摘要:

Aqueous tears were collected by glass capillary tubes at weekly intervals from 6–9 week old, Swiss-Webster mice. Mouse IgA, IgGI, IgG2a and IgM were quantitated in the tears using sandwich type enzyme linked immunosorbent assays (ELISAs). IgE was quantitated using an indirect two-step ELISA. The results established IgA as the predominant antibody in mouse tears at a level of 110µ;g/ml. This was determined using a mouse IgA standard which contained both polymeric and monomeric IgA. IgGI, IgG2a and IgM were detected in the tears at low levels, 13 ng/ml, 21 ng/ml and 442 ng/ml, respectively. IgE was not detectable in mouse tears with a lower limit of detection equal to 10 ng/ml. These studies establish the relative levels of IgA, IgG1, IgG2a and IgM in the tears of normal adult mice.

ISSN:0271-3683    

DOI:10.3109/02713688509003356

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

Reconstitution of the lens fiber cell protein known as MIP26 into liposomes composed of heterologous phospholipids was achieved; this protein renders the liposomes permeable to low molecular weight compounds. MIP26 from either bovine or human lenses was capable of forming channels in artificial membranes. The assay technique was sufficiently sensitive to allow reconstitution of MIP26 from single human lenses, enabling us to examine the function of channels from either cataractous or age-matched normal lenses. Decreases in pH can cause these channels to close, analogous to the hypothesized channel closing in thein vivosituation. The pH optimum of reconstituted channels in liposomes containing MIP26 from bovine lenses or normal human lenses is very sharp; but is substantially broadened if the liposomes contain MIP26 from cataractous human lenses. This latter result suggests a functional alteration in human lens membranes which is correlated with the development of human senile cataract.

ISSN:0271-3683    

DOI:10.3109/02713688509003357

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

A methodology was developed to determine the proportion of acetyl-(AChE) and butyrylcholinesterase (BuChE) in the albino and pigmented rabbit eye. It was found that BuChE contributed over 75% of the cholinesterase activity in all the ocular tissues but the corneal epithelium of the albino rabbit. This esterase was principally responsible for the parabolic chain length dependence of ocular hydrolysis of model naphthyl ester prodrugs reported previously. In contrast, when incubated with AChE, the rate of hydrolysis of these esters decreased monotonically with increasing ester chain length. Together these findings suggest that esters whose chain length exceeds 4 carbons will be hydrolyzed primarily by BuChE. It is suggested that the dominance of BuChE in ocular tissues is another factor which merits consideration in the design and evaluation of ocular ester prodrugs.

ISSN:0271-3683    

DOI:10.3109/02713688509003358

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

In the mouse, the homozygous presence of therdgene results in the genetically programmed death of the photoreceptor cells of the retina. Using congenic strains of mice and a novels sensitive, immunological approach for visualizing unique retinal proteins, we identified four bands of protein whose concentrations are regulated by the homozygous presence of the gene for retinal degeneration. Since these proteins (with apparent molecular weights of 23, 33, 55, and 69 kD) are present in normal adult mouse retinas and absent from rodless retinas, and from other mouse non-retinal tissues including brain, heart, kidney and liver, the data support the identification of these proteins as being retina specific. These proteins are not peculiar to the normal mouse retina; but rather, all four (23, 33, 55 and 69 kD) are common to rat retina; three (23, 33, and 55 kD) are common to bovine retina; and presently at least two, 23 and 69 kD, are clearly detectable in normal, adult human retina. The temporal appearance and disappearance of the four retinal specific protein bands coincide with the morphological maturation and degeneration of the photoreceptor cell population. Collectively, the present data suggest that one or more may be photoreceptor specific. These observations present the first step in the identification and characterization of specific soluble proteins correlated with the biochemical phenotype of therdgene and the death of photoreceptor cells of the retina.

ISSN:0271-3683    

DOI:10.3109/02713688509003359

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

The presence of surfactants in human tear samples was studied using methods employed in ELISA technology. Initial experiments whereby diluted tear samples were incubated with special polyacrylate ELISA microcuvettes showed no binding of tear-lactoferrin to the cuvettes whereas a marked binding of purified lactoferrin could be observed. In subsequent experiments it was shown that incubation of purified lactoferrin with increasing amounts of human tears resulted in a dose dependent inhibition of the binding of lactoferrin to the microcuvettes. Partial characterisation of the“coating inhibitory activity”in human tears was investigated by studying the effect of tears or isolated tear fractions on the non-specific binding of a peroxidase conjugated antibody onto the microcuvettes. Preincubation of microcuvettes with tears followed by a wash with phosphate buffered saline resulted in a marked inhibition of the adhesion of the peroxidase conjugate, suggesting that the coating inhibitory activity is caused by a binding of a factor in human tears to the solid phase. The findings reported here resemble earlier observations concerning lacrimal surfactants. The method described in this paper can be performed using a small quantity of human tears or isolated tear proteins and is therefore quite suitable for further biochemical analysis of tear surfactant activity as well as for clinical studies in patients with various tear film disorders.

ISSN:0271-3683    

DOI:10.3109/02713688509003360

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

We have conducted a correlative electrophysiological and morphological study of ceil to cell coupling in frog and rat lenses. Electrical impedance measurements from frog and rat lenses were curve fit to a model of lens structure to obtain a value for internal resistivity (Rj). The mean and standard deviation of Rj was 550±190 ohm-cm (n=7) in rat lenses and 3400±340 ohm-cm (n = 10) in frog lenses. These results indicate that the extent of cell to cell coupling is far more extensive in rat lenses than in frog lenses and therefore suggest that rat lens fiber cells are conjoined by greater numbers of communicating junctions than frog lens fiber cells. Freeze-etch replicas were made of fiber cells from rat and frog lenses of comparable size and from a comparable area (intermediate cortex) as that used in the electrophysiological study. A total of 987 and 1,393 square microns of replicated membrane were examined in rat and frog lenses, respectively. 1,573 communicating junctions were counted in rat lens replicas cumulatively measuring 313 square microns or 31.7% of the total membrane area. 604 communicating junctions were counted in frog lens replicas cumulatively measuring 163 square microns or 11.7% of the total membrane area. These results demonstrate that the amount of communicating junction predicted to be necessary to account for the more extensive electrotonic coupling between fiber cells in rat lenses than in frog lenses is qualitatively confirmed by morphological analysis.

ISSN:0271-3683    

DOI:10.3109/02713688509003361

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

Lens fibers are electrically coupled with each other and directly exchange dyes and metabolites. In most cells, this form of communication is mediated by gap junctions. Lens fibers lack typical gap Junctions. The lens junctions, although morphologically similar to gap junctions, differ from them structurally, chemically and immunologically. Nevertheless, recent evidence suggests that indeed lens junctions are communicating junctions. The lens junction protein, MIP26, displays structural characteristics similar to other channel proteins. Once incorporated into liposomes it forms channels permeable to molecules as heavy as 1.5 kDa. Like other communicating junctions, lens junctions assume crystalline arrays and uncouple with Ca++. The liposome incorporated channels close with Ca++and H+in the presence of calmodulin (CaM). Partial loss of gating competency occurs after proteolytic cleavage of the C-terminal arm of MIP26. The need for a unique type of communicating junction in lens is unclear. A possibility is that this tissue has some special cell-to-cell transport requirements, in terms of size and/or charge of permeants, not shared by coupled cells of other tissues.

ISSN:0271-3683    

DOI:10.3109/02713688509003362

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

Specific antibodies are versatile tools for analyzing cell surface proteins. This study involves the characterization of monoclonal antibodies which are specific for the junctional protein found in the lens fiber cell. This protein can be expected to include regions on the external membrane surface for junction formation, others on the cytoplasmic surface for regulation of junctional properties and, if cell-cell channels are indeed involved, transmembrane domains forming the hydrophilic connection between adjacent cytoplasms. Antibodies to these various regions would provide for an experimental analysis of the junctional protein, e.g., the identification of“active sites”for junction formation. Three monoclonal antibodies specific for the lens junctional protein in the chicken are described here. The first, termed B2, also recognizes the bovine junctional protein, MP26 (5). We have characterized the submolecular specificity of B2 and have found that it binds approximately ten amino acid residues from the C-terminus of MP26. In isolated lens junction preparations, B2 binds to the cytoplasmic surfaces of the lens junctions (both 12 nm and 16 ran thick forms). Thus, we consider MP26 a component of the lens junction. Monoclonal A4, the second antibody considered in detail here, was produced by immunization with lens membranes after treatment with low pH. We have found that lens junctional membranes are separated, or“split,”by treatment at pH 2.5–3.0. It appears that A4 binds to the external surface of the junctional membrane; EM studies to confirm this are in progress. In order to map the A4 binding site within the chicken junctional protein and to explore the arrangement of this protein within the membrane, a number of procedures were used to generate fragments of MP26. These included reactions with N-chlorosuccinimide and proteases after acid treatment. Antibody binding to fragments was evaluated with immunotransfer (“Western”) procedures. These studies mapped the A4 binding site to the center of the molecule and suggested that MP26 projected externally from the membrane at two different points. These results are consistent with a recent model, based on sequence data (6), for the arrangement of MP26 within the bovine lens membrane.

ISSN:0271-3683    

DOI:10.3109/02713688509003363

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

Fragments of intact rat, pig and bovine lenses and isolated membranes from bovine lenses were examined by freeze-fracture-etch electron microscopy employing samples ultra-rapidly frozen in the absence of fixatives or cryoprotectants. Complementary replicas of the bovine outer cortex clearly display small patches of square arrays (6.6 nm repeat) as structures distinct from gap junctions in the same membranes. In the outer cortex square arrays appear in single membranes where the extracellular space is not thinned, whereas gap junctions, as in other tissues, only occur where the extracellular space is greatly attenuated. Square arrays in the middle cortex appear as patches of varying size surrounded by smooth membrane fracture faces. Within the undulating membranes of tongue-and-groove interdigitations in the inner cortex and nucleus, the square arrays are extensive and are located specifically on the regions which have convex curvature toward the cytoplasm. Square crystalline regions alternate with non-crystalline regions within each membrane of an undulating pair. Across the extracellular space, crystalline regions are matched with non-crystalline regions, thus making it unlikely that the square array participates in intercellular communication. Because the undulations occur in isolated membranes following urea washing to remove cytoplasmic and extrinsic membrane proteins, the square arrays probably play a crucial role in the formation of the undulations and in the maintenance of membrane curvature. X-ray diffraction experiments show reflections from the crystalline square array for all twenty of our preparations of isolated membranes. These x-ray experiments indicate that the square arrays observed by freeze-fracture are abundant in lens fiber cell membranes.

ISSN:0271-3683    

DOI:10.3109/02713688509003364

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor

摘要:

The Main Intrinsic Polypeptide (MIP) of the ocular lens fiber cell plasma membrane was immunocytochemically localized at the ultrastructural level on ultrathin frozen sections of rat lens, and on extracted, gradient-purified bovine lens membranes. The results indicate that both the junctional and non-junctional membrane domains of the cortical lens fiber cell are MIP immunoreactive. Frozen thin section immunocytochemistry of the lens epithelium and hepatocytes, also using anti-MIP antibodies, revealed that these cells, and their intercellular junctions, are not MIP-immunoreactive. From these findings we conclude that 1) MIP, a putative fiber cell junctional protein, is present throughout the plasma membrane of the lens fiber cell, and is not confined to the fiber cell junctional domain, 2) MIP is not a detectable component of the lens epithelial cell membrane, or its intercellular junctions, 3) MIP is not detectable in gap junctions of hepatocytes.

ISSN:0271-3683    

DOI:10.3109/02713688509003365

出版商:Taylor&Francis    

年代:1985

数据来源: Taylor