A comparative study of three methods for intracellular loading of the calcium indicator aequorin in ferret papillary muscles

We compared the results of loading the bioluminescent Ca++ indicator aequorin by standard microinjection techniques to those obtained with two new chemical approaches to loading that utilize low concentrations of Ca++ chelator; i.e., 1) Immersion and 2) Macroinjection. After loading with the immersion and macroinjection methods, twitch tension returned to pre-load values indicating lack of damage to the muscles. The aequorin signals obtained with all three methods were similar and converted to similar quantitative values for [Ca++]i. Our data suggest that chemical loading (in particular macroinjection) may be preferable to microinjection, particularly in muscles with increased connective tissue content.     

Induction of IL-1 secretion from human monocytes by Fc region subfragments of human IgG1

Peripheral blood-derived human monocytes and the murine P388D1-monocytes-like cell line are induced to secrete IL-1 when stimulated with Fc region but not F(ab) region subfragments obtained from the cleavage of human IgG1 with papain or pepsin. The portion of the Fc region of IgG1 responsible for stimulation of IL-1 secretion appears to be located within the C gamma 3 domain of the molecule. This hypothesis is supported by the observation that the biologically active pepsin-derived pFc' subfragment is located within the C gamma 3 domain and the long-term papain digests containing predominately Fc' are also active. In contrast, short term papain digests containing mostly intact Fc fragments were found to be unable to induce IL-1 secretion.     

Histochemical localization of IGF-I and IGF-II mRNA in the rat between birth and adulthood

We describe the postnatal ontogeny and localization of insulin-like growth factors I and II (IGF-I and -II) in the rat. We have used oligodeoxyribonucleotide probes for in situ hybridization (hybridization histochemistry) and for Northern blotting. IGF-II mRNA is strongly expressed in liver, skeletal muscle, perichondrium, leptomeninges and choroid plexus of the newborn. Demonstrable levels fall dramatically in the liver at 18-20 days postnatally but persist for longer periods in muscle and remain undiminished throughout life in the pia/choroid plexus, indicating that different control mechanisms operate in these tissues. IGF-I mRNA is predominantly found in the liver. Its level in this organ rises well before levels of IGF-II fall. This suggests that distinct factors govern the expression of IGF-I and -II genes.     

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.     

Analysis of fluorescence transients of DCMU-treated leaves of Triticum species to provide estimates of the densities of photosystem II reaction centres

The fluorescence of the chlorophyll associated with photosystem II was studied in seedling and flag leaves of Triticum species. Seedling leaves of the diploid species T. urartu had higher values of t (the normalised area over the fluorescence induction curve of DCMU treated leaves) than those of the other species studied which included hexaploid T. aestivum. However this difference was not evident when leaves were grown in a low light intensity (40 µmol quanta of photosynthetically active radiation m^–2 s^–1). The smaller total number of chlorophyll molecules per photosystem II reaction centre (chl/RCII) in T. urartu (177) as compared with the other species (mean 234) was deduced from the observed differences in t. As a consequence of its lower chl/RCII, despite slightly lower chlorophyll content (mg m^–2), T. urartu had a greater density of reaction centres than the other species (2880 cf 2230 nmol m^–2 of leaf). Consistent with the lower chl/RCII of T. urartu, it had a higher chlorophyll a/b ratio than the other genotypes. Seedling leaves of T. urartu had higher light saturated rates of photosynthesis than those of the other species, when grown at high light, a difference associated with reaction centre density.In flag leaves, when the complications due to variable development and senescence patterns were eliminated, t of the diploid species including T. urartu was lower than that of T. aestivum. The lower apparent chl/RCII of T. urartu arose partly because the molar extinction coefficient of the chlorophyll in the leaves of T. urartu was greater than in T. aestivum. However, the density of PS II reaction centres was slightly lower for the diploid species studied because their chlorophyll contents were lower than the hexaploids.The validity of the method for estimating chl/RCII from fluorescence transients is discussed. The possibility is considered that the difference in apparent chl/RCII of flag and seedling leaves of R. urartu as compared to the other five genotypes is a consequence of its different adaptive response to the spectral quality of the light.     

Chelator-mediated iron efflux from reticulocytes

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.