Genetic evidence that the putative receptor binding domain of apolipoprotein B (residues 3130 to 3630) is not the only region of the protein involved in interaction with the low density lipoprotein receptor

We have searched for sequence differences in the region of the apolipoprotein B (apo B) gene encoding amino acids 3130-3630 in eight individuals with reduced affinity of low density lipoprotein (LDL) for the normal LDL-receptor. All individuals were hypercholesterolaemic and were selected either on the basis of reduced fractional catabolic rate (FCR) of autologous LDL or substantially reduced binding of their LDL to normal LDL-receptors determined by an in vitro cell growth assay using the U937 macrophage-like cell line. Segments of the apo B gene were amplified by the polymerase chain reaction. Using a combination of cloning and sequencing the amplified fragment, together with chemical cleavage mismatch analysis, no sequence differences were identified in this region of the gene. We therefore conclude that variation outside the region of the apo B gene that codes for amino acids 3130-3630 must be responsible for the reduced LDL clearance in these patients.     

The presence and actions of NPY in the canine endocrine pancreas

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

The canine sympathetic neuropeptide galanin: a neurotransmitter in pancreas, a neuromodulator in liver.

Our laboratory has investigated the role of the neuropeptide galanin in the sympathetic neural control of both the canine endocrine pancreas and liver. Galanin mRNA and peptide were found in the neuronal cell bodies of the celiac ganglion, which projects fibers to both organs. Galanin fibers formed dense networks around the islets. Galanin was released from these nerves and the amount released appeared sufficient to markedly inhibit basal insulin secretion. We therefore propose that galanin is a sympathetic neurotransmitter in canine endocrine pancreas. Galanin was also found in hepatic nerves usually co-localized with tyrosine hydroxylase, a sympathetic marker. Further, intraportal administration of the sympathetic neurotoxin, 6-hydroxydopamine, abolished galanin staining in the hepatic parenchyma. We evaluated the role of galanin in mediating the actions of sympathetic nerves to increase hepatic glucose production and decrease hepatic arterial conductance. Local infusion of synthetic galanin had little effect by itself, but it did potentiate the action of norepinephrine to stimulate hepatic glucose production, demonstrating a neuromodulatory action. In contrast, galanin had no effect on hepatic arterial blood flow. We therefore propose that in the liver galanin functions as a neuromodulator of norepinephrine's metabolic action.     

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species

C₄ photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C₄ and non-C₄ populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C₄ lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C₄ phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C₄ physiology away from its region of origin.     

Brood Ball-Mediated Transmission of Microbiome Members in the Dung Beetle,Onthophagus taurus (Coleoptera: Scarabaeidae)

Insects feeding on plant sap, blood, and other nutritionally incomplete diets are typically associated with mutualistic bacteria that supplement missing nutrients. Herbivorous mammal dung contains more than 86% cellulose and lacks amino acids essential for insect development and reproduction. Yet one of the most ecologically necessary and evolutionarily successful groups of beetles, the dung beetles (Scarabaeinae) feeds primarily, or exclusively, on dung. These associations suggest that dung beetles may benefit from mutualistic bacteria that provide nutrients missing from dung. The nesting behaviors of the female parent and the feeding behaviors of the larvae suggest that a microbiome could be vertically transmitted from the parental female to her offspring through the brood ball. Using sterile rearing and a combination of molecular and culture-based techniques, we examine transmission of the microbiome in the bull-headed dung beetle, Onthophagus taurus. Beetles were reared on autoclaved dung and the microbiome was characterized across development. A ~1425 bp region of the 16S rRNA identified Pseudomonadaceae, Enterobacteriaceae, and Comamonadaceae as the most common bacterial families across all life stages and populations, including cultured isolates from the 3^rd instar digestive system. Finer level phylotyping analyses based on lepA and gyrB amplicons of cultured isolates placed the isolates closest to Enterobacter cloacae, Providencia stuartii, Pusillimonas sp., Pedobacter heparinus, and Lysinibacillus sphaericus. Scanning electron micrographs of brood balls constructed from sterile dung reveals secretions and microbes only in the chamber the female prepares for the egg. The use of autoclaved dung for rearing, the presence of microbes in the brood ball and offspring, and identical 16S rRNA sequences in both parent and offspring suggests that the O. taurus female parent transmits specific microbiome members to her offspring through the brood chamber. The transmission of the dung beetle microbiome highlights the maintenance and likely importance of this newly-characterized bacterial community.     

Phylogenomics using low‐depth whole genome sequencing: A case study with the olive tribe

Species trees have traditionally been inferred from a few selected markers, and genome‐wide investigations remain largely restricted to model organisms or small groups of species for which sampling of fresh material is available, leaving out most of the existing and historical species diversity. The genomes of an increasing number of species, including specimens extracted from natural history collections, are being sequenced at low depth. While these data sets are widely used to analyse organelle genomes, the nuclear fraction is generally ignored. Here we evaluate different reference‐based methods to infer phylogenies of large taxonomic groups from such data sets. Using the example of the Oleeae tribe, a worldwide‐distributed group, we build phylogenies based on single nucleotide polymorphisms (SNPs) obtained using two reference genomes (the olive and ash trees). The inferred phylogenies are overall congruent, yet present differences that might reflect the effect of distance to the reference on the amount of missing data. To limit this issue, genome complexity was reduced by using pairs of orthologous coding sequences as the reference, thus allowing us to combine SNPs obtained using two distinct references. Concatenated and coalescence trees based on these combined SNPs suggest events of incomplete lineage sorting and/or hybridization during the diversification of this large phylogenetic group. Our results show that genome‐wide phylogenetic trees can be inferred from low‐depth sequence data sets for eukaryote groups with complex genomes, and histories of reticulate evolution. This opens new avenues for large‐scale phylogenomics and biogeographical analyses covering both the extant and the historical diversity stored in museum collections.     

Evaluating outcomes of restoration ecology projects on limited budgets: assessment of variation in sampling intensity and sampling frequency for four habitat types

While best practices for evaluating restoration ecology projects are emerging rapidly, budget constraints often limit postrestoration monitoring, which emphasizes the need for practical and efficient monitoring strategies. We examined the postrestoration outcome for an ENGO (Nature Conservancy of Canada) project, to assess retroactively how variation in intensity and frequency of sampling would have affected estimates of plant species composition, diversity, and richness over time. The project restored four habitat types (mesic forest, oak woodland, wet meadow, and sand barren) using sculptured seeding of tallgrass prairie and woody species. Species‐level plant cover was monitored annually for 10 years in 168 2 × 2–m quadrats. We performed randomization tests to examine estimates of species diversity and richness as a function of the number of quadrats sampled, and assessed the necessity of annual sampling for describing changes in species composition and successional trajectories. The randomization tests revealed that sampling 10–17 quadrats, depending on habitat type, was sufficient to obtain estimates of species diversity that were at least 95% of values obtained from the whole dataset. Species richness as a function of number of quadrats sampled did not plateau, which suggests that rather than increasing the number of sampling quadrats, richness could be estimated more efficiently using nonquadrat based sampling techniques. Nonmetric multidimensional scaling analysis revealed that plant species composition largely stabilized by 3–5 years postrestoration depending on habitat type. By that time, native, seeded species dominated the restoration, and the benefits of annual sampling for tracking changes in species composition diminished.