Pregnancy-associated plasma protein-A in pregnant cynomolgus monkeys (Macaca fascicularis): radioimmunoassay, normal levels, effect of RU 486, and preliminary characterization

Pregnancy-associated plasma protein-A (PAPP-A) is a human macromolecular glycoprotein produced by the trophoblast and possibly by the decidua. Its biological function is unknown, but in vitro, PAPP-A has been reported to be an inhibitor of granulocyte elastase. The present study was undertaken to see if pregnant cynomolgus monkeys could be an animal model sufficiently close to the human situation to study the physiology of PAPP-A. An antiserum to pregnant cynomolgus plasma was raised in rabbits. After adsorption with normal monkey plasma, this antiserum was used together with radioiodinated human PAPP-A to develop an heterologous radioimmunoassay for measurements of monkey PAPP-A. On polyacrylamide gel electrophoresis, it was shown that this polyspecific-antiserum bound the same molecular species of radioiodinated human PAPP-A as the available anti-human PAPP-A antiserum. The concentrations of cynomolgus PAPP-A (cPAPP-A) throughout pregnancy follow the same pattern as human PAPP-A (hPAPP-A) with an almost exponential increase up to term. The doubling time of cPAPP-A was similar to that of hPAPP-A. After RU 486-induced abortion or after spontaneous abortion, the levels of cPAPP-A decreased, with an apparent half-life of 2-3 days. Preliminary characterization of cPAPP-A revealed that although cPAPP-A was only immunologically related to hPAPP-A, it was biochemically very similar: they had the same PI and the same molecular weight, and both PAPP-As bound heparin. It is concluded that pregnant cynomolgus monkeys are a good model to study the physiology of PAPP-A.     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

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.      

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

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.