Low-frequency pulmonary impedance in rabbits and its response to inhaled methacholine.

We assessed pulmonary mechanics in six open-chest rabbits (3 young and 3 adult) by the forced oscillation technique between 0.16 and 10.64 Hz. Under control conditions, pulmonary resistance (RL) decreased markedly between 0.16 and 4 Hz, after which it became reasonably constant. Measurements of alveolar pressure from two alveolar capsules in each rabbit showed that the large decrease of RL with increasing frequency below 4 Hz was due to lung tissue rheology and that tissue resistance was close to zero above 4 Hz. Estimates of resistance and elastance, also obtained by fitting tidal ventilation data at 1 Hz to the equation of the linear single-compartment model, gave values for RL motion that were slightly higher than those obtained by forced oscillations at the same frequency, presumably because of the flow dependence of airways resistance. After treatment with increasing doses of aerosolized methacholine, RL and pulmonary elastance between 0.16 and 1.34 Hz progressively increased, as did the point at which the pulmonary reactance crossed zero (the resonant frequency). The alveolar pressure measurements showed the lung to become increasingly inhomogeneously ventilated in all six animals, whereas in the three younger rabbits lobar atelectasis developed at high methacholine concentrations and the alveolar capsules ceased to communicate with the central airways. We conclude that the low-frequency pulmonary impedance of rabbits exhibits the same qualitative features observed in other species and that it is a sensitive indicator of the changes in pulmonary mechanics occurring during bronchoconstriction.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

Zooplankton impacts on chlorophyll and transparency in Onondaga Lake,New York,USA

The transparency of polluted, hypereutrophic Onondaga Lake, New York, USA has improved substantially in the late 1980's as a result of reductions in phytoplankton biomass, in the absence of significant reductions in external phosphorus loading. Much of this improvement has been due to the occurrence of clearing events, e.g. sudden and dramatic increases in transparency. Field measurements, laboratory experiments, and modelling analyses were utilized to identify processes regulating phytoplankton standing crop during the spring to fall interval of 1987. Changes in the zooplankton community documented over the past decade support the conclusion that increased zooplankton grazing has contributed to improvements in transparency. Herbivores now represent a greater fraction of the zooplankton population and more efficient cladocerans are present in greater numbers. Biomanipulation practices, e.g. reestablishment of piscivorous species, designed to reduce the abundance of planktivorous fish species in Onondaga Lake, may serve to reduce pressure on the grazing community and thus result in further improvements in transparency.     

Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi.

Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.     

Protein surface charges and Ca2+ binding to individual sites in calbindin D9k: stopped-flow studies

The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k--a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin--have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods. The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein. These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions. Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported [Linse et al. (1988) Nature 335, 651-652]. The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, kon. The estimated maximum value of the association rate constant (kon(max) for Ca2+ binding to the wild-type protein is ca. 10(9) M-1 s-1. In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Interactive regulation of signalling pathways in bone cells: possible modulation of PGE2-stimulated adenylyl cyclase activity by protein kinase C

We have reported previously that tumour-promoting phorbol esters modulate both basal and vasoactive intestinal polypeptide (VIP)-stimulated adenylyl cyclase activity in GH3 (an established pituitary cell line). Here, we probe the receptor and cell specificity of this response. Experiments were performed in the presence of isobutylmethylxanthine. Unlike the response in GH3 cells, the tumour-promoting phorbol ester (tetradecanoyl phorbol acetate (TPA] did not affect either basal adenylyl cyclase activity nor VIP-stimulated activity in the rat osteosarcoma subclones UMR 106-01 and UMR 106-06. In addition, the cyclase responses to parathyroid hormone (PTH), and, in the case of UMR 106-06, to calcitonin were unaffected by tumour-promoting phorbol ester. However, prostaglandin E2-stimulated cyclase activity in both of these subclones was attenuated in a dose-dependent manner.     

Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings

Previous studies indicated that acute exposure of adrenal cells to adrenocorticotropic hormone (ACTH) markedly stimulates steroidogenic capacity in vitro but also inhibits cell proliferation. However, in vivo, ACTH is known to stimulate adrenal cell growth. To address this discrepancy, we determined the effect of long-term (9-11 days) continuous or intermittent exposure to ACTH on human fetal adrenal cell proliferation and steroidogenesis. Adrenal glands from fetuses 18-22 wk gestation were studied. Fetal zone cells were plated either on plastic or on an extracellular matrix (ECM) in the presence and absence of basic fibroblast growth factor (bFGF) (0.5 ng/ml) and 1 or 10 nM ACTH. As determined by cell counting, bFGF stimulated cell proliferation during 9 days in culture. In the presence of bFGF, the average doubling time decreased from 44 to 30 h on plastic and from 37 to 26 h on ECM. Under these conditions, ACTH did not inhibit cell proliferation. Proliferation of fetal adrenal corticosteroid-producing cells in the ACTH-treated cultures also was assessed by histochemical staining for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). The number of positive cells increased more than 4-fold between Days 5 and 9 in culture. Continuous treatment with 1 nM ACTH increased dehydroepiandrosterone sulfate (DHAS) production 5- to 10-fold during the first 5 days in culture. Thereafter, the stimulated hormone production decreased over time, although there was still a difference of almost 100-fold between the control and ACTH-treated cultures at the end of 9 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.