Peptide hormone degradation by a rat mast cell chymase-heparin complex

Material released from rat mast cells by compound $\text{48}\text{80}$ gave parallel responses using ACTH and β-endorphin radioimmunoassays. However, incubation of these labeled compounds under conditions of radioimmunoassay with released material and chromatography on Sephadex G-25 provided evidence that neither ACTH nor β-endorphin were present in the material released from mast cells, but represented an artifact produced by the presence of a protease. Analysis of the released enzyme on Sephadex G-75 under non-dissociative conditions yielded an active enzyme complex with a M_r > 150,000. Under dissociative conditions, the M_r of the enzyme was 25,000. The dissociated enzyme reassociated with purified rat mast cell heparin to form the high molecular weight complex. Further investigation of pH, substrate and inhibitor specificity showed that the peptide degradation is due to a chymotrypsin-like protease, the previously described mast cell chymase, which is active in degrading β-endorphin, ACTH, and ACTH^1–24.     

Increase in mast cell number and altered vascular permeability in thirsty rats.

The intravenous administration of 2M NaCl causes marked swelling, vacuolization and degranulation of rat mesenteric mast cells. 72 h of water deprivation (with food available) doubled the number of mast cells in the rat mesentery. Both experimental conditions induced venular labeling. $\text{In vitro}$, up to 300 mM NaCl did not elicit the release of amines from the mast cell. These results led us to infer the existence of some intermediary between hyperosmolarity and mast cell activation. Increased venular permeability, mast cell degranulation and proliferation are common features in inflammatory processes. Sodium salicylate, a non steroidal anti-inflammatory drug, was found to inhibit specifically cell dehydration thirst. A connection between inflammation and the peripheral mechanisms which trigger the central elaboration of the sensation of thirst is suggested.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

Reaction of peptide thiobenzyl esters with mammalian chymotrypsinlike enzymes: A sensitive assay method

Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin A_α with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m^?1 cm^?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values for these compounds.     

Recycling of mast cells following degranulation in vitro: An ultrastructural study

Mature mast cells, isolated from the rat peritoneal cavity, were placed into suspension culture, either as resting cells or after degranulation by exposure to compound $\text{48}\text{80}$, and were maintained for up to 63 hr. No mitotic cells were observed, and cell number was conserved. The culture conditions did not cause spontaneous degranulation and cell survival was better than 80%. However, with time in culture, an increasing percentage of cells acquired a vesiculated appearance, characterized by a Golgi area with distended cisternae, the accumulation of lysosomal or autophagic-like vesicles, and enlarged, irregular or fused secretory granules. In the degranulated group, about one-fourth of the cells recovered the morphological appearance of resting cells by 63 hr, indicating that they are capable of ‘recycling’. A cell type with a unique morphology, characterized by a large central vacuole containing secretory product, an eccentric nucleus, and mature secretory granules at the cell periphery appeared in the stimulated group after 22 hr of culture. It may be a possible intermediate stage in the mast cell regranulation process, based on its occurrence exclusively in the stimulated group, the correlation between its distribution and the recovery of mast cells to the resting state, and the morphological resemblance of its granule contents to stages in granule maturation in differentiating embryonic mast cells.     

Properties of [3H] diazepam binding to rat peritoneal mast cells

Benzodiazepine binding to rat peritoneal mast cells was investigated using [³H] diazepam as the radioactive probe. The specific binding of [³H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a K_D = 90 ± 10 nM and B_max of 261 ± 60 fmoles/10⁶ cells. The binding of [³H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [³H] diazepam is reversible with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2 ± 0.2}\text{min.}$ Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [³H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.     

Calcium requirement in compound 48/80 induced histamine release from rat mast cells in vitro

The effect of magnesium and EDTA on compound $\text{48}\text{80}$-induced histamine release and adenosine triphosphate (ATP) content of mast cells has been studied. Inhibition of histamine release after preincubation of the cells with or without EDTA in the absence of calcium and the reversal by calcium indicate that calcium is required for compound $\text{48}\text{80}$-induced histamine release. The presence of magnesium potentiate the inhibition caused by the lack of calcium. The inhibition of histamine release is not related to changes in cellular ATP content. The observations with EDTA suggest that calcium may be provided for the release process from intracellular sources.     

Pulmonary cell response to bacterial challenge in mutant mice with some hereditary alterations resembling cystic fibrosis

Animal models make it possible to perform studies that normally can not be done in human beings. In the present work, the cellular response pattern of the lungs both to the environment and to a challenger strain of bacteria were analyzed in two mouse mutations-cribriform degeneration ($\text{cri}$) and motheaten (me)-. The mice were submitted to an infective aerosol containing $\text{Staphylococcus aureus}$ and were killed either immediately or 4 h after exposure; pulmonary washings were performed in these animals as well as in non-infected controls. The results showed neither quantitative differences in the total cell count nor in cell viability between the different groups. However, there were qualitative changes in the differential cell content characterized by a neutrophilic response in $\text{cri}$/$\text{cri}$ mice and a giant cell response in $\text{me}$/$\text{me}$ mice. Since most of the cystic fibrosis patients suffer from chronic lung infection, with a persistently high proportion of neutrophils, these findings point to the $\text{cri}$/$\text{cri}$ mouse as a promising animal model for cystic fibrosis studies.     

Occurrence of proteinase a isoinhibitors in wild type yeast strains and commercial baker's yeast

The occurrence of the proteinase A inhibitors 2 and 3 was investigated in wild type strains of $\text{Saccharomyces}\text{cerevisiae}$ and $\text{Saccharomyces}\text{carlsbergensis}$ as well as in several strains of commercial baker's yeast. Haploid and diploid strains of $\text{Saccharomyces}\text{cerevisiae}$ contain only proteinase A inhibitor 3 whereas in $\text{Saccharomyces}\text{carlsbergensis}$ only proteinase A inhibitor 2 is found. Strains of commercial baker's yeast contain either proteinase A inhibitor 3 or both inhibitors in a constant ratio of 1:3. Single cell cultures isolated from a strain of commercial baker's yeast also contain a mixture of the two inhibitors. Therefore, baker's yeast is not a mixture of two different cell types but the genome for both inhibitors is present in each single cell. In general, the results indicate that the occurrence of the two proteinase A inhibitors is determined genetically and, therefore, they may be called “isoinhibitors”.     

Novel nucleotides from E.coli isolated and partially characterized

Two new nucleotides have been found in the formic acid extracts of $\text{Escherichia}\text{coli}$, $\text{Clostridium}\text{botulinum}$, $\text{Bacillus}\text{subtilis}$ and $\text{Rhodospirillum}\text{rubrum}$ isolated during log phase growth. In $\text{E.}\text{coli}$ the compounds are present at all times during cell growth but increase in amount during interruption of aeration and transition to stationary phase. They migrate close to ppGpp during one dimensional chromatography on PEI cellulose but are clearly separated from ppGpp by paper chromatography. The compounds are unstable on PEI cellulose and purification was effected by chromatography on A25 Sephadex ion exchange columns. Preliminary characterization indicates that the predominant compound is a dinucleoside polyphosphate and that both compounds contain a modified adenosine nucleoside.     

The accumulation of glycinamide ribotide by ade3 and ade8 mutants of Saccharomyces cerevisiae

The purine intermediate GAR is present in cell free extracts of $\text{ade3}$ and $\text{ade8}$ mutants of yeast. It is also detectable following acid hydrolysis of extracts of $\text{ade6}$ and $\text{ade7}$ which accumulate FGAR and FGAM respectively. GAR accumulation is repressed by growing cells in high levels of adenine. Neither $\text{ade4}$ nor $\text{ade5}$ accumulate GAR and both prevent accumulation of GAR in $\text{ade3}$ and $\text{ade8}$ and FGAR in $\text{ade6}$. Since $\text{ade3}$ is known to be defective in folate metabolism these results indicate that $\text{ade8}$ is blocked in the conversion of GAR→FGAR.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Mast cell histamine release induced by Portuguese man-of-war (Physalia) venom

Nematocyst venom from Portuguese Man-of War ($\text{Physalia}$ sp.) tentacles causes isolated rat peritoneal mast cells to release histamine. Extent of histamine release is dose-dependent (K_0.5 = 6.1 μg venom/ml) and attains 100% at high doses of venom. Release is independent of intra- and extracellular calcium levels and does not depend upon a cellular supply of ATP. The rate of histamine release is temperature-dependent and the extent of release is maximized broadly over the range of 10–30°C. The cytoplasmic marker lactate dehydrogenase, is released concomitantly with histamine but is more sensitive to the venom (K_0.5 = 2.1 μg/ml). Antimycin A, while it does not significantly affect venom-induced histamine release, increases the sensitivity of lactate dehydrogenase release (K_0.5 = 0.2 μg/ml). We conclude that $\text{Physalia}$ nematocyst venom induces the release of histamine from mast cells by a cytolytic mechanism and that this action is antagonized by an intracellular, energy-requiring process.     

Structure of leukotriene C. Identification of the amino acid part.

Leukotriene C, a “Slow Reacting Substance” (SRS) from mouse mast cell tumors, was earlier shown to be a derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid with a cysteine containing substituent in thioether linkage at C-6 (Murphy, R.C., Hammarström, S., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, $\text{76}$, 4275–4279 (1979)). The substituent has now been identified as γ-glutamylcysteinylglycine (glutathione).     

Comparison of circulating and peritoneal leukocyte cationic proteins in release of histamine from rat mast cells

Lysosomal cationic proteins which release histamine from rat peritoneal mast cells were prepared from circulating as well as peritoneal leukocytes of rabbits. The release of histamine by cationic proteins and by compound $\text{48}\text{80}$ was compared as a function of temperature, pH and concentration. Cationic protein-mediated histamine release appears to be a non-cytotoxic energy requiring process similar to compound $\text{48}\text{80}\text{-}\text{mediated release}$. It was inhibited by iodoacetate, n-ethylmaleimide, 2,4-dinitrophenol, malonate, oxamate, glutamate and slightly inhibited by 2-deoxyglucose. Pharmacologic inhibition of release by isoproterenol, aminophylline, dibutyryl cyclic AMP and prednisone was also demonstrated.     

The occurrence of pili associated with a plasmid of the W compatibility group

Using electron microscopy, it was found that the acquisition of the W group drug resistance plasmid S-a by normally pilusless bacterial strains was associated with the appearance of pili. The loss of drug resistance markers in presumed R^? revertants was accompanied by a loss of pili. The numbers of pili present on transconjugant strains of the three bacterial species tested were 3.2 pili/cell for $\text{Salmonella typhimurium}$, and 0.19 pili/cell for both $\text{Escherichia coli}$ and $\text{Pseudomonas aeruginosa}$. Negatively stained pili were about 12 nm thick and varied in length from 23 nm to 3,370 nm.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.     

Differences in aminoacylation in vivo and in vitro of lysine isoaccepting tRNAs from virus-transformed cells

When murine sarcoma virus-transformed cells are labeled with [³H]lysine $\text{in}\text{vivo}$ for various periods, 5 of 6 isoaccepting lysine tRNAs separable by RPC-5 chromatography are aminoacylated in 1 hr to the same extent that they are aminoacylated $\text{in}\text{vitro}$. The sixth isoacceptor, tRNA₆^Lys, is not aminoacylated $\text{in}\text{vivo}$ to a measurable extent in 1 hr, although it is present in the tRNA prepared from the cells. All six isoacceptors are aminoacylated with [³H]lysine $\text{in}\text{vivo}$ when the labeling period is 2 or 3 hr. These results further show that $\text{in}\text{vitro}$ correlations of the amount of tRNA₄^Lys with cell division accurately reflect the situation $\text{in}\text{vivo}$. Results of differential centrifugation indicate that tRNA₆^Lys occurs in mitochondria.     

Non-linear relationships between oxygen saturation and magnitude of fine structure of ultraviolet oxy versus deoxy difference spectrum in human hemoglobin

Ultraviolet difference spectra of fully oxygenated hemoglobin $\text{vs.}$ successively deoxygenated or reoxygenated hemoglobin were determined in the absence and presence of organic phosphates. Magnitude of fine structure in the difference spectrum around 290 nm, which is considered to be a partial reflection of oxygenation-induced changes in quaternary conformation of hemoglobin, was not linearly related to fractional oxygen saturation of hemoglobin of the reference cell. The non-linear feature was influenced by the organic phosphates as predicted by the allosteric model of Monod $\text{et al.}$ The present study suggests that the ultraviolet oxy $\text{vs.}$ deoxy difference spectrum measurements provide a useful way to examine the validity of the model.     

Increased synthesis of a low molecular weight protein in vincristine-resistant cells

A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells ($\text{DC-3F}\text{VCRd-5}$) has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of $\text{DC-3F}\text{VCRd-5}$, and only trace amounts were detected in control DC-3F cells. A similar protein (M_r = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in $\text{DC-3F}\text{VCRd-5}$ cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome.