The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

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.     

A sequential response to growth substances in coleoptiles from γ-irradiated wheat

Summary -irradiated wheat seed (500 kr) produces coleoptiles that grow without cell division or DNA synthesis. Apart from an initial 24-hr delay in growth, intact coleoptiles have a pattern of cell elongation similar to normal coleoptiles. The elongation of coleoptiles excised at a size of 2 mm, when the cells are small and just prior to entering a rapid elongation phase, is promoted by kinetin and gibberellic acid (GA₃). Elongation of coleoptiles excised at 8 mm, when the cells are larger and in the rapid elongation phase, is promoted by indoleacetic acid (IAA). This sequential response to growth substances in coleoptiles is remarkably similar to that in normal coleoptiles. The GA₃ response in excised coleoptiles is not inhibited by FUDR, confirming that DNA synthesis is not required for GA₃-induced elongation in coleoptiles.     

A fluorometric assay for γ-glutamyl transpeptidase: Demonstration of enzymatic activity in cultured cells of neural origin

A new fluorometric assay was developed for the measurement of -glutamyl transpeptidase (-GTP). The assay utilizes as a substrate the synthetic compound 7--glutamylamido-4-methyl coumarin which is cleaved by -GTP to yield the highly fluorescent product 7-amino-4-methyl coumarin. Optimal excitation and emission wavelengths for the assay are 345 nm and 470 nm, respectively, and the sensitivity of the assay is greatly enhanced by the high-pressure liquid chromatographic separation of the product from the substrate. The assay is minimally 25 times more sensitive than the conventional spectrophotometric assay and permits analysis of as little as 5000 cultured cells of neuronal and glial origin. Analysis of a variety of cultured cells of neuronal and glial origin with this assay suggests that -GTP is largely present in glia and to a lesser extent in neurons.     

Cytokine production in whole blood cell cultures of patients undergoing therapy with biological response modifiers or 5-fluorouracil

We have measured the levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-1, and IL-2 in the whole blood cell culture supernatants of 43 tumor patients undergoing a treatment with biological response modifiers or a conventional therapy with 5-fluorouracil and leucovorin. In the blood cell cultures of the 16 patients who received 5-fluorouracil and leucovorin IFN levels decreased (P0.01) and TNF levels rose (P0.05) during each therapy cycle. However, in the blood samples a declining number of total leukocytes and lymphocytes was measured (P0.05). Progressive disease could be correlated to a tendency towards lower IFN levels in the pretherapeutic cultures of these patients. The second group analyzed consisted of 8 patients receiving a low-dose IL-1 therapy. In this group we found either an unchanged or an augmented IFN production of the blood cells during treatment. In the group of 13 patients receiving low-dose recombinant IL-2 (4.5×10⁶IU m^–2 day^–1) significantly increasing IFN levels were seen in the blood cell cultures during the therapy (P0.05), although total leukocyte counts decreased. In this group, 4 had stable disease for at least 2 months and 9 patients had tumor progression under therapy. In the cultures of the latter a tendency towards lower IFN values was found. Finally, the cytokine production in the blood cell cultures of 6 patients receiving a combination therapy of IFN and high-dose IL-2 was studied. During this therapy a dramatically reduced production not only of IFN but also of all other measured cytokines was found. In this group all patients had tumor progression under therapy. It is concluded that the measurements of cytokine production in a reproducible whole blood culture system may be useful for monitoring immunological therapies and may help us to find out which doses of biological response modifiers have enhancing or suppressive effects on the functions of the immune cells.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

Modulation of GABAergic neurotransmission in the brain by dipeptides

The effects of endogenous and synthetic peptides containing GABA or its analogues on the GABA/benzodiazepine/chloride ionophore, complex, GABA_B receptor, Cl fluxes, GABA release and GABA uptake were studied using synaptic membranes, crude synaptoneurosomal preparations and slices prepared from the rat and mouse brain. The sodium-independent binding of GABA was strongly inhibited by GABA-histidine, followed by -glutamyl-homotaurine, GABA-glycine and -glutamyl-GABA. The binding of diazepam was slightly enhanced by the same peptides. The peptides alone had no effect on the chloride fluxes, but GABA-histidine, -glutamyl-GABA and GABA-glycine enhanced while -glutamyl-homotaurine and GABA-taurine inhibited GABA-stimulated chloride uptake. GABA-histidine was the most effective displacer of baclofen binding, but -glutamyl-homotaurine was entirely ineffective. The uptake of GABA was markedly inhibited in synaptosomal preparations by GABA-histidine, while all other peptides were less effective. -Glutamyl-taurine attenuated but -glutamyl-homotaurine and GABA-glycine enhanced the potassium-stimulated release of GABA. The present actions of GABA-histidine in vitro may be of significance for GABAergic neurotransmission in vivo.     

Comparative study of inhibitory effects by murine interferon γ and a new bisphosphonate (alendronate) in hypercalcemic,nude mice bearing human tumor (LJC-1-JCK)

The inhibitory effect of murine interferon (muIFN) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), muIFN was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

Comparative study of the antitumor effect of two types of murine recombinant interferons, (β) and (γ), against B16-F10 melanoma

Summary A comparative study of the antitumor effect of murine recombinant interferon() Mu-rIFN() and murine recombinant interferon() Mu-rIFN() on B16-F10 melanoma was conducted. Administration of Mu-rIFN() i.p. into C57BL/6 mice on days 1 to 7 produced a higher suppressive effect than Mu-rIFN() both on the growth of s.c. implanted tumor and on the formation of artificial pulmonary metastasis. Pharmacokinetic study of Mu-rIFN() demonstrated that high plasma levels were retained for a long time. In clonogenic assay, Mu-rIFN() at 1000 units/ml showed about 80% inhibition of colonies of B16-F10 melanoma. However, Mu-rIFN() hardly inhibited the colonies, even at 1000 units/ml. Augmentation of natural killer (NK) cytotoxicity was much greater with Mu-rIFN() than Mu-rIFN(), whereas Mu-rIFN() enhanced the cytotoxicity of peritoneal macrophages more strongly than Mu-rIFN(). Injection of Mu-rIFN() i.p. 1 day before tumor challenge also inhibited the formation of pulmonary metastasis of B16-F10 melanoma. However, pretreatment of mice with carrageenan significantly suppressed the inhibitory effect of Mu-rIFN(). From these results, it is suggested that the inhibitory effect of Mu-rIFN() on the tumor growth and metastases of B16-F10 melanoma is mediated partly by direct antitumor effect and partly by the activation of macrophages, and that the augmentation of NK activity contributes mainly to the antitumor effect of Mu-rIFN().     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma,epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase

The location of the endogenous inhibitor protein ( IF₁) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F₁I and intact F₁F₀I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of –IF₁, IF₁–IF₁, –IF₁, and –IF₁ cross-linkages in soluble F₁I and in native F₁F₀I complexes. Cross-linking blocked the release of IF₁ from its inhibitory site and therefore the activation of F₁I and F₁F₀I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF₁ is at a distance 12 Å,to and subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF₁ inhibits conformational changes of the catalytic subunits, the inhibitory mechanism of IF₁ may involve the interference with rotation of the central stalk.     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

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.     

The protein sequence homology of gamma-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of -crystallin were found to be N-terminally blocked. -Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified -crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between -crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp -II, rat 2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat -crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one // superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.