MS-based quantification of RhoA/B and RhoC ADP-ribosylation.

Mono ADP-ribosylation is a common characteristic of bacterial toxins resulting to aberrant activation or inactivation of target proteins. The C3 exoenzyme of Clostridium botulinum (C3bot) ADP-ribosylates the small GTPases RhoA, RhoB and RhoC, leading to inactivation of these important regulators and impaired down-stream signaling. Quantification of ADP-ribosylation using gel migration assays, antibodies, and radioactivity-based methods are limited. Therefore a novel LC-MS-based method to specifically determine and quantify ADP-ribosylation of Rho GTPases was established. A heavy labeled protein standard that contained ADP-ribosylation specific peptides in similar amounts in ADP ribosylated and non ADP ribosylated form was used for relative quantification in vivo. In a proof of principle experiment HT22 cells were treated with C3bot and the kinetics of RhoA/B and RhoC ADP-ribosylation were determined in vivo.

Glucosyltransferase-dependent and -independent effects of TcdB on the proteome of HEp-2 cells.

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase-dependent and -independent effect on treated HEp-2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase-deficient TcdBNXN and their proteomes were analyzed by LC-MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdBNXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to "GTPase mediated signaling" and the "cytoskeleton" while "chromatin" and "cell cycle" related proteins were altered by both, TcdB and TcdBNXN . The obtained dataset of HEp-2 cell proteome helps us to better understand glucosyltransferase-dependent and -independent mechanisms of TcdB and TcdBNXN , particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 (https://proteomecentral.proteomexchange.org/dataset/PXD006658).

Proteome Analysis of Human Perilymph Using an Intraoperative Sampling Method.

The knowledge about the etiology and pathophysiology of sensorineural hearing loss (SNHL) is still very limited. This study aims at the improvement of understanding different types of SNHL by proteome analysis of human perilymph. Sampling of perilymph was established during inner ear surgeries (cochlear implantation, vestibular schwannoma surgeries), and safety of the sampling method was determined by checking hearing threshold with pure-tone audiometry postoperatively. An in-depth shot-gun proteomics approach was performed to identify cochlear proteins and the individual proteome in perilymph of patients. This method enables the identification and quantification of protein composition of perilymph. The proteome of 41 collected perilymph samples with volumes of 1-12 µL was analyzed by data-dependent acquisition, resulting in overall 878 detected protein groups. At least 203 protein groups were solely identified in perilymph, not in reference samples (serum, cerebrospinal fluid), displaying a specific protein pattern for perilymph. Samples were grouped by patient's age and surgery type, leading to the identification of some proteins specific to particular subgroups. Proteins with different abundances between different sample groups were subjected to classification by gene ontology annotations. The identified proteins might serve as biomarkers to develop tools for noninvasive inner ear diagnostics and to elucidate molecular profiles of SNHL.

Toxin A of the nosocomial pathogen Clostridium difficile induces primary effects in the proteome of HEp-2 cells.

PURPOSE: This study was carried out to investigate the impact of high concentrations of Clostridium difficile toxin A (TcdA) on the proteome of human cells. It should also be examined whether a catalytically deficient mutant (TcdANXN ) has an effect on target cells. EXPERIMENTAL DESIGN: Proteome changes were investigated after treatment of HEp-2 cells with 20 nM TcdA for 8 h using a triplex SILAC labeling method and shotgun proteomics. Proteins from differently labeled and treated cells were combined for analysis using an HPLC coupled to an Orbitrap mass spectrometer. RESULTS: Nearly 4000 proteins were identified in each replicate and 3500 could be quantified by SILAC triplicate analysis. 51 proteins exhibited an altered abundance with 29 up-regulated and 22 down-regulated proteins. In contrast, TcdANXN had no provable impact on the protein profile of HEp-2 cells. Data analysis of regulated proteins revealed that mainly plasma membrane, cell death, cell proliferation and actin cytoskeleton proteins were affected by TcdA treatment. CONCLUSIONS AND CLINICAL RELEVANCE: This proteome analysis showed novel insights of TcdA impact onepithelial cells. Comparison with long-term treatment studies reveals distinctions in affected cellular processes that will improve the understanding of TcdA functions and might help to find new tools for diagnosis and treatment of CDI.

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

Vimentin mediates uptake of C3 exoenzyme.

Clostridium botulinum C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.

Cleavage of E-cadherin and ß-catenin by calpain affects Wnt signaling and spheroid formation in suspension cultures of human pluripotent stem cells.

The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active ß-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and ß-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of ß-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and ß-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological modulation of calpain activity prevents spheroid formation and causes disassembly of preexisting spheroids into single cells, thereby providing novel strategies for improving suspension culture conditions for human pluripotent stem cells in the future.

Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose.

The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)ß(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.

Anterior chamber depth and iridocorneal angle in healthy White subjects: effects of age, gender and refraction.

PURPOSE: Prior to phakic intraocular lens implantation, it is important to obtain precise knowledge of the anterior chamber depth (ACD). Accurate topographic evaluation of the iridocorneal angle is helpful in estimating risk for angle-closure glaucoma. This study investigated the use of the Orbscan II system to measure ACD and the iridocorneal angle in healthy subjects and assessed the influences of age, gender and spherical equivalent on these parameters. METHODS: The Orbscan II system was used to determine the ACD and iridocorneal angle in eight different positions in 390 healthy White subjects with a mean age of 41± 16years (range 10-80 years). The sample included 242 male and 148 female subjects. The influences of age, gender and spherical equivalent were assessed using multiple regression analysis. RESULTS: Mean ACD was 2.87 ± 0.04 mm in male subjects and 2.81±0.37mm in female subjects. The explanatory variables relevant to the ACD were age (partial regression coefficient B = -0.0115, p < 0.0001), spherical equivalent (B = - 0.0562, p< 0.0001) and gender (B = 0.0996, p=0.0036). The mean iridocorneal angle (MIA) was 30.7 ± 2.0 ° in male and 31.6 ± 2.1° in female subjects. The variables relevant to the MIA were gender (B =- 0.865, p < 0.0001), age (B =- 0.017, p = 0.0007) and spherical equivalent (B = - 0.121, p = 0.001). The superior iridocorneal angle displayed the strongest negative correlation with age, whereas the temporal angle exhibited the least correlation with age. CONCLUSIONS: The decline in ACD appears to be linear with age, amounting to a mean of 0.58 mm over 50 years. This may become clinically relevant in the use of phakic intraocular lenses. In addition, the angle is more severely constricted in the superior quadrant than in the temporal quadrant.

Cell function in the bovine mammary gland: a preliminary study on interdependence of healthy and infected udder quarters.

Udder defence mechanisms are not completely explained by current mastitis research. The anatomical construction of the udder implies that infection of one udder quarter does not influence the immune status of neighbouring quarters. To test this hypothesis, we compared the immune reactions of individual udder quarters in response to microbial attacks. In the course of immune reactions, polymorphonuclear leucocytes (PMN) release oxygen radicals, which can be determined by chemiluminescence (CL). Milk from 140 udder quarters of 36 cows was analysed for somatic cell count (SCC), differential cell count, viability and CL activity. Quarters with an SCC < 100,000 cells/ml and free of pathogens were defined as uninfected, all other quarters were categorized as infected. Three groups of cows were classified cytologically: group A (healthy, 11 animals, SCC limit < 100,000 cells/ml); group B (moderate mastitis, 8 cows, SCC > or = 100,000 and < 400,000 cells/ml in at least one quarter); and group C (severe mastitis, 17 cows, SCC > or = 400,000 cells/ml in at least one quarter). Infected and uninfected quarters in groups B and C were analysed separately. Viability of PMN leucocytes was significantly (P=0.0012) lower in group A (72.6%) than in healthy quarters of group C (84.0%). Lowering the SCC limit of healthy quarters to <50,000 cells/ml (group A: all quarters within the udder) revealed striking differences between samples of groups B and C: in addition to varying differential cell counts and viabilities, CL activity of group B<50 (2929 CL units/million PMN) was markedly lower than that of the other groups (5616 in group A<50 and 6445 CL units/million PMN in group C<50). These results allow the conclusion that the infection of one udder quarter influences the cell activity of neighbouring quarters. When the SCC threshold for healthy quarters was reduced to 50,000 cells/ml, greater differences in cell activities were detected between healthy udders and healthy quarters of infected udders.

Age-related changes in central and peripheral corneal thickness: determination of normal values with the Orbscan II topography system.

PURPOSE: The thickness of corneal tissue is an important parameter in refractive surgery, Goldmann applanation tonometry, and corneal diseases. The purpose of the study was to record system-specific normal values with the Orbscan II system and to investigate the influence of sex and age on central and peripheral corneal thickness to characterize more precisely the anatomy of the cornea. METHODS: The Orbscan II topography system is a computer-assisted slit-beam scanning technology that can map the anterior section of the eye. It was used to take 2 measurements at 10-minute intervals on 777 eyes of 390 normal white subjects ranging in age between 10 and 80 years. Two hundred forty-two participants were men and 148 were women. The central corneal thickness and the nasal, superior, inferior, and temporal peripheral corneal thickness at a distance of 3 mm from the center were analyzed. No correction factor was used. Using nonparametric test methods, we carried out a statistical analysis of the parameters of sex and age. RESULTS: The mean central corneal thickness of all 777 eyes was 595 +/- 41 microm. No sex-specific central difference was identifiable (P = 0.33), there was no significant difference between the sides (P = 0.23), and no significant difference was found between the first and second measurement. The group of 10- to 39-year-olds had a mean central corneal thickness of 591 +/- 41 microm, whereas that of 40- to 80-year-olds was 600 +/- 39 microm, which was a significant difference (P = 0.03). The mean peripheral corneal thickness was 689 +/- 46 microm nasally, 688 +/- 42 microm superiorly, 667 +/- 40 microm inferiorly, and 655 +/- 42 microm temporally. Nasally and superiorly, we found a significant negative correlation with age (Spearman rank correlation, P = 0.02). CONCLUSIONS: The normal values presented here are a suitable reference basis for future studies in whites. Peripheral corneal thickness is asymmetric and seems to undergo age-related anatomic changes. This information should be considered when planning penetrating keratoplasty and refractive surgery.

In-vitro toxicity of Ukrain against human Ewing tumor cell lines.

Ukrain is advertised by the manufacturer as a drug for alternative cancer cures with high activity against progressive Ewing tumors. Using the MTT assay, we compared the cytotoxicity of Ukrain with the cytotoxicity of N,N',N''-triethylenethiophosphoramide (thioTEPA), Chelidonium majus L. alkaloids, doxorubicin, cyclophosphamide and etoposide against four human Ewing tumor cell lines. In addition, we studied the cytotoxicity of thioTEPA combined with C. majus L. alkaloids after 48, 72 and 96 h. All compounds reduced the growth of Ewing tumor cell lines in a dose-dependent manner. The concentrations that reduced cell growth by 50% ranged between 6.2 and 31.1 micromol/l for Ukrain, 1.9 and 26.1 micromol/l for C. majus L. extract, and 1.7 and 448 micromol/l for thioTEPA. The sensitivity profile of Ukrain was comparable to that of the C. majus L. alkaloids, and different from that of thioTEPA, cyclophosphamide, etoposide and doxorubicin. Overall, doxorubicin was the most cytotoxic drug followed by cyclophosphamide. Ukrain and the C. majus L. alkaloids were slightly more cytotoxic than etoposide, while thioTEPA showed the lowest cytotoxicity. Co-exposure of thioTEPA with C. majus L. alkaloids resulted in additive but not in synergistic cytotoxicity. The in-vitro results indicate that the cytotoxicity of Ukrain against Ewing tumors is comparable to that of etoposide. While the latter can be used on the basis of broad clinical experience and known risk-benefit ratio, Ukrain for the present might be considered as a candidate for subsequent drug development by xenograft studies followed by systematic clinical trials.

[Resistance of mastitis pathogens in northern Germany]. / Resistenzen von Mastitiserregern im norddeutschen Raum.

The data of 1692 susceptibility tests acquired from April 2003 through March 2004 in the mastitis laboratory of the Institute for Food Quality and Safety were summarized in order to help veterinarians confronted with acute mastitis in choosing the appropriate antibiotic. Two thirds of the milk samples were infected with gram-positive cocci. One third of these were identified as Streptococcus (S.) uberis, one fourth as Staphylococcus (S.) aureus. All isolates (100%) of S. uberis, S. dysgalactiae, S. agalactiae and Arcanobacterium pyogenes were susceptible to Penicillin and Ampicillin. Concering S. aureus, nearly 100% of the isolates were susceptible to Oxacillin, Cephalothin, Cefacetril, Cefquinom and Neomycin, but only 88% of the isolates were sensitive to Penicillin, Ampicillin and Cefoperazon. The gram-negative rods (Escherichia (E.) coli, Klebsiella spp. and Pseudomonas spp.) displayed an irregular resistance pattern. More than 93% of all examined isolates including Pseudomonas spp. were susceptible to Colistin. The sensitivity of E. coli and Klebsiella spp.to Marbofloxacin, Enrofloxacin and Cefquinom exceeded 96%. Thus, the susceptibility of gram-positive mastitis pathogens to common antibiotics is favourable. Because the highly effective Colistin is no longer approved for local therapy, the situation for gram-negative bacteria is more difficult.

The influence of technical factors on differential cell count in milk.

Differential cell count of milk is a traditional parameter for the evaluation of udder health. The literature shows great variation in differential cell counts of the milk of healthy mammary glands: macrophages range from 0% to 80%, lymphocytes from 1.5% to 79.5%, polymorphonuclear neutrophils from 3% to 95%, and epithelial cells from 1% to 19%. We conducted three studies to seek explanations for such variation. In the first, we evaluated the impact of polyethylene and glass sampling bottles. The aim of the second study was to compare the results of differential cell counts performed by three different technicians. The third study evaluated two methods of smear preparation. When polyethylene plastic bottles were used, the macrophage population was minimized but lymphocytes remained unaffected. This was shown by an exemplary flow cytometric analysis using four monoclonal antibodies against three lymphocyte surface structures. There were significant differences in the differential cell counts of 40 smears made by three technicians despite identical operating procedures. For the sediment smear, milk was centrifuged once and the sediment spread by eye on a glass slide. For the "coffee grinder" smear method, the sample was subjected to four centrifugations and then placed on a cover glass in order to spread the sediment using centrifugal force. The coffee grinder procedure led to a reduction of lymphocytes and an enrichment of polymorphonuclear neutrophils without affecting the macrophage population. Both methods made it possible to distinguish different udder health classes. It can be concluded that differential cell counts are a useful tool for comparing and monitoring udder health only if: samples are taken in a glass bottle; smears are prepared with the identical technique; and the differential cell counts are performed by a single person.

White-to-white corneal diameter: normal values in healthy humans obtained with the Orbscan II topography system.

PURPOSE: The corneal horizontal diameter (white-to-white) is abnormal in diseases like microcornea, relative anterior microphthalmos, and corneal dystrophies. Because normal values are described imprecisely in the literature, the purpose of this study was to reevaluate the horizontal corneal diameter as a scientific parameter. METHODS: The horizontal corneal diameter was measured with the Orbscan II system in 370 right eyes and 373 left eyes of 390 healthy white subjects aged 10-80 years. There were 148 female subjects and 242 male subjects. Each measurement was repeated twice. Differences in gender, between right and left eyes, and age-related alterations were analyzed statistically. RESULTS: The average corneal diameter was 11.71 +/- 0.42 mm. The average corneal diameter was 11.77 +/- 0.37 mm in males compared with 11.64 +/- 0.47 mm in females. The resulting normal ranges were 11.04 to 12.50 for males and 10.70 to 12.58 mm for females. Differences in gender were not significant in the t test for independent samples (P = 0.071). There were no statistically significant differences between right and left eyes in the t test for dependent samples (P = 0.16). Corneal diameters decreased slightly with age. CONCLUSIONS: With the obtained normal values, more precise determination of microcornea and macrocornea will be possible in the future. The horizontal corneal diameter was not significantly greater in males than in females. Further studies are needed to show the reasons for the age-related decrease in measurements.

[Visual field in patients with arterial hypertension]. / Gesichtsfeldbefunde bei Patienten mit einer arteriellen Hypertonie.

BACKGROUND: The aim of this study was to investigate the quantitative changes of visual field in patients with arterial hypertension using standard white-on-white perimetry. MATERIALS AND METHODS: Sixty-two patients (f : m = 33 : 29) with arterial hypertension without damage in end-organs were included and divided into three groups: Group 1: systolic/diastolic blood pressure: 120 - 139/80 - 89 mm Hg (n = 18, f : m = 14 : 4, mean age 55 +/- 13 years), group 2: systolic/diastolic blood pressure: 140 - 159/90 - 99 mm Hg (n = 24; f : m = 9 : 15, mean age 55 +/- 12 years), group 3: systolic/diastolic blood pressure: 160 - 179/100 - 109 mm Hg (n = 20; f : m = 10 : 10, mean age 55 +/- 12 years). We compared these patients with an ophthalmological and healthy control group (systolic/diastolic blood pressure < 120/80 mm Hg, n = 20, f:m = 12 : 8, mean age 55 +/- 9 years). Beside the ophthalmological examinations (visual acuity, refraction, intraocular pressure, slit lamp and fundus examination) quantitative analysis of visual field by Octopus 1-2-3 perimeter was performed. Different parameters (mean deviation (MD), mean sensitivity (MS), loss variance (LV), pattern standard deviation (PSD) and the test-duration) were statistically compared using the Mann-Whitney-U test and the Wilcoxon test. RESULTS: Hypertensive retinopathy grade I - II was found in 10 patients with arterial hypertension. Statistic analysis showed no significant difference of MD, MS, LV, PSD and test duration between the patients with arterial hypertension and the control group (Mann-Whitney-U test: p > 0.05). There was also no significant difference between the three hypertonic groups (Mann-Whitney-U test: p > 0.05). CONCLUSIONS: The examination of visual field using standard achromatic automated perimetry based on investigation of the parvocellular system. Arterial hypertension, however, does not damage this parvocellular system enough to assess disturbances in the white-on-white perimetry. Further studies are necessary to clarify the value of Frequency Doubling Perimetry, which is based on the detection of early damage of the magnocellular system.

Preparation and characterization of 2-amino-5-thienyl-substituted multicharged methinium compounds.

[reaction: see text] A series of new 2-amino-5-thienyl-substituted multicharged methinium compounds have been prepared and characterized spectroscopically and electrochemically by reaction of lithiated species of N,N-disubstituted 2-aminothiophenes with alkyl derivatives of di- and tricarbonic acids and subsequent addition of perchloric acid.

Preparation and characterization of alpha,alpha'-bisdiarylamino-capped oligothiophenes.

[reaction: see text] A series of alpha,alpha'-bisdiarylamino-capped oligothiophenes C(n) were prepared by the palladium-catalyzed reaction of the dibromo compounds A(i) with diarylamines, N,N-diarylamino-substituted thiophenes or 2,2'-bithiophenes BX(j). These easily oxidizable compounds exhibit a high tendency to form amorphous glasses and characteristic electrochemical and spectroscopic properties that depend significantly on the number of their thiophene moieties.

Plasminogen activator inhibitor-1 mRNA expression in cultured pigmented ciliary epithelial cells of the porcine eye.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) is a main regulator of the fibrinolytic system. PAI-1 inhibits tissue plasminogen activator and urokinase-type plasminogen activator, resulting in reduced plasminogen activity and attenuated fibrinolysis and proteolysis. The present study was performed to determine the gene expression encoding for PAI-1 in cultured pigmented ciliary epithelial cells of the porcine eye and to detect PAI-1 activity in cell culture supernatants. METHODS: Total mRNA of respective confluent primary cultures of porcine ciliary epithelial cells, porcine liver cells and porcine kidney cells was isolated. Reverse transcribed PAI-1 mRNA was measured by real-time polymerase chain reaction (TaqMan PCR) with PAI-1 primers and probes deduced from the human PAI-1 gene. PAI-1 activity in supernatants of the cell cultures was determined by a specific chromogenic test (Coatest PAI). RESULTS: PAI-1 mRNA was localized in all samples of primary cultures of porcine pigmented ciliary epithelial cells. As a negative control we analyzed total mRNA of porcine kidney cells. PAI-1 mRNA was not detectable in these cells. On the other hand, we established PAI-1 mRNA in porcine liver cells as a positive control. High levels of PAI-1 activity were found in all samples of cell culture supernatants. CONCLUSIONS: Our results indicate that PAI-1 is produced and secreted by the porcine ciliary epithelium. We suggest that PAI-1 together with components of the fibrin/fibrinolytic system may be involved in aqueous humor outflow. Overproduction of PAI-1 may induce less fibrinolysis and extracellular proteolysis in aqueous humor and trabecular meshwork, which could result in an elevated intraocular pressure by increasing the outflow obstruction. Therefore stimulation of PAI-1 production may perhaps contribute to the pathogenesis of primary open-angle glaucoma.