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Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family.

Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. We used expression cloning to identify cDNAs encoding a wax synthase in the mouse preputial gland. The wax synthase gene is located on the X chromosome and encodes a member of the acyltransferase family of enzymes that synthesize neutral lipids. Expression of wax synthase in cultured cells led to the formation of wax monoesters from straight chain saturated, unsaturated, and polyunsaturated fatty alcohols and acids. Polyisoprenols also were incorporated into wax monoesters by the enzyme. The wax synthase had little or no ability to synthesize cholesteryl esters, diacylglycerols, or triacylglycerols, whereas other acyltransferases, including the acyl-CoA:monoacylglycerol acyltransferase 1 and 2 enzymes and the acyl-CoA:diacylglycerol acyltransferase 1 and 2 enzymes, exhibited modest wax monoester synthesis activities. Confocal light microscopy indicated that the wax synthase was localized in membranes of the endoplasmic reticulum. Wax synthase mRNA was abundant in tissues rich in sebaceous glands such as the preputial gland and eyelid and was present at lower levels in other tissues. Coexpression of cDNAs specifying fatty acyl-CoA reductase 1 and wax synthase led to the synthesis of wax monoesters. The data suggest that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.

Acyltransferases↗

[Viscoelastic behaviour of inlay waxes (part 1). Physical and dynamic viscoelastic properties of several raw material waxes (author's transl)].

Several raw material waxes used in the inlay waxes, such as paraffins, carnauba wax, beeswax and dammar were investigated by measurements of X-ray diffraction, dilatometry, differential thermal analysis and dynamic viscoelasticity. The relationships between the viscoelastic behaviour and the physical properties of these waxes were discussed. The solid-solid phase transition from orthorhombic to hexagonal occurred in paraffins and the difference of the crystal transition temperature was observed between the kinds of paraffins. The viscoelastic properties and the thermal expansion of paraffins changed considerably in the crystal transition temperature region. Carnauba wax was an orthorhombic material, but such a crystal transition as paraffins did not appear. The dynamic modules of carnauba wax was greater than that of paraffin and decreased slowly to 70 degrees C and the loss tangent was small in this region. It was found, therefore, that carnauba wax had an elastic property in the crystal transition region of paraffin. Beeswax was also an orthorhombic material. The dynamic modulus of this was smallest in these waxes and the loss tangent increased relatively slowly with increases in temperature. Dammar was an amorphous solid. The dynamic modulus and the loss tangent were approximately constant in the low temperature region and changed greatly in the glass transition region. The thermal expansion of dammar was smallest in these waxes.

Bees↗

Amended final report on the safety assessment of Oryza Sativa (rice) Bran Oil, Oryza Sativa (rice) Germ Oil, Rice Bran Acid,Oryza Sativa (rice) Bran Wax, Hydrogenated Rice Bran Wax, Oryza Sativa (rice)Bran Extract, Oryza Sativa (rice) Extract, Oryza Sativa (rice) Germ Powder, Oryza Sativa (rice) Starch, Oryza Sativa (rice) Bran, Hydrolyzed Rice Bran Extract, Hydrolyzed Rice Bran Protein, Hydrolyzed Rice Extract, and Hydrolyzed Rice Protein.

This report addresses the safety of cosmetic ingredients derived from rice, Oryza sativa. Oils, Fatty Acids, and Waxes: Rice Bran Oil functions in cosmetics as a conditioning agent--occlusive in 39 formulations across a wide range of product types. Rice Germ Oil is a skin-conditioning agent--occlusive in six formulations in only four product categories. Rice Bran Acid is described as a surfactant-cleansing agent, but was not in current use. Rice Bran Wax is a skin-conditioning agent--occlusive in eight formulations in five product categories. Industry did not directly report any use of Rice Bran Wax. Hydrogenated Rice Bran Wax is a binder, skin-conditioning agent--occlusive, and viscosity-increasing agent--nonaqueous in 11 formulations in six product categories. Rice Bran Oil had an oral LD50 of > 5 g/kg in white rats and Rice Wax had an oral LD50 of > 24 g/kg in male mice. A three-generation oral dosing study reported no toxic or teratologic effects in albino rats fed 10% Rice Bran Oil compared to a control group fed Peanut Oil. Undiluted Rice Bran Oil, Rice Germ Oil, and Hydrogenated Rice Bran Wax were not irritants in animal skin tests. Rice Bran Oil was not a sensitizer. Rice Bran Oil, Rice Germ Oil, Rice Wax, and Hydrogenated Rice Bran Wax were negative in ocular toxicity assays. A mixture of Rice Bran Oil and Rice Germ Oil had a ultraviolet (UV) absorption maximum at 315 nm, but was not phototoxic in a dermal exposure assay. Rice Bran Oil was negative in an Ames assay, and a component, gamma-oryzanol, was negative in bacterial and mammalian mutagenicity assays. Rice oils, fatty acids, and waxes were, at most, mildly irritating in clinical studies. Extracts: Rice Bran Extract is used in six formulations in four product categories. Rice Extract is a hair-conditioning agent, but was not in current use. Hydrolyzed Rice Extract is used in four formulations and current concentration of use data were provided for other uses. Hydrolyzed Rice Bran Extract, described as a skin-conditioning agent--miscellaneous, is used in two product categories. Use concentrations are in the 1% to 2% range. Rice Bran Extract is comprised of proteins, lipids, carbohydrates, mineral ash, and water. The content includes palmitic, stearic, oleic, and linoleic acids. Other components include antioxidants such as tocopherols. Rice Extract reduced the cytotoxicity of sodium chloride in male rats. Bran, Starch and Powder: Rice Bran (identified as rice hulls) is an abrasive and bulking agent in one formulation. Rice Starch is an absorbent and bulking agent in 51 formulations across a wide range of product categories. Rice Germ Powder is an abrasive and one manufacturer described an exfoliant use, but it was not reported to be used in 2002. Oral carcinogenicity studies done on components of Rice Bran (phytic acid and gamma-oryzanol) were negative. Rice Bran did not have an anticarcinogenic effect on 1,2-dimethylhydrazine-induced large bowel tumors. In cocarcinogenicity studies done using 1,2-dimethylhydrazine and other agents, with Rice Bran Oil and Rice Bran-derived hemicellulose and saccharide, tumor inhibition was observed; gamma-oryzanol did not inhibit the development of neoplasms. A decrease in cutaneous lesions in atopic dermatitis patients was reported following bathing with a Rice Bran preparation. Proteins: Hydrolyzed Rice Bran Protein and Hydrolyzed Rice Protein function as conditioning agents (hair or skin), but only the latter was reported to be used in a few products. An in vitro phototoxicity assay using UVA light found no photochemical toxicity. Rice bran protein hydrolysates are not acutely toxic, are not skin or ocular irritants in animals, are not skin sensitizers in guinea pig maximization tests, and are not irritating or sensitizing in clinical tests. Isolated cases of allergy to raw rice have been reported, but rice, in general, is considered non allergenic. The Cosmetic Ingredient Review (CIR) Expert Panel considered that safety test data available on certain of these ingredients could be extrapolated to the entire group. Although Rice Bran Extract does contain UV absorbing compounds at low concentrations, clinical experience suggested no phototoxicity would be associated with such materials. Rice derived ingredients generally are considered to be non allergenic. There were no safety test data available for Hydrolyzed Rice Extract and Hydrolyzed Rice Bran Extract, but their safety may be inferred from that of the extracts from which they are derived. Current levels of polychlorinated biphenyls (PCBs) and heavy metals in rice-derived ingredients used in cosmetics are not a safety concern. The Panel was concerned, however, that contaminants such as pesticides have been reported in Rice Bran Oil used for cooking. Pesticides and heavy metals should not exceed currently reported levels for rice-derived cosmetic ingredients. The CIR Expert Panel concluded that these rice-derived ingredients are safe as cosmetic ingredients in the practices of use and concentrations as described in this safety assessment.

Animals↗

[Viscoelastic behaviour of inlay waxes. (Part 3) Dynamic viscoelastic properties of ternary and quaternary mixtures of waxes (author's transl)].

The viscoelastic properties of ternary and quaternary mixtures added carnauba wax, dammar and beeswax to paraffin were investigated by measurement of dynamic viscoelasticity. The effects of the composition of waxes and the crystal transition temperature of paraffin to the viscoelastic behaviour of these waxes were discussed. The viscoelastic properties of ternary mixtures added carnauba wax and dammar to paraffin were similar to these of binary mixtures for paraffin and dammar in the region of lower temperatures, and similar to these of binary mixtures for paraffin and carnauba wax in the region of higher temperatures. In quaternary mixtures added carnauba wax, dammar and beeswax, tan delta was larger than that of ternary mixtures, so the effect of additions of dammar was disappeared in the region of lower temperatures. Additions of carnauba wax, like to the case of ternary mixtures, had an effect on the viscoelastic properties of quaternary mixtures, but the temperature dependence of G' and tan delta of these waxes were slightly broader than that of ternary mixtures in the region of higher temperature. For a same composition in ternary and quaternary mixtures, even if the paraffins with different transition temperature were used, the shapes of curves of G' and tan delta were so similar to one another that were approximately superposable.

Elasticity↗

Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1.

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5'His(6)WS/DGAT comprising an N-terminal His(6) tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5'His(6)atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein(-1) min(-1) was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5'His(6)WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1OmegaKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1OmegaKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.

Acinetobacter↗

Photodegradation of phosmet in wool wax models and on sheep wool: determination of wool wax bound phosmet by means of isotope ratio mass spectrometry.

The photochemical reactions of phosmet, an organophosphorus insecticide used for plant protection and for control of ectoparasites on productive livestock, were studied in the presence of wool wax. Induced by UV light, phosmet features numerous degradation pathways as well as photoaddition reactions with lipid structure moieties. In model irradiation experiments of phosmet in mixtures of solvents (cyclohexane, cyclohexene, 2-propanol) and fatty acid methyl esters (methyl stearate, methyl oleate, 12-hydroxymethyl stearate), both adjusted to the hydroxyl and iodine values of wool wax, half-lives were determined to be approximately 7 and 16 h, respectively. Irradiation of phosmet on crude sheep wool resulted in a degradation rate of 65% after 24 h. In tracer studies with stable isotope labeled phosmet ([15N]phosmet) in commercial lanolin and on raw sheep wool, employing a sunlight simulator and natural sunlight, wool wax bound phosmet was formed. After extraction and measurement by elemental analyzer/isotope ratio mass spectrometry, delta15N values of the phosmet-free wool wax fractions were notably increased as compared to the value of natural lanolin. Calculated from the delta15N values, an average of 13.9/15.6% (sunlight simulator/natural sunlight) was bound to wool wax lipids after irradiation of thin films of commercial lanolin. In experiments with sheep wool, 13.2 and 15.4%, respectively, were detected as wax-bound.

Animals↗

Arthritogenicity of wax D from various mycobacteria related to oil vehicle composition and to the combination with poly I:C, cord factor and acetylated wax D.

Most of wax D (peptidoglycolipid) used here appeared to be ineffective for production of arthritis when given in a water-in-oil emulsion, while the same wax D in squalane was very effective for production of arthritis. Arlacel A as an emulsifier appeared to suppress the arthritogenicity of wax D in squalane, probably through some interaction with the arthritogenic portion of wax D. Poly 1:C seemed to remarkedly enhance the arthritogenicity of wax D, even in water-in-oil emulsion. Acetylated wax D and cord factor (trehalsoe-dimycolate) were much less effective than poly 1:C. Delayed skin hypersensitivity to PPD, peptidoglycan and poly 1:C was also remarkedly affected by oil composition. However, there was no correlation between these delayed hypersensitivities and development of arthritis.

Adjuvants, Immunologic↗

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

Acyltransferases↗

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development.

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.

Amino Acid Sequence↗

Release from or through a wax matrix system. I. Basic release properties of the wax matrix system.

Release properties from a wax matrix tablet was examined. To obtain basic release properties, the wax matrix tablet was prepared from a physical mixture of drug and wax powder (hydrogenated caster oil) at a fixed mixing ratio. Properties of release from the single flat-faced surface or curved side surface of the wax matrix tablet were examined. The applicability of the square-root time law and of Higuchi equations was confirmed. The release rate constant obtained as g/min(1/2) changed with the release direction. However, the release rate constant obtained as g/cm2 x min(1/2) was almost the same. Hence it was suggested that the release property was almost the same and the wax matrix structure was uniform independent of release surface or direction at a fixed mixing ratio. However, these equations could not explain the entire release process. The applicability of a semilogarithmic equation was not as good compared with the square-root time law or Higuchi equation. However, it was revealed that the semilogarithmic equation was available to simulate the entire release process, even though the fit was somewhat poor. Hence it was suggested that the semilogarithmic equation was sufficient to describe the release process. The release rate constant was varied with release direction. However, these release rate constants were expressed by a function of the effective surface area and initial amount, independent of the release direction.

Algorithms↗

Release from or through a wax matrix system. II. Basic properties of release from or through the wax matrix layer.

In order to examine basic properties of release from and through wax matrix layer, reservoir device matrix tablet was prepared from a physical mixture of hydrogenated caster oil and drug that was the same one in the reservoir. Release process could be divided into two stages. The first stage was the formation process of water channel by dissolving the drug in the wax matrix layer, and dissolved drug was released from the matrix layer following the square-root-of-time law equation. Hence, the drug penetration coefficient and tortuosity in the matrix layer were estimated. The second stage was the zero order release process of drug in the reservoir through the wax matrix layer. The release rate constant was calculated from the slope of line. Hence, the drug permeability coefficient and tortuosity were estimated. Fundamentally, tortuosity can not be expressed by some meaningful factors, and is obtained as an experimental result. By preparing wax matrix system from a physical mixture other than melted granule method, it was suggested that the matrix structure was uniform three-dimensionally. As a result, tortuosity could be expressed by a function of porosity, because unrecognized factors such as the surface coverage and thickness of melted wax on the soluble component should not be involved.

Drug Delivery Systems↗

Release from or through a wax matrix system. VI. Analysis and prediction of the entire release process of the wax matrix tablet.

Analysis of the entire release process of the wax matrix tablet was examined. Wax matrix tablet was prepared from a physical mixture of drug and wax powder to obtain basic or clear release properties. The release process began to deviate from Higuchi equation when the released amount reached at around the half of the initial drug amount. Simulated release amount increase infinitely when the Higuchi equation was applied. Then, the Higuchi equation was modified to estimate the release process of the wax matrix tablet. The modified Higuchi equation was named as the H-my equation. Release process was well treated by the H-my equation. Release process simulated by the H-my equation fitted well with the measured entire release process. Also, release properties from and through wax matrix well coincident each other. Furthermore, it is possible to predict an optional release process when the amount of matrix and composition of matrix system were defined.

Tablets↗

[Rheological studies on deformation for dental waxes. 2. Stress relaxation behavior of inlay wax (author's transl)].

The rheological properties of inlay wax were investigated by experiment of stress relaxation, thermal expansion, thermal analysis and X-ray diffraction. The results obtained were as follows. The solid-solid phase transition caused by phase transition of paraffin was observed. The stress relaxation curves of inlay wax were obtained at various temperature, and from these curves the stress relaxation master curve was composed by application of time-temperature superposition principle. The temperature dependence of sift factor was devided into two regions of Arrhenius type, and the activation energy was about 60 kcal/mol at the temperature lower than 23 degrees C and 120 kcal/mol at higher temperature. This fact suggests that the relaxation mechanism of inlay wax can be classified into two different modes. The relaxation mechanism at lower temperature region is explained by crystalline relaxation, and at higher temperature is considered to depend upon the solid-solid phase transition process of inlay wax. Calculating from the activation energy, for each 2 degrees C rise in temperature, the rate of rheological change for inlay wax is approximately two-fold at lower temperature region, and about four-fold at higher temperature region.

Elasticity↗

[Viscoelastic behaviour of inlay waxes. (Part 2) Physical and dynamic viscoelastic properties for binary mixtures of waxes (author's transl)].

Binary mixtures of waxes added carnauba wax, beeswax or dammar to paraffin were investigated by measurements of X-ray diffraction, dilatometry, differential thermal analysis and dynamic viscoelasticity. The relationships between the viscoelastic behaviour and the physical properties of these waxes were discussed. Additions of carnauba wax to paraffin changed drastically viscoelastic properties of paraffin, that is, increased the dynamic modulus, G', and decreased the loss tangent, tan delta, in the region of higher temperatures including the crystal transition temperature region of paraffin. The possible explanation for this change of viscoelastic properties is that the presence of crystals of carnauba wax composed of longer chain molecules than that of paraffin rises interfacial interaction. The temperature dependence of viscoelastic properties for binary mixtures of paraffin and beeswax was approximately the same as that of paraffin. This is because paraffin and beeswax may form a sort of homogeneous phases. Additions of dammar to paraffin increased the elasticity of paraffin in the region of lower temperatures, but did not effected to change of G' and tan delta in the region of higher temperatures. Another effect of additions of dammar was to lower the thermal expansion of binary mixtures.

Elasticity↗