Dietary toxicity of calcium beta-hydroxy-beta-methyl butyrate (CaHMB).

HMB, 3-hydroxy-3-methyl butyrate, is of interest as a dietary supplement and a possible component of functional and medical foods. The purpose of this study was to evaluate the toxicity of the calcium salt of HMB, calcium 3-hydroxy-3-methyl butyrate (CaHMB, monohydrate, food grade), when administered daily in the diet of rats for at least 90 days. Male and female Crl:CD (SD)IGS BR animals were assigned to four groups. Each group received diets containing the carrier or 1%, 2%, or 5% of CaHMB mixed with diet. Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, and clinical and anatomic pathology evaluations. Administration of CaHMB in basal diet for 91 days was tolerated well. There were no unscheduled sacrifices or deaths. There were no CaHMB-related adverse effects on clinical observations, body weights, food consumption, clinical chemistry, hematology, absolute or relative organ weights, or macroscopic or microscopic observations. A statistically significant increase in inorganic phosphorous was observed in male animals in the 5% feeding group; however, this effect was not considered adverse. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 5% of CaHMB mixed with diet (3.49 g/kg BW for males and 4.16 g/kg BW for females).

Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system.

Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase. A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase. This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase. Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

Simulated microgravity and hypergravity attenuate heart tissue development in explant culture.

Exposure to altered gravity may disturb the cytoskeleton-cell surface-extracellular matrix (ECM) interface of embryonic cells. Development of organs such as the heart depends on dynamic interactions across cell surfaces. Fibronectin (FN), for example, a glycoprotein that links the ECM to the cytoskeleton through integrin surface receptors, is required for normal heart development. Thus, altered gravity may perturb organogenesis. We cultured precardiac explants from chick embryos in a rotating bioreactor vessel to simulate microgravity (microG), or in a tissue culture centrifuge, for 18 h during heart development. Bioreactor microG did not alter external morphology of explants, but did significantly reduce the proportion that developed contractions. Immunostaining for FN of explant sections showed that it also significantly reduced the linear extent of staining present in basement membrane regions. Analysis of ultrastructure revealed a significant reduction in the number of desmosomes per unit area and other differences. Hypergravity dramatically abolished development of contractions and altered morphogenesis. The results indicate a probable sensitivity of cardiomyogenic development involving FN to altered gravity.

Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids.

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids.

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

cDNA cloning and characterization of human Delta5-desaturase involved in the biosynthesis of arachidonic acid.

Two human expressed sequence tag (EST) cDNA sequences with identity with Delta(5)- and Delta(6)-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3' EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a Delta(5)-desaturase, as determined by the conversion of dihomo-gamma-linolenic acid (C(20:3,n-6)) into arachidonic acid (C(20:4,n-6)). The human Delta(5)-desaturase contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.

Polyunsaturated fatty acid-specific elongation enzymes.

We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.

Cloning of delta12- and delta6-desaturases from Mortierella alpina and recombinant production of gamma-linolenic acid in Saccharomyces cerevisiae.

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited delta6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the delta12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid. The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast.

Nurses' perceptions of chemical restraint use in long-term care.

The purpose of this pilot study is to validate the use of the Perceptions of Restraint Use Questionnaire (PRUQ) in assessing chemical restraint perceptions among nurses working in long-term care. The convenience sample includes 60 licensed nurses working in six long-term care facilities in Illinois. The reliability analysis for a modified version of the PRUQ, based on the research sample chosen, was found to have a Cronbach's coefficient alpha of .9450. Study findings reflect a moderately positive attitude toward chemical restraint use by nurses in long-term care.

Identification of Delta5-desaturase from Mortierella alpina by heterologous expression in Bakers' yeast and canola.

A DNA fragment with homology to Delta6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Delta6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Delta5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.

Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli.

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.

Three-year clinical evaluation of the Clearfil Liner Bond system.

Modern dental adhesive systems have improved the bond of restorative materials to mineralized tooth structures. The purpose of this study was to evaluate the clinical performance of composite restorations placed in abrasion and erosion lesions using the Clearfil Liner Bond dental adhesive system. Following ADA clinical guidelines for dentin and enamel adhesive materials, 62 facial class 5 smooth surface erosion or abrasion lesions with no undercuts and involving primarily root surfaces were restored in 25 adult male and female patients. The teeth were restored without preparations using Clearfil Liner Bond and Clearfil Photo Anterior composite resin. The clinical performance of the restorations was assessed by two examiners at baseline, 6 months, 1, 2, and 3 years using the following evaluative parameters: color match, marginal discoloration, and marginal integrity according to modified Ryge criteria; the presence or absence of recurrent decay; pre- and postoperative sensitivity; and restoration failure due to loss of retention or other causes. At the end of 3 years, four of the 55 restorations remaining in the study failed due to lack of retention (92.7% retention rate). The evaluations of the other clinical parameters demonstrated excellent performance by this system.

Laboratory evaluation of Amalgambond and Amalgambond Plus.

PURPOSE: To evaluate the shear bond strength (SBS) of a microfilled resin restorative material and an amalgam alloy to dentin using the Amalgambond Plus adhesive system with a variety of bonding regimens. MATERIALS AND METHODS: The study design incorporated bonding composite to both dry and moist dentin using open-ended plastic matrices and closed-ended gelatin capsules. In addition, the bond strength of amalgam to dry dentin was determined. RESULTS: The mean SBS of Silux Plus to dentin using Amalgambond without the HPA powder ranged from 17.55 +/- 1.96 MPa to 22.47 +/- 3.89 MPa. The SBS range observed when the microfilled composite was bonded to dentin with Amalgambond Plus (includes the HPA powder) was from 20.33 +/- 3.84 MPa to 22.50 +/- 3.69 MPa. There was no significant difference (P > 0.05) in the SBS of the microfilled resin restorative material to dentin between the groups that were bonded using an open-ended cylinder-shaped plastic matrix and those prepared using a closed-ended gelatin capsule matrix, nor was there a difference (P > 0.05) in the SBS when the microfilled resin restorative material was bonded to either wet or dry dentin surfaces. The use of multiple applications of Amalgambond adhesive did not markedly increase the SBS with this system. The mean bond strength of amalgam alloy (Dispersalloy) to dentin using Amalgambond Plus was 10.41 +/- 1.47 MPa.

Effect of porcelain surface treatments on bond strengths of composite resin bonded to porcelain.

A laboratory study was conducted to evaluate the bond strength of composite resin bonded to porcelain surfaces by use of a variety of treatment regimens with the All-Bond 2 adhesive system. There were significant differences in the 24-hour bond strengths between several of the surface treatment methods. The mean shear bond strength after 24 hours of water storage ranged from 10.6 +/- 2.3 MPa to 25.0 +/- 4.4 MPa. Nine of the surface treatment methods showed a significant decrease (p < 0.05) in bond strengths after 3 months of water storage and thermocycling. After 3 months, the bond strengths ranged 0.1 +/- 0.1 MPa to 17.4 +/- 2.0 MPa. Porcelain surface treatment with aluminum oxide air abrasion followed by hydrofluoric acid, a silane coupling agent, and an unfilled resin produced a bond strength after 3 months' water storage and thermocycling that was significantly greater (p < 0.05) than the other nine porcelain surface-treatment techniques. Visual examination of the debonded specimens generally showed cohesive failures in porcelain for the treatment groups with a mean bond strength above 13 MPa.

Preattentive and cognitive effects on perceptual completion at the blind spot.

Our findings indicate that preattentive processes, such as the filling in of homogeneously colored areas, discrete dots, or bars across the blind spot, take into account both the color and the form that stimulate the retina around the optic disk. Perceptual completion of the "junction" of two opposite colors facing each other on opposite sides of the blind spot was resolved by simultaneous segregation of the two colors at the location of a filled-in perpendicular line that suggested a boundary separating the two colors. Orientation preference and relative salience of one color versus the other determined which color was perceptually completed in a forced-choice situation that involved perceptual completion at the intersection of a cross formed by bars of opposite colors. A 1-min exposure to these stimuli presented an ambiguous situation for perceptual completion of either color within the blind spot, and resulted in a perceptual "flip-flop" from one color to the other, much like the phenomenon that occurs in figure reversal. Instructions to speed up this reversal process led to a fivefold reduction in latency to first reversal.

Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species.

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.

Oral Candida albicans in bone marrow transplant patients given chlorhexidine rinses: occurrence and susceptibilities to the agent.

The tongue and buccal mucosa of 26 bone marrow transplant recipients given three 0.12% chlorhexidine digluconate (CHX) oral rinses daily for 8 weeks were sampled weekly for oral Candida albicans. Putative C. albicans colony-forming units on selective bismuth sulfite glucose glycine yeast agar plates were identified with the API 20C system. The CHX minimum inhibitory concentrations (MICs) of oral C. albicans isolates obtained at all 8 sample weeks was determined with a microbroth dilution sensitivity assay. The CHX MIC range for yeast isolates selected randomly at all sample weeks was up to 2.5 to up to 20 micrograms/ml (mean MIC less than or equal to 8.5 micrograms/ml). The CHX MIC range for isolates at week 1 was less than or equal to 5 to less than or equal to 10 micrograms/ml (mean MIC less than or equal to 7.9 micrograms/ml) compared with less than or equal to 2.5 to less than or equal to 20 micrograms/ml at week 8 (mean MIC less than or equal to 8.8 micrograms/ml). Therefore the persistence of oral C. albicans in bone marrow transplant recipients using CHX rinses was due neither to low CHX susceptibilities nor to the development of resistance to the agent.

Gene transfer in marine water column and sediment microcosms by natural plasmid transformation.

We investigated the possibility for natural transformation in the marine environment by using broad-host-range plasmid multimers and a high-frequency-of-transformation (HFT) Vibrio strain as the recipient. Water and sediment samples were taken from Tampa Bay, the eastern Gulf of Mexico, the Florida Shelf near Miami, and the Bahamas Bank. In water column microcosms, transformation frequencies ranged from 1.7 x 10 to 2.7 x 10 transformants per recipient, with highest frequencies occurring when low levels of nutrients (peptone and yeast extract) were added. The presence of the ambient community either reduced transformation frequency by an order of magnitude or had no effect. In sterile sediments, nutrient additions had no consistent effect on transformation, with transfer frequencies similar to those observed in the water column. Transformation was not observed in any sediment experiment when the ambient microbial community was present. These findings are the first report of natural plasmid transformation in seawater and in the presence of the ambient microbial community. This process may be a mechanism for the acquisition of small, nonconjugative plasmids, which are commonly found in aquatic bacteria. Our data also suggest that natural transformation may be more likely to occur in the water column than in native marine sediments, contradicting prior conclusions based on studies with sterile sediments.

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.