Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

The effect of heat shock on primary cultures of brain capillary endothelium: inhibition of assembly of zonulae occludentes and the synthesis of heat-shock proteins

Subjecting primary cultures of bovine brain microvessel endothelial cells to thermal stress (heat shock) results in: (1) an inhibition of further tight junction assembly, (2) the disappearance and/or disassembly of tight junctions, (3) a 30-fold increase in the number of plasmic fracture (PF)-face intramembrane particles, and (4) the new and/or enhanced synthesis of at least three heat-shock polypeptides (HSPs) with molecular masses of approximately 100,000, 90,000 and 70,000. Endothelial cells which are heat-shocked and allowed to recover at 37 degrees C exhibit, within the first 2 h, a marked depression in the synthesis of HSPs and the new and/or enhanced synthesis of a 47,000 dalton "recovery" polypeptide. In later periods of recovery (2-4 h), the synthesis of this polypeptide is even more pronounced and is accompanied by the new and/or enhanced synthesis of a polypeptide(s) with a molecular mass of 35 to 37,000. The appearance of these "recovery protein(s)" in the endothelial cells is concomitant with a decrease in the number of PF-face intramembrane particles and the resumption of tight junction assembly. Results of this study suggest that some of the HSPs synthesized by thermally-stressed cultures of brain endothelial cells may activate or be directly involved in a mechanism(s) to ensure survival of these cells by decreasing membrane fluidity and stabilizing the plasma membrane of these cells. Moreover, our results also suggest that the recovery of these cells from the stress of heat shock is accompanied by the synthesis of "recovery" proteins which, in some manner, may be directly involved in, or necessary for, rapidly reversing the membrane-stabilizing effect of heat shock by promoting membrane fluidity and the apparent amplified synthesis and assembly and/or reassembly of tight junctions.     

Influence of protein intake and training status on nitrogen balance and lean body mass

The present study examined the effects of training status (endurance exercise or body building) on nitrogen balance, body composition, and urea excretion during periods of habitual and altered protein intakes. Experiments were performed on six elite bodybuilders, six elite endurance athletes, and six sedentary controls during a 10-day period of normal protein intake followed by a 10-day period of altered protein intake. The nitrogen balance data revealed that bodybuilders required 1.12 times and endurance athletes required 1.67 times more daily protein than sedentary controls. Lean body mass (density) was maintained in bodybuilders consuming 1.05 g protein.kg-1.day-1. Endurance athletes excreted more total daily urea than either bodybuilders or controls. We conclude that bodybuilders during habitual training require a daily protein intake only slightly greater than that for sedentary individuals in the maintenance of lean body mass and that endurance athletes require daily protein intakes greater than either bodybuilders or sedentary individuals to meet the needs of protein catabolism during exercise.     

Structure and Evolution of the Adh Genes of Drosophila Mojavensis

The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.     

Receptor binding and Nb2 cell mitogenic activities of glycosylated vs. unglycosylated porcine prolactin

Purified fractions of glycosylated (pGPrl) and unglycosylated (pUGPrl) porcine prolactin were prepared by affinity chromatography on Concanavalin A-Sepharose. The relative binding activities of these two forms of prolactin for receptors from porcine mammary, adrenal cortex and rabbit mammary, as well as their Nb2 cell mitogenic activity were determined. In both the porcine mammary and adrenal cortex receptor binding assays pGPrl had a 2-3 fold lower activity than pUGPrl. In the rabbit mammary binding assay pGPrl had a about a 5 fold lower activity than pUGPrl. Similarly, pGPrl had only about 20% of the activity of pUGPrl in the Nb2 cell proliferation assay.     

Gas exchange and SO2 fumigation studies with irrigated and unirrigated field grown Diplacus aurantiacus and Heteromeles arbutifolia

Summary Experiments were performed on an evergreen (Heteromeles arbutifolia) and a drought deciduous shrub (Diplacus aurantiacus) to determine, 1) whether approaches for evaluating SO₂ absorption by leaves in laboratory studies could be extended to field studies, 2) the effects of irrigation on metabolism and SO₂ responses of the study species during a season when water was limiting, 3) to interpret SO₂ responses on the basis of SO₂ flux rates. Laboratory-developed approaches for evaluating SO₂ absorption by leaves were found to be suitable for use with field plants, despite field plants having lower gas exchange rates. Supplementing water during times of deficit did not override all the biological and environmental factors that limited photosynthesis (A). Irrigation increased leaf longevity of D. aurantiacus, and stomatal conductance to water vapour (g); g was also shown to increase with H. arbutifolia on irrigation. Irrigation profoundly influenced plant response to SO₂. Unwatered D. aurantiacus had only a small g and therefore a reduced capacity to absorb SO₂ and respond to SO₂; which resulted in apparent SO₂ avoidance. Water availability and SO₂ both affect g and therefore, SO₂ flux rates into the mesophyll. Different ambient SO₂ concentrations of 8.3 and 26.2 mol m^-3 (0.2 and 0.6 ppm) were both found to result in similar SO₂ flux rates into the leaf, due to variations in g in response to water availability. Changes in g did not always result in changes in A, implying that carbon fixation may be little affected by some SO₂ exposures, although still potentially affecting such processes as maintenance of leaf water potential, transpirational cooling and nutrient uptake.Abbreviations SO₂ sulphur dioxide - A net photosynthesis - E transpiration - g stomatal conductance to water vapour - W Water vapour mole fraction difference between the leaf and air - WUE water use efficiency (mol CO₂ uptake per mol H₂O transpired)     

Long-term analysis of organelle translocation in isolated axoplasm of Myxicola infundibulum

Moving intra-axonal organelles demonstrate frequent variations in speed when viewed over several seconds. To evaluate these and other motion variations, a long-term analysis of organelle motion in isolated axoplasm of Myxicola infundibulum was carried out using differential interference contrast optics and analog and digital image enhancement techniques. Motion characteristics of individual organelles were analyzed for periods of up to 58 minutes. Three principle observations on organelle motion were made: 1) Classes of organelles of the same size demonstrated a 5- to 25-fold variation of speed, with the slowest speeds occurring most frequently; 2) organelle speeds over individual translocations (motion without stopping) are inversely proportional to their size, but the speeds calculated for the long-term analysis of organelle motion (total distance travelled/total observation time, including pauses) did not reflect this observation; and 3) organelles displayed variable trip lengths, durations, mean speeds, and pause durations, and the relationships between these variations showed no repetitive patterns. In contrast to reported observations of uniform velocities of organelles moving on isolated microtubule preparations, these observations suggest that a variety of factors must play a role in organelle translocation in Myxicola axoplasm.     

Global phylogeography of the avian malaria pathogen Plasmodium relictum based on MSP1 allelic diversity

Knowing the genetic variation that occurs in pathogen populations and how it is distributed across geographical areas is essential to understand parasite epidemiology, local patterns of virulence, and evolution of host‐resistance. In addition, it is important to identify populations of pathogens that are evolutionarily independent and thus ‘free’ to adapt to hosts and environments. Here, we investigated genetic variation in the globally distributed, highly invasive avian malaria parasite Plasmodium relictum, which has several distinctive mitochondrial haplotyps (cyt b lineages, SGS1, GRW11 and GRW4). The phylogeography of P. relictum was accessed using the highly variable nuclear gene merozoite surface protein 1 (MSP1), a gene linked to the invasion biology of the parasite. We show that the lineage GRW4 is evolutionarily independent of GRW11 and SGS1 whereas GRW11 and SGS1 share MSP1 alleles and thus suggesting the presence of two distinct species (GRW4 versus SGS1 and GRW11). Further, there were significant differences in the global distribution of MSP1 alleles with differences between GRW4 alleles in the New and the Old World. For SGS1, a lineage formerly believed to have both tropical and temperate transmission, there were clear differences in MSP1 alleles transmitted in tropical Africa compared to the temperate regions of Europe and Asia. Further, we highlight the occurrence of multiple MSP1 alleles in GRW4 isolates from the Hawaiian Islands, where the parasite has contributed to declines and extinctions of endemic forest birds since it was introduced. This study stresses the importance of multiple independent loci for understanding patterns of transmission and evolutionary independence across avian malaria parasites.