[Autokeratoplasty in perforation of the ectatic leukoma of the only functionally perspective eye]. / Autokeratoplastika pri perforatsii éktazirovannogo bel'ma edinstvennogo glaza.

Treatment and rehabilitation of patients with destructive lesions of the cornea is a serious problem for ophthalmologists due to severity of the outcome of the disease - the formation of leukomas with a persistent decrease in vision, or even a complete loss of visual functions. The article presents a clinical case of a patient with posttraumatic ectaticized leukoma of the functionally promising eye, which course was complicated by perforation. The authors, in the absence of the donor cornea, faced a complex clinical task - the risk of functional and anatomical death of the single eyeball in the absence of adequate care for the patient. After enucleation of the subatrophic blind right eye, we took a corneal specimen with a scleral rim of 12 mm trepan. To collect the material, we used an original method of introducing viscoelastic into the vitreal cavity of the hypotonic enucleated eyeball through the intersected optic nerve. This allowed to create the normotony of the enucleated eyeball and perform trepanation of the corneoscleral disc. Trepanation of the sharply ectatic cornea of the left eye was performed after paracentesis and massage of the dome of ectatic cornea with a spatula, which led to the 'subsidence' of the hyperinflated tissue. The corneoscleral disc was fixated with interrupted suture with a 9-0 thread. The postoperative period proceeded smoothly. The autograft has taken with active vascularization. For optical rehabilitation, the patient is scheduled to undergo a keratoprosthetic operation in a planned manner.

[Temporary biokeratoprostheses in total destruction of the cornea].

The paper presents an original method for temporary biokeratoprothetics in total destruction of the cornea, which consists of the following. In the operating room a 12-mm disc is cut out of patient's conchal cartilage by a trephine and then thinned down to 1 mm with a blade. The prepared autograft is placed in front of the iris completely overlapping corneal defect and sutured to the sclera with 10--12 U-shaped interrupted stitches using a 7/0 suture. Between the stitches 0.2--0.3 ml of viscoelastic are injected into the anterior chamber. Temporary blepharorrhaphy is done within the temporal one-third of the eyelids with a U-shaped suture 6/0. The authors present an own clinical observation of patient D., aged 46, with purulent corneal ulcer and total destruction of the cornea. In ophthalmic emergency, if no donor cornea is available, the described method allows to save the eyeball from its anatomical and functional loss. Further routine keratoplasty or optical keratoprosthetics may bring some functional improvement.

Activation of the human anaphase-promoting complex by proteins of the CDC20/Fizzy family.

The initiation of anaphase and exit from mitosis depend on the activation of the cyclosome/anaphase-promoting complex (APC) that ubiquitinates regulatory proteins such as anaphase inhibitors and mitotic cyclins [1-4]. Genetic experiments have demonstrated that two related WD40-repeat proteins--called Cdc20p and Hct1p/Cdh1p in budding yeast and Fizzy and Fizzy-related in Drosophila--are essential for APC--dependent proteolysis [5-11]. Human orthologs of these proteins--hCDC20/p55CDC [12] and hCDH1--have recently been found to associate with APC in a cell-cycle-dependent manner [13,14]. Here, we show that the amount of hCDC20 and hCDH1 bound to APC correlates with a high ubiquitination activity of APC and that binding of recombinant hCDC20 and hCDH1 can activate APC in vitro. Our results suggest that the association between hCDH1 and APC is regulated by post-translational mechanisms, whereas the amount of hCDC20 bound to APC may in addition be controlled by hCDC20 synthesis and destruction [15]. The temporally distinct association of hCDC20 and hCDH1 with APC suggests that these proteins are, respectively, mitosis-specific and G1-specific activating subunits of APC.

Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis.

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression.

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1-cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.

Mitotic regulation of the APC activator proteins CDC20 and CDH1.

The ordered activation of the ubiquitin protein ligase anaphase-promoting complex (APC) or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. Here, we show that CDC20 can only bind to and activate the mitotically phosphorylated form of the Xenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, we propose that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APC(CDC20) activation. We further show that CDH1 is phosphorylated in vivo during S, G2, and M phase and that CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APC(CDC20) and APC(CDH1); the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred.

Polymorphisms in the receptor for GDNF (RET) are not associated with Parkinson's disease in Southern Germany.

The aetiology of the selective neurodegeneration in Parkinson's disease (PD) is still unknown. Neurotrophic factors, e.g. glial cell line-derived neurotrophic factor (GDNF), have been shown to promote survival of dopaminergic neurons. Interestingly, aged mice lacking GDNF-receptor (RET) in their dopaminergic neurons show a phenotype similar to presymptomatic PD. We therefore were interested whether polymorphisms in the RET gene were associated with increased PD risk. Analyzing 25 SNPs in the RET region in 340 Southern German PD patients and 340 age- and sex-matched controls from Southern Germany (KORA S4), we did not find any significant association with PD, suggesting that the equilibrium of trophic factors in PD might be disturbed on other levels than the genomic encoding.

Ultramicroscopy: 3D reconstruction of large microscopical specimens.

Ultramicroscopy is a microscopical technique that allows optical sectioning and 3D reconstruction of biological and medical specimens. While in confocal microscopy specimen size is limited to several hundred micrometers at best, using ultramicroscopy even centimeter sized objects like whole mouse embryos can be reconstructed with micrometer resolution. This is possible by using a combination of a clearing procedure and the principle of lightsheet illumination. We present ultramicroscopic 3D reconstructions of whole immunohistochemically labelled mouse embryos and adult Drosophila, giving detailed insight into their anatomy. Its speed and simplicity makes ultramicroscopy ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons.

The anaphase-promoting complex/cyclosome (APC) is a tightly cell cycle-regulated ubiquitin-protein ligase that targets cyclin B and other destruction box-containing proteins for proteolysis at the end of mitosis and in G1. Recent work has shown that activation of the APC in mitosis depends on CDC20, whereas APC is maintained active in G1 via association with the CDC20-related protein CDH1. Here we show that the mitotic activator CDC20 is the only component of the APC ubiquitination pathway whose expression is restricted to proliferating cells, whereas the APC and CDH1 are also expressed in several mammalian tissues that predominantly contain differentiated cells, such as adult brain. Immunocytochemical analyses of cultured rat hippocampal neurons and of mouse and human brain sections indicate that the APC and CDH1 are ubiquitously expressed in the nuclei of postmitotic terminally differentiated neurons. The APC purified from brain contains all core subunits known from proliferating cells and is tightly associated with CDH1. Purified brain APC(CDH1) has a high cyclin B ubiquitination activity that depends less on the destruction box than on the activity of mitotic APC(CDC20). On the basis of these results, we propose that the functions of APC(CDH1) are not restricted to controlling cell-cycle progression but may include the ubiquitination of yet unidentified substrates in differentiated cells.

Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex.

We have discovered an early mitotic inhibitor, Emi1, which regulates mitosis by inhibiting the anaphase promoting complex/cyclosome (APC). Emi1 is a conserved F box protein containing a zinc binding region essential for APC inhibition. Emi1 accumulates before mitosis and is ubiquitylated and destroyed in mitosis, independent of the APC. Emi1 immunodepletion from cycling Xenopus extracts strongly delays cyclin B accumulation and mitotic entry, whereas nondestructible Emi1 stabilizes APC substrates and causes a mitotic block. Emi1 binds the APC activator Cdc20, and Cdc20 can rescue an Emi1-induced block to cyclin B destruction. Our results suggest that Emi1 regulates progression through early mitosis by preventing premature APC activation, and may help explain the well-known delay between cyclin B/Cdc2 activation and cyclin B destruction.

[The treatment of vascular optic neuropathies]. / K lecheniiu sosudistykh opticheskikh neiropatii.

The paper analyses results after treatment of 280 patients (467 eyes) with different forms of vascular optic neuropathies. Considering the method of treatment, the patients were divided into 5 groups. In group I--only traditional methods of treatment were used; in group II--inhalations of a 5% carbogen, 15 minutes 1-2 times a day, were added; in group III--hyperbaric oxygenation, 6-10 sessions, was added; in group IV--a blockade of sino-carotid zone by a 2% novocaine, 5 ml, 10 days, was added; in group V--a twenty-four-hour intracarotid infusion of medicinal preparation, 5-7 days, was made. The highest improvement of visual functions was recorded in group IV (90.2%), the lowest--in group I (41%). A conclusion is made that it is necessary to create wards of intensive therapy or vascular centers with a specially trained staff and a necessary store of infusional and other medicamentous preparations.

Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1.

CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G(1) and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G(1).