The γ phosphorylase kinase gene,Phkg, maps to mouse Chromosome 5 near Gus

Phosphorylase kinase is a multimeric regulatory enzyme in the glycogenolytic pathway. Interest in various types of phosphorylase kinase enzyme deficiency has focused attention on cloning and mapping the enzyme subunits. We report the mapping of the catalytic subunit gene, Phkg, to mouse Chromosome (Chr) 5 near -glucuronidase (Gus), between alpha fetoprotein (Afp) and erythropoietin (Epo). In addition, PCR-based polymorphism assays have been developed for the human (EPO) and mouse erythropoietin genes, and a unique recombinant inbred strain distribution pattern has been defined for Epo, a distal anchor marker on mouse Chr 5.     

Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.     

Plasma Digoxin Concentrations in Patients with Atrial Fibrillation

Plasma digoxin concentrations were measured by radioimmunoassay in 116 patients with atrial fibrillation on long-term oral treatment with the drug, and in 23 patients with digoxin toxicity. The mean concentrations were 1·4 ng./ml. and 3·1 ng./ml., respectively. Though an overlap occurred between the therapeutic and toxic ranges, toxicity is unlikely to occur below a level of 2 ng./ml. Plasma concentration showed a poor correlation with resting heart rate during atrial fibrillation. In patients with good renal function, however, a significant correlation was found between oral dose and plasma concentration. No evidence was obtained for increased sensitivity to therapeutic concentrations of the drug in elderly subjects, but the doses required to achieve these concentrations tended to be less than in younger patients.     

L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle

Background

In dystrophic mdx skeletal muscle, aberrant Ca²⁺ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca²⁺ currents. In regenerating mdx muscle, ‘revertant’ fibres restore dystrophin expression. Their functionality involving DHPR-Ca²⁺-channels is elusive.

Methods and Results

We developed a novel ‘in-situ’ confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified ‘mdx-like’. Some mdx fibres, however, had strong ‘wt-like’ dystrophin signals and were identified as ‘revertants’. Split mdx fibres were mostly ‘mdx-like’ and are not generally ‘revertants’. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in ‘revertants’. Using the two-micro-electrode-voltage clamp technique, Ca²⁺-current amplitudes (i_max) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely ‘revertants’, with amplitudes similar to wt or MinD fibres. Ca²⁺ current activation curves were similar in ‘wt-like’ and ‘mdx-like’ aged mdx fibres and are not the cause for the differences in current amplitudes. i_max amplitudes were fully restored in MinD fibres.

Conclusions

We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and ‘revertant’ mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated ‘revertant’ fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.     

Ultrastructural organization of amyloid fibrils by atomic force microscopy

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.     

The use of isotope tracers for identifying populations of migratory birds

 To determine whether stable isotopes can be used for identifying the geographic origins of migratory bird populations, we examined the isotopic composition of hydrogen (deuterium, δD), carbon (δ¹³C), and strontium (δ⁸⁷Sr) in tissues of a migratory passerine, the black-throated blue warbler (Dendroica caerulescens), throughout its breeding range in eastern North America. δD and δ¹³C values in feathers, which are grown in the breeding area, varied systematically along a latitudinal gradient, being highest in samples from the southern end of the species’ breeding range in Georgia and lowest in southern Canada. In addition, δD decreased from east to west across the northern part of the breeding range, from New Brunswick to Michigan. δ⁸⁷Sr ratios were highest in the Appalachian Mountains, and decreased towards the west. These patterns are consistent with geographical variation in the isotopic composition of the natural environment, i.e., with that of precipitation, plants, and soils for δD, δ¹³C, and δ⁸⁷Sr, respectively. Preliminary analyses of the δD and δ¹³C composition of feathers collected from warblers in their Caribbean winter grounds indicate that these individuals were mostly from northern breeding populations. Furthermore, variances in isotope ratios in samples from local areas in winter tended to be larger than those in summer, suggesting that individuals from different breeding localities may mix in winter habitats. These isotope markers, therefore, have the potential for locating the breeding origins of migratory species on their winter areas, for quantifying the degree of mixing of breeding populations on migratory and wintering sites, and for documenting other aspects of the population structure migratory animals – information needed for studies of year-round ecology of these species as well as for their conservation. Combining information from several stable isotopes will help to increase the resolution for determining the geographic origins of individuals in such highly vagile populations. Received: 24 April 1995 / Accepted: 2 June 1996     

Herbivore responses to nutrient enrichment and landscape heterogeneity in a mangrove ecosystem

Complex gradients in forest structure across the landscape of offshore mangrove islands in Belize are associated with nutrient deficiency and flooding. While nutrient availability can affect many ecological processes, here we investigate how N and P enrichment interact with forest structure in three distinct zones (fringe, transition, dwarf) to alter patterns of herbivory as a function of folivory, loss of yield, and tissue mining. The effects of nutrient addition and zone varied by functional feeding group or specific herbivore. Folivory ranged from 0 to 0.4% leaf area damaged per month, but rates did not vary by either nutrient enrichment or zone. Leaf lifetime damage ranged from 3 to 10% of the total leaf area and was caused primarily by the omnivorous tree crab Aratus pisonii. We detected two distinct spatial scales of response by A. pisonii that were unrelated to nutrient treatment, i.e., most feeding damage occurred in the fringe zone and crabs fed primarily on the oldest leaves in the canopy. Loss of yield caused by the bud moth Ecdytolopha sp. varied by zone but not by nutrient treatment. A periderm-mining Marmara sp. responded positively to nutrient enrichment and closely mirrored the growth response by Rhizophora mangle across the tree height gradient. In contrast, a leaf-mining Marmara sp. was controlled by parasitoids and predators that killed >89% of its larvae. Thus, nutrient availability altered patterns of herbivory of some but not all mangrove herbivores. These findings support the hypothesis that landscape heterogeneity of the biotic and abiotic environment has species-specific effects on community structure and trophic interactions. Predicting how herbivores respond to nutrient over-enrichment in mangrove ecosystems also requires an assessment of habitat heterogeneity coupled with feeding strategies and species-specific behavior measured on multiple scales of response.     

Carbon isotopes reveal soil organic matter dynamics following arid land shrub expansion

 Over the past century, overgrazing and drought in New Mexico’s Jornada Basin has promoted the replacement of native black grama (Bouteloua eriopoda Torr.) grass communities by shrubs, primarily mesquite (Prosopis glandulosa Torr.). We investigated the effects of shrub expansion on the distribution, origin, turnover, and quality of light (LFC) and heavy (HFC) soil organic matter (SOM) fractions using δ¹³C natural abundance to partition SOM into C₄ (grass) and C₃ (shrub) sources. Soil organic matter beneath grasses and mesquite was isotopically distinct from associated plant litter, providing evidence of both recent shrub expansion and Holocene plant community changes. Our δ¹³C analyses indicated that SOM derived from mesquite was greatest beneath shrub canopies, but extended at least 3 m beyond canopy margins, similar to the distribution of fine roots. Specific ¹⁴C activities of LFC indicated that root litter is an important source of SOM at depth. Comparison of turnover rates for surface LFC pools in grass (7 or 40 years) and mesquite (11 or 28 years) soils and for HFC pools by soil depth (∼150–280 years), suggest that mesquite may enhance soil C storage relative to grasses. We conclude that the replacement of semiarid grasslands by woody shrubs will effect changes in root biomass, litter production, and SOM cycling that influence nutrient availability and long-term soil C sequestration at the ecosystem level. Received: 17 May 1996 / Accepted: 12 November 1996     

A study of tolerance of ingested tannin in Schistocerca gregaria

A study has been made on the effects of ingestion of tannic acid on growth and development of Schistocerca gregaria. No deleterious effects were found on digestion or utilisation of food, even when food protein levels were very low. At high concentrations consumption rates were relatively low over the first day, but this effect was not sustained. The lack of ‘antidigestive’ effects is shown to be due partly to the hydrolysis of tannic acid to gallic acid and glucose, and partly to the adsorptive properties of the peritrophic membrane. Insects reared on food with high levels of tannic acid took a longer time to reach sexual maturity than did the controls, although fecundity was not affected thereafter.