Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2,4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30-40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G1----S). Since the latter type of effect most probably is Ca2+ dependent and since the spindle is sensitive to Ca2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase. All compounds were found to cause an increase of ATP with some of the applied concentrations. The results with diethyl maleate suggest that an increase of ATP is favored by an attack on mitochondrial reduced glutathione. The possible analogy between this effect and an increase of ATP and Ap4A in bacteria during oxidative stress is considered.     

Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells.

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.     

Linearity of the accumulation of various dosages of uranium in the major organs of mature male Japanese quail. Effect of various doses of estradiol-17 beta

1. Male Japanese quail were given 2.20 x 10(-4)-14.53 mg uranium/kg intravenously as uranyl ion (235U label). 2. The relationship between dosage and the 18-hr accumulation of U in leg bones, liver, kidneys and testes was linear. 3. Increases in U deposition with increased dosage were approximately 1:1, except for kidneys where 10-fold increases in dosage resulted in 25-fold increases in deposition. 4. Estradiol-17 beta increased U deposition in bones by 15-fold thereby providing some protection for the kidneys as now the ratio of dosage to accumulation was 1:1.     

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

Hemopexin-mediated heme uptake by liver. Characterization of the interaction of heme-hemopexin with isolated rabbit liver plasma membranes

Plasma membranes isolated from rabbit liver retain the ability to interact specifically with heme-hemopexin. In this system, apohemopexin does not compete effectively with heme-hemopexin for binding. The membranes bind heme-hemopexin complexes with high affinity (KD = 6.8 X 10(-7) M) and with an apparent capacity of 2.3 pmol/mg of membrane protein. These membranes also retain the ability to remove heme from heme-hemopexin. The release of heme reaches a plateau after 15-30 min at 30 degrees C and does not involve metabolic energy, proteolysis of hemopexin or pH gradients. The apohemopexin formed is rapidly released from the membranes. The accumulation of heme is saturable and is affected by pH and temperature with maximum uptake occurring between pH 5.5 and 6.5 and at 30 degrees C. Interestingly, much more heme (approximately 25 pmol/mg of membrane protein) is accumulated than hemopexin at saturation, implying that the receptor can turn over several times and that a heme-binding component exists in the rabbit liver plasma membrane.     

Effects of phorbol dibutyrate on M currents and M current inhibition in bullfrog sympathetic neurons

1. Effects of bath-applied phorbol dibutyrate (PDBu) on M currents (IM) and on the inhibition of IM by muscarine and luteinizing hormone-releasing hormone (LHRH) were recorded in voltage-clamped bullfrog lumbar sympathetic ganglion cells. 2. PDBu (0.1-30 microM) produced a slowly developing, irreversible and partial (less than or equal to 60%) inhibition of IM. This effect was not replicated by 4-alpha-phorbol or by vehicle. 3. After treatment with PDBu, residual IM showed a reduced sensitivity to inhibition by muscarine or LHRH but not by Ba2+. The reduced response to muscarine appeared to result from a 10-fold shift in the concentration dependence for inhibition. 4. PDBu did not clearly reproduce the ability of muscarine to inhibit the slow, Ca-activated K current IAHP or to increase the leak conductance at hyperpolarized potentials. The latter effect of muscarine was enhanced, rather than inhibited, by PDBu. 5. IM and IAHP were not inhibited by 1 mM dibutyryl cyclic AMP or by 20 microM forskolin. 6. It is concluded that activation of protein kinase C, but not protein kinase A, partly replicates the effect of muscarine on frog sympathetic neurons.     

Concentrations of glandular kallikrein in human nasal secretions increase during experimentally induced allergic rhinitis

We have previously demonstrated that a mixture of bradykinin and lysylbradykinin is generated in nasal secretions during the immediate allergic response to allergen. The present studies were performed to determine whether glandular kallikrein plays a role in kinin formation during the allergic reaction. Allergic individuals (n = 7) and nonallergic controls (n = 7) were challenged intranasally with appropriate allergen, and nasal lavages obtained before and after challenge were assayed for immunoreactive glandular kallikrein as well as for histamine, kinins, and N-alpha-tosyl-L-arginine methyl esterase (TAME-esterase) activity. The increase in postchallenge immunoreactive glandular kallikrein levels above baseline was significantly greater (p less than 0.01) for the allergic group (16.3 +/- 14 ng/ml; means +/- SD) than for the nonallergic controls (1.0 +/- 1.9 ng/ml). Increased levels of immunoreactive glandular kallikrein correlated with increases in kinins, histamine, and TAME-esterase activity and with the onset of clinical symptoms. Characterization of immunoreactive glandular kallikrein purified from postchallenge lavages by immunoaffinity chromatography confirmed the identity of this material as an authentic glandular kallikrein on the basis of its inhibition by protease inhibitors and by monospecific antibody to tissue kallikrein, its chromatographic behavior on gel filtration, and its ability to generate lysylbradykinin from highly purified human low m.w. kininogen. The specific activity of this purified material, in terms of kinin generation from kininogen, was very similar to that for authentic glandular kallikrein, suggesting that most if not all of the immunoreactive material purified from nasal lavages represented active enzyme. Inhibition studies by using pooled postchallenge lavages suggest that the majority of the kinin generating activity in these samples was due to glandular kallikrein. We conclude, therefore, that glandular kallikrein is secreted during the allergic response and can contribute to the formation of the lysylbradykinin produced during the allergic reaction.