[Co-exposure of carbon black and cadmium induces autophagy and inflammation in human bronchial epithelial cells via PERK pathway].

Objective: To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells. Methods: In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 µg/ml Cd, 100 µg/ml CBNPs, 100 µg/ml CBNPs+2 µg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results: There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined (P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group (P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression (P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group (P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group (P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene (P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 (P<0.05), but the interaction between the two chemicals had no statistical significance (P>0.05) . Conclusion: CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

[Analysis of risk factors for hyperkalemia in hemodialysis patients and establishment and verification of risk assessment model of hyperkalemia].

Objective: To explore risk factors for hyperkalemia in hemodialysis (HD) patients, and establish and verify a risk assessment model of hyperkalemia in HD patients. Methods: The clinical data of HD patients who were admitted to the Department of Nephrology of the First Affiliated Hospital of Zhengzhou University between April 2020 and January 2021 were retrospectively collected and divided into training dataset and validation dataset by using the conversion-random number generator. In the training dataset, multivariate logistic regression analysis was used to screen the risk factors for hyperkalemia in HD patients and the factors were scored to establish the risk assessment model. The validation dataset was substituted into the model and the receiver operating characteristic (ROC) curve was drawn and the area under the curve (AUC) was calculated to verify the effectiveness of the risk prediction model in predicting hyperkalemia. Results: A total of 502 HD patients were enrolled and further divided into training dataset (n=372) and validation dataset (n=130). There were 268 males and 234 females, with a mean age of (54±13) years. Multivariate logistic regression analysis showed that metabolic acidosis, high potassium diet, history of hyperkalemia, the change of electrocardiogram (ECG), disfunction of vascular access and time interval from last dialysis were risk factors for causing hyperkalemia in patients undergoing HD. Risk assessment model was established based on these risk factors. The AUC of the ROC curve was 0.799. Using 5 as the cut-off value, the sensitivity and specificity for predicting hyperkalemia events was 61.4% and 86.3%, respectively. Conclusion: The current study preliminarily established a risk assessment model for hyperkalemia in HD patients, which can help clinicians manage the potassium level of HD patients.

ERα-dependent stimulation of LCN2 in uterine epithelium during mouse early pregnancy.

Maintenance of a suitable uterine milieu is important for embryo development and subsequent implantation during early pregnancy. High estrogen level in proestrous and estrous stages is essential for uterine anti-bacterial activity during preimplantation period. Lipocalin-2 is an essential molecule which prevents bacterial infection by sequestering iron. In this study, the highest expression of lipocalin-2 is observed in the endometrial epithelium on day 1 of normal pregnancy and pseudopregnancy, which exhibit a similar hormone scenario. By injecting the agonists for estrogen receptor α and estrogen receptor ß in ovariectomized mice, we found estrogen receptor α is the dominant member for estrogen regulation on lipocalin-2 expression. Estrogen treatment in estrogen receptor α-knockout mice further confirmed the role of estrogen receptor α. Using published data from whole-genome estrogen receptor α binding site assay, significant estrogen receptor α recruitment peaks are found at the downstream of lipocalin-2 gene after estrogen treatment. Furthermore, to study the anti-bacterial activity of lipocalin-2 in uterus, Escherichia coli is injected to mimic bacterial infection. Our results showed an obvious induction of lipocalin-2 in Escherichia coli-treated group. Taken together, this study indicates estrogen regulation of lipocalin-2 in uterine epithelium is mediated by estrogen receptor α, and lipocalin-2 may have anti-bacterial activity during early pregnancy.

A survey of the blood supply in China during 2012-2014.

OBJECTIVES: The aim of this study was to identify the status of the blood supply in China during 2012-2014. BACKGROUND: China is a middle-income country, which contains more than 20% of the world population. Increasing the blood supply in China, along with increased healthcare coverage, involves many challenges. METHODS: A survey questionnaire regarding blood centre activities was sent to all of the blood centres in 32 provinces via the Internet. The data were collected from the responses and analysed using Microsoft Excel 2013. RESULTS: The total supply of whole blood and red blood cells (RBCs) in 2012 was 18 644 700 units; in 2013, 18 985 800 units; and in 2014, 19 658 800 units. A similar trend of the total platelet supply was also observed during the same period of 2012-2014, as follows: 1 019 100 units in 2012, 1 168 400 units in 2013 and 1 276 200 units in 2014. Similarly, the plasma supply was 27 529 300 units in 2012 and 27 657 600 units in 2013, which rose to 28 307 500 units in 2014. The total cryoprecipitate supply was 1 653 900, 1 891 300 and 2 366 500 units in 2012, 2013 and 2014, respectively. When the blood supply was analysed according to the geographic regional population, large differences in the rates of blood supply between regions were evident. CONCLUSIONS: The blood product supply in China is steadily increasing. Blood centres in China continue to face challenges regarding their ability to provide a sufficient blood supply in the future.

[Performance of matrix assisted laser desorption ionization time of flight biotyper system in clinical bacteria identification].

Objective: To evaluate the performance of MALDI Biotyper system in identification of clinically isolated pathogens so as to provide a new rapid identification method. Methods: Total 21 270 pathogens strains, isolated from the First Affiliated Hospital of Fujian Medical Universityduring Nov. 2015 to Dec. 2016, were identified by VITEK-Ⅱ, API and MALDI Biotyper system, respectively.The isolated strains were confirmed by DNA sequencing. Results: The identification of common bacteria with MALDI Biotyper and phenotypic system is highly consistent (>95% and >90%). Among 43 strains of anaerobic bacteria, MALDI Biotyper could identify 90.7% bacteria to species level and 97.7% bacteria to genus level with the statistical significance(χ(2)=6.76, P<0.01), while phenotypic system only identified 65.1% bacteria to species and 69.8% bacteria to genus. Also, no statistical significance was shown for Trichosporon and Candida(P>0.05). MALDI Biotyper could identify 76% filamentous fungi and all of Actinomycetes, Nocardia, Mycobacterium and Legionella to genus level. Conclusions: MALDI Biotyper is an easy-performed, sensitive method for the identification of clinically isolated pathogens. Additionally, the pretreatment and reference database has the effect on identification.

[Expressions of IL-1α, IL-1ß and IL-13 mRNA in Lung Tissue and Serum of Rats Drown in Fresh Water].

OBJECTIVES: To detect the changes of （interleukin, IL） -1α, IL-1ß and IL-13 mRNA in lung tissue and serum of drown rats, and to explore the potential value for the diagnosis of drowning in forensic practice. METHODS: Eighteen SD rats were randomly divided into drowning group, blank control group and myocardial infarction group （as control group）. The serum of right ventricular, the inferior lobe of right lung and the myocardium were taken from the rats in different groups. The expressions of IL-1α, IL-1ß and IL-13 mRNA in the lung tissue and the serum of right ventricular were detected by TaqMan probe method. RESULTS: The expression differences of IL-1α, IL-1ß and IL-13 mRNA in lung tissue between drowning group and blank control group, myocardial infarction group were not statistically significant （P>0.05）. The expression of IL-1ß and IL-13 mRNA in serum of right ventricular increased （ P<0.05）. The expression differences of IL-1α, IL-1ß and IL-13 mRNA in serum between blank control group and myocardial infarction group were not statistically significant (P>0.05). CONCLUSIONS: The changes of cytokines IL-1ß and IL-13 mRNA in the serum of right ventricular of drown rats are statistical significance, which are highly correlated with drowning.

Association of human platelet antigens polymorphisms with susceptibility to hepatitis C virus infection in Chinese population.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. Previous studies have identified a number of single nucleotide polymorphisms that are associated with HCV infection. Human platelet antigens (HPAs) polymorphisms play an important role in several diseases. Here, we demonstrated the association of the HPA-2, HPA-3, HPA-5 and HPA-15 polymorphisms with susceptibility to HCV infection in Chinese population. Overall, 118 patients with HCV and 167 controls were genotyped for HPAs. There were no significant differences in the allele and genotype frequency distribution for the HPA-3, HPA-5 and HPA-15 systems between the patients with chronic HCV infection and the healthy controls (p > .05). However, the genotype frequency of HPA-2aa was significantly lower, while HPA-2ab/bb was significantly higher in patients than that in the controls (p = .006). The allele frequency of HPA-2a in patients was significantly lower than that in the control group (p = .005). In contrast, HPA-2b in patients was significantly higher than that in the control group (p = .005). We conclude that HPA-2 polymorphism is associated with susceptibility to HCV infection, and individuals carrying the HPA-2b allele may have a higher risk of HCV infection compared with individuals carrying HPA-2a.

Association of paraoxonase polymorphisms with carotid artery atherosclerosis in essential hypertension patients.

We investigated the relationships between paraoxonase genetic polymorphisms and essential hypertension in carotid artery atherosclerotic patients. The study included 353 Han participants and 240 Uighur participants from Xinjiang; they were further divided into two groups: essential hypertension with carotid artery atherosclerosis (CAAD group) and essential hypertension without carotid artery atherosclerosis (control group). Genotypes were detected by PCR, followed by restriction analyses with specific endonucleases. In Han people, the M allele frequency was significantly higher in the CAAD group than in the control group. The CC/CS genotype and C allele frequencies were significantly higher in the CAAD group than in the control group. Logistic regression analysis demonstrated that PON1 55M allele [odds ratio (OR) = 1.889] and PON2 311C allele (OR = 1.692) are independent risk factors for CAAD. Combined genotype analysis showed that PON1 55M and PON2 311C alleles are independent risk factors for CAAD (OR = 1.428). In the Uighur population, the CC/ CS genotype and C allele frequencies were significantly higher in the CAAD group than in the control group. Logistic regression analysis demonstrated that the PON2 311C allele is an independent risk factor for CAAD. We conclude that the PON1 55M and PON2 311C alleles are independent risk factors for CAAD in essential hypertension patients from the Xinjiang Han population. We also conclude that the PON2 311C allele is a risk factor for CAAD in essential hypertension patients from the Xinjiang Uighur population.

Association between the TRAIL single nucleotide polymorphism rs1131580 and type 2 diabetes mellitus in a Han Chinese population.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is expressed in different tissues and cells, including the pancreas and lymphocytes, and it can selectively induce apoptosis in tumor cells but not in most normal cells. TRAIL plays critical roles in type 1 diabetes mellitus, and is involved in type 2 diabetes mellitus (T2DM). We recently discovered the association of nonalcoholic fatty liver disease, a risk factor for T2DM, with a single nucleotide polymorphism (SNP) in the TRAIL (TNFSF10) gene at site 1595C/T (rs1131580), indicating the possible association of T2DM with this TRAIL polymorphism. The aim of this study was to investigate the relationship of the TRAIL SNP at site 1595C/T (rs1131580) with T2DM susceptibility and the biometabolic parameters of T2DM in a Han Chinese population. The polymerase chain reaction-restriction fragment length polymorphism method was used to genotype SNP rs1131580 in 292 patients with T2DM and 266 healthy controls. We found that the frequency of the CC genotype and that of the C allele of rs1131580 were significantly higher in T2DM patients than in the control group. Additionally, the triglyceride and serum creatinine levels of T2DM patients with the CC genotype were significantly higher than those of patients with the TT genotype. Thus, the CC genotype of the TRAIL SNP at 1595C/T (rs1131580) confers increased susceptible to T2DM in a Han Chinese population from Shandong Province. These data suggest that the CC genotype at this SNP is related to diabetic severity and it might be a candidate for the prognostic assessment of T2DM.

[Effects of enhancing the expression of aryl hydrocarbon receptor in post-traumatic mice macrophages on the inflammatory cytokine level and bactericidal ability].

Objective: To explore the expression pattern of aryl hydrocarbon receptor (AhR) in mice peritoneal macrophages (PMs) after major trauma and analyze the effects of enhanced AhR expression on the inflammatory cytokine level and bactericidal ability after trauma. Methods: The experimental study method was used. Forty 6-8-week-old male C57BL/6J mice (the same mouse age, sex, and strain below) were divided into control group, post trauma hour (PTH) 2 group, PTH 6 group, and PTH 12 group according to the random number table (the same grouping method below), with 10 mice in each group. Mice in the latter 3 groups were constructed as severe trauma model with fracture+blood loss, while mice in control group were left untreated. The primary PMs (the same cells below) were extracted from the mice in control group, PTH 2 group, PTH 6 group, and PTH 12 group when uninjured or at PTH 2, 6, and 12, respectively. Then the protein and mRNA expressions of AhR were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction, respectively, and the gene expressions of AhR signaling pathway related molecules were analyzed by transcriptome sequencing. Twenty mice were divided into control group and PTH 6 group, with 10 mice in each group, and the PMs were extracted. The level of ubiquitin of AhR was detected by immunoprecipitation. Twelve mice were divided into dimethyl sulfoxide (DMSO) alone group, PTH 6+DMSO group, MG-132 alone group, and PTH 6+MG-132 group, with 3 mice in each group. After the corresponding treatment, PMs were extracted, and the protein expression of AhR was detected by Western blotting. Twenty mice were constructed as PTH 6 model. Then, the PMs were extracted and divided into empty negative control adenovirus (Ad-NC) group and AhR overexpression adenovirus (Ad-AhR) group. The protein expression of AhR was detected by Western blotting at 36 h after some PMs were transfected with the corresponding adenovirus. The rest cells in Ad-NC group were divided into Ad-NC alone group and Ad-NC+endotoxin/lipopolysaccharide (LPS) group, and the rest cells in Ad-AhR group were divided into Ad-AhR alone group and Ad-AhR+LPS group. The expressions of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in the cell supernatant were detected by enzyme-linked immunosorbent assay at 12 h after the corresponding treatment (n=6). Twenty mice were obtained to extract PMs. The cells were divided into control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group, and the intracellular bacterial load was detected by plate spread method after the corresponding treatment (n=6). Data were statistically analyzed with one-way analysis of variance, least significant difference test, analysis of variance for factorial design, and independent sample t test. Results: Compared with 1.16±0.28 of control group, the protein expressions of AhR in PMs in PTH 2 group (0.59±0.14), PTH 6 group (0.72±0.16), and PTH 12 group (0.71±0.17) were all significantly decreased (P<0.05). The overall comparison of the difference of AhR mRNA expression in PMs among control group, PTH 2 group, PTH 6 group, and PTH 12 group showed no statistical significance (P>0.05). The AhR signaling pathway related molecules included AhR, AhR inhibitor, cytochrome P450 family member 1b1, cytochrome P450 family member 11a1, heat shock protein 90, aryl hydrocarbon receptor-interaction protein, and heat shock protein 70 interaction protein. The heat shock protein 90 expression of PMs in PTH 2 group was higher than that in control group, while the expressions of other molecules did not change significantly after trauma. Compared with that in control group, the level of ubiquitin of AhR in PMs in PTH 6 group was increased. Compared with that in DMSO alone group, the protein expression of AhR in PMs in PTH 6+DMSO group was decreased, while that in PMs in MG-132 alone group had no significant change. Compared with that in PTH 6+DMSO group, the protein expression of AhR in PMs in PTH 6+MG-132 group was up-regulated. At transfection hour 36, compared with that in Ad-NC group, the protein expression of AhR in PMs in Ad-AhR group was increased. At treatment hour 12, compared with those in Ad-NC+LPS group, the expressions of IL-6 and TNF-α in PM supernatant of Ad-AhR+LPS group were significantly decreased (with t values of 4.80 and 3.82, respectively, P<0.05). The number of intracellular bacteria of 1×106 PMs in control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group was (3.0±1.8), (41.8±10.2), (1.8±1.2), and (24.2±6.3) colony forming unit, respectively. Compared with that in PTH 6+Ad-NC group, the number of intracellular bacteria of PMs in PTH 6+Ad-AhR group was significantly decreased (t=3.61, P<0.05). Conclusions: Ubiquitin degradation of AhR in PMs of mice after major trauma results in decreased protein expression of AhR. Increasing the expression of AhR in post-traumatic macrophages can reduce the expressions of LPS-induced inflammatory cytokines IL-6 and TNF-α, and improve the bactericidal ability of macrophages after trauma.

[Significance of the seated supine positioning nystagmus for the diagnosis of benign paroxysmal positional vertigo].

Objective:To observe and assess the significance of seated supine positioning nystagmus (SSPN) in the diagnosis of benign paroxysmal positional vertigo(BPPV).Method:Two hundreds patients who were diagnosed BPPV were tested with the seated supine positioning test(SSPT) to observe SSPN,then were tested Supine roll test(SRT) and Dix-Hallpike test(DHT). According to the result of SRT and DHT,patients were divided into different groups. The positive rate and feature of SSPN in different types of BPPV was analyzed.Result:Among the 200 patients,116 cases(58.0%) of them showed SSPN. Among the 116 cases who were divided to the posterior semicircular canal BPPV(PSC-BPPV) group,72 cases of them showed SSPN. Horizontal semicircular canal BPPV(HSC-BPPV) group were 60 cases,44 cases showed SSPN. Anterior semicircular canal BPPV(ASC-BPPV) group were 4 cases and none of them showed SSPN. The direction of SSPN was a combination of torsional nystagmus with the upper pole of the eyes beating toward the affected side combined with vertical nystagmus beating upward (toward the forehead) typically in the PSC-BPPV group. Canalolithiasis of HSC were 41 cases,and 28 cases showed SSPN,and SSPN was contralesional in 22 cases(78.6%) and ipsilesional in 6 cases. Cupulolithiasis of HSC were 19 cases and 16 cases showed SSPN,and SSPN was ipsilesional in 16 cases.Conclusion:Significance of the seated supine positioning nystagmus in different types of BPPV is different.

Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function.

Beclin 1 encodes a Bcl-2-interacting coiled-coil protein with autophagy and tumor suppressor function and is monoallelically deleted in 40-75% of sporadic human breast and ovarian cancers. Beclin 1 contains a leucine-rich nuclear export signal motif raising the possibility that its autophagy and/or tumor suppressor function may require regulated, CRM1-dependent, nucleocytoplasmic transport. In this study, we show that wild-type Beclin 1 colocalizes with both intracytoplasmic organelles and nuclei in COS7 monkey kidney and MCF7 human breast carcinoma cells. Inhibition of CRM1-dependent nuclear export with leptomycin B or mutation of the nuclear export signal motif of Beclin 1 results in predominantly nuclear localization. Unlike wild-type Beclin 1, the nuclear export mutant of Beclin 1 fails to promote nutrient deprivation-induced autophagy and fails to inhibit in vitro clonigenicity and in vivo tumorigenicity of MCF7 cells. Thus, beclin 1 has a leptomycin B-sensitive leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function. These findings suggest that the CRM1 nuclear export pathway may be important in the functional regulation of autophagic growth control.

Co-localization of the tumor-suppressor protein p53 and human papillomavirus E6 protein in human cervical carcinoma cell lines.

The loss of the tumor-suppressor activity of p53, either by mutation or by interaction with the human papillomavirus (HPV) E6 protein, is considered to be an important mechanism in the carcinogenesis of cervical cancer. We have studied the cytological distribution of these proteins in human cervical carcinoma cell lines using polyclonal anti-p53 and monoclonal anti-E6 antibodies. The antibody specificity was confirmed by immunoblot and immunocompetition analyses. The intracellular localization of p53 and E6 was detected using the techniques of conventional and three-dimensional confocal microscopy. In the HPV-18 or -16 integrated cell lines, HeLa, CaSki and SiHa, viral oncoprotein E6 and endogenous tumor-suppressor protein, p53, were observed by immunofluorescence in the cytoplasm; p53 also had a weak punctate staining in the nuclei of HeLa and CaSki cells. In the HPV-negative cervical carcinoma cell lines, C-33A and HT-3, which have mutated p53, p53 was localized predominantly to the nucleus, with C-33A cells having elevated levels of p53 compared with the other cell lines. High spatial resolution imaging, using confocal microscopy, was performed on the cells after double fluorescence staining for p53 (fluorescein) and E6 (rhodamine). The images showed that both p53 and E6 had similar cytoplasmic distributions, which implied that these two proteins may exist as a cytoplasmic complex. To substantiate this implication, fluorescence resonance energy transfer microscopy was performed, which provided direct evidence of a close association between p53 and E6 within individual HeLa cells. The results from this study support the theory that p53 protein binds HPV-16/18 E6 protein in the cell cytoplasm, thus preventing p53 from exerting its tumor-suppressor function in the nucleus. Hence, inactivation of wild-type p53 by p53-E6 complex formation in cervical cancer may be a critical step in malignant transformation.

Expression of a biologically active antiviral antibody using a sindbis virus vector system.

Monoclonal antibodies to the Sindbis virus E2 envelope glycoprotein protect mice against lethal encephalitis and mediate viral clearance from neurons. To facilitate structure-function analyses of anti-E2 mAbs, we developed an expression system that can be used for the construction of genetically engineered anti-E2 mAbs. We constructed recombinant Sindbis/immunoglobulin gene chimeric viruses that express heavy and light chains of an anti-E2 monoclonal antibody, R6. We used a PCR-based strategy to clone the entire rearranged heavy and light chain genes from R6 hybridoma cell cDNA into a double subgenomic Sindbis virus vector. The recombinant viruses, SIN/R6L and SIN/R6H, were generated by transfecting BHK-21 cells with in vitro transcribed RNA from Sindbis virus/R6 light chain and Sindbis virus/R6 heavy chain cDNA clones, respectively. Twelve hours after co-infection of BHK cells with SIN/R6L and SIN/R6H, the tissue culture supernatant contained up to 1.4 mg/ml of recombinant R6 IgG. The heavy and light chains of recombinant R6 were associated as judged by co-purification on protein A/G sepharose and co-electrophoresis of non-reduced proteins. The ELISA reactivity to Sindbis virus antigen was similar for recombinant R6 and R6 purified from ascites fluid. Furthermore, the in vivo biologic activity of recombinant R6 was similar to that of R6 purified from ascites; recombinant R6 treatment completely protected Balb/cJ mice from paralysis and death due to infection with neuroadapted Sindbis virus and also resulted in the clearance of infectious virus from the brains of immunodeficient scid mice persistently infected with wild-type Sindbis virus. Thus, the co-infection of BHK cells with SIN/R6L and SIN/R6H leads to the expression, assembly, and secretion of a biologically active recombinant antiviral antibody. Our results suggest that the Sindbis virus vector system is a simple and powerful tool for the production of functional, genetically engineered antibodies.

[Clonal origin of intrahepatic recurrence after resection of hepatocellular carcinoma].

Using integrated hepatitis B virus (HBV) DNA in the cancer cells as a genetic marker, clonal origin of intrahepatic recurrence of hepatocellular carcinoma was studied by Southern blot technique. Comparing 5 cases of postoperative recurrent hepatocellular carcinoma with their primary tumors, it was found that 1 case had identical HBV DNA integration patterns, 3 cases were of different clonality and 1 case with multiple nodular recurrences contained either a unicentric origin or a new distinctive clone origin. The results suggest that, in addition to recurrence from residual cancer cells, in some cases, hepatocellular carcinoma may develop repeatedly during a continuous process of carcinogen action, even after a radical resection.

Genotyping of human platelet antigen-15 by single closed-tube Tm-shift method.

INTRODUCTION: Genotyping of human platelet antigens (HPA) is useful for the diagnosis and prevention of platelet alloimmune syndromes. HPA-15 might play an important role in the development of platelet alloimmune syndromes. There are several disadvantages in the conventional methods for HPA-15 genotyping. The aim of this study was to develop a new method for HPA-15 genotyping by using single closed-tube melting temperature (T(m))-shift genotyping. METHODS: Two GC-rich tails of different lengths were attached to 5'-end of HPA-15 allele-specific PCR primers, such that HPA-15 alleles can be discriminated by the T(m)s of the PCR products. One hundred blood samples were genotyped for HPA-15 by the T(m)-shift and conventional polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: The comparison of the PCR-SSP and the T(m)-shift method showed four discordant results in one hundred samples tested. Confirmatory results demonstrated that the PCR-SSP produced several errors, whereas HPA-15 genotyping by T(m)-shift is correct. The retesting results of T(m)-shift method were consistent with those of the initial testing. CONCLUSION: The single closed-tube T(m)-shift method for HPA-15 genotyping is high-throughput, rapid, reliable, reproducible and cost-effective and it is superior to conventional PCR-SSP used in routine genotyping of HPA-15.

[Detection of serum Lp(a) level of coronary heart disease and its clinical significance].

OBJECTIVE: To study the association of human serum Lp(a) level and coronary heart diseases. METHODS: Objects examined were conposed of 2 groups: CHD (39 cases) and healthy controls (52 cases). Lp (a), TC, HDL-C, TG, apoB of two groups were determined and the results were done with statistic analysis. RESULTS: The mean serum Lp(a) concentrations (mg.L-1) in coronary heart disease group were shown higher significantly than that in the control group (P < 0.01). However, there were no significant correlation between the mean serum Lp(a) and the mean serum TC, HDL-C, TG, spoAI and aopB.(P > 0.05). CONCLUSIONS: Serum Lp(a) level is closely related to the occurrence of coronary heart disease. Lp(a) is a single risk factor for coronary heart disease. Detecting the determination of serum Lp(a) is extremely valuable to the clinical prediction and diagnosis of CHD.

Identification of the first trypanosome H/ACA RNA that guides pseudouridine formation on rRNA.

In trypanosomes small nucleolar RNA (snoRNA) genes are clustered, and the clusters encode for either single or multiple RNAs. We previously reported on a genomic locus in Leptomonas collosoma that encodes for multiple C/D snoRNAs whose expression is regulated at the processing level (Xu, Y., Liu, L., Lopez-Estraño, C., and Michaeli, S. (2001) J. Biol. Chem. 276, 14289-14298). In this study we have characterized, in the same genomic locus, the first trypanosome H/ACA RNA, which we termed h1. Having a length of 69 nucleotides, h1 has the potential to guide pseudouridylation on 28 S rRNA. The h1 is processed from a long polycistronic transcript that carries both the C/D and h1 snoRNAs. The h1/rRNA duplex obeys the rules for guiding pseudouridylation. Mapping of the pseudouridine site indicated that the predicted U is indeed modified. However, in contrast to all H/ACA RNAs, h1 consists of a single hairpin structure and is the shortest H/ACA RNA described so far.