scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

scholarly journals The Role of Apoptosis in the Regulation of Hematopoietic Stem Cells

2000 ◽  
Vol 191 (2) ◽  
pp. 253-264 ◽  
Author(s):  
Jos Domen ◽  
Samuel H. Cheshier ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Direct Role ◽  
Hematopoietic Stem

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2–transgenic mice have increased numbers of HSC in bone marrow (2.4× wild type), but fewer of these cells are in the S/G2/M phases of the cell cycle (0.6× wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

scholarly journals The side population phenomenon enriches for designated adrenocortical progenitor cells in mice

2012 ◽  
Vol 215 (3) ◽  
pp. 383-391
Author(s):  
Urs Lichtenauer ◽  
Igor Shapiro ◽  
Simone Sackmann ◽  
Jacques Drouin ◽  
Jürgen Scheele ◽  
...  
Keyword(s):  
Stem Cell ◽  
Side Population ◽  
Indirect Evidence ◽  
Stem Cell Marker ◽  
Wild Type ◽  
Acth Treatment ◽  
Cell Arrest

Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01–0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified.In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished inPbx1haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficientTpit−/−mice was significantly wider compared with wild-type adrenals (Tpit−/−259±10.7 vsTpit+/−100±12.3%;P<0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit−/−0.45±0.16 vsTpit+/−0.13±0.04%;P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%;P=0.33 and 0.09%), providing indirect evidence for a stem cell ‘arrest’ inTpit−/−mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.

Nanostructured Lipid Carriers (NLCs) for drug delivery: Role of Liquid lipid (oil)

2020 ◽  
Vol 17 ◽  
Author(s):  
Anisha D’Souza ◽  
Ranjita Shegokar
Keyword(s):  
Drug Delivery ◽  
Essential Oils ◽  
Drug Carrier ◽  
Drug Loading ◽  
Performance Criteria ◽  
Long Term Stability ◽  
Natural Oils ◽  
Optimal Drug

: In recent years, SLNs and NLCs are among the popular drug delivery systems studied for delivery of lipophilic drugs. Both systems have demonstrated several beneficial properties as an ideal drug-carrier, optimal drug-loading and good long-term stability. NLCs are getting popular due to their stability advantages and possibility to load various oil components either as an active or as a matrix. This review screens types of oils used till date in combination with solid lipid to form NLCs. These oils are broadly classified in two categories: Natural oils and Essential oils. NLCs offer range advantages in drug delivery due to the formation of imperfect matrix owing to the presence of oil. The type and percentage of oil used determines optimal drug loading and stability. Literature shows that variety of oils is used in NLCs mainly as matrix, which is from natural origin, triglycerides class. On the other hand, essential oils not only serve as a matrix but as an active. In short, oil is the key ingredient in formation of NLCs, hence needs to be selected wisely as per the performance criteria expected.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Suppressive Role of Bam32/DAPP1 in Chemokine-Induced Neutrophil Recruitment

2021 ◽  
Vol 22 (4) ◽  
pp. 1825
Author(s):  
Li Hao ◽  
Aaron J. Marshall ◽  
Lixin Liu
Keyword(s):  
Small Gtpases ◽  
Neutrophil Recruitment ◽  
Neutrophil Chemotaxis ◽  
Wild Type ◽  
Adaptor Molecule ◽  
Geranylgeranyltransferase I ◽  
And Migration ◽  
Rap1 Activation

Bam32 (B cell adaptor molecule of 32 kDa) functions in the immune responses of various leukocytes. However, the role of neutrophil Bam32 in inflammation is entirely unknown. Here, we determined the role of Bam32 in chemokine CXCL2-induced neutrophil chemotaxis in three mouse models of neutrophil recruitment. By using intravital microscopy in the mouse cremaster muscle, we found that transmigrated neutrophil number, neutrophil chemotaxis velocity, and total neutrophil chemotaxis distance were increased in Bam32−/− mice when compared with wild-type (WT) mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32−/− mice at 2 but not 4 h. The CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32−/− neutrophils in vitro were increased. We examined the activation of small GTPases Rac1, Rac2, and Rap1; the levels of phospho-Akt2 and total Akt2; and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 suppressed Rap1 activation without changing the activation of Rac1 and Rac2. The pharmacological inhibition of Rap1 by geranylgeranyltransferase I inhibitor (GGTI298) increased WT neutrophil chemotaxis. In addition, the deficiency of Bam32, as well as the inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. The inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32−/− mice. Together, our results reveal that Bam32 has a suppressive role in chemokine-induced neutrophil chemotaxis by regulating Rap1 activation and that this role of Bam32 in chemokine-induced neutrophil recruitment relies on the activation of PI3K effector Akt.

scholarly journals Glucose Transporter 8 (GLUT8) Regulates Enterocyte Fructose Transport and Global Mammalian Fructose Utilization

Endocrinology ◽  
2012 ◽  
Vol 153 (9) ◽  
pp. 4181-4191 ◽  
Author(s):  
Brian J. DeBosch ◽  
Maggie Chi ◽  
Kelle H. Moley
Keyword(s):  
Glucose Transporter ◽  
Serum Insulin ◽  
Density Lipoprotein ◽  
Wild Type ◽  
In Vivo Models ◽  
The Metabolic Syndrome ◽  
High Fructose

Enterocyte fructose absorption is a tightly regulated process that precedes the deleterious effects of excess dietary fructose in mammals. Glucose transporter (GLUT)8 is a glucose/fructose transporter previously shown to be expressed in murine intestine. The in vivo function of GLUT8, however, remains unclear. Here, we demonstrate enhanced fructose-induced fructose transport in both in vitro and in vivo models of enterocyte GLUT8 deficiency. Fructose exposure stimulated [14C]-fructose uptake and decreased GLUT8 protein abundance in Caco2 colonocytes, whereas direct short hairpin RNA-mediated GLUT8 knockdown also stimulated fructose uptake. To assess GLUT8 function in vivo, we generated GLUT8-deficient (GLUT8KO) mice. GLUT8KO mice exhibited significantly greater jejunal fructose uptake at baseline and after high-fructose diet (HFrD) feeding vs. wild-type mice. Strikingly, long-term HFrD feeding in GLUT8KO mice exacerbated fructose-induced increases in blood pressure, serum insulin, low-density lipoprotein and total cholesterol vs. wild-type controls. Enhanced fructose uptake paralleled with increased abundance of the fructose and glucose transporter, GLUT12, in HFrD-fed GLUT8KO mouse enterocytes and in Caco2 cultures exposed to high-fructose medium. We conclude that GLUT8 regulates enterocyte fructose transport by regulating GLUT12, and that disrupted GLUT8 function has deleterious long-term metabolic sequelae. GLUT8 may thus represent a modifiable target in the prevention and treatment of malnutrition or the metabolic syndrome.

Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury

2014 ◽  
Vol 307 (3) ◽  
pp. H337-H345 ◽  
Author(s):  
Lara Gotha ◽  
Sang Yup Lim ◽  
Azriel B. Osherov ◽  
Rafael Wolff ◽  
Beiping Qiang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Critical Role ◽  
Arterial Injury ◽  
Side Chains ◽  
Wild Type ◽  
Injury Response ◽  
Migratory Response

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2Δ3/Δ3 (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type ( P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB ( P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

scholarly journals GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

scholarly journals The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Paul White ◽  
Samuel F. Haysom ◽  
Matthew G. Iadanza ◽  
Anna J. Higgins ◽  
Jonathan M. Machin ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Antibody Fragment ◽  
Membrane Disruption ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Lipid Dynamics ◽  
Open Structures

AbstractThe folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

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