Specific role of NAD+ biosynthesis reduction mediated mitochondrial dysfunction in vascular endothelial injury induced by chronic intermittent hypoxia.

OBJECTIVE: Cardiovascular diseases (CVD) are prevalent among those with obstructive sleep apnea (OSA) and are the leading cause of death in these individuals. However, due to clinical confounders, the mechanism by which OSA induces CVD is still unclear. Previous studies have shown that chronic intermittent hypoxia (CIH) and high cholesterol diet (HCD) induce distinct characteristics of atherosclerotic plaques, highlighting the specific mechanisms involved in CIH-induced vascular endothelial injury. MATERIALS AND METHODS: This study aims to investigate whether nicotinamide adenine dinucleotide (NAD+) biosynthesis reduction-mediated mitochondrial dysfunction is responsible for vascular endothelial injury induced by CIH and to elucidate its specific role in this process. Models were established to stimulate human umbilical vein endothelial cells (HUVECs) with CIH and oxidized low-density lipoprotein (ox-LDL), and the NAD+ biosynthesis-related indicators, such as NAD+ levels and nicotinamide phosphoribosyltransferase (NAMPT) enzyme activity, were measured in this model. Additionally, interventions were performed by supplementing NAD+ levels with nicotinamide mononucleotide (NMN), inhibiting NAD+ synthesis with FK866, and evaluating mitochondrial function, oxidative stress status, vascular constriction and dilation function, and endothelial adhesion function in these models. A comparative study was conducted to assess the effects of these interventions. RESULTS: We found that under CIH conditions, NAMPT enzyme activity was inhibited, leading to a reduction in NAD+ biosynthesis and a decrease in NAD+/NADH ratio. At the same time, CIH caused mitochondrial dysfunction in HUVECs, including a decrease in adenosine triphosphate (ATP) content and mitochondrial membrane potential, as well as the activity of respiratory chain complex I and III, induced an increase in oxidative stress levels in endothelial cells, impaired vascular constriction and dilation function, and significantly increased expression of adhesion factors. The impact of CIH on endothelial cell-related mitochondrial function and endothelial function was restored by supplementing NMN. Although ox-LDL also causes multi-level endothelial injury, it does not involve the NAD+ pathway, as there were no significant changes in the related indicators, and the impaired endothelial function under ox-LDL conditions was not restored by supplementing NMN. CONCLUSIONS: CIH-induced vascular endothelial injury may be associated with NAD+ biosynthesis reduction-mediated mitochondrial dysfunction. Supplementing NAD+ precursors to increase its levels may be a potential intervention to ameliorate CIH-induced vascular endothelial injury, while it does not have a significant effect on endothelial injury caused by ox-LDL.

[The efficacy and safety profile of ixazomib/lenalidomide/dexamethasone in relapsed/refractory multiple myeloma: a multicenter real-world study in China].

Objective: To evaluate the efficacy and safety profile of ixazomib/lenalidomide/dexamethasone (IRd) in Chinese patients with relapsed/refractory multiple myeloma (MM) . Methods: This study comprising 14 medical centers in China included patients with relapsed/refractory MM who received at least. Ixazomib at an initial oral dose of 4 mg was administered. Seven patients had dose adjustment to 3 mg at the time of first dose. The lenalidomide doses were adjusted according to creatinine clearance rate. The efficacy and safety were evaluated every cycle. Results: In the study cohort of 74 patients, the median age was 65 years and 11 (14.9% ) patients received over three lines of therapy. Overall response rate (ORR) was 54.1% (40/74) , and 7 (9.5% ) , 14 (18.9% ) , and 19 (25.7% ) patients achieved stringent complete response or complete response, very good partial response, and partial response, respectively. The median progression-free survival and overall survival were 9.9 and 20 months, respectively. The median time to response was 1 month. The efficacy and survival outcome were similar to those reported in the Tourmaline-MM1 China Continuous Study. The ORR of patients refractory to bortezomib, lenalidomide, and bortezomib plus lenalidomide were 52.0% (13/25) , 57.1% (4/7) , and 33.3% (6/18) , respectively. The rate of grade 3-4 adverse events was 36.5% (27/74) . Common hematological toxicities were anemia, thrombocytopenia, lymphopenia, and neutropenia. Common non-hematological toxicities were fatigue, gastrointestinal symptoms, and infections. Two cases of grade 3 peripheral neuropathy were reported. The patients eligible for the Tourmaline-MM1 China Continuous Study had a higher ORR than the ineligible patients [77.8% (14/18) vs 46.4% (26/56) , P=0.020]. There was no difference in the rate of grade 3-4 adverse events [33.3% (6/18) vs 37.5% (21/56) , P=0.749]. Conclusion: The IRd regimen had good efficacy and acceptable toxicity in Chinese patients with relapsed/refractory MM.

Circ_0061140 promotes metastasis of bladder cancer through adsorbing microRNA-1236.

We detected some serious inaccuracies and mistakes. Therefore, the article, "Circ_0061140 promotes metastasis of bladder cancer through adsorbing microRNA-1236, by F. Feng, A.-P. Chen, X.-L. Wang, G.-L. Wu, published in Eur Rev Med Pharmacol Sci 2020; 24 (10): 5310-5319-DOI: 10.26355/eurrev_202005_21313-PMID: 32495864" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/21313.

Circ_0061140 promotes metastasis of bladder cancer through adsorbing microRNA-1236.

OBJECTIVE: The purpose of this study was to investigate the expression characteristics of circular RNA circ_0061140 in bladder cancer (BCa), and to further explore its effects on invasiveness and migration capacity of BCa cells, as well as its possible potential mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression level of circ_0061140 in tumor tissue samples and paracancerous ones collected from 42 patients with BCa, and the interplay between circ_0061140 level and the clinical indicators, as well as the prognosis of BCa patients were analyzed. Meanwhile, qRT-PCR was also used to verify circ_0061140 expression in BCa cell lines. In addition, a circ_0061140 knockdown model was constructed using Lentiviral in BCa cell lines, including T24 and 253j, and the effect of circ_0061140 on BCa cell functions and its underlying mechanisms were explored using Cell Counting Kit-8 (CCK-8), transwell, and cell wound healing assays. RESULTS: qPCR results showed that the expression level of circ_0061140 in tumor tissues of BCa patients was remarkably higher than that in adjacent tissues, and the difference was statistically significant. Compared with patients with low expression of circ_0061140, patients with high expression of circ_0061140 had worse prognosis and higher incidence of lymph node or distant metastasis. Compared with those in the negative control group, the proliferation and invasion, as well as the metastasis ability of BCa cells in the sh-circ_0061140 group, were remarkably attenuated. In addition, bioinformatics and Luciferase reporter gene assay demonstrated that circ_0061140 can specifically bind to microRNA-1236. At the same time, the results of qPCR revealed that the expression levels of circ_0061140 and microRNA-1236 were negatively correlated in the tumor tissues of BCa patients. Finally, cell recovery experiment indicated that silencing microRNA-1236 reversed the impact of the knockdown of circ_0061140 on the ability of BCa cells to proliferate and invade, suggesting that the two may regulate each other. CONCLUSIONS: Circ_0061140 level was found remarkably elevated in BCa tissues, as well as in cell lines, which was closely relevant to the incidence of lymph node or distant metastasis of BCa patients. In addition, circ_0061140 may enhance the proliferation rate and invasion ability of BCa cells through the modulation of microRNA-1236.

Pharmacokinetics of cefozopran by single and multiple intravenous infusions in healthy Chinese volunteers.

BACKGROUND AND OBJECTIVES: Cefozopran is a parenteral cephalosporin with a broad spectrum of activity against Gram-positive and Gram-negative bacteria. The objective of this study was to evaluate the pharmacokinetics of cefozopran after single- and multiple-dose intravenous administration in healthy subjects, to provide clinical guidance in its application. METHODS: This was a single-center, open-label, randomized, two-phase study conducted in 12 subjects. In the single-dose phase, subjects were randomly assigned to receive single doses of 0.5, 1.0 and 2.0 g of injected cefozopran hydrochloride in a three-way crossover design with a 5-day washout period between administrations. In the multiple-dose phase, subjects received 2.0 g every 12 h for 4 days. Plasma and urine pharmacokinetic samples were assayed by a validated high-performance liquid chromatography-tandem mass spectrometry method. Pharmacokinetic parameters were calculated and analyzed statistically. Safety assessments were conducted throughout the study. RESULTS: Twelve healthy volunteers (six males and six females) were enrolled and completed the study. Following a single 1-h intravenous infusion of 0.5, 1.0 or 2.0 g cefozopran, maximum plasma concentration (C max) and area under the plasma concentration-time curve from time zero to the time of the last measurable concentration (AUClast) increased in a dose-proportional manner. The mean half-life in plasma (t ½) was in the range of 1.20-2.80 h. Cefozopran was mainly excreted in its unchanged form, with no tendency for accumulation, via the kidney, and varied from 65.99 to 73.33 %. No appreciable accumulation of either drug occurred with multiple intravenous doses of cefozopran, and pharmacokinetic parameters for cefozopran were similar on days 1 and 4. No serious adverse events were reported. Adverse events were generally mild. CONCLUSION: Cefozopran was safe and well tolerated in the volunteers and displayed linear increases in the C max and AUClast values.

Effects of total glucosides of peony on AQP-5 and its mRNA expression in submandibular glands of NOD mice with Sjogren's syndrome.

OBJECTIVE: The aim of this study was to observe the effects of total glucosides of peony (TGP) on pathological change, Aquaporin-5 (AQP-5) and its mRNA expression in submandibular glands of non-obese diabetic (NOD) mice with Sjogren's Syndrome, to investigate its regulation on secretion of salivary glands. MATERIALS AND METHODS: 40 NOD mice were randomly divided into model group, TGP group, hydroxychloroquine group, combination group (n = 10). For TGP group, the mice were intragastrically administrated with 0.4 ml TGP dilution per day in accordance with 300 g/kg dose; for hydroxychloroquine group, the mice were intragastrically administrated with 0.4 ml hydroxychloroquine per day in accordance with 60 mg/kg dose; for the combination group, the mice were intragastrically administrated with 0.4 ml TGP dilution and 0.4 ml hydroxychloroquine. 8 weeks later, the mice were sacrificed, and submandibular glands were collected by anatomy. Pathological changes of submandibular gland were observed under a light microscope; AQP-5 protein in submandibular glands was detected by immunohistochemical staining; and AQP-5 mRNA expression in submandibular glands was detected by RT-PCR. RESULTS: The lymphocytic infiltration score of model mice was significantly higher than that of other groups. The pathological morphology and score of NOD mice were significantly improved after administration, and the combination group was superior to the hydroxychloroquine group and TGP group (p < 0.05). AQP-5 mRNA expression level of the model group was lower than other groups (p < 0.05); the expression levels in the TGP group and the combination group were higher than the hydroxychloroquine group (p < 0.05). CONCLUSIONS: TGP may improve pathological damage of submandibular glands of NOD mouse with Sjogren's syndrome by upregulating AQP-5 and its mRNA expression in submandibular glands.

Population-based estimate of the burden of acute gastrointestinal illness in Jiangsu province, China, 2010-2011.

To determine the burden and distribution of acute gastrointestinal illness (AGI) in the population, a cross-sectional, monthly face-to-face survey of 10 959 residents was conducted in Jiangsu province between July 2010 and June 2011. The adjusted monthly prevalence was 4.7% with 0.63 AGI episodes/person per year. The prevalence was the highest in children aged <5 years and lowest in persons aged ≥ 65 years. A bimodal seasonal distribution was observed with peaks in summer and winter. Regional difference of AGI prevalence was substantial [lowest 0.5% in Taicang, highest 15.1% in Xinqu (Wuxi prefecture)]. Healthcare was sought by 38.4% of the ill respondents. The use of antibiotics was reported by 65·2% of the ill respondents and 38.9% took antidiarrhoeals. In the multivariable model, gender, education, season, sentinel site and travel were significant risk factors of being a case of AGI. These results highlight the substantial burden of AGI and the risk factors associated with AGI in Jiangsu province, China.

Exhaustive swimming exercise related kidney injury in rats - protective effects of acetylbritannilactone.

The aim of this study was to investigate the protective effects of acetylbritannilactone (ABL) on renal injury induced by acute exhaustive exercise in the rat. The exhaustive exercise induced kidney injury in rats was established by exhaustive swimming (ES). ABL (26 mg/kg) or polyglycol (control) were administrated orally by gastric gavage 24 h before training. Renal function, biochemical index, renal histopathological change, oxidative stress indices, renal cell apoptosis and inflammatory molecules were checked after ES, for 6 h and 24 h. It was found that immediately after exhaustive swimming, the serum urea and creatinine were significantly higher in ES rats, and the same for serum creatine kinase. All the values were reduced in the ES rats treated with ABL. The increase of superoxide dismutase activity and decrease of malondialdehyde content in the kidney were found in rats with ABL treatment. Tubular cell apoptosis at different time points after ES were significantly reduced by the ABL treatment. The increased expression of TNF-α and NF-κB induced by ES was also significantly decreased by ABL treatment. Our results suggest that ABL protects rats from overtraining-induced kidney injury by inhibiting renal cell apoptosis and suppressing oxidative-stress generation and inhibiting inflammation.

Establishment of a cell-based drug screening model for identifying down-regulators of Protein Tyrosine Phosphatase 1B expression.

Protein Tyrosine Phosphatase 1B (PTP1B), an important negative regulator of insulin signaling, is thought to be an attractive therapeutic target for insulin resistance and type 2 diabetes. For the aim of screening PTP1B expression down-regulators, we established the drug screening cellular model based on transcriptional regulation of PTP1B. In this study, the promoter sequences of PTP1B were cloned into pGL3B-neo vector containing luciferase gene and neomycin resistance gene. The recombinant reporter gene vector pGL3B-neo /PTP1B was transfected into CV1 cells and therefore stable cell line, namely SPTP1B, was obtained. With the cell-based reporter gene assay, we detected more than one hundred compounds in microtiter wells. In the screening process, the compound CM107 which had extracted from the traditional Chinese medicinal herbs was identified to repress the activity of PTP1B promoter significantly in mode of dose-dependence.

Establishment of a cell-based drug screening system for identifying selective down-regulators of mPGES-1.

OBJECTIVE: To establish a drug screening system based on transcriptional regulation of microsomal PGE(2) synthase 1 (mPGES-1), cyclooxygenase 1 (COX-1) and 2 (COX-2) for discovering selective down-regulators of mPGES-1. METHODS: The upstream regulatory sequences of mPGES-1, COX-1, COX-2 were respectively cloned into pGL3B-neo vector containing luciferase gene and neomycin resistance gene (the pGL3B-neo vector had been previously constructed by cloning the neomycin resistance gene into the Sal I site of the pGL3-Basic vector). After that, the recombinant reporter gene vectors pGL3B-neo/mPGES-1, pGL3B-neo/COX-1, pGL3B-neo/COX-2 were respectively transfected into A549 cells and therefore stable cell lines, namely M 1, M 2 and M 3, were obtained. Samples were detected then by testing luciferase activity of M 1, M 2 and M 3 cells in microtiter wells to identify compounds that can selectively down-regulate mPGES-1 expression. Through luciferase activity testing, the compounds which had more than 40 % inhibition ratio on M 1 and less than 20 % inhibition ratio on M 2 and M 3 cells could be regarded as hits. RESULTS: Using the cell-based reporter gene assay, we screened compounds for selectively down-regulation of mPGES-1 expression and several compounds were discovered. CONCLUSION: A cell-based drug screening system was established to screen selective down-regulators of mPGES-1 expression, and compound CM188 was identified, which might become a lead compound for novel anti-arthritic drugs.

IFN-gamma is associated with risk of Schistosoma japonicum infection in China.

Before the start of the schistosomiasis transmission season, 129 villagers resident on a Schistosoma japonicum-endemic island in Poyang Lake, Jiangxi Province, 64 of whom were stool-positive for S. japonicum eggs by the Kato method and 65 negative, were treated with praziquantel. Forty-five days later the 93 subjects who presented for follow-up were all stool-negative. Blood samples were collected from all 93 individuals. S. japonicum soluble worm antigen (SWAP) and soluble egg antigen (SEA) stimulated IL-4, IL-5 and IFN-gamma production in whole-blood cultures were measured by ELISA. All the subjects were interviewed nine times during the subsequent transmission season to estimate the intensity of their contact with potentially infective snail habitats, and the subjects were all re-screened for S. japonicum by the Kato method at the end of the transmission season. Fourteen subjects were found to be infected at that time. There was some indication that the risk of infection might be associated with gender (with females being at higher risk) and with the intensity of water contact, and there was evidence that levels of SEA-induced IFN-gamma production were associated with reduced risk of infection.

Down-regulation of specific antigen-driven cytokine production in a population with endemic Schistosoma japonicum infection.

Schistosome antigen-driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL-4, IL-5, IL-10 and INF-gamma were all detected in the supernatants of whole-blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down-regulated in the presence of live, egg-laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA-specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool-positive than in those who were stool-negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti-SEA and IgM anti-SWAP antibody levels all fell significantly.

[Analysis of the mitochondria-related protein of Schistosoma japonicum and its antigen epitopes].

OBJECTIVE: To sequence the cloned gene Sj338 and to identify the encoded protein and its antigen epitopes. METHODS: The Sj338 gene fragment obtained from adult S. japonicum cDNA library amplified by PCR method was subcloned into pGEM-T vector for sequencing. The sequence of nucleotides and the characteristics of the encoded protein were analyzed by DNASIS Program and Goldkey DNA and Protein Analytical Program, and then the homology of the amino acid sequence was searched on the BLAST net. RESULTS: The cloned rSj338 gene was demonstrated to be 487 bp containing one 459 bp ORF, encoding a protein consisted of 153 amino acids with a molecular weight of 17.6 kDa. The amino acid sequence of the recombinant protein rSj338 shared 46% identity with that of the corresponding part of human mitochondrial import receptor and 44% identity with that of the Rattus sp. mitochondrial precursor receptor. The possible antigen epitopes were predicted within the peptide fragments of 26-32 aa, 37-46 aa and 147-151 aa. CONCLUSION: The protein encoded by rSj338 gene fragment might be the mitochondria-related protein of Schistosoma japonicum.

[Clonning and identification of the gene encoding specific IgE antibody-related antigen of Schistosoma japonicum].

OBJECTIVE: To obtain the gene encoding specific IgE-related antigen of Schistosoma japonicum from the cDNA library of adult Schistosoma japonicum. METHODS: The pooled sera from 15 individuals with high levels of specific IgE antibody against SWAP were absorbed with Protein-G and used for screening of IgE-related antigen from the adult worm cDNA library of Schistosoma japonicum. The inserted cDNA was amplified by PCR and sequenced. According to the first reading frame of the sequence, a pair of new probes, in which EcoR I and Not I sites were incorporated respectively, were designed and used to amplify the target gene. Then, the gene was cloned into vector pGEM-T and subcloned into expression vector pGEX-6p-1. The fusion protein was expressed, analysed by SDS-PAGE and identified by Western blotting with the specific IgE antibody, respectively. RESULTS: The inserted cDNA fragment from the positive clone was about 1,200 bp, with the ORF of 507 bp which encoded 169 amino acids. The deduced molecular weight of the recombinant protein was 19.3 kDa. The homology between the target gene (Sj43B) and other known DNA sequences was less than 40%. The fusion protein expressed by the recombinant vector pGEX-6p-1/Sj43B could be recognized by schistosome specific IgE antibody. CONCLUSION: Sj43B may encode the specific IgE-related antigen of Schistosoma japonicum. The successful construction of recombinant plasmid pGEX-6p-1/Sj43B lay the groundwork for further studies on immunological characteristics and protection immunity of the recombinant protein.

[Cloning and identification of deltamethrin-resistance or susceptibility associated genes of Culex pipiens pallens].

OBJECTIVE: To obtain deltamethrin-resistance or susceptibility associated genes of Culex pipiens pallens. METHODS: The differentially expressed genes were obtained by suppression subtractive hybridization (SSH), and identified by cDNA microarray and reverse Northern blotting. RESULTS: 523 and 286 clones were selected respectively in the two directional SSH. 155 and 42 genes were respectively expressed 2-3 and > 3 times higher in the insecticide-resistant strain than in the susceptible strain; 15 and 9 genes were respectively expressed 2-3 and > 3 times higher in the susceptible strain than in the resistant strain. There were 2 genes only expressed in the insecticide-resistant strain. 51 three times differentially expressed clones and 2 specially expressed clones were sequenced. 44 sequences were obtained which belong to 13 new genes. There were 8 over-expressed genes in resistant strain, 7 of which were similar respectively to mitochondrion rRNA gene, 60S ribosomal protein gene, 40S ribosomal protein S4 gene, trypsin gene, chymotrypsin A gene, ospin gene, and 16S ribosomal RNA gene. There were 5 over-expressed genes in susceptible strain, 2 of them being similar with 40S ribosomal protein S29 gene and myosin regulatory light chain 2 gene. In addition, 2 genes specially expressed in resistant strain were similar respectively to glycogen branching enzyme gene and ribosomal protein 46 gene. CONCLUSION: The differentially expressed genes may be associated with deltamethrin-resistance or susceptibility of Culex pipiens pallens.

[Studies of the genetic polymorphism of Schistosoma japonicum (Chinese mainland strains)].

In order to analyze the interspecies diversity and the extent of diversity among Schistosoma japonicum Chinese mainland strains, the genetic variation on gene level among 6 isolates collected from Jiangxi, Hunan, Hubei, Anhui, Sichuan, Yunnan and a cultured isolate from a laboratory were studied using molecular biological technique. There were only two different bases at position 112 and 143 in 536 bp sequence of 28S rDNA-D2 domain between Anhui and Yunnan isolates, the homology was 99.6%. The result could be explained the reason of why the migration rates of single chain 28S rDNA-D2 domain among the above 7 isolates were the same completely in PCR-SSCP. However, comparing with the sequences of the Philippine isolate of S. japonicum, S. mansoni and S. aematobium, there were 6, 94 and 93 different bases and the homologies were 98.9, 82.5 and 82.7 per cent separately. With 8 restriction endonucleases to analyze the ITS of rDNA obtained by PCR from the 7 isolates, the results showed that only 3 minor bands were different, e.g. 5.3% of total 58 fragments. It was suggested that the ITS of rDNA among 7 isolates were highly conserved. Using randomly amplified polymorphic DNA (RAPD) to analyze the genetic diversity of the genomes of the 7 isolates, the average genetic distance (D) calculated from total 284 amplified fragments was 0.22. The maximum D was 0.30 and existed between Anhui and Yunnan isolates. The minimum D was 0.13 and existed between Sichuan and Yunnan isolates. The clustering analysis of genetic distances showed that the 7 isolates could be gathered in one group. From above three results, it could be considered that the genetic diversity on gene level among S. japonicum Chinese mainland strains was very low.

[Studies on human cytokine responses before and after praziquantel chemotherapy in an endemic area of schistosomiasis japonica].

OBJECTIVE: To observe the cellular immune responses in a population of an endemic area of schistosomiasis japonica and the influence of praziquantel treatment. METHODS: Blood was taken from 129 residents (64 cases were egg-positive, 65 cases were egg-negitive) of an endemic area of Poyang Lake before and 45 days after praziquantel treatment. Cytokines induced by the schistosome soluble egg antigen (SEA) and soluble worm antigen preparations (SWAP) in the peripheral blood cells including IL-5, IL-10 and IFN-gamma were measured. RESULTS: Among 129 cases, the cytokine levels were found much higher in egg negative individuals than in egg-positive individuals. The cytokine levels induced by both antigens were increased significantly after praziqantel treatment especially IL-5 and IFN-gamma. CONCLUSION: The cellular immune responses in the population in schistosomiasis japonica endemic area exhibited a general trend of down-regulation and were elevated significantly after praziquantel treatment.

[Cloning and identification of cytochrome P450 resistance related genes in the mosquito, Culex pipiens pallens].

OBJECTIVE: To inquire into the relationship between cytochrome P450 and deltamethrin resistance. METHODS: 24 new cDNA sequences encoding cytochrome P450 were amplified respectively from deltamethrin-susceptible and -resistant strains of Culex pipiens pallens with a pair of degenerate primers according to the conservative amino acid sequences of CYP4 in insects by RT-PCR and the Direct Cloning Method, and then were identified by cDNA chip and reverse Northern. RESULTS: 112 positive clones were obtained, of which 24 were shown to be new sequences encoded for cytochrome P450. They have been lodged in GenBank and were appraised by the Nomenclature Committee of Cytochrome P450, belonging to the subfamily CYP4C, CYP4D, CYP4H and CYP4J in CYP4 family. The hybrid signal values of 6 P450 sequences (NYDS3, NYDS5, NYDR6, NYDR9, NYDR15 and NYDR17) were 3.1-9.7 times higher in the resistance probe than in the susceptible probe, and NYDR17 only reacted with the resistance probe. The result of reverse Northern in NYDR15 was similar to that of cDNA chip. CONCLUSION: CYP4 is related to deltamethrin resistance and the specific expression caused by point mutation of cytochrome P450 gene may exist in deltamethrin-resistant Cx. pipiens pallens.

[Gene cloning and characterization of mitochondria-related protein of Schistosoma japonicum].

OBJECTIVE: To subclone and characterize a cDNA clone coding for Schistosoma japonicum (S.j.) mitochondria-related protein. METHODS: The open reading frame of the fragment(Sj338/24) obtained from an adult worm cDNA library of S.j. was analysed, at the upstream and downstream of the open reading frame(ORF) the primers A and B were designed, respectively, and the cDNA fragment was used as PCR template. The Sj338 gene fragment obtained was amplified by PCR method and then subcloned into pGEM-T vector for sequencing. The gene sequence was analyzed and the target fragment was restrictedly digested and subcloned into expression vector pGEX-6P-1. The expressed recombinant protein was purified and characterized. RESULTS: The cloned Sj338 gene was demonstrated to be 487 bp long containing one 459 bp ORF, encoding a protein with a molecular weight of 17 kDa. The nucleotide sequence of the cloned gene Sj338 had higher homology with those genes coding for mitochondrial outer membrane protein of Homo sapiens and Rattus norvegicus. The recombinant construct of pGEX-6P-1/Sj338 could be expressed efficiently and the antigenicity of its product rSj338 has been demonstrated by Western blotting. CONCLUSION: Sj338 may be the gene coding for S.j. mitochondria-related protein and the recombinant protein may be used as a new vaccine candidate.

[Human parasite antigen-specific IFN-gamma response in schistosomitsis japonica endemic area].

OBJECTIVE: To explore the characteristics of human schistosome antigen-specific IFN-gamma response in a population in an area endemic for schistosomiasis japonica. METHODS: Three neighboring villages were chosen on Nanshan Island of Poyang Lake. 65 egg-negative persons and 64 egg-positive ones were selected randomly from the residents aged 14-41 years according to the egg counts by Kato-Katz thick smear method. IFN-gamma was measured in the whole blood culture supernatant after stimulated by the schistosome soluble egg antigen (SEA) and soluble worm antigen preparations (SWAP). Serum isotype-restricted antibody was detected by ELISA. RESULTS: IFN-gamma levels induced by both SEA and SWAP were increased significantly after praziquantel treatment. The SEA-specific IFN-gamma level of the uninfected group was much higher than that of the reinfected group. A negative correlation between IFN-gamma level and IgG4 production was found, reflecting that IFN-gamma might be associated with the resistance to schistosome reinfection. CONCLUSION: The changes in IFN-gamma level might play an important role in association with the resistance to schistosome reinfection.