Passive dual immunization against tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta maximally ameliorates acute aminonucleoside nephrosis.

Rats receiving a single dose (10 mg/100 g) of aminonucleoside of puromycin (PAN) develop heavy proteinuria and acute interstitial nephritis (AIN). Whole isolated glomeruli from rats injected with PAN secreted both TNF-alpha and IL-1 beta cytokines. TNF-alpha secretion was first and maximally detected on day 3, whereas IL-beta activity was found on day 7, when rats were heavily proteinuric and AIN developed. In vivo treatment with either anti-TNF-alpha or anti-IL-1 beta antibodies produced a drastic and simultaneous reduction in both levels of proteinuria and intensity of interstitial cell infiltrate. These effects improved when both antibodies were administered together. Our studies demonstrate the effectiveness of immunosuppressive therapy against these two cytokines in rats with PAN-induced nephrosis.     

Effects of Methyl Mercury on Protein Synthesis in Vitro

Abstract: Methyl mercury has been shown to interact with protein synthesis in vivo and in vitro. In the present paper a brain postmitochondrial supernatant was used for studies in vitro. Inorganic mercury (Hg²⁺) was shown to be a more potent inhibitor of protein synthesis than methyl mercury, puromycin or cycloheximide. The inhibitory effect of methyl mercury was potentiated by puromycin. It is thus possible that methyl mercury causes disintegration of polysomes in brain cells.     

ASCORBATE STIMULATION OF TYROSINE HYDROXYLASE FORMATION

Tyrosine hydroxylase is decreased in the adrenal glands of scorbutic guinea pigs. The enzyme, which has been purified and immunochemically assayed, is increased by L -ascorbate in vivo unless animals are treated with puromycin or actinomycin D to block protein biosynthesis.     

雷帕霉素对PAN肾病小鼠肾脏病变和VEGF及受体表达的影响

目的 探讨雷帕霉素对嘌呤霉素氨基核苷（PAN）肾病模型小鼠肾脏病变和肾小球VEGF及受体（VEGFR）表达的影响.方法 24只BALB/c小鼠随机分为PAN 组（单次尾静脉注射PAN造模,n=8）、雷帕霉素干预组（单次尾静脉注射PAN造模＋雷帕霉素干预,n=8）和对照组（单次尾静脉注射PBS,n=8）.各组动物留取24 h尿液,BCA蛋白定量试剂盒测定24 h尿蛋白排泄量.于造模后第10天处死动物取肾脏制备肾组织标本,透射电子显微镜观察各组肾小球足突结构改变;分别采用Real-time PCR、Western b1otting和免疫组织化学方法检测各组肾组织VEGF、VEGFRI、VEGFR2 mRNA以及VEGF、VEGFR2蛋白表达.结景24 h尿蛋白排泄量为PAN组〉雷帕霉素干预组〉对照组,组间差异均有统计学意义（P〈0.05）.电子显微镜观察发现PAN 组肾小球上皮细胞足突广泛融合.各组肾组织VEGF、VEGFRI、VEGFR2 mRNA及蛋白表达为PAN组〉雷帕霉素干预组〉对照组（P〈0.05）,且各组VEGF、VEGFRI、VEGFR2 mRNA及VEGF蛋白表达与24 h尿蛋白排泄量呈正相关（P〈0.05）.结论 雷帕霉素能减轻PAN肾病小鼠蛋白尿的程度,作用机制可能与下调肾小球VEGF及其受体基因表达有关.     

Effects of Cyclosporine on Puromycin Aminonucleoside-Induced Nephrotic Rats

Summary

The effects of cyclosporine (CY) were determined to see whether CY has the potency of reducing urinary protein excretions due to puromycin aminonucleoside (PAN) in nephrotic rats which are the model of minimal change nephrotic syndrome (MCNS). CY had a marked reduction of urine protein excretion during treatment with this drug, but it was observed that an increment in urinary protein excretion was introduced within a few days after the withdrawal of CY. Immunological and histological studies were performed to know the mechanisms of reducing urinary protein excretion in PAN rats. Firstly, the lymphocyte subsets of peripheral blood were evaluated using monoclonal antibodies of W3/25 and MRCOX/8. We found that the W3/25/MRCOX-8 ratio increased to 3.2 ±0.3 in the PAN rats but significantly decreased to 2.8±0.3 with CY treatment. From these data it was impossible to decide whether the effect with CY had direct activity or not, because the interleukin-2 (IL-2) activity was not measured in rats. Secondly, polyethyleneimine (PEI) staining was performed to detect die sites of anion charge on glomerular basement membrane (GBM), and anionic dots on GBM in the CY-treated rats were clearly defined as nearly normal.

Although there persist questions as to why nephrotic rats relapsed after the withdrawal of CY treatment, our results indicate that CY might reduce urinary protein excretion through immunological and non-immunological mechanisms.     

嘌呤霉素肾病模型肾组织微小RNA的差异性表达及雷至胶囊的干预作用

目的:观察嘌呤霉素氨基核苷(puromycin aminonucleoside,PAN)肾病模型鼠肾组织微小RNA(microRNAs,miR-NAs)差异性表达的特征及其与足细胞裂隙膜(slid diaphragm,SD)关键结构分子nephrin,podocin和骨架蛋白synaptopodin表达的关系;阐明雷至胶囊(Leizhi capsule,LZC)在体内通过调控模型鼠肾组织miRNAs差异性表达而改善足细胞nephrin,podocin,synaptopodin表达,减少蛋白尿的机制.方法:将50只雄性Wistar大鼠随机分为空白组(A)、模型组(B)、雷至胶囊组(C,5 mL·kg-1·d-1)、雷公藤多苷组(D,10mL·kg-1 ·d-1)和缬沙坦(E,7.5 mL· kg-1·d-1)组.除A组外,其余各组大鼠一次性经颈静脉注射PAN(100 mg·kg-1)而建立PAN肾病模型.造模后第2天灌胃给药,A,B组大鼠用生理盐水干预,共10 d.造模前及造模后第3,9天称量各组大鼠体重,并检测24h尿蛋白排泄量(urinary protein ration,Upro).造模后第11天,处死全部大鼠,采集血液和肾组织,观察血清生化指标血清白蛋白(albumin,Alb),血清肌酐(serum creatinine,Scr),血清尿素氮(blood urea nitrogen,BUN),肾小球超微结构(足细胞足突形态)以及肾组织dicer酶,nephrin,podocin,synaptopodin表达;同时,借助生物芯片(biochip)技术,分析肾皮质miRNAs差异性表达的特征,并且,借助荧光实时定量聚合酶链反应(Real-time PCR)验证mo-miR-23a,rno-miR-300-3p,rno-miR-24,rno-miR-30c等的差异性表达量.结果:在PAN诱导下,模型鼠出现蛋白尿、肾功能减退、低白蛋白血症、足细胞足突融合;在模型鼠肾组织中,dicer酶影响足细胞nephrin,podocin,synaptopodin表达;上调rno-miR-23a,mo-miR-300-3p表达,下调mo-miR-24,rno-miR-30c表达;差异性表达的miRNAs包括rno-miR-24,rno-miR-30c,rno-miR-23a,rno-miR-300-3p等.雷至胶囊能改善PAN肾病模型鼠的一般情况、蛋白尿、血清BUN、足细胞足突融合;还能减少模型鼠肾组织dicer酶表达,增加足细胞nephrin,podocin和synaptopodin表达;减弱在模型鼠肾组织中上调的rno-miR-23a,rno-miR-300-3p,增强在模型鼠肾组织中下调的rno-miR-24,rno-miR-30c等.结论:PAN在体内通过dicer酶/miRNAs差异性表达途径而影响模型鼠肾组织miRNAs表达,干预足细胞nephrin,podocin,synaptopodin表达,破坏足细胞结构和功能,产生蛋白尿;雷至胶囊可以减少PAN肾病模型鼠蛋白尿,其机制可能是干预dicer酶/miRNAs差异性表达途径,调控足细胞nephrin,podocin,synaptopodin表达,改善足细胞的结构与功能.     

MicroRNA-194过表达与抑制表达慢病毒载体的构建及对骨肉瘤细胞转染与筛选的方法学研究

目的：构建针对人microRNA—194过表达及抑制表达的慢病毒表达载体,寻找与探讨感染人骨肉瘤细胞系SO-SP-9607和U2-os的最佳步骤和方法。方法：利用PcR方法调取相应目的基因进行酶切,经电泳回收后与目的基因进行连接,产物转化细菌感受态细胞,对克隆进行PcR鉴定和测序对比分析后,构建相应microRNA—194过表达及抑制表达慢病毒表达载体;在人骨肉瘤细胞系SO-SP-9607和U2—os转染及筛选过程中,根据不同阶段及浓度设定相应实验组,并设相应对照组。倒置显微镜观察转染效率,筛选情况,进行比较。结果：PCR及测序结果证实重组慢病毒表达质粒构建正确。过表达及抑制表达重组慢病毒的滴度分别为15×10^8TU／ml及4×10^8TU／ml。感染复数（multiply of infection,MOI）值测定,实验组及对照组转染效率无明显差异,获得MOI值及感染时间数据。通过新的综合设计,经筛选后获得转染效率满意的目的克隆细胞。结论：成功构建了microRNA—194过表达及抑制表达慢病毒表达载体,并通过新的综合考虑设计,对人骨肉瘤细胞系SO-SP-9607和U2-os进行转染和筛选后,可较快和较高效率获得满意目的细胞。     

Protoribosome by quantum kernel energy method

Experimental evidence suggests the existence of an RNA molecular prebiotic entity, called by us the “protoribosome,” which may have evolved in the RNA world before evolution of the genetic code and proteins. This vestige of the RNA world, which possesses all of the capabilities required for peptide bond formation, seems to be still functioning in the heart of all of the contemporary ribosome. Within the modern ribosome this remnant includes the peptidyl transferase center. Its highly conserved nucleotide sequence is suggestive of its robustness under diverse environmental conditions, and hence on its prebiotic origin. Its twofold pseudosymmetry suggests that this entity could have been a dimer of self-folding RNA units that formed a pocket within which two activated amino acids might be accommodated, similar to the binding mode of modern tRNA molecules that carry amino acids or peptidyl moieties. Using quantum mechanics and crystal coordinates, this work studies the question of whether the putative protoribosome has properties necessary to function as an evolutionary precursor to the modern ribosome. The quantum model used in the calculations is density functional theory–B3LYP/3–21G*, implemented using the kernel energy method to make the computations practical and efficient. It occurs that the necessary conditions that would characterize a practicable protoribosome—namely (i) energetic structural stability and (ii) energetically stable attachment to substrates—are both well satisfied.A suggestion of a molecular entity, called “protoribosome,” which may have evolved and emerged from an RNA world before a subsequent evolution into the modern protein/nucleic acid world, has been reported (1–3). In contemporary cells the ribosomes translate the genetic information (stored in the DNA) into proteins. Ribosomes are gigantic complexes, which in prokaryotes are built of some 50 proteins and three RNA chains with a total of 4,500 nucleotides. Aptly referred to as the protein factory of all living cells, the ribosome is essential to the contemporary life, and its activity may have been crucial to the formation of life itself. Structural analysis identified an internal RNA region that exists in all known structures (1–5) and has universally conserved sequence (1), which contains the site of peptide bond formation, and thus may well be that of a remaining RNA world entity. Consistent with the findings that the main ribosomal functions—namely, the decoding of the genetic code, the formation of peptide bonds, and the creation of elongating proteins—are performed by ribosomal RNA and with the universality of this region among all kingdoms of life, we proposed that this region is a remnant of a prebiotic chemical entity with catalytic capabilities, and called it the “protoribosome.” Within the otherwise asymmetric ribosome, this region has a unique fold (6) and could have been the link to the modern world (7). It is characterized by a pseudotwofold symmetry with a highly conserved nucleotide sequence and seems to possess all of the assumed prerequisites for the formation of chemical bonds. This semisymmetric object could be a dimer of self-folding RNA units that formed a pocket within which two activated amino acids, as substrates, might be accommodated.A representation of a plausible sequence for spontaneous self-assembly of a protoribosome is shown in Fig. 1 (2). Here, we put forth the use of quantum mechanics to answer the following question: Is the suggested protoribosome structure a plausible reality? One may systematically remove—that is, mathematically—all surrounding parts of the modern ribosome and use the coordinates of a central symmetric pocket for constructing a putative protoribosome. Here we apply quantum mechanics to the structure of that protoribosome. The most fundamental inquiry followed in this article is that of the energetic stability of the proposed protoribosome. This is not presently known. And obviously if the structure is not energetically stable, it is not likely to be able to act as a biological catalyst, as would be required of a protoribosome. The protoribosome contains almost 200 nucleotides, namely thousands of atoms. Ab initio quantum calculations rise in difficulty as a high power of the number of atoms in the system. Therefore, quantum calculation of the protoribosome energy is a complex computational problem. Fortunately we are in possession of a recently discovered kernel energy method (KEM) (8–24), described below, which alleviates dramatically the computational difficulty of ab initio calculations. Importantly the KEM is highly accurate, as well as computationally efficient.Open in a separate windowFig. 1.The scheme by which small, self-folded RNA molecules dimerize to form a symmetrical pocket allowing accommodation of a pair of substrates. The A-site region (Areg) and the P-site region (Preg), respectively, (Upper Left) dimerize (Upper Right) to allow substrate accommodation. Reproduced by permission from ref. 2 [Davidovich et al. (2009) Research in Microbiology 160(7):487–492]. Copyright Elsevier Masson SAS.As an example of the large size of systems that can be studied with ab initio KEM, we have applied the method to a Hartree–Fock (HF) calculation of a 33,000-atom protein (16). It is entirely feasible to treat even larger molecules within the context of KEM capabilities. Therefore, we performed an ab initio KEM study of the protoribosome and showed that its existence is quite feasible. Using KEM we address the question of whether the basic symmetric structure of the folded dimer pocket that constitutes the protoribosome suggested previously (4) proves to be quantum mechanically stable. If so, the next question to address is: Can it accommodate a pair of amino acids bound to a chain of a few (1–3) nucleotides, representing the tRNA 3′−end, spatially and energetically? Furthermore, such calculations should indicate the energetic preferences for the length of the nucleotide chain and its correlation to the protoribome size, ranging between 120 and 180. If both questions would be validated quantum mechanically, that would be highly suggestive of the protoribosome as an actual remnant from the RNA world still functioning in the chemistry of life, in the modern DNA/RNA/protein world.     

THE EFFECT OF ATRIAL NATRIURETIC PEPTIDE INFUSION ON RENAL HAEMODYNAMICS AND PLASMA LIPOPROTEINS IN PUROMYCIN AMINONUCLEOSIDE NEPHROSIS IN RATS

1. The effect of continuous intravenous administration of 1 UmUg/h atrial natriuretic peptide (ANP) for 4 days was studied in normal male Sprague-Dawley rats and rats made nephrotic with puromycin aminonucleoside (PA). 2. ANP infusion significantly increased urinary sodium and potassium excretion by 3 days of infusion in control rats but not in PA-treated rats. ANP infusion significantly increased glomerular filtration rate in PA-treated rats, while effective renal plasma flow was similarly decreased compared with non-infused nephrotic rats. 3. Plasma high density lipoproteins (HDL) were significantly decreased and low density lipoproteins (LDL) were increased in PA-treated rats that received ANP; HDL were increased in normal rats infused with ANP. 4. Competitive binding studies demonstrated a lower density of specific ANP receptors in glomerular membranes from rats injected with PA, while binding affinity was unchanged. 5. Infusion with exogenous ANP did not promote natriuresis in PA nephrosis despite an enhancement of glomerular filtration rate (GFR), thus suggesting that sodium retention in this model is due to a post-glomerular defect. Plasma lipoprotein composition in both normal and nephrotic rats may be affected by ANP.     

Iimitations of podocyte adaptation for glomerular injury in puromycin aminonucleoside nephrosis

Glomerular synechiae that occurred in nephrotic rats with a single intraperitoneal injection of puromycin aminonucleoside were analyzed by immunohistochemistry, radiolabeled thymidine (PHI-thymidine) autoradiography, as well as light, electron and immunoelectron microscopy. To discriminate podocytes from parietal epithelial cells (PEC) and monocytes, monoclonal antibodies (mAb) against podocalyxin and ED1 were used. The cell kinetics of glomerular epithelial cells were autoradiographically assessed with isotope labeling procedures before and during nephrosis (co-labeled), and a mAb against proliferating cell nuclear antigen (PCNA). All the cell types except the podocyte of normal kidneys were labelled with rHI-thymidine at different rates. Detachment of degenerated podocytes from the outside of the glomerular basement membrane (GBM) is the first step of synechia, and detached sites are confronted by PEC that were hypertro-phied and frequently radiolabeled. Evidence that podocytes in glomeruli of nephrotic rats can proliferate was shown by the presence of mitoses, ^rHt]-thymidine uptake in the co-labeled experiment, and by PCNA staining, but reepithelialization over bare segments of the GBM with proliferated podocytes is doubtful. It was concluded that glomerular synechia resulted from the limits of podocyte adaptation to glomerular injuries.