Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Full-length sequence of the cDNA for human erythroid beta-spectrin

Spectrin is the major molecular consituent of the red cell membrane skeleton. We have isolated overlapping human erythroid beta-spectrin cDNA clones and determined 6773 base pairs of contiguous nucleotide sequence. This includes the entire coding sequence of beta-spectrin. The sequence translates into a 2137 amino acid, 246-kDa peptide. beta-Spectrin is found to consist of three distinct domains. Domain I, at the N terminus, is a 272-amino acid region lacking resemblance to the spectrin repetitive motif. Sequences in this region exhibit striking sequence homology, at both nucleotide and amino acid levels, to the N-terminal "actin-binding" domains of alpha-actinin and dystrophin. Between residues 51 and 270 there is 55% amino acid identity to human dystrophin, with only four single amino acid gaps in alignment. Domain II consists of 17 spectrin repeats. Several sequence variations are observed in typical repeat structure. Homology to alpha-actinin extends beyond domain I into the N-terminal portion of domain II. Domain III, 52 amino acid residues at the C terminus, does not adhere to the spectrin repeat motif. Combining knowledge of spectrin primary structure with previously reported functional studies, it is possible to make several inferences regarding structure/function relationships within the beta-spectrin molecule.     

14-Fluorobacteriorhodopsin and other fluorinated and 14-substituted analogues. An extra, unusually red-shifted pigment formed during dark adaptation

Five vinyl-substituted fluororetinal analogues (8-F, 10-F, 12-F, 14-F, and 13,14-F2) were found to give bacteriorhodopsin analogues with properties similar to those of the parent system. Of these, only 14-fluororetinal was found to give an extra red-shifted BR analogue (lambda max less than or equal to 680 nm) in equilibrium with the normal 587-nm pigment. The 680-nm pigment was enriched upon irradiation. It rearranged to the 587-nm pigment at room temperature (delta E [symbol: see text] = 20.8 kcal/mol). Chromophore extraction experiments revealed the all-trans geometry for the 680-nm pigment. 14-Chlororetinal gave a similarly red-shifted pigment while 14-methylretinal did not. A scheme for dark adaptation of the 14-halogenated bacteriorhodopsins has been proposed in which the new red-shifted pigment was assigned the all-trans, 15-syn geometry.     

The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus scutatus venom

Notexin from Notechis scutatus scutatus snake venom was modified with trinitrobenzenesulfonic acid, and the major trinitrophenylated (TNP) derivative was separated by high-performance liquid chromatography. Modification resulted in the incorporation of only one TNP group on the N-terminal alpha-amino group. The TNP derivative showed a precipitous decrease in enzymatic activity and lethal toxicity, whereas the antigenicity remained unchanged. However, trinitrophenylation did not significantly affect the secondary structure of the toxin molecule as revealed by the CD spectra. The results, that the modification reaction was accelerated by the Ca2+ and that the TNP derivative retains its affinity for Ca2+, indicate that the N-terminal alpha-amino group did not participate in the Ca2(+)-binding. The TNP derivative could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated notexin are almost the same as those of native notexin. These results suggest that the N-terminal alpha-amino group is essential for the phospholipase A2 activity and lethal toxicity of notexin, and that incorporation of the TNP group on the N-terminal alpha-amino group might give rise to a distortion of the active conformation of notexin.     

Lipolytic activities of a lipase immobilized on six selected supporting materials

Six different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase-coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45-55 degrees C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half-life of the PVC immobilized lipase operating at 30 degrees C in a packed-bed reactor was estimated to be about 400 h.     

Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Effector phenotype and immunologic specificity of T-cell-mediated adoptive therapy for a murine tumor that lacks intrinsic immunogenicity

The MCA 102 sarcoma has been defined by a variety of immunologic studies as a tumor lacking intrinsic immunogenicity. Nevertheless, we have recently demonstrated the feasibility of generating therapeutically effective lymphocytes for adoptive immunotherapy of this tumor. Procedures to achieve this required in vivo priming of syngeneic mice to elicit preeffector cells followed by in vitro sensitization (IVS) with tumor cells in the presence of IL-2. By selective depletion of T cell subsets in vivo, we identified the involvement of both CD4+ (L3T4+) and CD8+ (Lyt-2+) T cells in mediating tumor regression. The CD4+ cells exerted their helper function via the secretion of IL-2 because antitumor effects abrogated by depletion of CD4+ cells could be reconstituted by exogenous IL-2. In order to elicit preeffector cells with reactivity against the MCA 102 tumor, we found that in vivo sensitization could be accomplished with either the MCA 102 or MCA 106 tumor but not with the MCA 101 or MCA 105 tumor. Analysis of specificity of tumor stimulation during IVS of MCA 102 tumor-primed preeffector cells demonstrated cross-reactivity between not only the MCA 102 and MCA 106 tumors but also the MCA 105 tumor whereas the MCA 101 tumor was ineffective. In adoptive immunotherapy, transfer of IVS cells generated from MCA 102 tumor-primed and stimulated lymph node cells was able to mediate reductions of pulmonary metastases established from the MCA 102, MCA 105, and MCA 106 tumors but not from the MCA 101 tumor. We conclude that regression of the MCA 102 tumor is probably mediated through T cell recognition of a set of common tumor-associated Ag shared by several other syngeneic tumors. Immunologically, the tumor-associated Ag are characteristically different from classical tumor-specific transplantation Ag (TSTA) because immunity to TSTA on the MCA 105 or MCA 106 tumor does not cross-react with the MCA 102 tumor. Thus, this study demonstrates that Ag other than TSTA on chemically induced tumors can serve as target molecules for T cell-mediated adoptive immunotherapy.     

The nucleic acid content of some edible mushrooms

Summary An improved method for the determination of nucleic acid content in edible mushrooms is described. Details of tissue homogenization and extraction are also included. In regard to the limit suggested by the Protein Advisory Group of the United Nations System, the amount of nucleic acids found in Agaricus bisporus, Pleurotus cystidiosus, Pleurotus sajor-caju and Volvariella volvacea indicates that it is safe to consume mushrooms as daily vegetable. No significant changes have been found in the nucleic acid content of V. volvacea at different degrees of maturity. V. volvacea loses around 20% of its nucleic acids upon boiling for 10 min. Possible reasons for the discrepancy between the present finding and that given in an earlier report have been discussed.     

A complete separation of dimethylaminoazobenzenesulphonyl-amino acids. Amino acid analysis with low nanogram amounts of polypeptide with dimethylaminoazobenzenesulphonyl chloride.

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.