Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freund's complete adjuvant.     

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis Mushroom Crops in Spain

Diseased fruit bodies of Agaricus bitorquis, with similar symptoms to those caused by dry bubble on Agaricus bisporus, were observed in some Spanish crops during summer 1999. Isolates of Verticillium fungicola from A. bitorquis and A. bisporus were submitted to different temperatures and to prochloraz–Mn sensitivity tests. All the isolates collected from A. bitorquis and A. bisporus were identified as V. fungicola var. fungicola. Artificial infections of A. bisporus and A. bitorquis with V. fungicola var. fungicola are also described in the present study. The appearance of natural infections of V. fungicola var. fungicola in A. bitorquis crops could well be due to the growing temperatures used in Spain, which are considerably below those used in other countries.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Mesodern-inducing factors and mesodermal patterning

Identification of the signalling molecules involved in mesoderm formation in amphibian embryos still presents problems. None of the original candidates, such as activin, have been definitively ruled out, and the new factors, such as the nodal-related genes, have come on to the scene. Of the original candidates, activin has been definitively shown to act as a morphogen, whereas bone morphogenetic protein (BMP)-4 has emerged as a ventral inducer and an inhibitor of neural differentiation. The effects of BMP-4 are antagonized by chordin, a molecule related to the product of the Drosophila gene short gastrulation.     

A panel of antibodies useful in the cytologic diagnosis of metastatic melanoma.

Five antibodies, 2D.1 (pan-leukocyte), AE-1,3 (anti-keratin), B72.3 (anti-carcinoma), ME 1-14 (alpha-chondroitin sulfate proteoglycan) and polyclonal S-100 protein (P-S100), were tested to determine if this panel could be used immunocytochemically to differentiate melanoma from nonmelanoma. A total of 161 cases were evaluated: 145 fine needle aspirates of various body sites and 16 effusions, consisting of 52 melanomas, 41 adenocarcinomas, 11 squamous cell carcinomas, 14 undifferentiated carcinomas, 8 small cell carcinomas, 8 miscellaneous carcinomas, 8 primary central nervous system (CNS) tumors, 7 lymphomas/leukemias, 4 sarcomas and 8 benign effusions. The 52 melanomas were stained by ME 1-14 (in 31 cases) and by P-S100 (in 39 cases), but not by B72.3, AE-1,3 or 2D.1. The 82 carcinomas reacted with P-S100 (in 25 cases), B72.3 (in 37 cases), AE-1,3 (in 68 cases) and 2D.1 (in 1 case), but not with ME 1-14. Lymphomas were stained only by 2D.1 (5 of 7 cases). The eight primary CNS tumors reacted solely with ME1-14 (in 3 cases) and P-S100 (in 3 cases). The eight benign effusions exhibited staining by ME 1-14 (in 1 case), P-S100 (in 1 case), AE-1,3 (in 3 cases) and 2D.1 (in 8 cases), but not by B72.3. Thirty-six cases (including 11 melanomas) failed to stain with any antibody. In summary, 41 of 52 melanomas and 4 of 8 CNS tumors stained with ME1-14, P-S100 or both and were negative for B72.3, AE-1,3 and 2D.1. Only 2 of 101 other nonmelanomas exhibited this pattern. Thus, this panel distinguishes melanoma from other neoplastic and nonneoplastic processes in the majority of cases.