Ophthalmic Findings in Patients After Renal Transplantation.

The aim of the study was to perform complete ophthalmic examinations in patients after renal transplantation to determine ocular alterations and condition of the eyes. Moreover, ophthalmic findings were correlated with certain clinical characteristics related to transplantation such as post-operative renal functions and immunosuppressive regimen. The study was performed on 84 eyes of 42 patients who had received a renal transplant at least 6 months earlier. All patients underwent a complete ophthalmologic examination. In addition, in 33 (78.6%) patients peripapillary retinal nerve fiber layer (RNFL) thickness was determined using optical coherence tomography (Stratus OCT, Zeiss, Germany), which is a unique finding among renal transplantation patients. Recipients received immunosuppressive therapy consisting of tacrolimus, mycophenolate mofetil, and corticosteroid. Of 42 patients, 19 (45%) were women and 23 (55%) were men. The age of the patients ranged from 26 to 74 years, the mean age was 53.4 years. At least one ocular abnormality could be detected in 37 patients (88%), including impaired visual acuity (n = 31, 74%), keratoconjunctivitis sicca (n = 6, 14.3%), pinguecula (n = 3, 7.1%), arcus lipoides (n = 1, 2.4%), cataracts (n = 24, 57.1%), glaucoma (n = 2, 5%), retinal drusen (n = 6, 14.3%), and hypertensive or atherosclerotic retinopathy (n = 22, 52.4%). Twenty-five patients (75.8%) have reduced RNFL thickness. Cataract formation was positively correlated with age and usage of methylprednisolone. Moreover, RNFL thickness loss was correlated with transplantation duration and postoperative infections. Our study suggests that ocular disorders are frequent among renal transplantation patients. Besides immunosuppression and postoperative infection, aging is a high-risk factor in such cases.

[Acanthamoeba keratitis in patients with contact lens wear in the Department of Ophthalmology in Debrecen]. / Akanthamöbenkeratitis bei Kontaktlinsenträgern in der Patientenpopulation der Augenklinik Debrecen.

BACKGROUND: With increasing numbers of contact lens wearers, Acanthamoeba infections are also more frequent all over the world and also in Hungary. Despite improved diagnostic and therapeutic possibilities it can still cause serious visual loss. In this study the symptoms, signs, diagnostics and treatment options for Acanthamoeba keratitis (AK) in patients treated in our department are presented and compared to the literature. PATIENTS AND METHODS: Between 2001 and 2006 a total of 11 patients were treated in our department for Acanthamoeba keratitis. RESULTS: The mean age of the patients was 30 years and the 8 female and 3 male patients were all contact lens wearers. In six cases the right eye was affected and in five cases the left eye. Poor and improper contact lens hygiene was the cause of infection in all cases and 82% of the infections occurred in the summer period between June and September. In addition to long-term conservative treatment, perforating keratoplasty was performed in six cases. After the therapy the best visual acuity was 0.6, but 4 of the patients had a serious visual loss. Follow-up time ranged from 6 months to 5 years. CONCLUSIONS: Early diagnosis and long-term suitable local therapy are necessary for effective recovery. If the patient wears contact lens and there is severe pain with ring infiltration in the cornea, Acanthamoeba infection should be considered and suitable therapy should be given. Immediately performed perforating keratoplasty does not lead to visual improvement. Prevention by informing the patients about adequate hygiene and use of the cleaning solutions is one of the most important tasks of contact lens specialists.

[Limbo-conjunctival autografting in pterygium surgery]. / Autotransplantul limbo-conjunctival în chirurgia pterigionului.

PURPOSE: To evaluate the efficiency of the limbo-conjunctival autografting in pterygium surgery. MATERIALS AND METHODS: A study has been undertaken in two centers. The first one, which was a prospective observational one, went on for 20 months and included the patients operated with pterygium in the Ophthalmology Clinic in TgMures, RO. Two surgical techniques have been used: limbo-conjunctival autografting and the Arlt technique. The results of the two methods have been followed and compared, as well as the intra and post-surgery complications. The second study, a retrospective observational type of study, went on for 15 months and included the patients operated with pterygium in the Ophthalmology clinic in Debrecen, Hungary. We used 4 methods to treat the pterygium: the Arlt method, the McReynolds method, the sclero-corneal lamellar plasty and the limbo-conjunctival autografting. We analysed the obtained results using all these 4 methods. RESULTS: In the Ophthalmology Clinic in Targu-Mures we operated, during those 20 months, 106 patients with pterygium. Of these, 36 were operated using the limbo-conjunctival autografting, the rest using the Arlt technique. We noticed less relapse when the limbo-conjunctival autografting was used. In the Ophthalmology Clinic in Debrecen, during a 15 months period, there were 33 patients operated for pterygium. The largest number of relapses was noticed with the Arlt technique. CONCLUSIONS: When comparing the different methods used in the pterygium surgery in terms of relapse we noticed the increased efficiency of the limbo-conjunctival autografting. The encouraging results stimulate us in using and recommending this method.

Corneal pathology and indications for corneal transplant at the Tg. Mures Ophthalmology Clinic.

UNLABELLED: The purpose of this study was to evaluate the incidence of corneal pathology that presents indication for cornea/transplant, treatment and follow-up of these patients. MATERIALS AND METHODS: A retrospective analysis has been undertaken at the Tg. Mures Ophthalmology Hospital. In the study we have included all patients who had corneal pathology with intact deeper ocular structures. We have examined the treatment of these cases and their admittance to corneal transplantation. RESULTS: In 2008 in 99 cases of the 3246 hospitalized patients such a corneal condition has been described that presented indication for corneal transplantation. Most common causes were corneal leucomas, corneal ulcers, bullous keratopathy and corneal dystrophies and degenerations. Fourteen patients tried to benefit from corneal transplant abroad financed by the National Health Insurance Office; others went on their own expense. All together four patients appeared for postoperative control at our Hospital. CONCLUSIONS: We have found 99 cases of corneal transplant indication at our Hospital's patients and only a fraction of them have undergone surgery. We can point out a very low level of admittance and a great need for a regional corneal transplantation centers.

Cell proliferation and apoptosis in stromal corneal dystrophies.

The aim of our study was to evaluate corneal cell proliferation and apoptosis in cases of granular, macular and lattice dystrophy, and to provide evidence which may help to clarify whether apoptosis is a pathogenic factor in any of these dystrophies. The study group comprised 39 eyes (from 33 patients) which had undergone penetrating keratoplasty (PK) for stromal dystrophies: these comprised 12 eyes (from 9 patients, 55.5% males) with granular dystrophy, 13 eyes (12 patients, 33.3% males) with macular dystrophy, and 14 eyes (13 patients, 61.5% males) with lattice type I dystrophy. A further 4 corneal buttons from enucleated eyes of 4 patients with choroideal melanoma served as controls. Immunocytochemical analysis of Ki67 (DNAcon Kit, DakoCytomation A/S, Glostrup, Denmark) was used for evaluation of cell proliferation. Apoptosis was detected by use of the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling) assay method (Apoptag reagent, Q-Biogene, Strasbourg, France). Statistical comparisons were made using the Mann-Whitney test. No Ki67-positive cells were detected in the study-group or control corneas. In control corneas no apoptotic activity was found. In the study group the mean (normalised) apoptotic keratocyte number was 1.1+/-1.7 in granular dystrophy and 0.5+/-1.1 in lattice type I dystrophy (p = 0.36, 0.63 respectively). Compared to the controls, the difference was statistically significant only for macular dystrophy (1.6+/-1.2; p = 0.01). Keratocyte apoptosis seems to be a concomitant or pathogenic factor in macular dystrophy. However, the pathways that are triggered to result in increased apoptotic cell death remain to be clarified.

Comparison of divide and conquer and phaco-chop techniques during fluid-based phaco-emulsification.

PURPOSE: To determine whether, similar to ultrasound phaco-emulsification, applied energy and surgery time decrease using phaco-chop nucleus fragmentation method compared to divide and conquer technique using the fluid-based system. METHODS: This prospective, comparative, randomized clinical study included patients with cataract who were randomly assigned to use either standard divide and conquer technique (25 eyes of 25 patients, Group 1) or Nagahara phaco-chop maneuver (25 eyes of 25 patients, Group 2) during fluid-based phaco-emulsification. Surgical parameters were recorded and patients were examined 1 day, 10 days, and 1 month after surgery. Statistical analyses were performed using the paired test of Wilcoxon. RESULTS: Fluid-based time, mean fluid-based magnitude, effective fluid-based time, and the number of pulses were significantly less using phaco-chop technique compared to divide and conquer method (p<0.001). Surgery time was similar using the two nucleofractis techniques (p=0.97). Visual acuities showed no statistical differences between the two groups (p>0.05). CONCLUSIONS: Nuclear fragmentation can be performed with Nagahara phaco-chop technique using the fluid-based system as well. The applied fluid-based energy decreases compared to divide and conquer method. However, surgery time is not reduced due to the difficulties reaching the full occlusion necessary to hold the nucleus during the chop maneuver.

Corneal endothelial function after phacoemulsification using the fluid-based system compared to conventional ultrasound technique.

AIM: To quantitatively assess corneal endothelial changes after phacoemulsification with the fluid-based system compared to conventional ultrasound technique. METHODS: This prospective, randomized clinical study included patients with cataract who were randomly assigned either to have phacoemulsification with the fluid-based system (30 eyes of 30 patients--Group 1) or with traditional ultrasound (30 eyes of 30 patients--Group 2). Patients who were available at each follow-up visit (25 eyes in both groups) were enrolled in the statistical analysis. Endothelial function was evaluated by measuring central corneal thickness, central endothelial cell density (ECD), mean cell size, and coefficient of variation in cell size preoperatively, 10 days, 1 and 3 months, and 1 year after surgery. Statistical analyses were performed using two-way repeated measure ANOVA. RESULTS: An acute, reversible increase of central corneal thickness (CCT) was found 10 days after surgery, which was similar in both groups (P=0.35). ECD decreased, whereas mean cell size increased significantly immediately after surgery. However, the impairments were finished after 1 month. The alterations were similar in both groups (ECD: P=0.99; mean cell size: P=0.85). The coefficient of variation in cell size remained stable after surgery (P=0.08), and significant difference was not found between groups (P=0.99). The endothelial cell loss (ECL) was 6.5+/-8.4% in Group 1 and 6.5+/-11.7% in Group 2 (P=0.69). CONCLUSIONS: Corneal endothelial changes were similar using the fluid-based system compared to the traditional ultrasound technique. The fluid-based method proved to be as safe as conventional ultrasound in cataract surgery.

Quantitative determination of hyaluronan content in cerebral aneurysms by digital densitometry.

OBJECT: Hyaluronan (HA) is a highly hydrated macromolecule; it is one of the essential components of the extracellular matrix (ECM) of the arteries and plays an important role in maintaining the biomechanical features of blood vessels. Although the potential contribution of HA in aneurysms of different vessels has been studied intensively, no data are available about the alteration of the HA content in the extracellular matrix of intracranial aneurysms. The aim of the study was to determine the hyaluronan content in the wall of human cerebral arteries. METHODS: A biotinylated aggrecan fragment that binds specifically to HA was used to stain samples from cerebral aneurysms (n = 11) to compare the HA content to non-aneurysmal arteries of patients who had intracranial aneurysm (n = 11), and to histologically normal arteries of patients who had expired from non-vascular diseases (n = 14). Digital microscopic densitometry was used for the quantitative analysis of the hyaluronan content in these samples. RESULTS: The highest level (169.5 +/- 7.9) was detected in aneurysms, while the HA-level of non-aneurysmal vessels was lower (130.2 +/- 16.8). Both vessel groups contained significantly higher HA than the normal cerebral arteries (32.9 +/- 2.1). CONCLUSIONS: Results suggest that an elevated hyaluronan level in the extracellular matrix may affect the cerebral arterial wall architecture. It is reasonable to suppose that the increased hyaluronan content creates a viscoelastic ECM which might improve the biomechanical resistance of the thinned vessel wall.

[Clinicopathological characteristic of keratoglobus]. / Die klinisch-pathologischen Merkmale des Keratoglobus.

BACKGROUND: This study describes the clinical, topographical, pachymetrical, and histological features of a unique corneal disorder: keratoglobus. CASE REPORT: Ultrasound pachymetry proved that the structure of the cornea was evenly thinning towards the periphery. Histological examinations disclosed the absence of Bowman's membrane and the thinning of the stromal lamellae. CONCLUSION: On account of the thickness of the recipient's periphery, penetrating keratoplasty proved to be a reasonable surgical procedure in this case.

The expression pattern of hyaluronan synthase during human tooth development.

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.

[Tear examinations in primary peripheral lipid keratopathy]. / Untersuchungen der Tränenflüssigkeit im Falle einer primären peripheren Lipidkeratopathie.

The authors report a case of a bilateral peripheral lipid keratopathy, rising differential diagnostic points as well. After several year-long follow-up period the peripheral corneal opacity left the center unaffected. The diagnosis was confirmed by tear examinations which disclosed elevated level of fatty and phosphorous content compared to healthy control eyes.

Expression of aquaporin isoforms during human and mouse tooth development.

Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

Corneal thickness measurements with contact and noncontact specular microscopic and ultrasonic pachymetry.

PURPOSE: To evaluate the central corneal thickness values in normal and postkeratoplasty corneas with the new Topcon SP-2000P noncontact specular microscopic, contact specular microscopic, and the "common standard" ultrasonic pachymetry. METHODS: Central corneal thickness was determined in 119 eyes of 81 patients (73 normal eyes of 44 patients and 46 eyes after penetrating keratoplasty) first with a noncontact specular microscopic (Topcon SP-2000P; Topcon Corporation, Tokyo, Japan), then an ultrasonic (AL-1000; Tomey, Erlangen, Germany), and finally with a contact specular microscopic (EM-1000; Tomey, Erlangen, Germany) pachymetry two times each by the same investigator. RESULTS: Reliability of the central corneal measurements was equally high both in normal and in postkeratoplasty corneas with all of the instruments (Cronbach alpha = 0.99). Noncontact specular microscopic corneal thickness determination correlated significantly both with ultrasonic (r =.86, P <.0001) and contact specular microscopic pachymetry (r =.62, P <.0001). The ultrasonic pachymetry correlated well with the Tomey pachymetry (r =.69, P <.0001). The Topcon normal mean central corneal thickness value (542 +/- 46 microm) was 28 +/- 4 microm lower (P <.0001) compared with the ultrasonic data (570 +/- 42 microm), which was 68 +/- 1 microm lower (P <.0001) compared with Tomey thickness (638 +/- 43 microm). CONCLUSIONS: Central corneal thickness measurements with noncontact specular microscopic, contact specular microscopic, and ultrasonic pachymetry demonstrate that each of the instruments is reliable but cannot be simply used interchangeably.

Scanning-slit and specular microscopic pachymetry in comparison with ultrasonic determination of corneal thickness.

PURPOSE: To determine the central corneal thickness values in healthy eyes with the recently developed Orbscan scanning-slit system, contact and noncontact specular microscopic pachymetry and compare the results to conventional ultrasonic pachymetry. METHODS: In the following sequence, Orbscan, Topcon SP-2000P noncontact specular microscope, AL-1000 ultrasound, and Tomey contact specular microscope were used to record thickness values. Thirty-four healthy right corneas of 34 healthy subjects were investigated. RESULTS: Orbscan pachymetry correlated significantly with ultrasound (r = 0.64, p < 0.001), contact (r = 0.45, p < 0.001), and noncontact specular microscopy (r = 0.72, p < 0.001). Likewise, the Topcon SP-2000P noncontact specular microscopy pachymetry disclosed similar statistical results compared with ultrasound (r = 0.88, p < 0.001), and contact specular microscopy pachymetry (r = 0.76, p < 0.001). The mean central corneal thickness results were significantly higher ( p < or = 0.01) than ultrasonic values (580 +/- 43 microm) using the contact specular microscope (640 +/- 43 microm) or Orbscan system (602 +/- 59 microm) but were significantly lower ( p < 0.001) using the noncontact specular microscope (547 +/- 49 microm). CONCLUSIONS: The results indicate that the devices tested cannot be simply used interchangeably. For long-term patient follow-up, one specific instrument is recommended. Recently developed pachymetry machines are especially helpful when additional corneal data such as thickness profile, elevation maps, anterior chamber depth, and endothelial morphology are required.

Mosaicplasty for the treatment of osteochondritis dissecans of the talus: two to seven year results in 36 patients.

An osteochondral defect (OCD) is known as a symptomatic lesion causing pain, recurrent synovitis, and altered joint mechanics most commonly in a weight-bearing joint. Loose bodies may develop, which may then cause joint destruction and/or locking. The damage to the articular surface is most likely a precursor of ankle osteoarthritis. With the recent advances in diagnostic imaging, such as MRI, as well as the development of ankle arthroscopy, the identification and classification of these lesions has become much more precise. This allows more accurate staging and improves treatment recommendations. The assessment of a particular treatment is also improved. A variety of treatment alternatives are now available. These include arthroscopic procedures including debridement, retrograde drilling, and bone grafting. Compared to open treatment, arthroscopic procedures may be particularly advantageous in the treatment of small defects and stable OCD lesions. Until recently, however, favorable results have been less predictable for large or unstable osteochondral defects. We treat these more difficult lesions with a mosaic autogenous osteochondral transplantation. In our hands, this appears to provide an optimal treatment result. The present report evaluates the clinical outcome of 36 patients followed for two to seven years after a mosaicplasty autogenous osteochondral transplantation from a non or less weight bearing portion of the knee to the ipsilateral talus. Ankle function was measured by the Hannover scoring system and showed good to excellent results in 34 cases (94%) with no long term donor site morbidity. The encouraging clinical results are supplemented with radiographs and histology, which support the premise of lasting relief of symptoms and prevention of ankle arthrosis.

The distribution pattern of the hyaluronan receptor CD44 during human tooth development.

The aim was to investigate the expression pattern of the major cell-surface hyaluronan receptor CD44, as there are no existing data on its presence or absence in human dental structures at different developmental stages. Immunohistochemical localization of CD44 was studied using a monoclonal antibody, H3, that specifically recognizes an epitope in the common backbone of all CD44 isoforms. The dental lamina displayed a strong CD44 signal; the external enamel epithelium was negative. In the coronal region of the tooth germ the presecretory ameloblasts showed an intense reaction whereas the less differentiated inner enamel epithelial cells showed no signal at the cervical loop where they meet the external enamel epithelium. In the stellate reticulum a moderate reaction was detected. The secretory ameloblasts and the stratum intermedium showed a strong cell-surface CD44 signal. A strong signal was also observed on the odontoblasts and their processes. In the pulp, close to the odontoblastic layer, weak labelling was seen in the walls of capillary vessels. The distribution of CD44 in the human tooth germ corresponds to that of hyaluronan in most locations, suggesting that during tooth development this transmembrane protein plays an important part in hyaluronan-mediated events.

Accuracy of in vivo carotid B-mode ultrasound compared with pathological analysis: intima-media thickening, lumen diameter, and cross-sectional area.

BACKGROUND AND PURPOSE: This study aimed to determine the correlation of in vivo ultrasound measurements of intima-media thickening (IMT), lumen diameter, and cross-sectional area of the common carotid artery (CCA) with corresponding measurements obtained by gross pathology and histology. METHODS: Sixty-six moribund neurological patients (mean age 71 years) underwent B-mode ultrasound of the CCA a few days before death. During autopsy, carotid specimens were removed in toto. Carotid arteries were ligated and cannulated for injection of a hydrophilic embedding material under standardized conditions. The carotid bifurcation was frozen and cut manually in 3-mm cross slices. Digital image analysis was carried out to determine the diameter and the cross-sectional area of the frozen slices of the CCA. IMT was assessed by light microscope. Ultrasonic and planimetric data were compared. RESULTS: Mean measurements of lumen diameter and cross-sectional area were 7.13+/-1.27 mm and 0.496+/-0.167 cm(2), respectively, by ultrasound, and 7.81+/-1.45 mm and 0.516+/-0.194 cm(2), respectively, by planimetric analysis of the unfixed redistended carotid arteries (R(2)=0.389 and 0.497). The mean IMT was 1.005+/-0.267 mm by ultrasound and 0.67+/-0.141 mm histologically, resulting in a mean difference of -31%. CONCLUSIONS: Transcutaneous B-mode ultrasound provides a reliable approach for in vivo measurements of the cross-sectional area and, less exactly, of the lumen diameter of the CCA. Compared with histological results, in vivo ultrasound measurements of the IMT are systematically larger.

Factor XIII of blood coagulation as a nuclear crosslinking enzyme.

Intracellular localization and distribution of Factor XIII subunit A (FXIIIA) was investigated in association with monocyte-macrophage differentiation in a long term culture of human monocytes by light- and electron microscopical as well as biochemical and immunobiochemical techniques. To allow the detection of FXIIIA in cells with well-preserved ultrustructure, immunosera against glutaraldehyde-derivatized recombinant FXIIIA were developed in rabbits, then characterized and used in this study. In the early phase of macrophage differentiation intranuclear accumulation of FXIIIA was detected as a transient phenomenon in cells of the 2nd day culture by optical sectioning with 0,7 microm steps in laser scanning confocal microscopy and immunoblotting technique. FXIIIA could be detected by immunoelectron microscopic postembedding staining over electrodense DNA-containing areas. Fluoresceinated monodansylcadaverine incorporation assay was used to demonstrate that FXIIIA is not only present in the nuclei, but also expresses its transglutaminase activity. Our finding of the nuclear accumulation of FXIIIA in differentiating human macrophages is also unique in that a blood clotting factor has, for the first time, been localized in nuclei and has been shown to be an intracellular crosslinking enzyme. The possible role of nuclear FXIIIA in association with cellular processes involving chromatin structure remodeling, such as cell death, cell differentiation or cellular proliferation requires further in-depth investigation.

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures.

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H-thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that protein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal organisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

Primary structure of human matrilin-2, chromosome location of the MATN2 gene and conservation of an AT-AC intron in matrilin genes.

We isolated full-length cDNA clones for human matrilin-2, an oligomeric protein, which forms filamentous networks in the extracellular matrices of various tissues. The human matrilin-2 precursor is encoded by a 4.0-kb mRNA, it consists of 956 amino acids and shows 93% similarity to the mouse protein. Out of the two von Willebrand factor type A-like domains, the 10 epidermal growth factor-type modules, one unique sequence and the oligomerization module, the first A domain is the most conserved. RT-PCR demonstrated wide expression of the gene in human cell lines of fibroblastic or epithelial origin. Alternative splicing affected only 19 amino acids in a 75-moiety-long segment, unique to matrilin-2. Isolation and analysis of the 3' end of the gene revealed that the reason for alternative splicing is alternative 3' splice site selection. Further, we identified in the human matrilin-2 gene a U12 type AT-AC intron between the last two exons encoding the oligomerization domain. We mapped the matrilin-2 gene (MATN2) by fluorescence in situ hybridization at chromosome position 8q22.