Induction of an endogenous tumor necrosis factor in mice by murine recombinant interferon-γ combined with a lipid A subunit analog (GLA-60) of low toxicity

Summary We have investigated the endogenous production of a serum cytotoxic factor when recombinant interferon- (rIFN-) is combined with synthetic lipid A subunit analogs of low toxicity (GLA compounds). The cytotoxic activity of the serum was measured by the crystal violet staining method with L929 cells as a target. Intravenous administration of rIFN- followed by intravenous administration of lipopolysaccharide induced the endogenous production of a cytotoxic factor in the serum. The priming effect of rIFN- appeared immediately and persisted for approximately 20 h after the injection. Administration of lipopolysaccharide as a trigger enhanced the production of the cytotoxic factor in the serum maximally 2 h after the injection. The cytotoxic activity in the serum was completely inhibited by anti-(mouse tumor necrosis factor) (TNF) antibody. A synthetic lipid A subunit analog (GLA-60), which is much less toxic in its endotoxin activities than lipopolysaccharide or synthetic lipid A (compound 506), induced the endogenous production of serum TNF in rIFN--primed mice. GLA-60 entrapped within liposomes induced the production of serum TNF in rIFN--primed mice more effectively than GLA-60 solubilized in phosphate-buffered saline. Intravenous or intranasal administrations of rIFN- followed by intranasal administration of GLA-60 produced TNF in the lung washing fluid but not in the serum, indicating that TNF production can be induced locally rather than systemically by the alteration of the administration route of the primer and trigger. These results indicate that GLA-60, a lipid A subunit analog of low toxicity, is a beneficial triggering agent in the production of endogenous TNF, as well as having other immunopharmacological properties, and may provide a basis for cancer (metastases) treatment as a result of its ability to induce endogenous TNF.     

Purification and properties of gammagamma-enolase from pig brain

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as - (pI = 6.5), - (pI = 5.6), and -enolase (pI = 5.2). The pI of purified -enolase was also 5.2. The -enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55°C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%–80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, -enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K _m values for 2-PGA, PEP, and Mg²⁺ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K _m values of other vertebrate enolases. The amino acid composition of pig -enolase, as determined by amino acid analysis, shows strong similarity to the compositions of -enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig -enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig -enolase and the other -enolases are almost identical. Li⁺ proved to be a noncompetitive inhibitor with either 2-PGA or Mg²⁺ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2₁. An R _symm <5% was obtained for data between 50 and 3.65 Å, but was a disappointing 30% for data between 3.65 and 3.10 Å, indicating crystal disorder.     

Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds

In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with benzylaminopurine (BAP) or 6 --dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on half-strength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed.Abbreviations IAA Indole-acetic acid - IBA Indole-butyric acid - BAP Benzylaminopurine - 2iP 6 --dimethylallylamino purine     

Glutathione and γ-glutamyl transpeptidase are differentially distributed in the olfactory mucosa of rats

Summary Components of the -glutamyl cycle, including thiols, glutathione (GSH) and -glutamyl transpeptidase (-GT), were localized in the nasal mucosae of rats using histochemical and immunohistochemical methods. In olfactory mucosa, thiols were widely distributed, with intense staining in the mucociliary complex (MC), basal cells, acinar cells of Bowman's glands (BG), and olfactory nerve bundles, and with moderate staining in olfactory receptor neurons (ORNs). GSH was localized in MC, BG acinar cells, nerve bundles and, to a lesser extent, in ORNs. -GT immunoreactivity was restricted to the MC and to basolateral and apical membranes of BG acinar and duct cells. The basolateral membrane of BG acinar cells, located in close association with blood vessels and connective tissue, showed granule-like immunoreactivity. Inrespiratory mucosa, all three compounds were localized in the MC and acinar cells of respiratory glands (RG). In the MC, -GT immunoreactivity was associated primarily with brush borders of ciliated cells. Granular immunoreactivity was also apparent in the supranuclear region of RG acinar cells. These results demonstrate that components of the -glutamyl cycle are localized in olfactory and respiratory glands, and that they are secreted into the mucus, where they may mediate perireceptor events such as detoxification and/or solubilization of air-borne xenobiotics, toxicants and odorants.     

Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose ofγ-rays

The effect was studied of a low dose of-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.     

Chimeric B72.3 mouse/human (IgG1) antibody directs the lysis of tumor cells by lymphokine-activated killer cells

Summary Chimeric mouse/human B72.3 (cB72.3) antibodies having a human IgG1 (1) or IgG4 (4) constant region were compared to the native murine IgG1 B72.3 (nB72.3) monoclonal antibody (mAb) for their ability to participate with human effector cells in antibody-dependent cellular cytotoxicity (ADCC). Because the TAG-72 antigen recognized by B72.3 is poorly expressed on tissuecultured tumor cell lines, the xenografted OVCAR-3 human ovarian carcinoma ascites was used as a cytotoxicity target. The lytic activity of the cB72.3(1) mAb with peripheral blood lymphocytes was 1.5- to 50-fold greater than that of the nB72.3 mAb and usually the cB72.3(4) mAb. However, lymphocytes from some donors had similar ADCC activity with either the cB72.3(1) or cB72.3(4) mAb. The cB72.3(1) and the murine anti-colon carcinoma CO17-1A mAb had comparable activity in mediating ADCC against the OVCAR-3 tumor. Exposure of lymphoid cells to interleukin-2 (IL-2) (100–500 U/ml) for 24 h to generate lymphokine-activated killer (LAK) cells augmented ADCC mediated by the cB72.3(1) mAb 2- to 22-fold. By contrast, LAK cells from most donors expressed weak non-specific cytotoxicity against OVCAR-3 ascites tumor cells. The cB72.3(1), and to a lesser extent, the cB72.3(4) chimera also participated with monocytes in mediating ADCC, but the antibody-dependent lytic potency of monocytic effectors was much weaker than that of IL-2-activated lymphoid cells. These studies show that the cB72.3(1) mAb has appreciable ADCC-mediating properties, suggesting a potential role for its incorporation into treatment strategies utilizing adoptive killer cell and/or lymphokine therapy. Offprint requests to: J. Schlom, to whom reprint requests should be sent at 9000 Rockville Pike, Building 10, Room 8B07, Bethesda, MD 20892, USA     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Molecular cloning and chromosomal localization of a human skeletal muscle PP-1γ1 cDNA

Type-1-protein phosphatase (PP-1) activity is reduced in skeletal muscle from human subjects with insulin resistance (Kida et al. 1990). This reduced phosphatase activity probably leads to the abnormal insulin action for glucose storage observed in insulin-resistant subjects. In the present study, a human homolog of rat liver PP-11 cDNA was isolated from human skeletal muscle. The nucleotide sequence contains a 957-nucleotide open reading frame encoding an amino acid sequence identical to that encoded by rat liver PP-11 cDNA. Northern blot analysis shows PP-11-specific mRNA is expressed in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. PP-11 was localized to human Chromosome 12.     

Effect of di-n-propylacetate and γ-acetylenic GABA on hyperbaric oxygen-induced seizures and GABA metabolism

The administration of -acetylenic GABA or di-n-propylacetate to mice delayed the onset of hyperbaric oxygen-induced seizures in the animals. The former compound caused large increases in brain GABA content and strong inhibition of glutamate decarboxylase activity, whereas the latter compound brough about only moderate increases in brain GABA level and had little or no effect on the enzyme activity. It is suggested that the GABA system is not involved in the anticonvulsant mechanism of -acetylenic GABA but may play a role in the action of di-n-propylacetate.     

Synergistic induction of cytotoxicity in macrophages by murine interferon-γ and biological response modifiers derived from microorganisms

Summary The ability of recombinant murine interferon-gamma (rMuIFN-) to activate murine macrophages with or without several biological response modifiers (BRM), including synthetic muramyl dipeptide derivatives (MDPs), was investigated. Mouse peritoneal macrophages were activated by rMuIFN- alone to the cytostatic state, but not the cytolytic state. Other BRM as well as bacterial lipopolysaccharide (LPS), including a lyophilized preparation of an attenuated strain of Streptococcus hemolyticus, a cell wall skeleton of bacillus Calmette-Guerin and synthetic MDPs, were highly active in generating the synergism with rMuIFN-. Macrophages were endowed with the cytolytic activities by combinations of rMuIFN- and MDP-Lys(L18); the combination of 100U/ml of rMuIFN- with 10 ng/ml of MDP-Lys(L18) was sufficient to induce cytolytic activities in macrophages. The synergism was observed when the macrophages primed with rMuIFN- were treated with LPS or MDP-Lys(L18), but not when the sequence of treatment was reversed. The cytotoxicity of macrophages induced by rMuIFN- with MDP-Lys(L18) was suppressed by priming with MDP-Lys(L18). The suppressive effect was also observed by priming with LPS in combinations of rMUIFN- and LPS. The reason for the suppression of macrophage activation by priming with LPS and MDP-Lys(L18) is at present unknown.     

The in vitro development of cytotoxicity in response to granulocyte/macrophage-colony-stimulating factor or interferon γ in the peripheral blood monocytes of patients with solid tumors: Modulation by arachidonic acid metabolic inhibitors

Summary The capacity of granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interferon (IFN ) to elicit monocyte cytotoxicity in vitro in the peripheral blood monocytes of patients with solid tumors was investigated. The cytotoxicity elicited by IFN was significantly reduced in cancer patient monocytes compared to normal monocytes. The cytotoxicity elicited by GM-CSF, however, was comparable between cancer patient monocytes and normal monocytes, but was lower than that induced by IFN . Indomethacin, a cyclooxygenase inhibitor, significantly augmented IFN -elicited cytotoxicity in cancer patient monocytes, but not in normal monocytes. In contrast, indomethacin augmented GM-CSF-elicited cytotoxicity in both cancer patient monocytes and normal monocytes. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, was found to suppress cytotoxicity in response to IFN and GM-CSF in both cancer patient monocytes and normal monocytes. The addition of leukotrienes to NDGA-treated cultures restored the development of cytotoxicity. Thus there are differences in the in vitro response of cancer patient monocytes and normal monocytes to distinct biological activators. Furthermore, these responses can be manipulated by agents that modulate arachidonic acid metabolism.Supported in part by PHS grant CA 41741Fellow of the A. Onassis Foundation     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Production of interferon-β in a culture of fibroblast cells on some polymeric films

Normal human skin (NB1-RGB) cells were cultured in the presenceof polyinosinic and polycytidylic acids, diethylaminoethyldextran, cycloheximide and actinomycin D, which induced humaninterferon-. The simplest induction method, that requiredonly polyinosinic and polycytidylic acids and diethylaminoethyldextran was found to give the highest production ofinterferon- by the cells. The cell growth and productionof interferon- were investigated for NB1-RGB cellscultured on silk fibroin, poly(-methyl-L-glutamate),poly(-benzyl-L-glutamate) and collagen films prepared bythe Langmuir-Blodgett (LB) and casting methods. The cell densityof NB1-RGB cells cultured on the LB films was found to be higherthan that on the cast films made of the same polymer. Thisindicates that not only the chemical structure of the polymersused for the preparation of the films but the preparationmethods of the films, i.e., casting and LB methods, are also astrong factor affecting the cell growth. The production ofinterferon- per unit number of cells was found to behigher on the cast films than that on the LB films made of thesame polymer. This is explained by the fact that the optimalsuppressed growth of NB1-RGB cells on the cast films leads tothe enhanced production of interferon- on the cast filmscompared to those on the LB films prepared by the same polymer.     

A glycine-rich sequence in the catalytic site of F-type ATPase

Affinity labeling and genetic studies on the glycine-rich sequence of the subunit ofE. coli F-type ATPase are discussed. A model of the structure of the enzyme near the phosphate moiety is proposed.     

Regional distributions of γ-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and γ-aminobutyrate transaminase (GABA-T) in the central nervous brains of C57/BR,C3H/He,and F1 hybrid mice

The distributions of -aminobutyric acid (GABA), glutamate decarboxylase (GAD), and -aminobutyrate transaminase (GABA-T) have been studied in various brain areas of mice. These neurochemical markers, which are related to inhibitory neurotransmission, were investigated in different inbred strains of mice (C3H/He, C57/BR, and their F₁ hybrids). The regional distributions of GABA, GAD activity, and GABA-T activity in adult mice of these three strains were quite similar. No significant differences were found in any brain area for GAD or GABA-T activity. However, significant differences in GABA level were found in several brain areas among these strains of mice, especially in hypothalamus, hippcampus, olfactory bulb, and occipital cortex. These results provide further information to the possible influence of the GABAergic system in these brain areas.     

Influence of Triadimefon on the Metabolism of NaCl Stressed Radish

The effect of triadimefon (TDM) on various biochemical parameters was studied in NaCl stressed radish (Raphanus sativus L.). Stress imposed by 80 mM NaCl decreased the protein content and proline oxidase activity, and increased the proline and glycine betaine contents, and protease, -glutamyl kinase and ATPase activities. The TDM treatment alleviated the stress by increasing protein, and glycine betaine contents, and by decreasing proline accumulation, and proline oxidase and ATPase activities.     

Evolution of a protein superfamily: Relationships between vertebrate lens crystallins and microorganism dormancy proteins

Summary A search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the - and -crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The - and -crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single -crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of - and -crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.     

Unisite Hydrolysis of [γ32P]ATP by Soluble Mitochondrial F1ATPase and Its Release by Excess ADP and ATP. Effect of Trifluoperazine

Some of the characteristics of unisite hydrolysis of [³²P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [³²P]ATP bound at the catalytic sites of F₁ under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [³²P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [³²P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [³²P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [³²P]ATP/³²Pi bound at the catalytic site and a 50% diminution in the rate of ³²Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F₁ in the sense that in a fraction of F₁ molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [³²P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.     

An apparent correlation between the inhibition of induced ornithine decarboxylase activity by γ radiation and the capacity for DNA repair synthesis in normal and ataxia telangiectasia human fibroblasts: No correlation with cell survival

Summary Exposure of normal human fibroblasts (F107) in stationary phase to radiation inhibited the appearance of induced ornithine decarboxylase (ODC) activity. Skin fibroblasts derived from two ataxia telangiectasia (AT) patients (F184 and F182) displayed a similar response. The level of DNA repair synthesis was also similar in the three cell strains. Fibroblasts from another apparently normal donor (F196) were very sensitive to inhibition of induced ODC activity by radiation and were also deficient in radiation-induced DNA repair synthesis. However, the two strains derived from normal donors displayed the same degree of cellular sensitivity towards X-rays, whereas the two AT strains showed the typical hypersensitivity to the cytotoxic effect of X-irradiation. The results suggest a possible correlation between the inhibition of induced ODC activity by radiation and the extent of DNA repair synthesis at high radiation doses, but there is no correlation between these two parameters and cellular survival at low radiation doses.