Molecular clocks and the incompleteness of the fossil record

Molecular clocks can be evaluated by comparing absolute rates of evolution and by performing relative-rate tests. Typically, calculations of absolute rates are based on earliest observed occurrences in the fossil record. Relative-rate tests, on the other hand, merely require an unambiguous outgroup. A major disadvantage of relative-rate tests is their insensitivity to concomitant and equal rate changes in all lineages. Apparent differences in absolute rates, in turn, may be artifacts that are attributable to an incomplete fossil record.Recently developed methods in quantitative biostratigraphy recognize the incompleteness of the fossil record and allow us to place confidence intervals on the endpoints of taxon ranges. These methods are applicable to taxa whose fossil records are of markedly different quality. When we extend these methods and integrate molecular and paleontologic data, we can test the null hypothesis that seemingly disparate rates of molecular evolution are in fact equal under the simplifying assumption that fossils are randomly and independently distributed over their temporal ranges and that fossils can be accurately placed in a phylogenetic context. We can also estimate the range of ticking rates, if any, that are compatible with known fossil data. Ultimately, more accurate rate estimates for widely divergent taxa should allow for more meaningful comparisons of evolutionary rates.DNA hybridization data for monotremes and marsupials suggest a 17-fold difference for 14 different rate calculations with a mean value of approximately 1% divergence per million years. Variation among marsupials is sevenfold. However, when we apply appropriate statistical tests and make additional allowances for fossils of uncertain taxonomic assignment, etc., all 14 rates are compatible with a molecular clock ticking at approximately 0.4% divergence per million years. In addition, this analysis brings relative- and absolute-rate tests into accord.     

Phalaenopsis bellina (Rchb.f.) Christenson,a segregate from P. violacea Witte (Orchidaceae: Aeridinae)

A reevaluation of floral and vegetative morphology together with data from analysis of floral fragrance and flavonoid co-pigment chemistry suggest that specific status is warranted for the two kinds ofPhalaenopsis violacea Witte known informally as the “Bornean type” and the “Malayan type.”Phalaenopsis violacea var.bellina Rchb.f. is elevated to specific rank, providing a name for the “Bornean type” plants.     

GENETIC CONSTRAINTS ON MACROEVOLUTION: THE EVOLUTION OF HOST AFFILIATION IN THE LEAF BEETLE GENUS OPHRAELLA

We hypothesize that the evolution of an ecologically important character, the host associations of specialized phytophagous insects, has been influenced by limitations on genetic variation. Using as a historical framework a phylogenetic reconstruction of the history of host associations in the beetle genus Ophraella (Chrysomelidae), we have employed quantitative-genetic methods to screen four species for genetic variation in larval survival, oviposition (in one species only), and feeding responses to their congeners' host plants, in the Asteraceae. We here report results of studies of one species and evaluate the results from all four. Analysis of half-sib/full-sib families and of progenies of wild females of O. notulata, a specialist on Iva (Ambrosiinae), provided evidence of genetic variation in larval consumption of five of six test plants and in adult consumption of four of six. Larval mortality was complete on five plants; only on Ambrosia, a close relative of the natural host, was there appreciable, and genetically variable, survival. Oviposition on Ambrosia showed marginally significant evidence of genetic variation; a more distantly related plant elicited no oviposition at all. In compiling results from four Ophraella species, reported in this and two other papers, we found no evidence of genetic variation in 18 of 39 tests of feeding responses and 14 of 16 tests of larval survival on congeners' hosts. This result is consistent with the hypothesis that absence or paucity of genetic variation may constrain or at least bias the evolution of host associations. The lower incidence of genetic variation in survival than in feeding behavior may imply, according to recent models, that avoidance is a more common evolutionary response to novel plants than adaptation. The usually great disparity between mean performance on congeners' hosts and the species' natural hosts, and an almost complete lack of evidence for negative genetic correlations, argue against the likelihood that speciation has occurred by sympatric host shift. The presence versus apparent absence of genetic variation in consumption was correlated with the propinquity of relationship between the beetle species tested and the species that normally feeds on the test plant, suggesting that the history of host shifts in Ophraella has been guided in part by restrictions on genetic variation. It was also correlated with the propinquity of relationship between a test plant and the beetle's natural host. The contributions of plant relationships and insect relationships, themselves correlated in part, to the pattern of genetic variation, are not readily distinguishable, but together accord with phylogenetic evidence that these and other phytophagous insects adapt most readily to related plants. In this instance, therefore, the macroevolution of an ecologically important character appears to have been influenced by genetic constraints. We hypothesize that absence of the structural prerequisites for genetic variation in complex characters may affect genetic variation and the trajectory of evolution.     

Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of the cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRAPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type I turn involving the same amino acids in both glycosylated and unglycosylated peptides. The GalNAc attached to the threonine (T³) and serine (S⁴) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.     

Sigma-E is required for the production of the antibiotic actinomycin in Streptomyces antibioticus

The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus . The phsA promoter strikingly resembles a putative Streptomyces σ^E cognate promoter, and purified Eσ^E holoenzyme transcribed the phsA promoter in vitro . However, the phsA promoter was still active in an S. antibioticus sigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.     

Isolation and Characterization of Five Actin cDNAs from the Cestode Diphyllobothrium dendriticum: A Phylogenetic Study of the Multigene Family

Five cDNAs (pDidact2–pDidact6), representing different actin genes, were isolated from a Diphyllobothrium dendriticum cDNA library, and the DNA as well as the putative amino acid sequences were determined. The corresponding Didact2 and Didact4 genes code for peptides 376 amino acids long, with molecular weights 41,772 and 41,744 Da, respectively, while the deduced Didact3 protein is 377 amino acids long and weighs 41,912 Da. The pDidact5 and -6 cDNAs lack nucleotides corresponding to three to six amino acids at the amino-terminus. Two of the five cDNAs contain the conventional AATAAA as the putative polyadenylation signal, one has the common variant ATTAAA, whereas the hexanucleotide AATAGA is found 15 and 18 nucleotides, respectively, upstream of the poly(A) site in two of the cDNAs. Phylogenetic studies including 102 actin protein sequences revealed that there are at least four different types of cestode actins. In this study three of these types were found to be expressed in the adult D. dendriticum tapeworm. Structurally the cestode actin groupings differ from each other to an extent seen only among the metazoan actins between the vertebrate muscle and cytoplasmic isoforms. In the phylogenetic trees constructed, cestode actins were seen to map to two different regions, one on the border of the metazoan actins and the other within this group. It is, however, difficult to say whether the cestode actins branched off early in the metazoan evolution or if this position in the phylogenetic tree only reflects upon differences in evolutionary rate. Received: 19 June 1996 / Accepted: 20 August 1996     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.