LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells

Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress. The objectives of these studies are to determine how LEDGF controls PKC gamma activity in normal LECs: how this control of PKC gamma regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in LECs. A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF. PKC gamma protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng ml(-1). In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein-protein interaction between PKC gamma and Connexin 43. In the presence of LEDGF the activation of PKC gamma enhanced the phosphorylation of Connexin 43 by four-fold compared to the absence of LEDGF. The addition of LEDGF for 30 min resulted in a 65% decrease in gap junction Connexin 43 at the cell surface and a 70% decrease in gap junction activity. These results suggest that the activation of PKC gamma by LEDGF plays a major role in gap junction assembly/disassembly, which may enhance survival of LECs against osmolarity-stress induced by high sugar concentration.     

Potent inhibition of human folylpolyglutamate synthetase by a phosphinic acid mimic of the tetrahedral reaction intermediate

A phosphorous-containing pseudopeptide folate analog (Valiaeva et al., J Org Chem 2001;66:5146-54) was designed to mimic the tetrahedral intermediate formed in the ATP-dependent reaction catalyzed by folylpolyglutamate synthetase (FPGS). This analog, methotrexate-phosphinate (MTX-phosphinate; 4-amino-4-deoxy-10-methylpteroyl-L-Glu-gamma-[psi(P(O)(OH)-CH(2))]glutarate), is a highly potent (K(is), 3.1+/-0.5 nM), competitive inhibitor of recombinant human cytosolic FPGS. Within experimental limits, FPGS inhibition was not time-dependent, and preincubation of FPGS, inhibitor, and ATP did not potentiate the inhibition. These results suggest that slow phosphorylation to produce a more potent inhibitor form is not involved. MTX-phosphinate was not growth inhibitory to human CCRF-CEM leukemia cells at 1 microM (70-fold above the concentration of MTX giving 50% growth inhibition), probably because of poor transport. Because of its exceedingly high potency as an FPGS inhibitor, MTX-phosphinate represents a lead structure from which cell-permeable analogs may be developed to test the hypothesis that FPGS inhibition is therapeutically efficacious.     

Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.     

Effect of acute betaine administration on hepatic metabolism of S-amino acids in rats and mice

Alterations of hepatic glutathione level by betaine were observed previously. In this study effects of betaine administration (1000 mg/kg, i.p.) on S-amino acid metabolism in rats and mice were investigated. Hepatic glutathione level decreased rapidly followed by marked elevation in 24 hr. Concentrations of S-adenosylmethionine, S-adenosylhomocysteine, and methionine were increased whereas cystathionine decreased significantly, suggesting that homocysteine generated in the methionine cycle is preferentially remethylated to methionine rather than being utilized for synthesis of cysteine. Hepatic cysteine concentration declined immediately, but plasma cysteine increased. Effect of betaine on hepatic cysteine uptake was estimated from the difference in cysteine concentration in major blood vessels connected to liver. Cysteine concentration either in the portal vein or abdominal aorta was not altered, however, a significant increase was noted in the hepatic vein, indicating that hepatic uptake of cysteine was decreased by betaine treatment. Activities of glutamate cysteine ligase, cystathionine beta-synthase, and cystathionine gamma-lyase were elevated in 24 hr. Pretreatment with propargylglycine, an irreversible inhibitor of cystathionine gamma-lyase, did not abolish the betaine-induced reduction of hepatic glutathione in 4 hr, however, the elevation at t=24 hr was blocked completely. In conclusion the present results indicate that betaine administration induces time-dependent changes on hepatic metabolism of S-amino acids. Betaine enhances metabolic reactions in the methionine cycle, but inhibits cystathionine synthesis and cysteine uptake, leading to a decrease in supply of cysteine for glutathione synthesis. Reduction in glutathione is subsequently reversed due to induction of cysteine synthesis and glutamate cysteine ligase activity.     

Roles of protein kinase A and C in the opioid potentiation of the GABAA response in rat periaqueductal gray neuron

The periaqueductal gray (PAG) is the main target site of the opioid-induced analgesia. The present study was designed to examine the roles of protein kinase A (PKA) and C (PKC) in the opioid-induced modulation of the currents activated by an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). The PAG neurons were acutely isolated and voltage-clamped under the nystatin-perforated patch-clamp mode. The GABA-activated current was sensitively blocked by a GABA(A) receptor antagonist, bicuculline, and selectively carried by chloride ions. The GABA(A) receptor-activated Cl(-) current was potentiated by a mu-opioid receptor agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin acetate (DAMGO). The GABA response was also potentiated by phorbol-12-myristate-13-acetate (PMA). Pretreatment with PMA occluded the DAMGO potentiation. However, both chelerythrine and 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X) also potentiated the GABA response. Pretreatment with chelerythrine or GF109203X also occluded the DAMGO potentiation. Meanwhile, the GABA response was potentiated by N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89), while not altered by forskolin. Pretreatment with H-89 occluded the potentiation effect of DAMGO on the GABA response. In addition, the DAMGO effect was completely blocked by pretreatment with forskolin. From the result, it can be suggested that activation of mu-opioid receptor potentiates the GABA(A) response through the mediation of PKA inhibition, and that PKC is not directly involved in the action mechanism of DAMGO.     

Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC(3) receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH(2) (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC(3) receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC(3)/MC(4) receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1-24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC(3) receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC(3) receptors, the C-terminal Arg-Phe structure being relevant.     

Synthesis and activity of novel glutathione analogues containing an urethane backbone linkage

The new GSH analogues H-Glo(-Ser-Gly-OH)-OH (5), its O-benzyl derivative 4, and H-Glo(-Asp-Gly-OH)-OH (9), characterized by the replacement of central cysteine with either serine or aspartic acid, and containing an urethanic fragment as isosteric substitution of the scissile gamma-glutamylic junction, have been synthesized and characterized. Their ability to inhibit human GST P1-1 (hGST P1-1) in comparison with H-Glu(-Ser-Gly-OH)-OH and H-Glu(-Asp-Gly-OH)-OH, which are potent competitive inhibitors of rat GST 3-3 and 4-4, has been evaluated. In order to further investigate the effect of the isosteric substitution on the binding abilities of the new GSH analogues 4, 5 and 9, the previously reported cysteinyl-containing analogue H-Glo(-Cys-Gly-OH)-OH has been also evaluated as a co-substrate for hGSTP1-1.     

An ecological analysis of leukemia incidence around the highest 137Cs concentration in Poland

Cancer has long been known to be a hazard of exposure to ionizing radiation. However, the assessment of health effects from exposure to radiation is a matter of considerable controversy. This paper presents results of a retrospective study of leukemia incidence (203–207, ICD-9) around the highest ¹³⁷Cs pollution in Poland (as an effect of the Czarnobyl disaster and/or military bomb tests). The data relating to all the registered leukemias in males and females originated from the Regional Cancer Registry in Opole. The information on ¹³⁷Cs concentration rates in Opole province was derived from the state monitoring provided by the Polish Geological Institute in Warsaw. The spatial analysis – based on the random-effects Poisson regression model – was carried out via the Markov Chain Monte Carlo (MCMC) technique (Gibbs sampling) using BUGS software. The model incorporated epidemiological data and an ecological covariate – isotope concentrations – and provided a framework for estimating the strength of a dose–response relationship. The differences in incidence levels were quantified by traditional standardized morbidity ratios (SMRs) and presented in thematic maps as well as in combined charts of distance–disease–dose relations. Additionally, to assess spatial disease clustering, a Tango test was adopted. The results of this ecological study suggest that the ¹³⁷Cs concentrations did not have any negative influence on the exposed population.     

过敏性鼻炎患儿血清中白细胞介素-18、白细胞介素-12、干扰素-γ表达及其意义

①目的 探讨过敏性鼻炎患儿血清中白细胞介素-18（IL-18）、白细胞介素-12（IL-12）、干扰素-γ（IFN-γ）的表达及其意义.②方法 采用酶联免疫吸附法检测42例变应性鼻炎患儿和20例健康志愿儿血清中IL-18、IL-12、IFN-γ的含量.同时对比常年性鼻炎及季节性鼻炎患儿血清中IL-18、IL-12、IFN-γ的含量.③结果 鼻炎组血清IL-18含量为（97.02±20.67）ng/L、IL-12含量为（72.64±15.77）ng/L、IFN-γ为（0.54±0.19）ng/L,均低于正常对照组,IL-18含量为（212.75±32.96）ng/L、IL-12含量为（136.55±17.88）ng/L、IFN-γ为（1.41±0.22）ng/L,其差异具有统计学意义（P＜0.01）;且常年性变应性鼻炎患儿血清中IL-18、IL-12、IFN-γ水平低于季节变应性鼻炎患儿,其差异亦有统计学意义（P＜0.01）.④结论 IL-18、IL-12、IFN-γ在变应性鼻炎患儿血清中表达降低,说明其可能参与了对变应性炎症中抗炎抗过敏作用,且3种免疫因子之间有协同作用.     

联合注射外源性IFNγ和IGF-1治疗小鼠骨骼肌损伤

目的：观察局部联合注射外源性人胰岛素样生长因子-1（human insulin-like growth factor-1，hlGF-1）和人γ干扰素（human interferon γ，hIFNγ）对于小鼠急性钝挫伤骨骼肌修复过程中再生和纤维化的影响。方法：64只8周龄雄性C3H小鼠，制作右侧腓肠肌钝挫伤动物模型，随机分为4组，即A组（注射hIFNγ）、B组（注射hIGF-1）、C组（联合注射hIFNγ和hIGF-1）、D组（注射生理盐水）。挫伤后第10天，A、B、C、D组小鼠，腓肠肌损伤处分别注射不同药物进行干预。于干预前（伤后7d），和干预后4、18、32d，各组随机抽取4只小鼠进行损伤腓肠肌取材，以荧光定量PCR和免疫荧光化学染色方法检测不同时点Ⅱb型肌球蛋白重链（myosin heavy chain—Ⅱb，MHC—Ⅱb）及波形蛋白（Vimentin）的表达。结果：④干预后各时点，B、C组小鼠损伤骨骼肌局部MHC—Ⅱb表达较A、D组明显高；（爹干预后各时间点A、B、C组小鼠损伤骨骼肌局部Vimentin表达较D组低，其中以A组和B组更明显。结论：④骨骼肌急性损伤后，局部注射hIGF-1有明显促进骨骼肌纤维再生，和一定程度抑制纤维化的作用。②局部注射hIFN3,仅表现出抑制纤维化的作用，且比hlGF-1更加明显。⑧联合应用hlGF-1和hIFNγ，能够同时促进骨骼肌再生和抑制纤维化，有效地促进损伤骨骼肌的愈合。