Biomedical subjects

Nalin M Kumar

Publications and source records attributed to Nalin M Kumar.

5 recordsLinked to original sources

PubMed2005

Eye lens proteomics: from global approach to detailed information about phakinin and gamma E and F crystallin genes.

Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.

Amino Acid Sequence↗

PubMed2004

RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis.

PURPOSE: Transforming growth factor-beta(TGF-beta) is an important mediator of wound healing responses. In the eye, TGF-beta activity has been implicated in causing corneal haze after laser surgery and subconjunctival scarring following glaucoma surgery. The purpose of the study was to determine whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-beta (TbetaRII) could be used to suppress the TGF-beta action. METHODS: TbetaRII specific siRNAs designed from the human gene sequence were transfected into cultured human corneal fibroblasts. The protein and transcript levels of the receptor were determined by immunofluorescence, western blotting, and real time PCR. Immunofluorescence and immunoblotting were carried out to examine fibronectin assembly. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. In addition, the in vivo effects of TbetaRII siRNA were evaluated in a mouse model of ocular inflammation and fibrosis generated by subconjunctival injection of phosphate buffered saline and latex beads. Mouse TbetaRII siRNA was introduced into experimental eyes. Cellularity on tissue sections was evaluated after staining with hematoxylin and eosin. Collagen deposition was visualized by picrosirius red staining. RESULTS: TbetaRII siRNAs abrogated the receptor transcript and protein expression in cultured corneal fibroblasts. The gene knockdown inhibited fibronectin assembly and retarded cell migration. In the mouse model, introduction of TbetaRII specific siRNA significantly reduced the inflammatory response and matrix deposition. CONCLUSIONS: TbetaRII specific siRNAs were efficacious both in vitro and in vivo in knocking down the TGF-beta action. A direct application of siRNA into eyes to downregulate TbetaRII expression may provide a novel therapy for preventing ocular inflammation and scarring.

Adolescent↗

PubMed2003

Differences in expression patterns between mouse connexin-30.2 (Cx30.2) and its putative human orthologue, connexin-31.9.

A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9.

Amino Acid Sequence↗

PubMed2003

Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1).

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.

Animals↗

PubMed2002

Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1.

A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.9 gene localized to chromosome 17q21.2. Northern analysis of Cx31.9 showed a major 4.4-kilobase transcript, which was expressed at varying levels in all tissues analyzed. Two monoclonal antibodies generated against different domains of Cx31.9 recognized a 30-33-kDa protein from cells overexpressing Cx31.9. Immunofluorescence of overexpressing cells indicated the presence of Cx31.9 between adjacent cells, consistent with its localization to gap junctions. Double voltage clamp analyses of Cx31.9-overexpressing cells, and of paired Xenopus oocytes injected with Cx31.9 cRNA, demonstrated junctional currents indicative of gap junction channel formation. In contrast to previously characterized connexins, Cx31.9 showed no voltage-dependent gating within a physiologically relevant range. Cx31.9 was detected in human tissues by immunoblot analysis, and immunofluorescence localized Cx31.9 expression to vascular smooth muscle cells. Furthermore, it was demonstrated that Cx31.9 interacted with ZO-1. Thus, Cx31.9 represents a novel connexin gene that in vivo generates a protein with unique voltage gating properties.

Amino Acid Sequence↗