[Efficiency of vitrectomy in diabetic macular edema and morphometry of surgically removed of the internal limiting membrane]. / Význam vitrektomie u diabetického makulárního edému a morfometrie chirurgicky odstranené vnitrní limitující membrány.

AIM: To evaluate the efficiency of vitrectomy with peeling of the internal limiting membrane (ILM) in eyes with diffuse and/or cystoid nontractional diabetic macular edema (ME) refractory to macular laser photocoagulation. Histopathologic and morphometric analysis of the ILM in diabetic eyes was performed. Thickness of the ILM was correlated with the level of glykosylated hemoglobin (HbA1c) and other clinical factors. MATERIAL AND METHODS: The prospective study involved 56 eyes of 52 diabetic patients with a mean age of 63 +/- 7.6 years. Vitrectomy with trypan blue-assisted ILM peeling was performed in standard way. Mean follow-up period was 8.7 months (range 3 - 19 months). The ILM was fixed immediately after peeling in 2.5% glutaraldehyde and submitted for electron microscopic evaluation. The ILM was photographed in standard magnification (x 5000) with the scale of 1 microm in the shot. RESULTS: Statistical analysis of the postoperative visual acuity (VA) in the study group of 56 eyes proved a general improvement with prevalence of the resulted VA in intervals 0.1-0.2 and 0.5-1.0 related to ETDRS chart. The VA was improved by at least 2 lines in 29 eyes of 56 eyes (51.8%), one line in 6 eyes (10.7%) and remained unchanged in 11 eyes (19.6%). The postoperative VA deteriorated by one line in 2 eyes (3.6%) and at least 2 lines in 8 eyes (14.3%). Morphometric analysis demonstrated a significant thickening of the ILM in all eyes with a mean thickness of the ILM 3.61 +/- 1.22 micro m. It was found that a higher thickness of the ILM is related to elevated HbA1c by both types of diabetes mellitus (DM) (p = 0.040). We also found significant dependence of ILM thickness in relation to duration of DM by comparison of men and women (p = 0.026) and significant correlation between ILM thickness and the age of diabetic patients related to their gender (p = 0.029). CONCLUSIONS: Vitrectomy with peeling of the ILM in eyes with chronic diffuse and/or cystoid nontractional diabetic ME mildly improves the VA and extends a hope for its stabilization. We confirmed increased thickness of the surgically peeled ILM and statistically significant correlations to elevated HbA1c by both types of DM and to further clinical characteristics of case series. Morphometric and histopathologic analyses of the ILM contribute to more objective evaluation of ultrastructure of the vitreomacular interface.

[Malignant masquerade syndromes]. / Maligní maskující syndromy.

PURPOSE: To evaluate our experience with the diagnosis and treatment of malignant masquerade syndromes. METHODS: A retrospective study of 46 patients treated for malignant masquerade syndromes at our Department for Diagnosis and Treatment of Uveitis, 1st Faculty of Medicine in Prague, between 1995 and 2008, was performed. RESULTS: Eighty-nine patients with masquerade syndromes (7.2%) from all 1233 patients with uveitis were included. Malignant masquerade syndromes were recognized in 46 patients (22 females and 24 males, mean age 55 years). The most frequent cause of malignant masquerade syndromes was intraocular non-Hodgkin lymphoma (26 patients). The primary diagnosis was idiopathic uveitis in many cases. The most valuable diagnostic procedure was analysis of intraocular fluids. CONCLUSION: Diagnosis of masquerade syndromes should be considered in all patients with idiopathic corticosteroid-resistant chronic uveitis. Timely diagnosis and treatment may in case of malignant masquerade syndromes improve prognosis and sometimes gain control over this potentially lethal disease.

[TINU syndrome]. / TINU syndrom.

PURPOSE: To evaluate our experience with the diagnosis and the treatment of the TINU syndrome. The term TINU syndrome means the intraocular inflammation occurring in association with tubulointerstitial nephritis. The predominance of younger females was established. The uveitis is frequently chronic, moderate, and bilateral. The treatment with immunosuppressive drugs often has positive clinical response. METHODS: A retrospective study. RESULTS: Five patients, 4 women and 1 man, have been examined and treated for the TINU syndrome in our Department for Diagnosis and Treatment of Uveitis. The age ranged from 8 to 62 years. The treatment with immunosuppressive drugs caused reduction of the inflammatory reaction and visual acuity improvement. CONCLUSION: Immunosuppressive drugs were effective in all of our patients suffering from the TINU syndrome. The cooperation between ophthalmologist and nephrologist is crucial for the effective control of the disease activity and for drug regimen optimization.

[The frosted retinal vascular branch syndrome]. / Syndrom omrzlých vetví retinálních cév.

In January 2002, thirty-eight years old healthy man with unusual clinical signs of vasculitis was examined in the Center for Diagnosis and Treatment of Uveitis in our Department of Ophthalmology. The patient had a flu-like disease one month before the signs of the eye disease started. In the beginning of the disease, the visual acuity (VA) was 6/12 (20/40 or 0.5) for distance and Jaeger 8 for near and 6/6 (20/20 or 1.0) and Jaeger 1 respectively. The clinical appearance was similar to this of frosted branch angiitis. The thorough medical examination did not discover link between vasculitis and any systemic disease. Serologic examination discovered positivity of IgG immunoglobulin antibody against cytomegalovirus (CMV) only. The intraocular fluid sampling to confirm CMV antibody presence was denied by the patient. After starting the treatment with gancyclovirus and corticosteroids, the considerable improvement of clinical signs of the frosted branch angiitis was noticed. VA improved after five days of treatment. During one-year follow-up, neither recurrence of vasculitis nor the decline of VA of both eyes was marked. In Czech ophthalmologic literature, we did not find any article referring to frosted branch angiitis, so we took the liberty of offering the Czech term "syndrom omrzlých vetví retinálních cév".

[Intraocular lymphoma--a clinical study of 14 patients with non-Hodgkin's lymphoma]. / Nitroocní lymfom--klinická studie 14 pacientu s NHL.

AIM: To evaluate the course of clinical picture of intraocular lymphoma, possibilities of examination of this disease and the association with general symptoms of the non-Hodgkin lymphoma (NHL) in patients with manifestations of uveitis. PATIENTS AND METHODS: A retrospective study in 14 patients followed in the period of 1996-2001 in the Center for Diagnostics and Therapy of Uveitis, Ocular Clinic of General Faculty Hospital and 1st Medical Faculty, Charles University in Prague. RESULTS: The group included 7 women and 7 men at the average age of 57.3 years (18-82) with clinical picture of uveitis. The time period from the first symptoms to the diagnosis of NHL was 5-38 months (mean, 14.6 month). In two patients the diagnosis of systemic NHL preceded ocular symptoms, in four other patients the diagnosis of systemic or central nervous system NHL (CNS NHL) was established during ocular manifestations of uveitis. Intraocular lymphoma was the only first manifestation of CNS NHL in six patients for the period of 9-34 months (mean, 15.2). Two patients have been so far affected by primary intraocular lymphoma (PIL) for 8 and 14 months respectively, and presently do not display any signs of systemic or CNS NHL. Clinical signs of intraocular inflammation of both eyes were encountered in 71.4% of patients. Vitritis (85.7%) and tumor infiltration of retina (65.3%) were the most frequent manifestations of NHL. Intraocular NHL was diagnosed on the basis of cytological examination of samples of intraocular fluids in 8 patients (57.1%). In four patients radiotherapy was applied onto 5 eyes and in 5 patients radiotherapy of CNS was used. Nine patients were treated with chemotherapy. 50% of patients died until the end of 2001, the survival from the establishment of diagnosis was 20.6 months on the average. CONCLUSIONS: Intraocular lymphoma should be considered as the eye and life-threatening disease. Cytological examination of intraocular fluids in patients with uveitis who do not respond to the therapy with steroids in the usual way may give more precision and shorten the establishment of diagnosis in this masquerade syndrome. Early diagnosis and therapy may improve the prognosis of NHL.

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.

Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4'-bipyridine and by a coumarin derivative.

Inhibition of recombinant mouse wild type AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), and AChE peripheral site-directed mutants and human serum BChE variants by 4,4'-bipyridine (4,4'-BP) and the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) was studied. The enzyme activity was measured with acetylthiocholine as substrate. Enzyme-inhibitor dissociation constants for the catalytic and peripheral sites were evaluated from the apparent dissociation constants as a function of the substrate concentration. Inhibition by 4,4'-BP of AChE, BChE and the AChE mutant Y72N/Y124Q/W286A, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than for the catalytic site. CHMC displayed binding only to the catalytic site of Y72N/Y124Q/W286A mutant and only to the peripheral site of w.t. AChE and the human usual BChE. Modelling of the 4,4'-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic studies.

An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes.

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic.

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.