Monoclonal antibodies against alpha toxin of Clostridium perfringens

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

Parallel evolution in radiation ofOhomopterus ground beetles inferred from mitochondrial ND5 gene sequences

Molecular phylogenetic analyses using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences representing all 15 species and the majority of subspecies or races of theOhomopterus ground beetles from all over the Japanese archipelago have uncovered a remarkable evolutionary history. Clustering of the species in the molecular phylogenetic tree is linked to their geographic distribution and does not correlate with morphological characters. Taxonomically the same species or the members belonging to the same species-group fall out in more than two different places on the ND5 tree. Evidence has been presented against a possible participation of ancestral polymorphism and random lineage sorting or of hybrid individuals for the observed distribution of mitochondrial DNA haplotypes. The most plausible explanation of our results is that parallel evolution took place in different lineages. Most notably,O. dehaanii, O. yaconinus, andO. japonicus in a lineage reveal almost identical morphology with those of the same species (or subspecies) but belonging to the phylogenetically remote lineages.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with accession numbers D50711-DD-50733 and D87131-D87186.     

Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis

Abstract High level expression of the functional β-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing β-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora . Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from β-carotene is canthaxanthin (4,4'-diketo-β-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from β-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt . We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.     

Cloning and functional analysis of molecular chaperone genes from Bacillus stearothermophilus SIC1

Genes homologous to groES and groEL, which are recognized as molecular chaperone genes, from Bacillus stearothermophilus SIC1 were cloned and sequenced. By addition of GroES, GroEL and ATP in vitro, remaning activity of the alcohol dehydrogenase from Saccharomyces cerevisiae after heat treatment at 50°C for 6 min was improved from 55% to 90%. Furthermore, even though inclusion bodies were formed when a single chain Fv(sFv) was expressed in E. coli cells, during in vivo coexpression with molecular chaperone, a significant amount of the antibody protein could be recovered from the soluble fraction.     

Bilateral giant myelolipoma in the adrenal of a cotton-top tamarin (Saguinus oedipus)

Abstract: A rare case of bilateral adrenal myelolipomas in a female cotton-top tamarin is reported. Large bilateral masses in the adrenal glands were composed of mature adipose cells containing varying amounts of hematopoietic cells of the myeloid, erythroid, and megakaryocyte series. The gross and histologic features of this case closely resemble human “giant” adrenal myelolipomas.     

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B^*5101 binding self-peptides, 27 paptides bound to HLA-B^*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B^*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B^*5102 and B^*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B^*5102) and that of Gly for Trp at residue 167 (B^*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B^*5102 or HLA-B^*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.     

Effect of sizofiran on regional lymph nodes in patients with head and neck cancer

The immunomodulating effects of preoperative sizofiran (SPG) administration on regional lymph nodes were studied in patients with stage III or IV head and neck cancer, by comparing the immunofunction of peripheral blood. The regional lymph nodes were dissected surgically, and freshly obtained mononuclear cells were studied to investigate the interleukin-2 (IL-2) production, the LAK and NK activities, and the quantitative analysis of the surface phenotype of the mononuclear cells. The results indicated that SPG enhanced immunological activities in the regional lymph nodes, as shown by increased IL-2 production and cytotoxic activities of the effector cells (NK, LAK), and increased helper T lymphocytes (CD4⁺) in the tumor-uninvolved lymph nodes. The immunofunction following SPG administration was attenuated, but was still augmented in the regional lymph nodes with metastases. Therefore, SPG was found to be a biologic response modifier to enhance the immunofunctions of the regional lymph node in patients with head and neck cancer.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.     

Design,synthesis, and conformation of a model peptide of endothelin with cystine-stabilized α-helix motif

A model 16-peptide of endothelin-1 (MET-1), which has the minimized sequence homology to the corresponding pan of endothelin-1 (ET-1), was designed to confirm the cystine-stabilized α-helix motif. The model structure consists of an extended structure, a β-turn part, and an α-helix structure that is stabilized by two disulfide bonds. The α-helix segment was designed to emphasize the amphiphilic nature. In order to combine the extended structure and the α-helix segment, a D -Ala-Pro sequence was selected to fix the β-turn. The model endothelin 16-peptide amide was synthesized by solid-phase synthesis on a 4-methylbenzhydrylamine resin. Its conformation was examined by CD and two-dimensional (2D) ¹H-nmr measurements. MET-1 showed similar CD patterns to ET-1 in both buffer and 50% aqueous trifluoroethanol solution. The 2D nmr experiments in 50% aqueous ethylene glycol revealed that MET-1 closely resembles the conformation of ET-1 with an extended structure, an α-helix, and a β-turn unit in the same position of the sequence. Furthermore, model peptides without disulfide bond(s) could not assume a stable structure in aqueous solution, while they did have similar α-helical content in 50% trifluoroethanol with MET-1. When the two disulfide bridges were simultaneously formed, the peptide with the correct disulfide bonds (MET-1) was obtained in threefold excess to the isomer (apamin type. MET-2). These findings obtained by the modeling of ET-1 showed an important role for the stabilization of peptide conformation with disulfide bonds. © 1994 John Wiley & Sons, Inc.