Ability ofVibrio vulnificus to obtain iron from transferrin and other iron-binding proteins

The ability of virulent and avirulent strains ofVibrio vulnificus to overcome iron limitations by using iron bound to iron-binding proteins was examined. While no strains were able to obtain iron from lactoferrin or ferritin when these proteins were not fully saturated with iron, growth was enhanced by the iron-saturated form of these proteins. None of the strains was able to scavange iron from 30% saturated transferrin, but there were strain differences in the ability to obtain iron from the saturated form. The virulent strains were able to compete more efficiently with transferrin when it was fully saturated with iron than were the avirulent strains.     

An improved method for mRNA isolation and characterization of in vitro translation products by Western blotting

A method is described for isolation of messenger RNA (mRNA) from a rather intractable tissue source, calf stomach. The use of additional RNase inhibitors, vanadyl ribonucleoside complexes and proteinase K, which are used in conjunction with the guanidine thiocyanate/CsCl ultracentrifugation procedure traditionally employed for isolation of mRNA, is described. These modifications make the procedure universally applicable to a wide variety of tissues and cell types. The validity of the procedure is demonstrated by isolation of biologically active full-length preprochymosin mRNA. The integrity of the mRNA is measured by in vitro translation, Northern blot analysis, Southern blot analysis of preprochymosin cDNA using synthetic oligodeoxynucleotide probes and immunospecific identification of in vitro translation products using a modification of the Western blot which is described in this report.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.     

The production of floral oils byMonttea (Scrophulariaceae) and the function of tarsal pads inCentris bees

Plant species that secrete oil as their primary floral reward are rare and sporadically found in the angiosperms. We report here thatMonttea, a genus previously unsuspected of being an oil-plant, produces lipids from trichome elaiophores on the inside of the lower (anterior) lip. The discovery of the production of oils by species of this S. American genus explains the occurrence of unusual dual-function collecting structures in ArgentineCentris (Hymenoptera: Anthophoridae) and explains the presence of oil-collecting bees in regions where oil-secreting flowers were previously thought to be absent. The behavior of these centridine pollinators onMonttea flowers parallels that of oil-collecting bees onDiascia (Scrophulariaceae) in S. Africa.     

A refined linkage map for DNA markers around the pericentromeric region of chromosome 10

A refined genetic linkage map for the pericentromeric region of human chromosome 10 has been constructed from data on 12 distinct polymorphic DNA loci as well as the locus for multiple endocrine neoplasia type 2A (MEN 2A), a dominantly inherited cancer syndrome. The map extends from D10S24 (at 10p13-p12.2) to D10S3 (at 10q21-q23) and is about 70 cM long. Overall, higher female than male recombination frequencies were observed for this region, with the most remarkable female excess in the immediate vicinity of the centromere, as previously reported. Most of the DNA markers in this map are highly informative for linkage and the majority of the interlocus intervals are no more than 6 cM apart. Thus this map should provide a fine framework for future efforts in more detailed mapping studies around the centromeric area. A set of ordered cross-overs identified in this work is a valuable resource for rapidly and accurately localizing new DNA clones isolated from the pericentromeric region.     

Immunoblot analysis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in corpora lutea of cyclic and late-pregnant sheep

The specific contents of cytochrome P-450scc and adrenodoxin in corpora lutea of late pregnant sheep were, respectively, 1/5 and 1/8 that of corpora lutea of the oestrous cycle, suggesting lower steroidogenic enzyme capacity in the former. The contents of Complex V proteins were also lower in the corpora lutea of late pregnancy. It was observed in the immunoblots of both Complex V and cytochrome P-450scc that immunoreactive bands of molecular weights lower than the native proteins were present in the samples from corpora lutea of late pregnancy, indicative of degradation of the native enzymes. It is concluded that corpora lutea of sheep during late pregnancy have a much lower enzyme capacity for steroidogenesis than do those of the oestrous cycle (mid-luteal phase) due to a reduction in the content of cytochrome P-450scc and adrenodoxin. The reduction in the levels of steroidogenic enzyme proteins appears to be unspecific and probably reflects an overall demise in mitochondrial functions.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

Characterization of the stimulatory action of insulin on insulin-like growth factor II binding to rat adipose cells. Differences in the mechanism of insulin action on insulin-like growth factor II receptors and glucose transporters

Insulin is known to increase the number of cell surface insulin-like growth factor II (IGF-II) receptors in isolated rat adipose cells through a subcellular redistribution mechanism similar to that for the glucose transporter. The effects of insulin on these two processes, therefore, have now been directly compared in the same cell preparations. 1) Insulin increases the steady state number of cell surface IGF-II receptors by 7-13-fold without affecting receptor affinity; however, insulin stimulates glucose transport activity by 25-40-fold. 2) The insulin concentration required for half-maximal stimulation of cell surface IGF-II receptor number is approximately 30% lower than that for the stimulation of glucose transport activity. 3) The half-time for the achievement of insulin's maximal effect at 37 degrees C is much shorter for IGF-II receptor number (approximately 0.8 min) than for glucose transport activity (approximately 2.6 min). 4) Reversal of insulin's action at 37 degrees C occurs more rapidly for cell surface IGF-II receptors (t1/2 congruent to 2.9 min) than for glucose transport activity (t1/2 congruent to 4.9 min). 5) When the relative subcellular distribution of IGF-II receptors is examined in basal cells, less than 10% of the receptors are localized to the plasma membrane fraction indicating that most of the receptors, like glucose transporters, are localized to an intracellular compartment. However, in response to insulin, the number of plasma membrane IGF-II receptors increases only approximately 1.4-fold while the number of glucose transporters increases approximately 4.5-fold. Thus, while the stimulatory actions of insulin on cell surface IGF-II receptors and glucose transport activity are qualitatively similar, marked quantitative differences suggest that the subcellular cycling of these two integral membrane proteins occurs by distinct processes.