Monoclonal antibodies to alpha-chain regions of human fibrinogen that participate in polymer formation

Monoclonal antibodies have been generated against a cross-link-containing derivative of alpha polymer (alpha XLCNBr), isolated following CNBr digestion of fibrin [Sobel, J. H., Ehrlich, P. H., Birken, S., Saffran, A. J., & Canfield, R. E. (1983) Biochemistry (preceding paper in this issue)]. One cloned cell line (F-102) was chosen for characterization based on its apparent specificity for the A alpha-chain region A alpha 518-584 (CNBr X). A second line (F-103) was selected because of its anti-A alpha 241-476 (CNBr VIII) properties. These two regions of the A alpha chain have previously been implicated as major contributors to the cross-linking process that leads to alpha-polymer formation. Radioimmunoassays have been developed, employing the immunoglobulins produced by clones F-102 and F-103. These assays have been applied, in conjunction with high-performance liquid chromatography purified tryptic and chymotryptic derivatives of CNBr VIII and CNBr X, to localize the respective determinants involved in antibody binding. In each case, virtually full immunoreactivity was exhibited by both the CNBr fragment and a single tryptic or chymotryptic peptide originating from it. These findings indicate that sequence-specific, rather than conformational, determinants were operative in the generation of antibodies F-102 and F-103. The epitope recognized by F-102 was localized to the region of A alpha 540-554, while the F-103 binding site resided within A alpha 259-276. When these radioimmunoassays were applied to study the relative immunoreactivity exhibited by a variety of fibrinogen derivatives, the results obtained support earlier suggestions that the COOH-terminal portion of the A alpha chain contains regions of random conformation.     

Genetic and evolutionary relationships among Asian Macaques

Published gene frequency data, checked for consistency of allele definitions across laboratories and for comparability of geographically identical samples, were pooled into a data set containing frequencies at nine loci for each of 20 populations that encompassed 10 macaque species. Genetic distances were calculated by the methods of Kidd and Cavalli-Sforza (1974). These distances were used to construct phylogenetic trees and to evaluate the relationships between divergence times and effective population sizes. Inter-and intraspecific genetic distances and the groupings defined by phenetic tree analyses support Fooden’s (1976) classification of the genus Macacainto four species groups. A paleozoogeographical model of Asia including the known times of major sea-level changes allows us to explain Macacainto four species groups. A paleozoogeographical model of Asia including the known times of major sea-level changes allows us to explain qualitatively the inferred evolutionary relationships among macaque species. Many assumptions are required in order to estimate the variables necessary in the quantitative prediction of genetic differences for a comparison between any two populations. Examination of those assumptions demonstrates the need for more accurate genetic as well as paleozoogeographic information. An erratum to this article is available at .     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Tissue elastic properties of eight Hawaiian Dubautia species that differ in habitat and diploid chromosome number

Summary Eight Hawaiian Dubautia species grow in habitats as varied as exposed lava, dry scrub, mesic forest, wet forest, and bog. These species also differ in diploid chromosome number, with four species having 13 pairs of chromosomes and four species having 14 pairs. This ecological and chromosomal variation is paralleled by significant interspecific variation in tissue elastic properties. The four 13-paired species from dry habitats exhibit significantly lower tissue elastic moduli near full hydration (E _i) than the four 14-paired species from mesic to wet habitats. Values of E _i range from 2 to 4 MPa among the former species and from 9 to 18 MPa among the latter species. The turgor dependence of the elastic modulus also differs markedly between the two groups of species. As a result of these differences in tissue elastic properties, the capacity for maintaining high turgor pressures as tissue water content decreases is much greater in the 13-paired species from dry habitats than in the 14-paired species from mesic to wet habitats. These results indicate that the evolutionary diversification of the Dubautia species has been accompanied by a significant degree of change at the physiological level.     

What can economics contribute to the study of human evolution?

The revised edition of Paul Seabright’s The Company of Strangers is critically reviewed. Seabright aims to help non-economists participating in the cross-disciplinary study of the evolution of human sociality appreciate the potential value that can be added by economists. Though the book includes nicely constructed and vivid essays on a range of economic topics, in its main ambition it largely falls short. The most serious problem is endorsement of the so-called strong reciprocity hypothesis that has been promoted by several prominent economists, but does not pass muster with biologists.     

The role of humans in the diversification of a threatened island raptor

Background  

Anthropogenic habitat modifications have led to the extinction of many species and have favoured the expansion of others. Nonetheless, the possible role of humans as a diversifying force in vertebrate evolution has rarely been considered, especially for species with long generation times. We examine the influence that humans have had on the colonization and phenotypic and genetic differentiation of an insular population of a long-lived raptor species, the Egyptian vulture (Neophron percnopterus).     

Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Vitamin K-dependent oxygenase/carboxylase; differential inactivation by sulfhydryl reagents

Inhibition of vitamin K-dependent carboxylase and oxygenase by sulfhydryl reagents was compared. Formation of vitamin K epoxide and vitamin K-dependent carboxylation are both strongly (greater than 90%) inhibited by l mM p-hydroxy-mercuribenzoate, and this inhibition is reversed by dithiothreitol. Both activities are also effectively inhibited by N-ethylmaleimide (NEM). Preincubation with vitamin K hydroquinone prevents NEM inhibition of epoxide formation but not of carboxylation. These data argue that separate active sites are required to support vitamin K-dependent epoxide formation and carboxylation and that the binding site vitamin K oxygenase contains an active thiol group.