Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Plasma clearance of radiolabelled IGF-1 in the late gestation ovine fetus

We investigated the distribution of radiolabelled IGF-1 in the late gestation ovine fetus by exclusion gel chromatography following intravenous injection of 125I rh (recombinant human) met-IGF-1 into the chronically instrumented fetal lamb (120-130 days, n = 7). One minute after injection of 125I rh met-IGF-1 into the fetal femoral vein, 20.9 +/- 3.1% of the counts circulated in the 150K binding protein region, 55.0 +/- 3.7% in the 50K binding protein region and 18.7 +/- 0.6% in the free or 7K region. The chromatographic profiles obtained in the fetus were in general similar to those previously seen in the adult sheep. After an initial equilibration phase the half life of IGF-1 associated with the 150K binding fractions were 412.1 +/- 103.6 min. Two phases of clearance were observed for IGF-1 in association with the 50K binding fractions, an initial phase with a half life of 30.6 +/- 4.5 min followed by a second phase with a half life of 202.3 +/- 10.3 min. The 7K or 'free' form of IGF-1 had an initial half life of 12.6 +/- 5.1 min. Chromatography of samples of fetal tracheal fluid, fetal urine, amniotic fluid, maternal uterine venous plasma and maternal systemic plasma showed no movement of intact IGF-1 out of the fetal circulation into the fetal fluids or into the maternal circulation. However, when simultaneous samples were obtained from the fetal femoral artery and umbilical vein, higher radioactivity was consistently observed in the fetal femoral artery raising the possibility of placental uptake of IGF-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogold Localisation of the Intermediate Chain within the Protein Complex of Cytoplasmic Dynein

It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain.     

Development of a non-extracted ''two-site'' immunoradiometric assay for corticotropin utilizing extreme amino- and carboxy-terminally directed antibodies.

The development of a 'two-site' immunoradiometric assay (i.r.m.a.) for the direct estimation of human corticotropin-(1-39)-peptide in plasma is described. The assay is based on the simultaneous addition of 125I-labelled sheep anti-(N-terminal corticotropin) IgG (immunoglobulin G) antibodies and rabbit anti-(C-terminal corticotropin) antiserum to standards and unknowns (0.5 ml) followed by 18h incubation. The use of solid-phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of corticotropin-bound from free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) antiserum, which precipitates bound labelled antibody by complex-formation with rabbit anti-corticotropin antibodies, which are also hormone-bound. Several 125I-labelled sheep anti-(N-terminal corticotropin) IgG preparations were assessed in the i.r.m.a. Although each was derived from antisera raised to a thyroglobulin conjugate of synthetic corticotropin-(1-24)-peptide (Synacthen), purification of immunoglobulins before iodination by selective immunoadsorption resulted in preparations with distinct specificities which demonstrated marked differences in binding to intact human corticotropin-(1-39)-peptide. These preparations are compared in combination with two rabbit anti-(C-terminal corticotropin) antisera. A 'two-site' assay based on the use of 125I-labelled sheep anti-[ corticotropin-(2-16)-peptide] IgG and rabbit anti-[corticotropin-(34-39)-peptide] antiserum was optimized, since steric inhibition of antibody binding was avoided with this combination and because the measurement of only intact human corticotropin-(1-39)-peptide and not fragments was assured by the use of terminal antibodies. This i.r.m.a. is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 4% over the range 30-2200 pg/ml) and high sensitivity [8 pg of corticotropin/ml (95% confidence interval 3.7-12.0) (4 pg minimal detectable mass) can be detected directly in plasma].     

A liquid-phase two-site immunoradiometric assay for human prolactin.

The development of a 'two-site' immunoradiometric assay for human prolactin (hPrl) is described. The assay is based on the addition of radio-iodinated sheep anti-hPrl immunoglobulin G (IgG) and rabbit anti-hPrl serum to standards and unknowns followed by 3 h incubation. The use of solid phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of hPrl-bound and free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) serum which precipitates bound labelled antibody by complex formation with rabbit anti-hPrl antibodies which are also hPrl-bound. Varying the order of addition of specific antibodies had a pronounced effect on the 'operating range' and sensitivity of resultant assays. This was attributed to competition between labelled and unlabelled antibodies for binding sites on the hPrl molecule. The immunoradiometric assay employing 'simultaneous addition' of specific antibodies was compared to a 'simultaneous addition' hPrl radioimmunoassay developed using the same sheep antiserum as that used to prepare the radioiodinated sheep anti-hPrl IgG. This immunoradiometric assay is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 10% over the range 8-10000 mU/l), and high sensitivity (2.6 mU/l, 13 pg). In contrast, the hPrl radioimmunoassay required an incubation of 18 h, demonstrated a much reduced 'operating range' (the precision of dose estimates was less than 10% only over the range 25-1500 mU/l) and reduced sensitivity (9.8 mU/l, 49 pg).     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

A preliminary study suggests that nicotine and prefrontal dopamine affect cortico‐striatal areas in smokers with performance feedback

Nicotine and tonic dopamine (DA) levels [as inferred by catechol‐O‐methyl tranferase (COMT) Val158Met genotype] interact to affect prefrontal processing. Prefrontal cortical areas are involved in response to performance feedback, which is impaired in smokers. We investigated whether there is a nicotine × COMT genotype interaction in brain circuitry during performance feedback of a reward task. We scanned 23 healthy smokers (10 Val/Val homozygotes, 13 Met allele carriers) during two fMRI sessions while subjects were wearing a nicotine or placebo patch. A significant nicotine × COMT genotype interaction for BOLD signal during performance feedback in cortico‐striatal areas was seen. Activation in these areas during the nicotine patch condition was greater in Val/Val homozygotes and reduced in Met allele carriers. During negative performance feedback, the change in activation in error detection areas such as anterior cingulate cortex (ACC)/superior frontal gyrus on nicotine compared to placebo was greater in Val/Val homozygotes compared to Met allele carriers. With transdermal nicotine administration, Val/Val homozygotes showed greater activation with performance feedback in the dorsal striatum, area associated with habitual responding. In response to negative feedback, Val/Val homozygotes had greater activation in error detection areas, including the ACC, suggesting increased sensitivity to loss with nicotine exposure. Although these results are preliminary due to small sample size, they suggest a possible neurobiological mechanism underlying the clinical observation that Val/Val homozygotes, presumably with elevated COMT activity compared to Met allele carriers and therefore reduced prefrontal DA levels, have poorer outcomes with nicotine replacement therapy .     

The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host

Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole‐like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild‐type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.