Characterization of a mutant R11H αB-crystallin associated with human inherited cataract

Abstract αB-Crystallin plays an important part in cataract development. A novel mutation (R11H) was previously detected by our group. In the present study, we set out to investigate the possible molecular mechanism by which the R11H mutation causes cataract. We found that the mutant αB-crystallin exhibits folding defects, decreased surface hydrophobicity and enhanced chaperone-like activity compared with the wild-type αB-crystallin. The mutant protein shows nearly the same molecular mass and thermal stability as the wild-type form. Transfection studies revealed that the R11H mutant was remarkably similar to the wild-type protein in its subcellular distribution, but has an abnormal ability to induce cell apoptosis. These results suggest that the changes in hydrophobic exposure and the abnormal ability to induce programmed cell death of the mutant protein are likely to be responsible for the onset of cataract.

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Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occuring [sic] mutants associated with hyperphenylalaninemia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.93.9.4519-b ◽

1996 ◽

Vol 93 (9) ◽

pp. 4519-4519

Keyword(s):

Wild Type ◽

Type Form ◽

Wild Type Form

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Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.26.12384 ◽

1995 ◽

Vol 92 (26) ◽

pp. 12384-12388 ◽

Cited By ~ 13

Author(s):

G. Johnen ◽

D. Kowlessur ◽

B. A. Citron ◽

S. Kaufman

Keyword(s):

Wild Type ◽

Naturally Occurring ◽

Type Form ◽

Wild Type Form

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Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-Crystallin on Chaperone Function and Anti-Apoptotic Activity

International Journal of Molecular Sciences ◽

10.3390/ijms221910771 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10771

Author(s):

Sundararajan Mahalingam ◽

Srabani Karmakar ◽

Puttur Santhoshkumar ◽

Krishna K. Sharma

Keyword(s):

Deletion Mutant ◽

Mutant Protein ◽

Wild Type ◽

Chaperone Function ◽

Type Protein ◽

Wild Type Protein ◽

Cytotoxicity Studies ◽

Αb Crystallin ◽

Apoptotic Activity ◽

Subunit Organization

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

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Synergism of imipenem with fosfomycin associated with the active cell wall recycling and heteroresistance in Acinetobacter calcoaceticus-baumannii complex

Scientific Reports ◽

10.1038/s41598-021-04303-7 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Uthaibhorn Singkham-in ◽

Tanittha Chatsuwan

Keyword(s):

Cell Wall ◽

De Novo ◽

Acinetobacter Calcoaceticus ◽

Active Cell ◽

Wild Type ◽

Cell Wall Metabolism ◽

Carbapenem Resistant ◽

Type Form ◽

Wild Type Form ◽

Cell Wall Recycling

AbstractThe carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complex has become an urgent threat worldwide. Here, we determined antibiotic combinations and the feasible synergistic mechanisms against three couples of ACB (A. baumannii (AB250 and A10), A. pittii (AP1 and AP23), and A. nosocomialis (AN4 and AN12)). Imipenem with fosfomycin, the most effective in the time-killing assay, exhibited synergism to all strains except AB250. MurA, a fosfomycin target encoding the first enzyme in the de novo cell wall synthesis, was observed with the wild-type form in all isolates. Fosfomycin did not upregulate murA, indicating the MurA-independent pathway (cell wall recycling) presenting in all strains. Fosfomycin more upregulated the recycling route in synergistic strain (A10) than non-synergistic strain (AB250). Imipenem in the combination dramatically downregulated the recycling route in A10 but not in AB250, demonstrating the additional effect of imipenem on the recycling route, possibly resulting in synergism by the agitation of cell wall metabolism. Moreover, heteroresistance to imipenem was observed in only AB250. Our results indicate that unexpected activity of imipenem on the active cell wall recycling concurrently with the presence of heteroresistance subpopulation to imipenem may lead to the synergism of imipenem and fosfomycin against the ACB isolates.

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Unusual Clinical Presentation and Possible Rescue of a Novel Claudin-16 Mutation

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2006-0200 ◽

2006 ◽

Vol 91 (8) ◽

pp. 3076-3079 ◽

Cited By ~ 31

Author(s):

Dominik Müller ◽

P. Jaya Kausalya ◽

Detlef Bockenhauer ◽

Julia Thumfart ◽

Iwan C. Meij ◽

...

Keyword(s):

Mdck Cells ◽

Novel Mutation ◽

Surface Expression ◽

Blood Analysis ◽

Mutant Protein ◽

Molecular Defects ◽

Familial Hypomagnesemia ◽

Novel Therapeutic Strategies ◽

Urine And Serum

Abstract Context: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is caused by a dysfunction of Claudin-16 (CLDN16) and characterized by renal wasting of Mg2+ and Ca2+. Objective: The objectives of this study were to study the clinical parameters in suspected FHHNC patients, identify mutations in the CLDN16 gene, and analyze molecular defects associated with the mutant protein. Design, Setting, and Participants: CLDN16 genes from two siblings diagnosed with FHHNC were sequenced. Expression and characterization of the mutant protein in renal MDCK cells were studied. Outcome Measures: Standard urine and serum parameters to diagnose FHHNC were determined. Mutations in the CLDN16 gene were identified. The subcellular distribution of the mutant protein was analyzed by immunofluorescence microscopy. Results: Urine and blood analysis showed signs typical for FHHNC. One patient, in addition, presented with hypocalcemic tetany, a phenomenon so far not described for FHHNC. Both siblings carry a novel mutation in CLDN16, Y207X. The review of medical records showed that hypocalcemia is not uncommon in the early childhood of FHHNC patients. Expressed in MDCK cells, the Y207X mutant is not detected at tight junctions but instead is found in lysosomes and, to a lesser extent, the endoplasmic reticulum. Surface expression can be rescued by inhibiting clathrin-mediated internalization. Conclusions: We propose that mutations in CLDN16 are considered in childhood hypocalcemia. CLDN16 Y207X is transiently delivered to the plasma membrane but not retained and is rapidly retrieved by internalization. Inhibitors of endocytosis may provide novel therapeutic strategies.

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Inactivation of the selB Gene in Methanococcus maripaludis: Effect on Synthesis of Selenoproteins and Their Sulfur-Containing Homologs

Journal of Bacteriology ◽

10.1128/jb.185.1.107-114.2003 ◽

2003 ◽

Vol 185 (1) ◽

pp. 107-114 ◽

Cited By ~ 42

Author(s):

Michael Rother ◽

Isabella Mathes ◽

Friedrich Lottspeich ◽

August Böck

Keyword(s):

Gene Product ◽

Formate Dehydrogenase ◽

Selenium Supplementation ◽

Wild Type ◽

Methanococcus Maripaludis ◽

Translation Factor ◽

Type Form ◽

And Function ◽

Sulfur Containing ◽

Wild Type Form

ABSTRACT The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H2/CO2 is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.

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Seed Production and Germination of Eight Cultivars and the Wild Type of Ruellia tweediana: A Potentially Invasive Ornamental

Journal of Environmental Horticulture ◽

10.24266/0738-2898-21.3.137 ◽

2003 ◽

Vol 21 (3) ◽

pp. 137-143 ◽

Cited By ~ 4

Author(s):

Sandra B. Wilson ◽

Laurie K. Mecca

Keyword(s):

Seed Production ◽

Invasive Plant ◽

Temperature Treatment ◽

Native Plant ◽

Wild Type ◽

Type Form ◽

Storage Studies ◽

Exotic Pest ◽

Exotic Pest Plant ◽

Wild Type Form

Abstract Seed production and the influence of light and temperature on germination were determined for eight cultivars and the wild type form of Mexican petunia (Ruellia tweediana Griseb). The wild type form of R. tweediana has been ranked by the Florida Exotic Pest Plant Council as a Category I invasive plant, meaning it has invaded and is disrupting native plant communities in Florida. The cultivar ‘Purple Showers’ did not set any seed. Peak flowering and seed production for other cultivars generally occurred in August with ‘Morado Chi’, ‘Chi Chi’, and the wild type plants producing approximately three times the amount of seed produced by ‘Katie Pink’, ‘Katie Purple’, ‘Katie White’, and ‘Snow White’. For seed germination, significant cultivar x light interactions occurred for each temperature treatment. Some germination was observed at each temperature except for ‘Katie Purple’, ‘Katie Variegated’, and ‘Katie White’ at 33C (91.4F) without light. Regardless of cultivar or provision of light, 94-100% germination occurred at 30/20C (86/68F), with the exception of ‘Katie Purple’ (65%), ‘Katie Variegated’ (54%), and ‘Katie White’ (83%) without light. At 15, 24 or 33C (59, 75.2, and 91.4F), germination was generally greatest for ‘Chi Chi’ (with or without light) and ‘Morado Chi’ (with light). In parallel greenhouse studies, where seeds were germinated in pots containing a soilless medium, highest germination was achieved by ‘Chi Chi’, ‘Katie Pink’, ‘Katie Variegated’ and ‘Morado Chi’, while significantly lower germination was achieved by wild type plants (55%) by day 14. Storage studies demonstrated that germination of seed collected from the wild type and ‘Chi Chi’ plants began to decrease between 6 and 12 months when maintained at 24C (75.2F) but had equal (‘Chi Chi’) or higher (wild type) germination when maintained at 4C (39.2F) for 12 months.

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Biofilm-Constructing Variants of Paraburkholderia phytofirmans PsJN Outcompete the Wild-Type Form in Free-Living and Static Conditions but Not In Planta

Applied and Environmental Microbiology ◽

10.1128/aem.02670-18 ◽

2019 ◽

Vol 85 (11) ◽

Cited By ~ 1

Author(s):

Marine Rondeau ◽

Qassim Esmaeel ◽

Jérôme Crouzet ◽

Pauline Blin ◽

Isabelle Gosselin ◽

...

Keyword(s):

Wild Type ◽

In Planta ◽

Free Living ◽

Iron Sulfur Cluster ◽

Content Type ◽

Iron Sulfur ◽

Type Form ◽

Static Conditions ◽

Wild Type Form

ABSTRACT Members of the genus Burkholderia colonize diverse ecological niches. Among the plant-associated strains, Paraburkholderia phytofirmans PsJN is an endophyte with a broad host range. In a spatially structured environment (unshaken broth cultures), biofilm-constructing specialists of P. phytofirmans PsJN colonizing the air-liquid interface arose at high frequency. In addition to forming a robust biofilm in vitro and in planta on Arabidopsis roots, those mucoid phenotypic variants display a reduced swimming ability and modulate the expression of several microbe-associated molecular patterns (MAMPs), including exopolysaccharides (EPS), flagellin, and GroEL. Interestingly, the variants induce low PR1 and PDF1.2 expression compared to that of the parental strain, suggesting a possible evasion of plant host immunity. We further demonstrated that switching from the planktonic to the sessile form did not involve quorum-sensing genes but arose from spontaneous mutations in two genes belonging to an iron-sulfur cluster: hscA (encoding a cochaperone protein) and iscS (encoding a cysteine desulfurase). A mutational approach validated the implication of these two genes in the appearance of variants. We showed for the first time that in a heterogeneous environment, P. phytofirmans strain PsJN is able to rapidly diversify and coexpress a variant that outcompete the wild-type form in free-living and static conditions but not in planta. IMPORTANCE Paraburkholderia phytofirmans strain PsJN is a well-studied plant-associated bacterium known to induce resistance against biotic and abiotic stresses. In this work, we described the spontaneous appearance of mucoid variants in PsJN from static cultures. We showed that the conversion from the wild-type (WT) form to variants (V) correlates with an overproduction of EPS, an enhanced ability to form biofilm in vitro and in planta, and a reduced swimming motility. Our results revealed also that these phenotypes are in part associated with spontaneous mutations in an iron-sulfur cluster. Overall, the data provided here allow a better understanding of the adaptive mechanisms likely developed by P. phytofirmans PsJN in a heterogeneous environment.

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