Cow Lens Research Articles

Fluid Circulation Determined in the Isolated Bovine Lens

In 1997, a theoretical model was developed that predicted the existence of an internal, Na(+)-driven fluid circulation from the poles to the equator of the lens. In the present work, we demonstrate with a novel system that fluid movement can be measured across the polar and equatorial surface areas of isolated cow lenses. We have also determined the effects of ouabain and reduced bath [Na(+)]. Lenses were isolated in a chamber with three compartments separated by two thin O-rings. Each compartment, anterior (A), equatorial (E), and posterior (P), was connected to a vertical capillary graduated in 0.25 μL. Capillary levels were read every 15 minutes. The protocols consisted of 2 hours in either open circuit or short circuit. The effects of ouabain and low-Na(+) solutions were determined under open circuit. In 21 experiments, the E capillary increased at a mean rate of 0.060 μL/min while the A and P levels decreased at rates of 0.044 and 0.037 μL/min, respectively, closely accounting for the increase in E. The first-hour flows under short circuit were approximately 40% larger than those in open-circuit conditions. The first-hour flows were always larger than those during the second hour. Preincubation of lenses with either ouabain or low-[Na(+)] solutions resulted in reduced rates of fluid transport. When KCl was used to replace NaCl, a transitory stimulation of fluid transport occurred. These experiments support that a fluid circulation consistent with the 1997 model is physiologically active.

Surface and Volume Changes in the Lens during Accommodation

I was excited by the results presented in the excellent paper by Sheppard et al.1 showing a 2.3% increase in lens volume and a decrease of 1.82% in surface during accommodation in human volunteers. I was very gratified that they acknowledged that we had predicted similar changes in human lens volume and surface based on mathematical modeling of human lens dimensions, using the Pappus theorem as well as our experimentally induced changes in lens dimensions of isolated cow lenses under conditions mimicking accommodation.2,3

Mechanical stretching forces oppose osmotic lens swelling

Mechanical stretching forces oppose osmotic lens swelling

Comparative analysis of crystallins and lipids from the lens of Antarctic toothfish and cow

Animal model systems of senile cataract and lens crystallin stability are essential to understand the complex nature of lens transparency. Our aim in this study was to assess the long-lived Antarctic toothfish Dissostichus mawsoni (Norman) as a model system to understand long-term lens clarity in terms of solubility changes that occur to crystallins. We compared the toothfish with the mammalian model cow lens, dissecting each species' lens into a cortex and nuclear region. In addition to crystallin distribution, we also assayed fatty acid (FA) composition by negative ion electrospray ionization mass spectrometry (ESI-MS). The majority of toothfish lens crystallins from cortex (90.4%) were soluble, whereas only a third (31.8%) from the nucleus was soluble. Crystallin solubility analysis by SDS-PAGE and immunoblots revealed that relative proportions of crystallins in both soluble and urea-soluble fractions were similar within each species examined and in agreement with previous reports for bovine lens. From our data, we found that both toothfish and cow crystallins follow patterns of insolubility that mirror each animals lens composition with more γ crystallin aggregation seen in the toothfish lens nucleus than in cow. Toothfish lens lipids had a large amount of polyunsaturated fatty acids that were absent in cow resulting in an unsaturation index (I(U)) four-fold higher than that of cow. We identified a novel FA with a molecular mass of 267 mass units in the lens epithelial layer of the toothfish that accounted for well over 50% of the FA abundance. The unidentified lipid in the toothfish lens epithelia corresponds to either an odd-chain (17 carbons) FA or a furanoid. We conclude that long-lived fishes are likely good animal models of lens crystallin solubility and may model post-translational modifications and solubility changes better than short-lived animal models.

Changes in rabbit and cow lens shape and volume upon imposition of anisotonic conditions

In vivo, mammalian lenses have the capacity to effect fully reversible changes in shape, and possibly volume, during the accommodation process. Isolated lenses also change shape by readily swelling or shrinking when placed in anisotonic media. However, the manner by which the lens changes its shape when its volume is changed osmotically is not firmly established. Putatively, the lens could swell or shrink evenly in all directions, or manifest distinctive swelling and/or shrinking patterns when exposed to anisotonic media. The present study measured physical changes in lenses consistent with the latter alternative using methods we developed for determining rapid changes in lens shape and volume. It was found in isolated rabbit and cow lenses that the length of the axis between the anterior and posterior poles (A–P length) primarily increases under hypotonic conditions (−40 to −100 mOsM), with smaller, or no changes, in equatorial diameter (ED). Hypertonic conditions (+50 to +100 mOsM) on rabbit lenses elicited a predominant reduction in ED, while the A–P length was only marginally reduced. Hypertonic solutions of +150 mOsM were required to obtain similar changes in cow lens shape. The ratio of the A–P length to the ED was taken as a measure of “circularity”. This ratio increased gradually in rabbit and cow lenses bathed in hypotonic solutions because of the increase in the A–P length. The calculated lens volume increased in tandem with the increase in “circularity”. Lens circularity also increased under hypertonic conditions due to the decrease in ED, but this increase in circularity during shrinkage was not as pronounced as that which occurred during swelling. As such, the lens has a tendency upon swelling to change its shape by approaching the structure of a globular spheroid (as occurs during accommodation for near focusing), but lens shrinkage does not result in a flatter lens with a reduced A–P length as occurs during dis-accommodation for distance focusing. Moreover, osmotically evoked shape changes appear irreversible, in contrast to the mechanically elicited shape changes of accommodation.

Surface change of the mammalian lens during accommodation

Classical theories suggest that the surface area of the crystalline lens changes during accommodation while the lens volume remains constant. Our recent work challenged this view by showing that the lens volume decreases as the lens flattens during unaccommodation. In this paper we investigate 1) the magnitude of changes in the surface of the in vitro isolated cow lens during simulated accommodation, as well as that of human lens models, determined from lateral photographs and the application of the first theorem of Pappus; and 2) the velocity of the equatorial diameter recovery of prestretched cow and rabbit lenses by using a custom-built software-controlled stretching apparatus synchronized to a digital camera. Our results showed that the in vitro cow lens surface changed in an unexpected manner during accommodation depending on how much tension was applied to flatten the lens. In this case, the anterior surface initially collapsed with a reduction in surface followed by an increase in surface, when the stretching was applied. In the human lens model, the surface increased when the lens unaccommodated. The lens volume always decreases as the lens flattens. An explanation for the unexpected surface change is presented and discussed. Furthermore, we determined that the changes in lens volume, as reflected by the speed of the equatorial diameter recovery in in vitro cow and rabbit lenses during simulated accommodation, occurred within a physiologically relevant time frame (200 ms), implying a rapid movement of fluid to and from the lens during accommodation.

Reply to “Letter to the editor: ‘Ocular lens does not change volume during accommodation’”

reply: Thank you for providing a forum to answer the comments of Drs. Schachar and Liao ([5][1]). #### General comments. The number of experiments was not 7; it was a total of 20 divided in 3 sets: 7 relaxed-stretched, 6 relaxed-stretched-relaxed (see Table 1 in Ref. [2][2]), and 7 under oil (see

Effect of age on ocular irritancy as measured with in vitro bovine lenses

This project studied the effect of age on optical quality of cultured bovine lenses exposed to a number of common surfactants and alcohol. Lenses from calves (8-18 months) and cows (2-3 years) were isolated aseptically and studied optically for 96 h after treatment with various commercial surfactants and an alcohol. Potential eye irritancy was evaluated using a scanning laser in vitro assay system which records the change in focal characteristics (back vertex distance variability or BVDV) of the bovine lenses. Lenses were divided into a total of 14 groups. Both calf and cow lenses (a total of 257 lenses were used) were arranged into control, 0.01% BAK, 1% SDS, 1.0% Triton X-100, 100% ethanol, 10% Tween-20 and 1.0% Tween-20 treatment groups. The cationic surfactant BAK caused the most amount of optical change to the bovine lenses, followed by SDS, Triton X-100, ethanol and then Tween-20. There was also a significant difference in BVDV between the cow and calf groups for all the treated groups, except for Tween-20, with the calf lenses showing greater optical damage. In the case of 10% Tween-20, both cow and calf lenses show equal optical damage while at 1.0% both groups show no effect and are no different from the untreated control lenses. Younger bovine lenses are more sensitive to the surfactants and alcohol tested when compared to their older counterparts, indicating that younger eyes may be more sensitive to these chemicals. The results further indicate that age is a factor that should be taken into account in assessing ocular risk.

Cold-stable eye lens crystallins of the Antarctic nototheniid toothfishDissostichus mawsoniNorman

The eye lenses of the Antarctic nototheniid fishes that inhabit the perennially freezing Antarctic seawater are transparent at -2 degrees C, whereas the cold-sensitive mammalian and tropical fish lenses display cold-induced cataract at 20 degrees C and 7 degrees C, respectively. No cold-cataract occurs in the giant Antarctic toothfish Dissostichus mawsoni lens when cooled to temperatures as low as -12 degrees C, indicating highly cold-stable lens proteins. To investigate this cold stability, we characterised the lens crystallin proteins of the Antarctic toothfish, in parallel with those of the sub-tropical bigeye tuna Thunnus obesus and the endothermic cow Bos taurus, representing three disparate thermal climes (-2 degrees C, 18 degrees C and 37 degrees C, respectively). Sizing chromatography resolved their lens crystallins into three groups, alpha/betaH, beta and gamma, with gamma crystallins being the most abundant (>40%) lens proteins in fish, in contrast to the cow lens where they comprise only 19%. The upper thermal stability of these crystallin components correlated with the body temperature of the species. In vitro chaperone assays showed that fish alpha crystallin can protect same-species gamma crystallins from heat denaturation, as well as lysozyme from DTT-induced unfolding, and therefore are small Heat Shock Proteins (sHSP) like their mammalian counterparts. Dynamic light scattering measured an increase in size of alphagamma crystallin mixtures upon heating, which supports formation of the alphagamma complex as an integral part of the chaperone process. Surprisingly, in cross-species chaperone assays, tuna alpha crystallins only partly protected toothfish gamma crystallins, while cow alpha crystallins completely failed to protect, indicating partial and no alphagamma interaction, respectively. Toothfish gamma was likely to be the component that failed to interact, as the supernatant from a cow alpha plus toothfish gamma incubation could chaperone cow gamma crystallins in a subsequent heat incubation, indicating the presence of uncomplexed cow alpha. This suggests that the inability of toothfish gamma crystallins to fully complex with tuna alpha, and not at all with the cow alpha crystallins, may have its basis in adaptive changes in the protein that relate to the extreme cold-stability of the toothfish lens.

Glycero- versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth

The human lens differs from other mammalian lenses in its very slow growth and unusual phospholipid composition of its cell membranes. Dihydrosphingomyelins (DHSMs) make up about half of all phospholipids in adult human fiber membranes. In all other membranes, sphingomyelins(SMs) with a trans double bond in their backbone, are prevalent. In our quest to understand the biological implications of such elevated DHSM levels, we analyzed membranes from various regions of human, elephant, giraffe, polar bear, pig and cow lenses. The levels of DHSMs were minor in non-human lens membranes. A strong correlation was observed between growth rate and relative contents of phosphatidylcholines(PCs) in epithelia and outer cortical fibers. Sphingomyelins became increasingly predominant in differentiated fibers and this increase was age dependent. Indeed, nuclear fiber membranes of aged non-human mammals were composed, almost exclusively, of (SMs). Although human lens membranes followed comparable compositional trends, the magnitude of the changes was much smaller. We postulate that the high relative contents of DHSMs provide a biochemically inert matrix in which only small amounts of PCs and SMs and their metabolites, known to promote and arrest growth, respectively, are present. This compositional difference is proposed to contribute to the slow multiplication and elongation of human lens cells.

Lipid composition of lens plasma membrane fractions enriched in fiber junctions.

Little is known about the lipid environment of lens fiber junctions, the plasma membrane structure proposed to be responsible for passage of low molecular weight metabolites between adjacent lens fiber cells. Plasma membranes of the ocular lens are especially rich in fiber junctions. The resistance of junctional domains to disruption by detergent or alkali treatment provides the opportunity to isolate a lens plasma membrane fraction enriched in fiber junctions. When examined by electron microscopy, the fiber junction fraction prepared from bovine lenses was enriched with junctional structures by about twofold when compared to total plasma membrane. We compared the protein, phospholipid, and cholesterol concentration of total plasma membrane with fiber junctional membrane from rat and cow lens and from aged normal cataractous human lenses. The principal finding was that junctional membrane contained 20-40% more total lipid than that of the total plasma membrane. This was due to a proportionate increase in the relative content (mg/mg protein) of both phospholipid and cholesterol. Exclusive of one exception (nucleus of bovine lens), the cholesterol/phospholipid molar ratios of the two fractions were similar. In the bovine nucleus, the cholesterol/phospholipid molar ratio was substantially higher in the fiber junctional-enriched membrane fraction than in the total plasma membrane, suggesting a special association of cholesterol with bovine nuclear fiber junctions. The relative lipid compositions of the plasma membrane and fiber junction-enriched fractions from human normal and cataractous lenses were similar, suggesting that human senile cataractogenesis involves changes in the lens plasma membrane more subtle than would be reflected by gross changes in the membrane lipid composition.

High-molecular-weight protein aggregates of calf and cow lens: Spectroscopic evaluation

To gain insight into the molecular features of the high-molecular-weight (HMW) fraction of soluble lens proteins and their changes in aging, we isolated this fraction from the nucleus of calf and cow lenses and measured fluorescence and circular dichroism (CD) properties of the samples. Not only was there an increase in the HMW fraction in the older lens, but there was also an agerelated difference in tertiary structure that was clearly manifested in the fluorescence and CD parameters. The far-u.v. CD of low- and high-molecular-weight proteins do not differ significantly in band position and magnitude, but the near-u.v. CD of HMW protein does differ distinctly from that of all other crystallins (α, β and γ); the entire CD spectrum of this protein is displayed in the negative region. Millipore filtration further revealed that HMW aggregates are essentially a polydisperse population of different conformation (tertiary structure) and that these aggregates are associated by non-convalent interactions. This association is caused mainly by the apolar (hydrophobic) nature of the constituent protein, α-Crystallin has more hydrophobic domain along the peptide chain than do other crystallins and thus is likely to be the predominant protein in HMW aggregates.

Fluorescence polarization studies of fluorescein isothiocyanate conjugates of bovine lens crystallins

Bovine lens alpha-, beta H- and gamma-crystallin were labeled with the amine-specific fluorescent probe, fluorescein isothiocyanate (FITC) and studied with steady-state polarization measurements. Rotational relaxation times (rho) were estimated for various crystallins and were compared with calculated values. The observed rho value is considerably faster for alpha- and beta H-crystallin conjugate than the calculated value, indicating existence of a segmental motion of the probe on these two crystallins. The segmental flexibility may result from a less tightly folded structure in these crystallins. alpha-Crystallin isolated from the cow lens nucleus shows a smaller rho value than the young cortical alpha-crystallin. The protein partial unfolding process appears to be age-related, and a possible consequence is that crystallin becomes more susceptible to chemical modifications.

Front surface fluorometric study of lens insoluble proteins.

Although the quantity of insoluble lens proteins increases with aging and cataracts, it is unknown whether the quality, such as protein structure, also changes correspondingly. In this study front surface fluorometry was used to study powdered samples of insoluble proteins isolated from young calf and old cow lenses, as well as from young clear and old cataractous human lenses. Tryptophan (Trp) fluorescence shows that there is a difference in protein conformation between young and old bovine samples. The old sample appears to have more open structure. This is demonstrated by a red shift in Trp emission maxima. The water-insoluble fraction shows 3-4 nm shift, while its urea-soluble and urea-insoluble fraction show 1-2 nm shift. The unfoldedness found in soluble crystallins is thus retained when they become insoluble. Both young and old human insoluble samples give a long Trp emission wavelength without showing any difference. Another major change that can be measured by fluorescence is the appearance of the 370/440 nm peak in old bovine insoluble powdered samples. Human samples also give this peak, and its position shifts to a longer wavelength in senile lenses.

The occurrence of glutathione—insulin transhydrogenase (protein—disulfide interchange enzyme) in the lens

Glutathione-insulin transhydrogenase (GIT, thiol:protein-disulfide isomerase/oxidoreductase, E.C. 5.3.4.1/1.8.4.2) catalyzes via sulfhydryldisulfide interchange, the scission as well as formation of disulfide bonds in many diverse proteins. Using insulin as a substrate, the lens epithelial layer of cows, rats and rabbits was found to contain GIT activity. The enzyme's activity is activated by GSH and inhibited by N-ethylmaleimide. Subcellular distribution of bovine lens epithelial homogenates showed that the majority of GIT activity is located in the insoluble fraction (10,000 g pellet) and in the high molecular weight fraction (60,000 g pellet). Lens epithelial extracts were subjected to SDS-PAGE followed by Western blot, and probed with a polyclonal antibody to rat liver GIT, or with either of two monoclonal antibodies directed against different epitopes of the enzyme. Lens epithelium was found to contain two forms of GIT, one with the same molecular weight as the purified enzyme (Mr 56Kd), and a second having an Mr of 67Kd. Immunoblots using polyclonal antibodies revealed an additional major immunoreactive band of 32Kd in the cow lens epithelial layer as well as in the isolated cortical and nuclear portions. Rat lenses showed no immunoreactive 32Kd band. Using a bovine cortical/nuclear fraction the 32Kd reactivity was found to be associated with the beta H-crystallin fraction, but the extract failed to show GIT activity with the insulin substrate. This suggests that beta H-crystallin may share a common epitope with GIT.

Tryptamine in the vertebrate lens

The normal ionic balance and transparency of the lens is regulated by Na +−K + pumps in the lens epithelium and lens fibers. Inhibition of the (Na ++K +)-ATPase and ion transport results in osmotic swelling and opacification. We have previously shown that 10 −4–10 −3 M tryptamine has an inhibitory effect on both ion transport and ATPase activity in the lens. In this paper we show that isolated lenses incubated in Ringer's solution containing tryptamine exhibit Na + and K + concentration reversal. Agents that affect tryptophan metabolic pathways, such as pargyline and p-chlorophenylalanine, were tested for their effect on lens tryptamine levels and Na + and K + concentrations. Toad lenses cultured 10 days in Medium-199 with tryptophan, p-chlorophenylalanine and pargyline, each at a concentration of 1 mM, had increased tryptamine concentrations relative to lenses incubated in control medium. The lenses with the larger increase in tryptamine content also showed a decrease in K + concentration and an increase in Na +. The endogenous tryptamine levels in toad, frog, rabbit and cow lenses were measured, and varied from 0.065 μg/g in the rabbit to 0.219 μg/g in the cow. Tryptamine levels were also determined in senile cataractous and in non-cataractous human lenses. The lenses diagnosed as cataractous had a significantly higher tryptamine concentration, with the highest levels found in the cataractous lenses which were dark brown on inspection. Tryptophan, which has recently been shown to be elevated in the plasma of patients with cataracts, is actively transported into the lens where it may be decarboxylated to tryptamine. The accumulation of the amine may be a contributing factor in the development of senile cataracts.

Immunocytochemical localization of the lens main intrinsic polypeptide (MIP26) in communicating junctions.

Plasma membranes of vertebrate lens fiber cells contain a major intrinsic polypeptide with an apparent molecular weight of 26,000 (MIP26). These plasma membranes are extremely rich in communicating junctions, and it has been suggested that MIP26 is a component of them. MIP26 was purified from cow lenses using preparative SDS gel electrophoresis followed by hydroxylapatite column chromatography. From gel electrophoresis patterns and aggregational properties it was concluded that the MIP26 preparation was homogeneous. The purified MIP26 was used to produce monospecific antibodies in rabbits as assessed by double immunodiffusion and crossed immunoelectrophoresis of purified MIP26 and solubilized lens plasma membranes against the antiserum. Indirect immunocytochemical studies were performed on open and closed lens plasma membrane vesicles by incubation in anti-MIP antiserum followed by ferritin-conjugated goat antirabbit IgG. The conjugate bound unequivocally to lens communicating junctions, indicating that MIP26 is a component of these structures.

Study of Trace Metallic Elements in the Lens

In calf lenses, cow lenses and human senile cataractous lenses, the contents of iron, copper, zinc, magnesium and calcium were analyzed by the atomic absorption spectrophotometry. Bovine lenses showed regional variations in the elementary contents, in that the zinc and iron contents were highest in the anterior cortex of both calf and cow lenses and lowest in the nucleus, and that copper, magnesium and calcium contents in the superficial parts were higher than those in the nucleus in both calf and cow lenses. Bovine lenses showed such variations in elementary contents, tending to increase with age, but copper, magnesium and calcium contents registered no variations specifically associated with age, with no significant difference between the calf and cow lenses. Magnesium content was found to be the highest of alle elements in both calf and cow lenses. In human cataractous lenses, calcium and zinc contents were shown to be higher in completely cataractous lenses than in partially cataractous lenses. Calcium content was found to be particularly high in the former group. No significant difference was found in iron, copper or magnesium content between the two groups.

Lipids in tissues of the eye. X. Molecular species of sphingomyelins from different parts of calf lens in relation to differentiation and aging

Sphingomyelins have been isolated from epithelium, cortex and nucleus of calf lens. The ceramides obtained by phospholipase hydrolysis were characterized in detail as intact molecules and after degradation, mainly by thin-layer chromatography, gas chromatography and mass spectrometry. Sphingosine ( d- erythro-1,3-dihydroxy-2-amino- trans-4-octadecene) was the only long-chain base found. Saturated normal fatty acids with 16, 18, 22 and 24 carbon atoms and a monounsaturated 24 carbon fatty acid with a cis double bond were dominant in the different parts of the lens. No branched paraffin chains were present. Sphingomyelins with a high C 24:1 C 24:0 fatty acid ratio occurred in lens cortex and nucleus. During differentiation from epithelial cell to cortex fibre the C 24:1 content of sphingomyelins increased strongly, which was mainly compensated by a decrease in C 16 and C 18 fatty acids. Thus a marked increase in mean chain length and mono-unsaturation of the paraffin chains occurred. Only slight differences were detected between the fatty acid composition of cortical and nuclear sphingomyelins, and of calf and cow lens sphingomyelins. The data point to an intensive net biosynthesis of sphingomyelins in the equator and to metabolic stability in cortex and nucleus.

Free isoelectric focusing of bovine lens γ-crystallins

A method is described for separation of γ-crystallin components from the major bulk of crystallin residues of the bovine lens cortex. Carrier-free isoelectric focusing in a pH gradient from 3 to 10 of bovine γ-crystallins in a coil of polyethylene tubing is used to purify a cow lens cortex γ-crystallin fraction obtained from Sepharose 6B chromatography. Purity of the γ-crystallin preparation thus obtained is checked by polyacrylamide thin layer isoelectric focusing, immunoelectrophoresis, and two-dimensional antigen/antibody crossed electrophoresis against a polyvalent and a specific antiserum. Free isofocusing appears to be an excellent method by which to separate γ-crystallin components in purest form from a preparation containing 75% β-crystallins. Isoelectric points of protein components are determined by polyacrylamide thin layer isoelectric focusing on an analytical scale. It is defined that β-crystallins have isoelectric points of 7·00 and lower, and that γ-crystallins have isoelectric points over 7·00. The proteins from the cortex of the adult bovine lens contain about 1% of a crystallin which is identical to the γ-crystallins of the nucleus of the lens, which were originally of embryonic origin.