drugs in the liver but only for lovastatin in the lens. After 4 days of once-daily oral doses, synthesis in the lens was induced two to threefold by lovastatin but not by pravastatin. When the drug was included in the continuous diet for 4-5 days, lovastatin caused increases in cholesterol synthesis in the lens whereas lenses from pravastatin-treated animals were identical to controls. This was not a species-specific effect since a similar tissue selectivity was observed in the hamster. The increase in cholesterol synthesis in lenses observed in lovastatin-treated rats was accompanied by an increase in the activity of HMG-CoA reductase enzyme. These studies demonstrate that non-selective HMG-CoA reductase enzyme inhibitors can inhibit cholesterol synthesis in the lens, and following this inhibition a marked induction in the cholesterol biosynthetic pathway develops in the lens and this induction is associated with an increase in HMG-CoA reductase enzyme activity.

Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver.

Mevalonate kinase [ATP:(R)-mevalonate 5-phosphotransferase, EC 2.7.1.36] may be a regulatory site in the cholesterol biosynthetic pathway, and a mutation in the gene coding for this enzyme is thought to cause the genetic disease mevalonic aciduria. To characterize this enzyme, a rat liver cDNA library was screened with a monospecific antibody, and a 1.7-kilobase cDNA clone coding for mevalonate kinase was isolated. The complete DNA sequence was determined, and the longest open reading frame coded for a protein containing 395 amino acids with a deduced molecular weight of 41,990. Identification of the cDNA clone was confirmed by expression of enzyme activity in yeast and by protein sequence data obtained from sequencing purified rat mevalonate kinase. The deduced amino acid sequence of mevalonate kinase contained a motif for the ATP-binding site found in protein kinases, and it also showed sequence homology to the yeast RAR1 protein. The size of mevalonate kinase mRNA in rat liver was approximately 2 kilobases. Treatment with diets containing cholesterol-lowering agents caused an increase in both mevalonate kinase activity and mRNA levels, whereas diets containing 5% cholesterol lowered the levels of both enzyme activity and mRNA. These data indicate that long-term regulation of enzyme activity in rat liver is controlled by changes in the levels of mevalonate kinase mRNA.

Purification and regulation of mevalonate kinase from rat liver.

Mevalonate kinase may play a key role in regulating cholesterol biosynthesis because its activity may be regulated via feedback inhibition by intermediates in the cholesterol biosynthetic pathway. To study the regulation of mevalonate kinase, the enzyme was purified to homogeneity from rat liver, and monospecific antibody against mevalonate kinase was prepared. The purified mevalonate kinase had a dimeric structure composed of identical subunits, and the Mr of the enzyme determined by gel chromatography was 86,000. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit Mr was 39,900. The pI for mevalonate kinate was 6.2. The levels of mevalonate kinase protein and enzyme activity were determined in the livers of rats treated with either cholesterol-lowering agents (cholestyramine, pravastatin, and lovastatin) or with dietary modifications. Diets containing cholestyramine alone or cholestyramine and either pravastatin or lovastatin increased mevalonate kinase activity 3-6-fold. Mevalonate kinase activity decreased approximately 50% in rats treated with diets containing either 5% cholesterol or 5% cholesterol and 0.5% cholic acid. Fasting did not significantly change mevalonate kinase activity. The amount of mevalonate kinase protein in the liver was quantitated using immunoblots, and the changes in the levels of kinase activity induced by either drug treatment or by cholesterol feeding were correlated with similar changes in the levels of mevalonate kinase protein. Therefore, under these experimental conditions, mevalonate kinase activity in the liver was regulated principally by changes in the rates of enzyme synthesis and degradation.

Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosynthesis in lens.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was observed with pravastatin even at very high doses. This tissue-selective inhibition of sterol synthesis by pravastatin was likely due to the inability of pravastatin to enter the intact lens since pravastatin and lovastatin acid were equally effective inhibitors of HMG-CoA reductase enzyme activity in whole lens homogenates. We conclude that pravastatin is tissue-selective with respect to lens and liver in its ability to inhibit cholesterol synthesis.

In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes.

We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.

An antibody against 100- to 116-kDa polypeptides in coated vesicles inhibits triskelion binding.

There is considerable evidence that the 100- to 116-kDa polypeptides in calf brain coated vesicles are involved in the assembly of clathrin triskelions to form coated vesicles. We have raised polyclonal antibodies against these polypeptides. By Western blot analysis, these antibodies bind to a distinct subset of the six polypeptides in the region 100-116 kDa. Whole cell homogenates from calf brain, calf liver, and rat liver also show immunoreactivity in the 100-kDa region with no other cross reactivity. Isolated coated vesicles from calf liver, rat brain, and soybean roots also cross-react. Stripped coated vesicles, which are depleted of clathrin but which retain the 100- to 116-kDa polypeptides, quantitatively rebind 125I-triskelions. This binding is inhibited in a dose-dependent manner by 100- to 116-kDa antibody but not by nonimmune serum or by anti-clathrin polyclonal antibody. These studies indicate that (1) specific sites on the 100- to 116-kDa polypeptides are required for assembly of coated vesicles, and (2) this antibody will be useful in clarifying more precisely the role of the 100- to 116-kDa polypeptides in coated vesicle recycling.

Proteolysis and lectin histochemistry.

Lectin binding to formalin-fixed, paraffin-embedded tissue can often be enhanced by pre-treatment of the sections with proteolytic enzymes. However, the pattern of staining may be profoundly influenced by the type of enzyme preparation which is used. Sites of binding of thirteen different lectins to murine ovary and thyroid gland were studied after exposure of tissue sections to crude trypsin, purified trypsin, purified alpha-chymotrypsin, pepsin, protease VII, papain, bromelain, thermolysin or elastase. With most lectins, the results obtained were similar regardless of which enzyme was used for proteolytic digestion. However, the pattern of binding of soy bean lectin to the ovary and of concanavalin A and common pea lectin to the thyroid gland was highly dependent upon the enzyme used to pre-treat the sections. In both tissues, the staining pattern seen in untreated frozen sections was similar to that found in formalin-fixed, paraffin-embedded material digested with purified trypsin, but was different from that observed after exposure of processed sections to crude trypsin. The location of binding sites after treatment of paraffin sections with chymotrypsin was the same as that after digestion with crude trypsin. Results obtained after the use of other proteolytic enzymes varied according to the tissue being studied. These findings imply that the effect of treatment with crude trypsin is due to contaminating chymotrypsin, and demonstrate that the use of purified trypsin may have advantages over other proteolytic enzymes in lectin histochemistry. The observations may also apply to other related cytochemical techniques such as immunocytochemistry.

Elimination of the non-specific binding of avidin to tissue sections.

A simple procedure is described for eliminating non-specific staining with avidin-peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 micrograms/ml biotinylated Pisum sativum agglutinin. Avidin-peroxidase conjugates (5 micrograms/ml), diluted in standard 0.05 M tris-buffered saline, pH 7.6, containing 0.139 M NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125 M Tris-buffered saline (containing 0.347 M NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.

A combination fluorescence assay and Folin-Ciocalteau phenol reagent assay for the detection of paralytic shellfish poisons.

Paralytic shellfish poison (PSP) profiles of crude shellfish extracts were determined by linear gradient elution from a Bio-Rad AG-50-X4 strong cation-exchange resin mini-column. STX, GTX2 and GTX3 were detected by fluorescence assay. NeoSTX and GTX1/GTX4 were detected with a Folin-Ciocalteau phenol reagent assay. The major toxicity associated with extracts of Mytilus edulis and Mya arenaria collected during a 1972 red tide off Hampton, New Hampshire, was due to the presence of GTX1/GTX4, with some activity associated with neoSTX, GTX2 and GTX3. STX was also present. Correlations to mouse toxicity are provided.

Receptor-mediated delivery of photoprotective agents by low-density lipoprotein.

Low density lipoprotein (LDL) has been used to deliver toxic molecules to cells by receptor-mediated endocytosis. In these studies, the cholesteryl ester core of LDL was replaced with a lipophilic, toxic molecule. We now report that photoprotective azo dyes can be stably incorporated into LDL, and that this reconstituted LDL protects cells from the photosensitizing action of pyrene methanol (PM) in a receptor-dependent process. The photoprotective action of the azo dye is due to its ability to scavenge singlet oxygen that is produced by the photosensitive agent in response to UV light.

Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3 -methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) converts HMG-CoA to mevalonate, a key intermediate in the formation of cholesterol and several nonsterol isoprenoid compounds. Using the bromodeoxyuridine/bisbenzimide photosensitization technique, we isolated a mutant clone of Chinese hamster ovary cells that requires mevalonate for growth. This mutant, designated UT-2 cells, expresses 2-5% of the HMG-CoA reductase activity of parental Chinese hamster ovary cells, even after growth for 9 months in the absence of selective pressure. By immunoblotting, no cross-reactive HMG-CoA reductase protein was detected in UT-2 cells. Incorporation of [14C]acetate and [14C]pyruvate into cholesterol was less than 5% of that in parental Chinese hamster ovary cells. In contrast, [3H]mevalonate incorporation into cholesterol was normal. The activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, the two enzymes that precede HMG-CoA reductase in the cholesterol biosynthetic pathway, were normal or slightly elevated in UT-2 cells. No gross deletions or rearrangements in the gene for HMG-CoA reductase were apparent when DNA from UT-2 cells was digested with restriction endonucleases, subjected to Southern blotting, and probed with a 32P-labeled cDNA for HMG-CoA reductase. We conclude that UT-2 cells have a mutation that specifically prevents the production of normal amounts of HMG-CoA reductase.