Disruption ofGja8(α8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation

Development ◽  
2002 ◽  
Vol 129 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Pei Rong ◽  
Xin Wang ◽  
Ingrid Niesman ◽  
Ying Wu ◽  
Lucio E. Benedetti ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Cell Communication ◽  
Cell Maturation ◽  
Wild Type ◽  
Lacz Reporter ◽  
Lacz Reporter Gene ◽  
Gap Junction Channels ◽  
Protein Levels ◽  
Lens Growth ◽  
And Control

The development of the vertebrate lens utilizes a sophisticated cell-cell communication network via gap junction channels, which are made up of at least three connexin isoforms, α8 (Cx50), α3 (Cx46) and α1 (Cx43), and which are encoded by three different genes. In a previous study, we reported that, with a disruption of Gja3 (α3 connexin), mice developed nuclear cataracts with a normal sized lens. We show that Gja8tm1 (α8–/–) mice develop microphthalmia with small lenses and nuclear cataracts, while the α8 heterozygous (+/–) mice have relatively normal eyes and lenses. A comparative study of these α3 and α8 knockout mice showed that the protein levels of both α3 and α8 were independently regulated and there was no compensation for either the α3 or α8 protein from the wild-type allele when the other allele was disrupted. More interestingly, western blotting data indicated that the presence of α8 in the lens nucleus is dependent on α3 connexin, but not vice versa. The staining of the knock-in lacZ reporter gene showed the promoter activity of α8 connexin is much higher than that of α3 connexin in embryonic lenses and in adult lens epithelium. More importantly, a delayed denucleation process was observed in the interior fibers of the α8–/– lenses. Therefore, α8 connexin is required for proper fiber cell maturation and control of lens size.

scholarly journals Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook ofSalmonella

1999 ◽  
Vol 181 (18) ◽  
pp. 5808-5813 ◽  
Author(s):  
Kazumasa Muramoto ◽  
Shigeru Makishima ◽  
Shin-Ichi Aizawa ◽  
Robert M. Macnab
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Basal Bodies ◽  
Wild Type ◽  
Hook Length ◽  
Capping Protein ◽  
Protein Levels ◽  
Junction Protein ◽  
And Control ◽  
Control Protein

ABSTRACT The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, afliC (flagellin) mutant, or a fliD(hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

scholarly journals 5 S rRNA is involved in fidelity of translational reading frame.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 95-105
Author(s):  
J D Dinman ◽  
R B Wickner
Keyword(s):  
Rdna Locus ◽  
Wild Type ◽  
Lacz Reporter ◽  
Lacz Expression ◽  
Lacz Reporter Gene ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Episomal Vector ◽  
Electrophoretic Mobilities ◽  
Insertion Mutations

Abstract Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frameshifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof- phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof- phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.

scholarly journals Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cynthia Shu ◽  
Ariana N. Kaxon-Rupp ◽  
Judd R. Collado ◽  
Robert Damoiseaux ◽  
Rachelle H. Crosbie
Keyword(s):  
Gene Expression ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Calcium Channel ◽  
Muscle Cell ◽  
High Throughput ◽  
Promoter Activity ◽  
Muscle Cells ◽  
Wild Type ◽  
Protein Levels

Abstract Background Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Methods Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. Results We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. Conclusions We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

Acute-phase responses in transgenic mice with CNS overexpression of IL-1 receptor antagonist

1999 ◽  
Vol 276 (3) ◽  
pp. R644-R651 ◽  
Author(s):  
Johan Lundkvist ◽  
Anna K. Sundgren-Andersson ◽  
Susanne Tingsborg ◽  
Pernilla Östlund ◽  
Catherine Engfors ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Receptor Antagonist ◽  
Interleukin 1 ◽  
Serum Levels ◽  
Type I ◽  
Wild Type ◽  
Specific Expression ◽  
Protein Levels ◽  
Transgenic Phenotype ◽  
And Control

The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1α and IL-1β by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to ∼50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1β (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1β (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 μg/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 μg/kg ip; 1 and 6 h after injection) IL-1β and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1β and IL-6 plasma levels.

scholarly journals Functions ofCandida albicanscell wall glycosidasesDfg5pandDcw1pin biofilm formation and HOG MAPK pathway

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5685 ◽  
Author(s):  
Ryan Mancuso ◽  
Jennifer Chinnici ◽  
Charlene Tsou ◽  
Sujay Busarajan ◽  
Raveena Munnangi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Osmotic Stress ◽  
Biofilm Formation ◽  
Imaging Analysis ◽  
Wild Type ◽  
Microscopy Imaging ◽  
Protein Levels ◽  
Hyphal Morphogenesis ◽  
Mutant Strains ◽  
And Control

BackgroundCandida albicansis a commensal fungus that inhabits the oral mucosal surface and causes oral and systemic candidiasis. Oral candidiasis most commonly occurs in patients with AIDS, denture wearers and newborn children. Systemic candidiasis occurs mainly in immunocompromised patients and patients admitted to hospitals for prolonged periods.C. albicanshomologous genes,DFG5andDCW1, encode for two closely related cell wall proteins with putative glycosyltransferase enzyme activity and C-terminal GPI-anchors. Past studies have shown that individualDFG5andDCW1mutations are viable but simultaneous deletion ofDFG5andDCW1inC. albicansresults in lethality. However, the exact functions of these cell wall based enzymes, which represent potential drug targets, are not understood.MethodsC. albicansDFG5/DCW1heterologous and conditional double mutant strains were assessed for growth and biofilm formation in comparison to wild type and parental strains. Cell wall and heat stress susceptibility of the mutant and control strains were assessed using agar spotting assays. Growth was assessed under normal and osmotic stress conditions along with light microscopy imaging. Biofilm dry weight and microscopic imaging analysis of biofilms was performed. Hypha formation in response to serum was analyzed using light microscopy imaging. Western blot analysis of mutant strains and control strains was performed to assess Hog1 basal levels and phosphorylation status.ResultsAnalysis of the heterologous mutants indicated that Dfg5p is more important for growth while Dcw1p appeared to play a role in cell wall integrity response. The conditional double mutant was observed to be less resistant to cell wall stress. However, growth of the mutants was similar under control and osmotic stress conditions. The mutants were also able to grow similar to wild type under heat stress. Biofilm formation was reduced in the mutants whereDFG5was deleted or suppressed. Hyphal morphogenesis was reduced although germ tube formation was observed in the biofilms of the mutant strains. Basal Hog1 protein levels were reduced or absent in theDFG5andDCW1mutants. However, osmotic stress was able to induce Hog1 protein levels comparable to wild type. Hog1 phosphorylation appeared to be slightly reduced although not significantly. In addition to biofilm assays, serum dose response imaging analysis indicated that hyphae formation inDFG5andDCW1mutants was defective.ConclusionsThese data indicate thatDFG5andDCW1are required for hyphal morphogenesis and biofilm formation inC. albicans. These functions may be regulated via basal Hog1 MAPK which is required for transcriptional regulation of chitin synthesis.

Conjugated Linoleic Acid Causes a Marked Increase in Liver α-Tocopherol and Liver α-Tocopherol Transfer Protein in C57BL/6 J Mice

2010 ◽  
Vol 80 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Pei-Min Chao ◽  
Wan-Hsuan Chen ◽  
Chun-Huei Liao ◽  
Huey-Mei Shaw
Keyword(s):  
Antioxidant Enzymes ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Thiobarbituric Acid ◽  
Control Group ◽  
Thiobarbituric Acid Reactive Substances ◽  
Control Groups ◽  
Protein Levels ◽  
Transfer Protein ◽  
And Control

Conjugated linoleic acid (CLA) is a collective term for the positional and geometric isomers of a conjugated diene of linoleic acid (C18:2, n-6). The aims of the present study were to evaluate whether levels of hepatic α-tocopherol, α-tocopherol transfer protein (α-TTP), and antioxidant enzymes in mice were affected by a CLA-supplemented diet. C57BL/6 J mice were divided into the CLA and control groups, which were fed, respectively, a 5 % fat diet with or without 1 g/100 g of CLA (1:1 mixture of cis-9, trans-11 and trans-10, cis-12) for four weeks. α-Tocopherol levels in plasma and liver were significantly higher in the CLA group than in the control group. Liver α-TTP levels were also significantly increased in the CLA group, the α-TTP/β-actin ratio being 2.5-fold higher than that in control mice (p<0.01). Thiobarbituric acid-reactive substances were significantly decreased in the CLA group (p<0.01). There were no significant differences between the two groups in levels of three antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and catalase). The accumulation of liver α-tocopherol seen with the CLA diet can be attributed to the antioxidant potential of CLA and the ability of α-TTP induction. The lack of changes in antioxidant enzyme protein levels and the reduced lipid peroxidation in the liver of CLA mice are due to α-tocopherol accumulation.

Pengaruh Lama Pengasinan Terhadap Kadar Protein Putih Telur Itik

2014 ◽  
Vol 1 (1) ◽  
pp. 36 ◽  
Author(s):  
Siti Fatimah ◽  
Muji Rahayu ◽  
Siti Aminah
Keyword(s):  
Protein Level ◽  
Egg White ◽  
Special Treatment ◽  
Egg White Protein ◽  
Protein Levels ◽  
Duck Egg ◽  
Egg Period ◽  
Graph Presentation ◽  
And Control ◽  
The Impact

Background : Egg  is one of the animal protein source, which has delicious taste, easy to digest and highly nutritious. Besides its affordable price, its supply availability is unquestionable as well. However, due to its short storability, it requires special treatment, such as preserving, to store it for long period. One way to preserve the egg is by pickling egg, which generally requires seven to ten days of marinating. During the process of marinating, there will be a visual change of egg white and yolk. Their structures  will be more solid (the occurrence of thickening process) because salinization will lead to protein denaturalization. Consequently, it has an influence as well towards the content of egg white protein of duck egg. This study is aimed to explore the impact of various time of pickling egg towards egg white protein of duck egg. Method  : The study where takes place in a laboratories, is a true experimental study for the reason that the researcher must provide intervention, hence all of potentially confounding variables are manageable. Samples that had been used in this study are duck eggs which were bought from North Brebes. This study is expected to generate data from four various time of pickling egg and control (no treatment). Since there are four samples, accordingly the number of data resulted are twenty. The resulted data will be descriptively presented in table, graph, presentation, and narration. Result  : Protein level examination within duck white egg shows changes  in protein levels that occurs in every variation of pickling egg time, where the average results of the assay of duck egg white protein is 14.94% without treatment (control), in five days of pickling time is 13.68%, in seven days of pickling time is 13.29%, in nine days of pickling time is 12.87% and eleven days of pickling time is 12.78%. Conclusion  : There is a significant impact among the period of pickling time to the protein level degradation of duck white egg. Keywords : Duck egg, period of pickling time, level protein of duck white egg.

UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6

2020 ◽  
Vol 20 (12) ◽  
pp. 1487-1496 ◽  
Author(s):  
Midori Murakami ◽  
Hiroto Izumi ◽  
Tomoko Kurita ◽  
Chiho Koi ◽  
Yasuo Morimoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Anticancer Agent ◽  
Cisplatin Resistance ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Molecular Target ◽  
Transcriptional Level ◽  
And Control ◽  
Resistant Cells

Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

scholarly journals Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lidan Liu ◽  
Chaim Z. Aron ◽  
Cullen M. Grable ◽  
Adrian Robles ◽  
Xiangli Liu ◽  
...  
Keyword(s):  
Proinflammatory Cytokine ◽  
Inflammatory Cytokine ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Mrna Levels ◽  
Wild Type ◽  
Cytokine Mrna ◽  
Protein Levels ◽  
Cytokine Levels

AbstractLevels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

scholarly journals Interactions of Drosophila Ultrabithorax Regulatory Regions With Native and Foreign Promoters

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Fernando Casares ◽  
Welcome Bender ◽  
John Merriam ◽  
Ernesto Sánchez-Herrero
Keyword(s):  
P Element ◽  
Lacz Gene ◽  
Bithorax Complex ◽  
Lacz Reporter ◽  
Normal Pattern ◽  
Lacz Reporter Gene ◽  
Regulatory Regions ◽  
Long Range Interactions ◽  
In Trans ◽  
Dependent Interaction

The Ultrabithorax (Ubx) gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Two P-element “enhancer traps” have been recovered within the locus that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lucZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome.

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