scholarly journals Mutations in Rotavirus Nonstructural Glycoprotein NSP4 Are Associated with Altered Virus Virulence

1998 ◽  
Vol 72 (5) ◽  
pp. 3666-3672 ◽  
Author(s):  
Mingdong Zhang ◽  
Carl Q.-Y. Zeng ◽  
Yanjie Dong ◽  
Judith M. Ball ◽  
Linda J. Saif ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Structural Changes ◽  
Biological Activities ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Nonstructural Proteins ◽  
Neonatal Mice ◽  
Life Threatening ◽  
Cloned Gene

ABSTRACT Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in children and animals. One of the nonstructural proteins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticulum-specific glycoprotein. Recently, our laboratory has shown that NSP4 causes diarrhea in 6- to 10-day-old mice by functioning as an enterotoxin. To confirm the role of NSP4 in rotavirus pathogenesis, we sequenced gene 10 from two pairs of virulent and attenuated porcine rotaviruses, the OSU and Gottfried strains. Comparisons of the NSP4 sequences from these two pairs of rotaviruses suggested that structural changes between amino acids (aa) 131 and 140 are important in pathogenesis. We next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression system and compared the biological activities of the purified proteins. NSP4 from OSU-v virus increased intracellular calcium levels over 10-fold in intestinal cells when added exogenously and 6-fold in insect cells when expressed endogenously, whereas NSP4 from OSU-a virus had little effect. NSP4 from OSU-v caused diarrhea in 13 of 23 neonatal mice, while NSP4 from OSU-a caused disease in only 4 of 25 mice (P < 0.01). These results suggest that avirulence is associated with mutations in NSP4. Results from site-directed mutational analyses showed that mutated OSU-v NSP4 with deletion or substitutions in the region of aa 131 to 140 lost its ability to increase intracellular calcium levels and to induce diarrhea in neonatal mice, confirming the importance of amino acid changes from OSU-v NSP4 to OSU-a NSP4 in the alteration of virus virulence.

scholarly journals Genetic basis and molecular biology of cardiac arrhythmias in cardiomyopathies

2020 ◽  
Vol 116 (9) ◽  
pp. 1600-1619 ◽  
Author(s):  
Ali J Marian ◽  
Babken Asatryan ◽  
Xander H T Wehrens
Keyword(s):  
Molecular Biology ◽  
Cardiac Arrhythmias ◽  
Genetic Basis ◽  
Structural Changes ◽  
Causal Role ◽  
Ion Currents ◽  
Pathogenic Variants ◽  
Life Threatening ◽  
Shared Genetic

Abstract Cardiac arrhythmias are common, often the first, and sometimes the life-threatening manifestations of hereditary cardiomyopathies. Pathogenic variants in several genes known to cause hereditary cardiac arrhythmias have also been identified in the sporadic cases and small families with cardiomyopathies. These findings suggest a shared genetic aetiology of a subset of hereditary cardiomyopathies and cardiac arrhythmias. The concept of a shared genetic aetiology is in accord with the complex and exquisite interplays that exist between the ion currents and cardiac mechanical function. However, neither the causal role of cardiac arrhythmias genes in cardiomyopathies is well established nor the causal role of cardiomyopathy genes in arrhythmias. On the contrary, secondary changes in ion currents, such as post-translational modifications, are common and contributors to the pathogenesis of arrhythmias in cardiomyopathies through altering biophysical and functional properties of the ion channels. Moreover, structural changes, such as cardiac hypertrophy, dilatation, and fibrosis provide a pro-arrhythmic substrate in hereditary cardiomyopathies. Genetic basis and molecular biology of cardiac arrhythmias in hereditary cardiomyopathies are discussed.

scholarly journals Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system

1999 ◽  
Vol 342 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Corné H. W. KLAASSEN ◽  
Petra H. M. BOVEE-GEURTS ◽  
Godelieve L. J. DECALUWÉ ◽  
Willem J. DEGRIP
Keyword(s):  
Membrane Proteins ◽  
Large Scale ◽  
Structural Changes ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Scale Production ◽  
Protein Activation ◽  
Bovine Rhodopsin ◽  
Lipid Environment

Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni2+-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

Preparation of Recombinant Human GDNF by Baculovirus Expression System and Analysis of Its Biological Activities

2000 ◽  
Vol 273 (3) ◽  
pp. 902-906 ◽  
Author(s):  
Zhe-Yu Chen ◽  
Jian-Xin Sun ◽  
Jian-Hong Li ◽  
Cheng He ◽  
Chang-Lin Lu ◽  
...  
Keyword(s):  
Biological Activities ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Baculovirus Expression

scholarly journals Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System

Insects ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 517
Author(s):  
Takeru Ebihara ◽  
Jian Xu ◽  
Yoshino Tonooka ◽  
Takumi Nagasato ◽  
Kohei Kakino ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Biological Activities ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Silkworm Larvae ◽  
Baculovirus Expression ◽  
Factor Α ◽  
Necrosis Factor

The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.

Isoform specificity of Na-K-ATPase-mediated ouabain signaling

2008 ◽  
Vol 294 (4) ◽  
pp. F859-F866 ◽  
Author(s):  
Sandrine V. Pierre ◽  
Yoann Sottejeau ◽  
Jean-Michel Gourbeau ◽  
Gladis Sánchez ◽  
Amjad Shidyak ◽  
...  
Keyword(s):  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Critical Event ◽  
Kinase Activation ◽  
Baculovirus Expression ◽  
Erk Phosphorylation ◽  
A Cell ◽  
Isoform Specificity

The ion transporter Na-K-ATPase functions as a cell signal transducer that mediates ouabain-induced activation of protein kinases, such as ERK. While Na-K-ATPase composed of the α1-polypeptide is involved in cell signaling, the role of other α-isoforms (α2, α3, and α4) in transmitting ouabain effects is unknown. We have explored this using baculovirus-directed expression of Na-K-ATPase polypeptides in insect cells and ERK phosphorylation as an indicator of ouabain-induced signaling. Ouabain addition to Sf-9 cells coexpressing Na-K-ATPase α1- and β1-isoforms stimulated ERK phosphorylation. In contrast, expression of the α1- and β1-polypeptides alone resulted in no effect, indicating that the αβ-complex is necessary for Na-K-ATPase signaling. Moreover, the ouabain effect was sensitive to genistein, suggesting that Na-K-ATPase-mediated tyrosine kinase activation is a critical event in the intracellular cascade leading to ERK phosphorylation. In addition, the Na-K-ATPases α3β1- and α4β1-isozymes, but not α2β1, responded to ouabain treatment. In agreement with the differences in ouabain affinity of the α-polypeptides, α1β1 required 100- to 1,000-fold more ouabain to signal than did α4β1 and α3β1, respectively. These results confirm the role of the Na-K-ATPase in ouabain signal transduction, show that there are important isoform-specific differences in Na-K-ATPase signaling, and demonstrate the suitability of the baculovirus expression system for studying Na-K-ATPase-mediated ouabain effects.

scholarly journals Psoralen Derivatives: Recent Advances of Synthetic Strategy and Pharmacological Properties

2020 ◽  
Vol 19 (3) ◽  
pp. 222-239 ◽  
Author(s):  
Joazaizulfazli Jamalis ◽  
Faten Syahira Mohamed Yusof ◽  
Subhash Chander ◽  
Roswanira Abd. Wahab ◽  
Deepak P. Bhagwat ◽  
...  
Keyword(s):  
Structural Changes ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Ring System ◽  
Related Disorder ◽  
Antifungal Effect ◽  
Synthetic Strategy ◽  
Tricyclic Compounds ◽  
Psoralen Derivatives

Psoralen or furocoumarin is a linear three ring heterocyclic compound. Psoralens are planar, tricyclic compounds, consisting of a furan ring fused to a coumarin moiety. Psoralen has been known for a wide spectrum of biological activities, spanning from cytotoxic, photosensitizing, insecticidal, antibacterial to antifungal effect. Thus, several structural changes were introduced to explore the role of specific positions with respect to the biological activity. Convenient approaches utilized for the synthesis of psoralen skeleton can be categorized into two parts: (i) the preparation of the tricyclic ring system from resorcinol, (ii) the exocyclic modification of the intact ring system. Furthermore, although psoralens have been used in diverse ways, we mainly focus in this work on their clinical utility for the treatment of psioraisis, vitiligo and skin-related disorder.

The C-terminal tail domain of neurofilament protein-H (NF-H) forms the crossbridges and regulates neurofilament bundle formation

2000 ◽  
Vol 113 (21) ◽  
pp. 3861-3869 ◽  
Author(s):  
J. Chen ◽  
T. Nakata ◽  
Z. Zhang ◽  
N. Hirokawa
Keyword(s):  
Network Formation ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Sf9 Cells ◽  
Tail Domain ◽  
Neurofilament Protein ◽  
Baculovirus Expression ◽  
10 Nm Filaments ◽  
Parallel Arrays

In order to study the role of NF-H in a neurofilament network formation in neurons, we coexpressed NF-H with neurofilament protein-L (NF-L) in Sf9 cells using the baculovirus expression system. Electron microscopy observations revealed that parallel arrays of 10 nm filaments with frequent crossbridges between adjacent filaments were formed in the cytoplasm of Sf9 cells infected with the recombinant virus that co-expressed NF-L and NF-H. To explore the function of the C-terminal tail domain of NF-H, various deletion mutants lacking portions of the tail domain were constructed, and each of them was coexpressed with NF-L. The results show that the tail domain of NF-H is a structural component of crossbridges and is involved in parallel bundle formation of neurofilaments, as core filaments of the axon. The last 191 amino acids of the C-terminal tail domain of NF-H play a key role in crossbridge formation.

scholarly journals Rotavirus VP6 as an Adjuvant for Bivalent Norovirus Vaccine Produced in Nicotiana benthamiana

Pharmaceutics ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 229 ◽  
Author(s):  
Maria Malm ◽  
André Diessner ◽  
Kirsi Tamminen ◽  
Markus Liebscher ◽  
Timo Vesikari ◽  
...  
Keyword(s):  
Immune Responses ◽  
Nicotiana Benthamiana ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Vaccine Antigen ◽  
Adjuvant Effect ◽  
Efficient Uptake ◽  
Rotavirus Vp6

Rotaviruses (RVs) and noroviruses (NoVs) are major causes of childhood acute gastroenteritis. During development of a combination vaccine based on NoV virus-like particles (VLP) and RV VP6 produced in baculovirus expression system in insect cells, a dual role of VP6 as a vaccine antigen and an adjuvant for NoV-specific immune responses was discovered. Here the VP6 adjuvant effect on bivalent GI.4 and GII.4-2006a NoV VLPs produced in Nicotiana benthamiana was investigated. BALB/c mice were immunized intradermally with suboptimal (0.3 µg) dose of each NoV VLP alone or combined with 10 µg of VP6, or equal doses of NoV VLPs and VP6 (1 µg/antigen). NoV-specific serum IgG antibodies and their blocking activity were analyzed using vaccine-homologous and heterologous NoV VLPs. Immunization with 0.3 µg NoV VLPs alone was insufficient to induce NoV-specific immune responses, but with co-administration of 10 µg of VP6, antibodies against vaccine-derived and heterologous NoV genotypes were generated. Furthermore, corresponding adjuvant effect of VP6 was observed with 1 µg dose. Efficient uptake and presentation of VP6 by dendritic cells was demonstrated in vitro. These results show that adjuvant effect of VP6 on bivalent NoV VLP vaccine is independent of the cell source used for vaccine production.

scholarly journals Point mutations in bovine opsin can be classified in four groups with respect to their effect on the biosynthetic pathway of opsin

1996 ◽  
Vol 320 (3) ◽  
pp. 807-815 ◽  
Author(s):  
Godelieve L. J. DeCALUWÉ ◽  
Willem J. DeGRIP
Keyword(s):  
Structural Changes ◽  
Expression System ◽  
Point Mutations ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Heterogeneous Distribution ◽  
Functional Consequences ◽  
Transfer Signal ◽  
Translational Process

Expression in vitro with the recombinant baculovirus expression system showed correct biosynthesis and post-translational processing of ‘wild-type’ bovine opsin with regard to translocation, glycosylation, palmitoylation and targeting. However, several of these processes were severely affected by point mutations. From the overall results of 16 mutants reported here, four groups were distinguished. One group significantly affected neither biosynthesis nor folding of opsin (D83N, P291A, A299C-V300A-P303G). A second group produced a truncated protein (R69H, Y301F), suggesting that these positions are essential for a correct translational process. A third group affected membrane translocation as well as glycosylation, which can be interpreted as interference with the function of a transfer signal. Substitutions at positions Glu-113, Glu-122, Glu-134, Arg-135 and Lys-248 belong to this category. A fourth group induced structural changes in the protein that led to heterogeneous distribution in the plasma membrane (E113Q/D, W265F, Y268S). Taking any functional consequences of these mutations into consideration, it seems that point mutations can have mosaic effects and therefore should be examined at several levels (folding, targeting, functional parameters).

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