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).     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

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.     

POPULATION VARIABILITY IN DANTHONIA CAESPITOSA (GRAMINEAE) IN RESPONSES TO INCREASING DENSITY UNDER THREE TEMPERATURE REGIMES

The objective of this research was to compare the responses to increasing density of different natural populations of Danthonia caespitosa, a perennial grass of southern Australia, under different temperature regimes. Seeds were collected from four populations along a 167-km N–S transect beginning north of Deniliquin, N.S.W., and ending near Heathcote, Victoria. Seedlings from each population were planted in loamy sand at three densities and grown under day/night temperature regimes of 15/10, 24/19, and 33/28 C in the Canberra Ceres Phytotron. Results indicate that one natural population (or one temperature regime) can not be used to characterize this species' responses to increasing density. The effects of density and temperature on tillering, plant height, leaf length, and leaf width varied significantly among populations, so that populations achieved comparable shoot weights by different relative responses to density. Variability in biomass among individuals (as indicated by coefficients of variation) increased from low to intermediate density; however, size variability at the highest density was either greater, lesser, or approximately the same as that of plants at the intermediate density, depending upon the population and the temperature regime. The combinations of density and temperature which produced the maximum shoot growth per pot varied markedly among populations; at 24/19 C, populations from frequently disturbed areas of low perennial plant cover had greater shoot weights per pot at the intermediate density, while those populations from more stable grassland sites had their greatest shoot weights at the highest density. Flowering occurred in all populations at the low density in 15/10 C; however, only for the population from the ungrazed grassland was there any flowering in the other temperature regimes or at higher densities. It is concluded that a “response to density stress” could be differentially described for each population at each temperature regime, even though the four study populations represented only 1/10 of the latitudinal range of D. caespitosa.