Membrane-tethered mucins are large glycoproteins present in the glycocalyx along the apical surface of all wet-surfaced epithelia of the body, including that of the ocular surface. Originally thought to function only in epithelial surface lubrication and hydration, data now indicate that the mucins are multifunctional molecules, each having unique as well as common functions. This review summarizes current knowledge regarding the three major membrane mucins of the ocular surface, MUC1, MUC4, and MUC16. The mucins vary in their ocular surface distribution, size, structural motifs, and functions. The ectodomains of each are released into the tear film and are, thus, a component of the soluble mucins of the tear film. Both animal and in vitro models for their study are herein described, as are alterations of the mucins in ocular surface disease.

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.

We report that protein adducts of iso [4]levuglandin E 2 (iso [4]LGE 2 ), a highly reactive product of free radical-induced lipid oxidation, accumulate in human glaucomatous trabecular meshwork (TM) but not in controls. Reactive oxygen species play a pathogenic role in primary open angle glaucoma by fostering changes that reduce permeability of the TM tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure. IsoLGs covalently modify proteins and are especially effective in causing protein-protein crosslinking. We found elevated levels of calpain-1 in glaucomatous TM. However, calpain activity in glaucomatous TM is only about 50% to that in controls. This paradox is explicable by the fact that modification by isoLGs renders calpain-1 inactive. Thus, treatment of calpain-1 with iso [4]LGE 2 in vitro results in covalent modification, inactivation, the formation of high molecular weight aggregates (as determined by Western and dynamic light scattering analyses), and resistance to proteasomal digestion. Iso [4]LGE 2 -modified calpain-1 undergoes ubiquitination and its loading impairs the cellular proteasome activity consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. These data suggests that interference with proteasomal activity, owing to protein modification by isoLGs could contribute to glaucoma pathophysiology by decreasing the ability of the TM to modulate outflow resistance. KeywordsGlaucoma; Intraocular pressure; isolevuglandin; Primary open angle glaucoma; Retinal ganglion cell; Reactive oxygen species; Trabecular meshwork Glaucomas are a group of irreversible blinding neurodegenerative diseases that are late onset and progressive in nature. They are designated primary, when they occur without a known cause, or secondary, when onset can be attributed to any other illness or injury. World wide about 70 million people suffer from glaucoma 1 . Primary open angle glaucoma (POAG) is characterized by changes that reduce permeability of the trabecular meshwork (TM) tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure (IOP). Mounting evidence supports the hypothesis that reactive oxygen species (ROS) play a pathogenic role in POAG 2 . Elevated levels of hydrogen peroxide in the aqueous humor promote TM degeneration and outflow resistance. Protection against the deleterious effects of ROS should be provided by the antioxidant activities of superoxide dismutase and glutathione Address Correspondence to: Sanjoy K. Bhattacharya, Bascom Palmer Eye, Institute, 1638 NW 10 th Avenue, Room 706A, University of Miami, Miami, FL,; E-mail: Sbhattacharya@med.miami.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 15. Published in final edited form as:Biochemistry. 2008 January 15; 47(2): 817-825. doi:10.1021/bi701517m. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript peroxidase that are elevated in the aqueous humour of...

Streptococcus pneumoniae is a pathogen associated with a range of invasive and noninvasive infections. Despite the identification of the majority of virulence factors expressed by S. pneumoniae, knowledge of the strategies used by this bacterium to trigger infections, especially those originating at wet-surfaced epithelia, remains limited. In this regard, we recently reported a mechanism used by a nonencapsulated, epidemic conjunctivitis-causing strain of S. pneumoniae (strain SP168) to gain access into ocular surface epithelial cells. Mechanistically, strain SP168 secretes a zinc metalloproteinase, encoded by a truncated zmpC gene, to cleave off the ectodomain of a vital defense component – the membrane mucin MUC16 – from the apical glycocalyx barrier of ocular surface epithelial cells and, thereby invades underlying epithelial cells. Here, we compare the truncated SP168 ZmpC to its highly conserved archetype from S. pneumoniae serotype 4 (TIGR4), which has been linked to pneumococcal virulence in previous studies. Comparative nucleotide sequence analyses revealed that the zmpC gene corresponding to strain SP168 has two stretches of DNA deleted near its 5’ end. A third 3 bp in-frame deletion, resulting in the elimination of an alanine residue, was found towards the middle segment of the SP168 zmpC. Closer examination of the primary structure revealed that the SP168 ZmpC lacks the canonical LPXTG motif – a signature typical of several surface proteins of gram-positive bacteria and of other pneumococcal zinc metalloproteinases. Surprisingly, in vitro assays performed using recombinant forms of ZmpC indicated that the truncated SP168 ZmpC induces more cleavage of the MUC16 ectodomain than its TIGR4 counterpart. This feature may help explain, in part, why S. pneumoniae strain SP168 is better equipped at abrogating the MUC16 glycocalyx barrier en route to causing epidemic conjunctivitis.

Glaucoma is a group of irreversible blinding eye diseases affecting over 70 million people worldwide. Systemic delivery of calpain-1 inhibitors was proposed as a neuroprotection strategy for the prevention of progressive optic nerve damage in glaucoma. We present a general review of calpain-1 and an account of vast differences in processing of calpain-1 in the trabecular meshwork (TM) and the optic nerve. Calpain-1 accumulates in the glaucomatous TM tissues in vivo. However, calpain-1 activity is substantially lower in the glaucomatous TM compared to controls, apparently owing to partial degradation, and modification by lipid oxidation products such as iso[4]levuglandin E2 (iso [4]LGE 2 ). Treatment of calpain-1 with iso [4]LGE 2 in vitro results in covalent modification, inactivation, and resistance to protease digestion. iso [4]LGE 2 -modified calpain-1 appeared to undergo ubiquitination in the TM by cellular degradation machinery mediated by ubch1-2, ubch5,6 and E6-AP, E2 and E3 enzymes respectively. In the TM, iso [4]LGE 2 -modified calpain-1 loading impairs the cellular proteasome activity consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. In contrast, higher calpain-1 activity, that appears to be under translational control, was observed in glaucomatous optic nerve compared to control. Therapeutic neuroprotection strategies using calpain-1 inhibitors will require consideration of such anatomic differences in its activity and biosynthesis.

After the adoption of green revolution, India has combined action of cypermethrin and methanolic extracts of Coriandrum sativum, Piper nigrum observed for their larvicidal and pupicidal activites against Aedes aegypti. When analyzed individually, Cypermethrin were found to be most effectual against the first instar larvae of Aedes aegypti, methanolic extracts of Piper nigrum, sativum being least effective. The LC obtained with Cypermethrin and methanolic extracts of Piper nigrum, Coriandrum sativum against the first instar larvae were 0.61, 0.71 and 0.86%, respectively and the LC 90 values were 1.32, 2.73 and 3.71% respectively. The combination of Cypermethrin and Coriandrum sativum was studied at mixed with Cypermethrin 0.5% and Coriandrum sativum 1.5, 2.0 and 2.5. Similar mixtures were also used for the combination of Cypermethrin and The Cypermethrin and Coriandrum sativum extract combination acted antagonistically. The combination of Cypermethrin and Piper nigrum extract acted synergistically against the target organisms at a first instar larvae, which showed the best results of: LC 50 0.58 and LC 90 2.40% at 24 hours, respectively. The present study will be helpful in developing in a commercial formulation for effe vector management.