Can Genetic Estimators Provide Robust Estimates of the Effective Number of Breeders in Small Populations?

The effective population size (N_e) is proportional to the loss of genetic diversity and the rate of inbreeding, and its accurate estimation is crucial for the monitoring of small populations. Here, we integrate temporal studies of the gecko Oedura reticulata, to compare genetic and demographic estimators of N_e. Because geckos have overlapping generations, our goal was to demographically estimate N_bI, the inbreeding effective number of breeders and to calculate the N_bI/N_a ratio (N_a = number of adults) for four populations. Demographically estimated N_bI ranged from 1 to 65 individuals. The mean reduction in the effective number of breeders relative to census size (N_bI/N_a) was 0.1 to 1.1. We identified the variance in reproductive success as the most important variable contributing to reduction of this ratio. We used four methods to estimate the genetic based inbreeding effective number of breeders N_bI(gen) and the variance effective populations size N_eV(gen) estimates from the genotype data. Two of these methods - a temporal moment-based (MBT) and a likelihood-based approach (TM3) require at least two samples in time, while the other two were single-sample estimators - the linkage disequilibrium method with bias correction LDNe and the program ONeSAMP. The genetic based estimates were fairly similar across methods and also similar to the demographic estimates excluding those estimates, in which upper confidence interval boundaries were uninformative. For example, LDNe and ONeSAMP estimates ranged from 14–55 and 24–48 individuals, respectively. However, temporal methods suffered from a large variation in confidence intervals and concerns about the prior information. We conclude that the single-sample estimators are an acceptable short-cut to estimate N_bI for species such as geckos and will be of great importance for the monitoring of species in fragmented landscapes.     

Direct Observations of Shifts in the β-Sheet Register of a Protein-Peptide Complex Using Explicit Solvent Simulations

Using explicit solvent molecular dynamics simulations, we were able to obtain direct observations of shifts in the hydrogen-bonding register of an intermolecular β-sheet protein-peptide complex. The β-sheet is formed between the FHA domain of cancer marker protein Ki67 (Ki67FHA) and a peptide fragment of the hNIFK signaling protein. Potential encounter complexes of the Ki67FHA receptor and hNIFK peptide are misregistered states of the β-sheet. Rearrangements of one of these misregistered states to the native state were captured in three independent simulations. All three rearrangements occurred by a common mechanism: an aromatic residue of the peptide (F263) anchors into a transient hydrophobic pocket of the receptor to facilitate the formation of native hydrogen bonds. To our knowledge, these simulations provide the first atomically detailed visualizations of a mechanism by which nature might correct for errors in the alignment of intermolecular β-sheets.     

Cα and Cβ Carbon-13 Chemical Shifts in Proteins From an Empirical Database

We have constructed an extensive database of 13C Cα and Cβ chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical α- helix and β-sheet geometries for Cα, and of ca. 2 ppm for Cβ. The side-chain dihedral angle χ1 has an effect of up to 0.5 ppm on the Cα shift, particularly for amino acids with branched side-chains at Cβ. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different φ,ψ shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and χ1 angle, is 0.96 ppm for both Cα and Cβ. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.     

Chemical and biological consequences ofβ-decay

Summary In Part 1 of this article physical and chemical effects of-decay in labelled molecules were reviewed and their potential importance for breaking predetermined and specific bonds were pointed out. After incorporation of labelled biomolecules in living systems, such as viruses, phages or cells, the radioactive decay of the label alters the biological behaviour of the system, in the extreme case causing loss of the ability to reproduce, the extent of these consequences depending strongly on the type of radioisotope.Now Part 2 includes a brief discussion of biological effects associated with-decay, emphasizing the relative importance of local transmutation and internal radiation effects from the decay of³H,¹⁴C,³²P,³³P,³⁵S and¹²⁵I. Attempt is also made, whenever possible at the present stage of understanding, to correlate biological effects with chemical processes on a molecular level.     

Chemical and biological consequences of β-decay

Summary Radioactive decay in a labelled molecule leads to specific chemical and biological consequences which are due to local transmutation effects such as recoil, electronic excitation, build-up of charge states and change of chemical identity, as well as to internal radiolytic effects. In the present paper these effects are reviewed emphasizing the relation of the chemical alterations on a molecular level to the biological manifestation. Potential importance of this type of research for biomedical applications is pointed out. In part 1 we review the underlying physical and chemical principles and consequences of -decay of³H,¹⁴C,³²P,³³P,³⁵S and¹²⁵I for gaseous and simple condensed organic systems. Part 2 which will appear in the next issue will include the discussion of biological effects associated with -decay.     

Coordinated Processing of 3′ Slipped (CAG)n/(CTG)n Hairpins by DNA Polymerases β and δ Preferentially Induces Repeat Expansions

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3′-slipped hairpin using its 3′-5′ proofreading activity when the hairpin contains no immediate 3′ complementary sequences. However, in the presence of pol β, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol β incorporates several nucleotides to the hairpin 3′-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3′-slipped (CAG)_n/(CTG)_n hairpins by polymerases δ and β on during DNA synthesis induces CAG/CTG repeat expansions.     

Assignment of 13C Chemical Shifts of α-D-Ribonucleoside Sugar Carbons by 1J(CH) Coupling Constants

Abstract

The ¹J(CH) coupling constant of C-1 in nucleosides is increased compared to those of the other carbons of the sugar moiety. Applying this to several D-ribonucleosides the signals C-4′/C-1′of these a-anomers are reversed to those of the 8-counterparts (C-1′/C-4′). This phenomenon and the broadening of the C-3′ signal compared to that of C-2′ establishes the seauence C-4′,1′,2′,3′,5′ (increasing field) for a number of α-D-ribonucleosides.     

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β₂-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.     

Comparison of α and β keratin in reptiles

Summary The different patterns of keratin formation that have evolved in the class Reptilia are all variations of a common process. In Squamata (snakes and lizards), a sequence of layers composed of or keratin is formed periodically, after which the old epidermal generation is shed. In Chelonia (turtles and tortoises), the epidermis of the shell is composed of only keratin, whereas the skin of the neck and leg is composed exclusively of keratin. Molting in toto does not occur and shedding is a continuous process comparable to that in avian and mammalian epidermis. In Crocodilia (crocodiles, caimans, alligators) there is only a single layer of cornified cells, but the composition of the layer varies in different parts of the scale. The hinge regions have many of the morphological characteristics of and keratin whereas the center resembles keratin. The living cells beneath contain accumulations of keratohyalin.There are four ultrastructural characteristics of a keratinized layer: 1) cellular outlines remain distinct, 2) a thickened plasma membrane forms during keratinization, 3) 80 Å filaments embedded in an amorphous matrix can be seen, and 4) PAS-positive material accumulates in extracellular spaces between the desmosomes.The layer exhibits none of these features. Instead the cells more or less (depending on species) coalesce into a compact layer which becomes attenuated in the hinge regions. A 30 Å filament pattern can be seen.The mesos layer of squamates resembles the hinge region of crocodilians, exhibiting a combination of the characteristics of both and keratin.This study constitutes publication No. 464 from the Oregon Regional Primate Research Center, supported in part by NIH Grant No. FR-00163.     

Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.     

Production and characterization of Clostridium perfringens recombinant β toxoid

In this work, we produced and evaluated a vaccine based on a β toxoid of Clostridium perfringens type C produced in Escherichia coli (rBT). The non-toxic rBT was innocuous for mice and induced 14 IU mL(-1) of β antitoxin in rabbits, complying with the European Pharmacopeia and CFR9 - USDA guidelines.     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

Isolation of monotreme T-cell receptor α and β chains

Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the mammalian immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR -chain cDNAs (TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR -chain cDNA (TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.     

Noradrenaline and “Chemical Coding” of Hypothalamic Neurones

FIRING rates of single neurones in the “feeding system”—the perifornical and ventromedial areas of the hypothalamus—are altered by the systemic administration of an anorexigenic agent, such as amphetamine or glucose^1–4. Using the micro-iontophoretic technique which involves releasing chemicals directly on individual neurones, Oomura et al. confirmed that glucose can alter the spontaneous firing rates of some neurones in the hypothalamus of the rat⁵. We wish to report that micro-iontophoretic applications of glucose, amphetamine and noradrenaline to hypothalamic neurones yield a pattern of results not readily reconcilable with the current views of the role of adrenergic substances as “transmitters” in the regulation of hypothalamic feeding function.     

Modulation of the β-Adrenergic Receptor-Coupled Adenylate Cyclase by Chemical Inducers of Differentiation: Effects on β Receptors and the Inhibitory Regulatory Protein Gi

Abstract

Several drugs known to induce differentiation in tumor cells were analyzed for their effects on the β-adrenergic receptor-coupled adenylate cyclase system in two human carcinoma cell lines, HeLa and A431. Each of the drugs was tested alone or in combination with sodium butyrate (NaBu), a known inducer of this signal transduction system. Puromycine amino nucleoside (PMAN) caused the largest increase in β-adrenergic receptors in HeLa cells followed by hexamethylenebisacetamide (HMBA) whereas 5′-azacytidine (5AZC) was ineffective. In addition, PMAN but not the others acted together with NaBu to elevate receptor levels 12-fold over control values. In contrast, HMBA and 5AZC were much more effective on A431 cells, PMAN caused only a slight increase in β receptors and none of the drugs acted in concert with NaBu. The increase in β receptors was usually accompanied by a corresponding increase in isoproterenol-stimulated adenylate cyclase activity. These effects of the drugs appeared to require protein synthesis as they were blocked by cycloheximide. In addition, some of the drugs caused a substantial decrease in basal adenylate cyclase activity. This effect on basal activity was abolished in cells treated with pertussis toxin, which ADP-ribosylates the inhibitory GTP-binding protein, G_i. Both HeLa and A431 cells contained a 41 kDalton substrate for the toxin which corresponds to the α; subunit of G_i. The G_i subunit was ADP-ribosylated by the toxin to a similar extent in membranes from control and drug-treated cells. Thus, the drugs appear to induce quantitative changes in β-adrenergic receptors and qualitative changes in G_i which results in a highly responsive β-adrenergic-stimulated adenylate cyclase.