Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel

When excised inside-out membrane patches are bathed in symmetrical Cl--rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl- current at +100 mV was 74 +/- 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl- current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 +/- 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl- channel and not the result of pore block.     

FE65 as a link between VLDLR and APP to regulate their trafficking and processing

Background

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson??s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results

In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1?/? cells recapitulate the respiratory defect in isolated mitochondria from PINK1?/? mouse brains, suggesting that these PINK1?/? cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1?/? cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1?/? cells, and this genotypic difference between PINK1?/? and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1?/? cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1?/? and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1?/? compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions

Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1?/? cells.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution.     

Characterization of the Ryanodine Receptor-Ca2+ Release Channel from the Thoracic Tissues of the Lepidopteran Insect Heliothis virescens

The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [³H]-ryanodine binding activity of 1.19 ± 0.21 pmol/mg protein (mean ±se; n= 4). [³H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a K _D of 3.82 nm (n= 4). [³H]-ryanodine binding was dependent on [Ca²⁺] in a biphasic manner and was stimulated by 1 mm ATP. Millimolar caffeine did not stimulate [³H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca²⁺. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca²⁺-sensitive, Ca²⁺-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca²⁺ conductance of ∼110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nm ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [³H]-ryanodine binding properties of a native insect RyR. Received: 3 July 2000/Revised: 17 October 2000     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.