Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

Characterization of β-endorphin- and α-MSH-related peptides in rat heart

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that β-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to β-endorphin- and α-MSH-related peptides. Ion exchange HPLC analysis revealed that β-endorphin(1–31) was further processed to α-N-acetyl-β-endorphin(1–31), which comprised 35.9 ± 0.1% of total immunoreactivity, and smaller amounts of β-endorphin(1–27), β-endorphin(1–26), and their α-N-acetylated derivatives. The predominant α-MSH immunoreactive peptides coeluted with α-MSH and N,O-diacetyl-α-MSH by reverse-phase HPLC, although small amounts of ACTH(1–13)-NH₂ were also present. Thus, multiple forms of β-endorphin and α-MSH are localized in rat heart. β-Endorphin(1–31) is a minor constituent, however, indicating that nonopioid β-endorphin peptides predominate.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Intrinsic ligand binding properties of the human and bovine α,-interferon receptors

The Type I interferon receptor (IFN-αR) interacts with all IFN-αs, IFN-β and IFN-ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-αA and IFN-αB. Thus, the IFN-αR1 polypeptide most likely plays a direct role in ligand binding.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Factors influencing α-tocopherol synthesis in pepper fruits

Determination of vitamin E in the fruit pericarp of green, yellow and red varieties of Capsicum annuum L. from the local market points to a parallel accumulation in pepper fruits of α-tocopherol with secondary carotenoids and triacylglycerols enriched in unsaturated fatty acids. Highest α-tocopherol concentrations of about 400 nmol per g of dry weight have been found in red fruits. Ripe yellow and red pepper fruits grown under greenhouse conditions were smaller and contained lower α-tocopherol contents than corresponding ones from the local market. An approximation to the α-tocopherol levels in market fruits has been observed, however, if the green plants had been treated with the bleaching herbicide norflurazone before fruit ripening, affecting the carotenoid pathway. Optimum herbicide efficiency has been obtained via watering of the green plants. In ripe fruits of the yellow and red varieties α-tocopherol contents were paralleled by increasing γ-tocopherol methyltransferase activities. In chromoplast preparations from pepper, methylation capacities have been found for γ- and δ-tocopherol as well as for the structurally related tocotrienols, the diterpene side chain of which consists of a geranylgeranyl- instead of the reduced phytyl residue found in tocopherols. β-Tocopherol was not methylated, which supports the position-specific methylation of prenylquinones at the 5 position of the tocopherol aromatic headgroup.     

5α,6α-Epoxy-cholestan-3β-ol (cholesterol α-oxide): A specific substrate for rat liver glutathione transferase B

A semi-micro assay was developed for the conjugation of 5α,6α-epoxy-cholestan-3β-ol (cholesterol α-oxide) with glutathione. The soluble supernatant of rat liver homogenate catalysed the reaction at a rate of 0.2–0.5 pmol.min⁻¹ .mg protein⁻¹ with 4μM cholesterol α-oxide, while the reaction in the presence of GSH alone was barely detectable. Enzymic activity in the soluble supernatant was due equally to the two forms of glutathione transferase B (100 pmol.min⁻.mg protein⁻¹), glutathione transferases AA, A, C and E being unreactive. The activity of purified glutathione transferase B was about 5-times that expected from the activity of the soluble supernatant. Complex enzyme kinetics were obtained suggestive of substrate inhibition.     

Enzymatic synthesis of α- -fucosyl-N-acetyllactosamines and 3′-O-α- -fucosyllactose utilizing α- -fucosidases

An α- -fucosidase from porcine liver produced α- -Fuc-(1→2)-β- -Gal-(1→4)- -GlcNAc (2′-O-α- -fucosyl-N-acetyllactosamine, 1) together with its isomers α- -Fuc-(1→3)-β- -Gal-(1→4)- -GlcNAc (2) and α- -Fuc-(1→6)-β- -Gal-(1→4)- -GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl α- -fucopyranoside and β- -Gal-(1→4)- -GlcNAc. The enzyme formed the trisaccharides 1–3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. α- -fucosidase led to the regioselective synthesis of trisaccharides containing a (1→3)-linked α- -fucosyl residue. When β- -Gal-(1→4)- -GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and α- -Fuc-(1→3)-β- -Gal-(1→4)- -Glc (3′-O-α- -fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

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.     

Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

α1-Antitrypsin and α2-macroglobulin do not inhibit the kinin-releasing activity of kallikreins from human urine and saliva

α₁-Antitrypsin, α₂-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva.α₁-Antitrypsin and α₂-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did non inhibit or bind with urinary and salivary kallikreins.     

The α2δ subunit augments functional expression and modifies the pharmacology of CaV1.3 L-type channels

The auxiliary Ca_Vα₂δ-1 subunit is an important component of voltage-gated Ca²⁺ (Ca_V) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca_V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca_Vα₂δ-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca_V1.3 channels in HEK-293 cells. Co-expression of Ca_Vα₂δ-1 significantly increased macroscopic currents and conferred the Ca_V1.3α₁/Ca_Vβ₃ channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca_Vα₂δ-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane δ domain of the protein, reduced functionality in terms of enhancement of Ca_V1.3α₁/Ca_Vβ₃ currents. In addition, co-expression of the δ domain drastically inhibited macroscopic currents through recombinant Ca_V1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca_Vα₂δ-1 auxiliary subunit may interact with Ca_V1.3 channels and regulate their functional expression.