[Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes].

When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be detrimental. In the study, the streptavidin monomer fragment (S1-116) was fused with the intein N-terminal (termed precursor S1-116-IN), and S1-116-IN was expressed in E. coli (BL21). Meanwhile, the SA117-160 fragment was fused with the C-terminal intein, and then this chimeric polypeptide was expressed on magnetosomes by fusion with magnetosome membrance protein MamF. In the in vitro protein splicing system, the purified engineered magnetosomes (BMP-SA117-160-IC) and the S1-116-IN precursor were mixed. Intein-mediated trans-splicing reaction was induced to produce the functional magnetic beads BMP-SA. Our results indicate that intein-mediated protein trans-splicing may lead to efficient synthesis of the recombinant streptavidin on the magnetosomes, showing its promising potential to produce other functional magnetic nanoparticles.

Mur (MNS 10) screening with a novel loop-mediated isothermal amplification assay in Zhongshan, China.

BACKGROUND: The distribution of the Mur blood group antigen is 5-7% in the south of China, and a much higher prevalence is observed in some areas of the region. Anti-Mur can cause hemolytic disease of the newborn and severe transfusion reactions. OBJECTIVES: Genetic testing is more ideal than conventional serological tests because antibodies for detection are usually not available. METHODS: In this study, a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Mur blood group antigen was established. RESULTS: Fifteen of 275 (5·5%) samples were confirmed by LAMP as Mur antigen positive. All the Mur antigen-positive samples were GP.Mur subtype which was confirmed with sequencing. CONCLUSION: The LAMP method has identical results with conventional serology method but more suitable for large-scale screening.

The relationship between the TGF-beta1 gene -509C/T polymorphism and tubulointerstitial damage resulting from primary nephrotic syndrome.

BACKGROUND: The aim of this study was to investigate the correlation between the transforming growth factor (TGF)-beta1 gene -509C/T polymorphism and the susceptibility to primary nephrotic syndrome (PNS), and in particular to the severe degree of tubulointerstitial damage (TID) seen in Chinese. METHODS: Ninety-eight PNS patients and 128 normal controls were studied. The extent of tubulointerstitial changes was evaluated and patients were divided into two groups according to the severe or mild degree of TID. The TGF-beta1gene -509C/T polymorphism was detected with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, and the serum level of TGF-beta1 was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: No statistical differences in genotype or allele frequency of the TGF-beta1 gene -509C/T were found between PNS and normal subjects. However, T allele and CT + T T genotype frequency were higher in the PNS with severe TID than the mild TID and controls. Additionally, the serum concentration of TGF-beta1 was significantly higher in the PNS with severe TID group than the other two groups and in the T T genotype individuals than the CC and CT genotype individuals. A logistic regression analysis demonstrated that TGF-beta1 gene -509C/T genotype was the risk factor of TID in PNS patients [OR (odd ratio) 2.34, confidence interval (CI) 0.98-3.46, p = 0.012]. CONCLUSION. TGF-beta1 gene -509C/T polymorphism was associated with severe TID. The higher value in serum concentration of TGF-beta1 was also associated with severe TID and the T T genotype/T allele. T allele gene might be the important risk factor for susceptibility.

[Urinary RBP and glucocorticoid hormone therapy sensibility in adult primary nephrotic syndrome].

OBJECTIVE: To clarify the relationship between the renal tubular function and the efficacy of glucocorticoid, in adult patients with primary nephrotic syndrome. METHODS: Pro- and post-therapy urinary RBP and NAG were determined double antibodies sandwich ELISA and color comparimetry with p-nitrate reductase, respectively, in eighty adult patients with primary nephritic syndrome, according to the concentrations of urinary protein, these patients were divided into three no remission group, partial remission group and fully remission group, fifty-one normal persons as control group. Renal tubular function parameters among groups were compared before and after therapy, and the results were analysed when those parameters were used to predict the sensibility of glucocorticoid therapy. RESULTS: 1. There were no significant differences in urinary RBP, NAG and protein levels before treatment among patient groups (P > 0.05); 2. There were significant decreases in urinary RBP and NAG following glucocorticoid therapy in those response to glucocorticoid (P < 0.01); 3. There were significant differences in urinary protein, RBP and NAG among three groups of patients with primary nephrotic syndrome after treatment. CONCLUSION: Determination of urinary RBP may predict the sensibility of the therapy in the adult patients with primary nephrotic syndrome and its diagnostic efficiency is better than urinary NAG.

[Relationship between protein loss and dialysis duration results of PET and KT/Vurea in CAPD patients].

The study focused on the relationship between protein loss and dialysis duration, results of PET and KT/Vurea in two groups of CAPD patients. 16 patients of one group were included in CAPD for only 30 days while 19 patients of the other group were included in CAPD for more than one year. The results showed that value of protein loss had no obvious significance between both groups. Compared with the patients with value of PET D/Pcr < 0.65, the patients with value of PET D/Pcr > or = 0.65 had no significant protein loss. Between the patients whose index of urea clearance > or = 2.0, and those index of uera clearance < 2.0 the protein loss had no difference. It is suggested that, in routine dialysis duration peritoneal transport of small solutes and dialysis adequency. The conclusion shows furtherly that transportion of large protein and small solutes through different ways.

[Culture and characterization of human peritoneal mesothelial cells].

OBJECTIVE: To establish a reproducible model for culture of human peritoneal mesothelial cells (HPMCs), and observe the expressions of extracellular matrix (ECM) protein and interleukin-8 (IL-8) in HPMC. METHODS: Mesothelial cells were isolated from human omenta by trypsin EDTA disaggregation. HPMCs were identified by morphology and streptomyces protein-peroxidase (SP) method. In the same way, expressions of fibronectin, collagen type III, V and transforming growth factor beta 1 (TGF beta 1) were measured. Expression of IL-8 mRNA and FN mRNA in the HPMCs was detected by reverse transcription (RT) and polymerase chain reaction (PCR) amplification. RESULTS: Confluent HPMCs appeared multipolar under microscope; numerous surface microvilli and an abundant endoplasmic reticulum were observed. Cultured HPMCs coexpressed cytokeratin and vimentin, and synthesized fibronectin, TGF beta 1 and collagen types III (instead of type V). Expressions of IL-8 mRNA and FN mRNA were also observed in HPMCs. CONCLUSION: Establishment of cultured HPMCs model will provide the basis of the research in preventment of peritoneal fibrosis and roles of IL-8 in peritoneal dialysis.

Nephrotoxicity of high- and low-osmolar contrast media. The protective role of amlodipine in a rat model.

PURPOSE: To evaluate the nephrotoxicity of high- and low-osmolar contrast media (HOCM, LOCM) on kidneys in Sprague-Dawley rats. The protective role of amlodipine was studied. MATERIAL AND METHODS: Forty rats of both sexes were randomly divided into 5 groups (n=8/group) and glycerine for inducing renal failure was given to all rats except controls. RESULTS: In diatrizoate-injected rats, blood urea nitrogen (BUN) and serum creatinine (SCr) were increased; levels of phospholipase A2 (PLA2), lipid peroxide (LPO) and calcium were also increased in renal tissues. There was no significant difference between LOCM (iohexol) animals and glycerol controls either in the renal levels of PLA2, LPO and calcium or in the levels of BUN and SCr. The histologic changes were milder in the LOCM animals than in the HOCM animals. In the group pretreated with amlodipine, no increase in the levels of BUN or SCr was discovered and the renal content of PLA2, LPO and calcium were significantly lower than in the HOCM group; the renal injuries induced by diatrizoate were alleviated. CONCLUSION: The HOCM, diatrizoate, was more toxic to rat kidneys than the LOCM iohexol; PLA2, LPO and calcium load played a role in producing renal function impairment induced by diatrizoate meglumine; amlodipine protected the renal tissue from nephrotoxicity induced by diatrizoate.

[The effect of increasing ventral movement on the small solutes transport during peritoneal dialysis].

OBJECTIVE: To investigate whether the small solutes transport and the efficiency of peritoneal dialysis can be improved through ventral movement. METHODS: Eighteen continuous ambulatory peritoneal dialysis (CAPD) patients and 6 male New Zealand rabbits peritoneal dialysis models were observed. Through self and pre-post control, we compared the concentration ratio of the solutes in effluent dialyste(D) to the solutes in plasma(P), mass transfer area coefficient and the drained volume. RESULTS: The animal experiments showed that the D/P value for BUN was different prominently when comparing the vibration and non-vibration groups in 45 min and 60 min separately(P < 0.05). On the contrary, no obvious difference existed between the two groups when comparing the D/P values for protein and drained volume. The clinical study showed: D/P value for BUN at the 2 and 4 hr and D/P value for creatinine at the 2 hr increased obviously either in the low or high frequency group when compared with the control group(P < 0.01, P < 0.01, P < 0.05, respectively). D/P for protein and drained volume had no difference compared with the control group(P > 0.05). CONCLUSION: The ventral movement and vibration could efficiently increase the transport of small solutes such as BUN and creatinine while had no influence on the transport of large molecular solutes such as protein and the ultrafiltration volume. It suggested that increasing the movement of CAPD patients can improve the efficiency of peritoneal dialysis.

[Effects of nitroprusside and vibration on peritoneal transport of solutes in continuous ambulatory peritoneal dialysis patients].

To explore the relationship between peritoneal transport of solutes and permeability of the peritoneum capillary as well as peritoneal stagnant fluid layer within dwell time, we observed the effects of nitroprusside and vibration on peritoneal transport of solutes in continuous ambulatory peritoneal dialysis(CAPD) patients. Twelve stable, routine CAPD patients were involved, who had no peritonitis for at least 4 weeks before the test. Standard peritoneal equilibrium tests(PETs) were performed, and mass transfer area coefficiency(MTAC) were calculated after adding nitroprusside to the dialysate or vibrating the patients's peritoneum. The concentrations of total protein, albumin and immunity globulin G in total drained dialysate were examined, and total drained volumes were recorded. Compared with the control, the MTACs value of BUN, Creatinine(Cr) increased significantly both in the nitroprusside group and vibration group(P < 0.05); the concentration of immunoglobin G in the total drained fluid was higher in the nitroprusside group than that in the control(P < 0.05); However, there was no significant difference in the total drained volume among the three groups. We conclude that nitroprusside and vibration can increase the peritoneal transport of small molecular solutes, and vibration has less influence on the loss of protein in CAPD. It suggests that moderated movement may improve the removal of the small molecules in CAPD patients.

Assessment of urinary endothelin-1 and nitric oxide levels and their relationship with clinical and pathologic types in primary glomerulonephritis.

To determine the relationship between the urinary endothelin (ET-1), nitric oxide (NO) levels and the clinical, pathologic types of primary glomerulonephritis (GN) patients, urinary levels of ET-1 and NO were detected in 27 patients with biopsy-proven primary GN and 12 normal controls by radioimmunoassay and by copper-plated and cadmium column reduction assay, respectively. The results showed that urinary ET-1 levels in the patients with primary GN were significantly higher than in normal controls (p < 0.01), while the urinary ET-1 levels in patients with moderate mesangial proliferation GN were significantly higher than those in patients with mild mesangial proliferation GN (p < 0.05). Urinary ET-1 levels in patients whose clinical feature was nephrotic syndrome were found to be higher than in patients whose clinical feature was nephritic syndrome. However, urinary NO levels were to the contrary (p < 0.05). The ratio of ET-1/NO in primary GN patients was significantly higher than that in normal controls, and it positively correlated with the 24-hour urinary excretion of protein. These results suggest that urinary ET-1 levels are related to the proliferation of mesangial cells. The imbalance between ET-1 and NO may be related to the pathogenesis of primary GN and the occurrence of proteinuria.

Assessment of renal function in the early stages of nephrotoxicity induced by iodinated contrast media.

To determine whether there are early renal function parameters (RFP) which can be monitored to rapidly detect nephrotoxicity induced by contrast media (CM), we observed RFP in 16 patients with normal renal function before and after administration of CM. Forty-eight hours after diatrizoate meglumine administration, blood urea nitrogen (BUN) and serum creatinine (SCr) increased (p < 0.05). In all patients, acute tubular damage was revealed by early urinary RFP. Increases in levels of serum angiotensin-I-converting enzyme (ACE), beta(2)-microglobulin (beta(2)M) and urinary albumin (Alb) were associated with alterations in glomerular function. The changes in early RFP occurred earlier than those of BUN and SCr. The present study demonstrates that serum ACE, beta(2)M, urinary Alb, gamma-glutamyl-transpeptidase and N-acetyl-beta-D-glucosidase are sensitive parameters for the early assessment of subclinical nephrotoxicity induced by CM.