Establishment of an appropriate animal model for lacritin studies: cloning and characterization of lacritin in monkey eyes.

Lacritin is a mitogen of human salivary gland cells as well as a stimulator of human corneal epithelial cells. It is expected to be an important factor in maintaining the surrounding ocular surface. The monkey would be a relevant animal model in which to study the role of lacritin in ophthalmic physiology and pathology. However, to our knowledge, no cDNA cloning or functional analysis of monkey lacritin has been performed. Thus, the purposes of this study were: (1) to clone the monkey ortholog of lacritin; (2) to characterize lacritin in tears from several species; and (3) to determine the tissues where lacritin is produced and secreted. cDNA for lacritin from rhesus macaque contained 547 bp, with 411 bp in an open reading frame (ORF) encoding a protein of 137 amino acids. Monkey lacritin showed 89% amino acid homology with human lacritin; one amino acid was deleted in all three monkey strains. The predicted MW of mature lacritin was 12.2 kDa, and the isoelectric point was 4.99. Lacritin showed anomalous migration at approximately 21.0 kDa on SDS-PAGE, as confirmed by immunoblotting and amino acid sequencing. Similar to native lacritin in monkey tears, a 21 kDa band was also detected in human tears. In contrast, no lacritin was observed at a similar position on SDS-PAGE in rat, rabbit and dog tears. In the monkey, lacritin mRNA was expressed highly in the lacrimal gland, moderately in the conjunctiva and the meibomian gland, and weakly in corneal epithelium. In primates, lacritin was produced in the lacrimal gland and secreted into tear fluid. These results suggest that lacritin might be important for the maintenance of the ocular surface in higher animals, such as monkeys and humans.

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.

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.

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.

Use of beta-methylphenylalanine (beta MeF) residues to probe the nature of the interaction of substance P with its receptor: effects of beta MeF-containing substance P analogs on rabbit iris smooth muscle contraction.

The effects of substituting (2S,3S)-beta-methylphenylalanine (S-beta MeF) or (2S,3R)-beta-methylphenylalanine (R-beta MeF) for the Phe7 and/or Phe8 residues of the tachykinin substance P (SP, RPKPQQFFGLM-NH2) upon the ability of SP to stimulate contraction of the rabbit iris smooth muscle were investigated. The eight beta MeF-containing SP analogs (four monosubstituted analogs, four disubstituted analogs) 1-8 were synthesized and found to be agonsts of SP in the smooth muscle contraction assay, having EC50 values ranging from 0.15 to 10.0 nM. Three analogs are significantly more active than SP [8R-(beta MeF)SP (4), 7S,8S-(beta MeF)2SP (5), and 7R,8S-(beta MeF)2SP (6)], three analogs are approximately equipotent with SP [7S-(beta MeF)SP (1), 7R-(beta MeF)SP (2), and 7S,8R-(beta MeF)2SP (8)], and two analogs are significantly less active than SP [8S-(beta MeF)SP (3) and 7R,8R-(beta MeF)2SP (7)]. The effects of the beta MeF substitutions upon the activity of SP are not additive and cannot be explained using simple conformational models which focus only on the side chain conformations of the beta MeF residues. It is postulated that the beta MeF residues induce minor distortions in the peptide backbone with resultant consequences upon peptide-receptor binding which are not dictated soley by the side chain conformations. This idea is consistent with 1H-NMR data for the monosubstituted analogs 1-4, which imply that the beta MeF substitutions cause slight distortions in the peptide backbone and that the beta MeF side chains are assuming trans or gauche(-) conformations.

Phospholpid studies of marine organisms: 2.(1) Phospholipids, phospholipid-bound fatty acids and free sterols of the spongeAplysina fistularis (Pallas) formafulva (Pallas) (=Verongia thiona)(2). Isolation and structure elucidation of unprecedented branched fatty acids.

The free sterols and phospholipids of the demospongeAplysina fistularis were isolated and analyzed. The free sterols consisted mainly of the unusual 26-methylated sterols aplysterol (53%) and 24(28)-dehydroaplysterol (7%) together with 7 commonly occurring sterods. The major phospholipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and diphosphatidylglycerol. The major fatty acyl components of the phospholipids consisted of 85% C14-C20 acids, including the unprecedented 2,6,10-trimethyl-5-tetradecenoic acid and 11-methyloctadecanoic acid. The remaining 15% were C27-C30 demospongic acids, including 2 novel acids tentatively assigned the structures 5,9,23-octacosatrienoic acid and 5,9,23-nonacosatrienoic acid, and 3 novel acids proven to be 5,9,21-octacosatrienoic acid, Z,Z-20-methyl-5,9-hexacosadienoic acid and Z,Z-22-methyl-5,9-octacosadienoic acid. The biosyntheses of the novel demospongic acids are proposed to occur by chain elongation of monoenoic or branched precursors followed by desaturation. The large quantities of typically bacterial phospholipids and fatty acids found implied the presence of bacteria in the sponge, in agreement with microscopic studies. Analysis of the phospholipid-bound fatty acids in a sponge cell-enriched fraction indicated that the demospongic acids, including the 2 branched structures, were the major acids of the sponge cells. The presence inA. fistularis of demospongic acids containing membrane disordering groups-methyl branches or double bonds-on the ω7 carbon is proposed to be due to the need by the sponge for membranes possessing fluidity near the middle of the phospholipid bilayer. It is also proposed that the C26 methyl group of aplysterol causes disordering of the phospholipid bilayer in the same region, and thus also evolved in response to this need.