Anti-insulin antibody structure and conformation. I. Molecular modeling and mechanics of an insulin antibody.

A knowledge-based three-dimensional model of an anti-insulin antibody, 125, was constructed using the structures of conserved residues found in other known crystallographic immunoglobulins. Molecular modeling and mechanics were done with the 125 amino acid sequences using QUANTA and CHARMm on a Silicon Graphics 4D70GT workstation. A minimal model was made by scaffolding using crystallography coordinates of the antibody HyHEL-5, because it had the highest amino acid sequence homology with 125 (84% light chain, 65% heavy chain). The three hypervariable loop turns that are longer in 125 than in HyHEL-5 (L1, L3, and H3) were modeled separately and incorporated into the HyHEL-5 structure; then other amino acid substitutions were made and torsions optimized. The 125 model maintains all the structural attributes of an antibody and the structures conserved in known antibodies. Although there are many polar amino acids (especially serines) in this site, the overall van der Waals surface shape is determined by positions of aromatic side chains. Based on this model, it is suggested that hydrogen bonding may be key in the interaction between the human insulin A chain loop antigenic epitope and 125.     

The function of host discrimination and superparasitization in parasitoids

Summary Host discrimination, i.e. the ability to distinguish unparasitized hosts from parasitized ones, and to reject the latter for egg laying is present in many parasitic wasp species. This property is classically considered as an example of contest competition, and is supposed to have a number of functions. However, different species do not react to each other's marks and lay eggs in hosts parasitized by the other species. Apparently the marks used for recognition are specific.Multiparasitization is the best strategy when hosts are scarce and the egg supplies of the parasitoids are not limited. Interspecific host discrimination is not an ESS.Superparasitization within one species would have selective advantage if the number of unparasitized hosts is small and the wasp has a reasonable chance to lay her egg in a host that is not parasitized by herself, and if the chance for her offspring to survive the competitive battle with the first parasitoid larva is not too small. This is shown to be the case.However, marks are not individual and wasps cannot distinguish hosts parasitized by themselves from those parasitized by others. The hypothesis is tested that the egg laying strategy (i.e. the decision to superparasitize) of wasps is dependent on the number of conspecifics that is searching simultaneously for hosts, since this determines the chance that a parasitized host encountered by a wasp is parasitized by herself.It is shown that host discrimination cannot be regarded as a case of contest competition. Other aspects of superparasitization, related to interference and population regulation, sex allocation and encapsulation are briefly discussed.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.