Proteolytic enzymes and arachidonic acid metabolites produced by MRC-5 cells on various microcarrier substrates

Summary Human diploid fibroblasts were cultured on microcarriers made from DEAE-dextran, denatured collagen, DEAE-dextran linked to denatured collagen, and glass. Cells grown on these four substrates were examined for the production of proteolytic enzymes and arachidonic acid metabolites. Culture fluids from cells grown on the DEAE-dextran microcarriers contained the highest amounts of proteolytic enzyme activity. Both plasminogen-independent and plasminogen-dependent fibrinolytic activities were present and the plasminogen-dependent activity seemed to result from the presence of both urokinase and tissue plasminogen activator. Culture fluid from the cells grown on the glass microcarriers contained the least amount of protease activity, and nearly all of the plasminogen-activator activity seemed to be of the urokinase type. Protease activity in the culture fluids of cells grown on the other two substrates were intermediate. With regard to arachidonic acid metabolites, cells grown on the DEAE-dextran microcarriers produced the highest amounts of cyclooxygenase products but very low levels of lipoxygenase metabolites. Cells grown on the other three substrates produced comparable amounts of various cyclooxygenase products (lower than that produced by cells on the DEAE-dextrans substrate). Cells grown on the glass microcarriers also produced detectable amounts of two lipoxygenase metabolites—leukotriene B₄ and leukotriene C₄. Inasmuch as both proteolytic enzymes and arachidonic acid metabolites regulate basic cell properties, the differential amount of these metabolites observed in the culture fluids on the various substrates may contribute to the biological differences that exist on these substrates. This study was supported in part by grants R44 CA 36656 and IK08HL01332-01 from the Public Health Service, U. S. Department of Health and Human Services and by grant BC-512 from the American Cancer Society. JDH is a research fellow of the American Lung Association.     

Reflex cardiovascular responses caused by stimulation of pulmonary C-fibers with capsaicin in dogs

The purpose of these studies was to determine quantitatively the reflex cardiovascular responses to stimulation of the pulmonary C-fibers in dogs. We used a preparation in which the airway, pulmonary artery, and the pulmonary veins to the left lung were cannulated in situ. Ventilation and perfusion of the right lung maintained the animal in relatively normal homeostasis. Capsaicin, a decylenic acid amide of vanillylamine that selectively stimulates nerve endings of unmyelinated fibers (C-fibers), was injected into the left pulmonary artery in 5-ml boluses. Maximal reflex responses were obtained with concentrations as low as 0.8-1.6 X micrograms-1 X kg-1. Heart rate, hindlimb resistance, and left ventricular contractility were lowered transiently (the maximal responses showing declines of 40, 13, and 15.2%, respectively). As a result of these changes, combined with vasodilation in other resistance vessels, cardiac output fell 28% and blood pressure fell 35%. Interrupting the afferent neural pathway by severing the ipsilateral cervical vagus nerve eliminated these responses, confirming the distribution of their reflex origin. Although the role of these reflexes in homeostasis has not been decided, the results of this study suggest that the lungs of dogs, if appropriately stimulated, potentially can exert a major inhibitory influence on the neural regulation of cardiovascular function.     

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

Distribution,biology and hybridization of Scaphirhynchus albus and S. platorynchus in the Missouri and Mississippi rivers

Synopsis Scaphirhynchus albus and S. platorynchus were studied in Missouri during 1978–1979 to assess their distribution and abundance, to obtain information on their life histories, and to identify existing or potential threats to their survival. S. platorynchus was collected in substantial numbers (4355 specimens) at all 12 sampling stations in the Missouri and Mississippi rivers, while only 11 S. albus were captured from 6 stations. Twelve specimens identified in the field as hybrids between the two species were captured from 4 stations. Morphometric and meristic comparisons of presumed hybrids with the parent species, using cluster and principal components analyses, demonstrated intermediacy of most specimens identified in the field as hybrids. Aquatic insects comprised most of the diet of S. platorynchus and S. albus, but S. albus and the hybrids had consumed considerable quantities of fish. S. albus grew more rapidly than S. platorynchus, while the growth of hybrids was intermediate. Hybridization appears to be a recent phenomenon, resulting from man-caused changes in the big-river environment. Hybridization may be a threat to survival of S. albus in the study streams.     

Possible evidence for novel metabolism of nitrogen gas by a green alga

We report the isolation of a cukaryotic green alga ( Chlorella , strain WPI-2) which accumulates large stores of nitrogen (N) during growth in N-free medium and seems to incorporate¹⁴N₂, yet does not reduce acetylene to ethylene. Total N accumulation during growth on N-free medium and in gases free of combined N was measured by three methods: Kjeldahl, oxidative pyrolysis via chemiluminescence (Antek N analyzer), and Dumas (Coleman N analyzer). Increases in N ranging from 22–64%± 1% were observed. Isotope dilution studies using cells labelled with ¹⁵NO ₃- and then shifted to ¹⁴N₂ in N-free medium showed dilution of the ¹⁵N isotope by ¹⁴N from 5.67 to 5.32%± 0.05%. Using a variety of conditions, we were unable to demonstrate the reduction of acctylene to ethylene by WPI-2, although diazotrophic cyanobacteria gave positive results. Although the data on WPI-2 are not conclusive in establishing this alga as a diazotroph, the data do suggest that within the Chlorophyceae there may exist a novel form of nitrogen gas metabolism.     

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate

Summary The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues). This work was supported by NIH grants CA 37589 and HL 33847, and grant 1718 from the Council for Tobacco Research. Editor’s Statement This paper is the first report of the comparison of the hormone requirements of primary cultures of normal and tumor prostate epithelial cells from the same system.     

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

Changes in left ventricular geometry during spontaneous breathing

The purpose of these experiments was to determine the effects of a spontaneously generated inspiration on the size and shape of the left ventricle (LV) in anesthetized supine dogs. We implanted markers in the LV to establish three perpendicular axes and recorded the motion of these markers using biplane cinefluoroscopy at 60 Hz. The primary changes in LV size that accompanied inspiration occurred at end diastole (ED). The largest change in LVED dimension was a 2.46-mm narrowing of the septal-lateral wall dimension, but the apex-base dimension decreased also, by 0.74 mm. The anteroposterior dimension actually widened by 1.07 mm. The septal-lateral narrowing was caused by both a 1.0-mm narrowing of the distance between the septal marker and the apex-base axis, as well as by a 1.4-mm narrowing between the apex-base axis and the lateral wall marker. Narrowing of the septal portion seemed expected because of presumed enhanced right ventricular filling during inspiration. Narrowing of the lateral portion of the LV, while the anteroposterior dimension widened, was surprising because a change in LVEDV shape is implied. Assuming ventricular homogeneity, this change in LVED shape implies that the forces applied to the epicardial surface were not uniform. There must have been a retraction on the anterior and posterior surface that was not experienced by the lateral LV wall. The net effect of these dimensional changes of the LV at end diastole (estimated from the product of the three ED axes) was a 3.5-cm3 reduction in LVED volume.(ABSTRACT TRUNCATED AT 250 WORDS)