Enantioselective inhibition of TNF-α release by thalidomide and thalidomide-analogues

The question whether the immunomodulating activity of rac-thalidomide resides in either the (−)-(S)- or the (+)-(R)-enantiomer was addressed by synthesis and separation of pure enantiomers of thalidomide-analogues which carry a methyl-group at the asymmetric carbon atom and are thus prevented from racemization. The effect of the pure enantiomers of the thalidomide-analogues and also of the enantiomers of thalidomide on relapse of TNF-α was tested in vitro by using stimulated peripheral mononuclear blood cells. Both enantiomers of thalidomide inhibited the release of TNF-α equally well at low concentrations (5 and 12.5 μg/ml) but at higher concentrations (25 and 50 μg/ml) there was a weak but statistically significant selectivity towards the (−)-(S)-enantiomer. In the case of the configuration-stable thalidomide-analogues there was a very pronounced and statistically significant enantioselectivity towards the (S)-form even at lower concentrations (≥5 μg/ml). The (S)-enantiomers of the thalidomide-analogues differed in their inhibitory potency from (−)-(S)-thalidomide suggesting that the introduction of the methyl-group increases the TNF-α-inhibitory activity while the reduction of one of the carbonyl-functions in the glutarimide-moiety to a methylene-group decreases activity. The effect of these small molecular alterations on activity and the enantioselectivity towards the (S)-enantiomers may indicate that thalidomide and its analogues directly interact with one or several cellular target-proteins. © 1996 Wiley-Liss, Inc.     

Involvement of PKC-β in PTH, TNF-α, and IL-1β Effects on IL-6 Promoter in Osteoblastic Cells and on PTH-Stimulated Bone Resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-β, LY379196, was used to determine the role of the PKC-β isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced translocation of PKC-α and -β_I to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-β_I translocation by PTH, TNF-α or IL-1β was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-α-, or IL-1β-stimulated translocation of PKC-α. PTH, TNF-α, and IL-1β increased luciferase expression in UMR-106 cells transiently transfected with a −224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-β_I is a component of the signaling pathway that mediates PTH-, TNF-α-, and IL-1β-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon-γ and interleukin-1α

Articular chondrocytes from rheumatoid joints have been shown to express class II major histocompatibility (MHC) antigens that were correlated with the presence of interferon-gamma (IFN-γ) in the inflamed joint. Chondrocytes expressing MHC antigens function as antigens function as antigen presenting cells and thus stimulate lymphocyte proliferation. These responses suggest a powerful role for the IFN-γ stimulation of chondrocytes. The present studies were designed to examine the functional role of chondrocytes exposed to IFN-γ during cartilage degradation that occurs in synovial disease. Destruction of cartilage in arthritis is partially attributable to metalloproteinases released by the chondrocytes in response to interleukin-1 (IL-1). Bovine articular chondrocytes treated with interleukin-1 alpha (IL-1α) produced enhanced levels of stromelysin mRNA, however, Northern blots could not determine the percentage of cells responding. Exposure of bovine articular chondrocytes to IFN-γ induced the expression of bovine HLA-DR (boHLA-DR) antigen in 50% of the cells. Using a modified cell sorting technique, chondrocytes that expressed class II MHC antigens produced two fold greater stromelysin mRNA than chondrocytes that did not express this antigen. In contrast, collagen type II mRNA levels were similar in chondrocytes, regardless of the expression of class II MHC antigens. In situ hybridization studies showed that less than half of all cartilage chondrocytes were induced to synthesize stromelysin mRNA. These observations suggest that IFN-γ stimulates specific subpopulations of chondrocytes to be functionally active in inflammation-induced metalloprotease secretion. © 1993 Wiley-Liss, Inc.     

Conformational study of poly(α,β-L-aspartic acid)

The conformation of several samples of poly(α,β-L -Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L -Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm^?1 was shifted to 620 cm^?1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.     

Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.     

Hepatotoxin rubratoxin B induced the secretion of TNF-alpha, IL-8, and MCP-1 in HL60 cells

Induction of cytokine secretion by rubratoxin B was investigated using HL60 cells. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 were secreted from 40 and 80 microg/ml rubratoxin B-treated cells. In 20 and 40 microg/ml rubratoxin B-treated samples, monocyte chemotactic protein (MCP)-1 was released. These rubratoxin B-induced cytokines are known to promote liver myelocytic cell infiltration, and activate cytokine-recruited cells. As a result, recruited myelocytic cells are considered to contribute to hepatic injury. We investigated the effects of tyrosine kinase inhibitors genistein and emodin. Genistein reduced the release of all three cytokines from rubratoxin B-treated cells. Likewise, emodin diminished the secretion of MCP-1. Alternatively, emodin reversed on the secretion of TNF-alpha, and the release of IL-8 was not affected. Since emodin did not impede rubratoxin B-caused TNF-alpha and IL-8 secretion, they appeared to be regulated differently from MCP-1 secretion, suggesting that rubratoxin B exerts its toxicity using two or more signal transduction pathways.     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Increased excretion of 5α,7α-dihydroxy-11-ketotetranor-prostanoic acid on anaphylaxis in the guinea-pig

The major urinary metabolite of PGF_1α and PGF_2α, 5α,7α-dihydroxy-11-ketotetranor-prostanoic acid was measured in guinea-pigs by multiple-ion analysis after chromatographic purification and conversion into the trimethylsilys ether-O-methyloxime derivative of the methyl ester. The basal excretion amounted to 0.5–1.1 μg/24 hours. In three of four non-anaesthetized guinea-pigs sensitized to ovalbumin the excretion of the metabolite was almost doubled on inhalation of the antigen. In one of four animals this was also seen on inhalation of histamine. Peroral pretreatment with indomethacin (25–50 mg/kg) reduced the basal excretion of the PGF-metabolite and abolished the expected rise on challenge with antigen. Nonetheless the animals reacted with respiratory distress and convulsions. The findings indicate that the prostaglandins are not the major mediators of anaphylactic bronchoconstriction in the guinea-pig.     

Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Conformational characterization of peptides rich in the cycloaliphatic Cα,α-disubstituted glycine 1-amino-cyclononane-1-carboxylic acid

A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C^α,α,-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac₉c) and two Ala/Ac₉c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and ¹H-NMR. The molecular structures of the amino acid derivatives mClAc-Ac₉c-OH and Z-Ac₉c-OtBu, the dipeptide pBrBz-(Ac₉c)₂-OtBu, the tetrapeptide Z-(Ac₉c)₄-OtBu, and the pentapeptide Z-( Ac₉c)₅-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac₉c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac₉c residue is a strong β-turn and helix former. A comparison with the structural propensity of α-aminoisobutyric acid, the prototype of C^α,α-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3−8) is made and the implications for the use of the Ac₉c residue in conformationally constrained analogues of bioactive peptides are briefly examined. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci. 3: 367–382 No. of Figures: 10. No. of Tables: 6. No. of References: 62     

Inhibitory effect of β-diketones and their metal complexes on TNF-α induced expression of ICAM-1 on human endothelial cells

Recent reports show that the natural β-diketone curcumin displays important biological properties regarding the intercellular adhesion molecule-1 (ICAM-1), which plays a critical role in the immune responses and inflammation. In this study the ICAM-1 inhibitory activity of β-diketone compounds, which are curcumin models lacking aromatic peripheral hydroxyl and methoxy groups, along with some metal derivatives is investigated. β-Diketones are systematically more active than metal complexes and the best obtained inhibition is 75% for both groups. The best inhibitors are 4-benzoyl-3-methyl-1-phenyl-pyrazol-5-one (HQ^Ph) among the ligands, and sodium benzoylacetonato among metal derivatives. These results appear in line with the reported antitumor activity of related species. Since 4-acyl-5-pyrazolones posses four tautomeric forms, those corresponding to HQ^Ph were investigated using density functional theory. Docking of all HQ^Ph tautomers on ICAM-1 protein was performed suggesting one keto-enol form favored to act in biological environment.     

Effects of P450 inhibition and induction on the olfactory toxicity of β, β′-iminodipropionitrile (IDPN) in the rat

In addition to the neurotoxic effects of β, β′-iminodipropionitrile (IDPN) which have been previously reported by other investigators, the olfactory toxicity of this compound has recently been uncovered in this laboratory. Due to the apparently conflicting observations that the IDPN-induced lesion in the olfactory mucosa is very focal in nature (suggesting site-specific activation) and the observation by other investigators that the behavioral effects of IDPN appear to be due to the parent compound, we initiated studies into the possible role of the cytochrome P450 enzymes in the olfactory toxicity of IDPN. Immunohistochemical studies with antibodies raised against several different P450 isoforms revealed good correlation between IDPN-induced olfactory mucosal degeneration and the localization of a protein immunoreacting with an antibody to P450 2E1. Enzymatic studies revealed that there is approximately fivefold more ρ-nitrophenol hydroxylation activity in the olfactory mucosa than in the liver on a per milligram microsomal protein basis. Administration of 1% acetone in the drinking water increased the levels of olfactory mucosal 2E1, and the increase in enzyme levels corresponded to increased olfactory toxicity of IDPN; inhibition of P450 activities with either metyrapone or carbon tetrachloride eliminated or significantly decreased the olfactory toxicity of IDPN, respectively. These studies suggest a role for cytochrome P450, specifically the 2E1 isoform, in the activation of IDPN within the nasal mucosa.     

Studies on growth hormone secretion VIII effects of α,β-methylene-ATP on prostaglandin induced GH release and cyclic AMP accumulation in rat anterior pituitary glands

α, β-methylene-ATP, a competitive inhibitor of adenylate cyclase of liver and fat cell membrane preparations, caused a dose related inhibition of PGE₁ and PGE₂-induced cyclic AMP accumulation in rat anterior pituitary explants. At the same time, this ATP analog potentiated PGE₁ and PGE₂-promoted growth hormone secretion. The possible functional role of prostaglandins and cyclic nucleotides in the regulation of growth hormone secretion remains to be defined.     

Differential Subcellular Localization of Protein Phosphatase-1 α, γ1, and δ Isoforms during Both Interphase and Mitosis in Mammalian Cells

Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, α, γ1, and δ, have nearly identical catalytic domains, but they vary in sequence at their extreme NH₂ and COOH termini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 α associates with the nuclear matrix, PP-1 γ1 concentrates in nucleoli in association with RNA, and PP-1 δ localizes to nonnucleolar whole chromatin. During mitosis, PP-1 α is localized to the centrosome, PP-1 γ1 is associated with microtubules of the mitotic spindle, and PP-1 δ strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.     

Differential inhibition by α- and β-tocopherol of human erythroleukemia cell adhesion: role of integrins

The effect of α- and β-tocopherol on human erythroleukemia cell (HEL) adhesion induced by phorbol 12-myristate 13-acetate (PMA) has been studied. Adhesion induced by PMA stimulation was prevented by 44.5% by physiological concentrations of α-tocopherol. Under the same experimental conditions, β-tocopherol, an analogue of α-tocopherol, produced 11% inhibition of adhesion. Cell response gradually increased from 0 to 24 h of α-tocopherol treatment. Only a slight time dependency of β-tocopherol inhibition was observed. Another human erythroleukemia cell line (K562) and the human monocyte tumor cell line U937 showed 5.0 and 11.2% inhibition, respectively. Similar to α-tocopherol, the protein kinase C inhibitor, Calphostin C, and the MAPK inhibitor, PD98059, prevented PMA-induced cell adhesion. An inhibition of ERK-1 phosphorylation was observed for α-tocopherol only in HEL, implying that MAP kinase pathway is involved in this cell line. Fluorescence-activated cell sorting (FACS), by using various integrin-specific monoclonal antibodies, has shown that α (1–6), β1, and αv integrins are less expressed at the cell surface after α-tocopherol treatment. Beta-tocopherol treatment was less effective.