Characterization of a recombinant porcine follistatin in a heat shock expression system

2002 ◽  
Vol 82 (3) ◽  
pp. 295-304
Author(s):  
C. R. Christensen ◽  
J. Kowalski ◽  
P. J. Chedrese ◽  
V. Misra ◽  
B. Laarveld ◽  
...  
Keyword(s):  
Heat Shock ◽  
Ovarian Function ◽  
Expression System ◽  
Porcine Granulosa Cells ◽  
Molecular Masses ◽  
Estradiol 17Β

The role of follistatin in ovarian function has yet to be fully elucidated; it is present in low concentration in vivo and it is difficult to obtain suitable quantities of follistatin for characterization. We have cloned porcine follistatin cDNA in an expression system that uses the heat shock protein promoter BoHSP70. Recombinant follistatin with apparent molecular masses of 39, 46, 48, 50 kDa was expressed and secreted into culture medium at concentrations of 400–500 μg × 20 mL-1 × 150 cm-2 flask (4 × 107 cells). In ligand blots, the recombinant follistatin was shown to be immunologically similar to native follistatin and to bind to recombinant activin A. Porcine granulosa cells dissected from 1–3 mm follicles from immature gilts were cultured with recombinant follistatin or with a follistatin monoclonal antibody to examine the activity of the recombinant follistatin. At a physiologically relevant concentration of 1 μg mL-1 the recombinant porcine follistatin suppressed the accumulation of estradiol-17β (P < 0.05). There was a trend for estradiol-17β accumulation in the presence of 10 μg mL-1 of monoclonal anti-follistatin antibody (P = 0.1). This expression system consistently produced large quantities of recombinant porcine follistatin, which is immunologically and biologically similar to follistatin, and is capable of independently inhibiting estratiol-17βproduction in vitro. Key words: Porcine, follistatin, heat shock promoter, glycoprotein, ovary, estradiol

scholarly journals Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60

2005 ◽  
Vol 391 (2) ◽  
pp. 185-190 ◽  
Author(s):  
Renu Wadhwa ◽  
Syuichi Takano ◽  
Kamaljit Kaur ◽  
Satoshi Aida ◽  
Tomoko Yaguchi ◽  
...  
Keyword(s):  
Heat Shock ◽  
Molecular Interactions ◽  
Heat Shock Protein 60 ◽  
Hsp60 Expression ◽  
Mitochondrial Hsp70 ◽  
Shock Proteins ◽  
First Time

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

scholarly journals Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform

2019 ◽  
Vol 476 (10) ◽  
pp. 1521-1537 ◽  
Author(s):  
Emma J. Goldberg ◽  
Katherine A. Buddo ◽  
Kelsey L. McLaughlin ◽  
Regina F. Fernandez ◽  
Andrea S. Pereyra ◽  
...  
Keyword(s):  
Acid Oxidation ◽  
Valuable Insight ◽  
Keto Acid ◽  
Isolated Mitochondria ◽  
Mouse Tissues ◽  
Branched Chain

Abstract Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism—evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

In vivo and in vitro response to octreotide LAR in a TSH-secreting adenoma: characterization of somatostatin receptor expression and role of subtype 5

Pituitary ◽  
2010 ◽  
Vol 14 (2) ◽  
pp. 141-147 ◽  
Author(s):  
Federico Gatto ◽  
Federica Barbieri ◽  
Lara Castelletti ◽  
Marica Arvigo ◽  
Alessandra Pattarozzi ◽  
...  
Keyword(s):  
Somatostatin Receptor ◽  
Receptor Expression ◽  
Octreotide Lar ◽  
In Vitro Response

scholarly journals Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

2010 ◽  
Vol 78 (6) ◽  
pp. 2370-2376 ◽  
Author(s):  
Louise M. Temple ◽  
David M. Miyamoto ◽  
Manju Mehta ◽  
Christian M. Capitini ◽  
Stephen Von Stetina ◽  
...  
Keyword(s):  
Whooping Cough ◽  
Bioinformatic Analysis ◽  
Tracheal Ring ◽  
Tracheal Cell ◽  
Bordetella Avium ◽  
Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

Mammalian lymphocytes: stress-induced synthesis of heat-shock proteins in vitro and in vivo

1985 ◽  
Vol 63 (7) ◽  
pp. 711-722 ◽  
Author(s):  
David Rodenhiser ◽  
Jack H. Jung ◽  
Burr G. Atkinson
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
White Blood Cells ◽  
Sodium Dodecyl ◽  
Molecular Masses ◽  
Febrile Episodes ◽  
Shock Proteins ◽  
Mouse Lymphocytes

Mammalian (human, mouse, and rabbit) white blood cells (lymphocytes) maintained in culture respond to a brief incubation at an elevated temperature (at or above 41 °C) by (i) the new and (or) enhanced synthesis of a small number of proteins (the so-called heat-shock proteins; HSPs) having molecular masses of approximately 110 000, 100 000, 90 000, 70 000, 65 000, and 26 000 daltons and (ii) the depressed synthesis of proteins normally made at 37 °C. The HSPs synthesized in culture by human, rabbit, and mouse (peripheral and splenic) lymphocytes are similar in number, molecular mass, and distribution on two-dimensional (isoelectric focusing and sodium dodecyl sulfate – polyacrylamide) electrophoretic gels to those synthesized in vivo by lymphocytes in hyperthermic mice. Since the level of hyperthermia used to induce HSP synthesis in mouse lymphocytes in vitro and in vivo is of a magnitude (41 °C) also used to promote thermotolerance in mice and is similar to temperatures attained during febrile episodes in rabbits and in humans, we suggest that the in vitro and in vivo synthesis of HSPs by mouse lymphocytes, demonstrated in this study, represents a relevant, physiological response which mammalian lymphocytes may normally use to survive periods of thermal stress.

scholarly journals NLRP3- and AIM2-autonomy in a mouse model of MSU crystal-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1[beta] expression as relevant therapy for gout patients.

2019 ◽  
Author(s):  
Alexandre Mariotte ◽  
Aurore Decauwer ◽  
Chrystelle Po ◽  
Cherine Abou-Faycal ◽  
Angelique Pichot ◽  
...  
Keyword(s):  
Mouse Model ◽  
Acute Inflammation ◽  
Joint Inflammation ◽  
Inflammatory Action ◽  
Recognition Receptor ◽  
Msu Crystals

The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1b in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1b; secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Here, we provide an extensive clinical, biological and molecular characterization of the acute uratic inflammation mouse model induced by subcutaneous injection of MSU crystals, which accurately mimics human gout. Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities, among which the use of topical application of imiquimod to promote interferon-dependent anti-inflammatory action maybe relevant.

scholarly journals Characterization of AcMNPV with a deletion of ac69 gene

2011 ◽  
Vol 2 (1) ◽  
pp. 4
Author(s):  
Jianhao Ke ◽  
Jinwen Wang ◽  
Riqiang Deng ◽  
Lin Lin ◽  
Bei Jinlong ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Trichoplusia Ni ◽  
Virus Production ◽  
Viral Infectivity ◽  
Methyltransferase Activity ◽  
Budded Virus

<p>ORF69 (Ac69) of <em>Autographa californica</em> multiple nucleopolyhedrovirus (Ac<em>M</em>NPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in <em>Spodoptera frugiperda</em> Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an <em>ac69</em> knockout Ac<em>M</em>NPV bacmid mutant and analyses of the influence of <em>ac69</em> deletion on the viral infectivity in Sf-9 cells and <em>Trichoplusia ni</em> larvae so as to investigate the role of <em>ac69 in the viral life cycle. Results indicated that ac69</em> deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect Ac<em>M</em>NPV infectivity for <em>Trichoplusia ni</em> larvae in LD<sub>50</sub> and LT<sub>50</sub> bioassay when administered orally. These results suggest that <em>ac69</em> may be dispensable for viral infectivity both in vitro and in vivo.</p>

scholarly journals Antigenic and genetic characterization of a divergent African virus, Ikoma lyssavirus

2014 ◽  
Vol 95 (5) ◽  
pp. 1025-1032 ◽  
Author(s):  
Daniel L. Horton ◽  
Ashley C. Banyard ◽  
Denise A. Marston ◽  
Emma Wise ◽  
David Selden ◽  
...  
Keyword(s):  
Laboratory Mouse ◽  
Rabies Vaccine ◽  
Molecular Techniques ◽  
Phenotypic Characteristics ◽  
Nucleotide Divergence ◽  
The Brain

In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between the genome of IKOV and those of other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spillover event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone, however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in vitro and in vivo characterization of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains and (3) poorly neutralized by sera from humans and animals immunized against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.

In Vitro and In Vivo Characterization of Scleral Implant of Indomethacin: Role of Plasticizer and Cross-Linking Time

Drug Delivery ◽  
2003 ◽  
Vol 10 (4) ◽  
pp. 269-275 ◽  
Author(s):  
M. Thilek Kumar ◽  
C. Rajeswari ◽  
J. Balasubramaniam ◽  
J. K. Pandit ◽  
S. Kant
Keyword(s):  
Cross Linking ◽  
In Vivo Characterization
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