Differential ligand binding by two subunits of the rat liver asialoglycoprotein receptor

The rat liver asialoglycoprotein receptor consists of two typesof subunits, a predominant polypeptide designated rat hepaticlectin 1 (RHL-1) and a minor polypeptide, RHL-2/3, that comesin two differentially glycosylated forms. The exact stoichiometryand arrangement of the subunits in the RHL oligomer are notknown. The carbohydrate-recognition domain of RHL-2/ has beenprepared by limited proteolysis of the liver receptor so thatits properties can be compared with those of the correspondingdomain of RHL-1 previously produced in a bacterial expressionsystem. Binding studies indicate that while RHL-1 binds N-acetylgalactosaminewith approximately 60-fold higher affinity than it binds galactose,RHL-2/ has only 2-fold selectivity for N-acetylgalactosamine.In general, the pattern of monosaccharide-binding specificityfor RHL-2/ is similar to RHL-1, but the discrimination of varioussugars relative to galactose is reduced substantially. Limitedproteolysis and crosslinking studies demonstrate that RHL- 2/is easily removed from the RHL oligomer in detergent solutionand that RHL-1 remains at least trimeric following removal ofRHL-2/. These studies suggest that RHL-1 forms a ligand-bindingcore while RHL-2/ acts more as an accessory subunit contributingto selective binding of certain oligosaccharide structures. asialoglycoprotein receptor binding carbohydrate recognition lectin proteolysis     

In vivo binding of 2,3,6,2′,3′,6′-hexachlorobiphenyl and 2,4,5,2′,4′,5′-hexachlorobiphenyl to mouse liver macromolecules

The role of metabolic activation in the binding of polychlorinated biphenyls (PCBs) to cellular macromolecules was investigated in vivo by comparing the relative binding of 2,4,5,2′,4′,5′-[U-¹⁴C]hexachlorobiphenyl (2,4,5), a slowly metabolized PCB, with that of 2,3,6,2′,3′,6′-[U-¹⁴C]hexachlorobiphenyl (2,3,6), a rapidly metabolized PCB, and the appropriate controls. Each hexachlorobiphenyl was administered to mice, orally for 5 days (7.28 mg/kg/day). Following the dosing schedule, animals were killed at 1, 5 and 8 days. The concentration of each PCB was determined in liver, muscle and kidney and in purified macromolecules isolated from those tissues. The concentration of 2,4,5 was consistently higher than the concentration of 2,3,6 in all tissues studied. However, the amount of 2,3,6 bound to the purified macromolecules was consistently at least one order of magnitude greater than that of 2,4,5. The greatest binding was observed in RNA followed by protein and DNA, respectively. The purity of the macromolecules and the presence of PCB-derived radioactivity at the monomer level were confirmed. This is the first report of ¹⁴C-labeled PCB being bound to purified RNA, DNA, and proteins isolated from the tissues of animals treated in vivo. The binding is thought to be covalent and to be the result of metabolic activation.     

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.     

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.     

A male poecillid's sexually dimorphic body plan, behavior, and nervous system

Here we review the literature of a male poecillid's sexually dimorphic body plan, behavior, and nervous system, including work dating from the mid 1800s to the mid 1990s as well as work in press or in preparation for publication. Rosa-Molinar described the remodeling of the sexually dimorphic anal fin appendicular support, confirmed earlier claims about the development of the male and female secondary sex characteristics in the Western Mosquitofish, Gambusia affinis and provided for the first time direct embryonic evidence suggesting that remodeling of the sexually dimorphic anal fin appendicular support is biphasic. The first process begins in embryos and proceeds similarly in immature males and females; the second process occurs only in males and results in the anterior transposition of the anal fin and its appendicular support to the level of vertebra 11 [Rosa-Molinar E, Hendricks SE, Rodriguez-Sierra JF, Fritzsch B. 1994. Development of the anal fin appendicular support in the western mosquitofish, Gambusia affinis (Baird and Girard, 1854): a reinvestigation and reinterpretation. Acta Anat 151:20-35.] and the formation of a gonopodium used for internal fertilization. Studies using high-speed video cameras confirmed and extended Peden's and others' observations of copulatory behavior. The cameras showed that circumduction is a complex movement combining in a very fast sequence abduction, extension and pronation, S-start-type fast-start (defined as torque-thrust), and adduction movements. Recent work on the nervous system demonstrated dye-coupling between motor neurons and interneurons via gap junctions, suggesting an attractive substrate for the rapid motions involved in poecillid copulatory reflexes.     

Regional-Scale Drivers of Forest Structure and Function in Northwestern Amazonia

Field studies in Amazonia have found a relationship at continental scales between soil fertility and broad trends in forest structure and function. Little is known at regional scales, however, about how discrete patterns in forest structure or functional attributes map onto underlying edaphic or geological patterns. We collected airborne LiDAR (Light Detection and Ranging) data and VSWIR (Visible to Shortwave Infrared) imaging spectroscopy measurements over 600 km² of northwestern Amazonian lowland forests. We also established 83 inventories of plant species composition and soil properties, distributed between two widespread geological formations. Using these data, we mapped forest structure and canopy reflectance, and compared them to patterns in plant species composition, soils, and underlying geology. We found that variations in soils and species composition explained up to 70% of variation in canopy height, and corresponded to profound changes in forest vertical profiles. We further found that soils and plant species composition explained more than 90% of the variation in canopy reflectance as measured by imaging spectroscopy, indicating edaphic and compositional control of canopy chemical properties. We last found that soils explained between 30% and 70% of the variation in gap frequency in these forests, depending on the height threshold used to define gaps. Our findings indicate that a relatively small number of edaphic and compositional variables, corresponding to underlying geology, may be responsible for variations in canopy structure and chemistry over large expanses of Amazonian forest.