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Biomedical subjects

T R Shearer

Publications and source records attributed to T R Shearer.

At least 19 recordsLinked to original sources
PubMed2006

Amelioration of retinal degeneration and proteolysis in acute ocular hypertensive rats by calpain inhibitor ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester.

BACKGROUND: Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins. METHODS: Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate alpha-spectrin was analyzed by immunoblotting. RESULTS: Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species. CONCLUSION: Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.

Animals↗
PubMed2006

Low activity by the calpain system in primate lenses causes resistance to calcium-induced proteolysis.

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Aged↗
PubMed2005

Involvement of Egr-1 in lens epithelial cell death induced by selenite.

Selenite-overdose cataract in young rats may be caused by an initial insult to the lens epithelial cells. Our previous DNA array analysis revealed a significant increase in the expression of mRNA for early growth response protein-1 (Egr-1) in lens epithelial cells after injection of selenite. This suggested that up-regulation of Egr-1 mRNA may be involved in lens epithelial cell death. The purpose of the present experiment was to further clarify the involvement of Egr-1 in lens epithelial cell death induced by selenite. Rat lens epithelial explants were cultured with sodium selenite. Selenite caused epithelial explants to leak LDH into the medium. During LDH leakage, increased expression of mRNA for Egr-1 was observed by RT-PCR. To further test the involvement of Egr-1 in selenite-induced cell death, mouse lens epithelial cell line (alpha-TN4 cells) was treated with antisense oligonucleotide for Egr-1. Antisense oligonucleotide for Egr-1 significantly diminished expression of Egr-1 protein and leakage of LDH. These results suggested that increased activity of Egr-1 may be a factor in lens epithelial cell death induced by selenite.

Animals↗
PubMed2005

Degradation of human aquaporin 0 by m-calpain.

Opacities (cataracts) in the lens of the eye are a leading cause of preventable blindness. Aquaporins function as water channels, and the C-terminus is postulated as a regulatory domain. The C-terminal domain of aquaporin 0 (AQP0) develops numerous truncation sites during lens aging. The purpose of the present experiment was to determine if the calcium-activated protease m-calpain (EC 3.4.22.17) was responsible for truncation of human AQP0. AQP0 was isolated from young human donors, incubated with recombinant m-calpain, and the cleavage sites on the released peptides were determined by on-line electrospray ionization mass spectrometry. We found that four cleavage sites on human AQP0 could be tentatively assigned to m-calpain. This is the first evidence for possible calpain activity in human lens. Because the cause(s) of 17 other cleavage sites was unknown, the data also suggested that other, as yet unknown, proteases or non-enzymatic mechanisms are more active than calpain in human lens.

Amino Acid Sequence↗
PubMed2005

Biochemical properties of lens-specific calpain Lp85.

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K(50%act)=20 microM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on alphaA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

Animals↗
PubMed2003

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite.

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.

Animals↗
PubMed2001

Involvement of phosphorylation of myosin phosphatase by ROCK in trabecular meshwork and ciliary muscle contraction.

The control of smooth muscle contraction is an important factor in maintaining normal intraocular pressure. However, the specific factors causing changes in control by phosphorylation/dephosphorylation schemes in the eye are not well-defined. The purposes of this experiment were to (i) determine the localization of ROCK (Rho-associated, coiled coil-forming kinase) in monkey and rabbit eye tissues and (ii) measure phosphorylation of ROCK substrate during trabecular meshwork or ciliary muscle contraction induced by carbachol. We found that mRNAs for both ROCK I and II were expressed in most eye tissues from rabbit and monkey. Proteins for ROCK I and II were present in all eye tissues studied except lens. When trabecular meshwork or ciliary muscle were incubated with carbachol to induce contraction, phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase, a substrate for ROCK, started within 1 min and continued for at least 1 h. This phosphorylation was well correlated with contraction of trabecular meshwork or ciliary muscle. These results suggested that ROCK might regulate contraction of trabecular meshwork or ciliary muscle through phosphorylation of MBS of myosin phosphatase.

Animals↗
PubMed2001

Different expression patterns for ubiquitous calpains and Capn3 splice variants in monkey ocular tissues.

The purpose of the present investigation was to compare the expression of ubiquitous and tissue-specific calpains in ocular tissues from the Macaca fascicularis monkey. Calpain isoforms in retina and corneal epithelium from adult M. fascicularis monkeys were characterized by RT-PCR, cDNA cloning and sequencing. Calpain isoform activities in ocular tissues were investigated by fractionation on DEAE-HPLC, immunoblotting, and casein zymography. Capn3 splice variants in the ocular tissues from rat, rabbit and monkey were compared after RT-PCR. RT-PCR analysis revealed that numerous splice variants of Capn3 were expressed in the epithelium from monkey cornea. The variants contained deletions or insertions in or around the IS1, IS2, and NS regions. The cDNAs for Capn3 variants were highly conserved, yet the expression patterns of the Capn3 isoforms were widely different among the mammalian species. In contrast, the expression patterns of ubiquitous calpains in ocular tissues were conserved among the mammalian species, and similarities between monkey and human cDNAs for Capn1 (mu-calpain) and Capn2 (m-calpain) were 98 and 99%, respectively. These results suggested that differences in expression patterns of Capn3 variants might be related to the function of each variant in a particular tissue or species.

Alternative Splicing↗
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↗
PubMed2001

Deamidation of human beta B1 alters the elongated structure of the dimer.

The purpose of these experiments was to determine if truncation and deamidation alter the structure of a human lens protein, beta B1-crystallin. Recombinant wild type and a deamidated form of recombinant beta B1 were expressed in Escherichia coli. Wild type beta B1 was also enzymatically cleaved to generate a physiologically-relevant truncated beta B1. Purity and size of the expressed proteins were confirmed by SDS-PAGE and electrospray ionization mass spectrometry. Size exclusion chromatography and light scattering were used to determine aggregation states of beta B1. Protein conformations were predicted from sedimentation velocity analysis. Molecular weights of 49,000 and 54,000 Da were obtained for wild type beta B1 by sedimentation equilibrium and light scattering, respectively. A sedimentation coefficient of 2.7 S was determined for wild type beta B1. Molecular weights of 54,000 and 60,000 Da were determined for deamidated beta B1 by sedimentation equilibrium and light scattering, respectively. However, deamidated beta B1 eluted earlier than wild type beta B1 on size exclusion chromatography, with an estimated molecular weight between 78,000 and 116,000 Da. Loss of the extensions of beta B1 caused abnormal association of the protein with the stationary phase during size exclusion chromatography. Wild type beta B1 was predicted to form a dimer with an elongated structure. The earlier elution of the deamidated beta B1 dimer on size exclusion chromatography suggested the dimer was less compact. Truncation caused abnormal column interactions suggesting an altered conformation. These changes are important because truncation and deamidation occur extensively in aging human lenses and may be important for senile cataract formation.

Animals↗
PubMed2001

Purification and characterization of lens specific calpain (Lp82) from bovine lens.

Ubiquitous type m-calpain and lens specific Lp82 calpain were separated and partially purified from fetal bovine lens and the enzymatic characteristics were compared. Lens m-calpain required 200 microM calcium for 1/2 maximal activity, while Lp82 required 30 microM. Both types of calpains were inhibited by 0.1 mM E64, and 5 mM iodoacetamide, but not by 1 mM phenylmethylsulfonyl fluoride. Lp82 was insensitive to 1 microM calpastatin peptide while m-calpain was effectively inhibited. In the presence of calcium, m-calpain lost most of its activity within 2 hr, while Lp82 was continually active for 18 hr. Both calpains cleaved the natural substrates betaA3 and alphaB crystallins in a similar manner. However, incubation of alphaA crystallin with m-calpain removed ten amino acid residues from its C-terminus, while incubation with Lp82 removed only five residues. The latter truncation product of alphaA was also found in vivo. These data suggested that Lp82 may have a more important role than m-calpain in modification of crystallins during lens maturation.

Animals↗
PubMed2001

Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats.

PURPOSE: 1) To measure the amount of calpain inhibitor SJA6017 taken up by lenses of young rats after administration; and 2) To test efficacy of SJA6017 against selenite cataract in regard to amelioration of proteolysis of lens protein and prevention of lens nuclear opacity. METHODS: Selenite nuclear cataracts were produced by subcutaneous injection of an overdose of sodium selenite to 16-day-old rats. SJA6017 was administered daily using intraperitoneal injections at 100 mg/kg body weight/day for 4 days. Lenses were observed and photographed by slit lamp biomicroscopy, and scored into one of three stages. Enucleated lenses were also scored into one of four stages and lens opacities in the nuclear region were quantified by image analysis. Proteolysis of crystallins was detected by SDS-PAGE. The amount of SJA6017 taken up by the lens was detected with a column switching HPLC system. RESULTS: Nuclear cataracts were visible in 31% of the animals receiving only selenite, while the frequency of nuclear cataract in the Se+SJA6017 group was reduced to only 16%. This effect of SJA6017 was confirmed by densitometric analysis as a reduction in the density of the nucleus. Similar proteolytic changes of crystallins occurred at all stages of selenite cataract formation. The amount of SJA6017 in the lens was detected at the level of 0.03 microM. CONCLUSIONS: Systemic SJA6017 was taken up by the lens, and SJA6017 ameliorated in vivo selenite cataract formation. These studies are important because they partially validate the biochemical rationale for developing non-surgical, drug treatments for cataract prevention in man.

Animals↗
PubMed2001

Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses.

PURPOSE: To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. METHODS: Total soluble proteins from bovine, human, and rodent lenses, betaH crystallin, or recombinant betaB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Proteolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. RESULTS: The in vitro cleavage sites produced by m-calpain on the N-termini of human betaB1, betaA3, and betaB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of alpha- and beta-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. CONCLUSIONS: Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.

Amino Acid Sequence↗
PubMed2001

Calpain-induced light scattering in young rat lenses is enhanced by UV-B.

The purpose of this study is to determine if UV-B enhances light scattering after proteolysis of crystallins by calpains, and to determine if lens-specific calpain Lp82 is involved, along with m-calpain, in the mechanism of in vitro precipitation. Lens soluble proteins from young rats were hydrolyzed for 24 hr by endogenous lens calpains, and the proteins were further incubated for up to 7 days with periodic irradiation by UV-B. Light scattering was measured daily at 405 nm. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, activation of calpains, and formation of high molecular weight aggregations. Appreciable light scattering occurred in lens soluble proteins after proteolysis of crystallins by m-calpain and Lp82. UV-B markedly enhanced this light scattering and the formation of higher molecular weight aggregates consisting of proteolyzed alpha- and beta- and intact gamma-crystallins. Calpain inhibitor E64 and antioxidants DTE or GSH prevented the light scattering. These results show that calpain-induced light scattering is enhanced by the natural oxidant UV-B. Activation of Lp82, along with m-calpain, contributed to the light scattering. The linkage between proteolysis and oxidation is important because both oxidation and truncation of crystallins are found in aged human lenses, which are constantly exposed to UV irradiation.

Animals↗
PubMed2000

Evidence for apoptosis in the selenite rat model of cataract.

The purposes of this experiment were (1) to determine if apoptosis was accelerated during formation of selenite cataract, and (2) to determine the role of calpains and caspases in lens apoptosis. Evidence for apoptosis in selenite-injected rats included: approximately 7-8% of epithelial cells in germinative zone were positive, disappearance of the nuclear membrane, condensation of the chromatin, and breakdown of PARP. Activation of calpains was indicated by characteristic limited proteolysis of crystallins, breakdown of alpha-spectrin to 150/145 kDa fragments, hydrolysis of vimentin, and autolytic breakdown of m-calpain. Selenite cataract did not have an appreciable effect on the mRNA levels for caspase-3, calpains, and calpastatin. This indicated the increased enzyme activity of m-calpain and caspase-3 in selenite cataract occurred at the enzyme level rather than by upregulation of mRNAs. Increased calpain and caspase activity may be linked to the selenite-induced apoptosis. Such data are important because they indicate that apoptosis may be a fairly early event in selenite cataract.

Animals↗
PubMed2000

Involvement of cysteine proteases in bFGF-induced angiogenesis in guinea pig and rat cornea.

Overexpression and activation of matrix metalloprotease (MMP) have been implicated in angiogenesis. However, the involvement of cysteine proteases, such as calpains (EC 34.22.17), is obscure. Thus, the purpose of this experiment was to study the involvement of cysteine proteases in angiogenesis induced by basic fibroblast growth factor (bFGF) in guinea pig and rat corneas using cysteine protease inhibitors. Sustained-release polymers containing bFGF were implanted into guinea pig and rat corneas to induce angiogenesis. For treatment of corneal angiogenesis, polymers containing cysteine protease inhibitors, leupeptin or SJA6017, were also implanted into corneas. Using the slit lamp, the corneas were observed for nine days after polymer implantation. Soluble proteins and albumin levels were used as markers of corneal injury by angiogenesis. bFGF induced angiogenesis in guinea pig and rat corneas. In guinea pig cornea, wet weight, the amount of soluble protein and albumin was highest at four days after bFGF-containing pellet implantation. In rat cornea, the amount of soluble protein and albumin was highest at six days, and wet weight increased within four days. One hundred nmole of leupeptin showed a tendency to reduce bFGF-induced angiogenesis in guinea pig cornea, and 10 nmole of SJA6017 was effective in reducing bFGF-induced angiogenesis in rat cornea, although SJA6017 showed a stronger effect than leupeptin. Ten nmole of SJA6017 significantly reduced the number of new blood vessels. These data suggested involvement of cysteine proteases in angiogenesis in guinea pig and rat cornea.

Albumins↗
PubMed2000

Lp85 calpain is an enzymatically active rodent-specific isozyme of lens Lp82.

PURPOSES: To clone and sequence the cDNA for Lp85 calpain from young rat lens, and to test for Lp85 protein expression and proteolytic activity. METHODS: RT-PCR and molecular cloning were performed on total RNA from 12 day-old rats. Lp85 protein expression was visualized by immunoblotting using a specific antibody developed to the unique peptide sequence in Lp85. Proteolytic activity was assessed by casein zymography. Transient expression of Lp85 and previously characterized lens-specific calpain Lp82 were separately performed in mammalian COS-7 cells. RESULTS: The 2410-bp cDNA for rat lens Lp85 encoded a protein of 737 amino acid residues with a calculated molecular weight of 85.0 kDa and a predicted pI of 5.67. The amino acid sequence of Lp85 was identical to Lp82 except for an insert region of 28 amino acids in domain IV of the calcium-binding region. mRNA and protein for Lp85 were present only in rat and mouse lenses and not in other tissues or species. Lp85 protein concentrations were highest in the nuclear region, most concentrated in the insoluble fraction, disappeared with lens maturation, and Lp85 exhibited migration similar to Lp82 on native PAGE gels. Lp85 was enzymatically active when expressed in COS-7 cells. CONCLUSIONS: Lp85 is a newly classified, lens- and rodent-specific, enzymatically active, member of the AX1 (alternative exon 1) subclass of calpains. In conjunction with Lp82 and m-calpain in lens, Lp85 may be responsible for proteolysis during normal lens development and maturation or during cataract formation in young rodents.

Amino Acid Sequence↗