Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

scholarly journals Characterization of the anthranilate degradation pathway in Geobacillus thermodenitrificans NG80-2

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Xueqian Liu ◽  
Yangpeng Dong ◽  
Xiaomin Li ◽  
Yi Ren ◽  
Yanxia Li ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Degradation Pathway ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Geobacillus Thermodenitrificans ◽  
Reservoir Conditions ◽  
Important Intermediate ◽  
First Time

Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH2-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the anthranilate degradation gene cluster in Geobacillus thermodenitrificans NG80-2 were functionally characterized in vitro. GTNG_3159 produces FAD to be reduced by GTNG_3158 and the reduced FAD (FADH2) is utilized by GTNG_3160 to convert anthranilate to 3-hydroxyanthranilate (3-HAA), which is further degraded to acetyl-CoA through a meta-cleavage pathway also encoded in the gene cluster. Utilization of this pathway for the degradation of anthranilate and tryptophan by NG80-2 under physiological conditions was confirmed by real-time RT-PCR analysis of representative genes. This is believed to be the first time that the degradation pathway of anthranilate via 3-HAA has been characterized in a bacterium. This pathway is likely to play an important role in the survival of G. thermodenitrificans in the oil reservoir conditions from which strain NG80-2 was isolated.

scholarly journals Identification and Characterization of Avian Retroviruses in Chicken Embryo-Derived Yellow Fever Vaccines: Investigation of Transmission to Vaccine Recipients

2003 ◽  
Vol 77 (2) ◽  
pp. 1105-1111 ◽  
Author(s):  
Althaf I. Hussain ◽  
Jeffrey A. Johnson ◽  
Marcos da Silva Freire ◽  
Walid Heneine
Keyword(s):  
Western Blot ◽  
Yellow Fever ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Serum Samples ◽  
Rna Sequences ◽  
Western Blot Assay ◽  
Specific Pcr ◽  
Show Evidence

ABSTRACT All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.

scholarly journals Overexpression of P-glycoprotein, MRP2, and CYP3A4 impairs intestinal absorption of octreotide in rats with portal hypertension

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaoyu Sun ◽  
Shunxiong Tang ◽  
Binbin Hou ◽  
Zhijun Duan ◽  
Zhen Liu ◽  
...  
Keyword(s):  
Liver Cirrhosis ◽  
Portal Hypertension ◽  
Western Blot ◽  
In Vitro Experiments ◽  
Rt Pcr ◽  
P Glycoprotein ◽  
Intestinal Microsomes ◽  
First Pass

Abstract Background Portal hypertension (PH) is the main cause of complications and death in liver cirrhosis. The effect of oral administration of octreotide (OCT), a drug that reduces PH by the constriction of mesenteric arteries, is limited by a remarkable intestinal first-pass elimination. Methods The bile duct ligation (BDL) was used in rats to induce liver cirrhosis with PH to examine the kinetics and molecular factors such as P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and cytochrome P450 3A4 (CYP3A4) influencing the intestinal OCT absorption via in situ and in vitro experiments on jejunal segments, transportation experiments on Caco-2 cells and experiments using intestinal microsomes and recombinant human CYP3A4. Moreover, RT-PCR, western blot, and immunohistochemistry were performed. Results Both in situ and in vitro experiments in jejunal segments showed that intestinal OCT absorption in both control and PH rats was largely controlled by P-gp and, to a lesser extent, by MRP2. OCT transport mediated by P-gp and MRP2 was demonstrated on Caco-2 cells. The results of RT-PCR, western blot, and immunohistochemistry suggested that impaired OCT absorption in PH was in part due to the jejunal upregulation of these two transporters. The use of intestinal microsomes and recombinant human CYP3A4 revealed that CYP3A4 metabolized OCT, and its upregulation in PH likely contributed to impaired drug absorption. Conclusions Inhibition of P-gp, MRP2, and CYP3A4 might represent a valid option for decreasing intestinal first-pass effects on orally administered OCT, thereby increasing its bioavailability to alleviate PH in patients with cirrhosis.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

scholarly journals Identification and Functional Characterization of the CVOMT and EOMT Genes Promoters from Ocimum Basilicum L

2021 ◽  
Author(s):  
Fatemeh Khakdan ◽  
Zahra Shirazi ◽  
Mojtaba Ranjbar
Keyword(s):  
Water Deficit ◽  
Transcriptional Control ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Water Deficit Stress ◽  
Specific Expression ◽  
Stress Dependent ◽  
First Time ◽  
Dependent Mechanism

Abstract Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced by the water deficit stress-dependent mechanism. Here, for the first time, pObCVOMT and pObEOMT promoters were extracted by the genome walking method. They were then cloned into the upstream of the β-glucuronidase (GUS) reporter gene to identify the pattern of GUS water deficit stress-specific expression. Histochemical GUS assays showed in transgenic tobacco lines bearing the GUS gene driven by pObCVOMT and pObEOMT promoters, GUS was strongly expressed under water deficit stress. qRT-PCR analysis of pObCVOMT and pObEOMT transgenic plants confirmed the histochemical assays, indicating that the GUS expression is also significantly induced and up-regulated by increasing density of water deficit stress. This indicates these promoters are able to drive inducible expression. The cis-acting elements analysis showed that the pObCVOMT and pObEOMT promoters contained dehydration or water deficit-related transcriptional control elements.

Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1453-1464 ◽  
Author(s):  
M. Gabriela Bowden ◽  
Wei Chen ◽  
Jenny Singvall ◽  
Yi Xu ◽  
Sharon J. Peacock ◽  
...  
Keyword(s):  
Cell Wall ◽  
Staphylococcus Epidermidis ◽  
Tertiary Structure ◽  
Genomic Sequence ◽  
Human Infection ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Antibody Titres

Staphylococcus epidermidis is a ubiquitous human skin commensal that has emerged as a major cause of foreign-body infections. Eleven genes encoding putative cell-wall-anchored proteins were identified by computer analysis of the publicly available S. epidermidis unfinished genomic sequence. Four genes encode previously described proteins (Aap, Bhp, SdrF and SdrG), while the remaining seven have not been characterized. Analysis of primary sequences of the Staphylococcus epidermidis surface (Ses) proteins indicates that they have a structural organization similar to the previously described cell-wall-anchored proteins from S. aureus and other Gram-positive cocci. However, not all of the Ses proteins are direct homologues of the S. aureus proteins. Secondary and tertiary structure predictions suggest that most of the Ses proteins are composed of several contiguous subdomains, and that the majority of these predicted subdomains are folded into β-rich structures. PCR analysis indicates that certain genes may be found more frequently in disease isolates compared to strains isolated from healthy skin. Patients recovering from S. epidermidis infections had higher antibody titres against some Ses proteins, implying that these proteins are expressed during human infection. Western blot analyses of early-logarithmic and late-stationary in vitro cultures suggest that different regulatory mechanisms control the expression of the Ses proteins.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

300 ESTABLISHMENT AND CHARACTERIZATION OF RAT MESENCHYMAL STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 247
Author(s):  
T. H. Kim ◽  
B. G. Jeon ◽  
S. L. Lee ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcriptional Factors ◽  
Skin Tissue ◽  
Rt Pcr ◽  
Alternative Source ◽  
Differentiated Cells ◽  
Fetal Bovine

Mesenchymal stem cells (MSC) are regarded as an attractive source for tissue engineering and regeneration, and bone marrow extract has been commonly used as a source of pluripotent MSC. However, skin tissue has recently been identified as a convenient alternative source of MSC. The present study was focused on the effect of characterised MSC derived from rat on expression of early transcriptional factors, alkaline phosphate (AP) activity, and in vitro differentiation into selected cell lineages. The MSC were isolated from 8-week-old s.d. rat’s ear skin and cultured in advanced DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. To evaluate AP activity, cells were fixed with 3.7% formaldehyde solution and stained with Western Blue® (Promega, Madison, WI, USA). Expressions of early transcriptional factors (Oct-4, Sox2, and Nanog) were evaluated by RT-PCR. Differentiation into distinct mesenchymal lineages such as adipogenic, osteogenic, and neuron was done by following previously described protocols and assessed by lineage-specific stains. The specific genes in the osteocytes (osteocalcin, osteonectin, osteopontin, and Runx2), adipocytes (pparγ2, adiponectin, and aP2) or neuron (nestin, neurogenin 1, β-tublin, and nerve growth factor) were characterised by RT-PCR. The MSC were positive for AP activity and expressed Oct-4, Sox2, and Nanog. Following induction, MSC were successfully differentiated into adipocytes, osteocytes, and neurons. As adipocytes markers, aP2, pparγ2, and adiponectin were strongly detected in the adipocyte induced cells. Osteonectin, osteocalcin, Runx2, and osteopontin were expressed in the adipocyte induced cells. Futhermore, neuron-specific markers were clearly expressed in the neuronal differentiated cells. In conclusion, MSC have the capability of differentiation into multilineages including adipocytes, osteocytes, and neurons under the specific induction conditions. Skin tissue in rat can serve as an easily accessible and expandable alternative source for MSC harvesting and preclinical applications using an animal model. This work was supported by Grant No. 2007031034040 from Bio-organ and 200908FHT010204005 from Biogreen21, Republic of Korea.

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