Thymosin beta4 and its posttranslational modifications.

Thymosin beta(4) as well as the other members of the beta-thymosin family are important G-actin sequestering peptides. The chemical properties, the biosynthesis, and posttranslational modifications (PTMs) of these peptides are discussed. During biosynthesis of thymosin beta(4) the initiator methionine is removed and the N-terminus is acetylated. Research on proteomics revealed several acetylated lysine residues and two phosphorylated threonine residues. The enormous number of phosphorylable and acetylable sites in the human proteome raises the question about the biological significance of these PTMs in the context of beta-thymosins. Presently, this question cannot be answered because neither the concentration of these modified beta-thymosins in cells is known nor the consequences of the modifications on the biological function(s) of beta-thymosins have been studied yet. Thymosin beta(4) is also posttranslationally modified by transglutaminase forming covalent bonds with other molecules. Prolyl oligopeptidase generates ac-SDKP from thymosin beta(4). The concentration of C-terminal peptide fragments of thymosin beta(4) is elevated in the blood of patients with rheumatoid arthritis.

Determination of thymosin beta4 and protein in human wound fluid after abdominal surgery.

Wound fluids were collected up to 60 h after abdominal surgery. Immediately after obtaining the wound fluid by Robinson drainage, wound fluid was centrifuged to remove blood cells and inflammatory cells. The concentration of total protein as well as of thymosin beta(4) was determined in the cell-free supernatant solution. Total protein concentration decreased from about 50 g/L to 30 g/L within 60 h after surgery. After surgery we observed a concentration of up to 20 mg thymosin beta(4) per liter decreasing to about 1 mg/L with time. Neither thymosin beta(10) nor oxidized thymosin beta(4) was detected in human wound fluid.

beta-Thymosins.

The development of thymosin beta(4) from a thymic hormone to an actin-sequestering peptide and back to a cytokine supporting wound healing will be outlined. Thymosin fraction 5 consists of a mixture of polypeptides and improves immune response. Starting with fraction 5, several main peptides (thymosin alpha(1), polypeptide beta(1), and thymosin beta(4)) were isolated and tested for biological activity. However, none of the isolated peptides were really thymic hormones. They are all biological important peptides with diverse functions. Polypeptide beta(1) is identical to ubiquitin truncated by two C-terminal glycine residues. Several peptides related to thymosin beta(4) were isolated and sequenced from various species. Large amounts of thymosin beta(4) were found in many cells. It was postulated that the beta-thymosins might have a general function. The identification of a biological function of thymosin beta(4) was tedious. In 1990, Dan Safer and his colleagues recognized that thymosin beta(4) sequesters G-actin. The dissociation constant of the complex in the micromolar range allows for fast binding and release of G-actin. beta-Thymosins are the main intracellular G-actin-sequestering peptides in most vertebrate cells. Thymosin beta(4) is unstructured but folds into a stable conformation on binding to G-actin. It is present in the nucleus as well as the cytoplasm and might be responsible for sequestering nuclear actin. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to ac-SDKP possibly generated from thymosin beta(4). However, very little is known about molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

Chemotherapeutic drugs change actin skeleton organization and the expression of beta-thymosins in human breast cancer cells.

PURPOSE: Elevated expression of the beta-thymosin isotypes T beta(4), T beta(10), and T(15) appears to be involved in the manifestation of a malignant phenotype of human tumor cells, including those of mammary carcinomas. This has evoked an interest in these peptides as diagnostic/prognostic tumor markers. If increased levels of beta-thymosins correspond to tumor malignancy, the question arises whether tumor growth inhibition induced by chemotherapeutic drugs would reduce their expression. METHODS: Two human breast cancer cell lines, the estrogen receptor(ER)-positive MCF-7 and the ER-negative MDA-MB231, were thus analyzed for the amount of beta-thymosin mRNAs by RNase protection assay and for the respective peptide levels by HPLC following different hormonal and drug treatments. RESULTS: Both cell lines, growing in medium with 10% FCS, contain T beta(4) (400-500 fg/cell) and Tbeta(10) (about 100 fg/cell), but no T beta(15). Incubating MCF-7 cells with tamoxifen (1 microM) for 5 days resulted in about 80% growth inhibition and in reduction of intracellular T beta(4) and T beta(10) concentrations by about 40%. Levels of T beta(4) and T beta(10)-mRNA were reduced by about 60%. In contrast, cisplatin (2 microM) changed neither the peptide concentrations nor the mRNA levels of beta-thymosins, in spite of marked growth inhibition. In addition, no changes in beta-thymosin expression were observed in MDA-MB231 cells treated with either drug. MCF-7 cells maintained in estrogen-poor medium (10% horse serum) or stimulated to grow with estradiol (1 nM) had Tbeta(4) and T beta(10) concentrations reduced by about 30%, but changes in T beta(4)- and T beta(10)-mRNA levels did not correspond to those of the peptide. CONCLUSION: Expression of T beta(4) and T beta(10) mRNAs and their peptides is differentially regulated and does not correlate with growth. Instead, reduced beta-thymosin expression may be linked to more intensive TRITC-phalloidin staining of F-actin lining the membrane at sites of intimate cell-cell contacts, while increased beta-thymosin levels appear in cells with more extensive substrate adhesion. This suggests that beta-thymosins play a role in cell surface dynamics.

beta-Thymosins, small acidic peptides with multiple functions.

The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin.

Thymosin beta(4) possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin beta(4) serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin beta(4) may serve as a useful tool for further investigations in cell biology. Thymosin beta(4) could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin beta(4) concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin beta(4) may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.

A novel phosphopantetheine:protein transferase activating yeast mitochondrial acyl carrier protein.

In Saccharomyces cerevisiae, the low molecular weight acyl carrier protein (ACP) of mitochondrial type II fatty acid synthase (FAS) and the cytoplasmic type I FAS multienzyme contain 4'-phosphopantetheine as a prosthetic group. Sequence alignment studies with the recently isolated phosphopantetheine:protein transferase (PPTase), Ppt1p, from Brevibacterium ammoniagenes revealed the yeast open reading frame, YPL148C, as a potential PPTase gene (25% identical and 43% conserved amino acids). In accordance with this similarity, pantetheinylation of mitochondrial ACP was lost upon disruption of YPL148C. In contrast, biosynthesis of cytoplasmic holo-FAS remained unaffected by this mutation. According to these characteristics, the newly identified gene was designated as PPT2. Similar to ACP null mutants, cellular lipoic acid synthesis and, hence, respiration were abolished in PPT2 deletants. ACP pantetheinylation, lipoic acid synthesis, and respiratory competence were restored upon transformation of PPT2 mutants with cloned PPT2 DNA. In vitro, holo-ACP synthesis was achieved by incubating apo-ACP with coenzyme A in the presence of purified Ppt2p. The homologous yeast enzyme could be replaced, in this assay, by the ACP synthase (EC 2.7.8.7) of Escherichia coli but not by the type I FAS-specific PPTase of B. ammoniagenes, Ppt1p. These results conform with the inability of Ppt2p to activate the cytoplasmic type I FAS complex of yeast.

The dipyridyls paraquat and diquat attenuate the interaction of G-actin with thymosin beta4.

Beta-thymosins sequester G-actin and preserve a pool of monomers of actin which constitute an important prerequisite for cellular function of the microfilament system. To study the influence of paraquat binding to G-actin on the interaction of G-actin with thymosin beta4 we determined the apparent dissociation constant of the G-actin-thymosin beta4 complex in the absence or presence of paraquat using an ultrafiltration assay. Paraquat (1,1'-dimethyl-4,4'-dipyridylium dichloride) attenuates this interaction in a concentration- and time-dependent manner. When exposed to 10 mM paraquat, the apparent dissociation constant increased 10-85-fold within 15 min to 24 h. After incubation for 24 h even a paraquat concentration as low as 100 microM increased the dissociation constant of the G-actin-thymosin beta4 complex from 0.66 microM to 0.82 microM (P < 0.05). Diquat (1,1'-ethylene-2,2'-dipyridylium dibromide) similarly weakens the interaction of G-actin and beta-thymosins. In none of the experiments was oxidation of the methionine residue or any other modification of thymosin beta4 detected. Therefore we conclude that the dipyridyls paraquat and diquat directly interact with G-actin and thereby impede the interaction between G-actin and thymosin beta4.

Plant profilin induces actin polymerization from actin : beta-thymosin complexes and competes directly with beta-thymosins and with negative co-operativity with DNase I for binding to actin.

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin beta4 and beta9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tbeta4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tbeta4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle alpha-actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross-linking indicated that birch profilin competes with Tbeta4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross-linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co-operativity between the profilin and DNase I binding sites on actin.

C-terminal truncation of thymosin beta10 by an intracellular protease and its influence on the interaction with G-actin studied by ultrafiltration.

Two beta-thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen. The portion of this peptide increased when the tissue was first frozen and then thawed at 4 degrees C. Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid. By means of amino acid analysis and MALDI-mass spectroscopy this peptide was identified to be a C-terminally truncated form of thymosin beta10. Having studied the formation in more detail we found that after a 4-h thaw at 4 degrees C all thymosin beta10 was truncated to thymosin beta10(1-41), which was further degraded during the next 20 h. On the other hand, thymosin beta4Ala, the second beta-thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin beta10 is formed by a carboxydipeptidase while thymosin beta4Ala is rather stable against proteolytic modification. By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G-actin and these three beta-thymosins to be 0.28, 0.72, and 0.94 microM for thymosin beta4Ala, beta10, and thymosin beta10(1-41), respectively. The complex with beta4Ala is unambiguously more stable than the complex with beta10 or beta4 (0.81 microM). The change in the dissociation constant generated by the truncation of the two C-terminal amino acid residues of beta10 is small but statistically significant. This demonstrates that even the very last amino acid residues at the C-terminus of beta-thymosins are involved in the interaction with G-actin.

Mapping the binding site of thymosin beta4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin.

The beta-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin beta4 was determined by analysing the binding of thymosin beta4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin beta4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin beta4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin beta4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin beta4 was obtained only when using the chemically cross-linked actin-thymosin beta4 complex, indicating that thymosin beta4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin beta4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

Microheterogeneity of the hydrophobic and hydrophilic part of the glycosylphosphatidylinositol anchor of alkaline phosphatase from calf intestine.

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl-(Etn-P)2-glycosyl-PtdIns with HF generated 8 glycosyl-Ins species the largest of which (G1 and G2) have the following proposed structures: [sequence: see text] G3 and G5 are lower homologues of G1 and G2, respectively, being one alpha 1-2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N-acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic ManxGlc-Ins species are either substituted with an N-acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular-mass determination by matrix-assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO3. Purification of the glycosylinositol species was achieved by a novel reverse-phase HPLC technique using fluorescent fluoren-9-yl-methoxy-carbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level. We observed recently that native alkaline phosphatase separates on octyl-Sepharose into four distinct fractions of increasing hydrophobicity (F1-F4). Here we show that all four fractions contain G1-G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10% which is in contrast to most glycosyl-PtdIns--anchored proteins of mammalian origin.

Targeting of liver metastases of colorectal cancer with IgG, F(ab')2, and Fab' anti-carcinoembryonic antigen antibodies labeled with 99mTc: the role of metabolism and kinetics.

The aim of this study was to investigate targeting of the liver metastases by directly 99mTc-labeled complete (IgG) and fragmented antibodies [F(ab')2 and Fab'] in relation to their kinetics and metabolic fate. A total of 127 patients with metastatic colorectal cancer were examined [IgG1, BW 431/26 (Behringwerke, Marburg, Germany) n = 50; F(ab')2, F023C5 (Sorin Biomedica, Saluggia, Italy) n = 58; Fab', IMMU-4 (Immunomedics, Morris Plains, NJ) n = 19]. Native monoclonal antibodies (MAbs), serum samples from 10 min to 24 h postinjection (p.i.), and urine were analyzed by gel filtration chromatography. Kinetic data were deduced from whole-body and single-photon emission computed tomographic scans, performed 10 min to 24 h p.i. (region-of-interest technique). In BW 431/26, 96% of injected activity was labeled IgG1; in F023C5, 29% was F(ab')2, and 71% was Fab'; and in IMMU-4, 92% was Fab', and 8% was F(ab')2. Serum half-lives were: IgG1, 36 h (liver uptake predominant); F(ab')2, 16 h; and Fab', 4 h (renal uptake predominant). All MAbs were metabolized, fragments more rapidly than IgG, to low-molecular-weight products and excreted into the urine (e.g., Tc-cystine). In targeting liver metastases, sensitivities were found to be higher for fragments (44.1, 72.5, and 80% for BW 431/26, F023C5, and IMMU-4, respectively) but at significantly lower tumor:background ratios than with IgG (1.78 +/- 0.29 versus 1.29 +/- 0.11 and 1.43 +/- 0.53; P < 0.01). With IgG, there was a continuous tumor uptake over 24 h, whereas with fragments, the maximal uptake occurred mostly within 1 h, with subsequent clearance being slower for antigen-bound activity than for nonspecific background. Hence, diagnosis was possible mostly after 4 h with fragments but often not before 24 h with IgG. These results show that the higher sensitivity of fragments in liver lesion targeting at earlier p.i. times does not rely on an increased antibody uptake but on a more rapid clearance of nonspecific background activity due to faster metabolism and excretion. Intact MAbs show a slow, continuous uptake, leading to higher tumor:background ratios at later p.i. times, often beyond the imaging possibilities of 99mTc.

Interactions of beta-thymosins, thymosin beta 4-sulfoxide, and N-terminally truncated thymosin beta 4 with actin studied by equilibrium centrifugation, chemical cross-linking and viscometry.

All beta-thymosins studied interact with G-actin in a bimolecular complex and inhibit the polymerization to F-actin under high salt conditions. The interactions between actin and beta-thymosins have been studied under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equilibrium centrifugation of unlabeled G-actin, viscometry, and chemical cross-linking to investigate the interactions with several beta-thymosins, oxidized thymosin beta 4 and N-terminally truncated beta 4. The apparent dissociation constants for actin from bovine heart and beta-thymosins were 2.5, 0.1, and 2.7 microM for thymosin beta 4, [Ala1]beta 4(beta Ala4), and beta 10, respectively. Comparable apparent dissociation constants were obtained for the interaction of G-actin from rabbit skeletal muscle and thymosin beta 4 or beta Ala4. In rabbits thymosin beta Ala4 replaces beta 4 being different in amino acid residue 1 only. The apparent dissociation constant of thymosin beta 10 with actin from rabbit skeletal muscle, however, is about 10% of the value obtained with actin from bovine heart. Oxidation of thymosin beta 4 at Met6 (beta 4-sulfoxide) as well as truncation of 6 [beta 4-(7-43)] or 12 [beta 4-(13-43)] amino acid residues from the N-terminus increase apparent dissociation constants to 38-53 microM. Truncation of the first 23 amino acid residues [beta 4-(24-43)] abolishes interaction with G-actin completely. Therefore, amino acid residues between position 13 and 24 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl)-carbodiimide cross-linking of G-actin. In spite of comparable apparent dissociation constants between actin and thymosin beta 4-sulfoxide or beta 4-(7-43) or beta 4-(13-43), only beta 4-sulfoxide and not the truncated beta-thymosins inhibits actin polymerization, however, only at a 20-fold higher concentration than beta 4. Thus the first six amino acid residues are indispensable to inhibit salt-induced actin polymerization as analyzed by viscometry. While the apparent dissociation constant of the actin/thymosin beta 4 complex generated from a preformed actin/DNase-I complex is 160 microM, a fivefold excess of DNase I over the preformed actin/thymosin-beta 4 complex is necessary to observe a comparable dissociation constant.

The blood-aqueous barrier in eyes with pseudoexfoliation syndrome.

UNLABELLED: Eyes with pseudoexfoliation syndrome (PEX) frequently show clinical signs of impairment of the blood-aqueous barrier. Herein we give an overview of recent studies that analyzed the blood-aqueous barrier in eyes with PEX. METHODS: The authors review and summarize recent studies including quantification of aqueous flare in eyes with PEX using the laser flare cell meter (LFCM; Kowa FC-1000) in comparison with normal eyes and eyes with primary open-angle glaucoma (POAG), quantification of aqueous flare in eyes with PEX with and without secondary open-angle glaucoma (SOAG), and quantitative biochemical determination of total aqueous protein concentration in PEX eyes. In addition, studies of noninvasive quantification of the blood-aqueous barrier breakdown following trabeculectomy and following phacoemulsification with intraocular lens implantation in eyes with and without PEX are reviewed. RESULTS: In eyes with manifest PEX, both aqueous flare and aqueous protein concentration were significantly increased in comparison with normal control eyes and eyes with POAG. Flare values in PEX eyes with SOAG were not significantly different from flare values in PEX eyes without SOAG. Following trabeculectomy as well as following cataract surgery, breakdown of the blood-aqueous barrier as determined by quantification of aqueous flare was significantly higher in eyes with PEX than in eyes without PEX. CONCLUSIONS: Impairment of the blood-aqueous barrier with increase in aqueous protein concentration is a feature of PEX and may be quantified both by flare measurement and by biochemical protein determination. The extensive blood-aqueous barrier breakdown in eyes with PEX following intraocular surgery is an important risk factor for early or late postoperative complications. The alterations of the blood-aqueous barrier should be considered in the medical and surgical treatment of eyes with PEX.

Structural modifications of monoclonal antibodies following direct versus indirect labelling with 99Tcm: does fragmentation really occur?

In this study, the influence of direct and indirect 99Tcm-labelling on the molecular structural integrity of monoclonal antibodies and other immunoglobulin preparations was investigated. Molecular composition of antibody preparations [two IgG monoclonal antibodies, one F(ab')2 fragment (all directly labelled), one indirectly labelled polyclonal human immunoglobulin preparation] and of serum samples after antibody injection were studied using polyacrylamide gel electrophoresis (PAGE; non-reducing and reducing conditions) and gel filtration chromatography. With PAGE, depending on the conditions used, a variety of lower molecular weight products could be detected. When analysing the same antibody preparations by gel filtration chromatography, all complete antibody preparations appeared as homogenous proteins of IgG molecular weight (150 kD). In F(ab')2 fragments, some further fragmentation to Fab' was noticed. Neither in vitro nor in vivo (serum) evidence of smaller fragments could be detected by gel filtration, despite their presence in PAGE. We therefore conclude that through the reductive step of direct 99Tcm-labelling, interchain disulphide linkages are broken but the polypeptide chains of complete IgG remain associated by non-covalent linkages, whereas (F(ab')2 is fragmented further to form essentially Fab'. The protein-denaturating conditions of PAGE (even if performed non-reducingly) seem to produce artifacts, not representing the real in vivo condition. PAGE results should therefore be interpreted only with great care.

[Tyndallometry with the laser flare cell meter and biochemical protein determination in the aqueous humor of eyes with pseudoexfoliation syndrome]. / Tyndallometrie mit dem Laser-flare-cell-Meter und biochemische Proteinbestimmung im Kammerwasser bei Augen mit Pseudoexfoliationssyndrom.

UNLABELLED: Slit-lamp examination and flare measurement in eyes with pseudoexfoliation (PSX) syndrome frequently reveal signs of impairment of the blood-aqueous barrier. We studied both aqueous flare and aqueous protein concentration in PSX eyes to verify and quantify these alterations. PATIENTS: Aqueous flare was measured using the Laser Flare-Cell Meter Kowa FC-1000 in 158 normal control eyes (age 56.0 +/- 12.0 years), in 60 PSX eyes (age 72.5 +/- 9.4 years), and in 84 eyes with primary chronic open-angle glaucoma (PCOAG) (age 56.3 +/- 12.4 years). Total aqueous protein concentration was determined biochemically in 12 "normal" control eyes with senile cataracts (age 57.6 +/- 20.4 years), in 27 PSX eyes (age 72.6 +/- 9.1 years), and in 25 PCOAG eyes (age 63.3 +/- 13.5 years). After the primary aqueous humor was aspirated during cataract extraction or trabeculectomy, a modified BCA-Peirce method was employed. RESULTS: In PSX eyes, both aqueous flare (12.3 +/- 8.2 photon counts/ms) and aqueous protein concentration (0.42 +/- 0.16 mg/ml total protein) were significantly increased in comparison with normal control eyes (4.3 +/- 1.1 photon counts/ms and 0.22 +/- 0.08 mg/ml) and with PCOAG eyes (4.7 +/- 1.6 photon counts/ms and 0.26 +/- 0.09 mg/ml) (P < 0.0001). Differences between normal control eyes and PCOAG eyes were not significant (P > 0.05). There was a high correlation between aqueous flare measurements and aqueous protein concentration in eyes with PSX (y = 0.27x + 0.29, r = 0.65, P = 0.0006, n = 24). CONCLUSIONS: Our results indicate that impairment of the blood-aqueous barrier with an increase in aqueous protein concentration is a feature of PSX and may be quantified both by flare measurement and biochemical protein determination. These alterations should be considered in the pharmacological and surgical treatment of PSX eyes.

Induction of the polymerization of actin from the actin:thymosin beta 4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin.

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin beta 4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked+ends. Free, slowly growing-ends were unable to induce polymerization of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin beta 4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin beta 4. This Kd was found to be 0.4 microM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin beta 4 complex; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.

Protein quantification and electrophoresis in aqueous humor of pseudoexfoliation eyes.

PURPOSE: Pseudoexfoliation (PSX) eyes frequently show clinical signs of blood-aqueous barrier impairment. To analyze these alterations, the authors examined aqueous humor of human eyes with and without PSX. METHODS: After aqueous humor samples had been obtained during cataract or filtering glaucoma surgery, a modified Pierce-bicin choninic acid assay was used to quantify total aqueous protein concentration in 27 PSX eyes and 37 eyes without clinical signs of PSX (12 cataract eyes and 25 eyes with primary open-angle glaucoma). In addition, aqueous protein composition was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis, silver staining, and laser densitometry in 27 PSX eyes and 59 eyes without PSX. RESULTS: Aqueous protein concentration was significantly higher in PSX (mean 0.42 +/- 0.16 mg/ml) than in normal cataract eyes (0.22 +/- 0.08 mg/ml, P < 0.0001) and in eyes with open-angle glaucoma (0.26 +/- 0.09 mg/ml, P < 0.0001, Wilcoxon-Mann-Whitney test). Electrophoresis revealed a characteristic increase of a 12.5-kDa band in 15 of 27 PSX eyes but in only 1 of 59 eyes without PSX (P < 0.00001, chi-square test). CONCLUSIONS: These results substantiate increased aqueous protein concentration and aqueous barrier impairment in PSX. The additional finding of an increased 12.5-kDa band in 56% of PSX eyes may be related to the pathogenesis of PSX in the anterior ocular segment.

Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine.

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.