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N A Reed

Publications and source records attributed to N A Reed.

5 recordsLinked to original sources
PubMed2001

An immunohistochemical method for the detection of proteins in the vertebrate lens.

Fluorescence immunohistochemistry has traditionally been difficult or impossible to perform on the vertebrate lens because of its extremely high protein content. Described here is a robust and rapid method for preparing and labeling vertebrate eyes for confocal microscopy. This technique has successfully been applied to localize proteins in the lens epithelium and capsule, as well as the primary and secondary fibers. This technique preserves tissue morphology and coupled with double and triple labeling, has allowed localization of proteins bound to plasma membrane, basement membrane, nucleus, endoplasmic reticulum as well as sub-nuclear compartments. In addition, the present technique has proven useful for fluorescent immunohistochemical analysis of diverse tissues including whole embryos, adult muscle, pancreas, and liver. This procedure allowed us to successfully localize a wide variety of antigens on diverse vertebrate tissues including the more challenging vertebrate lens.

Animals↗
PubMed2001

Characterization and expression of calpain 10. A novel ubiquitous calpain with nuclear localization.

Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to hydrolyze specific substrates important in calcium-regulated signaling pathways. Recently, an atypical member of the calpain family, calpain 10, was described, and genetic variation in this gene was associated with an increased risk of type II diabetes mellitus in humans. In the present report, a polyclonal antibody directed against rat calpain 10 was developed. This antibody was used to monitor the expression of calpain 10 protein in tissues from rats, mice, and humans. Calpain 10 protein was found to be present in all tissues examined by Western blotting including the lens, retina, brain, heart, and skeletal muscle. Although some calpain 10 was detectable in the water-soluble protein fraction of these tissues, it was preferentially found in the water-insoluble fraction. In the lens, immunohistochemistry revealed that calpain 10 was predominately located in the cytoplasm of epithelial and newly differentiating lens fibers at the transition zone. However, calpain 10 was found to be associated with the plasma membrane of differentiated lens fiber cells and the sarcolemma of skeletal muscle. In the lens epithelium-derived cell line, alphaTN4-1, the calpain 10 protein was found in a punctate distribution in the cell nucleus as well as the cytoplasm. After the elevation of intracellular calcium levels with ionomycin, calpain 10 protein levels in the nucleus of alphaTN4-1 cells increased markedly, whereas those in the cytoplasm decreased. In the lens, the elevation of intracellular calcium levels after selenite administration resulted in increased levels of calpain 10 RNA within 1 day and a loss of calpain 10 protein from the lens nucleus coincident with the onset of selenite cataract. In conclusion, calpain 10 seems to be a ubiquitous calpain, the expression level and subcellular distribution of which are dynamically influenced by calcium.

Adult↗
PubMed2000

Influence of specific regions in Lp82 calpain on protein stability, activity, and localization within lens.

PURPOSE: To determine the influence of specific regions within Lp82 calpain on protein stability, enzymatic activity, and localization within lens and to test the influence of an Lp82 knockout mouse on normal maturational proteolysis in lens. METHODS: DNA constructs for Lp82 and Lp82-related proteins were subcloned into the pcDNA 3.1 vector. The constructs contained a substitution of the novel sequence (NS) region from p94 for the AX1 N-terminal region of Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient expression of these Lp82-related proteins was performed in COS-7 mammalian cells. Immunoblotting and casein zymography were used to measure protein stability and enzymatic activity of the expressed proteins. Homologous recombination was used to knock out p94 gene expression and p94 splice variants such as Lp82 and Lp85 in the lenses of 10-day-old mice. Confocal microscopy revealed the immunohistochemical localization Lp82 and Lp85 within lens. RESULTS: Insertion of IS1 into Lp82 resulted in a lack of stable protein and loss of enzymatic activity. In contrast, substitution of the NS region for AX1 and insertion of IS2 into Lp82 had no effect on the stability of the Lp82-related proteins. p94 knockout mice at 10 days of age exhibited a total absence of Lp82 activity in the lens but normal activity for the separate mu- and m-calpain gene products. Calcium-induced in vitro proteolysis was retarded in these Lp82/p94 knockout lenses. Lp82 and Lp85 immunostaining was intense throughout the cytoplasm of the cortical and nuclear fibers of newborn mouse lenses with little staining in the epithelium. In contrast, immunostaining for the ubiquitous m-calpain was highest in the epithelium and bow region, with much lower levels in the nucleus. The naturally occurring IS3 insert in Lp85 also promoted the association of Lp85 with the perinuclear region of the nucleated lens fibers. CONCLUSIONS: The lack of the IS1 region in Lp82 accounts for the stability and abundance of enzymatically active Lp82 protein in rodent lenses. Conversely, the presence of the IS1 region is responsible for the lability of p94 and Rt88 calpains in muscle and retina, respectively. The insert in Lp85 may promote membrane association. A consequence of the specific loss of Lp82 in the lens may be to retard normal maturational proteolysis.

Animals↗