KCNQ2-Associated Neonatal Epilepsy: Phenotype Might Correlate With Genotype

We analyzed the KCNQ2 wild-type gene and 3 mutations to highlight the important association between the KCNQ2 phenotype and genotype. The clinical phenotypes of 3 mutations (p.E515D, p.V543 M, and p.R213Q) were compared. KCNQ2, wild-type, and mutant KCNQ2 alleles were transfected into HEK293 cells before whole-cell patch-clamp analysis. Neurodevelopmental outcomes were worst in patients with the p.R213Q mutation, better in patients with the p.E515D mutation, and best in patients with the novel p.V543 M mutation. The currents in p.E515D and in p.V543 M were significantly lower than in the wild type in homomeric and heteromeric transfected HEK293 cells ( P < .05). The opening threshold shifted to values that were more positive, and the maximal current induced by strong depolarization was higher in cells with the p.E515D and p.R213Q mutations. We provide evidence that genotype is involved in determining clinical phenotype, including the seizure frequency and outcome.

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Plasmid DNA Production in Proteome-Reduced Escherichia coli

Microorganisms ◽

10.3390/microorganisms8091444 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1444

Author(s):

Mitzi de la Cruz ◽

Elisa A. Ramírez ◽

Juan-Carlos Sigala ◽

José Utrilla ◽

Alvaro R. Lara

Keyword(s):

Escherichia Coli ◽

Plasmid Dna ◽

Type Strain ◽

Wild Type Strain ◽

The Novel ◽

Wild Type ◽

Batch Mode ◽

Cell Factories ◽

Starting Point ◽

In The Wild

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

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Genetic and demographic effects of single wild-type immigrants on a mutant population of Tribolium castaneum

Canadian Journal of Genetics and Cytology ◽

10.1139/g86-139 ◽

1986 ◽

Vol 28 (6) ◽

pp. 1003-1008 ◽

Cited By ~ 1

Author(s):

Michael A. Delgado ◽

DuWayne C. Englert

Keyword(s):

Tribolium Castaneum ◽

Wild Type ◽

Gene Frequencies ◽

Treatment Groups ◽

Female Immigrant ◽

Rate Of Increase ◽

Wild Type Male ◽

Wild Type Female ◽

In The Wild ◽

Type Gene

The effects of single wild-type immigrants on populations of Tribolium castaneum initially homozygous for the antennapedia (ap) allele were examined in reference to gene frequencies and age structures. One population received a wild-type male, another received a wild-type female, and the control population received no wild-type immigrant. The rate of increase in the wild-type gene frequency was significantly higher in the female immigrant population. Rapid increase in heterozygosity for this population resulted in a higher average number of adults than for the other two treatment groups. No significant differences in the numbers of larvae and pupae were observed. Results indicated increased larval survivability to be the major factor in establishment of the wild-type gene and the sex of the immigrant in the rate of increase.Key words: Tribolium, population, selection, immigration, antennapedia.

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NsdC and NsdD Affect Aspergillus flavus Morphogenesis and Aflatoxin Production

Eukaryotic Cell ◽

10.1128/ec.00069-12 ◽

2012 ◽

Vol 11 (9) ◽

pp. 1104-1111 ◽

Cited By ~ 66

Author(s):

Jeffrey W. Cary ◽

Pamela Y. Harris-Coward ◽

Kenneth C. Ehrlich ◽

Brian M. Mack ◽

Shubha P. Kale ◽

...

Keyword(s):

Aspergillus Flavus ◽

Strong Association ◽

Aflatoxin Production ◽

Expression Data ◽

Aflatoxin Biosynthesis ◽

Wild Type ◽

Content Type ◽

Conidium Formation ◽

In The Wild ◽

Type Gene

ABSTRACT The transcription factors NsdC and NsdD are required for sexual development in Aspergillus nidulans . We now show these proteins also play a role in asexual development in the agriculturally important aflatoxin (AF)-producing fungus Aspergillus flavus . We found that both NsdC and NsdD are required for production of asexual sclerotia, normal aflatoxin biosynthesis, and conidiophore development. Conidiophores in nsdC and nsdD deletion mutants had shortened stipes and altered conidial heads compared to those of wild-type A. flavus . Our results suggest that NsdC and NsdD regulate transcription of genes required for early processes in conidiophore development preceding conidium formation. As the cultures aged, the Δ nsdC and Δ nsdD mutants produced a dark pigment that was not observed in the wild type. Gene expression data showed that although AflR is expressed at normal levels, a number of aflatoxin biosynthesis genes are expressed at reduced levels in both nsd mutants. Expression of aflD , aflM , and aflP was greatly reduced in nsdC mutants, and neither aflatoxin nor the proteins for these genes could be detected. Our results support previous studies showing that there is a strong association between conidiophore and sclerotium development and aflatoxin production in A. flavus.

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Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

Journal of Bacteriology ◽

10.1128/jb.183.2.528-535.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 528-535 ◽

Cited By ~ 16

Author(s):

Hsien-Ming Lee ◽

Shiaw-Wei Tyan ◽

Wei-Ming Leu ◽

Ling-Yun Chen ◽

David Chanhen Chen ◽

...

Keyword(s):

Gene Cluster ◽

Mutant Strain ◽

Type Strain ◽

Xanthomonas Campestris ◽

Wild Type Strain ◽

Type Ii ◽

Wild Type ◽

In The Wild ◽

Steady State Levels ◽

Type Gene

ABSTRACT The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestrispv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from thexpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into thexpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsNgene or by introducing extra copies of wild-type xpsL orxpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL orxpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with thexpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of thexpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

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Gentamicin Inhibits Ca2+ Channel TPRV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway

Journal of the American Society of Nephrology ◽

10.1681/asn.2021030392 ◽

2022 ◽

pp. ASN.2021030392

Author(s):

Wouter van Megen ◽

Megan Beggs ◽

Sung-Wan An ◽

Patrícia Ferreira ◽

Justin Lee ◽

...

Keyword(s):

Patch Clamp ◽

Hek293 Cells ◽

Luciferase Reporter ◽

Thick Ascending Limb ◽

Wild Type ◽

Distal Nephron ◽

Proximal Tubular ◽

Claudin 14

Background Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal calcium wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. Methods We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase-reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5), as determined by patch-clamp in HEK293 cells. Results Gentamicin increased urinary Ca2+ excretion in wild-type mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/-mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and that chronic gentamicin administration downregulated distal nephron Ca2+ transporters. Conclusions Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into calcium wasting in patients treated with gentamicin.

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Abstract 16067: Phosphorylation of Lamin-A/C Modulates Peak Sodium Current in a Patient With Cardiac Conduction Disease

Circulation ◽

10.1161/circ.132.suppl_3.16067 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Michael Olaopa ◽

Katherine Spoonamore ◽

Peng-Sheng Chen ◽

Tomohiko Ai ◽

Matteo Vatta

Keyword(s):

Patch Clamp ◽

Western Blot ◽

Potential Role ◽

Sodium Current ◽

Hek293 Cells ◽

Cardiac Conduction ◽

Lamin A ◽

Wild Type ◽

Potential Therapeutic Target ◽

Constitutive Phosphorylation

Introduction: Deleterious variants in the LMNA gene, which encodes for Lamin-A/C, have been shown to be associated with cardiac conduction diseases (CCDs) and cardiomyopathy. We previously reported that two LMNA variants found in patients presenting with CCDs can prevent the LMNA -mediated activation of peak sodium current (I Na ). In addition, western blot analyses showed phosphorylation of Lamin at serine 22 (S22) was reduced in HEK293 cells expressing either variant. Thus, we sought to investigate what potential role S22 plays on modulating Nav1.5 function. In this study, we focused our analyses on one of the two variants, which is c.1634G>A (p.R545H) in LMNA . We tested the hypothesis that S22 affects peak I Na . Methods and Results: We generated two plasmids that mimic unphosphorylated wild type LMNA ( p.S22A ) and constitutively phosphorylated mutant LMNA ( p.S22D-p.R545H ). We measured and compared peak I Na using patch clamp techniques in HEK293 cells transfected with each LMNA and SCN5A plasmids. Our study showed that p.S22A significantly prevented the LMNA -mediated activation of peak I Na by 63% compared to wild type ( p.S22A : -121 ±11 pA/pF, N =10 vs. wild type LMNA : -340 ±45 pA/pF, N=10, Mann-Whitney U =2, p<0.0005 one-tailed). On the contrary, p.S22D-p.R545H partially restored the reduced peak I Na by 44% ( p.S22D-p.R545H : -137 ±43 pA/pF, N=7 vs. p.R545H : -95 ±15 pA/pF, N=10, Mann-Whitney U =27, p<0.1 one-tailed). Conclusions: Our data indicate the loss of phosphorylation at S22 significantly reduces the LMNA -mediated activation of peak I Na . In the disease state, constitutive phosphorylation partially restores, yet fails to normalize peak I Na . Our observation suggests that S22 could represent a potential therapeutic target in patients with LMNA -mediated CCD. To our knowledge, this is the first study to functionally link phosphorylation of Lamin-A/C at S22 to peak I Na levels and Nav1.5 function.

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Microarray analysis of Arabidopsis WRKY33 mutants in response to the necrotrophic fungus Botrytis cinerea

10.31226/osf.io/rw2av ◽

2018 ◽

Author(s):

Synan AbuQamar ◽

Khaled Moustafa

Keyword(s):

Transcription Factor ◽

Botrytis Cinerea ◽

Defense Responses ◽

Plant Responses ◽

Wild Type Plant ◽

The Novel ◽

Wild Type ◽

Genes Encoding ◽

In The Wild ◽

Molecular Components

The WRKY33 transcription factor was reported for resistance to the necrotrophic fungus Botrytis cinerea. Using microarray-based analysis, we compared Arabidopsis WRKY33 overexpressing lines and wrky33 mutant that showed altered susceptibility to B. cinerea with their corresponding wild-type plants. In the wild-type, about 1660 genes (7% of the transcriptome) were induced and 1054 genes (5% of the transcriptome) were repressed at least twofold at early stages of inoculation with B. cinerea, confirming previous data of the contribution of these genes in B. cinerea resistance. In Arabidopsis wild-type plant infected with B. cinerea, the expressions of the differentially expressed genes encoding for proteins and metabolites involved in pathogen defense and non-defense responses, seem to be dependent on a functional WRKY33 gene. The expression profile of 12-oxo-phytodienoic acid- and phytoprostane A1-treated Arabidopsis plants in response to B. cinerea revealed that cyclopentenones can also modulate WRKY33 regulation upon inoculation with B. cinerea. These results support the role of electrophilic oxylipins in mediating plant responses to B. cinerea infection through the TGA transcription factor. Future directions toward the identification of the molecular components in cyclopentenone signaling will elucidate the novel oxylipin signal transduction pathways in plant defense.

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Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function

International Journal of Molecular Sciences ◽

10.3390/ijms222413652 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13652

Author(s):

Eleonora Persoons ◽

Sara Kerselaers ◽

Thomas Voets ◽

Joris Vriens ◽

Katharina Held

Keyword(s):

Calcium Imaging ◽

Synergistic Action ◽

Sex Hormone ◽

Wild Type ◽

Neurodevelopmental Disease ◽

Whole Cell Patch Clamp ◽

Channel Opening ◽

In The Wild ◽

Increased Sensitivity ◽

Increased Activity

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

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Neonates with the KCNQ2 Y755C Variants: Not Associated with Neonatal Epileptic Encephalopathy

Advances in Neurology and Neuroscience ◽

10.33140/an.04.03.02 ◽

2021 ◽

Vol 4 (3) ◽

Keyword(s):

Electrical Current ◽

Epileptic Encephalopathy ◽

Hek293 Cells ◽

Functional Study ◽

Neonatal Seizure ◽

Pediatric Epilepsy ◽

Wild Type ◽

Functional Changes ◽

Investigation Methods ◽

In The Wild

Background: Pediatric epilepsy caused by a KCNQ2 gene mutation usually manifests the phenotype of a neonatal seizure. KCNQ2 encephalopathy in newborns continues to be reported on. Objectives: The exact mechanism and phenotype of the KCNQ2 mutation still require investigation. Methods: One hundred twenty-one patients with childhood epilepsy without an identified cause underwent KCNQ2 sequencing. KCNQ2 mutation variants were transfected into human embryonic kidney 293 (HEK293) cells to investigate functional changes. Results: Two patients with the c.2264G>G/A (p.Y755C) variant had neonatal epileptic encephalopathy: one had electroencephalography (EEG) burst suppression and the other had multiple focal spikes. However, the mutation was not found in the 80 healthy adult claiming without ever seizures before. A functional study showed that p.Y755C currents were not different from those in the wild-type and from those in the benign (p.N780T) polymorphism in homomeric and heteromeric (wild-type KCNQ2: mutant = 1:1) transfected HEK293 cells. Electrical current differences between HEK293 cells with wildtype mutations and cells transfected with the wild-type KCNQ2, KCNQ3, and p.Y755C mutations in a 1:2:1 ratio were not significant. Their seizures remitted after they turned 1 year old. Conclusion: We suggest that patients with the KCNQ2 p.Y755C mutations are not associated with neonatal epileptic encephalopathy

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