A decrease of the rabbit's physiologic IOP after the application of specific amino acids and double combination of antiglaucomatics mixture.

PURPOSE: To compare the effect of amino acids mixtures to a double combination of antiglaucomatics on the physiologic intraocular pressure (IOP) in rabbits. METHODS: Experimental evaluations were performed on 5 female rabbits of the New Zealand White species. RESULTS: 1) After instillation of 10 % L-arginine. HCl in the double combination of antiglaucomatics: a) 0.5 % Timolol with 0.005 % Xalatan mixture, the biphasic IOP decrease was measured. The mean decrease in 24 hours was - 2.9 torr; b) 2 % Trusopt with 0.005 % Xalatane mixture in 24 hours, the biphasic decrease of the IOP was measured. The mean decrease in 24 hours was - 4.2 torr; c) Fixed combination COSOPT the mean IOP value decrease was - 1.8 torr. 2) The IOP decrease achieved during 24 hours by instillation of the amino acid 10 % L-taurine.HCl and the double combination of antiglaucomatics: 0.5% Timolol with 0.005% Xalatane mixture was in average - 2.8 torr. The pupilar diameter was not changed. CONCLUSIONS: We assume that after the interaction of the selected amino acids 10 % L-arginin or 10 % L-taurin with the double combination of antiglaucomatics (Trusopt and Xalatan, COSOPT or Timoptol and Xalatan) a new substance was formed. The effect of this substance is not separate or additive but acts as a newly formed substance. In vivo, it is only a weak interaction with the free amino acids of the conjunctival sac and the antiglaucomatics. The amino acids interacted in vitro with the double combination of antiglaucomatics resulting in a new "bio-antiglaucomatic" better penetrating into the target area. This new substance was responsible for a more significant decrease and regulation IOP after the mixture application compared to the antiglaucomatics alone (Fig. 1, Ref. 23).

Mechanical injuries of the eye.

BACKGROUND: Traumatic mechanical damage of the eye can cause serious morphological and functional changes in eye structures. Thorough examination and appropriate approach are essential in successful treatment and maintenance of visual functions. MATERIAL AND METHODS: In the years 2005-2007, 78 patients with mechanical eye injury were treated at the Department of Ophthalmology, Faculty of Medicine, Comenius University, Bratislava. We treated 17 eyes with globe contusions, 19 eyes with penetrating injuries, 16 eyes with intraocular foreign bodies, 2 eyes with perforating injuries and 24 eyes with globe ruptures. RESULTS: Final visual acuity better than 5/50 was achieved in 7 eyes (41.2%) with contusion, 15 eyes (78.9%) wih penetrating injuries, 10 eyes (62.5%) with intraocular foreign bodies and in 5 eyes (20.8%) with globe ruptures. Twelve eyes (50.0%) from the group of patients with globe ruptures were without light perception. CONCLUSION: Traumatic mechanical damage of the eye can result in serious morphological and functional impact on eye tissue structures. Globe rupture often causes a visual loss despite advances in diagnostic and surgical procedures. Proper examination and appropriate approach are essential in successful treatment and saving the visual functions. Modern diagnostic and surgical procedures can save many eyes and maintain their useful function (Tab. 9, Fig. 2, Ref. 12).

Optical coherence tomography--a new imaging method in ophthalmology.

An improvement of examination methods in ophthalmology, technical digitalisation and knowledge of validity of examinations in various diseases contributes to early diagnostics, thereby leading to an opportunity for early treatment of eye disorders. Standard introduction of the so-called optical coherence tomography into the ophthamological clinical practice facilitated new options for a detailed analysis of pathological processes in the particular layers of the retina (Fig. 2, Ref. 5). Full Text (Free, PDF) www.bmj.sk.

Heterogeneous amino acids in Ras and Rap1A specifying sensitivity to GAP proteins.

Guanosine triphosphatase (GTPase) activity of Ras is increased by interaction with Ras-GAP (GTPase-activating protein) or with the GAP-related domain of the type 1 neurofibromatosis protein (NF1-GRD), but Ras is not affected by interaction with cytoplasmic and membrane forms of Rap-GAP; Rap1A, whose effector function can suppress transformation by Ras, is sensitive to both forms of Rap-GAP and resistant to Ras-GAP and NF1-GRD. A series of chimeric proteins composed of portions of Ras and Rap were constructed; some were sensitive to Ras-GAP but resistant to NF1-GRD, and others were sensitive to cytoplasmic Rap-GAP but resistant to membrane Rap-GAP. Sensitivity of chimeras to Ras-GAP and cytoplasmic Rap-GAP was mediated by amino acids that are carboxyl-terminal to the effector region. Residues 61 to 65 of Ras conferred Ras-GAP sensitivity, but a larger number of Rap1A residues were required for sensitivity to cytoplasmic Rap-GAP. Chimeras carrying the Ras effector region that were sensitive only to Ras-GAP or only to cytoplasmic Rap-GAP transformed NIH 3T3 cells poorly. Thus, distinct amino acids of Ras and Rap1A mediate sensitivity to each of the proteins with GAP activity, and transforming potential of Ras and sensitivity of Ras to Ras-GAP are at least partially independent properties.

Rabbit's intraocular pressure after instillation of timolol and aminoacid lysine, arginine, glycine or taurine mixture.

AIM: Presented experimental work was aimed to examine a pharmacokinetic efficiency of 0.5% Timolol mixtures with 4 free amino acids, present in conjunctival sac: lysine, arginine, glycine or taurine on the IOP physiological values in rabbits. METHODS: The experimental work was performed on 5 female rabbits of the New Zealand White species. After instillation at 8.00 a.m. into the left conjunctival sac: a) the 10% L-lysine x 2HCl x 2H2O in 0.5% Timolol; b) the 10% L-arginine x HCl in 0.5% Timolol; c) the 10% L-glycine x HCl in 0.5% Timolol; d) the 10% L-taurine x HCl in 0.5% Timolol, the IOP was measured before and in 5th, 15th, 30th, 60th, 120th, 180th and 240th min and in 24 hours. The right eye of the same rabbit was used as control with the instillation of both 0.5% Timolol and amino acids alone into the conjunctival sac. RESULTS: a) The IOP has decreased after mixture of 10% lysine in 0.5% Timolol with two spikes--not significant decrease up to 60th min and a high significant decrease between 120th to 240th min; b) The mixture of 10% arginine in 0.5% Timolol decreased the IOP values with a high significance (from 2.1 to 4 torr); c) The 10% glycine in 0.5% Timolol showed a significant IOP decrease (excluding measurement in 5th min) in all measured times, the biggest IOP decrease was observed in 60th min (mean value 8.5 torr), the decrease was 3.3 torr also in 24th hours; d) The 10% taurine in 0.5% Timolol showed also a significant IOP decrease (excluding measurement in 5th min) in all measured times with maximum in 180th min (with a decrease to 6.7 torr) and the decrease showed a significantly low level--3.3 torr--also in 24 hours. CONCLUSIONS: The results proved that the mixture of antiglaucomatic 0.5% Timolol and amino acid (lysine, arginine, glycine or taurine) contains a new biologically active substance, the "specific bioantiglaucomatic" created by interaction. Compared to the substances alone, mixture of amino acid in the antiglaucomatic decreased the physiologic IOP in rabbits with a high significance. The effect on IOP based on the interaction of the mixture of amino acid with antiglaucomatic is specific and its efficacy together with time was changed depending on the type of amino acid. Our in vitro produced bioantiglaucomatic fulfilled the physiological criteria for the IOP reduction (Fig. 4, Ref 12). Full Text (Free, PDF) www.bmj.sk.

Novel ion exchange chromatography method for nca arsenic separation.

A high-performance liquid chromatography (HPLC) device equipped with an anion exchange column was used to isolate nca 77As from reactor irradiated natGeO2 targets. The oxidation states of the isotope 77As during the process was verified by thin layer chromatography. The radionuclidic purity of the separated fractions was checked by gamma measurements and it was found to be 99.91% for the As fraction. The elaborated method was applied to separate the isotope 74As from cyclotron irradiated natGeO2 targets too.

Founder effect is responsible for the p.Leu131Phe heparin-binding-site antithrombin mutation common in Hungary: phenotype analysis in a large cohort.

BACKGROUND: Antithrombin (AT) is a key regulator of the coagulation. In type II deficiency, the heparin-binding-site defect (type II HBS) is considered to be relatively low thrombosis risk. OBJECTIVES: Our aims were to search for SERPINC1 mutation(s) and to describe the clinical and laboratory phenotype of a large number of AT Budapest3 (ATBp3, p.Leu131Phe) carriers and confirm the presence of a founder effect. PATIENTS/METHODS: AT-deficient patients were recruited and carriers of ATBp3, n = 102 (63 families) were selected. To investigate the founder effect, eight intragenic single nucleotide polymorphisms, a 5'-length dimorphism, and five microsatellite markers were detected. Clinical and laboratory data of the patients were collected and analyzed. RESULTS: In AT deficiency, 16 different causative mutations were found, and the great majority of patients were of type II HBS subtype. Most of them (n = 102/118, 86.5%) carried the ATBp3 mutation. The ATBp3 mutant allele was associated with one single haplotype, while different haplotypes were detected in the case of normal allele. The anti-factor Xa-based AT activity assay that we used could detect all ATBp3 patients with high sensitivity in our cohort. ATBp3 homozygosity (n = 26) was associated with thrombosis at a young age and conferred a high thrombotic risk. Half of the heterozygotes (n = 41/76, 53.9%) also had venous and/or arterial thrombosis, and pregnancy complications were also recorded. CONCLUSION: In Hungary, the founder mutation, ATBp3, is the most common AT deficiency. Our study is the first in which the clinical characterization of ATBp3 mutation was executed in a large population.

Differential effects of activation of protein kinase C and cyclic-AMP-dependent protein kinase on sodium-dependent phosphate uptake in NIH 3T3 cells.

Activation of protein kinase C (PKC) by phorbol ester (PMA), or by diacylglycerol analogue (OAG) treatment of NIH 3T3 cells resulted in the rapid (within 2-5 min) stimulation (approx. 2-fold) of sodium-dependent phosphate (Pi) transport. Conversely, preincubation of these cells with forskolin and cholera toxin, or incubation with 8-bromo-cAMP, to activate cAMP-dependent protein kinase (PKA), resulted in a decrease in Na+/Pi transport. Activation of either PKC or PKA did not change the Vmax of Pi uptake. However, activation of PKC did result in an increase, while activation of PKA caused a decrease, in the affinity for Pi. These results indicate that there is differential regulation of Na+/Pi uptake in NIH 3T3 cells by activators of PKC (stimulated) and PKA (inhibited) as a consequence of changes in the affinity of the transporter for Pi.

Differential effects of free and liposome-associated 1-O-octadecyl-2-O-methylglycerophosphocholine on protein kinase C.

Incorporation of ET-18-OCH3 into well-characterized liposomes known as ELL-12 has eliminated its gastrointestinal and hemolytic toxicity without loss of growth inhibiting activity. ET-18-OCH3, but not ELL-12, blunted the increase in membrane protein kinase C (PKC) activity induced by 12-O-tetradecanoylphorbol 13-myristate (TPA) and markedly reduced levels of PKC alpha in NIH 3T3 fibroblasts. Furthermore, prolonged treatment with ELL-12 neither inhibited TPA-induced translocations of PKC alpha and PKC delta to the particulate fraction nor caused down-regulation, and did not affect the cellular distribution of TPA-insensitive PKC zeta. In Jurkat T cells, where ELL-12 markedly induced apoptosis that was blocked by an inhibitor of caspase-3-like activities, it had no effect on PKC activity or translocation induced by TPA. Thus, it seems unlikely that PKC is involved in the therapeutic effects of ELL-12.

Early X-irradiation of rats-4. Decrease in phosphorylation of low molecular weight proteins in the olfactory bulb accompanies the loss of GABAergic microneurons.

X-irradiation of neonatal rats by low, fragmented and repeated doses reduces or eliminates several phosphoproteins (16, 18 and 19 Da) in an extract of the olfactory bulb. This coincides with a dramatic reduction of the number of GABAergic granule cells. The phosphorylation of these proteins was specifically stimulated in control animals by Ca(2+)/calmodulin, but not by cyclic adenosine triphosphate. The reduced inhibitory function, observed previously in other "granuloprived" bulbs, might be the consequence of loss of those cells expressing these low molecular weight phosphoproteins. To assess the cellular changes caused by X-irradiation, low molecular weight phosphoproteins can serve as specific markers in the olfactory bulb.

Sphingosine-1-phosphate in cell growth and cell death.

Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and Fas ligand, sphingosine-1-phosphate (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, nerve growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and sphingosine kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP, and consequent regulation of different family members of mitogen-activated protein kinases (JNK versus ERK), is an important factor that determines whether a cell survives or dies.

Inhibition by H-7 of the protein kinase C prevents formation of brain edema in Sprague-Dawley CFY rats.

The effect of the protein kinase C enzyme inhibitor H-7 was examined on the brain edema formation evoked by bilateral occlusion of the common carotid arteries in Sprague-Dawley rats of CFY strain. Brain edema was assessed by measurement of water and electrolyte contents of the brain. The results showed that pretreatment with H-7 reduced the extent of brain edema formation in a dose-dependent manner. The fact that H-7 treatment prevented the accumulation of water and certain electrolytes in the brain indicates that the protein kinase C may be activated not only in the neuronal structures but also in the microvessels during ischemia, which can lead directly or via certain calcium-mediated mechanisms to the opening of tight junctions resulting in the development of brain edema.

Immunohistochemical localization of raf protein kinase in dendritic spines and spine apparatuses of the rat cerebral cortex.

The ultrastructural localization of raf protein (product of the raf protooncogene) in the neocortex, pyriform cortex and hippocampus of the rat has been investigated by means of pre-embedding immunohistochemistry. Specificity of the antiserum was tested with Western blotting. Besides the immunoreactivity of the dendrites, remarkably strong immunostaining of the dendritic spines and spine apparatuses was noted in each of the investigated areas. The postsynaptic densities were also stained. Since raf proteins are serine/threonine-specific protein kinases, our findings could be important steps toward the understanding of dendritic spine plasticity.

Epidural resiniferatoxin induced prolonged regional analgesia to pain.

Adequate treatment of cancer pain remains a significant clinical problem. To reduce side effects of treatment, intrathecal and epidural routes of administration have been used where appropriate to reduce the total dose of agent administered while achieving regional control. Resiniferatoxin (RTX), an ultrapotent capsaicin analog, gives long-term desensitization of nociception via C-fiber sensory neurons. We evaluate here the analgesic effect on rats of epidurally administered RTX, using latency of response to a thermal stimulus in unrestrained animals. Results were compared with those for systemically administered RTX. Vehicle or graded doses of RTX were injected subcutaneously (s.c.) or through an indwelling lumbar (L4) epidural catheter as a single dose. Both routes of application of RTX produced profound thermal analgesia, reaching a plateau within 4-6 h and showing no restoration of pain sensitivity over 7 days. Vehicle was without effect. For the epidural route, the effect was selective as expected for the targeted spinal cord region, whereas the subcutaneous administration of RTX had a generalized analgesic effect. At doses yielding a tripling of back paw withdrawal latency, epidural treatment was 25-fold more effective than the subcutaneous route of application. Consistent with the regional selectivity of the lumbar epidural route, the front paws showed no more effect than by systemic RTX treatment. Binding experiments with [3H]RTX provided further evidence of the segmental desensitization induced by epidural RTX. We conclude that epidural administration of RTX at the lumbar spinal level produces profound, long-lasting, segmental analgesia to C-fiber mediated pain in the rat.

Transient increase of raf protein kinase-like immunoreactivity in the rat dentate gyrus during long-term potentiation.

Altered levels of cellular raf proteins (products of the raf protooncogenes) have been shown in the neurons of the dentate fascia of rats in response to high-frequency stimulation, with light microscopic immunohistochemistry by using polyclonal antibodies. No raf-1-like staining was seen in unstimulated tissue, while the pan-raf antibodies revealed immunoreactivity in the cytoplasm of neurons in the Ammon's horn and dentate fascia of rats and guinea pigs. The induction of long-term potentiation in the dentate fascia of freely-moving rats triggered the appearance of raf-1-like staining and increased the number of granule cells with pan-raf-like immunoreactivity. Since these proteins are serine/threonine-specific protein kinases, their appearance in long-term potentiation may indicate the activation of important cell membrane - nucleus transduction pathways.

New chimera proteins for fluorescence correlation spectroscopy.

A new class of chimera proteins has been developed. They are ideally suited for detection by fluorescence correlation spectroscopy (FCS), a new technology to analyze molecular interactions. The molecular structure of these chimera proteins consists of four domains: a N-terminal (His)6-tag for affinity chromatography followed by an eight amino acid epitope for immunodetection, a polypeptide affinity domain (ADF) for target specific interaction and a C-terminal Green Fluorescent Protein (GFPuv) for reporting of interaction with the target by FCS. We designed, prepared and characterized a prototype of ADF-GFP proteins capable of specific interaction with DNA fragments bearing nuclear factor (NF)-kappaB sites. ADF NF-kappaB p50 and a non-DNA-binding deletion mutant (p35) combined with GFPuv were inserted in a procaryotic vector and expressed in E. coli. Following affinity purification the fluoroproteins p50-GFPuv and p35-GFPuv were employed in specific protein-protein and protein-DNA interaction studies. FCS analysis as well as EMSA showed that p50-GFPuv revealed a fully functional ADF. We present a model for the preparation of GFP fusion proteins capable of specific interaction with proteins, lipids or nucleic acids. The rational design allows any polypeptide fragment to be incorporated into the chimeric protein. So a new series of bio-molecules with different binding specificities and assays can be developed.

Fusion of the binding domain of Raf-1 kinase with green fluorescent protein for activated Ras detection by fluorescence correlation spectroscopy.

Ras proto-oncogenes play a central role in cell proliferation by the regulation of signal transduction pathways from receptors of the outer cell membrane to the nucleus via the activation of transcription factors. Wild-type Ras cycles between the activated GTP-bound and the inactivated GDP-bound state, and the GTPase reaction is a timer for the interaction between Ras-GTP and effector molecules such as Raf-1 protein kinase. Mutations of ras resulting in the loss of the intrinsic GTPase activity result in autonomous proliferation. Mutated Ras is found in a variety of human tumors. Therefore, monitoring of GTP-loaded conformation of Ras related proteins could be utilised in cancer diagnosis. To develop a fluorescence based bioassay we have coupled the gene for the N-terminal Ras binding domain (RBD) of Raf-1 protein kinase with the gene for the green fluorescent protein (GFP). The chimeric fusion protein RBDGFP was identified by immunoblotting and subsequently investigated by fluorescence correlation spectroscopy (FCS), a new analytical technology allowing the measurement of characteristic diffusion times of fluorescently labeled molecules. Molecular interactions increase the molecular weight and influence the diffusion time of RBDGFP. FCS diffusion value of the recombinant protein was in coincidence with the molecular weight of the construct. Fluorimetric measurements of RBDGFP versus GFP showed clearly that the recombinant protein contains functional GFP. Increased FCS transition times indicated the interaction of RBDGFP with its corresponding antibody. Suboptimal binding of the fusion protein to activated Ras, how ever, resulted in a modest influence on the diffusion value. Taken together our rational design and construct shows the way for a ready characterisation of novel GFP-connected fusion proteins employing FCS.

[Reaction of the fibrose eyeball covering upon the suture material synthetic and absorbable "Dexon". (Experimental study) (author's transl)]. / Réaction du tissu conjonctif scléro cornéen à un matériel de suture synthétique et résorbable "Dexon". Etude expérimentale.

The present paper deals with the results of 20 eyes of experimentally operated laboratory animals, whose perforating wounds (of cornea and those of sclerocorneal region) have been suturated with a new type of absorbable synthetic suture material "Dexon" thickness being 6-0. The synthetic absorbable suture material "Dexon" is made of polyglycol acid, and it has been introduced to market by the firm Davis and Geck (U.S.A.). The inflammatory tissue reaction to the presence of the suture material "Dexon" is prominent and can by compared to the reaction to chromic resorbable suture materials (catgut or collagen). The suture material "Dexon" start to resorb in the course of the 2nd-3rd weeks after operation. The suture material "Dexon" can be used at an advantage in the operation where it is possible to cover the knots by a conjuctival lobe (in sclerocorneal region or in strabismus surgery) in this way the tendency to overgrowing of epithelial cells along the suture channels can be prevented. The "Dexon" material is sufficiently flexible and firm and has no antigenic properties, therefore it appears very prospective for ophthalmosurgery.

[Changes in the retinal enzyme system in age-related macular degeneration]. / Zmeny enzymových systémov sietnice pri VPMD.

The contribution interprets latest findings in retinal pathophysiology from the view of the retinal enzymatic system function in age related macular degeneration and mutual interaction between the atrophic degenerative process of the retinal pigmented epithelium, the Bruch's membrane and choriocapillaris at the macula.

[The occurrence and evidence of malignant intraocular tumors in Slovakia (1968-1985)]. / Výskyt a evidencia malígnych vnútroocných nádorov v SR (1968-1985).

The paper presents a survey of the incidence of notified malignant intraocular tumors, code 190 of the International Classification of Diseases (ICD-9). It analyzes the incidence, aspecific incidence and mortality rate also according to the 4th subclass of code 190 (ICD-9), covering the territory of the Slovak Republic in the period from 1968-1985. The incidence of intraocular tumors over the period studied was found to be 8.2/100,000 inhabitants. The recorded data correspond to values given for other regions.