Clinical analysis of 61 patients with thrombotic thrombocytopenic purpura / 北京大学学报(医学版)

OBJECTIVE@#To analyze and summarize the clinical features, diagnosis, treatment and prognosis of 61 patients with thrombotic thrombocytopenic purpura (TTP), so as to improve the ability of diagnosis and treatment.@*METHODS@#The clinical data of 61 TTP patients admitted to Peking University People's Hospital from January 2004 to March 2019 were retrospectively analyzed, and the clinical manifestations, blood routine, hemolysis indicators, and von Willebrand factor lyase (von Willebrand factor-cleaving protease, vWF-CP, also known as ADAMTS13) of these patients were observed. According to the outcome at the time of discharge, they were divided into survival group and death group, and the differences in clinical characteristics, neutrophil to lymphocyte ratio (NLR) and plasma exchange between the two groups were compared. The PLASMIC scores were calculated and compared with ADAMTS13 to determine the accuracy of the PLASMIC score in predicting ADAMTS13.@*RESULTS@#Among the 61 TTP patients, 22 were males and 39 were females, with an average age of (48±17) years. In the study, 48 cases had pentalogy, only 9 had triad, and the remaining 4 had no neuropsychiatric symptoms. Twenty-seven cases (44.3%) died and 34 cases (55.7%) survived. Among the 61 TTP patients, the platelet count was (12.9±9.5)×109/L, the hemoglobin (66.5±20.7) g/L, the percentage of erythrocyte fragments 3% (2%, 7%), and the plasma free hemoglobin increased to 360 (200, 457) mg /L, and the lactate dehydrogenase 1 508 (811, 2 133.8) U/L. The blood clotting was basically normal. The ADAMTS13 value of 30 patients was 49.0 (40.8, 61.3) μg/L, the ADAMTS activity of 10 patients was < 5%, and the remaining 21 patients were not checked. The PLASMIC score was 6-7 in 58 cases, 5 in 2 cases, and 4 in 1 case. The PLASMIC score predicted the decreased activity or the reduction of ADAMTS with a sensitivity as high as 97.5%. The NLR in the death group was higher than that in the survival group, but the difference was not statistically significant (P>0.05). The total amount and frequency of plasma exchange (PEX) in the death group were significantly less than those in the survival group, and the difference was statistically significant (P < 0.05). There was no significant difference in the treatment of glucocorticoids and human immunoglobulin between the two groups (P>0.05).@*CONCLUSION@#PEX can significantly improve the survival rate of TTP patients. PLASMIC score can easily and quickly predict the possibility of ADAMTS13 activity reduction, which is beneficial to the early diagnosis of TTP and PEX treatment. NLR can reflect the systemic inflammatory process, but its significance in TTP needs further study.

Structural insights into leishmanolysins encoded on chromosome 10 of Leishmania (Viannia) braziliensis

BACKGROUND Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.

Immunoprotective effect of combined pneumococcal endopeptidase O and pneumococcal surface adhesin A vaccines against Streptococcus pneumoniae infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins.</p><p><b>METHODS</b>Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination.</p><p><b>RESULTS</b>The target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups.</p><p><b>CONCLUSIONS</b>Combined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.</p>

Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide / 生物工程学报

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.

Effect of Irradiation on Microparticles in Red Blood Cell Concentrates

Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×10(9)/L (±22.7×10(9)/L), and the total number of MPs ranged from 2.6×10(9)/L to 96.9×10(9)/L. The mean number of MPs increased to 22.6×10(9)/L (±31.6×10(9)/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×10(9)/L) and 2.2% (263×10(6)/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×10(9)/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×10(6)/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Contexto social del debate sobre el control natal en Colombia en las décadas de 1960 y 1970: política, medicina y sociedad / The social context of the birth control debate in Colombia in the 1960s and 1970s: politics, medicine and society

El artículo busca presentar el contexto y aproximación preliminares necesarios para comprender y abordar el debate sobre el control natal en Colombia en las décadas de 1960 y 1970. Recoge las principales posturas en conflicto en dicho período, y los discursos y lógicas que permearon la llegada de los programas de planificación norteamericanos a América Latina como forma de control político de los movimientos revolucionarios.

The article seeks to present the necessary context and a preliminary approach to understanding and addressing the birth control debate in Colombia in the 1960s and 1970s. It covers the main conflicting positions during that period and the discourses and logics permeating the arrival of North American family planning programs to Latin America as a form of political control of revolutionary movements.

Bacteroides fragilis Toxin Induces IL-8 Secretion in HT29/C1 Cells through Disruption of E-cadherin Junctions

Enterotoxigenic Bacteroides fragilis (ETBF) is a human gut commensal bacteria that causes inflammatory diarrhea and colitis. ETBF also promotes colorectal tumorigenesis in the Min mouse model. The key virulence factor is a secreted metalloprotease called B. fragilis toxin (BFT). BFT induces E-cadherin cleavage, cell rounding, activation of the beta-catenin pathway and secretion of IL-8 in colonic epithelial cells. However, the precise mechanism by which these processes occur and how these processes are interrelated is still unclear. E-cadherin form homophilic interactions which tethers adjacent cells. Loss of E-cadherin results in detachment of adjacent cells. Prior studies have suggested that BFT induces IL-8 expression by inducing E-cadherin cleavage; cells that do not express E-cadherin do not secrete IL-8 in response to BFT. In the current study, we found that HT29/C1cells treated with dilute trypsin solution induced E-cadherin degradation and IL-8 secretion, consistent with the hypothesis that E-cadherin cleavage causes IL-8 secretion. However, physical damage to the cell monolayer did not induce IL-8 secretion. We also show that EDTA-mediated disruption of E-cadherin interactions without E-cadherin degradation was sufficient to induce IL-8 secretion. Finally, we determined that HT29/C1 cells treated with LiCl (beta-catenin activator) induced IL-8 secretion in a dose-dependent and time-dependent manner. Taken together, our results suggest that BFT induced IL-8 secretion may occur by the following process: E-cadherin cleavage, disruption of cellular interactions, activation of the beta-catenin pathway and IL-8 expression. However, we further propose that E-cadherin cleavage per se may not be required for BFT induced IL-8 secretion.

Construct a molecular switch based on bacterial quorum sensing / 生物工程学报

Engineering the existing or manual assembling biosynthetic pathways involves two important issues: the activity and expression level of key enzymes in the pathway. Concerning the enzyme expression study, the conventional approach is to use strong promoter to initiate the overexpression of the target protein. The excessive expression of the target protein usually result in the intracellular accumulation of a large number of inactive inclusion bodies, thereby seriously affect the physiological state of the cell and the effective functioning of the relevant biological pathways. To solve this problem, we would like to design a molecular switch to precisely manipulate the expression level of key enzymes in the biosynthetic process, which has important practical value for the study of metabolic rhythm of the biosynthetic pathway and to promote the efficiency of the biosynthetic pathway. Based on the basic principles of quorum sensing existing in the bacterial community and combining the dynamic characteristics of the enzymatic catalysis, we first established cell-cell communication mechanisms mediated by signal molecule homoserine lactone (AHL) in the E. coli community and target protein EGFP was expressed under the control of the promoter P(lux1). In the process of cell growth, AHL accumulated to a certain concentration to start the expression of target gene egfp. At the different cell growth stages, AHL-degrading enzyme AiiA was induced and resulted in the degradation of AHL molecule in a controlled environment, thereby controlling the transcription efficiency of target gene egfp and ultimately achieve the precise control of the level of expression of the target protein EGFP. The detection of cell growth state, the mRNA level and protein expression level of the target gene showed the artificially designed molecular switch can control the level of expression of a target gene in a convenient and efficient manner with a spatial and temporal regulation of rigor. The molecular switch is expected to be widely used in the field of metabolic engineering and synthetic biology research areas.

Posible papel de Bacteroides fragilis enterotoxigénico en la etiología de la vaginitis infecciosa / Possible role of enterotoxigenic Bacteroides fragilis in the etiology of infectious vaginitis

La vaginitis es un trastorno ginecológico frecuente producido por distintas causas, algunas de las cuales permanecen desconocidas. Bacteroides fragilis es el anaerobio más importante en bacteriología clínica. Algunas cepas son enterotoxigénicas y se asocian con síndromes intestinales y extraintestinales. Recientemente han sido aisladas de pacientes con vaginitis. En este trabajo se planteó investigar la posible asociación de B. fragilis enterotoxigénico con la vaginitis infecciosa. Fueron procesadas 265 muestras de exudado vaginal. 202 de mujeres sintomáticas y 63 mujeres sanas. La identificación de los microorganismos se realizó por métodos convencionales. En 31,2% de las pacientes sintomáticas se identificaron: Gardnerella vaginalis, Candida albicans, Mobiluncus, Mycoplasma hominis, Ureaplasma urealyticum y Streptococcus agalactiae. En 27 pacientes sintomáticas y en 5 mujeres sanas se identificó B. fragilis. Estas cepas fueron cultivadas en medio líquido e incubadas durante 48 h a 36° C en anaerobiosis. La toxicidad en los sobrenadantes se ensayó en células HT-29. 18 cepas de B. fragilis aisladas de pacientes sintomáticas fueron enterotoxigénicas, ya que indujeron alteraciones en la monocapa celular y en las células. No se identificó en mujeres sanas (P<0,05). 77,7% de las cepas de B. fragilis enterotoxigénicas no se encontraron asociadas con otros patógenos específicos. Este hecho sugiere que pudiera ser un agente causante de vaginitis, ya que el efecto de la enterotoxina sobre la E-cadherina del epitelio vaginal podría facilitar la invasión y su posible papel patógeno en la vagina. Esta es la primera investigación que asocia a Bacteroides fragilis enterotoxigénico como posible causa de vaginitis infecciosa.

Vaginitis is a common gynecologic disorder. It is due to several causes, some even unknown. Bacteroides fragilis is the most important anaerobe in clinical bacteriology, some strains of this group are notable for being enterotoxigenic and they have been associated with intestinal and extraintestinal syndromes. They have recently been isolated from patients with vaginitis. The purpose of this study was to investigate a possible association of enterotoxigenic B. fragilis with infectious vaginitis. 265 samples of vaginal exudate were processed, 202 from symptomatic patients and 63 healthy women. The identification of the microorganisms was carried out by conventional methods. In 31.2% of symptomatic patients were identified: Gardnerella vaginalis, Mobiluncus, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum and Streptococcus agalactiae. B. fragilis was identified in 27 symptomatic patients and 5 healthy women. These strains were cultivated in liquid medium and incubated during 48 h at 36°C in anaerobe chambers. Supernatant activity was assayed in HT-29 cells. Eighteen B. fragilis strains isolated from symptomatic patients were enterotoxigenic, because induced alterations in target cell morphology. It was not identified in healthy women (P<0.05). 77.7% of enterotoxigenic B. fragilis strains were not associated with other specific pathogens. This fact suggests that enterotoxigenic B. fragilis could be a cause for vaginitis. The effect of enterotoxin on E-cadherin of vaginal epithelium could facilitate invasion and its possible pathogenic role in the vagina. This is the first report that associates enterotoxigenic Bacteroides fragilis as a possible cause of infectious vaginitis.

Plutella xylostella granulovirus PP31 interacts with two host proteins / 病毒学报

Protein-protein interactions between viruses and hosts are common during viral infection and replication. In this study, a cDNA library from larvae of Plutella xylostella was constructed and used for screening of genes encoding proteins interacting with Plutella xylostella granulovirus (PlxyGV) proteins. Two cDNA clones containing genes encoding proteins interacting with PlxyGV PP31 were identified by yeast two-hybrid assays. Sequence analysis showed that the genes encoded homologues of receptor for activated protein C kinase (RACK) and methionine aminopeptidase 2 (MetAP2), respectively. The P. xylostella rack gene and the PlxyGV pp31 was expressed in an E. coli strain to produce proteins fused with a 6-His or a GST tag. It was shown that the rack was expressed as a 38kD peptide as prospected. The 38kD His-tagged peptide was co-purified with GST-PP31 by GST-bind resin in GST-pulldown assays, confirming interaction between the PlxyGV PP31 and the RACK protein of P. xylostella.

High mobility group box 1 protein: possible pathogenic link to atrial fibrillation / 中华医学杂志(英文版)

Atrial fibrillation (AF) is the most common sustained dysrhythmia in clinical practice. The bulk of evidence suggests that inflammatory processes, oxidative stress and matrix metalloproteinase are associated with development of AF. However, these agents may be involved in high mobility group box 1 protein (HMGB1). We hypothesized that HMGB1 may be a possible pathogenic link to AF. A growing body of evidence supports these hypotheses. First, the level of serum HMGB1 is significantly increased in patients with AF including paroxysmal and persistent AF. Second, HMGB1 has been identified as a new pro-inflammatory cytokine in cardiovascular diseases, along with tumor necrosis factor (TNF)-α, interleukin (IL)-6, and C-reactive protein, and there is cross-talk between HMGB1 and inflammatory cytokines. Third, oxidative stress is involved in the release of the pro-inflammatory cytokine, HMGB1, indicating there is cross-talk between oxidative stress and inflammation, and oxidative stress may reinforce the effect of inflammation on the pathogenesis of AF and inflammation may play a more important role in the pathogenesis of AF. Fourth, HMGB1 can promote matrix metalloproteinase-9 upregulation and activation. Fifth, HMGB1 receptors (receptor for advanced glycation end products, Toll-like receptor-2,4) may mediate the atrial structural remodeling or be up-regulated in patients with non-valvular AF. These results suggest that HMGB1 may participate in the pathogenesis of AF and provide a potential target for pharmacological interruption of AF.

Characteristics of protein from red crab (Chionoecetes japonicus) shell by commercial proteases

This study was performed to examine the characteristics of protein of red crab (Chionoecetes japonicus) shell powder hydrolyzed by commercial proteases. Red crab shell was digested by commercial proteases, such as Protamex (P), Neutrase (N), Flavourzyme (F), Alcalase (A), Protease M (PM) and Protease A (PA). Protein yield analyzed by Biuret assay, absorbance at 280 nm and brix revealed that PA was the enzyme having the highest proteolytic activity. SDS PAGE showed that molecular weight of proteins produced by protease treatments was various and below 150 kDa. Combinational treatment of proteases (PA + P, PA + PM, PA + F, PA + A) was tried whether these increase protein hydrolysis from red crab shell powder compared to a PA single treatment. Soluble protein content was similar, but amino acid concentration by combinational treatments was higher than PA single treatment [PA + P 247.4 mg/g > PA + F (206.4 mg/g) > PA + A (133.4 mg/g) > PA + PM (59.1 mg/g) > PA (54.9 mg/g)]. Amino acid composition by combinational treatments was slightly different. Most abundant essential amino acids were phenylalanine, glycine, alanine, and leucine, whereas tyrosine and cystine were not detected.

Biological and molecular characteristics of zymv isolates from cucumber [cucumis sativus] plant grown in Riyadh, KSA

Zucchini yellow mosaic virus[ZYMV] is potyvirus with a worldwide distribution. The virus is the most prevalent in cucurbits. Samples of cucumber plants [Cucumls sativus L.] that show symptoms of virus infection were collected during the 18-May-2009 growing season from field-grown cucumber in Riyadh Saudi Arabia. Based on DAS-ELISA with a polyclonal antiserum against ZYMV and reverse transcription polymerase chain reaction [RT-PCR] analysis using two primer pairs, the potyvirus was identified as strain of ZYMV. The anti-ZYMV antibodies in dot-blot, confirming the identity of the isolated viral particles as a potyvirus immunogenically related to ZYMV. Nucleotides Sequence analysis confirms that the isolated virus belong to potyvirus and its gen bank is GU808330.1. This is the first report on ZYMV as causal virus infecting cucumber in KSA

[Effects of calcitonin nasal spray on serum bone resorptive marker CTX and other laboratory findings in osteopenic menopausal women: a clinical trial]

Various therapeutic options such as calcitonin have been suggested for patients with low bone density, despite uncertain efficacy in most patients. C-telopeptide of type I collagen [CTX] is a new bone marker used for the assessment of bone resorption. The aim of this study was to evaluate the therapeutic effects of nasal spray calcitonin in women with osteopenia via serum CTX and other laboratory tests. We conducted a self controlled clinical trial in 2009 on 105 women of menopausal age diagnosed in Baqiyatallah Hospital Clinic with osteopenia based on a bone mineral density score of 1.5 SD lower than peak bone mass. The patients were assigned to receive nasal spray calcitonin [200 IU/day], calcium [1000 mg/day] and Vit-D [400 IU/day] for 6 months. Serum CTX and other laboratory parameters were measured before and after the treatment. The data were analyzed by SPSS, version 17, using t-tests and a P<0.05 was considered statistically significant. Fifty-two patients completed the study and the mean CTX level decreased significantly from 3.10 +/- 2.03 to 2.61 +/- 1.82 pmol/lit [P<0.001], but total serum levels of PTH, Ca, AST, ALT and Alkaline Ph decreased insignificantly. It seems that nasal spray of calcitonin is significantly effective in preventing disease progression and treatment of low bone density by inhibiting bone tissue resorption indicated by CTX although further studies with larger samples sizes and inclusion of control groups are warranted

Clinical research on Sanmiao powder combined fibrinogenase for injection in treatment of acute deep venous thrombosis of lower limbs / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the clinical effectiveness of Sanmiao Powder (SP) combined fibrinogenase for injection in treatment of acute deep venous thrombosis (ADVT) of lower limbs.</p><p><b>METHODS</b>Eighty patients with ADVT were randomly assigned to two groups according to the disease course (within 7 days or 7-28 days), 40 in each group. Every time phase was also divided into two groups, i. e., one group treated with fibrinogenase for injection alone (Group A and C) and another group treated with fibrinogenase for injection + SP administration (Group B and D) , 20 in each group. The clinical effectiveness was observed after 2-week treatment.</p><p><b>RESULTS</b>The fibrinogenase for injection + SP administration showed better effects in alleviating the swelling of limbs, relieving pain, and lowering fibrinogen. Better effects were obtained in the group with the disease course less than 7 days.</p><p><b>CONCLUSION</b>Better effect on ADVT was obtained by integrative medicine.</p>

Effect of Small Hairpin RNA Targeting Endothelin-Converting Enzyme-1 in Monocrotaline-Induced Pulmonary Hypertensive Rats

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.

Characterization of protein secretion based on structural fusion degree / 生物工程学报

Selection of suitable signal peptides is an important factor for efficient secretion of heterologous proteins. We defined structural fusion degree (SFD) as the compatibility degree of target proteins and signal peptides by a bioinformatics approach. We mathematically analyzed the interaction of fused signal peptides and adjacent residues of proteins, and proposed a mathematical model of extended signal region and the protein. SFD Features was extracted from this model to characterize the secretability of heterologous proteins. Simulation tests showed that SFD features can effectively discriminate high secretory proteins from poor ones in the host Bacillus subtilis. Results from this research will be useful in signal peptide selection and have a better guiding significance for the optimization of heterologous protein secretion.

Characterization of a Novel Monoclonal Antibody (27H2) Recognizing Human CD34 Class III Epitope

BACKGROUND: Monoclonal antibodies (mAbs) recognizing Class III epitope of CD34 are essential for flow cytometric diagnosis of leukemia. METHODS: 27H2 mAb was developed from a mouse alternatively immunized with human acute leukemia cell lines, KG1 and Molm-1. Using flow cytometric analysis of various leukemic cell lines and peripheral blood, immunohistochemical study of frozen tonsil, we characterized 27H2 mAb. Antigen immunoprecipitated with 27H2 mAb immunobloted with anti-CD34 mAb. A case of bone marrow sample of acute lymphoblastic leukemia (ALL) patient was obtained at CBNU Hospital. For epitope identification enzyme treatment with neuraminidase and O-sialoglycoprotein endopeptidase (OSGE) and blocking assay with known classIII mAb (HPCA-2) were done. RESULTS: Only KG1 and Molm-1 revealed positive immunoreactivity. Immunohistochemical staining disclosed strong membranous immunoreactivity on high endothelial venules. Antigen immunoprecipitated by 27H2 mAb showed approximately 100 kDa sized band immunoblotted with anti-CD34 under non-reducing conditions. Epitope recognized by 27H2 mAb disclosed resistancy to both neuraminidase and OSGE treatment and completely blocked with known class III mAb preincubation. CD34 positive leukemic cells in BM of pre B cell ALL patient detected by FITC-conjugated 27H2 and HPCA-2 were identified with similar sensitivity. CONCLUSION: A novel murine mAb recognizing class III epitope of human CD34 with high affinity, which is useful for flow cytometric diagnosis of leukemia, was developed.

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro.