Targeting of superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.     

Morphological phylogenetics of theAsteraceae-Asteroideae,with notes on character evolution

The relationships within theAsteraceae-Asteroideae are scrutinized by means of cladistic analyses of morphological and chemical data. New data are presented, and novel interpretations of features previously used in cladistic analyses are made. Examples are insertion of anther filaments in the corolla, and morphology of disc floret styles, respectively. The results include a polyphyletic or paraphyleticInuleae s. l., and a paraphyleticHeliantheae s.l. in which theEupatorieae constantly form a subclade. Comparisons are made with previous cladistic analyses of morphological data, and those acquired from molecular data. Speculations about the evolution of certain organs are presented.     

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.     

Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

THE MARINE DINOFLAGELLATE ALEXANDRIUM OSTENFELDII: PARALYTIC SHELLFISH TOXIN CONCENTRATION,COMPOSITION, AND TOXICITY TO A TINTINNID CILIATE1

The composition of paralytic shellfish toxins in the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen grown in unialgal culture was determined by high-performance liquid chromatography. The toxin profile revealed that the low-potency sulfamate toxin B₂ was dominant (90 molar % of total toxins), but small amounts of the weakly toxic 21-N-sulfocarbamoyl derivatives C₁₊₂ and trace amounts of the carbamate toxins GTX₂ and GTX₃ were also present. The mammalian toxicity was confirmed by a modification of the conventional AOAC mouse bioassay (0.6–1.4 pg STX_eq· cell^-1). The acute toxicity to a potential predator, the tintinnid ciliate Favella ehrenbergi (Clap, et Lach.) Jörg., was also investigated. The ciliate was able to graze on A. ostenfeldii when the cell concentration of the dinoflagellate was low (<2000 cells · mL^-1). At higher concentrations the ciliate was affected by exudates (presumably PSP toxins) that induced backward swimming followed by swelling and lysis of the cell. Fluorescence microscopy of calcofluor-stained cells was employed as an easy and rapid method to identify this and other thecate dinoflagellates.     

Omphalina foliacea, a new basidiolichen from America

A relatively large Leptogium -like lichen overgrowing mosses in wet paramos has been discovered in the northern Andes and Costa Rica. It has, however, a green photobiont and basidiomycete hyphae. No fruitbodies have been observed, only asexual reproduction by fragmentation of the fragile thallus and by goniocysts formed on the ecorticate lower surface. Some aspects of the taxonomy of Omphalina are briefly discussed.     

POPULATION STRUCTURE IN AN INSHORE CETACEAN REVEALED BY MICROSATELLITE AND mtDNA ANALYSIS: BOTTLENOSE DOLPHINS (TURSIOPS SP.) IN SHARK BAY, WESTERN AUSTRALIA

We examined population substructure of bottlenose dolphins ( Tursiops sp). in Shark Bay, Western Australia, using 10 highly polymorphic microsatellite loci, and mitochondrial DNA (mtDNA). For microsatellite analysis, 302 different animals were sampled from seven localities throughout the bay. Analysis of genetic differentiation between sampling localities showed a significant correlation between the number of migrants ( Nm ) calculated from F _ST, R _ST and private alleles, and distance between localities–a pattern of isolation-by-distance. For mtDNA, 220 individuals from all seven localities were sequenced for a 351 base pair fragment of the control region, resulting in eight haplotypes, with two distinct clusters of haplotypes. Values of F _ST and (φ)_ST for mtDNA yielded statistically significant differences, mostly between localities that were not adjacent to each other, suggesting female gene flow over a scale larger than the sampled localities. We also observed a significant correlation between the number of female migrants calculated from F _ST and φ_ST and the distance of sampling localities. Our results indicate that dispersal in female dolphins in Shark Bay is more restricted than that of males.     

DIET OF HARBOR PORPOISES IN THE KATTEGAT AND SKAGERRAK SEAS: ACCOUNTING FOR INDIVIDUAL VARIATION AND SAMPLE SIZE

Stomach contents of 112 bycaught harbor porpoises ( Phocoena phocoena ) collected between 1989 and 1996 in the Kattegat and Skagerrak seas were analyzed to describe diet composition and estimate prey size, to examine sample size requirements, and to compare juvenile and adult diets. Although porpoises preyed on a variety of species, only a few contributed substantially to the diet. Atlantic herring ( Clupea harengus ) was the dominating prey species for both juveniles and adults. Our results, in combination with those from previous studies, suggest that where herring is a dominant food source, porpoises prey primarily on size classes containing mature or maturing individuals. Further, we also show that Atlantic hagfish ( Myxine glutinosa ) may be an important food resource, at least for adult porpoises. Examination of sample size requirement showed that, depending on the taxonomic level used to describe the diet, a minimum of 35–71 stomachs are needed to be confident that all common prey species will be found.