Mechanisms involved in α6β1-integrin-mediated Ca signalling

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca²⁺ signalling. Both Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and capacitative Ca²⁺ entry via Ca²⁺ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca²⁺ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca²⁺-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP₃) and cADPR.     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

C nuclear magnetic resonance study of 17α-substituted estradiols

We report the ¹³C NMR data for 20 compounds bearing a substituent (alkyl, alkenyl, alkynyl, alkylamide, spiro-γ-lactone, phenyl, benzyl, naphthyl, etc.) at the 17α-position of estradiol. The carbon assignments were done using 1D and 2D NMR experiments (distortionless enhancement by polarization transfer, homonuclear correlated spectroscopy, heteronuclear shift correlation, and heteronuclear shift correlation via long-range couplings). Only the chemical shifts of carbons 12–18, which surround the substitution site, were affected by the addition of a substituent. The magnitude of the effects (shielding or deshielding) was influenced by the 17α-substituent. The individual effects at these carbons were sufficiently distinctive to identify specific centers and should be valuable for signal assignment of a variety of 17α-derivatives of estradiol. In addition to carbon-skeleton assignment, we also report the carbon-substituent assignments.     

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.     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

17α-Methyl testosterone is a competitive inhibitor of aromatase activity in Jar choriocarcinoma cells and macrophage-like THP-1 cells in culture

17α-Methyl testosterone is a synthetic androgen with affinity for the androgen receptor. 17α-Methyl testosterone is used widely as a component of hormone replacement therapy. Previous reports have indicated that contrary to testosterone, 17α-methyl testosterone is not aromatized. However, 17α-methyl testosterone still could affect local estrogen formation by regulating aromatase expression or by inhibiting aromatase action. Both possibilities have important clinical implications. To evaluate the effect of 17α-methyl testosterone on the expression and activity of aromatase, we tested the choriocarcinoma Jar cell line, a cell line that express high levels of P₄₅₀ aromatase, and the macrophage-like THP-1 cells, which express aromatase only after undergoing differentiation. We found that in both cell lines, 17α-methyl testosterone inhibits aromatase activity in a dose-related manner. The curve of inhibition parallels that of letrozole and gives complete inhibition at 10⁻⁴ M 17α-methyl testosterone, determined by the tritium release assay. 17α-Methyl testosterone does not have detectable effects on aromatase RNA and protein expression by Jar cells. Undifferentiated THP-1 cells had no aromatase activity and showed no effect of 17α-methyl testosterone, but differentiated THP-1 (macrophage-like) cells had a similar inhibition of aromatase activity by 17α-methyl testosterone to that seen in Jar cells. The Lineweaver–Burke plot shows 17α-methyl testosterone to be a competitive aromatase inhibitor. Our results show for the first time that 17α-methyl testosterone acts as an aromatase inhibitor. These findings are relevant for understanding the effects of 17α-methyl testosterone as a component of hormone replacement therapy. 17α-Methyl testosterone may, as a functional androgen and orally active steroidal inhibitor of endogenous estrogen production, also offer special possibilities for the prevention/treatment of hormone-sensitive cancers.     

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.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots: fate of 6α- and 6β-hydrogens of lathosterol

The fate of 6α- and 6β-hydrogens of lathosterol during the transformation into 20-hydroxyecdysone was chased by feeding [3α,6β-²H₂]- and [3α,6α-²H₂]-lathosterols to hairy roots of Ajuga reptans var. atropurpurea. The behavior of 6β-hydrogen, which mostly migrated to the C-5 position of 20-hydroxyecdysone, was in agreement with that of C-6 hydrogen of cholesterol. The results strongly supported the view that cholesterol and lathosterol are first metabolized into 7-dehydrocholesterol, which is then converted into 20-hydroxyecdysone via 7-dehydrocholesterol 5α,6α-epoxide in the hairy roots.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

The m6A methyltransferase METTL3 modifies PGC-1α mRNA promoting mitochondrial dysfunction and oxLDL-induced inflammation in monocytes

Mitochondrial biogenesis and energy metabolism are essential for regulating the inflammatory state of monocytes. This state is partially controlled by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a coactivator that regulates mitochondrial biogenesis and energy metabolism. Disruption of these processes can also contribute to the initiation of chronic inflammatory diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis. Methyltransferase-like 3 (METTL3)-dependent N⁶-methyladenosine (m⁶A) methylation has recently been shown to regulate a variety of inflammatory processes. However, the role of m⁶A mRNA methylation in affecting mitochondrial metabolism in monocytes under inflammation is unclear, nor is there an established relationship between m⁶A methylation and PGC-1α. In this study, we identified a novel mechanism by which METTL3 acts during oxidized low-density lipoprotein (oxLDL)-induced monocyte inflammation, where METTL3 and YTH N⁶-methyladenosine RNA binding protein 2 (YTHDF2) cooperatively modify PGC-1α mRNA, mediating its degradation, decreasing PGC-1α protein levels, and thereby enhancing the inflammatory response. METTL3 coordinated with YTHDF2 to suppress the expression of PGC-1α, as well as that of cytochrome c (CYCS) and NADH:ubiquinone oxidoreductase subunit C2 (NDUFC2) and reduced ATP production and oxygen consumption rate (OCR). This subsequently increased the accumulation of cellular and mitochondrial reactive oxygen species (ROS) and the levels of proinflammatory cytokines in inflammatory monocytes. These data may provide new insights into the role of METTL3-dependent m⁶A modification of PGC-1α mRNA in the monocyte inflammation response. These data also contribute to a more comprehensive understanding of the pathogenesis of monocyte-macrophage inflammation-associated diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis.     

Analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors using liquid chromatography–electrospray mass spectrometry

Employing high-performance liquid chromatography–electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC–MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC₅₀ values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.     

Metal-ion-dependent hydrophobic-interaction chromatography of α-lactalbumins

α-Lactalbumins from bovine, human, goat, sheep, and horse milk bind to phenyl-Sepharose in the presence of EDTA and can be eluted by addition of Ca²⁺ (0.001–100 m ). This property has been utilized to purify these proteins in a one-step purification from milk whey. α-Lactalbumin purified in this manner has the same ultraviolet and proton nuclear magnetic resonance spectra as that purified by other methods. Using binding to phenyl-Sepharose as an assay, the conformation of bovine α-lactalbumin upon the addition of several metal ions that are known to interact with this protein was investigated. Lanthanides, Mn²⁺, Mg²⁺, and Cd²⁺ can substitute for Ca²⁺, whereas Zn²⁺, Al³⁺, and Co²⁺ cannot. Surprisingly, whereas lower concentrations of La³⁺, Mn²⁺, and Cd²⁺ (1 m and less) caused elution from the hydrophobic support, higher concentrations (10 m ) were ineffective. These observations can be rationalized assuming the presence of two distinct metal-ion binding sites with different specificities.     

Estradiol -17-β enhances the formation of {H}-PGF2α from {H}-PGE2 in the uterus isolated from ovariectomized rats

Formation of {³H}-PGF_2α and {³H}-13,14,dihydro-15-keto-PGF_2α from {³H}-PGE₂ by the supernatant of uterine homogenates from estrous and ovariectomized rats, was studied, using the reaction system PGE₂ + NADPH + {³H}-PGE₂ + supernatant. Enzymatic conversion was lower in uterine supernatants from spayed rats than in uterine homogenates of rats at natural estrus.Spayed animals were injected with progesterone (P) or with estradiol-17-β (E₀) at a dose of 1.0 or 50.0 ug. Conversion of {³H}-PGF_2α to {³H}-PGE₂ or to {³H}-13,14,dihydro-15-keto-PGF_2α did not differ in control ovariectomized or ovariectomized rats receiving P or 1.0 ug E₀. However, 50.0 ug E₀ induced a significant oversion after 30 (P < 0.01) and 60 (P < 0.001) min of incubation.It is concluded that E₀, at the 50.0 ug dose, but not the 1.0 ug dose of E₀, nor progesterone, stimulated conversion of {³H}-PGE₂ into {³H}-PGF_2α or {³H}-13, 14,dihydro-15-keto-PGF_2α, presumably through the activity of the enzyme PGE₂-9-keto-reductase.     

Assembly of the CD8α/p56 protein complex in stably expressing rat epithelial cells

We have previously characterized the biogenesis of the human CD8α protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8α/p56^lck protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56^lck, or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8α, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8α established hetero-oligomeric complexes with p56^lck, as revealed by co-immunoprecipitation assays performed with anti-CD8α antibody. Moreover, p56^lck bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56^lck binds rapidly to CD8α and most of the p56^lck is bound to CD8α at steady state; (2) CD8α/p56^lck protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56^lck reaches the cell surface.     

β1 Integrins Mediate Chondrocyte Interaction with Type I Collagen, Type II Collagen, and Fibronectin

Chondrocytes isolated from the cephalic region of sterna from 14-day-old chick embryos used β1 integrins and required either Mg²⁺ or Mn²⁺ for attachment to plates coated with type I collagen, type II collagen, and fibronectin. β1 integrin was concentrated in adhesion plaques of the chondrocytes plated on type I collagen, type II collagen, and fibronectin substrates. Chondrocytes expressed at least 3 α-subunits, including α3, α5, and putative α2. α5, but not α3, had a higher molecular weight in chondrocytes than in fibroblasts. Levels of α3 and α5 were about 25-30% of that in fibroblasts. When the chondrocytes were cultured in the presence of ascorbate in suspension, the cells aggregated into clusters. This aggregation was dependent on β1 integrin and type II collagen.     

Solution structure investigations of olefin Pd(II) and Pt(II) complex ion pairs bearing α-diimine ligands by F, H-HOESY NMR

Complexes [M(η¹,η²-C₈H₁₂OMe)((2,6-(R)₂---C₆H₃)N=C(R′)---C(R′)=N((2,6-(R)₂---C₆H₃))]PF₆ (where M=Pd, R=H and R′₂=Me₂ (1), M=Pd, R=Me and R′₂=Me₂ (2), M=Pd, R=Et and R′₂=Me₂ (3), M=Pd, R=iPr and R′₂=Me₂ (4), M=Pd, R=iPr and R′₂=An (5), M=Pt, R=iPr and R′₂=An (6)) were synthesized by the reaction of [M(η¹,η²-C₈H₁₂OMe)Cl]₂ with the appropriate α-diimine ligand in the presence of NH₄PF₆. Their ion pair structure in solution was investigated by detecting dipolar interactions between protons belonging to the cation and fluorine nuclei of the anion (interionic contacts) in the ¹⁹F, ¹H-HOESY NMR spectra. In complexes 1–4, the anion in solution is located close to the peripheral protons of the α-diimine ligand and it interacts with the R′ protons and with the R protons that point toward the R′ groups. The steric protection of apical position exerted by the R substituents is clearly illustrated by the absence of interionic contacts between any protons of the cycloctenylmethoxy-moiety and the anion for R≥Me in 1–4. In complexes 5 and 6 the interactions between the anion and the peripheral N,N protons also predominate but other anion–cation orientations are significantly present and, consequently, the interionic structure is less specific.