Determination of β-glucosidase enzymatic function of the Histoplasma capsulatum H antigen using a native expression system

Gene ◽  
2000 ◽  
Vol 247 (1-2) ◽  
pp. 191-197 ◽  
Author(s):  
Kimber L. Fisher ◽  
Jon P. Woods
Keyword(s):  
Histoplasma Capsulatum ◽  
Expression System ◽  
Enzymatic Function

EFFECT OF HISTAMINE ON GASTRIC PEPSINOGEN, PEPSIN SECRETION, AND BLOOD PEPSINOGEN IN GUINEA PIGS

1957 ◽  
Vol 35 (1) ◽  
pp. 729-732
Author(s):  
K. Kowalewski
Keyword(s):  
Body Weight ◽  
Gastric Mucosa ◽  
Guinea Pig ◽  
Gastric Juice ◽  
Guinea Pigs ◽  
Gastric Tissue ◽  
Pepsin Secretion ◽  
Enzymatic Function ◽  
Guinea Pig Stomach

The endocrine and exocrine activity of guinea pig stomach was measured by the determination of pepsinogen in gastric tissue and in plasma. Gastric juice pepsin was also studied.A significant increase of both pepsinogen and pepsin was found in animals treated with a dose of histamine (75 mg. per kg. of body weight). These results give further evidence that the zymogenic cells of gastric mucosa may be stimulated by histamine. The determination of pepsinogen in gastric tissue seems to permit a direct approach to the enzymatic function of zymogenic cells.

scholarly journals An Alkane-Responsive Expression System for the Production of Fine Chemicals

1999 ◽  
Vol 65 (6) ◽  
pp. 2324-2332 ◽  
Author(s):  
Sven Panke ◽  
Andreas Meyer ◽  
Caroline M. Huber ◽  
Bernard Witholt ◽  
Marcel G. Wubbolts
Keyword(s):  
Specific Activity ◽  
Styrene Oxide ◽  
Expression System ◽  
Regulatory System ◽  
Dry Weight ◽  
Pseudomonas Oleovorans ◽  
Structural Genes ◽  
Biotechnological Processes ◽  
Terminal Amino

ABSTRACT Membrane-located monooxygenase systems, such as thePseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of alkS, whose activated gene product positively regulates the transcription of the structural genes alkBFGHJKL, on a 3.7-kb SalI-HpaI OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.

DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
M Ph Verbeet ◽  
R F Evers ◽  
A Leyte ◽  
H L Lamain ◽  
A J J Van Ooyen ◽  
...  
Keyword(s):  
Factor Viii ◽  
Recombinant Proteins ◽  
Cleavage Site ◽  
Specific Activity ◽  
Expression System ◽  
Full Length ◽  
Procoagulant Activity ◽  
Expression Vectors ◽  
Recombinant Fviii

Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.

scholarly journals GPD1L links redox state to cardiac excitability by PKC-dependent phosphorylation of the sodium channel SCN5A

2009 ◽  
Vol 297 (4) ◽  
pp. H1446-H1452 ◽  
Author(s):  
Carmen R. Valdivia ◽  
Kazuo Ueda ◽  
Michael J. Ackerman ◽  
Jonathan C. Makielski
Keyword(s):  
Sodium Channel ◽  
Sodium Current ◽  
Expression System ◽  
Sudden Cardiac Arrest ◽  
Sudden Infant ◽  
Cell Physiology ◽  
Glycerol Phosphate ◽  
Enzymatic Function ◽  
Cardiac Sodium Channel ◽  
Glycerol Phosphate Dehydrogenase

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current ( INa) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease INa through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased INa. These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to INa and excitability and may be important more generally in cardiac ischemia and heart failure.

Regulation of Na+-K+-Cl− cotransporter type 2 by the with no lysine kinase-dependent signaling pathway

2019 ◽  
Vol 317 (1) ◽  
pp. C20-C30 ◽  
Author(s):  
Andrée-Anne Marcoux ◽  
Samira Slimani ◽  
Laurence E. Tremblay ◽  
Rachelle Frenette-Cotton ◽  
Alexandre P. Garneau ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ion Transport ◽  
Transmembrane Domain ◽  
Expression System ◽  
Xenopus Laevis Oocyte ◽  
Thick Ascending Limb ◽  
Cell Shrinkage ◽  
Enzymatic Function ◽  
Wnk Kinase

Na+-K+-Cl− cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl−] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.

scholarly journals Methods for the Refolding of Disulfide-Rich Proteins

2017 ◽  
Author(s):  
Christopher N Haggarty-Weir ◽  
Roma Galloway ◽  
William Godfrey
Keyword(s):  
Genetic Manipulation ◽  
Small Scale ◽  
Resonance Spectroscopy ◽  
E Coli ◽  
Enzymatic Function ◽  
Sds Page ◽  
Reduced And Oxidized Glutathione ◽  
Scale Test ◽  
Bacterial Cytoplasm

AbstractEschericia coli remains the workhorse producing recombinant proteins given its ease of handling, access and genetic manipulation using standard laboratory techniques. However, disulfide-rich proteins can be difficult to produce in E. coli, in large part due to the reducing environment of the bacterial cytoplasm. Refolding from insoluble inclusion bodies can be a viable strategy for generating substantial quantities of disulfide-rich protein. For the best chance of successfully refolding a protein, it is vital to carry out a variety of small-scale test refolds under a swathe of conditions including altering the concentration of urea, salts, reduced and oxidized glutathione, temperature, length of refold time and protein dilution factor. Once a protein has undergone refolding it is vital to determine that the final product is natively folded given the chance of soluble misfolded protein. For determination of correct folding, a variety of techniques can be employed, and ideally, numerous should be used together. For proteins that possess enzymatic function the gold standard to assess correct folding is an activity assay. Non-enzymatic proteins can be assessed using a combination of circular dichroism and nuclear magnetic resonance spectroscopy. These techniques should be utilized alongside mass spectrometry, Western blotting and SDS-PAGE.

EFFECT OF HISTAMINE ON GASTRIC PEPSINOGEN, PEPSIN SECRETION, AND BLOOD PEPSINOGEN IN GUINEA PIGS

1957 ◽  
Vol 35 (9) ◽  
pp. 729-732 ◽  
Author(s):  
K. Kowalewski
Keyword(s):  
Body Weight ◽  
Gastric Mucosa ◽  
Guinea Pig ◽  
Gastric Juice ◽  
Guinea Pigs ◽  
Gastric Tissue ◽  
Pepsin Secretion ◽  
Enzymatic Function ◽  
Guinea Pig Stomach

The endocrine and exocrine activity of guinea pig stomach was measured by the determination of pepsinogen in gastric tissue and in plasma. Gastric juice pepsin was also studied.A significant increase of both pepsinogen and pepsin was found in animals treated with a dose of histamine (75 mg. per kg. of body weight). These results give further evidence that the zymogenic cells of gastric mucosa may be stimulated by histamine. The determination of pepsinogen in gastric tissue seems to permit a direct approach to the enzymatic function of zymogenic cells.

scholarly journals Determination of the Nucleotide Sequence ofBombyx mori Cytoplasmic Polyhedrosis Virus Segment 9 and Its Expression in BmN4 Cells

1998 ◽  
Vol 72 (7) ◽  
pp. 5762-5768 ◽  
Author(s):  
Kyoji Hagiwara ◽  
Masahiro Tomita ◽  
Kenta Nakai ◽  
Jun Kobayashi ◽  
Shigetoshi Miyajima ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Immunoblot Analysis ◽  
Homology Search ◽  
Cytoplasmic Polyhedrosis Virus ◽  
Reading Frame ◽  
Baculovirus Expression ◽  
Human Rotavirus ◽  
Polyhedrosis Virus

ABSTRACT Cloning and sequencing of segment 9 of Bombyx moricytoplasmic polyhedrosis virus (BmCPV) strains H and I were performed. The segment consisted of 1,186 bp harboring 5′ and 3′ noncoding regions and an open reading frame from positions 75 to 1037, encoding a protein with 320 amino acids, termed NS5. Comparison of the nucleotide sequences of NS5 for the two strains indicated 37 point differences resulting in only six amino acid replacements. Homology search showed that NS5 has localized similarities to human poliovirus RNA-dependent RNA polymerase and human rotavirus NS26. By Western blot analysis, NS5 was found in BmCPV-infected midgut cells, but not in polyhedra or virus virions, and was mainly detectable in the nucleus in BmCPV-infected BmN4 cells. Immunoblot analysis with anti-NS5 and antipolyhedrin antibodies displayed marked differences in the period of expression of NS5 and polyhedrin: the polyhedrin molecule was first detected 2 or 3 days after infection with BmCPV, whereas the expression of NS5 was initiated within a few hours. In addition, the level of polyhedrin increased as the infection developed, whereas the amount of NS5 remained essentially constant. When segment 9 was expressed with a baculovirus expression system, the resulting NS5 protein possessed the ability to bind to the double-stranded RNA genome. These results suggest that NS5 is expressed in early stages of infection and contributes to regulation of genomic RNA function.

scholarly journals Purification and characterization of embryo-specific soy urease (Glycine max) and its antifungal potential against Paracoccidioides brasiliensis

2021 ◽  
Vol 46 (1SI) ◽  
pp. 41-52
Author(s):  
Elisângela Andrade Ângelo ◽  
Tainá Michelle Da Cruz ◽  
José Renato Pattaro Júnior ◽  
Daniele Maria Zanzarin ◽  
Franciele Abigail Vilugron Rodrigues ◽  
...  
Keyword(s):  
Glycine Max ◽  
Biochemical Characterization ◽  
Paracoccidioides Brasiliensis ◽  
Maximum Velocity ◽  
Enzymatic Function ◽  
Potential Applications ◽  
First Time

Ureases are amidohydrolases that catalyze the hydrolysis of urea to ammonia and carbamate. In addition to the enzymatic function, ureases have fungitoxic and insecticidal function, which are independent of their catalytic activity. Soy (Glycine max) has two main urease isoforms: ubiquitous and embryo-specific, the latter is present in beans. In view of the potential applications of ureases, this work aimed to extract, purify, characterize the structure, activity and fungitoxic activity of soy urease against Paracoccidioides brasiliensis. The biochemical characterization was performed, in terms of optimal pH and temperature, as well as the determination of the Michaelis�Menten constant (KM) and maximum velocity (Vmax). The protein sequence was identified by mass spectrometry and used in computational modeling of the biological structure. The optimum pH and temperature of the enzyme were 6.5 and 65 �C, respectively, KM 526 mmol L-1 and Vmax 7.4 mmol L-1NH3׵gurease-1�s-1 and biological unity as a trimer. The antifungal activity assays (in vitro) were promising, showing a fungicidal profile of the urease, with a minimum inhibitory concentration of 10 �g�mL-1. This work demonstrated, for the first time, the fungitoxic activity of embryo-specific soy urease against the Pb18 strain of P. brasiliensis.

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