Chemo-enzymatic preparation of copolymeric polythioesters containing branched-chain thioether groups

Branched-chain copolymeric polythioesters (PTE) were formed in good yield (∼87%) by chemoenzymatic reactions including thiyl radical-induced addition of 1,6-hexanedithiol to the >C=C< double bond of dimethyl 1,18-octadec-9-enedioate and transthioesterification of polyfunctional dimethyl 1,18-octadec-9-enedioate with bifunctional 1,6-hexanedithiol catalyzed by immobilized lipase from Rhizomucor miehei. The reactions were performed in vacuo at 80°C without a solvent. PTE was extracted from the reaction mixture using methyl-t-butylether and precipitated from i-hexane. The polymer structure of the i-hexane-insoluble PTE precipitate was elucidated by GPC/SEC showing an average molecular mass (M _w) of 1,857 Da corresponding to a molecular weight range of up to 24,000 Da and a maximum degree of polymerization of up to 50 monomer units. Chemical derivatization with TMSH demonstrated the formation of up to ∼58 mol% of a branched-chain thio(S)ether, i.e., dimethyl S-9-(6-mercaptohexylthio)-1,18-octadecanedioate, and small proportions (∼8 mol%) of a dimeric disulfide formed therefrom. The chemical structures of various low-molecular weight (<900 Da) reaction products formed by transthioesterification, addition reaction or disulfide formation of the reactants or reaction intermediates, e.g., 1,18-octadec-9-enedioic acid methyl(O)ester 6′-S-mercaptohexyl thio(S)ester, dimethyl S-9-(6-mercaptohexylthio)-1,18-octadecanedioate, were elucidated by GC–MS. Similarly, dimethyl S-9-(6-S-methylthiohexylthio)-1,18-octadecanedioate and dimethyl 11,18,19,26-tetrathia-10,27-di-(7-carboxymethyl-heptyl)hexatriacontane-1,36-dioate were detected in the reaction mixtures after derivatization with trimethylsulfonium hydroxide.     

Chymotrypsin responsive hydrogel: application of a disulfide exchange protocol for the preparation of methacrylamide containing peptides

Methacrylamide groups were selectively coupled to cysteine residues in the presence of amines and alcohols by utilizing a disulfide exchange reaction in aqueous, acidic buffer. The tetrapeptide sequence, CYKC, was used as a cross-linker to create poly(acrylamide) hydrogels that dissolved when subjected to either a flowing or stationary solution of alpha-chymotrypsin. Control hydrogels that were cross-linked with the tetrapeptide, CSKC, were not affected by the same protease solution. In contrast, dissolution of both the CYKC and CSKC cross-linked hydrogel structures was accomplished by using the disulfide reducing agent tris(2-carboxyethyl) phosphine (TCEP). The chemoselective conjugation technique described could have utility for more advanced protease-responsive hydrogels as well as other hybrid materials composed of synthetic and biomacromolecules.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

MALDI-MS-imaging of whole human lens capsule

The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens.     

Lamins of ocular lens epithelial cells

Experiments were performed to characterize a prominent nuclear matrix (NM) protein isolated from tissue cultured mouse lens epithelial cells. This NM protein was separated by SDS-PAGE and the stained gel band was analyzed by mass spectroscopy. Blast analysis of the amino acid sequence derived by mass spectroscopy revealed the presence of Lamin C in the NM of the mouse lens epithelial cells. We also examined nuclear proteins of adult and fetal human lenses. Data collected from these experiments showed the presence of Lamin C in both adult and fetal lens cells. However fetal lens cells only show Lamin C dimers, whereas adult human lens contained dimers, monomers and degraded Lamin C. Early and late passaged tissue cultured mouse lens epithelial cells also contained Lamin C in the nucleus with a preponderance of the dimer in the early passaged cells. The biological significance of the presence of dimers in human fetal lens cells and early passaged mouse lens cells is not known. However, it could suggest an enhanced docking capability of Lamin C dimers for other physiologically important nuclear proteins.     

Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel

Mechanical properties of a polyacrylamide gel with reversible DNA crosslinks are presented. In this system, three DNA strands replace traditional chemical crosslinkers. In contrast to thermoset chemically crosslinked polyacrylamide, the new hydrogel is thermoreversible; crosslink dissociation without the addition of heat is also feasible by introducing a specific removal DNA strand. This hydrogel is characterized by a critical crosslink concentration at which gelation occurs. Below the critical point, a characteristic temperature exists at which a transition in viscosity is observed. Both temperature-dependent viscosity and elastic modulus of the material are functions of crosslink density.     

A reversible hydrogel membrane for controlling the delivery of macromolecules

Glucose-sensitive hydrogel membranes have been synthesized and characterized for their rate-of-delivery of macromolecules. The mechanism for changing this rate is based on variable displacement of the affinity interaction between dextran and concanavalin A (con A). Our main objective was to characterize the diffusion of model proteins (insulin, lysozyme, and BSA) through the membrane, in response to changes in environmental glucose concentrations. Membranes were constructed from crosslinked dextrans to which con A was coupled via a spacer arm. Changes in the porosity of the resulting hydrogel in the presence of glucose led to changes in the diffusion rate observed for a range of proteins. Gels of specified thickness were cast around to nylon gauze support (pore size, 0.1 mm) to improve mechanical strength. Diffusion of proteins through the gel membrane was determined using a twin-chamber diffusion cell with the concentrations being continuously monitored using a UV-spectrophotometer. Changes in the transport properties of the membranes in response to glucose were explored and it was found that, while 0.1M D-glucose caused a substantial, but saturateable, increase in the rates of diffusion of both insulin and lysozyme, controls using glycerol or L-glucose (0.1M) had no significant effect. Sequential addition and removal of external glucose in a stepwise manner showed that permeability changes were reversible. As expected, diffusion rates were inversely proportional to membrane thickness. A maximum increase in permeability was observed at pH 7.4 and at 37 degrees C. The results demonstrate that this hydrogel membrane functions as a smart material allowing control of solute delivery in response to specific changes in its external environment.     

Identification of plasma proteins containing sulfite-reactive disulfide bonds

Plasma protein S-sulfonate compounds (RS-SO-3) have previously been shown to form, presumably by sulfitolysis of disulfide bonds, as a result of exposure to sulfite. In the investigations reported here, we identify two proteins in rabbit plasma, namely albumin and plasma fibronectin, which contain reactive sites for S-sulfonate formation. Separation and identification of these proteins following in vitro and in vivo exposure to sulfite was accomplished primarily by column chromatographic and electrophoretic techniques. In addition, the structure of presumed S-sulfonate groups was confirmed by the identification of cysteinyl-S-sulfonate residues in protein hydrolysates generated by enzymatic digestion. The molar ratio of RS-SO-3 in both albumin and plasma fibronectin was less than one. Data from our experiments suggest that the mixed disulfide site of non- mercaptalbumin is the reactive site for S-sulfonate formation. The site(s) of formation within the plasma fibronectin molecule was not investigated. The possible physiological significance of disulfide sulfitolysis of albumin and plasma fibronectin is discussed.     

Spatial distribution of glycerophospholipids in the ocular lens

Knowledge of the spatial distribution of lipids in the intraocular lens is important for understanding the physiology and biochemistry of this unique tissue and for gaining a better insight into the mechanisms underlying diseases of the lens. Following our previous study showing the spatial distribution of sphingolipids in the porcine lens, the current study used ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) to provide the whole lipidome of porcine lens and these studies were supplemented by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) of the lens using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to determine the spatial distribution of glycerophospholipids. Altogether 172 lipid species were identified with high confidence and their concentration was determined. Sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines were the most abundant lipid classes. We then determined the spatial and concentration-dependent distributions of 20 phosphatidylcholines, 6 phosphatidylethanolamines, and 4 phosphatidic acids. Based on the planar molecular images of the lipids, we report the organization of fiber cell membranes within the ocular lens and suggest roles for these lipids in normal and diseased lenses.     

Volume change of the ocular lens during accommodation

During accommodation, mammalian lenses change shape from a rounder configuration (near focusing) to a flatter one (distance focusing). Thus the lens must have the capacity to change its volume, capsular surface area, or both. Because lens topology is similar to a torus, we developed an approach that allows volume determination from the lens cross-sectional area (CSA). The CSA was obtained from photographs taken perpendicularly to the lenticular anterior-posterior (A-P) axis and computed with software. We calculated the volume of isolated bovine lenses in conditions simulating accommodation by forcing shape changes with a custom-built stretching device in which the ciliary body-zonulae-lens complex (CB-Z-L) was placed. Two measurements were taken (CSA and center of mass) to calculate volume. Mechanically stretching the CB-Z-L increased the equatorial length and decreased the A-P length, CSA, and lens volume. The control parameters were restored when the lenses were stretched and relaxed in an aqueous physiological solution, but not when submerged in oil, a condition with which fluid leaves the lens and does not reenter. This suggests that changes in lens CSA previously observed in humans could have resulted from fluid movement out of the lens. Thus accommodation may involve changes not only in capsular surface but also in volume. Furthermore, we calculated theoretical volume changes during accommodation in models of human lenses using published structural parameters. In conclusion, we suggest that impediments to fluid flow between the aquaporin-rich lens fibers and the lens surface could contribute to the aging-related loss of accommodative power. lens volume calculation; intralenticular fluid movement; presbyopia; mammalian lens     

Kinetics of Myo-inositol transport in rat ocular lens

Myo-inositol (MI) influx as a function of concentration in rat lens consisted of a saturable component, fit by a rectangular hyperbola, and a linear component which was more distinct at high myo-inositol concentrations suggesting passive diffusion. The hyperbolic component was half-maximally saturated (K_t) at 61.3 μM and had a maximal transport rate (J_max) of 44.6 μMol/kg wet wt/h. The linear component had an apparent permeability coefficient of 1.44 × 10^?6 s^?1. Sorbitol, which distributed rapidly in the extracellular space (6.83 ml/100 g wet wt), also appeared to enter the intracellular space with a permeability coefficient of 1.37 × 10^?6 s^?1, similar to that of myo-inositol. The influx of myo-inositol was critically dependent on the concentration of extracellular sodium consistent with a sodium-myo-inositol contransport. The kinetics of influx activation by sodium suggested an apparent 2:1 coupling ratio for sodium and myo-inositol. When potassium was used as sodium substitute, a significantly stronger influx inhibition was observed than with nondepolarizing sodium substitutes, indicating that myoinositol was driven by the electrochemicl gradient of sodium rather than the chemical gradient only. Reducing the extracellular Na concentration increased the MI concentration at which transport was half-maximally activated, suggesting an ordered binding sequence of Na followed by MI. Myo-inositol influx was competitively inhibited by phlorizin with an inhibitory coefficient (K_i) of 35 μM. Phloretin also was capable of inhibition but with a much lesser efficacy. Myoinositol desaturates from the lens at a rate of 0.00862 h^?1. Approximately 19% of the efflux can be inhibited with phlorizin, suggesting that it represents carrier-mediated flux. The phlorizin insensitive flux has a rate of 0.00695 h^?1 or 1.93 × 10^?6 s^?1, similar to the Na-independent passive influx. MI influx is due to a Na-dependent, phlorizin-sensitive active transport while the efflux consists largely of a phlorizin-independent passive leakage. © 1995 Wiley-Liss, Inc.     

Molecular dynamics simulations of the human ocular lens with age and cataract

The human ocular lens consists primarily of elongated, static fibers characterized by high stability and low turnover, which differ dramatically in their composition and properties from other biological membranes. Cholesterol (Chol) and sphingolipids (SL) are present at high concentrations, including saturated SLs, such as dihyrosphingomyelin (DHSM). Past molecular dynamics simulations demonstrated that the presence of DHSM and high Chol concentration contributes to higher order in lipid membranes. This current study simulated more complex models of human lens membranes. Models were developed representing physiological compositions in cataractous lenses aged 74 ± 6 years and in healthy lenses aged 22 ± 4, 41 ± 6, and 69 ± 3 years. With older age, Chol and ceramide concentrations increase and glycerophospholipid concentration decreases. With cataract, ceramide concentration increases and Chol and glycerophospholipid concentrations decrease. Surface area per lipid, deuterium order parameters (S_CD), sterol tilt angle, electron density profiles, bilayer thickness, chain interdigitation, two-dimensional radial distribution functions (2D-RDF), lipid clustering, and hydrogen bonding were calculated for all simulations. All systems exhibited low surface area per lipid and high bilayer thickness, indicative of strong vertical packing. S_CD parameters suggest similarly, with saturated tails in the hydrophobic core of the membrane having elevated order. Vertical packing and acyl tail order increased with both age and cataract condition. Lateral diffusion decreased with age and cataracts, with the older and cataractous models demonstrating increased long-range structure by the 2D-RDF analysis. In future work examining the membrane proteins of the lens, these models can serve as a physiologically accurate representation of the lens lipidome.     

Multiaxial mechanical behavior of the porcine anterior lens capsule

The biomechanics of the lens capsule of the eye is important both in physiologic processes such as accommodation and clinical treatments such as cataract surgery. Although the lens capsule experiences multiaxial stresses in vivo, there have been no measurements of its multiaxial properties or possible regional heterogeneities. Rather all prior mechanical data have come from 1-D pressure–volume or uniaxial force-length tests. Here, we report a new experimental approach to study in situ the regional, multiaxial mechanical behavior of the lens capsule. Moreover, we report multiaxial data suggesting that the porcine anterior lens capsule exhibits a typical nonlinear pseudoelastic behavior over finite strains, that the in situ state is pre-stressed multiaxially, and that the meridional and circumferential directions are principal directions of strain, which is nearly equibiaxial at the pole but less so towards the equator. Such data are fundamental to much needed constitutive formulations.     

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.