Receptor binding characteristics and cytotoxicity of insulin-methotrexate

AIM:To characterize the receptor binding affinity andcytotoxicity of insulin-methotrexate (MTX) for the potentialutilization of insulin as carriers for carcinoma target drugs.METHODS:MTX was covalently linked to insulin.Insulin-MTX conjugate was purified by Sephadex G-25 columnand analyzed by high performance liquid chromatography.Hepatocellular carcinoma cell membrane fractions wereisolated by sucrose density gradient centrifugation.Competitive displacement of ~(125)I-insulin with insulin andinsulin-MTX binding to insulin receptors were carried out.Cytoreductive effect of insulin-MTX on human hepatomaBEL7402 cells and human hepatocyte cell line HL7702 wasevaluated using the MTT assay.RESULTS:Insulin-MTX competed as effectively as insulinwith ~(125)I-insulin for insulin receptors.The values of Kd forinsulin-MTX and insulin were 93.82 19.32 nmol/L and5.01 1.24 nmol/L,respectively.The value of Kd for insulin-MTX was significantly increased in comparison with insulin(t=7.2532,n=4,P<0.005).Insulin-MTX inhibited the growthof human hepatoma cells (BEL7402) almost as potently asMTX.The inhibitory effect reached a peak on the 5 th daywhen the growth of cells was inhibited by 79% at aconcentration of 5.0μg/mL insulin-MTX.Treatment with5.0μg/mL of MTX and 5.0μg/mL of insulin-MTX merelyresulted in inhibition of HL7702 cells by 31.5% and 7.8%on the 5 th day.CONCLUSION:Insulin-MTX specifically recognizes insulinreceptors and inhibits the growth of BEL7402 cells.Theseresults suggest that insulin can be used as a carrier inreceptor mediated carcinoma-targeting therapy.     

Role of β-Adrenergic Receptor Subtypes in Lipolysis

In vitro lipolysis stimulated by low (-)-isopre-naline concentrations (30 nM) in epididymal white adipo-cytes from Sprague-Dawley rats was inhibited at least 60–80% by the specific ₁-antagonists LK 204-545 and CGP 20712A (1 M), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via ₁-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for ₁-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, ₃-adrenergic receptors were fully activated and were the dominant -adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the ₁-antagonists, demonstrating that the ₃-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively ₂-selective agonist formoterol in the presence of ₁-blockade (1 M CGP 20712A) demonstrated the inability of the ₂-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 1 M. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (500-fold lower than its ₂-adrenergic receptor pA ₂, 7.80 ± 0.21), suggesting that formoterol was not acting via ₂-adrenergic receptors. These data are consistent with ₁-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that ₂-adrenergic receptors play no obvious direct role in mediating -adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a ₃-selective antagonist), was found to be a non-selective antagonist at the three -adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.     

Protein Kinase Activity of Phosphoinositide 3-kinase Regulates-adrenergic Receptor Endocytosis

Phosphoinositide3-kinase(PI(3)K)isauniqueenzymecharacterizedbybothlipidandproteinkinaseactivities.Here,wedemonstratearequirementfortheproteinkinaseactivityofPI(3)Kinagonist-dependent-adrenergicreceptor(βAR)internalization.UsingPI(3)Kmutantswitheitherproteinorlipidphosphorylationactivity,weidentifythecytoskeletalproteinnon-muscletropomyosinasasubstrateofPI(3)K,whichisphospho-     

Genetic Variants of Peroxisome Proliferator-Activated Receptor δ Are Associated with Gastric Cancer

Background

Peroxisome proliferator-activated receptors (PPAR) are implicated in pathogenesis of insulin resistance and cancers of the digestive system.

Aim

We investigated the associations of single nucleotide polymorphisms (SNPs) of PPAR δ and γ with gastric cancer and explored interactions with risk factors of gastric cancer.

Methods

We conducted our analysis in a case–control study of 196 gastric cancer patients and 397 controls residing in the Taixing region of Jiangsu, China. Six SNPs in the PPARδ (rs2076167, rs3734254) and PPARγ genes (rs10865710, rs1801282, rs3856806, rs13306747) were genotyped. We employed logistic regression to evaluate the association between each genotype and gastric cancer and tested for gene–environment interaction with Helicobacter pylori (H. pylori) infection, smoking status, and meat and salt intake.

Results

We found that the G/G variant rs2076167, in tight linkage disequilibrium with rs3734254 (R ² = 0.97), was associated with increased risk of gastric cancer in a recessive model (OR 2.20, 95 % CI 1.12, 4.32). The association between G/G variant of rs2016167 and gastric cancer was particularly strong among those with higher salt intake (OR 5.11, 95 % CI 1.11, 23.5), but did not vary by H. pylori infection or smoking status.

Conclusion

We found that genetic variants of PPARδ were associated with gastric cancer. If the association is confirmed in larger studies, it may implicate a role for PPARδ activators, such as insulin-sensitizing agents, in prevention of gastric cancer.     

Interaction of Bacteriophage λ with Its E. coli Receptor,LamB

The initial step of viral infection is the binding of a virus onto the host cell surface. This first viral-host interaction would determine subsequent infection steps and the fate of the entire infection process. A basic understating of the underlining mechanism of initial virus-host binding is a prerequisite for establishing the nature of viral infection. Bacteriophage λ and its host Escherichia coli serve as an excellent paradigm for this purpose. λ phages bind to specific receptors, LamB, on the host cell surface during the infection process. The interaction of bacteriophage λ with the LamB receptor has been the topic of many studies, resulting in wealth of information on the structure, biochemical properties and molecular biology of this system. Recently, imaging studies using fluorescently labeled phages and its receptor unveil the role of spatiotemporal dynamics and divulge the importance of stochasticity from hidden variables in the infection outcomes. The scope of this article is to review the present state of research on the interaction of bacteriophage λ and its E. coli receptor, LamB.     

Diverse Regulation of Cardiac Expression of Relaxin Receptor by α1- and β1-Adrenoceptors

Purpose

Relaxin, a new drug for heart failure therapy, exerts its cardiac actions through relaxin family peptide receptor 1 (RXFP1). Factors regulating RXFP1 expression remain unknown. We have investigated effects of activation of adrenoceptors (AR), an important modulator in the development and prognosis of heart failure, on expression of RXFP1 in rat cardiomyocytes and mouse left ventricles (LV).

Methods

Expression of RXFP1 at mRNA (real-time PCR) and protein levels (immunoblotting) was measured in cardiomyocytes treated with α- and β-AR agonists or antagonists. RXFP1 expression was also determined in the LV of transgenic mouse strains with cardiac-restricted overexpression of α_1A-, α_1B- or β₂-AR. Specific inhibitors were used to explore signal pathways involved in α₁-AR mediated regulation of RXFP1 in cardiomyocytes.

Results

In cultured cardiomyocytes, α₁-AR stimulation resulted in 2–3 fold increase in RXFP1 mRNA (P?<?0.001), which was blocked by specific inhibitors for protein kinase C (PKC) or mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK). Activation of β₁-, but not β₂-AR, significantly inhibited RXFP1 expression (P?<?0.001). Relative to respective wild-type controls, RXFP1 mRNA levels in the LV of mice overexpressing α_1A- or α_1B-AR were increased by 3- or 10-fold, respectively, but unchanged in β₂-AR transgenic hearts. Upregulation by α₁-AR stimulation RXFP1 expression was confirmed at protein levels both in vitro and in vivo.

Conclusions

Expression of RXFP1 was up-regulated by α₁-AR but suppressed by β-AR, mainly β₁-AR subtype, in cardiomyocytes. Future studies are warranted to characterize the functional significance of such regulation, especially in the setting of heart failure.     

Cardioprotective Effect of Angiotensin Ⅱ Receptor Antagonist on Perfused Ischemic Reperfusion Injury of Whole Isolated Rat Hearts

Objectives Investigated the eardioproteetive and mechanisms of losartan on whole isolated isehemie reperfused rat heart. Methods Langendorff perfused systems was used to investigate losartan effect on whole isolated rat hearts in CPK, LDH, MDA, SOD, ang Ⅱ and arrhythmia. Resuits Losartan decreased incidence of arrhythmia,improved atrial ventrieular block recovery in reperfusion period, during isehemie period, CPK and LDH in I/R group increased significantly compared with control group, 51.33±27.02 vs 22.42±13.33, 31.80±4. 56 vs 22.28±15.96, respectively, but greatly decreased in losartan group compared with I/R group,23.90±21.74 vs 51.33±27.02 and 11.50±13.20vs 31.80±4.56, respectively. During reperfusion period CPK, LDH increased significantly in I/R group compared with control group, 49.11 ±20.63 vs 12.14±5.92 and 28.70±4.69 vs 23.10±21.38, respectively, but decreased greatly in losartan group compared with I/R group, 39.40±9.60 vs 49. 11±20.63 and 14.50±13.75 vs 28.70±4.69. The content of MDA, ang II in I/R group myoeytes is higher than control group‘s , 26.± 9. 25 vs 17.2 ±3.37 and 8.43 ±3.81 vs 4.80±0.20. However the content of SOD in two groups has no significantly change, 148.20±8.72 vs 145.08±6.82. the content of MDA in losartan group myocardial tissue is much lower than control group, 15.92±4. 05 vs26.80±9.25 and the content of ang Ⅱ in losartan group myocardial tissue is much higher than I/R group, 12.44±6. 09 vs 8.43±3.21. The department of cardiology of second hospital of Tianjin medical university Tianjin 300211 However, SOD has no significant change in two groups, 143.47±7.91 vs145.08±6.82. Conclusions Losartan against isehemie-reperfusion injury of whole isolated rat hearts, those beneficial effects are mediate primarily by the inhibited of angiotensin Ⅱ binding with its receptor and inhibited oxygen free radical scavenging potential.     

Therapeutic Value of Estrogen Receptor α in Hepatocellular Carcinoma Based on Molecular Mechanisms

The incidence of hepatocellular carcinoma (HCC) is signifi-cantly lower in women than men, implying that estrogen receptors (ERs) may play an important role in ...     

Cardioprotective Effect of Angiotensin n Receptor Antagonist on Perfused Ischemic Reperfusion Injury of Whole Isolated Rat Hearts

Objectives Investigated the cardioprotective and mechanisms of losartan on whole isolated ischemic reperfused rat heart. Methods Langendorff perfused systems was used to investigate losartan effect on whole isolated rat hearts in CPK, LDH, MDA, SOD, ang II and arrhythmia. Results Losartan decreased incidence of arrhythmia, improved atrial ventricular block recovery in reperfu-sion period, during ischemic period, CPK and LDH in I/R group increased significantly compared with control group, 51. 33±27. 02 vs 22. 42 ± 13. 33, 31. 80 ±4.56 vs 22. 28 ± 15. 96, respectively, but greatly decreased in losartan group compared with I/R group, 23. 90±21.74 vs 51. 33 ±27. 02 and 11. 50 ±13. 20 vs 31. 80 ±4. 56, respectively. During reperfusion period CPK, LDH increased significantly in I/R group compared with control group, 49. 11 ± 20. 63 vs 12. 14 ±5.92 and 28. 70±4. 69 vs 23. 10±21. 38, respectively, but decreased greatly in losartan group compared with I/R group, 39. 40 ± 9. 60 vs 49. 11 ± 20.63 an     

Does Vitamin D Receptor and Calcium Receptor Activation Therapy Play a Role in the Histopathologic Alterations of Parathyroid Glands in Refractory Uremic Hyperparathyroidism?

Background and objectives: Vitamin D receptor activation by vitamin D sterols and calcium-sensing receptor stimulation by cinacalcet are the most powerful treatments of secondary hyperparathyroidism. This study was aimed to assess a possible association between histopathologic changes of parathyroid tissue and treatment modality.Design, setting, participants, & measurements: Studies were performed on 82 parathyroids of 22 adult white hemodialysis patients undergoing first parathyroidectomy. The type of hyperplasia and the distribution of chief and oxyphil cells, expressed as oxyphil/chief cell ratio, were assessed. Three groups could be studied according to treatment modality: group A consisted of 6 patients who were treated with cinacalcet, intravenous calcitriol, and phosphate binders; group B consisted of 6 patients who were treated with intravenous calcitriol and phosphate binders, and group C consisted of 10 patients who were treated with phosphate binders alone.Results: Sixty-eight (82.9%) out of 82 glands removed showed nodular hyperplasia. It was more frequent in groups A and B than in group C. A stepwise forward logistic regression model showed that the probability of nodular hyperplasia was higher in patients who were on calcitriol and/or cinacalcet therapy, in female gender and in patients with a higher body mass index. Oxyphil/chief cell ratio also was significantly different among the three groups. Cinacalcet treatment was the only predictor of this ratio.Conclusions: An association was found between calcitriol and/or cinacalcet therapy and a high prevalence of nodular hyperplasia, and between cinacalcet therapy and high oxyphil/chief cell ratio. The meaning of the observed associations remains uncertain.A large number of patients with uremia develop secondary hyperparathyroidism (SHPT) (1,2). Receptor therapy is the most adequate treatment of SHPT: Calcitriol and vitamin D analogues act on vitamin D receptors (VDR) in the intestine to increase calcium (Ca) absorption and on VDR in parathyroid cells to inhibit parathyroid hormone (PTH) mRNA synthesis (3); a cell surface receptor located on parathyroid cells, the calcium-sensing receptor (CaR), has been recognized as the primary mechanism that mediates the effects of ionized calcium (Ca²⁺) on PTH secretion (4,5). The CaR seems to play a key role in the excessive cell proliferation that occurs in parathyroid hyperplasia (5). Drugs that mimic or potentiate the action of Ca²⁺ at this receptor, calcimimetics, have become available for treatment of dialysis patients who are affected by SHPT (2,6–8); however, in a minority of them, parathyroid overfunction persists, progressively escapes medical control, and eventually becomes extremely severe, requiring surgical parathyroidectomy (PTx). In a time when effective treatment strategies are increasingly available, PTx could be viewed as indicating a medical failure. The reasons for the failure of response of parathyroid glands to the wide and powerful therapeutic armamentarium (namely receptor therapy) that is available must be looked for in several domains, such as intrinsic factors linked to the large volume of the glands themselves with nodular hyperplasia (NH), a reduced density of VDR and CaR, and a persistent hyperphosphatemia (1,2). The main purpose of this study was to assess in hemodialysis patients who were affected by refractory SHPT whether an association exists between the histopathologic alterations of parathyroid glands (NH versus diffuse hyperplasia [DH] and the expression of cell phenotypes) and the treatments with intravenous calcitriol and/or cinacalcet and/or phosphate (P) binders.     

Up and Down Expression of Androgen Receptor, Estrogen Receptor beta and Platelet Derived Growth Factor beta by Testosterone in Aortic Vascular Smooth Muscle Tissues

INTRODUCTIONCoronary heart disease (CHD) is the primary cause of death and disability. Numerous clinical and epidemiological reports demonstrate that men are gener- ally more susceptible to CHD than their female counter- parts. Increasing evidence accumulates that estrogen and perhaps progesterone have a favorable effect on fe- male cardiovascular risk and atherogenic processes. 1 - 4 Much less work, however , has addressed the possi- ble effects of androgens on atherogenesis , 5 and th…     

Association of β2-Adrenergic Receptor Polymorphisms with Asthma in a North Indian Population

Background

β₂-Adrenergic receptor (β₂AR), a G-protein coupled receptor, is present on the bronchial smooth muscle cells and results in bronchodilation upon activation. The genetic factors determining β₂AR expression and function may not only alter the response of an individual to the therapy but also may serve as predictive markers for response to the agonists used in the therapy. The present study aimed at evaluating the role of β₂AR-16 and β₂AR-27 gene polymorphisms in asthma.

Methods

A case–control study was performed with a total of 824 adult subjects, including 410 asthmatics and 414 healthy controls from regions of North India. The β₂AR-16 and β₂AR-27 polymorphisms were genotyped by PCR–RFLP.

Results

Statistical analysis for the β₂AR-16 polymorphism revealed that the mutant Gly16 allele was significantly associated with asthma, with OR?=?0.80, 95?% CI?=?0.65–0.99, and P?=?0.032. The Gly16/Gly16 mutant genotype also confers decreased risk toward asthma, with OR?=?0.65, 95?% CI?=?0.41–1.02, and P?=?0.049. However, the β₂AR-27 polymorphism was not associated with asthma as it did not reach statistical significance, with OR?=?0.86, 95?% CI?=?0.69–1.07, and P?=?0.163.

Conclusion

The β₂AR-16 polymorphism confers a decreased risk toward asthma while the β₂AR-27 polymorphism is not associated with asthma in the studied North Indian population.     

Pharmacology and Pharmacokinetics of A Novel Nonpeptide Angiotensin II Receptor Antagonist–DMP 811

DMP 811 exhibited high binding affinity for the angiotensin II subtype receptor AT₁ in rat adrenal tissues with an IC₅₀ of 6 nM, but not for the subtype receptor AT₂. In the isolated rabbit aorta, DMP 811 inhibited the contractile response to angiotensin II selectively and noncompetitively with a K_B value of 0.1 nM. In conscious renal hypertensive rats, DMP 811 decreased blood pressure with i.v. and p.o. ED3_30s of 0.005 and 0.03 mg/kg, respectively (p.o. ED₃₀ for losartan=0.59 mg/kg). In conscious furosemide-treated dogs, DMP 811 given either at 0.3 or 1 mg/kg p.o. decreased blood pressure. DMP 811 has oral bioavailabilities of 7 and 29% in rats and dogs, respectively, after a solution dose and 8 and 13%, respectively, after a suspension or capsule dosing. Our study indicates that DMP 811 is a selective and insurmountable AT₁ receptor antagonist and is a 20-fold more potent orally-active antihypertensive agent than losartan.     

Contribution of Complementarity-Determining Region 3 of the T-Cell Receptor Vδ2 Chain to the Recognition of Aminobisphosphonates by Human γδ T-Cells

Human gammadelta T-lymphocytes expressing Vgamma2Vdelta2 T-cell receptors (TCRs) can be stimulated by aminobisphosphonates and can kill certain tumor cells. Although germline-encoded lysine residues on the Jgamma1.2 segment have been demonstrated to be essential for the recognition of nonpeptide antigens by human gammadelta T-cells, the role of the junctional sequences of the TCR delta chain in the recognition of aminobisphosphonates by Vgamma2Vdelta2+ T-cells remains unknown. We examined the structure of complementarity-determining region 3 (CDR3) of Vdelta2 chains expressed by aminobisphosphonate-stimulated human gammadelta T-cells. CDR3 size-spectratyping analysis of Vdelta2 chains revealed that risedronate did not induce a CDR3 size distribution pattern of Vdelta2 cells that was distinct from that of Vdelta2 cells cultured without risedronate. The clonality of risedronate-expanded Vdelta2 T-cells was also determined by sequencing analysis, with the result that no particular consensus sequences were observed. However, most Vdelta2 T-cells freshly isolated from peripheral blood carried a distinctive junctional motif consisting of a hydrophobic amino acid residue (valine, leucine, or isoleucine [Val/Leu/Ile]) at conserved position 97, and this feature was not altered by risedronate stimulation. A significant proportion of Vdelta1 T-cells from the same individual had Leu at position 97, but Vdelta1 T-cells did not expand in response to risedronate. Moreover, Vdelta2 T-cells from the nonresponder against risedronate also carried a Val/Leu/Ile amino acid residue at position 97. These results suggest that the presence of a hydrophobic amino acid residue at position 97 in CDR3 of the TCR delta chain is not sufficient to account for the recognition of aminobisphosphonate by human gammadelta T-cells.     

Dual-Acting Angiotensin Receptor–Neprilysin Inhibition

Lowering blood pressure by pharmacologic intervention reduces the incidence of cardiovascular events. Nevertheless, despite the widespread availability of effective antihypertensive medications, the vast majority of hypertensive patients worldwide continue to have inadequate blood pressure control. The development of new antihypertensive drugs could contribute to improving the hypertension control rate, and the blockade of new pathophysiologic pathways involved in blood pressure regulation would offer additional benefits. The dual inhibition of the angiotensin II receptor and neprilysin could provide clinical benefits in a range of cardiovascular diseases, including hypertension and heart failure.