Ca stimulated neutral lipase activity in castor bean lipid bodies

The membranes of lipid bodies from the endosperm of seeds of Ricinus communis have long been known to contain an acid lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3). The means by which fat hydrolysis is initiated and controlled in the endosperm of the young seedling are not yet understood, although it is generally assumed that the acid lipase is the enzyme responsible for the conversion of stored triacylglycerols to fatty acids and glycerol. However, the enzyme from seeds is not an effective catalyst at cytoplasmic pH since it has a pH optimum at 4.5 and is virtually inactive above pH 6.0. The results described in this paper show that during early growth of castor seeds the lipid bodies acquire a lipase which is active at neutral pH values. The lipase is absent from dry seeds, appears at day 3, and increases rapidly in activity until day 5. The pattern of appearance of the lipase mirrors that of other enzymes involved in the conversion of fat to sugar. The lipase is stimulated 40-fold by 30 micromolar free Ca²⁺ and the activity at pH 7.0 to 7.5 adequately accounts for the known rate of triacylglycerol hydrolysis in vivo.     

Substrate specificities of lipases in view of kinetic resolution of unsaturated fatty acids

Several commercially available lipases have been evaluated with regard to their substrate specificity in the esterification of fatty acids having specific positions of cis double bonds, e.g. petroselinic acid (n-12 18:1), alpha-linolenic acid (n-3 18:3), gamma-linolenic acid (n-6 18:3), stearidonic acid (n-3 18:4), dihomogamma-linolenic acid (n-6 20:3), eicosapentaenoic acid (n-3 20:5) and docosahexaenoic acid (n-3 22:6), with n-butanol. A common feature of most lipases, e.g. those from Penicillium cyclopium, Candida cylindracea, Mucor miehei, Rhizopus arrhizus and Penicillium sp. is that fatty acids having the first double bond from the carboxyl end as a cis-4 (n-3 22:6), cis-6 (n-12 18:1, n-6 18:3, n-3 18:4) or a cis-8 (n-6 20:3) double bond are strongly discriminated against compared to the other fatty acids, such as myristic acid (14:0), the reference standard, and n-3 18:3. In the case of the lipase from porcine pancreas, however, the discrimination against the above fatty acids is not as strong as with the other lipases. In contrast, the lipase from Chromobacterium viscosum shows a preference for n-12 18:1, n-6 18:3 and n-3 18:4. The observed substrate specificities can be utilized for enrichment of particular fatty acids by lipase-catalysed kinetic resolution from fatty acid mixtures, derived from naturally occurring fats and other lipids.Dedicated to Prof. David A. Walker, Robert Hill Institute, Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK, on the occasion of his sixty-fifth birthday on 18 August 1993 Correspondence to: K. D. Mukherjee     

Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.)

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.     

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

Drying of ethanol vapors by adsorption on corn meal

Experiments have been carried out on a laboratory-scale adsorber packed with 3.3 kg cornmeal. The effects of varying particle size, feed concentrations and flow rates, and regeneration conditions on the product quality and breakthrough times were investigated. It was found that 85% (w/w) vapor feed gave the best product, and product yields of up to 99.8% (w/w) alcohol were obtained at energy costs as low as 4.0 MJ kg(-1). It is suggested how this figure could be improved.     

The crystalline glycoprotein cell wall of the green alga Chlorogonium elongatum: a structural analysis.

Members of the Chlamydomonaceae, mostly single-celled green algae, have been shown to contain a crystalline glycoprotein cell wall component. Most of the species examined fall into a class of algae whose walls have an identical crystalline unit cell. Chlorogonium elongatum has been chosen as a representative of this class in order to investigate in more detail its cell wall structure. The alga has a spindleshaped cell wall which retains its asymmetric shape on isolation. Sections from walls fized in the presence of tannic acid clearly reveal a regular subunit monolayer, about 20 nm thick, within the wall. Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis shows the presence of at least 2 major glycoprotein species in the wall. Negatively stained purified cell walls demonstrate the crystalline nature of the cell wall. Optical diffraction of bright-field images and direct electron diffraction both give clear diffraction patterns whose spacings extend out to 3 nm and fall on a reciprocal lattice whose vectors describe a 2-dimensional unit cell within the wall 21.5 nm X 7.0 nm and an included angle of 80 degrees. Lattice defects within the cell wall are revealed by both negative staining and surface replication. Through-focal series were used to choose images with the optimal degree of underfocus for image processing. Linear integration and optical filtering of such images gave essentially the same result. A similar image was also obtained by computing the autocorrelation function of the amplitudes in the electron-diffraction pattern and the optical-diffraction pattern of the in-focus image. On the basis of these data a 2-dimensional model of the crystalline cell wall layer is presented.     

Self-assembly of a plant cell wall in vitro

A method has been developed by which the cell wall of Chlamydomonas reinhardi may be dissociated into its components, and then reassembled in vitro into a product that is chemically and structurally identical to the original cell wall. Chaotropic agents, such as lithium chloride and sodium perchlorate, separate the wall into two fractions, an insoluble amorphous inner wall layer, which retains its integrity (7.5% by weight of the complete wall) and a salt-soluble fraction containing the homogeneous glycoproteins responsible for the outer crystalline layers of the cell wall. Removal of the salt from dissociated walls by dialysis leads to the rapid recovery of complete reassembled cell walls. The conditions necessary for successful reconstitution of the cell wall in vitro include the presence of a suitable surface, across which a decreasing salt gradient exists, and the presence of both the salt-insoluble and the salt-soluble components. The salt-soluble glycoproteins alone can self-assemble under various conditions to form fragments that have the crystalline structure characteristic of the outer layers of the complete cell wall. Both the inner wall layer and the salt-soluble glyco-proteins have similar bulk amino acid and sugar (arabinose, galactose, mannose) compositions and both contain hydroxyproline. On the basis of the in vitro reconstitution of the cell wall we discuss certain aspects of in vivo cell wall morphogenesis. This communication describes the first case in which a plant cell wall has been reconstructed in vitro, and indicates that components of very large cellular structures are capable of being built by a simple self-assembly process.     

A correlation between BCL-2 modifying factor,p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.