Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Fertilization and embryo loss in Booroola Merino x South Australian Merino ewes: Effect of the F gene

The effects of Booroola genotype (F+, ++); the number of ovulations per ewe (one, two or three); and the age of a ewe (2.5 yr vs 3.5 to 6.5 yr) on the percentage of ova fertilized, embryo loss and fetal loss were examined in Booroola x South Australian Merino ewes slaughtered on Days 4, 21 and 90 after insemination. Ewes slaughtered on Day 90 were examined by real-time ultrasound imaging (RUI) on Day 45. Fertilization failure was independent of ewe genotype, ovulation rate and age of ewe, and it was not an important source of wastage (F+, 9.4%; ++, 6.7%). Most embryo loss occurred during the first 21 d (F+, 54.7%; ++, 40.3%). Interpretation of the effects of genotype and ovulation rate on embryo wastage measured on Days 21, 45 and 90 was obscured by significant (P < 0.05) genotype and ovulation rate interactions with the day of slaughter/RUI. The effect of age on embryo loss was not significant (P > 0.05). Reasons for the high rate of wastage observed in this experiment require further study.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Mytilid bivalveLithophaga in Upper Triassic coralPamiroseris from Zlambach beds compared with cretaceousLithophaga Alpina

Summary The mytilid genusLithophaga is confirmed for the Upper Triassic. The Rhaetian specimen, boring the dead part of coral, is compared with the SenonianL. alpina, which is associated with live coral.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Interactions of proteins and cholesterol with lipids in bilayer membranes.

Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.