A comparison of the in vitro binding of α-tocopherol to microsomes of lung,liver, heart and brain of the rat

The in vitro binding of α-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of α-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of α-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for α-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain > liver ≈ heart > lung. The differences in affinity did not correlate with total fatty acid content (r = − 0.39), total unsaturated fatty acid content (r = − 0.26), or with the content of fatty acids containing two or more double bonds (r = − 0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r = + 0.96). Since lung microsomes contain approx. 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.     

Antimicrobial activity and mechanism of action of a novel cationic α-helical dodecapeptide,a partial sequence of cyanate lyase from rice

CL(14-25), a dodecapeptide, that is a partial region near N-terminus of cyanate lyase (CL, EC 4.3.99.1) from rice (Oryza sativa L. japonica), contains three arginine and two lysine residues. It was a novel cationic α-helical antimicrobial peptide. The antimicrobial activity of CL(14-25) against Porphyromonas gingivalis, a periodontal pathogen, was quantitatively evaluated by a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentration of CL(14-25) against P. gingivalis cells was 145 μM. CL(14-25), even at a concentration of 800 μM, had no hemolytic activity. When giant unilamellar vesicles (GUVs) that mimic the membrane composition of Gram-negative bacteria were used, microscopy image analysis suggested that CL(14-25) disrupted GUVs in a detergent-like manner. Therefore, CL(14-25) appears to exhibit antimicrobial activity through membrane disruption. To investigate the contribution of cationic amino acid residues in CL(14-25) to its antimicrobial activity, we synthesized four truncated CL analogs, in which one or two cationic amino acid residues were deleted from the N- and C- termini of CL(14-25). The degrees of calcein leakage from large unilamellar vesicles (LUVs) and 3,3′-dipropylthiadicarbocyanine iodide (diSC₃-5) release from P. gingivalis cells induced by truncated CL analogs were closely related to their antimicrobial activities. CL analogs, which were truncated by removing an arginine residue from the N-terminus and a lysine residue from the C-terminus maintained their antimicrobial activity. However, CL analogs, which were further truncated by removing two arginine residues from the N-terminus, and an arginine and a lysine residue from the C-terminus, rarely exhibited antimicrobial activity.     

Identification and expression analyses of a novel serotonin receptor gene, 5-HT2β, in the field cricket, Gryllus bimaculatus

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2β gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2β gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.     

Molecular cloning,expression, and biochemical characterization of a novel cold-active α-amylase from Bacillus sp. dsh19-1

A novel gene (ANK58566) encoding a cold-active α-amylase was cloned from marine bacterium Bacillus sp. dsh19-1 (CCTCC AB 2015426), and the protein was expressed in Escherichia coli. The gene had a length of 1302 bp and encoded an α-amylase of 433 amino acids with an estimated molecular mass of 50.1 kDa. The recombinant α-amylase (AmyD-1) showed maximum activity at 20 °C and pH 6.0, and retained about 35.7% of activity at 4 °C. The AmyD-1 activity was stimulated by Ca²⁺ and Na⁺. However, the chelating agent, EDTA, inactivated the enzyme. Moreover, AmyD-1 displayed extreme salt tolerance, with the highest activity in the presence of 2.0 M NaCl and 60.5% of activity in 5.0 M NaCl. The K_m, V_max and k_cat of AmyD-1 in 2.0 M NaCl were 2.8 mg ml⁻¹, 21.8 mg ml⁻¹ min⁻¹ and 933.5 s⁻¹, respectively, at 20 °C and pH 6.0 with soluble starch as substrate. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) revealed that the end products of starch hydrolysis by AmyD-1 were glucose, maltose, maltotriose, maltotetraose, and malt oligosaccharides. Thus, AmyD-1 is one of the very few α-amylases that can tolerate low temperatures and high salt concentrations, which makes it to be a potential candidate for research in basic and applied microbiology.

     

Angiopeptin,a somatostatin-14 analogue,decreases adhesiveness of rat Leukocytes to unstimulated and IL-1β-activated rat heart endothelial cells

We have previously demonstrated that somatostatin-14 and its octapeptide analogue, angiopeptin, decrease the ability of rat heart endothelial cells to bind leukocytes [Leszczynski, et al., Reg. Pept. 43 (1993) 131–140]. Here, we examined whether exposure of leukocytes to angiopeptin modifies their adhesiveness to the unstimulated and to IL-1β-activated endothelium. Monolayers of unstimulated endothelial cells bind 274 ± 12 leukocytes/mm². Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 μM) reduced significantly (p < 0.05) adhesion of leukocytes from 274 ± 12 to 188 ± 10, 185 ± 8 and 172 ± 3 cells/mm², respectively. Stimulation of endothelial cells with IL-1β (100U/ml) for 24 hours increased endothelial adhesiveness from 274 +- 12 to 381 ± 17 adhering leukocytes/mm². Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1μM) reduced significantly (p < 0.05) binding of leukocytes to IL-1β-activated endothelium from 381 ± 17 to 237 ± 8, 254 ± 11 and 248 ± 13 cells/mm², respectively. Angiopeptin had no effect on the expression of lymphocyte function-associated molecule-1 (LFA-1; CDlla/CD18) by leukocytes, as assessed by flow cytometry. This suggests that angiopeptin modulates adhesive properties of leukocytes by (1) altering the expression of other than LFA-1 adhesion molecule(s) and/or (2) modulating the affinity of adhesion molecule(s) expressed by leukocytes. In conclusion, our results demonstrate that angiopeptin reduces leukocyte adhesiveness to unstimulated and to IL-1β-activated endothelium. It suggests that angiopeptin may suppress immune response via modulation of the leukocyte-endothelial interaction.     

Purification and characterization of AHPM,a novel non-hemorrhagic P-IIIc metalloproteinase with α-fibrinogenolytic and platelet aggregation-inhibition activities,from Agkistrodon halys pallas venom

A novel non-hemorrhagic metalloproteinase, AHPM, was purified from the venom of Agkistrodon halys pallas by a combination of ion-exchange and gel filtration chromatography. AHPM is a dimeric glycoprotein with multiple pIs around pH 7.9 and has a molecular mass of 110 kDa with two blocked N-terminuses. Partial sequence of AHPM obtained by LC-MS/MS analysis together with its dimeric nature reveals that it is a P-IIIc snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. AHPM has a conserved DECD sequence in the disintegrin-like domain. AHPM hydrolyzes casein and fibrinogen and also dissolves fibrin clots and the proteolytic activity is abolished by EDTA, but not by PMSF, suggesting that it is a metalloproteinase. The protease hydrolyzes rapidly the Aα-chain of fibrinogen followed by the Bβ-chain and does not cleave the γ-chain. AHPM contains endogenous Zn²⁺ and Ca²⁺ ions at a molar ratio of 1:1.9 and 1:4.2, respectively, and Zn²⁺ ions are essential for its proteolytic activity. AHPM inhibits collagen-and ADP-induced platelet aggregation with half maximal inhibitory concentrations of 200 ± 8 nM and 280 ± 10 nM, respectively. EDTA markedly attenuates the inhibition of ADP-induced platelet aggregation by AHPM, indicating that the fibrinogenolytic activity of AHPM is involved in its inhibition of ADP-induced platelet aggregation. AHPM is devoid of hemorrhagic activity when injected (up to 30 μg) subcutaneously into mice. AHPM is so far identified as first non-hemorrhagic P-IIIc SVMP which has both fibrinolytic and platelet aggregation-inhibition activities. The bifunctional enzyme may have a potential clinical application as a thrombolytic agent.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

Synthesis of a novel class of glycocluster with a cyclic α-(1→6)-octaglucoside as a scaffold and their binding abilities to concanavalin A

The synthesis of small glycoclusters with high affinity toward lectins is one of the important subjects in glycotechnology. Although cyclic α-(1→6)-d-octaglucoside (CI8) is an attractive scaffold on which to put glycosyl pendants, the compound has only secondary hydroxyl groups, which are relatively unreactive for substitution reactions. The oxidation of the vicinal diols of CI8 and reductive amination of the resultant dialdehydes with 2-aminoethyl mannoside gave mannose-CI8 conjugates with a variety of average mannose incorporation numbers (2-7). The average numbers were deduced from MALDI-TOF mass and ¹H NMR spectroscopy. The binding ability of mannose-CI8 conjugates to concanavalin A increased with the increasing numbers of average mannose incorporation, reaching a plateau at tetravalence, as estimated from a latex bead-based agglutination lectin assay. Toxicity tests demonstrated the biocompatibility of mannose-CI8 conjugates.     

Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3β to unravel a novel C-terminal scaffold-binding region

The specificity and regulation of GSK3β are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3β docking and appeared that GSK3β Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3β and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3β through the single-point mutation of four corresponding sites within GSK3β (residues 260–300) as scaffold-binding region I (designated SBR-I_260–300). Our data showed that these three binding proteins shared similar binding sites on GSK3β. We also found that the binding of GSK3β V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3β L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3β. Taken together, our data revealed that in addition to the core kinase domain, SBR-I_260–300, another novel C-terminus helix region, designated SBR-II_339–383, also appeared to participate in the recognition and specificity of GSK3β in binding to other specific proteins.     

Antibodies directed to restricted sequences of the c-Erb a α hinge domain interfere with hormone or dna binding to recombinant α-type triiodothyronine receptor (c-Erb a α1) and detect structural changes

Abstract

In a previous study we showed that antibodies directed to restricted sequences in the hinge domain of α-type triiodothyronine (T3) receptor (TR) (aminoacids 150-166, 172-191, 144-162) selectively recognized and immunoprecipitated α-type TR in tissues. Furthermore, antibodies to peptide 172-191 (anti-α 172) strongly impaired T3 binding to natural TR α. To get more precise informations on the ability of these antibodies to recognize the TR, alter TR properties and/or detect conformational changes in TR, TR αl was produced in E. coli as a non-mutated, non-fusion protein from a rat c-erb A α1 cDNA inserted into pTrc99A vector. The recombinant c-Erb A αl protein, after solubilization with 5 M guanidine and progressive refolding, presented the main characteristics of TR α: unique or largely dominant band of 46 KDa in Western blots with the different anti-c-Erb A α antibodies; binding to DNA and to T3. Binding to DNA was markedly attenuated by anti-α 144 but not by anti-α 150 and anti-α 172. Binding to T3 was modified by anti-α 150 and -α 172 with different characteristics whether recombinant TR was previously bound to T3 or not, and with marked differences in comparative studies with natural TR. When liganded to T3, recombinant and natural TR αl present the same pattern of interaction with both antibodies: immunoprecipitation without any dissociation of T3 by anti-α 150; marked dissociation of bound T3 by anti-α 172. By contrast unliganded recombinant and natural TR are oppositely altered by these antibodies in their ability to bind T3: strong impairment restricted to anti-α 172 for natural TR, and to anti-α 150 for recombinant TR. Anti-α 144 did not interfere. These results lay emphasis on: 1/ the existence and biological relevance of different conformational states of TR α hinge domain, particularly whether TR is liganded or not to T3 and whether it is in a nuclear environment or bacterially-produced; 2/ an important role of the C-terminal part of hinge domain for efficient hormone binding, this involving a region that overlaps the α 150 and α 172 sequences.     

Fusion of Epstein-Barr virus with epithelial cells can be triggered by αvβ5 in addition to αvβ6 and αvβ8, and integrin binding triggers a conformational change in glycoproteins gHgL

Fusion of herpesviruses with their target cells requires a minimum of three glycoproteins, namely, gB and a complex of gH and gL. Epstein-Barr virus (EBV) fusion with an epithelial cell requires no additional virus glycoproteins, and we have shown previously that it can be initiated by an interaction between integrin αvβ6 or αvβ8 and gHgL. We now report that integrin αvβ5 can also bind to gHgL and trigger fusion. Binding of gHgL to integrins is a two-step reaction. The first step, analyzed by surface plasmon resonance, was fast, with high association and low dissociation rate constants. The second step, detected by fluorescence spectroscopy of gHgL labeled at cysteine 153 at the domain I-domain II interface with the environmentally sensitive probes acrylodan and IANBD, involved a slower conformational change. Interaction of gHgL with neutralizing monoclonal antibodies or Fab' fragments was also consistent with a two-step reaction involving fast high-affinity binding and a subsequent slower conformational change. None of the antibodies bound to the same epitope, and none completely inhibited integrin binding. However, binding of each decreased the rate of conformational change induced by integrin binding, suggesting that neutralization might involve a conformational change that precludes fusion. Overall, the data are consistent with the interaction of gHgL with an integrin inducing a functionally important rearrangement at the domain I-domain II interface.     

Determination of a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist (LY300164) and its N-acetyl metabolite in mouse,rat, dog,and monkey plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor antagonist LY300164 (compound I) and its N-acetyl metabolite (compound II) in plasma was developed. The assay utilized solid-phase extraction on a C₁₈ Bond Elut cartridge followed by reversed-phase HPLC with UV detection at 310 nm. The method exhibited a large linear range from 0.05 μg/ml to 50 μg/ml with an intra-sassay accuracy for compound I and compound II ranging from 89.0% to 114.5% and intra-assay precision ranging from 0.5 to 15.3% in mouse, rat, dog, and monkey plasma. The inter-assay accuracy of compound I and compound II was 93.3% to 101.8% and the inter-assay precision was 1.6% to 11.2% in dog plasma. The lower limit of quantitation was 0.05 μg/ml for compound I in plasma from all species tested. The lower limit of quantitation for compound II was 0.05 μg/ml in dog and monkey plasma and 0.1 μg/ml in mouse and rat plasma. Extracts of compound I and II from dog plasma were shown to be stable for 24 h at room temperature, and both compounds were stable when spiked into rat and monkey plasma frozen at −70°C for 27 days. The method has shown to be useful in the investigation of the pharmacokinetics of the parent compound (I) and metabolite (II) in preclinical studies.     

NMK-BH2, a novel microtubule-depolymerising bis (indolyl)-hydrazide-hydrazone,induces apoptotic and autophagic cell death in cervical cancer cells by binding to tubulin at colchicine – site

BackgroundMicrotubules, the key components of the eukaryotic cytoskeleton and mitotic spindle, are one of the most sought-after targets for cancer chemotherapy, especially due to their indispensible role in mitosis. Cervical cancer is a prevalent malignancy among women of developing countries including India. In spite of the remarkable therapeutic advancement, the non-specificity of chemotherapeutic drugs adversely affect the patients' survival and well-being, thus, necessitating the quest for novel indole-based anti-microtubule agent against cervical cancer, with high degree of potency and selectivity.MethodsFor in vitro studies, we used MTT assay, confocal microscopy, fluorescence microscopy, flow cytometry and Western blot analysis. Study in cell free system was accomplished by spectrophotometry, fluorescence spectroscopy and TEM and computational analysis was done by AutodockTools 1.5.6.ResultsNMK-BH2 exhibited significant and selective anti-proliferative activity against cervical cancer HeLa cells (IC₅₀ = 1.5 μM) over normal cells. It perturbed the cytoskeletal and spindle microtubules of HeLa cells leading to mitotic block and cell death by apoptosis and autophagy. Furthermore, NMK-BH2 targeted the tubulin-microtubule system through fast and strong binding to the αβ-tubulin heterodimers at colchicine-site.ConclusionThis study identifies and characterises NMK-BH2 as a novel anti-microtubule agent and provides insights into its key anti-cancer mechanism through two different cell death pathways: apoptosis and autophagy, which are mutually independent.General significanceIt navigates the potential of the novel bis (indolyl)-hydrazide-hydrazone, NMK-BH2, to serve as lead for development of new generation microtubule-disrupting chemotherapeutic with improved efficacy and remarkable selectivity towards better cure of cervical cancer.     

Effect of U50,488H, a κ-opioid receptor agonist on myocardial α-and β-myosin heavy chain expression and oxidative stress associated with isoproterenol-induced cardiac hypertrophy in rat

Both oxidative stress and β-MHC expression are associated with pathological cardiac hypertrophy. β-adrenergic receptor stimulation plays an important role in cardiac hypertrophy. Recent studies have reported a negative interplay between opioid receptors and adrenoceptors in heart. This study investigated the effect of U50,488H (a selective κ-opioid receptor agonist) on myocardial oxidative stress and α- and β-MHC expression in isoproterenol-induced cardiac hypertrophy. Male Wistar rats were administered normal saline (control), isoproterenol (ISO) (5 mg/kg BW s.c. OD), and isoproterenol with U50,488H (0.4 and 0.6 mg/kg BW, i.p. OD) for 14 days. In a separate group, nor-binaltorphimine (nor-BNI) (0.5 mg/kg, BW, i.p.) (κ-receptor antagonist) was administered along with ISO and U50,488H. ISO administration caused significant increase in left ventricular (LV) wall thicknesses, LV mass in echocardiography, heart weight to body weight ratio, and myocyte size as compared to control. Both the doses of U50,488H offered significant protection against these changes. The higher dose of U50,488H significantly prevented ISO-induced increase in myocardial lipid peroxidation and depletion of myocardial antioxidants (glutathione, superoxide dismutase, and catalase), while a similar trend (although not significant) was observed with the lower dose also. ISO-induced myocardial fibrosis was also significantly attenuated by both the doses of U50,488H. Isoproterenol-induced β-MHC expression in the hypertrophied heart was not altered by either doses of U50,488H, however, the latter prevented the loss of myocardial α-MHC expression. All these effects of U50,488H were blocked by nor-BNI. This study provides the evidence that U50,488H reduced oxidative stress and preserved expression of α-MHC in isoproterenol-induced cardiac hypertrophy.