Identification of the preferentially targeted proteins by carbamylation during whole lens incubation by using radio-labelled potassium cyanate and mass spectrometry

AIMTo attempt to identify the primary targets of carbamylation in bovine lenses incubated under physiological condition.

Carbamylation of Human Lens γ-crystaUins:Relevance to Cataract Formation

Cigarette smoking is a main source of cyanide in human body,which can be taken as a risk factor of cataract formation.In this study,combined gas chromatography and mass spectrum(GC/MS)was used todetermine the amino acid hydantoin after the incubation of soluble humanlensγ-crystallins with cyanate.The carbamylated amino acids obtained bythis procedure are alanine and hlycine,which are N-terminal amino acids ofγ-crystallin,and leucine.The aggregate,which can be observed incarbamylatedγ_1-crystallin on SDS-PAGE,may be related to the formation of disulfide and non-disulfide covalent bonds,and it seems that Y₂and Yy-crystallins can not be aggregated to any great extend.The results in this study indicate that the GC/MS is an effective method for analyzing the carbamylation of lens proteins;y-crystallin may play a very important role in the formation of cataract associated with accumulation of cyanate in human body,such as heavy smoking.

Carbamylated lipoproteins in diabetes

Diabetic dyslipidemia is characterized by quantitative and qualitative abnormalities in lipoproteins.In addition to glycation and oxidation,carbamylation is also a post-translational modification affecting lipoproteins in diabetes.Patients with type 2 diabetes(T2D)exhibit higher levels of carbamylated low-density lipoproteins(cLDL)and high-density lipoproteins(cHDL).Accumulating evidence suggests that cLDL plays a role in atherosclerosis in diabetes.cLDL levels have been shown to predict cardiovascular events and all-cause mortality.cLDL facilitates immune cell recruitment in the vascular wall,promotes accumulation of lipids in macrophages,and contributes to endothelial dysf-unction,endothelial nitric oxide-synthase(eNOS)inactivation and endothelial repair defects.Lastly,cLDL induces thrombus formation and platelet aggregation.On the other hand,recent data have demonstrated that cHDL serum level is independently associated with all-cause and cardiovascular-related mortality in T2D patients.This relationship may be causative since the atheroprotective properties of HDL are altered after carbamylation.Thus,cHDL loses the ability to remove cholesterol from macrophages,to inhibit monocyte adhesion and recruitment,to induce eNOS activation and to inhibit apoptosis.Taken together,it seems very likely that the abnormalities in the biological functions of LDL and HDL after carbamylation contribute to atherosclerosis and to the elevated cardiovascular risk in diabetes.

THE IMMUNOHISTOCHEMISTRY AND IN SITU cDNA-mRNA HYBRIDIZATION OF CARBAMYL PHOSPHATE SYNTHETASE I IN ENZYME-ALTERED LIVER CELLS DURING CARCINOGENESIS

The changes of carbamyl phosphate synthetase I(CPS 1)in diethylnitrosamine-(DEN)-inducedenzyme-altered liver cells were studied by means of immunohistochemical(PAP)and in situcDNA-mRNA hybridization methods.The experimental rats were treated with DEN,2-acetylaminofluorene(2-AAF)and 2/3 hepatectomy according to Solt-Farber’s protocol andwere further promoted by oral daily administration of 0.05% phenobarbital in drinking water.The results showed that the average number of lesions showing abnormal expression of CPS1 was relatively constant over the course of the experiment(8 months),while the numberof normally expressing lesions gradually decreased.The former lesions were also largerin volume than the latter ones.We conclude that in DEN-initiated lesions the abnormallyexpressed CPS 1 lesions may grow continuously,thus leading to the formation of largernodules.We also suspect that some of these lesions have increased tendencies to developinto tumors.

Nonhematopoietic erythropoietin derivative protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis

Objective：Carbamylated EPO（CEPO） is a derivative of erythropoietin（EPO） by subjecting it to carbamylation. It does not stimulate erythropoiesis, but effectively protects tissue from injury. The present study was to investigate the effect of CEPO treatment using in vitro models of hypoxia/reoxygenation（H/R）. Methods：Cardiomyocytes were exposed to hypoxia（95% N2 and 5% CO2） for 1 hour followed by 4 hours of reoxygenation（95% O2 and 5% CO2）. CEPO was administered after hypoxia, just before reoxygenation. The apoptotic cardiomyocytes were determined by flow cytometry. The level of protein was assessed by western blot analysis. Results： CEPO treatment significantly decreased the apoptotic cardiomyocytes by 54.20% compared with H/R group. Western blot analysis showed that CEPO administration increased the level of Bcl-2（an antiapoptotic protein） by 62.22% compared with H/R group. Conclusion： Acute administration of CEPO protected cardiomyocytes from H/R-induced apoptosis. CEPO protected cardiomyocytes with a concomitant upregulation of Bcl-2 after H/R injury.

Carbamylated Erythropoietin Alleviates Kidney Damage in Diabetic Rats by Suppressing Oxidative Stress

The oxidative stress response plays an important role in the occurrence and development of diabetic kidney disease(DKD).It has become a new treatment target for DKD.In the current study,the effects of carbamylated erythropoietin(CEPO)on renal oxidative stress and damage in diabetic rats were examined.Thirty Sprague Dawley rats were intraperitoneally administered with 60 mg/kg streptozotocin to establish the diabetes model.The diabetic rats were randomly allocated into 4 groups(n=6 each):diabetes model group(DM group),DM+CEPO treatment group(DC group),DM+CEPO+EPO receptor(EPOR)blocking peptide treatment group(DCEB group),and DM+CEPO+CD131 blocking peptide treatment group(DCCB group).Meanwhile,a normal control group(NC group,n=6)was set up.Kidney tissues and blood samples were obtained for evaluation of oxidative stress and renal function.The results showed that diabetic rats exhibited increased oxidative stress in the kidney and early pathological changes associated with DKD.Treatment with CEPO reduced oxidative stress and attenuated renal dysfunction.However,diabetic rats treated with the combination of CEPO and EPOR blocking peptide or CD131 blocking peptide showed increased oxidative stress and reduced renal function when compared with CEPO treatment alone group.These results suggested that CEPO can protect against kidney damage in DKD by inhibiting oxidative stress injury via EPOR-CD131 heterodimers.

Experimental bio-artificial liver:Importance of the architectural design on ammonia detoxification performance

AIM To determine the influence of the construction design over the biological component's performance in an experimental bio-artificial liver(BAL) device.METHODS Two BAL models for liver microorgans(LMOs) were constructed. First, we constructed a cylindrical BAL and tested it without the biological component to establish its correct functioning. Samples of blood and biological compartment(BC) fluid were taken after 0, 60, and 120 min of perfusion. Osmolality, hematocrit, ammonia and glucose concentrations, lactate dehydrogenase(LDH) release(as a LMO viability parameter), and oxygen consumption and ammonia metabolizing capacity(as LMO functionality parameters) were determined. CPSI and OTC gene expression and function were measured. The second BAL, a "flat bottom" model, was constructed using a 25 cm2 culture flask while maintaining all other components between the models. The BC of both BALs had the same capacity(approximately 50 cm3) and both were manipulated with the same perfusion system. The performances of the two BALs were compared to show the influence of architecture.RESULTS The cylindrical BAL showed a good exchange of fluids and metabolites between blood and the BC, reflected by the matching of osmolalities, and glucose and ammonia concentration ratios after 120 min of perfusion. No hemoconcentration was detected, the hematocrit levels remained stable during the whole study, and the minimal percentage of hemolysis(0.65% ± 0.10%) observed was due to the action of the peristaltic pump. When LMOs were used as biological component of this BAL they showed similar values to the ones obtained in a Normothermic Reoxygenation System(NRS) for almost all the parameters assayed. After 120 min, the results obtained were: LDH release(%): 14.7 ± 3.1 in the BAL and 15.5 ± 3.2 in the NRS(n = 6); oxygen consumption(μmol/min?g wet tissue): 1.16 ± 0.21 in the BAL and 0.84 ± 0.15 in the NRS(n = 6); relative expression of Cps1 and Otc: 0.63 ± 0.12 and 0.67 ± 0.20, respectively, in the BAL, and 0.86 ± 0.10 and 0.82 ± 0.07, respectively, in the NRS(n = 3); enzymatic activity of CPSI and OTC(U/g wet tissue): 3.03 ± 0.86 and 222.0 ± 23.5, respectively, in the BAL, and 3.12 ± 0.73 and 228.8 ± 32.8, respectively, in the NRS(n = 3). In spite of these similarities, LMOs as a biological component of the cylindrical BAL were not able to detoxify ammonia at a significant level(not detected vs 35.1% ± 7.0% of the initial 1 mM NH4+ dose in NRS, n = 6). Therefore, we built a second BAL with an entirely different design that offers a flat base BC. When LMOs were placed in this "flat bottom"device they were able to detoxify 49.3% ± 8.8% of the initial ammonia overload after 120 min of perfusion(n = 6), with a detoxification capacity of 13.2 ± 2.2 μmol/g wet tissue.CONCLUSION In this work, we demonstrate the importance of adapting the BAL architecture to the biological component characteristics to obtain an adequate BAL performance.

Effect of carbamylated erythropoietin on retinopathy of diabetic rats

Objective:To study the effect of carbamylated erythropoietin (CEPO) on retinopathy of diabetic rats.Methods: Male SD rats were selected as experimental animals and randomly divided into control group, DM group and CEPO group, and diabetic animal models were established and then given CEPO intervention. 2 weeks after intervention, the retina was collected to detect the expression of angiogenesis molecules, apoptosis molecules and oxidative stress pathway molecules.Results: HIF-1α, VEGF, Ang-1, Bax, Caspase-3, Nrf-2, ARE, HO-1 and NQO-1 mRNA expression in retina of DM group were significantly higher than those of control group while TKLK, PEDF, Bcl-2 and Survivin mRNA expression were significantly lower than those of control group;HIF-1α, VEGF, Ang-1, TKLK and PEDF mRNA expression in retina of CEPO group were not significantly different from those of DM group, Bcl-2, Survivin, Nrf-2, ARE, HO-1 and NQO-1 mRNA expression were significantly higher than those of DM group, and Bax and Caspase-3 mRNA expression were significantly lower than those of DM group.Conclusion:CEPO can reduce the apoptosis and oxidative stress injury of the retina tissue in diabetic rats without affecting the angiogenesis.

DETERMINATION OF THE TRANSCRIPTION INITIATION SITE OF-THE CARBAMYL PHOSPHATE SYNTHETASE I GENE

Carbamyl phosphate synthetase I (CPSI for short )is the first key enzyme of the urea cycle and mainly located in mitochondria of hepatocytes. It is the tissue specific enzyme of liver and involved in the differentiation of hepatocytes. We have observed the decrease of the activity of CPSI during hepatocarcinogenesis. A series of experiments suggest that CPSI gene be regulated at transcriptional level. Transcription regulation of eukaryotic genes is mediated by interaction of trans-acting factors and cis-acting elements. Cis-acting elements are

STUDIES ON DNA SEQUENCE SPECIFIC BINDING PROTEINS FOR 5' FLANKING UPSTREAM REGION OF CPSI GENE

Two specific carbamyl phosphate synthetase Ⅰ gene binding nuclear proteins (M. W. 109kD and 74 kD) have been determined in the rat liver by the protein blotting technique (South-western blot assay). The result shows that they are not present in the normal rat spleenand F-26 rat hepatoma cell. The Ba131 nuclease deletion in the CPSI gene 5’ upstream regionproves that the binding sites for 109 kD and 74 kD are respectively located in the regions of- 38 bp to - 4 bp and - 113 bp to -38 bp. The binding proteins may be the liver-specific onesof the CPSI gene, which are related to hepatocyte differentiation and hepatocarcinogenesis.

The spin delocalization substituent parameter σ——8. The spin-delocalizing abilities of the nitro, cyano, carbomethoxy, carboxy and carbamyl groups

The rate constants (k) of the homo-cycloaddition reactions of five substituted α, β, β-tri- fluorostyrenes (TFS’s), i.e. p-nitrotrifluorostyrene (4), p-cyanotrifluorostyrene (5), p-carbomethoxytri- fluorostyrene (6), p-carboxytrifluorostyrene (7) and p-carbamyltrifluorostyrene (8), have been measured in the temperature range of 110-160℃. The ~σmb polar substituent parameters of these TFS's cal- culated from ^(19) F NMR chemical shifts are: NO_2, 0.86; CN, 0.86; CO_2CH_2, 0.40; CO_2H, 0.31; CONH_2, 0.10. The spin delocalization substituent parameters ~σT of NO_2, CN, CO_2 CH_2, CO_2H and CONH_2 are 0.32, 0.38, 0.31, 0.37 and 0.37 respectively. Thus all these electron-pair attracting groups are also very effective spin-stabilizers.