Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

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.      

Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations

The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm.     

Essential function in chloroplast recognition of the ferredoxin transit peptide processing region

Molecular Genetics and Genomics - Plant ferredoxin is a nuclear-encoded chloroplast protein that is synthesized in the cytoplasm as a transit peptide-containing precursor molecule. To identify...     

Functional polymorphisms of Fc receptors in human monocyte-mediated cytotoxicity towards erythrocytes induced by murine isotype switch variants

Human monocytes can be triggered to antibody-dependent cell-mediated cytotoxicity (ADCC) by murine antibodies. In this study, a series of H chain isotype switch variant antibodies against glycophorin A on human RBC was used to study the influence of isotype on the induction of ADCC. Furthermore, it was studied whether the functional heterogeneity in responsiveness to IgG1 and IgG2b anti-CD3 antibodies, as found among different donors in T cell proliferation induction experiments, was reflected in ADCC. Whereas IgG2a induced ADCC to the same extent in monocytes from all donors, IgG1 showed a heterogeneous pattern, which corresponded to the heterogeneity in T cell proliferation studies. IgG1 anti-CD3 nonresponder monocytes could, however, be induced to ADCC by IgG1 antiglycophorin, although they needed a much higher antibody density on the target cell than did responder monocytes. IgG2b antiglycophorin at a high density induced ADCC in monocytes from all donors irrespective of responsiveness to IgG2b anti-CD3, whereas IgE and IgA antiglycophorin were barely effective in monocytes from all donors. By specific blocking with mAb, the FcR that were involved in ADCC directed by the various isotypes were characterized. ADCC by IgG2a was predominantly mediated by FcRI and could be specifically enhanced by culturing the monocytes with rIFN-gamma. ADCC by IgG1 was predominantly mediated through FcRII in both anti-CD3 responder and nonresponder monocytes. FcRII was also involved in ADCC by IgG2b, although other receptors seemed to contribute significantly to ADCC. When FcRII or FcRI were blocked, IgG1 and IgG2a could also functionally interact with FcRI and FcRII, respectively, provided that the target cells were sensitized to a high degree. These findings indicate that FcRI and both forms of FcRII can mediate cytotoxicity and that the specificity of human FcR for murine isotypes is relative.     

Nitrogen losses from perennial grass species

Nitrogen losses from plants may occur through a variety of pathways, but so far, most studies have only quantified losses of nutrients by above-ground litter production. We used ¹⁵N pulse labelling to quantify total nitrogen losses from above- and below-ground plant parts. Using this method we were able to include also pathways other than above-ground litter production. To test the hypothesis that species from nutrient-poor habitats lose less nitrogen than species from more fertile soils, six perennial grasses from habitats with a wide range of nutrient availability were investigated: Lolium perenne, Arrhenatherum elatius, Anthoxanthum odoratum, Festuca rubra, F. ovina and Molinia caerulea. The results of an experiment carried out in pots in a green-house at two fertility levels show that statistically significant losses occur through pathways other than above-ground litter production. In the low fertility treatment, most (70%) losses from L. perenne occurred by litter production, but in Ar. elatius, F. rubra, F. ovina and M. caerulea, more than 50% of labelled N losses took place by root turn-over, leaching or exudation from roots. When nutrient supply increased, the ¹⁵N losses in above-ground dead material increased in all species and in Ar. elatius, A. odoratum and F. rubra the ¹⁵N losses via other pathways decreased. Ranked according to decreasing turnover coefficient the sequence of species was: L. perenne, A. odoratum, F. rubra, F. ovina, Ar. elatius, M. caerulea. These results suggest that species adapted to sites with low availability of nutrients lose less nitrogen (including above- and below-ground losses) than species adapted to more fertile soils.