Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding.

The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity.

North Carolina macular dystrophy: clinicopathologic correlation.

PURPOSE: To describe the clinical and histopathologic findings of a 72-year-old female with North Carolina macular dystrophy. METHODS: Observational case report with histopathologic correlation. Clinical examination includes slit-lamp biomicroscopy, indirect ophthalmoscopy, color fundus photography, and focal electroretinography. Histopathologic examination of the enucleated left eye performed with light microscopy. RESULTS: Light microscopy demonstrated a discrete macular lesion characterized by focal absence of photoreceptor cells and retinal pigment epithelium with attenuation of the Bruch membrane and focal atrophy of the choriocapillaris. Adjacent to the macular lesion, some lipofuscin was identified in the retinal pigment epithelium. CONCLUSION: North Carolina macular dystrophy has both clinical and microscopic appearances of a well-demarcated lesion confined to the macula, which involves the retina, pigment epithelium, and choriocapillaris.

Acanthamoeba keratitis associated with fungal keratitis.

PURPOSE: To report fungal infection complicating Acanthamoeba keratitis. METHODS: Case report. A 45-year-old woman with contact lens-related bilateral Acanthamoeba keratitis developed corneal ulcer, corneal perforation, and mature cataract in the left eye, which was managed by penetrating keratoplasty, lensectomy, and vitrectomy. RESULTS: Histopathologic examination of the keratoplasty specimen from the left eye revealed extensive lamellar stromal necrosis with the coexistence of both empty cysts and branching hyphae. Cultures from the keratoplasty specimen grew Scedosporium apiospermum. CONCLUSION: Keratomycosis caused by S. apiospermum may complicate protracted Acanthamoeba keratitis.

Ocular involvement in patients with posttransplant lymphoproliferative disorder.

OBJECTIVES: To describe ocular disease in 3 patients with posttransplant lymphoproliferative disorder (PTLD) and to identify the frequency of such ocular involvement. METHODS: Medical record reviews. Using Kaplan-Meier analysis, we calculated the frequency of ocular involvement among pediatric patients with systemic PTLD after liver transplantation. RESULTS: Each patient had bilateral anterior chamber cells. Biopsy of an iris nodule from a patient who had undergone cardiac transplantation confirmed the diagnosis of PTLD, but no signs of systemic PTLD were found. The other 2 patients had systemic PTLD after liver transplantation; 1 presented with iris nodules in both eyes and a subretinal mass in the left eye, while the other had bilateral anterior chamber cells only. Ocular signs improved slowly after reduction of immunosuppressive drug therapy. Ophthalmological examinations were performed on 22 of 25 pediatric patients with PTLD after liver transplantation; 2 had ocular disease. Kaplan-Meier analysis indicated a 20% risk of ocular involvement at 3 years after development of PTLD (95% confidence intervals, 0%-50%). CONCLUSIONS: Posttransplant lymphoproliferative disorder should be considered in the differential diagnosis of uveitis after organ transplantation. Anterior chamber cells and iris nodules are the most common ocular signs, but the posterior segment can be involved. Ocular involvement can occur without evidence of systemic disease and can be asymptomatic. Reduction of immunosuppressive drug therapy is an appropriate treatment.

North Carolina macular dystrophy: clinicopathologic correlation.

PURPOSE: To describe the clinical and histopathologic findings in a 72-year-old woman with North Carolina macular dystrophy. METHODS: Clinical examination was performed by slit-lamp biomicroscopy, indirect ophthalmoscopy, color fundus photography, and focal electroretinography. Histopathologic examination of the enucleated left eye consisted of light microscopy. RESULTS: Light microscopy demonstrated a discrete macular lesion characterized by focal absence of photoreceptor cells and retinal pigment epithelium. Bruch's membrane was attenuated in the center of the lesion and associated with marked atrophy of the choriocapillaris. Adjacent to the central lesion, some lipofuscin was identified in the retinal pigment epithelium. CONCLUSIONS: North Carolina macular dystrophy has both clinical and microscopic appearances of a well-demarcated retinal and pigment epithelial lesion confined to the macula. This is consistent with the clinical impression that it is a focal macular dystrophy.

Functional cavity dimensions of tear lipocalin.

PURPOSE: We calibrated the cavity of tear lipocalin with a series of fluorescent labeled lipids of increasing chain length and varying diameter. METHODS: Cavity length was assessed with competitive fluorescent assays in which DAUDA was displaced from apo-tear lipocalin with ligands of increasing carbon chain lengths from C12-C24. The concentrations of competitors that inhibit 50% of the binding of DAUDA (IC(50)) were compared. Functional diameters of tear lipocalin and beta-lactoglobulin were estimated with fatty acids bearing fluorescent labels of various diameters. The cavity dimensions of other lipocalins were derived from their published crystal structure coordinates. RESULTS: In tear lipocalin, the binding affinities of fatty acids increased up to a carbon chain length of 18 (22.5 A) but remained constant from C18-C24. The cavity length of other lipocalins in crystal form were similar to tear lipocalin in solution. Tear lipocalin showed decreased binding affinities with progressively increasing ring dimensions of the ligand. In contrast to beta-lactoglobulin and retinol binding protein, tear lipocalin bound DAUDA and cholesterol in the calyx. Neither tear lipocalin nor beta-lactoglobulin bound P646 in their respective cavities. The calculated inter-sheet distances at the mouth of the crystallized lipocalins ranged from 16-22A. CONCLUSIONS: Tear lipocalin is more promiscuous than beta-lactoglobulin or retinol binding protein because of a greater functional diameter. Differences in ligand specificity of the various lipocalins can not be explained simply by variation in cavity length or the intersheet distances at the calyx mouths as determined by crystal structure. Other factors may influence ligand specificity such as size and/or dynamic motion of loops between the beta strands.

Bilateral blindness and lumbosacral myelopathy associated with high-dose carmustine and cisplatin therapy.

PURPOSE: To report the early ocular pathologic findings associated with high-dose carmustine and cisplatin therapy. METHODS: A patient with metastatic breast carcinoma developed an acute onset of branch retinal artery occlusion, bilateral blindness, and a myelopathy involving the lower extremities after high-dose chemotherapy and bone marrow transplant. RESULTS: Histopathologic examination of the eye and optic nerves at autopsy disclosed nerve fiber layer infarction secondary to right inferior temporal retinal artery thrombosis. Patchy necrosis of both optic nerves, medulla oblongata, and spinal cord was associated with focal small-vessel thrombosis. CONCLUSIONS: The syndrome of retinal vascular occlusion, optic neuropathy, and myelopathy is associated with the high-dose chemotherapeutic agents carmustine and cisplatin. The distribution of necrosis suggests an ischemic event rather than direct neurotoxic effects.

Chronic exposure of hydroxyapatite orbital implants: cilia implantation and epithelial downgrowth.

PURPOSE: To describe previously unreported histologic findings in two patients who developed chronic implant exposure and abscess formation within hydroxyapatite orbital implants. METHODS: Surgically removed implant specimens were processed for histopathologic examination and stained for microorganisms. Each patient's clinical course, socket appearance, and exposure management were reviewed. RESULTS: Histopathologic examination of case 1 showed a channel of necrosis leading from the anterior surface of the implant to its center. Hair shafts were discovered embedded within this channel. Histopathologic examination of the site of chronic exposure in case 2 showed epithelial ingrowth into the pores of the implant. Both spheres in this report indicated limited fibrovascular ingrowth and abscess formation. CONCLUSIONS: Chronic exposure of hydroxyapatite implants allows a portal of entry for extraneous hair shafts and also can lead to epithelial downgrowth. Both of these may be contributing factors in the development of serious implant infections.

Corneal transplantation in a patient with mucopolysaccharidosis type VII (Sly disease).

PURPOSE: To illustrate a good visual outcome following penetrating keratoplasty in a patient with Sly disease, a rare mucopolysaccharidosis (MPS) caused by a deficiency of beta-glucuronidase. METHODS: A 15-year-old male with progressive bilateral corneal opacification had a complete medical, genetic, and ophthalmic evaluation followed by a penetrating keratoplasty. RESULTS: The cornea has remained clear for two years following surgery. Histopathology of the corneal button demonstrated vacuoles and granular inclusions consistent with this lysosomal storage disease. CONCLUSION: While research is ongoing in the fields of enzyme replacement and bone marrow transplantation, these treatments may not alleviate or reverse the corneal clouding. This case illustrates that cornea transplantation may be a valuable treatment option for visually rehabilitating such patients.

Endonuclease activity in lipocalins.

Several lipocalins contain conserved amino acid sequences similar to the phosphodiester bond cleavage domain of sugar non-specific magnesium-dependent nucleases of the Serratia marcescens type. His-89 and Glu-127 of the S. marcescens endonuclease are believed to have a role in the active catalytic site by the attack of a water molecule at the phosphorus atom of the bridging phosphate. Tear lipocalin contains both amino acids in analogous regions, and is active as a nuclease. Two forms of beta-lactoglobulin contain only Glu-134 (analogous to Glu-127 of the Serratia nuclease) yet retain nuclease activity equal to or greater than that of tear lipocalin. However, retinol-binding protein lacks both of these motifs and shows no detectable activity. DNA-nicking activity is decreased by 80% in the mutant of tear lipocalin that replaces Glu-128 but is unchanged by mutations of His-84. The endonuclease activity of tear lipocalin is dependent on the bivalent cations Mg(2+) or Mn(2+) but is decreased at high concentrations of NaCl. These findings indicate that some lipocalins have non-specific endonuclease activity similar in characteristics to the Mg(2+)-dependent nucleases and related to the conserved sequence LEDFXR (where 'X' denotes 'any other residue'), in which the glutamic residue seems to be important for activity.

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.

Secretory lipophilins: a tale of two species.

Secretory lipophilins are "lipid-loving" proteins that are major constituents of several mammalian secretions, including the prostatic fluid of rats and the tears of humans and rabbits. These proteins form covalent heterodimers that are stabilized by three intramolecular cystine disulfide bonds. The heterodimers, some of which are glycosylated, may undergo additional non-covalent assembly to form tetramers. The peptide components found in secretory lipophilins are from two subfamilies: lipophilins A/B and lipophilin C. The C subfamily members described in this report are three rabbit and one human lipophilin, plus human mammaglobin and the C3 subunit of rat prostatein. Human A/B and C lipophilins are expressed by many tissues and are especially prominent in endocrine-responsive organs. The gene for human lipophilin B resides at chromosome 10q22-23. This region harbors the PTEN/MMAC1 gene and is believed to contain additional tumor suppressor genes. Although the functions of secretory lipophilins are imperfectly understood, their abundance in glandular secretions and in hormone-responsive tissues suggests that they deserve considerably more attention than they have received to date.

Tear lipocalins: potential lipid scavengers for the corneal surface.

PURPOSE: To investigate the dynamic effect of tear lipocalins (TLs), the major lipid-binding protein in tears, at aqueous-cornea and lipid-aqueous interfaces, and their potential contribution to surface tension in the tear film. METHODS: Human apo- and holo-TLs were applied to the aqueous subphase in a Langmuir trough, and changes in surface pressure were measured. Changes in the contact angle of tear components were observed on Teflon and ferric-stearate-treated surfaces. A nitroxide-labeled derivative of lauric acid and a fluorescence-labeled derivative of palmitic acid were used to monitor the dynamic interaction of lipid removed from a hydrophobic surface by the major tear components in solution. RESULTS: TLs increase the surface pressure at the aqueous-air interface by penetrating, spreading, and rearranging on the surface. Apo-TLs show a longer diffusion-dependent induction time than holo-TLs due to more extensive oligomerization of the apoprotein. Kinetic analysis of relaxation time suggests that apo-TLs have more rapid surface penetration and rearrangement than holo-TLs, indicative of a more flexible structure in apo-TLs. TLs reduce the contact angle of solutions on lipid films, a property that is greater with TLs than other tear proteins. TLs, unlike lysozyme and lactoferrin, remove labeled lipids from hydrophobic surfaces and deliver them into solution. CONCLUSIONS: TLs are potent lipid-binding proteins that increase the surface pressure of aqueous solutions while scavenging lipids from hydrophobic surfaces and delivering them to the aqueous phase of tears. These data suggest important functional roles for TLs in maintaining the integrity of the tear film.

Interaction of tear lipocalin with lysozyme and lactoferrin.

The interaction of human tear lipocalin with lysozyme and lactoferrin was studied by electron paramagnetic resonance (EPR) spectroscopy. TL mutants I98C and F99C were spin labeled with MTSL and its derivative. The spectra demonstrated that at sites C98 and C99 the mobility of the nitroxides was reduced in the presence of lysozyme, lactoferrin, but not albumin. The reduced mobility was manifested as a reduction in side chain motion and backbone fluctuations. The overall correlation time of tear lipocalin, measured by MTSL derivative-labeled F99C, was prolonged in the presence of lysozyme and lactoferrin indicating that the interaction involves direct contact. The effect was mitigated at high salt concentration suggesting an electrostatic interaction of the molecules. The reduction in side chain mobility at C98 and C99 of tear lipocalin was observed in tears. Taken together, the data indicate that tear lipocalin interacts with both lysozyme and lactoferrin and suggest that they may function in concert with one another.

Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling.

Side chain mobility, accessibility, and backbone motion were studied by site-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and oxygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K(3)Fe(CN)(6), exhibited two nitroxide populations for most residues. The motionally constrained population was relatively less accessible to K(3)Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contribution of the immobile component was greater, indicating a larger population with tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound within the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MTSL derivative (I) attached to F99C resulted in the lack of side chain motion and therefore reflects the overall rotation of the TL complex. The correlation time of F99C in tears (13.5 ns) was the same as that in buffer and indicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, indicating that the fatty acids are not rigidly anchored in the cavity, but move within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDDA were performed to determine the orientation and depth of a series of fatty acid derivatives in the cavity of TL. Fatty acids are oriented with the hydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the nitroxide varied according to position such that nitroxides near the mouth had greater mobility than those located deep in the cavity. Nitroxides positioned up to 16 carbon units from the hydrocarbon tail of the ligand are motionally restricted and inaccessible, indicating the cavity extends to at least this depth. EPR spectra obtained with and without sucrose showed that the intracavitary position of lauric acid in TL is similar to that in beta-lactoglobulin. However, unlike beta-lactoglobulin, TL binds 16-doxyl stearic acid, suggesting less steric hindrance and greater promiscuity for TL.

Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity.

The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine+ were substituted for TRP 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss os secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.

Bilateral periopticointrascleral hemorrhages associated with traumatic child abuse.

PURPOSE: To report a case of bilateral periopticoscleral hemorrhages associated with traumatic child abuse. METHODS: Postmortem gross examination and histopathologic studies of both eyes and the optic nerves of a 6-month-old infant who died from subdural hematoma. RESULTS: Gross examination and histopathologic step sections disclosed bilateral intrascleral hemorrhages around both optic nerves. In addition, bilateral diffuse multilayered retinal, vitreous, and sublaminar (beneath the internal limiting membrane) hemorrhages were present. CONCLUSION: Periopticointrascleral hemorrhages are characteristic of blunt head trauma and may constitute important forensic evidence in cases of suspected child abuse.

Lipophilins: human peptides homologous to rat prostatein.

Lipophilin components A, B and C are human homologues of prostatein, the major secreted protein of rat prostate. This report describes their cDNA sequences, tissue expression and chromosomal localization. Lipophilin gene products were widely expressed in normal tissues, especially in endocrine-responsive organs. The gene for lipophilin C (also called mammaglobin b) is located on chromosome 11q12-q13.1, near the mammaglobin gene, a homologue overexpressed in many breast cancers. The lipophilin B gene resides on chromosome 10q23, a region deleted in many tumors, and the lipophilin A gene is on chromosome 15q12-q13.

Nonvascular contractile cells in sclera and choroid of humans and monkeys.

PURPOSE: To investigate by histochemistry and immunohistochemistry the distribution and innervation of nonvascular contractile cells in the sclera and choroid of humans and monkeys. METHODS: Globes were obtained from 2 macaque monkeys and 19 human cadavers that ranged in age from fetal life to 94 years. Immunohistochemistry was performed using monoclonal antibody against human smooth muscle (SM) alpha-actin and tyrosine hydroxylase (TH). The nicotinamide-adenine dinucleotide phosphate (NADPH)- diaphorase reaction was used as a marker for nitric oxide synthase. RESULTS: The scleras of all but fetal, newborn, and infant globes exhibited myofibroblasts, amelanotic, fibroblastlike cells having SM alpha-actin immunoreactivity. In the choroid of all but fetal eyes, SM cells were present in the suprachoroidal layer, forming a reticulum of flattened laminae, and in the choriocapillaris where ovoid-to-spindle-shaped SM cells were arrayed in parallel layers immediately adjacent to Bruch's membrane. Contractile cells in the sclera and choroid were most concentrated subfoveally and were sparse anteriorly. Nerve terminals positive for NADPH- diaphorase were colocalized with SM alpha-actin-positive cells in the sclera and choroid, whereas TH-positive nerve terminals colocalized with SM cells in the choroid. Clusters of ganglion cells were present on the posterior surface of globes near SM cells. CONCLUSIONS: The posterior choroid and sclera of humans and monkeys contain nonvascular contractile cells. The presence of nerve terminals and adjacent ganglion cells suggests neural control of these contractile cells. The absence of such contractile cells in fetal, newborn, and infant eyes is an argument against a major role of these cells in promoting ocular enlargement. These contractile cells may instead participate in regulation of refractive state by maintenance of ocular size in the face of intraocular pressure or in intermediate-term regulation of choroidal thickness.