Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.     

Propagation of Geotrichum candidum in Acid Brine

Geotrichum candidum completely neutralized the acid brine and reduced its biochemical oxygen demand (BOD) by 88%. Yield of dry mycelium was 62 g per 100 g of BOD utilized.     

Cleavage of territrems by alkaline hydrogen peroxide: Isolation and characterization of the aromatic moiety of territrems

The chemical reaction of cleavaging territrem B to give 3,4,5-trimethoxy benzoic acid by alkaline hydrogen peroxide was investigated. The method was applied for confirmation of the chemical structure of the aromatic moiety of territrem A, A’, B, and B’. The physicochemical properties of the aromatic cleavage product of territrem Aindicated the structure as 3,4-methylendioxy, 5-methoxy benzoic acid (or 4-methoxy, 6-carboxy, 1, 3-benzodioxole). The experiment also gave the evidences that territrem A and A’, on the other hand territrem B and B’ have the identical aromatic moieties on their structures.     

Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane.

The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.     

Calorimetric studies of the effects of cholesterol on the phase transition of C(18):C(10) phosphatidylcholine.

Differential scanning calorimetry (DSC) has been employed to study the effects of cholesterol on the phase transition of C(18):C(10) phosphatidylcholine (C(18):C(10)PC). C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated lipid bilayers above the transition. Three types of samples were used. The treated sample is the lipid dispersion that had undergone three freeze-thaw cycles and stored at 4 degrees C for more than 48 h. The untreated sample was made by vortexing the dry lipid in 50 mM KCl, without the above-mentioned pretreatment. The cold-treated sample was prepared by incubating the treated sample at -20 degrees C for 15 d. There is no apparent difference in the DSC curves between the treated and cold-treated samples. The data derived from the treated samples seem to be more reproducible. The DSC curves between the cholesterol/C(18):C(10)PC and cholesterol/symmetric diacylphosphatidylcholine mixtures are different in three aspects: overall appearance, the cholesterol dependence of delta H, and the effect of cholesterol on the maximal transition temperature Tm, the onset temperature To, and the completion temperature Tc. for both the treated and untreated samples, the total enthalpy change delta H of the phase transition of C(18):C(10)PC decreases with increasing cholesterol content, approaching zero at approximately 25 mol%. This level is lower than the total enthalpy changes reported previously for the cholesterol/symmetric diacylphosphatidylcholine mixtures. Both the heating and cooling thermograms show that Tm, To, and Tc decrease with increasing cholesterol content. The decreasing rates of these temperatures with cholesterol are in the neighborhood of -0.24 degree per mol% of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the enantiomeric composition of (R*,S*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius

The enantiomeric composition of sitophilate, the granary weevil [Sitophilus granarius (L.)] male-produced aggregation pheromone [(R^*,S^*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate)], was determined by three methods: (1) bioassaying the synthetic (2S,3R) and (2R,3S) enantiomers of the active (R^*,S^*) diastereomer; (2) ¹H NMR spectroscopy of Mosher ester derivatives of the natural pheromone and synthetic (2S,3R)-and (2R,3S)-sitophilate; and (3) capillary GLC comparisons of the retention times of derivatized natural pheromone and the two synthetic enantiomers. The combined methods confirmed the (2S,3R) enantiomer as the active form of sitophilate. Male granary weevils were shown to produce >96% (2S,3R)-sitophilate. No significant attraction of S. granarius by the (2R,3S) enantiomer was observed. Rice and maize weevils [S. oryzae (L.) and S. zeamais Motschulsky] were not attracted by (2S,3R)-sitophilate. S. granarius L. est un déprédateur important des grains stockés. Le (R^*,S^*)-1-éthylpropyl 2-méthyl-3-hydroxypentanoate a été identifié en 1987 comme le principal composé du sitophilate, la phéromone mâle d'agrégation de S. granarius. La composition énantiométrique du sitophilate a été déterminée par 3 méthodes:

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.