Human prorenin has "gate and handle" regions for its non-proteolytic activation

We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 degrees C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 degrees C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degrees C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degrees C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degrees C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degrees C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degrees C. These results indicate that prorenin has "gate" (T7PFKR10P) and "handle" (I11PFLKR15P) regions critical for its non-proteolytic activation.     

The dominant role of the prosegment of prorenin in determining the rate of activation by acid or trypsin: studies with molecular chimeras

Human prorenin activation by acid or trypsin is faster than rat prorenin by two orders of magnitude. No plausible mechanism exists to explain the difference. Two chimeric mutant prorenins were produced in CHO cells. A chimera, hPro/rRen, composed of human prorenin prosegment and rat active renin segment, was activated as fast as wild-type human prorenin at pH 3.3 and 25 degrees C or by trypsin (1 microg/ml). The other chimera, rPro/hRen, composed of rat prorenin prosegment and human active renin segment, was activated as slowly as wild-type rat prorenin at pH 3.3 and 25 degrees C or by trypsin (50 microg/ml). These results indicate that the rate of activation of prorenin is predominantly determined by the N-terminal pro-sequence. Plausible mechanisms are discussed.     

Receptor-dependent prorenin activation and induction of PAI-1 expression in vascular smooth muscle cells

Although elevated plasma prorenin levels are commonly found in diabetic patients and correlate with microvascular complications, the pathological role of these increases, if any, remains unclear. Prorenin/renin binding to the prorenin/renin receptor [(p)RR] enhances the efficiency of angiotensinogen cleavage by renin and unmasks prorenin catalytic activity. We asked whether plasma prorenin could be activated in local vascular tissue through receptor binding. Immunohistochemical staining showing localization of the (p)RR in the aorta to vascular smooth muscle cells (VSMCs). After cultured rat VSMCs were incubated with 10(-7) M inactive prorenin, cultured supernatant acquired the ability to generate ANG I from angiotensinogen, indicating that prorenin had been activated. Activated prorenin facilitated angiotensin generation in cultured VSMCs when exogenous angiotensinogen was added. Small interfering RNA (siRNA) against the (p)RR blocked this activation and subsequent angiotensin generation. Prorenin alone induced dose- and time-dependent increases in mRNA and protein for the profibrotic molecule plasminogen activator inhibitor (PAI)-1, effects that were blocked by siRNA, but not by the ANG II receptor antagonist saralasin. When inactive prorenin and angiotensinogen were incubated with cells, PAI-1 mRNA increased a striking 54-fold, 8-fold higher than the increase seen with prorenin alone. PAI-1 protein increased 2.75-fold. These effects were blocked by treatment with siRNA + saralasin. We conclude that prorenin at high concentration binds the (p)RR on VSMCs and is activated. This activation leads to increased expression of PAI-1 via ANG II-independent and -dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may contribute to the progression of fibrotic disease.     

Possible activation of human prorenin by contact with the cell membrane

Semipurified human kidney prorenin was exposed in vitro to a mixture of lipids mimicking the composition of the inner leaflet of the cell membrane, in the presence of semipurified human angiotensinogen at a concentration of 1/4 Km. Prorenin was activated in a time-dependent manner over a period of 60 min. This lipid-dependent activation was completely reversed thereafter. Pre-incubation with anti-renin serum completely prevented this activated prorenin-dependent generation of angiotensin I. Our data suggest that human prorenin can be reversibly activated by contact with the cell membrane.     

Simultaneous activation of PLA2 and PLC are required to promote acrosomal reaction stimulated by progesterone via G-proteins

The acrosome reaction (AR) is a special exocytotic process promoted by signal transduction pathways studied in many laboratories. Progesterone (P4) is one of the trigger molecules proposed. Upon the binding of P4 to its receptor, several molecules could be activated, including G-proteins, phospholipase A(2) (PLA(2)), and phospholipase C (PLC). The role of these molecules was analyzed in this study using the Chlortetracycline (CTC) protocol to detect and quantify the AR. Incubation of capacitated sperm cells with GTPgammas (GTPgammas, a mimetic of G-protein activation), arachidonic acid (AA, product of PLA(2) action), or phorbol ester (PMA, an activator of PLC) for 15 min increased the AR to a similar percentage as P4. Conversely, a decrease in the AR was detected when sperm cells were incubated with P4 after preincubation with: GDPbetaS (GDP, an inhibitor of G-protein activation), ONO RS-82 (ONO, an inhibitor of PLA(2)), or neomycin (Neo, an inhibitor of PLC) for 15 min. To analyze the activation sequence of G proteins, PLA(2), and PLC combinations of these mimetic/inhibitors were used during successive incubation periods. Inhibition promoted by GDP, ONO, and Neo were overcome by 15-min incubation with GTPgammas, AA, or PMA, respectively. But GTPgammas or P4 did not reverse the inhibition due to incubation with Neo and ONO. Interestingly, this dual inhibition was reverted by another 15-min incubation with AA or PMA. Results presented here could indicate that the AR triggered by P4 is driven by activation of G-proteins, that in turn activate PLA(2) and PLC simultaneously, that finally promote acrosomal exocytosis.     

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.     

Hydrolysis by plasma kallikrein of fluorogenic peptides derived from prorenin processing site

Human plasma kallikrein (HPK) activates plasma prorenin to renin, and the physiological significance of this activation is still unknown. In this paper we investigated the efficiency and the cleavage pattern of the hydrolysis by HPK of the internally quenched fluorescent peptides (qf-peptides) derived from the amino acid sequence of human prorenin cleavage site. The peptide Abz-F-S-Q-P-M-K-R-L-T-L-G-N-T-T-Q-EDDnp (Abz=ortho-aminobenzoic acid, and EDDnp=N-[2,4-dinitrophenyl]-ethylene diamine), that corresponds to the amino acid sequence P(7) to P(7)' of human prorenin cleavage site, is hydrolyzed at the correct processing site (R-L bond) with k(cat)/K(m)=85 mM(-1) s(-1). Alanine was scanned in all positions from P(5) to P(5)' in order to investigate the substrate specificity requirements of HPK.The qf-peptides derived from the equivalent segment of rat prorenin, that has Lys-Lys as basic amino acid pair, and the peptide Abz-NVTSPVQ-EDDnp that contains the proposed cleavage site of rat prorenin have very low susceptibility to hydrolysis by rat plasma kallikrein. These data are according to the previously reported absence of rat plasma prorenin activation by rat plasma kallikrein (RPK), and with the view that prorenin activation in rat requires alternative enzymes and/or mechanism.All the obtained peptides described in this paper were also assayed with bovine trypsin that was taken as a reference protease because it is commonly used to activate prorenin.     

Refolding and activation of human prorenin expressed in Escherichia coli: application of recombinant human renin for inhibitor screening

Human prorenin was expressed in Escherichia coli as a fusion protein of thioredoxin. The chimeric protein, which accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by an arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the recombinant human renin activity. Using the recombinant human renin, we screened its inhibitory activity in fermented soybean paste (miso) and demonstrated that miso contained renin inhibitory activity derived from soybean. The IC(50) values for soybean and steamed soybean extracts were determined to be 1.9 and 1.6 mg/ml, respectively. This is the first demonstration of renin inhibitory activity in miso and soybean.     

Trypsin activation of human and cat prorenin: a comparative study.

Prorenin can be converted to renin by limited proteolysis with trypsin. In the current study we compared conditions for activation of human renal and ovarian prorenin and cat renal prorenin with either liquid-phase trypsin or trypsin bound to sepharose (solid phase). Higher concentrations of trypsin were required to activate cat prorenin than human prorenin. Human prorenin was destroyed by high concentrations of trypsin, while cat prorenin was not destroyed by up to 2 mg/mL solid-phase trypsin. For both human and cat prorenin, addition of the competitive serine protease inhibitor benzamidine--HCl increased the concentration of trypsin needed to activate prorenin, resulting in slightly higher levels of human prorenin but lower levels of cat prorenin. For human samples, activation with solid-phase trypsin resulted in slightly higher estimates of prorenin than liquid-phase trypsin. These results demonstrate species differences in the susceptibility of prorenin to trypsin cleavage. Cat prorenin requires more trypsin to be activated and is less susceptible to destruction than human prorenin.     

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.     

(Pro)renin Receptor Mediates Both Angiotensin II-Dependent and -Independent Oxidative Stress in Neuronal Cells

The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.     

HPV16 L1蛋白在大肠杆菌中的表达、纯化及免疫原性研究

用IPTG诱导目的工程菌pQE31-HPV16L1/M15(pREP4),对表达产物进行SDSPAGE和Western blot分析;用表达的L1蛋白免疫BAL B/C小鼠得到抗血清后,利用真核源性的VLP粗提物验证小鼠抗血清的特异性.利用IMAC金属亲和层析柱纯化L1蛋白.SDSPAGE结果显示表达产物在约57 ku处有蛋白条带;Western blot结果证实此条带可与HPV16 L1蛋白的单克隆抗体反应;纯化后的L1蛋白也同样保留免疫特异性;小鼠抗血清可与HPV16L1 VLP(病毒样颗粒)发生特异性反应,证实重组表达的L1蛋白具有免疫原性.本实验表明HPV16 L1蛋白在工程菌M15(pREP4)中高效表达,为研制HPV16预防性基因工程疫苗和感染的诊断试剂提供了物质基础和技术方法.     

Isolation of completely inactive plasma prorenin and its activation by kallikreins. A possible new link between renin and kallikrein.

Existing views on prorenin are conflicting and its physiological activation mechanism is not clear. In an attempt to obtain clearcut views on the molecular properties of prorenin in human plasma, the renin zymogen (prorenin) was separated from active renin by two steps of affinity chromatography and it was demonstrated that prorenin is a completely inactivate zymogen contrary to the existing information. Inactive prorenin has an apparent molecular of 56,000 contrary to 46,000-43,000 of partially active prorenin. Isolated and acid-treated human prorenin was shown to be activated by kallikreins from human urine and plasma. This activation was completely blocked by Trasylol. Hog pancreatic kallikrein also activated human prorenin. The kallikrein mediated activation of prorenin indicates the existence of a new link between the vasoconstricting renin-angiotensin system and the vasodilating kallikreinkinin system.     

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1, 3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.     

Enhanced intrarenal receptor-mediated prorenin activation in chronic progressive anti-thymocyte serum nephritis rats on high salt intake

Despite suppression of the circulating renin-angiotensin system (RAS), high salt intake (HSI) aggravates kidney injury in chronic kidney disease. To elucidate the effect of HSI on intrarenal RAS, we investigated the levels of intrarenal prorenin, renin, (pro)renin receptor (PRR), receptor-mediated prorenin activation, and ANG II in chronic anti-thymocyte serum (ATS) nephritic rats on HSI. Kidney fibrosis grew more severe in the nephritic rats on HSI than normal salt intake. Despite suppression of plasma renin and ANG II, marked increases in tubular prorenin and renin proteins without concomitant rises in renin mRNA, non-proteolytically activated prorenin, and ANG II were noted in the nephritic rats on HSI. Redistribution of PRR from the cytoplasm to the apical membrane, along with elevated non-proteolytically activated prorenin and ANG II, was observed in the collecting ducts and connecting tubules in the nephritic rats on HSI. Olmesartan decreased cortical prorenin, non-proteolytically activated prorenin and ANG II, and apical membranous PRR in the collecting ducts and connecting tubules, and attenuated the renal lesions. Cell surface trafficking of PRR was enhanced by ANG II and was suppressed by olmesartan in Madin-Darby canine kidney cells. These data suggest the involvement of the ANG II-dependent increase in apical membrane PRR in the augmentation of intrarenal binding of prorenin and renin, followed by nonproteolytic activation of prorenin, enhancement of renin catalytic activity, ANG II generation, and progression of kidney fibrosis in the nephritic rat kidneys on HSI. The origin of the increased tubular prorenin and renin remains to be clarified. Further studies measuring the urinary prorenin and renin are needed.     

Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.     

Cytolytic activity of Ia-restricted T cell clones and hybridomas: evidence for a cytolytic mechanism independent of interferon-gamma, lymphotoxin, and tumor necrosis factor-alpha

Ten different helper T cell (Th) hybridomas that are specific to Ia or antigen plus Ia were found to express nonspecific cytolytic activity toward the cytotoxin (CT)-resistant P815 cells upon activation with either Con A or a monoclonal anti-T3 antibody (T3-mAb). In contrast to cytolytic Th1 clones which secrete high levels of interferon-gamma (IFN-gamma) and cytotoxin (CT) (lymphotoxin (LT, also known as TNF-beta) or tumor necrosis factor-alpha (TNF-alpha], these Th hybridomas produce low or undetectable levels of IFN-gamma and CT. No inhibitory activity of IFN-gamma and CT was observed in culture supernatants of activated Th hybridomas. Double-chamber experiments demonstrated that CT-sensitive L929 cells when physically separated from activated Th1 clones were killed by membrane-permeable CT. Under identical experimental conditions, lysis of P815 cells did not occur. Moreover, activation of Th hybridomas directly in wells containing the CT-sensitive L929 cells failed to induce target cell lysis. This confirms that these Th hybridomas produce little CT and argues against high local concentrations of CT being responsible for Th hybridoma-mediated killing of P815 cells. Finally, a polyclonal rabbit antiserum to rTNF-alpha, which strongly and specifically inhibited CT-mediated and Th1 clone-mediated killing of L929 cells, failed to inhibit P815 lysis by activated Th1 clones and Th hybridomas. These observations establish that a cytolytic mechanism independent of IFN-gamma, LT, and TNF-alpha is responsible for lysis of CT-resistant target cells.     

Expression of Galpha 13 (Q226L) induces P19 stem cells to primitive endoderm via MEKK1, 2, or 4.

Galpha13 mediates the ability of the morphogen retinoic acid to promote primitive endoderm formation from mouse P19 embryonal carcinoma stem cells, a process that includes the obligate activation of Jun N-terminal kinase. Expression of the constitutively activated (Q226L) GTPase-deficient form of Galpha13 mimics retinoic acid and was used to investigate the signaling upstream of primitive endoderm formation. Jun N-terminal kinase 1 activity, MEK1,2, MKK4, and MEKK1 were constitutively activated in clones stably transfected to express Q226L Galpha13. Dominant negative forms of MEKK1 and MEKK4 were expressed stably in the clones harboring Q226L Galpha13. Expression of dominant negative versions of either MEKK1 or MEKK4 effectively blocks both the activation of Jun N-terminal kinase as well as the formation of primitive endoderm. Depletion of MEKK1, -2, or -4 by antisense oligodeoxynucleotides suppressed signaling from Q226L Galpha13 to JNK1 and primitive endoderm formation. We demonstrate that the signal linkage map from Galpha13 activation to primitive endoderm formation in these stem cells requires activation at three levels of the mitogen-activated protein kinase cascade: MEKK1, -2, or -4 for MAP kinase kinase kinase; MKK4 and/or MEK1 for MAP kinase kinase; and JNK1 for MAP kinase.     

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1,3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.     

The fate of prorenin during granulopoiesis in epithelioid cells

Summary Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2 A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH₂ terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex.After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme.The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a some-what earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.The data presented are consistent with the assumption that the secretion of active renin takes place by the exocytosis of mature secretory granules, while the secretion of inactive renin, which is a truncated version of intact prorenin, is mediated by the exocytosis of juvenile and intermediate granules.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg