[Practical application of the Paris system for reporting urinary cytology].

Objective: To validate the diagnostic performance of the Paris system for reporting urinary cytology (TPS). Methods: A total of 7 046 urine cytology samples from 3 402 patients collected in the Department of Pathology, Beijing Hospital, China from January 2020 to January 2022 were analyzed. 488 patients had a biopsy or resection specimen during the follow-up period of 6 months. The sensitivity, specificity, risk of malignancy (ROM) and risk of high-grade malignancy (ROHM) of the TPS were evaluated using histological diagnosis as the golden standard. Results: Among the 7 046 samples, high-grade urothelial carcinoma (HGUC) accounted for 5.7% (399/7 046), suspicious for high-grade urothelial carcinoma (SHGUC) for 3.2% (227/7 046), atypical urothelial cells (AUC) for 8.4% (593/7 046), and negative for high-grade urothelial carcinoma (NHGUC) for 72.9% (5 139/7 046) including low-grade urothelial neoplasm (LGUN) for 0.8% (59/7 046) and insufficient samples for 9.8% (688/7 046). 488 patients had a bladder biopsy or resection in the follow-up of six months, including 314 males and 174 females, aged 27 to 92 years (average, 66 years). The ROHM of TPS was 94.7% in HGUC, 83.3% in SHGUC, 41.3% in AUC and 18.8% in NHGUC. The sensitivity and specificity of urine cytology were 70.1% (169/241) and 97.0% (162/167), respectively. The negative predictive value of NHGUC was 69.2% (162/234). Conclusions: The study has shown that TPS classification has high sensitivity and specificity, high ROHM for HGUC and SHGUC, and high negative predictive value for NHGUC. The application of TPS reporting system can better interpret the clinical significance of cytology samples, improve the accuracy of urine cytopathology and ensure continuous diagnostic consistency.

[A case of acute inhalation dinitrogen tetroxide poisoning].

Dinitrogen tetroxide is often used as an oxidant in rocket propellant and has strong irritant and corrosive properties. This paper analyzes the clinical data of a patient with dinitrogen tetroxide poisoning admitted in the 63710 Army Hospital of Chinese People's Liberation Army, so as to further explore the poisoning mechanism, clinical characteristics and key points of acute inhaled dinitrogen tetroxide poisoning.

[The malignant phenotype of calcineurin B homologous protein 2 in gastric cancer and its clinical significance].

Objective: To study the expression and clinical significance of calcineurin B homologous protein 2 (CHP2) in gastric cancer (GC) and its effect on malignant phenotype of GC cells. Methods: The protein expression of CHP2 in 297 GC tissue and 198 normal gastric tissue samples were detected by immunohistochemistry. The relationship between the expression of CHP2 and clinicopathologic parameters of GC were analyzed. CHP2-overexpression plasmids and CHP2-interference plasmids were transfected into GC cell lines respectively. Wound healing assay and Transwell experiment was used to detect the invasion and migration ability of GC cells, and cell counting kit-8 (CCK-8) method was used to detect the proliferation ability of GC cells. Results: The positive expression rate of CHP2 in GC was 68.7% (204/297), which was higher in benign margin (34.1%) (31/91), chronic gastritis (59.1%) (13/22), intestinal metaplasia (34.2%) (13/38), low-grade intraepithelial neoplasia (40.0%) (12/30) and high-grade intraepithelial neoplasia (41.2%)(7/17). The positive expression of CHP2 was correlated with tumor, node and metastasis (TNM) stage, lymph node metastasis and distant metastasis (all P<0.05), but not with gender, age, Laurén classification, human epidermal growth factor receptor 2 (HER2) levels, depth of invasion, carcinoembryonic antigen (CEA) level and CEA 19-9 level (all P>0.05). The results of multivariate analysis showed that high expression of CHP2 and TNM stage were both independent parameters for predicting GC patient prognosis (both P<0.05). Interference of CHP2 expression in HGC-27 cells suppressed proliferation and migration significantly (P<0.05). However, over-expression CHP2 in AGS cells promoted proliferation, and migration significantly (P<0.05). Conclusion: CHP2 plays an important role in the development of GC, which is expected to be a molecular marker for patient prognosis and a potential target of targeted therapy for GC patients.

[Protective effects of berberine against exhaustion exercise induced myocardial injury in rats].

Objective: To investigate the effect and possible mechanism of berberine (Ber) on myocardial injury induced by exhaustion exercise (Ee). Methods: Forty healthy male SPF Sprague-Dawley rats were randomly divided into 5 groups using the random unit group design method: control group, Ee group and Ee plus Ber group (low: 50 mg·kg(-1)·d(-1), medium: 100 mg·kg(-1)·d(-1) and high dose: 150 mg·kg(-1)·d(-1), n=8 each). Ber (1.5 ml) or equal volume saline was given per gavage for 14 days. Rats assigned to Ee groups underwent Ee swimming once daily and rats in control group remain sedentary. After 14 days, echocardiographic measurements were performed and left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), left ventricular diastolic diameter (LVIDd) and left ventricular systolic diameter (LVIDs) were obtained. The morphological structure of heart was detected by HE and Masson staining. Serum superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Cardiomyocytes apoptosis was detected by TUNEL method. The protein expression of myocardial hypertrophy marker protein B-type natriuretic peptide (BNP) and apoptotic marker protein (Bcl-2, Bax) in rat myocardial tissue was detected by Western blot. Results: (1) Both LVFS and LVEF were significantly lower, and LVIDs and LVIDd were significantly larger in Ee group than those in control group (all P<0.01). The LVFS and LVEF in medium dose of Ber and high-dose Ber groups were significantly higher, and the LVIDs and LVIDd were significantly smaller than those in Ee group (all P<0.01). (2) The results of HE staining showed that the myocardial cells in control group were closely arranged, regular, normal in morphology, clear in structure, and uniform in staining. The myocardial cells of rats in Ee group were disarranged, cell staining was uneven, and vacuoles appeared in the cytoplasm. The disorder of myocardial cell arrangement and unequal staining in the medium dose of Ber were attenuated than in Ee group. The Masson staining results showed that the myocardial cells in control group were closely arranged, regular, normal in shape, clear in structure, and rarely blue-stained (fibrosis). Myocardial cells in rats in Ee group showed obvious fibrosis. The myocardial cell fibrosis in rats with medium dose of Ber was significantly reduced than exercise group. (3) MDA content in myocardial tissue of rats in Ee group was significantly higher than that of control group, and MDA content in myocardial tissue of rats in medium dose of Ber group was significantly lower than in Ee group (P<0.01). The SOD activity of myocardial tissue in rats was significantly lower than that of control group, while that of rats with medium dose of Ber was significantly higher than that of rats in Ee group (P<0.01). (4) TUNEL staining results showed that only a small amount of apoptosis myocardial cells were seen in control group, and a large number of apoptosis myocardial cells were seen in rats in Ee group. However, the number of apoptotic cardiomyocytes in medium dose of Ber was significantly lower than that in Ee group. The AI of rat cardiomyocytes was significantly higher than that of control group (P<0.01), and the AI of rat cardiomyocytes in median dose of Ber group was significantly lower than in Ee group (P<0.01). (5) BNP and Bax protein expression in the myocardial tissues of rats in Ee group were significantly higher than in control group (P<0.01). BNP and Bax protein expression in the myocardial tissues in median dose of Ber group were significantly lower than that of Ee group (P<0.01). The myocardial protein expression level of Bax was significantly higher, and the myocardial protein level of Bcl-2 was significantly lower in Ee group than in control group (both P<0.01), treatment with median dose of Ber could partly reverse above changes (both P<0.01). Conclusion: Ber can attenuate exhaustion exercise induced myocardial injury and remodeling in rats, and the beneficial effects of Ber might possibly be mediated by reducing free radical release and cardiomyocytes apoptosis.

[Value of endobronchial ultrasound-guided transbronchial needle aspiration of enlarged mediastinal lymph nodes and analysis of false negative results].

Objective: To explore the diagnostic value of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the diagnosis of enlarged mediastinal lymph nodes, and to analyze the reasons of false negative results of EBUS-TBNA. Methods: A retrospective analysis of the clinical and imaging data of 389 patients who underwent EBUS-TBNA for sampling enlarged hilar and mediastinal lymph nodes from October 2009 to October 2015 in Shandong Tumor Hospital, to evaluate its significance in the diagnosis of enlarged mediastinal lymph nodes, and to analyze the reasons of false negative results. Results: Among the 389 collected cases, positive biopsy results were obtained in 362 cases, and false negative results in 27 cases. The sensitivity, specificity, positive predictive value and negative predictive value were 92.9%, 100%, 100% and 25.0%, respectively. There was a positive correlation between the size of lymph node and biopsy positive rate (P=0.021). The subcarinal lymph nodes had the highest positive rate (97.7%), followed by the paratracheal lymph nodes (91.2%), with a statistically significant difference (P=0.006). The positive rates obtained by cytology (88.7%) and pathology (92.5%, ) showed no significant difference (P=0.065). The positive rate of EBUS-TBNA was not correlated with pathological types (P=0.932). Needle types (21G, 22G) had no significant effect on diagnosis accuracy (P=0.142). Conclusions: EBUS-TBNA is a practical technology for diagnosis of enlarged mediastinal lymph nodes, with unique characteristics such as minimally invasiveness, local anesthesia, good safety and repeatability. Along with the accumulation of surgical experience, improvement of operative skills, more close cooperation between surgeons, cytologists and pathologists, false negative results will be reduced and positive rate of EBUS-TBNA examination will be further improved.

[Mechanism of alpha-lipoic acid in treating TNBS-induced colitis in mice].

Objective: To investigate whether α-lipoic acid (ALA) regulates autophagy in the pathogenesis of colitis through the mammalian target of rapamycin (mTOR) signaling pathway. Methods: Balb/c mouse colitis model was induced with 2, 4, 6-trinitrobenzenesulfonic acid (TNBS). Male Balb/c mice (22-25 g) were divided into four groups: control group, ALA group, colitis group, and ALA treatment group, with 10 mice in each group. ALA was administered at a dose of 80 mg/kg once a day for 7 days. The colon weight index, the disease activity index (DAI) and the histologic degeneration score (HDS) of colon tissue in each group were calculated. The autophagy gene Beclin-1 mRNA level was detected by RT-PCR. The levels of mTOR, phosphorylated mTOR (p-mTOR), and Beclin-1 proteins were detected by Western blot. Results: Compared with the control group, there was no statistically significant difference in colon weight index in the ALA group[(9.21±0.57)vs(8.91±0.91)g/kg, P>0.05], but increased in the colitis group [(12.65±1.33)g/kg, P<0.05] and decreased in the ALA treatment group [(10.04±1.02)g/kg, P<0.05]; there were no significant differences in DAI or HDS in the ALA group, but significantly increased in the colitis group, and decreased in the ALA treatment group (all P<0.05). The Beclin-1 mRNA and protein levels showed no significant differences between the control and the ALA groups (1.00±0.12 vs 1.05±0.05, 1.00±0.11 vs 1.00±0.06). However, the expression of Beclin-1 was significantly decreased in the colitis group compared to the control group (0.51±0.07 vs 1.00±0.12, 0.52±0.07 vs 1.00±0.11, both P<0.05), but significantly increased in the ALA treatment group compared to the colitis group (1.75±0.09 vs 0.51±0.07, 1.82±0.14 vs 0.52±0.07, both P<0.05). The mTOR total protein levels were not significantly different among the four groups, but the p-mTOR level was significantly higher in the colitis group than in the control group (3.07±0.20 vs 1.00±0.07), and reduced in the ALA treatment group than in the colitis group (1.49±0.11 vs 3.07±0.20) (all P<0.05). Conclusion: ALA may improve the TNBS-induced colitis in mice by inhibiting the phosphorylation of mTOR to promote autophagy.

Aquaporin in the proliferation and apoptosis of diabetic myocardial cells.

The aim of this study was to explore the effect of aquaporin on the molecular mechanism of human diabetic myocardial cell apoptosis. The methylthiazolyle tetrazolium assay was used to detect the inhibitory effect of different concentrations of aquaporin on cell growth. The rate of aquaporin-induced myocardial cell apoptosis was detected by flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide double-stained cells. We also attempted to quantify the expression of Bcl-2, Bax, caspase-3, and survivin in diabetic myocardial cells by western blot analysis. Aquaporin was found to inhibit the proliferation of diabetic myocardial cells in a concentration-dependent manner; the increase in aquaporin concentration led to an increase in Bax (apoptosis protein) expression, decrease in Bcl-2 expression (anti-apoptosis protein), increase in the Bax/Bcl-2 ratio, and a decrease in caspase-3 and survivin expression (P < 0.05). Therefore, aquaporin significantly inhibits the proliferation of diabetic myocardial cells and cell apoptosis in a dose-dependent manner by upregulating the ratio of Bax/Bcl-2 protein expression, activating the caspase-3 protein cascade, and regulating the expression of survivin.

The therapeutic efficiency of extracorporeal magnetic innervation treatment in women with urinary tract dysfunction following radical hysterectomy.

Data on 32 consecutive women with demonstrable urinary tract dysfunction for at least 6 months following radical hysterectomy (RH) for uterine cervical cancer, who received 24 sessions of extracorporeal magnetic innervation (ExMI) treatment twice-weekly were collected. The 1-h pad test weight decreased from 27.2 g pre-treatment to 12.1 g post-treatment (p < 0.05). Both the median UDI-6 and IIQ-7 scores showed statistically significant improvements (p < 0.001) at every stage of the ExMI treatment and in the 24.2 months mean follow-up duration after treatment. The symptoms of frequency, stress incontinence, urge incontinence and voiding difficulty showed statistically significant improvements (p < 0.001) after 8 and 24 sessions of ExMI treatment. The urodynamic parameters between pre-treatment and post-treatment after 24 sessions revealed no statistically significant changes. Based on the objective and subjective measures observed in this study, 24 sessions of twice-weekly ExMI treatment is an additional non-invasive therapy option for patients with the symptoms of lower urinary tract following RH.

Clonotypic heterogeneity in experimental interstitial nephritis. Restricted specificity of the anti-tubular basement membrane B cell repertoire is associated with a disease-modifying crossreactive idiotype.

Experimental anti-tubular basement membrane (anti-TBM) disease is an autoimmune interstitial nephritis elicited in susceptible rodents after immunization with renal tubular antigen. The nephritogenic antigen in the immunizing preparation is 3M-1, a 48,000 Mr noncollagenous glycoprotein. The hallmarks of the renal lesion are the presence of anti-TBM antibodies (anti-TBM-Ab) and a dense mononuclear cell infiltrate. The anti-TBM B cell repertoire in this disease was analyzed using a library of 22 anti-TBM mAbs generated in a prototypically susceptible Brown Norway rat. These anti-TBM mAbs were all demonstrated to be 3M-1 specific and their characterization formed the basis for the following observations: (a) The size of the anti-TBM B cell population is estimated at 58 distinct clones; (b) by competitive inhibition criteria, all anti-TBM mAbs recognize the same (or spatially close) epitope(s) on 3M-1. This focused recognition was maintained in spite of considerable variability in affinity. Epitopic dominance could also be demonstrated in human polyclonal anti-TBM antisera from a patient with anti-TBM disease; and (c) a crossreactive idiotype was documented, and antisera directed toward this set of variable region determinants was shown to be effective as a prophylactic regimen to abrogate disease, and as a therapeutic modality to arrest the progression of disease; (d) analysis of VH gene families suggested biased usage of Q52- and 7183-like families, although at least three gene families are used in the anti-TBM-Ab response. Thus, the anti-TBM B cell compartment in BN rats is moderately large, but is primarily focused to a single epitope on the nephritogenic antigen and is associated with a disease-modifying crossreactive idiotype.

Characterization of a cis-acting regulatory element which silences expression of the class II-A beta gene in epithelium.

Class II major histocompatibility complex (MHC) genes encode for alpha/beta chain pairs that are constitutively expressed principally on mature B cells and dendritic cells in mice. These gene products are easily induced on macrophages with cytokines, and may also aberrantly appear on the surface of epithelium during immune injury. The appearance of class II determinants in parenchymal tissue potentially renders these somatic cells capable of antigen presentation to circulating CD4+ T lymphocytes, and their absence may be protective for normal tissues expressing self-antigens. The low surface class II expression observed on parenchymal cells generally correlates with low levels of mRNA, suggesting that transcription rate is a major element in class II regulation. To understand the transcriptional mechanism maintaining low basal surface expression of class II in somatic cells, we transiently transfected mini-gene reporter constructs to study the regulation of the murine A beta promoter in a cultured renal epithelial cell line. We describe here a negative cis-acting regulatory region located between -552 and -489 bp upstream of the A beta cap site that silences the transcriptional activity of the A beta promoter in epithelial cells in an orientation-dependent manner, and is also able to silence a heterologous promoter. This region is not active in class II-expressing B cells (BAL-17) in culture, but is functional in two other murine class II-negative cell lines, fibroblasts and thymoma T cells. Using competition electrophoretic mobility shift assays, we have localized the core protein binding site within this region to an 8-10-bp response element, designated A beta NRE, at -543 to -534 bp. A nuclear extract from BAL-17 cells does not bind to this element. Mutation of this site abrogates the transcriptional silencing activity of the region. We conclude that the transcription of class II-A beta in parenchymal cells, and some lymphocytes, can be actively repressed by an upstream silencing element.

Immunohistochemical localization of glucagon and pancreatic polypeptide on rat endocrine pancreas: coexistence in rat islet cells.

We used immunofluorescence double staining method to investigate the cellular localization of glucagon and pancreatic polypeptide (PP) in rat pancreatic islets. The results showed that both A-cells (glucagon-secreting cells) and PP-cells (PP-secreting cells) were located in the periphery of the islets. However, A-cells and PP-cells had a different regional distribution. Most of A-cells were located in the splenic lobe but a few of them were in the duodenal lobe of the pancreas. In contrast, the majority of PP-cells were found in the duodenal lobe and a few of them were in the splenic lobe of the pancreas. Furthermore, we found that 67.74% A-cells had PP immunoreactivity, 70.92% PP-cells contained glucagon immunoreactivity with immunofluorescence double staining. Our data support the concept of a common precursor stem cell for pancreatic hormone-producing cells.

[HIV infection status and characteristics of non-remunerated blood donors in Hangzhou, 2008-2017].

Objective: To understand HIV infection status and characteristics of non-remunerated blood donors in Hangzhou. Methods: HIV antibody test were conducted for non-remunerated blood donors in Hangzhou and their demographic and epidemiological information were collected from 2008-2017. χ(2) test for trend (liner by liner association chi square test) was used for the comparison of the HIV infection trends in each year. Results: A total of 1 461 129 non-remunerated blood donors were surveyed in Hangzhou during 2008-2017, and 260 blood donors were HIV positive. Most HIV infected blood donors were males (96.5%, 251/260) and aged 18-34 years (72.7%, 189/260). Among 260 HIV positive blood donors, those reporting repeated non-remunerated blood donation accounted for 36.9% (96/260), those reporting homosexual transmission accounted for 53.5% (139/260) and those reporting heterosexual transmission accounted for 44.6% (116/260). The HIV infected persons reporting homosexual behaviors were mainly aged 18-34 years (82.0%, 114/139) and unmarried (71.2%, 99/139). Most HIV infected students reported homosexual transmission (88.4%, 23/26). The crude HIV positive rate was 0.8/10 000-2.5/10 000, the differences in annual HIV positive rate had no significance (trend χ(2)=2.355, P=0.125). The crude HIV positive rate in male blood donors aged 18-24 years increased from 1.1/10 000 in 2008 to 3.7/10 000 in 2017, the difference was significant (trend χ(2)=5.175, P=0.023). Standardized HIV positive rate was 0.9/10 000-2.4/10 000. Conclusions: HIV infection rate was low in non-remunerated blood donors in Hangzhou during 2008-2017. Most HIV infected persons were males and aged 18-34 years. Heterosexual and homosexual contacts were the major transmission routes. The HIV positive rate in males aged 18-24 years showed an increase trend.

Comparative distribution of the alpha 1(IV), alpha 5(IV), and alpha 6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients.

We have shown previously that the 5' ends of the genes for the alpha 5(IV) and alpha 6(IV) collagen chains lie head-to-head on Xq22 and are deleted in patients with Alport syndrome (AS)-associated diffuse leiomyomatosis. In this study, we raised a rabbit anti-human alpha 6(IV)chain antibody, demonstrated its specificity by the analysis of recombinant NC1 domains af all six type IV chains, and studied the distribution of the alpha 6(IV) chain in relation to the alpha 1(IV) and alpha 5(IV) chains in human adult and fetal tissues involved in AS and diffuse leiomyomatosis. The alpha 6(IV) chain colocalizes with the alpha 5(IV) chain in basement membranes (BMs) of many tissues, but not in glomerular BM. These data exclude the alpha 6(IV) chain as a site for AS mutations. The head-to-head genomic pairing of the alpha 5(IV) and alpha 6 (IV) genes implies coordinate transcription of the two genes. Differential localization of the alpha 5(IV) and alpha 6(IV) chains shows that the two chains are not always coordinately regulated. The alpha 6(IV) chain, together with the alpha 3(IV)-alpha 5(IV) chains, was absent from all renal BMs in eight patients with X-linked AS while the alpha 1(IV) and alpha 2(IV) chains were increased. The data support the existence of two independent collagen networks, one for the alpha 3(IV)-alpha 6(IV) chains and one for the alpha 1(IV) and alpha 2(IV) chains.

The application value of continuous nursing for home oxygen therapy of patients in the stable phase of chronic obstructive pulmonary disease.

OBJECTIVE: To analyze the application value of continuous nursing in improving the effects of home oxygen therapy for patients in the stable phase of chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: Patients in the stable phase of COPD (n=106) were selected and divided into the control group and observation group based on parity digit of their admission number. There were 53 cases in each group. The patients in the control group received COPD health education at discharge, while the observation group received continuous nursing. The effect of home oxygen therapy in both groups after 3 months was compared. RESULTS: The compliance in the observation group for home oxygen therapy was significantly higher than that in the control group. Blood gas analysis and various indicators of pulmonary function in the control group at follow-up visits were not changed compared with those before. In contrast, partial pressure of blood oxygen and blood oxygen saturation of the observation group were lower than those before discharge. With the increasing partial pressure of carbon dioxide in arterial blood, the indicators of pulmonary function became lower than before. Comparing the various indexes between both groups at follow-up visits, the differences were statistically significant (p<0.05). The self-care ability and quality of life scores of patients in the observation group were higher than those of the control group, and the differences were statistically significant (p<0.05). CONCLUSIONS: By establishing health records and network platforms, continuous nursing can provide continuous health education and supervision for patients with COPD, which can effectively improve oxygen therapy compliance, self-care ability and quality of life. It has good application and promotional value.

Enamel matrix proteins regulate hypoxia-induced cellular biobehavior and osteogenic differentiation in human periodontal ligament cells.

Hypoxia is a crucial microenvironment for inflamed periodontal tissue and periodontal wound healing. Enamel matrix proteins (EMPs) potentially can promote the formation of new periodontium. The effects of EMPs on periodontal ligament cells under hypoxia, however, remain unclear. We investigated the effects of EMPs on cellular biobehavior and osteogenic differentiation of human periodontal ligament cells (hPDLCs) under hypoxia. Under cobalt chloride (CoCl2)-induced hypoxia, cellular biobehavior of hPDLCs, including proliferation, attachment, spreading, and migration with or without EMPs, was evaluated by 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), cell counting, spreading area measurement and wound scratch assay. The osteogenic activity of hPDLCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining (ARS). The expressions of osteogenic genes including runt related transcription factor 2 (Runx2), ALP, osteocalcin (OCN) and collagen type I (Col-I) were detected using real time quantitative PCR, western blot and immunocytochemistry assays. The biobehavior and osteogenic differentiation of hPDLCs were inhibited significantly under hypoxia. EMPs have no effect on cell proliferation under mimicked hypoxia. EMPs partly reversed the inhibitory effects of hypoxia, however, for other cellular biobehavior including attachment, spreading and migration, and markedly up-regulated osteogenic differentiation activities including ALP, mineralization ability and the expressions of osteogenic genes such as Runx2, ALP, osteocalcin, and collagen type I in hPDLCs under hypoxia. EMPs attenuate the hypoxic injury to cellular biobehavior and osteogenic differentiation in hPDLCs under hypoxia.

Dual activities of human prolidase.

The cDNA encoding human liver prolidase derived from a healthy adult's liver was cloned into the expression vector pPIC9K of Pichia pastoris to construct the recombination expression vector pPIC9K-P. The pPIC9K-P was digested by restriction enzyme Pme I, and then transformed into P. pastoris GS115 by electroporation. Transformants (the insertion recombinant) were induced by methanol to express the recombination protein. The optimal induction conditions (medium pH, methanol concentration and induction time) of the insertion transformant with the highest enzymatic activity were estimated by orthogonal experimental design L9(3(4)). The SDS-PAGE of the recombinant human prolidase (rh-prolidase) in induction medium showed a molecular weight of 73 kDa. The activities of the rh-prolidase and organophosphoric acid anhydrolases (OPAA) were assayed by colorimetric methods. The recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions, the maximal activities of prolidase and OPAA came to 44.1 and 54.8 nmol/min/mg protein respectively in the medium supernatant. The rh-prolidase purified from the supernatant by ion exchange gradient chromatography (DEAE-Sepharose Fast Flow) and gel filtration chromatography (Sephacryl S-200 High Resolution) showed a single band by SDS-PAGE analysis. The purified rh-prolidase could decompose soman via hydrolytic reaction in vitro.

A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.

Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain.

Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4 kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.

Lipoperoxidative injury to macrophages by oxidatively modified low density lipoprotein may play an important role in foam cell formation.

Both oxidatively and malondialdehyde modified low density lipoprotein (Ox-LDL and MDA-LDL) could be recognized by the scavenger receptor and induce intracellular cholesteryl ester accumulation of macrophage. The cholesteryl ester accumulation caused by MDA-LDL could be largely cleared by high density lipoprotein (HDL3), but that caused by Ox-LDL could not be. Further studies showed that Ox-LDL and MDA-LDL all could decrease the binding capacity of HDL3 and increase intracellular thiobarbituric acid reactive substances (TBARS). When macrophages were first cultured with MDA-LDL and then in medium without LDL, the decreased binding capacity of HDL3 was somewhat recovered and the intracellular TBARS did not increase any more. However, if macrophages were first cultured with Ox-LDL, the binding capacity of H DL3 continued to decrease and intracellular TBARS continued to increase. There was a negative correlation (r = -0.81, P < 0.01) between the decreased binding capacity of HDL3 and the increased intracellular TBARS caused by Ox-LDL. These results imply that lipid peroxidative injury to macrophages caused by Ox-LDL play an important role in foam cell formation.

High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical-pharmacological characteristic study of its product.

The human butyrylcholinesterase (BChE, EC 3.1.1.8) gene was highly expressed in Bombyx mori using baculovirus vector, and the biochemical-pharmacological properties of its product were studied. BChE cDNA was cloned into transfer vector pBn96 and co-transfected with wild-type Bombyx mori nucleopolyhedrovirus (BmNPV) DNA into BmN cells. The recombinant virus with the highest enzyme activity was sorted out and purified. Once the BmN cells or silkworm larvae had been infected with the recombinant virus, recombinant human BChE (rhBChE) could be secreted into the culture medium or the hemolymph of the larvae at levels of 1.5 mg x L(-1) and 35 mg x L(-1), respectively. Western blot and enzymatic staining of the electrophoresis gel of non-denatured protein showed that rhBChE manifested similar antigenicity and enzyme activity to native human BChE (nhBChE). The production of rhBChE in the hemolymph was 23-fold higher than that in BmN cells and about 280-fold that in Chinese hamster overy cells (125 microg x L(-1)). This is the first report of human BChE expression in silkworm with the highest level of yield so far. rhBChE was highly similar to nhBChE in respect to substrate affinity, inhibitor sensitivity, and reactivity of the inhibited enzyme. It is suggested that rhBChE functions as well as nhBChE and has potential practical value.