Gene characterization of two digestive serine proteases in Sitodiplosis mosellana: implications for alternative control strategies

2010 ◽  
Vol 142 (6) ◽  
pp. 532-545
Author(s):  
Lourdes D. Arrueta ◽  
Richard H. Shukle ◽  
Ian L. Wise ◽  
Omprakash Mittapalli
Keyword(s):  
Serine Proteases ◽  
Expressed Sequence Tag ◽  
Fat Body ◽  
Control Strategies ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Alternative Control ◽  
Sitodiplosis Mosellana

AbstractTwo full-length cDNA sequences encoding digestive serine proteases (designated as SmPROT-1 and SmPROT-2) were recovered from the midgut of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), in an ongoing expressed sequence tag project. The deduced amino acid sequences shared homology with digestive serine proteases from insect and non-insect species, including conserved regions such as the catalytic triad, active pocket, and conserved structural motifs. Secretory signal peptides in both proteases at the N-terminals indicate that these proteins could function as midgut digestive serine proteases. A phylogenetic analysis grouped SmPROT-1 and SmPROT-2 with trypsin-like and chymotrysin-like serine proteases, respectively. Quantitative real-time PCR analysis showed that SmPROT-1 and SmPROT-2 were expressed predominantly in the midgut rather than in other tissues (fat body and salivary glands). Expression analyses revealed high mRNA levels for the feeding instars (1st- and 2nd-instar larvae) compared with other stages (neonate, 3rd instar, pupa, and adult). These results provide new insights into the biology of S. mosellana and are discussed in the context of developing alternative control strategies.

Cloning and functional expression of rat kidney dipeptidyl peptidase II

2001 ◽  
Vol 353 (2) ◽  
pp. 283-290 ◽  
Author(s):  
Kayoko M. FUKASAWA ◽  
Katsuhiko FUKASAWA ◽  
Koichi HIGAKI ◽  
Naoki SHIINA ◽  
Michiaki OHNO ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Expressed Sequence Tag ◽  
Specific Activity ◽  
Rat Kidney ◽  
Functional Expression ◽  
Dipeptidyl Peptidase ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Calculated Molecular Mass

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4µmol/min per mg of protein for Lys-Ala-β-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400Da, which was lower than the value (about 60000Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

scholarly journals Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils.

1990 ◽  
Vol 172 (6) ◽  
pp. 1709-1715 ◽  
Author(s):  
D Campanelli ◽  
M Melchior ◽  
Y Fu ◽  
M Nakata ◽  
H Shuman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Serine Protease ◽  
Serine Proteases ◽  
Specific Activity ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Predicted Amino Acid Sequence ◽  
Proteinase 3

Closely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of approximately 29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre- and pro-sequences. The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments. The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization of the full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Activation of Apoptosis in a βB1-CTGF Transgenic Mouse Model

2021 ◽  
Vol 22 (4) ◽  
pp. 1997
Author(s):  
Maximilian Weiss ◽  
Sabrina Reinehr ◽  
Ana M. Mueller-Buehl ◽  
Johanna D. Doerner ◽  
Rudolf Fuchshofer ◽  
...  
Keyword(s):  
Open Angle Glaucoma ◽  
Bipolar Cells ◽  
Nerve Degeneration ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Pcr Analysis ◽  
Progression Rate ◽  
Suitable Model ◽  
Mrna Levels ◽  
Gabaergic Synapse ◽  
Slow Progression

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5–9/group) and quantitative real-time PCR analysis (n = 5–7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma.

scholarly journals Molecular detection and genetic diversity of Leucocytozoon sabrazesi in chickens in Thailand

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Runglawan Chawengkirttikul ◽  
Witchuta Junsiri ◽  
Amaya Watthanadirek ◽  
Napassorn Poolsawat ◽  
Sutthida Minsakorn ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Egg Production ◽  
Control Strategies ◽  
Amino Acid Sequences ◽  
Economic Losses ◽  
Nested Polymerase Chain Reaction ◽  
Blood Samples ◽  
Tropical Regions ◽  
Vaccination Programs ◽  
High Entropy

AbstractLeucocytozoon sabrazesi is the intracellular protozoa of leucocytozoonosis, which is transmitted by the insect vectors and affects chickens in most subtropical and tropical regions of the globe, except South America, and causing enormous economic losses due to decreasing meat yield and egg production. In this study, L. sabrazesi gametocytes have been observed in the blood smears, and molecular methods have been used to analyse the occurrence and genetic diversity of L. sabrazesi in blood samples from 313 chickens raised in northern, western and southern parts of Thailand. The nested polymerase chain reaction (nested PCR) assay based on the cytb gene revealed that 80.51% (252/313) chickens were positive of L. sabrazesi. The phylogenetic analysis indicated that L. sabrazesi cytb gene is conserved in Thailand, showed 2 clades and 2 subclades with similarity ranged from 89.5 to 100%. The diversity analysis showed 13 and 18 haplotypes of the sequences from Thailand and from other countries, respectively. The entropy analyses of nucleic acid sequences showed 26 high entropy peaks with values ranging from 0.24493 to 1.21056, while those of amino acid sequences exhibited 5 high entropy peaks with values ranging from 0.39267 to 0.97012. The results; therefore, indicate a high molecular occurrence of L. sabrazesi in chicken blood samples with the associated factors that is statistically significant (p < 0.05). Hence, our results could be used to improve the immunodiagnostic methods and to find appropriate preventive control strategies or vaccination programs against leucocytozoonosis in order to mitigate or eliminate the harmful impact of this infection on chicken industry.

scholarly journals Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yali Liu ◽  
Ling Zhang ◽  
Shaofeng Wei ◽  
Jinyang Cai ◽  
Zhenzhong Zang ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Human Intestinal Epithelial Cells ◽  
Pulsatilla Chinensis ◽  
Dependent Transport ◽  
Cytotoxic Studies

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

scholarly journals Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiumei Miao ◽  
Lijing Zhang ◽  
Xiaowei Hu ◽  
Shuzhen Nan ◽  
Xiaolong Chen ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linoleic Acid ◽  
Gene Family ◽  
Fatty Acid Biosynthesis ◽  
Family Members ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Acid Biosynthesis ◽  
Desert Shrub ◽  
Artemisia Sphaerocephala

Abstract Background Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. Results Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2–1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2–1, − 10, and − 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2–1 and AsFAD2–10 could also produce palmitolinoleic acid. Conclusions This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

scholarly journals Enhanced activity of ventricular Na+-HCO3− cotransport in pressure overload hypertrophy

2007 ◽  
Vol 293 (2) ◽  
pp. H1254-H1264 ◽  
Author(s):  
Taku Yamamoto ◽  
Takeshi Shirayama ◽  
Tomohiko Sakatani ◽  
Tomosaburo Takahashi ◽  
Hideo Tanaka ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Single Cells ◽  
Ischemia Reperfusion ◽  
Pressure Overload ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Aortic Constriction ◽  
Ph Indicator ◽  
Adult Rat ◽  
Acid Extrusion

The Na+-HCO3− cotransporter (NBC) plays a key role in intracellular pH (pHi) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pHi was measured in single cells using the fluorescent pH indicator 2′,7′-bis(2-carboxyethyl)5-( 6 )carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ( JNBC) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na+/H+ exchange was also increased in hypertrophied myocytes, the relative enhancement of JNBC was larger, 6) membrane depolarization markedly increased JNBC in hypertrophied myocytes, and 7) losartan, an ANG II AT1 receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na+ overload, which may render the ventricles more vulnerable to Ca2+ overload during ischemia-reperfusion.

scholarly journals Biological and Molecular Characteristics of a Novel Partitivirus Infecting the Edible Fungus Lentinula edodes

Plant Disease ◽  
2017 ◽  
Vol 101 (5) ◽  
pp. 726-733 ◽  
Author(s):  
Mengpei Guo ◽  
Yinbing Bian ◽  
Jinjie Wang ◽  
Gangzheng Wang ◽  
Xiaolong Ma ◽  
...  
Keyword(s):  
Lentinula Edodes ◽  
Control Strategies ◽  
Phylogenetic Analyses ◽  
Pcr Analysis ◽  
Molecular Characteristics ◽  
Rt Pcr ◽  
Edible Fungus ◽  
Qpcr Analysis ◽  
Disease Diagnostics ◽  
Wild Strains

A new partitivirus named Lentinula edodes partitivirus 1 (LePV1) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePV1 dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePV1 to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePV1, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePV1. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes.

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