scholarly journals Effects of α-crystallin gene knockout on zebrafish lens development

2021 ◽  
Author(s):  
Mason Posner ◽  
Kelly L. Murray ◽  
Brandon Andrew ◽  
Stuart Brdicka ◽  
Alexis Butterbaugh-Roberts ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Lens Development ◽  
Differential Interference Contrast Microscopy ◽  
Fiber Cells ◽  
Interference Contrast Microscopy ◽  
Developmental Role ◽  
Genetic Compensation ◽  
Shock Proteins ◽  
And Function ◽  
Vertebrate Eye

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataract, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three α-crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed by mass spectrometry and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either αBa- or αBb-crystallin produced no significant lens defects. Mutation of each α-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for αA-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.

Direct visualization of the isolated and perfused macula densa

1985 ◽  
Vol 248 (6) ◽  
pp. F890-F894 ◽  
Author(s):  
K. L. Kirk ◽  
P. D. Bell ◽  
D. W. Barfuss ◽  
M. Ribadeneira
Keyword(s):  
Structural Changes ◽  
Macula Densa ◽  
Thick Ascending Limb ◽  
Direct Visualization ◽  
Differential Interference Contrast Microscopy ◽  
Intercellular Spaces ◽  
Small Water ◽  
Nacl Concentration ◽  
Interference Contrast Microscopy ◽  
And Function

Direct examination of the structure and function of the macula densa is compromised by the relative inaccessibility and small size of this cell plaque. We report the isolation and perfusion of rabbit nephron segments with attached glomeruli and the direct visualization of the macula densa with differential interference-contrast microscopy. We used this technique to examine the structural sensitivity of the macula densa to perturbations in luminal osmolality and NaCl concentration. Reducing luminal osmolality from 290 to 70 mosmol/kg by removing NaCl resulted in a dilation of the lateral intercellular spaces that was both reversible and specific to the region of the macula densa. Associated with the dilation of the intercellular spaces was a small (congruent to 10%), but statistically significant, increase in the height of the macula densa cells. These structural events were related to the reduction in luminal osmolality, since isosmotic replacement of NaCl with mannitol resulted in no detectable structural changes. Thus, the macula densa may represent a small water-permeable plaque of cells within the remaining water-impermeable thick ascending limb of Henle's loop.

scholarly journals The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation

2021 ◽  
Vol 22 (5) ◽  
pp. 2591
Author(s):  
Pengfei Ma ◽  
Jie Li ◽  
Lei Qi ◽  
Xiuzhu Dong
Keyword(s):  
Heat Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Small Heat Shock Protein ◽  
Phase Cell ◽  
Molecular Weights ◽  
Oxidative Inactivation ◽  
Methionine Residues ◽  
Shock Proteins ◽  
And Function

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70–90% and 86–100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an “oxidant sink” effect, i.e., to consume the oxidants in environments via aspartate oxidation

NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Katia Urso ◽  
Arantzazu Alfranca ◽  
Sara Martínez-Martínez ◽  
Amelia Escolano ◽  
Inmaculada Ortega ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Gene Knockout ◽  
Activated T Cells ◽  
Knock Down ◽  
And Function

Abstract The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.

scholarly journals Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

2003 ◽  
Vol 160 (5) ◽  
pp. 671-683 ◽  
Author(s):  
Alexey Khodjakov ◽  
Lily Copenagle ◽  
Michael B. Gordon ◽  
Duane A. Compton ◽  
Tarun M. Kapoor
Keyword(s):  
Three Dimensional ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Formation ◽  
Capture Mechanism ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5 ◽  
Dependent Mechanism

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living α-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

scholarly journals The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

2021 ◽  
Vol 22 (7) ◽  
pp. 3700
Author(s):  
Junna Hayashi ◽  
Jennifer Ton ◽  
Sparsh Negi ◽  
Daniel E. K. M. Stephens ◽  
Dean L. Pountney ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Aggregation ◽  
Molecular Chaperone ◽  
Cross Linking ◽  
Αb Crystallin ◽  
The Cross ◽  
Shock Proteins ◽  
And Function

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

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