Nuclear-encoded subunits of human cytochrome c oxidase: SstI restriction fragment length polymorphism

A DNA polymorphism of the nuclear-encoded subunit Va of the human cytochrome c oxidase (COX), a mitochondrial respiratory enzyme, is reported. No polymorphism was detected in genes for the subunits IV and Vb of the same enzyme.     

Seed storage protein variation in Arachis species.

Seventy-two accessions, representing 22 species from sections Arachis, Erectoides, Extranervosae, and Triseminalae of the genus Arachis, were screened for seed storage protein polymorphism. Variation was detected between sections, between genome types, between species, and in some cases between different accessions of the same species or different seeds of the same accession. Arachis duranensis and one accession of A. cardenasii were found to have identical protein patterns. The greatest dissimilarity was found between species of the section Extranervosae and species of the section Triseminalae. Those of section Erectoides showed much similarity with some species of section Arachis. Protein polymorphism was shown to distinguish the two subspecies of A. hypogaea (fastigiata and hypogaea) in 27 of 28 cases. The seed protein profile of A. monticola was a combination of seed protein profiles from the two A. hypogaea subspecies. The relatedness between the various species was calculated and those that had the greatest similarity with A. hypogaea were A. spegazzinii and A. batizocoi.     

Acetate inhibition on growth of recombinant E. coli and expression of fusion protein TGFaα-PE40

Summary Acetate was inhibitory to the growth of early induced E. coli cells and their expression of fusion protein, transforming growth factor--Pseudomonas exotoxin 40 (TGF-PE40), but the inhibitory level was strain dependent For E. coli JM109 (pTAC-TGF57-PE40), 2 g/L of added acetate (3 g/L of total acetate in the medium) decreased TGFa-PE40 production by 38.0%. Acetate was less inhibitory to E. coli RR1, and RR1 was not affected by adding 2 g/L of acetate. However, 5 g/L of added acetate (6.7 g/L of total acetate in the medium) decreased TGF-PE40 production by 21.2%. These results indicate that higher acetate concentration was associated with inhibition of TGF-PE40 expression of E. coli JM109 during late induction.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain.

To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed.     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

Identifying historical and future global change drivers that place species recovery at risk

Ecosystem management in the face of global change requires understanding how co-occurring threats affect species and communities. Such an understanding allows for effective management strategies to be identified and implemented. An important component of this is differentiating between factors that are within (e.g. invasive predators) or outside (e.g. drought, large wildfires) of a local manager's control. In the global biodiversity hotspot of south-western Australia, small- and medium-sized mammal species are severely affected by anthropogenic threats and environmental disturbances, including invasive predators, fire, and declining rainfall. However, the relative importance of different drivers has not been quantified. We used data from a long-term monitoring program to fit Bayesian state-space models that estimated spatial and temporal changes in the relative abundance of four threatened mammal species: the woylie (Bettongia penicillata), chuditch (Dasyurus geoffroii), koomal (Trichosurus vulpecula) and quenda (Isoodon fusciventor). We then use Bayesian structural equation modelling to identify the direct and indirect drivers of population changes, and scenario analysis to forecast population responses to future environmental change. We found that habitat loss or conversion and reduced primary productivity (caused by rainfall declines) had greater effects on species' spatial and temporal population change than the range of fire and invasive predator (the red fox Vulpes vulpes) management actions observed in the study area. Scenario analysis revealed that a greater extent of severe fire and further rainfall declines predicted under climate change, operating in concert are likely to further reduce the abundance of these species, but may be mitigated partially by invasive predator control. Considering both historical and future drivers of population change is necessary to identify the factors that risk species recovery. Given that both anthropogenic pressures and environmental disturbances can undermine conservation efforts, managers must consider how the relative benefit of conservation actions will be shaped by ongoing global change.     

The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures – apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.