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1.
JCI Insight ; 9(1)2024 Jan 09.
Article in English | MEDLINE | ID: mdl-37971880

ABSTRACT

Syndromic ciliopathies and retinal degenerations are large heterogeneous groups of genetic diseases. Pathogenic variants in the CFAP418 gene may cause both disorders, and its protein sequence is evolutionarily conserved. However, the disease mechanism underlying CFAP418 mutations has not been explored. Here, we apply quantitative lipidomic, proteomic, and phosphoproteomic profiling and affinity purification coupled with mass spectrometry to address the molecular function of CFAP418 in the retina. We show that CFAP418 protein binds to the lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but does not form a tight and static complex with proteins. Loss of Cfap418 in mice disturbs membrane lipid homeostasis and membrane-protein associations, which subsequently causes mitochondrial defects and membrane-remodeling abnormalities across multiple vesicular trafficking pathways in photoreceptors, especially the endosomal sorting complexes required for transport (ESCRT) pathway. Ablation of Cfap418 also increases the activity of PA-binding protein kinase Cα in the retina. Overall, our results indicate that membrane lipid imbalance is a pathological mechanism underlying syndromic ciliopathies and retinal degenerations which is associated with other known causative genes of these diseases.


Subject(s)
Ciliopathies , Retinal Degeneration , Mice , Animals , Retinal Degeneration/genetics , Proteomics , Membrane Proteins/genetics , Membrane Lipids
2.
Genes (Basel) ; 12(6)2021 05 25.
Article in English | MEDLINE | ID: mdl-34070435

ABSTRACT

Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient's healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient's iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.


Subject(s)
Extracellular Matrix Proteins/genetics , Gene Editing/methods , Induced Pluripotent Stem Cells/metabolism , Primary Cell Culture/methods , Usher Syndromes/genetics , CRISPR-Cas Systems , Cells, Cultured , Extracellular Matrix Proteins/metabolism , Female , Gene Deletion , Humans , Usher Syndromes/metabolism , Usher Syndromes/pathology
3.
Curr Biol ; 29(6): 921-934.e4, 2019 03 18.
Article in English | MEDLINE | ID: mdl-30827920

ABSTRACT

The transduction compartment of inner ear hair cells, the hair bundle, is composed of stereocilia rows of graded height, a property essential for sensory function that remains poorly understood at the molecular level. We previously showed that GPSM2-GNAI is enriched at stereocilia distal tips and required for their postnatal elongation and bundle morphogenesis-two characteristics shared with MYO15A (short isoform), WHRN, and EPS8 proteins. Here we first performed a comprehensive genetic analysis of the mouse auditory epithelium to show that GPSM2, GNAI, MYO15A, and WHRN operate in series within the same pathway. To understand how these functionally disparate proteins act as an obligate complex, we then systematically analyzed their distribution in normal and mutant bundles over time. We discovered that WHRN-GPSM2-GNAI is an extra module recruited by and added to a pre-existing MYO15A-EPS8 stereocilia tip complex. This extended complex is only present in the first, tallest row, and is required to stabilize larger amounts of MYO15A-EPS8 than in shorter rows, which at tips harbor only MYO15A-EPS8. In the absence of GPSM2 or GNAI function, including in the epistatic Myo15a and Whrn mutants, bundles retain an embryonic-like organization that coincides with generic stereocilia at the molecular level. We propose that GPSM2-GNAI confers on the first row its unique tallest identity and participates in generating differential row identity across the hair bundle.


Subject(s)
Cell Cycle Proteins/genetics , GTP-Binding Protein alpha Subunit, Gi2/genetics , Hair Cells, Auditory, Inner/physiology , Stereocilia/physiology , Animals , Cell Cycle Proteins/metabolism , GTP-Binding Protein alpha Subunit, Gi2/metabolism , HEK293 Cells , Humans , Mice
4.
J Neurosci ; 38(13): 3160-3176, 2018 03 28.
Article in English | MEDLINE | ID: mdl-29440555

ABSTRACT

C8ORF37 is a causative gene for three different clinical forms of incurable retinal degeneration. However, the completely unknown function of C8ORF37 limits our understanding of the pathogenicity of C8ORF37 mutations. Here, we performed a comprehensive phenotypic characterization of a C8orf37 KO mouse line, generated using CRISPR/Cas9 technology. Both C8orf37 KO male and female mice exhibited progressive and simultaneous degeneration of rod and cone photoreceptors but no non-ocular phenotypes. The major ultrastructural feature of C8orf37 KO photoreceptors was massive disorganization of the outer segment (OS) membrane discs starting from the onset of disc morphogenesis during development. At the molecular level, the amounts of multiple OS-specific membrane proteins, including proteins involved in membrane disc organization, were reduced, although these proteins were targeted normally to the OS. Considering the distribution of C8ORF37 throughout the photoreceptor cell body, the normal structure of the KO photoreceptor connecting cilium, and the absence of defects in other ciliary organs of the KO mice, our findings do not support the previous notion that C8ORF37 was a ciliary protein. Because C8ORF37 is absent in the photoreceptor OS, C8ORF37 may participate in the secretory pathway of OS membrane proteins in the photoreceptor cell body and thus maintain the homeostasis of these proteins. This study established a valid animal model for future therapeutic studies of C8ORF37-associated retinal degeneration. This study also shed new light on the role of C8ORF37 in photoreceptors and on the pathogenic mechanism underlying retinal degeneration caused by C8ORF37 mutations.SIGNIFICANCE STATEMENT Inherited retinal degeneration is a group of incurable conditions with poorly understood underlying molecular mechanisms. We investigated C8ORF37, a causative gene for three retinal degenerative conditions: retinitis pigmentosa, cone-rod dystrophy, and Bardet-Biedl syndrome. C8ORF37 encodes a protein with no known functional domains and thus its biological function is unpredictable. We knocked out the C8ORF37 ortholog in mice, which resulted in a retinal phenotype similar to that observed in patients. We further demonstrated that C8ORF37 is required for photoreceptor outer segment disc formation and alignment, a process that is critical for photoreceptor function and survival. This study advances our understanding of the pathogenesis of retinal degeneration and establishes a valuable mouse model for future therapeutic development.


Subject(s)
Homeostasis , Intracellular Signaling Peptides and Proteins/metabolism , Retinal Degeneration/genetics , Retinal Photoreceptor Cell Outer Segment/metabolism , Animals , Cell Line , Female , Intracellular Signaling Peptides and Proteins/genetics , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Morphogenesis , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Photoreceptor Cell Outer Segment/ultrastructure
5.
Neural Dev ; 10: 12, 2015 Apr 27.
Article in English | MEDLINE | ID: mdl-25927996

ABSTRACT

BACKGROUND: Vertebrate retinal development is a complex process, requiring the specification and maintenance of retinal identity, proliferative expansion of retinal progenitor cells (RPCs), and their differentiation into retinal neurons and glia. The homeobox gene Vsx2 is expressed in RPCs and required for the proper execution of this retinal program. However, our understanding of the mechanisms by which Vsx2 does this is still rudimentary. To define the autonomy requirements for Vsx2 in the regulation of RPC properties, we generated chimeric mouse embryos comprised of wild-type and Vsx2-deficient cells. RESULTS: We show that Vsx2 maintains retinal identity in part through the cell-autonomous repression of the retinal pigment epithelium determinant Mitf, and that Lhx2 is required cell autonomously for the ectopic Mitf expression in Vsx2-deficient cells. We also found significant cell-nonautonomous contributions to Vsx2-mediated regulation of RPC proliferation, pointing to an important role for Vsx2 in establishing a growth-promoting extracellular environment. Additionally, we report a cell-autonomous requirement for Vsx2 in controlling when neurogenesis is initiated, indicating that Vsx2 is an important mediator of neurogenic competence. Finally, the distribution of wild-type cells shifted away from RPCs and toward retinal ganglion cell precursors in patches of high Vsx2-deficient cell density to potentially compensate for the lack of fated precursors in these areas. CONCLUSIONS: Through the generation and analysis of genetic chimeras, we demonstrate that Vsx2 utilizes both cell-autonomous and cell-nonautonomous mechanisms to regulate progenitor properties in the embryonic retina. Importantly, Vsx2's role in regulating Mitf is in part separable from its role in promoting proliferation, and proliferation is excluded as the intrinsic timer that determines when neurogenesis is initiated. These findings highlight the complexity of Vsx2 function during retinal development and provide a framework for identifying the molecular mechanisms mediating these functions.


Subject(s)
Homeodomain Proteins/physiology , Microphthalmia-Associated Transcription Factor/physiology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Retina/embryology , Transcription Factors/physiology , Animals , Cell Division , Chimera , Embryo Transfer , Female , Genes, Reporter , Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/physiology , Male , Mice , Mice, Transgenic , Microphthalmia-Associated Transcription Factor/biosynthesis , Microphthalmia-Associated Transcription Factor/genetics , Mosaicism , Neuroglia/cytology , Organ Specificity , Retina/cytology , Retinal Ganglion Cells/cytology , Transcription Factors/deficiency , Transcription Factors/genetics
6.
J Neurosci ; 33(30): 12197-207, 2013 Jul 24.
Article in English | MEDLINE | ID: mdl-23884928

ABSTRACT

The LIM-Homeodomain transcription factor Lhx2 is an essential organizer of early eye development and is subsequently expressed in retinal progenitor cells (RPCs). To determine its requirement in RPCs, we performed a temporal series of conditional inactivations in mice with the early RPC driver Pax6 α-Cre and the tamoxifen-inducible Hes1(CreERT2) driver. Deletion of Lhx2 caused a significant reduction of the progenitor population and a corresponding increase in neurogenesis. Precursor fate choice correlated with the time of inactivation; early and late inactivation led to the overproduction of retinal ganglion cells (RGCs) and rod photoreceptors, respectively. In each case, however, the overproduction was selective, occurring at the expense of other cell types and indicating a role for Lhx2 in generating cell type diversity. RPCs that persisted in the absence of Lhx2 continued to generate RGC precursors beyond their normal production window, suggesting that Lhx2 facilitates a transition in competence state. These results identify Lhx2 as a key regulator of RPC properties that contribute to the ordered production of multiple cell types during retinal tissue formation.


Subject(s)
Gene Expression Regulation, Developmental , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/physiology , Neural Stem Cells/physiology , Neurogenesis/physiology , Retina/embryology , Transcription Factors/genetics , Transcription Factors/physiology , Animals , Cell Differentiation/physiology , Female , Gene Knock-In Techniques , Male , Mice , Mice, Mutant Strains , Neural Stem Cells/cytology , Pregnancy , Retina/cytology , Retina/growth & development , Retina/physiology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/physiology , Retinal Rod Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/physiology
7.
Dev Dyn ; 241(5): 941-52, 2012 May.
Article in English | MEDLINE | ID: mdl-22434780

ABSTRACT

BACKGROUND: The cell-cycle regulator Cyclin D1 is expressed in embryonic retinal progenitor cells (RPCs) and regulates their cell-cycle rate and neurogenic output. We report here that Cyclin D1 also has important functions in postnatal retinal histogenesis. RESULTS: The initial production of Müller glia and bipolar cells was enhanced in Cyclin D1 knockout (Ccnd1(-/-) ) retinas. Despite a steeper than normal rate of depletion of the RPC population at embryonic ages, postnatal Ccnd1(-/-) retinas exhibited an extended window of proliferation, neurogenesis, and gliogenesis. Cyclin D3, normally confined to Müller glia, was prematurely expressed in Ccnd1(-/-) RPCs. However, Cyclin D3 did not compensate for Cyclin D1 in regulating cell-cycle kinetics or neurogenic output. CONCLUSIONS: The data presented in this study along with our previous finding that Cyclin D2 was unable to completely compensate for the absence of Cyclin D1 indicate that Cyclin D1 regulates retinal histogenesis in ways not shared by the other D-cyclins.


Subject(s)
Cell Cycle/physiology , Cell Differentiation/physiology , Cell Proliferation , Cyclin D1/genetics , Retina/physiology , Animals , Cyclin D1/metabolism , Cyclin D2/genetics , Cyclin D2/metabolism , Cyclin D3/genetics , Cyclin D3/metabolism , Mice , Mice, Knockout , Retina/cytology , Stem Cells/cytology , Stem Cells/physiology
8.
Anal Bioanal Chem ; 397(7): 2939-47, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20549489

ABSTRACT

A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that allowed cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from approximately 6,200 cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 +/- 2.4 microM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 x 10(-5) nmol cell(-1) min(-1). Upon pharmacological treatment with a beta-adrenergic agonist to stimulate lipolysis, a 6.9 +/- 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions in near real-time.


Subject(s)
Adipocytes/chemistry , Adipocytes/metabolism , Fatty Acids, Nonesterified/analysis , Microfluidic Analytical Techniques/methods , 3T3-L1 Cells , Animals , Cell Culture Techniques , Fatty Acids, Nonesterified/metabolism , Lipolysis , Mice , Microfluidic Analytical Techniques/instrumentation , Online Systems/instrumentation
9.
Invest Ophthalmol Vis Sci ; 50(8): 3996-4003, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19324864

ABSTRACT

PURPOSE: Müller glia are essential for maintaining retinal homeostasis and exhibit neuroprotective and deleterious responses during retinal degeneration. Having the ability to visualize and genetically manipulate Müller glia in vivo will facilitate a better understanding of how these cells contribute to these processes. The goal of this study was to determine whether regulatory elements of the retinaldehyde binding protein 1 (Rlbp1; formerly Cralbp) gene can drive robust Müller glial gene expression in vivo. METHODS: Transgenic mice were generated by pronuclear injection of a construct carrying a 3-kilobase (kb) region of the Rlbp1 gene and 5'-flanking sequences linked to the enhanced green fluorescent protein (GFP) cDNA. GFP expression was analyzed by immunohistology in regions of the central nervous system in which RLBP1 protein is expressed, in retinas from wild-type and retinal degeneration 1 (rd1) mice, and during retinal development. RESULTS: Three transgenic lines were generated, and the one with the strongest and most consistent GFP expression was characterized further. Müller glia displayed robust GFP expression at all postnatal developmental stages and in the rd1 retina. Onset of expression occurred by birth in retinal progenitor cells. CONCLUSIONS: Regulatory elements in a restricted region of the Rlbp1 gene are sufficient to drive GFP expression in vivo. This transgenic line provides robust GFP expression that can be used to visualize retinal progenitor cells during postnatal development and Müller glia during their differentiation and in the healthy or degenerating adult retina.


Subject(s)
Carrier Proteins/genetics , Gene Expression Regulation/physiology , Green Fluorescent Proteins/genetics , Neuroglia/metabolism , Promoter Regions, Genetic/physiology , Retinal Degeneration/genetics , Retinal Neurons/metabolism , Animals , Female , Fluorescent Antibody Technique, Indirect , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Microscopy, Confocal , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Neurons/pathology , Retinaldehyde/physiology
10.
Anal Chem ; 81(6): 2350-6, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19231843

ABSTRACT

A dual-chip microfluidic platform that coupled perfusion of cultured adipocytes with on-line fluorescence-based enzyme assay was developed to monitor glycerol secretions in real time from cultured adipocytes. The perfusion cell chip, which could be reversibly sealed to allow reloading of cells and reuse of the chip, was shown by modeling to generate low shear stress on the cells under study. Effluent from the perfusion chip was pumped into an enzyme assay chip for monitoring of secretion from the cells. The on-line enzyme assay had a limit of detection (LOD) of 4 microM glycerol. The temporal resolution of the combined system for detecting changes in glycerol concentration was 90 s. The microfluidic device was used to continuously monitor glycerol secretion from murine 3T3-L1 adipocytes, grown and differentiated on glass coverslips, for at least 2 h. An average basal glycerol concentration of 28 microM was detected in the effluent. Pharmacological treatment with a beta-adrenergic agonist to stimulate lipolysis evoked a 3-fold increase in glycerol secretion followed by sustained release that was 40% higher than basal concentration. The ability to monitor changes in cellular secretion over time may provide insight into adipocyte metabolism and the dysregulation that occurs with obesity-related disorders.


Subject(s)
Adipocytes/metabolism , Enzyme Assays/methods , Glycerol/analysis , 3T3-L1 Cells , Adrenergic beta-Agonists/pharmacology , Animals , Enzyme Assays/instrumentation , Fluorescent Dyes/chemistry , Mice , Microfluidic Analytical Techniques , Time Factors
11.
Dev Biol ; 317(2): 560-75, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18417110

ABSTRACT

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.


Subject(s)
Eye Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Hedgehog Proteins/metabolism , Homeodomain Proteins/metabolism , Retina/embryology , Signal Transduction/physiology , Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Cell Proliferation , Immunohistochemistry , In Situ Hybridization , Mice , Retina/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology
12.
Brain Res ; 1192: 99-113, 2008 Feb 04.
Article in English | MEDLINE | ID: mdl-17919464

ABSTRACT

Chx10/Vsx2 and Vsx1 are the only Paired-like CVC (Prd-L:CVC) homeobox genes in the mouse genome. Both are expressed in the retina and have important but distinct roles in retinal development. Mutations in Chx10/Vsx2 cause reduced retinal progenitor cell (RPC) proliferation and an absence of bipolar cells, while mutations in Vsx1 impair differentiation of cone bipolar cells. Given their structural similarities and importance in retinal development, we sought to determine if a regulatory interaction exists between these genes and whether inactivation of both genes blocks initiation of retinal development. We found that Chx10/Vsx2 binds to a specific sequence in the Vsx1 5'-intergenic region and represses the activity of a luciferase reporter under the control of the Vsx1 promoter. This is consistent with our observation that there is an inverse relationship between the levels of Chx10/Vsx2 and Vsx1 immunostaining within the bipolar cell class. Furthermore, Vsx1 mRNA is upregulated in the RPCs of Chx10/Vsx2 deficient mice and zebrafish embryos injected with a chx10/vsx2 morpholino. In mice deficient for both Chx10/Vsx2 and Vsx1 and zebrafish embryos co-injected with chx10/Vsx2 and vsx1 morpholinos, the changes in embryonic retinal development and marker expression are similar in magnitude to embryos with Chx10/Vsx2 loss of function only. From these studies, we propose that Vsx1 is a direct target of Chx10/Vsx2-mediated transcriptional repression. Although Vsx1 mRNA is upregulated in Chx10/Vsx2 deficient RPCs, Vsx1 does not genetically compensate for loss of Chx10/Vsx2, demonstrating that Prd-L:CVC genes, although important, are not absolutely required to initiate retinal development.


Subject(s)
Eye Proteins/genetics , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Retina/embryology , Retina/metabolism , Stem Cells/metabolism , Transcription Factors/genetics , Animals , Cell Differentiation/genetics , Cell Line , Conserved Sequence/genetics , Down-Regulation/genetics , Evolution, Molecular , Genes, Homeobox/genetics , Humans , Mice , Mice, Knockout , Promoter Regions, Genetic/genetics , RNA, Messenger/metabolism , Regulatory Elements, Transcriptional/genetics , Repressor Proteins/genetics , Retina/cytology , Stem Cells/cytology , Up-Regulation/genetics , Vertebrates/embryology , Zebrafish
13.
Lab Chip ; 6(10): 1355-61, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17102849

ABSTRACT

Separation quality on glass microfluidic devices fabricated from photomasks of different optical resolutions was compared by measuring the dispersion (apparent diffusion) coefficients of a set of standard compounds separated on these devices. Currently, the channel manifolds of most microfluidic devices are patterned using chrome photomasks. A much cheaper, more robust alternative to chrome photomasks are laser photoplotted masks. The primary disadvantage to using laser photoplots is that the optical resolution of these masks is not as high as that of chrome masks, and this feature increases the side-wall roughness of etched channel manifolds patterned using such masks. The increased wall roughness may affect the fluid flow within the channels and, therefore, the separation quality. To determine the effect of increased sidewall channel roughness, microchip channel manifolds were patterned in soda lime glass using a chrome photomask and laser photoplots printed at resolutions of 620, 1240, 3100 and 6200 dots per centimetre (dpc). Separations were performed on these devices using dilute solutions of fluorescently labeled amino acids. The peak variances of the amino acids were calculated at increasing distances down the separation channel and plotted as a function of migration time. From this plot, dispersion coefficients of the analytes were measured. This allowed for a reliable, relatively easy, direct separation analysis among microchips fabricated from the various photomasks. After multiple separations using microchips fabricated from each resolution mask, we found that the change in sidewall surface roughness did not significantly affect the dispersion coefficients measured, and thus the separation quality. The lower mask resolution limit, rather, was governed by the fidelity to which the mask could capture the original CAD design.

14.
Arch Neurol ; 60(1): 97-103, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12533095

ABSTRACT

CONTEXT: Polyglutamine-mediated neurodegeneration in spinocerebellar ataxia type 7 (SCA7) involves specific central nervous system structures despite widespread expression of the mutant ataxin-7 protein. OBJECTIVE: To determine whether expression of multiple gene products could contribute to selective neurodegeneration in SCA7. RESULTS: We identified a novel SCA7 transcript and protein, both of which are enriched within the central nervous system. An isoform-specific antibody revealed that the novel ataxin-7 variant, in contrast with the previously described protein, localizes to neuronal cytoplasm and not to inclusion bodies present within the tissues of patients with SCA7. CONCLUSIONS: In addition to expanding our understanding of SCA7 gene expression, identification of a novel ataxin-7 protein enriched in the central nervous system suggests that expression of multiple polyglutamine-containing proteins may play a role in generating the neurodegenerative patterns characteristic of SCA7 and other polyglutamine expansion diseases.


Subject(s)
Nerve Tissue Proteins/genetics , Spinocerebellar Ataxias/genetics , Ataxin-7 , Base Sequence , Brain Chemistry/genetics , Humans , Molecular Sequence Data , Nerve Degeneration/genetics , Nerve Tissue Proteins/analysis , Spinocerebellar Ataxias/pathology
15.
Plant Physiol ; 131(1): 114-28, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12529520

ABSTRACT

Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification of PP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function.


Subject(s)
Magnoliopsida/genetics , Plant Lectins/genetics , Amino Acid Sequence , Apium/genetics , Apium/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Conserved Sequence/genetics , Cucumis/genetics , Cucumis/metabolism , Gene Expression Regulation, Plant , Magnoliopsida/metabolism , Molecular Sequence Data , Multigene Family/genetics , Phylogeny , Plant Lectins/metabolism , Sequence Homology, Amino Acid
16.
Ann Neurol ; 52(5): 654-7, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12402266

ABSTRACT

A naturally occurring mutation of the mass1 (monogenic audiogenic seizure-susceptible) gene recently has been reported in the Frings mouse strain, which is prone to audiogenic seizures. The human orthologous gene, MASS1, was mapped to chromosome 5q14, for which we previously have reported significant evidence of linkage to febrile seizures (FEB4). We screened for MASS1 mutations in individuals from 48 families with familial febrile seizures and found 25 DNA alterations. None of nine missense polymorphic alleles was significantly associated with febrile seizures; however, a nonsense mutation (S2652X) causing a deletion of the C-terminal 126 amino acid residues was identified in one family with febrile and afebrile seizures. Our results suggest that a loss-of-function mutation in MASS1 might be responsible for the seizure phenotypes, though it is not likely that MASS1 contributed to the cause of febrile seizures in most of our families.


Subject(s)
Codon, Nonsense/genetics , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Receptors, G-Protein-Coupled , Seizures, Febrile/genetics , Seizures/genetics , Base Sequence/genetics , Child , Female , Humans , Male , Pedigree
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