Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

<p>Abstract</p> <p>Background</p> <p>ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of <it>Pseudomonas fluorescens </it>SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of <it>P. aeruginosa </it>alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.</p> <p>Results</p> <p>The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD) to three types of proteins; green fluorescent protein (GFP), epidermal growth factor (EGF) and cytoplasmic transduction peptide (CTP). These fusion proteins were expressed in <it>Escherichia coli </it>together with ABC transporter of either <it>P. fluorescens </it>or <it>Erwinia chrysanthemi</it>. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of <it>E. coli </it>with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant.</p> <p>Conclusion</p> <p>The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in <it>E. coli </it>supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in <it>Pseudomonas </it>using LARDs or TliA as a C-terminal signal sequence.</p

Brucellosis among ruminants in some districts of Bangladesh using four conventional serological assays

Brucellosis causes a great economic loss to the livestock industries through abortion, infertility, birth of weak and dead offspring, increased calving interval and reduction of milk yield and it is endemic in Bangladesh. The present study was performed to know the seroprevalence of brucellosis for 1000 ruminants (135 Buffaloes, 465 cattle, 230 goats and 170 sheep) in five different districts of Bangladesh by four conventional serological tests such as: Rose Bengal Plate Test (RBT), tube agglutination test (TAT), competitive enzyme-linked immunosorbent assay (C-ELISA), and Fluorescent polarization assay (FPA). Sheep has the highest prevalence (8.24%) of brucellosis. The seroprevalence of brucellosis was significantly higher in animals with previous abortion record in case of buffaloes, cattle, goats and sheep than that with no abortion record. C-ELISA can be the most suitable choice for extensive use in many kinds of livestocks and accurate estimation of Brucella antibodies in ruminants in Bangladesh

Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea

<p>Abstract</p> <p>Background</p> <p>A Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to <it>B. abortus </it>isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates.</p> <p>Results</p> <p>A total of 177 isolates originating from 105 cattle farms for the period 1996 to 2008 were selected as representatives for the nine provinces of South Korea. A dendrogram of strain relatedness was constructed in accordance with the number of tandem repeat units for 17 loci so that it was possible to trace back in the restricted areas. Even in a farm contaminated by one source, however, the <it>Brucella </it>isolates showed an increase or decrease in one TRs copy number at some loci with high DI values. Moreover, those 17 loci was confirmed in stability via <it>in-vitro </it>and <it>in-vivo </it>passage, and found to be sufficiently stable markers that can readily identify the inoculated strain even if minor changes were detected. In the parsimony analysis with foreign <it>Brucella </it>isolates, domestic isolates were clustered distinctively, and located near the Central and Southern American isolates.</p> <p>Conclusion</p> <p>The MLVA assay has enough discrimination power in the <it>Brucella </it>species level and can be utilized as a tool for the epidemiological trace-back of the <it>B. abortus </it>isolates. But it is important to consider that <it>Brucella </it>isolates may be capable of undergoing minor changes at some loci in the course of infection or in accordance with the changes of the host.</p

In Utero Development of the Fetal Gall Bladder in the Korean Population

OBJECTIVE: To provide reference ranges of the fetal gall bladder in the Korean population. MATERIALS AND METHODS: Fetal gall bladder development was evaluated in well-dated, non-anomalous fetuses in the Korean population between February and April 2003 and the visualization rate and reference values were determined from the obtained data. RESULTS: The visualization rate of the fetal gall bladder increased as gestation advanced to a plateau above 90%, which was maintained between 16 and 34 weeks. The measured parameters from the fetal gall bladder had a significant positive relationship with gestational age (p = 0.000 for all cases), and the correlation of length and area with the gestational age (r = 0.741 and r = 0.690, respectively) was better than the correlation of width, height, and volume with gestational age. The repeatability coefficients and coefficients of variation between the two operators were 5.56 mm and 12.9% for the length and 344.11 mm(2) and 33.52% for the area. The median length of the fetal gall bladder in the Korean population was not significantly different from the mean length of gall bladders in the Caucasian and African-American populations (p = 0.915). CONCLUSION: We have provided reference values for the fetal gall bladder throughout the gestation period in the Korean population

Loss of Nuclear BAP1 Expression Is Associated with High WHO/ISUP Grade in Clear Cell Renal Cell Carcinoma

Background BRCA1-associated protein 1 (BAP1) mutations are frequently reported in clear cell renal cell carcinoma (ccRCC); however, very few studies have evaluated the role of these mutations in other renal cell carcinoma (RCC) subtypes. Therefore, we analyzed BAP1 protein expression using immunohistochemistry in several RCC subtypes and assessed its relationship with clinicopathological characteristics of patients. Methods BAP1 expression was immunohistochemically evaluated in tissue microarray blocks constructed from 371 samples of RCC collected from two medical institutions. BAP1 expression was evaluated based on the extent of nuclear staining in tumor cells, and no expression or expression in < 10% of tumor cells was defined as negative. Results Loss of BAP1 expression was observed in ccRCC (56/300, 18.7%), chromophobe RCC (6/26, 23.1%), and clear cell papillary RCC (1/4, 25%), while we failed to detect BAP1 expression loss in papillary RCC, acquired cystic disease-associated RCC, or collecting duct carcinoma. In ccRCC, loss of BAP1 expression was significantly associated with high World Health Organization (WHO)/International Society of Urological Pathology (ISUP) grade (p = .002); however, no significant correlation was observed between loss of BAP1 expression and survival in ccRCC. Loss of BAP1 expression showed no association with prognostic factors in chromophobe RCC. Conclusions Loss of BAP1 nuclear expression was observed in both ccRCC and chromophobe RCC. In addition, BAP1 expression loss was associated with poor prognostic factors such as high WHO/ISUP grade in ccRCC

Permanent Pacemaker for Syncope after Heart Transplantation with Bicaval Technique

Sinus node dysfunction occurs occasionally after heart transplantation and may be caused by surgical trauma, ischemia to the sinus node, rejection, drug therapy, and increasing donor age. However, the timing and indication of permanent pacemaker insertion due to sinus node dysfunction following heart transplantation is contentious. Here, we report a case of a permanent pacemaker insertion for syncope due to sinus arrest after heart transplantation, even with a bicaval technique, which has been known to associate with few incidences of sinus node dysfunction