[Incomplete Wolfram syndrome. Clinical case report].

We describe clinical presentation of Wolfram syndrome and follow-up data in a child. Diagnostics of Wolfram syndrome takes time because clinical symptoms develop not at the time of disease manifestation, but usually several years later. The sequence of manifestations also varies. According to the literature, sensorineural hearing loss occurs in the 2nd decade, and bladder atony develops only by the 3rd decade. In the presented case, initial manifestations of bladder innervation disorders in the form of its dysfunction developed as early as the first year, and sensorineural hearing loss formed by the 4th year of the disease. As in other studies, the patient developed optic disc atrophy within the first year after diabetes onset. This clinical case confirms variability in the clinical symptoms of Wolfram syndrome. The sequence in which the disease picture develops (in this case, there was an incomplete form of syndrome - the absence of diabetes insipidus) does not always coincide with the classic course of syndrome, which complicates timely diagnosis.

[ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES IN LIPOPOLYSACCHARIDE-PROMOTED ACCUMULATION OF INTRACELLULAR LIPID DROPLETS].

It is known that in various cell types bacterial lipopolysaccharide (LPS) causes mitochondrial disfunction and promotes accumulation of triglycerides in intracellular lipid droplets. The precise mechanisms which mediate LPS-induced neutral lipid deposition remain poorly understood. In the present work performed on primary cultured epithelial cells isolated from the frog urinary bladder we studied the possible role of mitochondrial reactive oxygen species (mROS) in LPS-in-duced alteration of lipid metabolism. It was shown that LPS stimulated ROS production, decrea- 705 sed fatty acids oxidation, enhanced intracellular triglyceride deposition and promoted the formation of lipid droplets visualized by Nile red staining. Pretreatment of cells with mitochondrial-tar-geted antioxidant MitoQ at dose 25 nM for 2 h almost completely eliminated all the above effects of LPS. In contrast to MitoQ, a-tocopherol was ineffective. The data obtained indicate that increase of mROS level is a critical factor that mediates LPS-induced intracellular deposition of neutral lipids in epithelial cells.

[EFFECT OF LIPOPOLYSACCHARIDE ON NEUTRAL LIPID METABOLISM AND CELLULAR ENERGETICS IN FROG URINARY BLADDER EPITHELIAL CELLS].

It was shown previously that colonization of the frog urinary bladder by gram-negative bacteria leads to decreased ability of antidiuretic hormone to reabsorb water from the urinary bladder (Fock et al. J. Exp. Zool., 2013, 319A: 487-494). In the present work performed on epithelial cells isolated from the frog urinary bladder the influence of E. coli lipopolysaccharide (LPS) on neutral lipid metabolism and cellular energetics was studied. It was shown that incubation of cells with LPS led to decrease of fatty acids oxidation and to retention of triacylglycerols (TAG) followed by an increase of the cytoplasmic lipid droplets content and cellular amount of TAG. Fatty acid composition of TAG was not changed under LPS. LPS did not alter mitochondrial membrane potential, however, LPS decreased oxygen consumption rate both in basal and uncoupling conditions. Cellular ATP production was also reduced in the presence of LPS. The data obtained indicate that a decreased ability of antidiuretic hormone to reabsorb water from the urinary bladder induced by bacterial pathogens could be related to inhibition of fatty acids oxidation and impaired energy metabolism.

[Deposition of exogenous and endogenously generated unsaturated fatty acids in lipid droplets triacylglycerol as a mechanism of its sequestration in epithelial cells].

Neutral lipids are deposited in intracellular compartments called lipid droplets, which are known to be critically implicated in regulation of cellular lipid metabolism. These organelles consist of a core of neutral lipids, mainly triacylglycerol (TAG) and cholesteryl esters, surrounded by phospholipid monolayer. Using Nile red lipid staining and [3H]-arachidonic and [3H]-oleic acids as precursors for lipid biosynthesis, we have evaluated the mechanisms of lipid body induction elicited by exogenous fatty acids within primary cultured epithelial cells from the frog urinary bladder. It was found that arachidonic and oleic acids at concentrations 10-50 tM stimulated lipid droplets formation accompanied by accumulation of TAG and by the significant increase of incorporation of fatty acids into TAG indicating an enhanced TAG biosynthesis. No changes of cholesteryl esters content were observed under these conditions. In cells, prelabelled with [3H]-oleic acids, etomoxir, an inhibitor of O-carnitine palmitroyltansferase 1, decreased oxidation of oleic acid and increased its incorporation into TAG leading to intracellular TAG accumulation. In cells, prelabelled with [3H]-arachidonic acid, diclofenac, an inhibitor of cyclooxygenase 1 and 2, led to significant decrease in cellular PGE2 production and to reesterification of free arachidonic acid to TAG but not to phospholipids. Taking together, these data evidence that in isolated frog urinary bladder epithelial cells, reacylation of unsaturated free fatty acids into TAG is a main route of their metabolic conversion under the conditions of the increased cytosolic level of free fatty acids.

[Gram-negative bacteria influence on the ability of the arginine-vasotocin to stimulate osmotic permeability of the frog urinary bladder epithelium].

The influence of endogenous gram-negative bacteria colonizing the mucosal epithelium of frog Rana temporaria L. urinary bladders (FUB) on arginine-vasotocin AVT-stimulated osmotic water flow in isolated urinary bladders was investigated. 170 animals were examined and only 40% were contaminated with gram-negative bacteria (about 10(3)-10(6) CFU per hemibladder). Several Enterobacteriaceae species were identified (Hafnia alvei, 36.7%, E. coli, 32.3%, Serratia marcescens, 8.8%, Citrobacter freundii, 4.4% etc.). Basal osmotic water flow level was invariable in "clean" and contaminated FUB, whereas bacterial contamination resulted in considerable decrease in AVT-stimulated water flow ("clean": 2.53 +/- 0.13, n = 59, contaminated: 1.21 +/- 0.17 me/min/cm2, n = 38, p < 0.001, within first 15 min of incubation with 5 x 10(-10)M AVT). Gentamycin protection assay revealed predominantly adhesive forms of bacteria. Thus our data indicated that the presence of gram-negative bacteria colonizing the mucosal epithelium of the urinary bladder results in decreased adility of ADH to rise osmotic water permeability which in turn could impair body osmoregulation.

[Regulatory interconnections of cyclooxigenase and inducible no-synthase in urinary bladder epithelial cells of the frog Rana temporaria under action of bacterial stimuli].

Earlier we have shown that in epithelial cells of the frog urinary bladder under action of bacterial lipopolysaccharides (LPS) there is activated expression of inducible NO-synthase (iNOS) and there is increased the NO production, which can play an important role in providing protective cell reactions from pathogens. The goal of the present work consisted in study of cyclooxigenase (cOG) products and mechanisms of their regulatory effect on expression of iNOS under action of LPS. In experiments on urinary bladder epithelial cells on the frog Rana temporaria it has been shown that incubation of the cells for 21 h with LPS leads to a rise in production of PGE2 and nitrites, stable NO metabolites. Inhibitor of iNOS 1400W decreased sharply production of nitrites, but did not affect the PGE2 level. Both the basal and the LPS-stimulated level of PGE2 and nitrites were inhibited in the presence of selective cOG inhibitors--SC-560 (cOG-1) and NS-398 (cOG-2). The IC50 value amounted to 90, 220, and 470 microM for NS-398, SC-560, and diclofenac (unspecific inhibitor of both isoforms), respectively. PGE2 and butaprost, the EP2-receptor agonist, but not agonists of EP1/EP3 or EP1 receptors, partially eliminated the inhibitory action of diclofenac on production of nitrites. Action of PGE2 was accompanied by an increase in the intracellular cAMP. Analysis of expression of iNOS mRNA in the epithelial cells incubated with LPS or LPS + inhibitor of cOG has shown the LPS-stimulated rise in expression of iNOS mRNA to decrease sharply in the presence of SC-560 or NS-398. Thus, the epithelial cells of the frog urinary bladder have the effectively functioning system of the congenital immune protection against bacterial pathogens, the most important component of this system being PGE2 and NO. Analysis of mechanisms of regulatory interactions of cOG and iNOS indicates that in this cell type the main regulators of iNOS expression and of the nitrogen oxide level are products of the cOG catalytic activity.

[Lipopolysaccharide E. coli inhibits the arginine-vasotocin-induced increase of osmotic water permeability in the frog urinary bladder].

Since Gram-negative bacteria are known to be present in the cavity of urinary bladders in amphibian species, it was interesting to study the effect of bacterial endotoxins on epithelial signaling network which provides the arginine-vasotocin-induced increase of osmotic water permeability (OWP). The effect of LPS E. coli on AVT-induced OWP was studied in isolated frog Rana temporaria L. urinary bladder incubated during 20-21 hours in modified L-15 culture medium in sterile conditions. The LPS (25 microg/ml) was added into the mucosal solution. It was shown that exposure to LPS caused a strong suppression of the increase of OWP under AVT (0.5 nM), forskolin (35 microM) or IBMX (200 microM). Moreover, LPS induced more than 2-folds decrease both ofbasal and AVT-stimulated content of cAMP in the bladder tissue. The inhibitory effect of LPS on AVT-induced increase of OWP was eliminated in the presence of ODQ, 20 microM, a cytosolic guanylate cyclase inhibitor. With the use of RT-PCR it was shown that the expression of mRNA iNOS was 10-fold increased in 6 hours after LPS administration. These findings demonstrate the ability of frog bladder mucosal epithelial cells to recognize bacterial LPS and initiate antipathogen immune response related to increased production of nitric oxide. The activation of signal transduction cascade mediated by the LPS-induced immune response leads to a decrease of intracellular cAMP and down-regulates AVT-stimulated OWP acting at least in part through NO/cGMP-dependent signaling pathway.

[No-synthase activity in primary cultured frog urinary bladder epithielial cells and no-dependent antimicrobial effect].

We have shown previously that endogenous NO modulates the effect of arginine-vasotocin on the increase in the osmotic water permeability of the frog urinary bladder epithelium. The aim of the present work was to develop a procedure of cultivation of epithelial cells from the frog urinary bladder as a primary culture in order to study in vitro the cellular production of NO and its regulation. Isolated cells were cultivated in modified L-15 medium with 10% FBS and gentamycin (40 microg/ml) at room temperature. Under these conditions, at least 50% cells kept their viability until 8 days of incubation. NO-synthase (NOS) activity was estimated as nitrite (NO2-) accumulation in culture medium; NO2- concentration in the presence of L-NAME, inhibitor of all NOS types, was considered as NOS-independent and was subtracted from each value. The nitrite accumulation was linear in time during 3 days of cultivation and was inhibited by 1400W, inducible NOS (iNOS) inhibitor, and 7-nitroindazole, constitutive NOS's inhibitor, at doses 5-50 and 10-200 microM, respectively. One-day incubation of he cells in the medium with low concentration of gentamycin (1 or 2 microg/ml) led to the significant increase in amount of bacterial in cultured fluid identified as E. coli and Acinetobacter sp. Addition of L-NAME (5 - 103 M) to the medium potentiated the bacteria growth 1.5- and 2.5-times in the presence of 2 and 1 microg gentamycin/ml, respectively. Thus, epithelial cells form the frog urinary bladder possess NO-dependent antibacterial effect which is probably provided by induction of iNOS expression. Taken together, these data demonstrate that the primary culture of the frog urinary bladder epithelial cells is a perspective experimental model for the study of regulation of NOS activity and NO production being of particular interest in relation to the defense effect of NO in epithelia.

[Arginase activity in the frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production].

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate, arginase can affect the NO synthesis. In the present work, the properties of arginase from the frog Rana temporaria L. urinary bladder epithelial cells possessing the NO-synthase activity were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme had the temperature optimum in the range of 55-60 degrees C, K(m) for arginine 23 mM, and V(max) about 10 nmol urea/mg protein/min, and its activity was effictively inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10(-6) to 10(-4) M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney >> brain > urinary bladder (epithelium) > heart > testis. The arginase activity in the isolated urinary bladder epithelial cells was 3 times higher than that in the intact urinary bladder. To evaluate the role of arginase in the regulation of NO production, epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO2-, the stable NO metabolite, was determined in the culture fluid after 18-20 h of cells incubation. The vast majority of the produced nitrites are associated with the NOS activity, as L-NAME, the NOS-inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in 199 medium. BEC (10(-4) M) increased the nitrite production by 18.0 % +/- 2.7 in the L-15 medium and by 24.2 +/- 3.5 in the 199 medium (p < 0.05). The obtained data indicate a relatively high arginase activity in the frog urinary bladder epithelium and its involvement in regulation of NO production by epithelial cells.

[The presence of nitric oxide synthase in mucosal epithelial cells of the frog urinary bladder].

In experiments on frog Rana temporaria L. urinary bladder, we investigated localization of NO-synthase (NOS) in urinary bladder slices and measured NOS activity in the suspension of mucosal epithelial cells. Intensive NADPH-diaphorase staining which is widely used as an indicator of NOS activity was found in mucosal epithelium. Almost all mucosal epithelial cells isolated in Ca2+ -free conditions demonstrated positive NADPH-diaphorase reactivity. Direct measurement of NOS activity in suspension of mucosal cells determined by the rate of conversion of L-arginine to L-citrullin showed that the enzyme activity was reduced in absence of external Ca2+ and was inhibited by L-NAME: non-specific NOS inhibitor, and 1400 W: a highly selective iNOS inhibitor (control: 754 +/- 184; L-NAME, 1 mM 329 +/- 87; 1400 W, 20 mM: 547 +/- 25; Ca2+ -free/EDTA: 490 +/- 184 cpm [3H]-citrullin/10(6) cells per 45 min, p < 0.05, n = 7-8). The data obtained demonstrate that frog urinary bladder mucosa epithelial cells provided antidiuretic hormone-induced increase of osmotic water permeability contain nitric oxide synthase. The presence of inducible (iNOS) as well as constitutive isoform(s) revealed in these cells allows to suggest involvement of NOS in intracellular signaling pathways regulated water transport across the epithelium.

[Atrial natriuretic factor stimulates the frog urinary bladder osmotic permeability in presence of a cyclic nucleotide phosphodiesterase inhibitor]. / Atrial'nyi natriureticheskii faktor stimuliruet osmoticheskuiu pronitsaemost' mochevogo puzyria liagushki v prisutstvii ingibitora fosfodiésteraz tsiklicheskikh nukleotidov.

The role of atrial natriuretic factor (ANF) in regulation of osmotic water permeability was studied in isolated frog Rana temporaria L. urinary bladder. It was found that ANF (rANF, 1-28) added to the serosal solution at concentrations 5 x 10(-8) M and higher dosedependently stimulated the arginine-vasotocin (AVT)-induced increase of osmotic water permeability. The effect of ANF was revealed only in presence of 3-isobuthyl-1-methylxantine (180 microM) and was accompanied by significant elevation of cGMP level in urinary bladder homogenate and isolated mucosal epithelial cells. C-ANF (des[Gln18, Ser19, Gly20, Leu21, Gly22]-ANF-(4-23)-NH2), a specific agonist of NPR-C receptor, exerted no effect on osmotic water permeability. ANF induced a significant increase of cAMP in urinary bladder homogenates (AVT, 5 x 10(-11) M: 52.3 +/- 10.6; AVT + ANF, 10(-7) M: 114.2 +/- 26.9 pmol/mg protein, n = 5, p < 0.05). The activity of adenylate cyclase in crude plasmatic membrane fraction was not changed. Milrinone, a specific inhibitor of phosphodiesterase 3, at concentrations from 25 to 80 microM, enhanced both the hydroosmotic response to AVT and AVT-stimulated cAMP production. Altogether these data demonstrate that, in the frog urinary bladder, ANF stimulates the AVT-induced increase of osmotic water permeability acting probably through NPR-A receptor-coupled mobilization of cGMP and cGMP-dependent inhibition of phosphodiesterase 3.

[Effect of cGMP-dependent signaling system in regulation of osmotic permeability in the frog urinary bladder]. / Rol' tsGMF-zavisimoi signal'noi sistemy v reguliatsii osmoticheskoi pronitsaemosti mochevogo puzyria liagushki.

In frogs' isolated urinary bladders, contribution of cytosolic guanylate cyclase and cGMP-dependent protein kinase to regulation of osmotic permeability was studied. ODQ (25-100 microM), an inhibitor of cytosolic guanylate cyclase induced an increase of vasotocin-activated osmotic permeability but had no effect on the hormone-activated transepithelial urea transport. In isolated mucosal epithelial cells ODQ (50 microM) decreased the concentration of intracellular cGMP. In these cells L-NAME (0.5 nM), an inhibitor of NO synthase, also decreased the level of cGMP whereas cAMP was significantly increased. 8-pCPT-cGMP (25 and 50 microM), a permeable cGMP analogue which selectively activates protein kinase G, inhibited vasotocin-induced increase of water transport along osmotic gradient indicating that protein kinase G is involved in regulation of water reabsorption. The data obtained show that NO/cGMP signalling system in the frog urinary bladder appears to be a negative modulator of vasotocin-activated increase of osmotic permeability.

[Various effects of prostaglandin E2 on reabsorption of water and urea in the amphibia osmosis-regulating epithelium]. / Razlichie vliianii prostaglandina E2 na reabsorbtsiiu vody i mocheviny v osmoreguliruiushchem épitelii amfibii.

Principal similarities between molecular pathways providing the enhancement of water and urea reabsorption under the action of argininvasotocin (AVT) in amphibian urinary bladder suggest that prostaglandin E2 (PGE2) could be a negative regulator of urea transport. To analyse this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladder. The urea permeability (Pu) was determined from the rate of efflux of (14) Curea from mucosal to serosal solution in isoosmotic conditions. The water permeability was measured in separate experiments in presence of an osmotic gradient. In contrast to water permeability, we were unable to demonstrate any inhibitory effect of 10-1000 nM PGE2 on AVT-stimulated urea transport using a variety of protocols. It was found that basolateral PGE2 exposure (10 nM-1 microM) caused an increase in Pu with no effect on osmotic water flow. The PGE2 effect was markedly inhibited by phloretin, a specific inhibitor of urea transporter. Sulprostone, an EP1/EP3 prostaglandin E2 receptor agonist, had no effect on Pu suggesting the contribution of EP2/EP4 receptor subtypes. In presence of osmotic water flow, the AVT-induced urea transport was significantly higher. This water flow-dependent urea permeability was inhibited by PGE2 although the inhibitory effect was less pronounced in comparison to the action of PGE2 on osmotic water flow. On the basis of these results we can make a conclusion that PGE2 has different role in regulation of water and urea transport in the frog urinary bladder. PGE2 could be considered as a stimulator of urea transport and an inhibitor of osmotic water flow activated by the AVT. The ability of PGE2 to regulate various types of cAMP-dependent transport by different mechanisms seems to be based on the presence of multiple basolateral PGE2 receptor subtypes in amphibian osmosis-regulatory epithelium.

[The role of prostaglandin E2 and its receptors in the regulation of osmotic permeability of the frog bladder epithelium]. / Rol' prostaglandina E2 i ego retseptorov v reguliatsii osmoticheskoi pronitsaemosti épiteliiia mochevogo puzyria liagushki.

Arginine-vasotocin-induced enhancement of osmotic water flow was inhibited by PGE2, sulprostone, M&B 28767 and 17-phenyl-omega-trinor PGE2. The inhibitory action of these agents except M&B 28767 was more obvious when the osmotic water flow was activated by dibutyryl-cAMP. The findings suggest that, in the frog urinary bladder, the inhibitory effect of the PGE2 on arginine-vasotocin-induced water flow appears to be mediated via two receptor subtypes coupled with different secondary messengers. The inhibition-sensitive targets are localised both on pre- and post-cAMP steps of the hormonal signal transduction pathway.

[Comparative study of the action of antineoplastic platinum compounds with varying nephrotoxic effects]. / Sravnitel'noe issledovanie deistviia protivoopukholevykh soedinenii platiny, obladaiushchikh razlichnym nefrotoksichnym éffektom.

Studying the action of the two antitumour platinum compounds--cisplatin capable of exerting a nephrotoxic action and cycloplatam which has no damaging effect on the kidney, it was found that 3 h after the administration of cycloplatam the content of platinum in the kidney was 2 times lower than in the cfse of cisplatin. Due to different dynamics of the excretion of platinum compounds from the kidney 5 lays after their addition the content of platinum in the kidney was the same in both cases. The content of platinum in the nuclei, mitochondria and supernatant with respect to a total content in the kidney cortex was almost equal for both compounds. Inhibition of nephrotoxic effect of cisplatin after the animals were pretreated with choline chloride or paraaminohippurate is not connected with a decrease of platinum in the kidney either 3 h, or 5 days after the injection of these preparations. The mechanisms of nephrotoxic action of cisplatin and its prevention are discussed.

[The content of electrolytes (Na, K, Ca, Mg) in vertebrate otoliths and otoconia]. / Soderzhanie élektrolitov (Na, K, Ca, Mg) v otolitakh i otokoniiakh pozvonochnykh.

Na, K, Ca and Mg concentrations have been determined in the otoliths and otoconia of the lamprey, skate, guppy and hen. Lamprey otoliths sharply differ from the otoliths and otoconia of other animals with respect to their K and Ca content. In all the animals investigated excluding lamprey, only traces of K were found. Na concentration in the otilithic apparatus of the investigated animals varies within 0.475-1.04 mM/g. Na presumably is bound to proteins of the organic matrix and plays an important role in the growth of otoliths and otoconia. Concentration of Ca in the otoliths and otoconia of all animals excluding the lamprey was equal to 9.09-9.63 mM/g: organic substances constitute 4-10 per cent of their mass. In the lamprey, Ca concentration in the otoliths was found to be 5.33 mM/g, the content of organic matter amounting to more than 40 per cent. In constantly growing otoliths of fish, concentration of Mg was significantly lower than in otoconia of fish and other animals. This is presumably due to the fact that Mg inhibits crystalline growth of calcium salts.

Renal function and renal platinum content in uninephrectomized rats following cisplatin administration.

The changes in renal function and renal platinum content were assessed in uninephrectomized and sham-operated female Wistar rats on the third day after treatment with 2.5 mg/kg BW or 5 mg/kg BW cisplatin. Treatment of control and nephrectomized rats with 2.5 mg/kg BW cisplatin resulted in indices of renal function which were not significantly different from those of animals which had received no cisplatin, though the renal platinum contents in nephrectomized rats were practically the same as in two-kidney animals given 5 mg/kg BW cisplatin. Treatment with 5 mg/kg cisplatin resulted in much less severe changes in kidney weight and renal function compared to two-kidney animals, in spite of much more substantial (by 49-58%) platinum accumulation.

[Body fluid and electrolyte content in rat tissues after space flight in the "Space-2044" spacecraft]. / Soderzhanie vody i élektrolitov v tkaniakh krys posle poleta na biosputnike "Kosmos-2044".

Contents of water, sodium, calcium and magnesium in different tissues of the rats stayed in space for 2 weeks in healing period of bone fracture following graded trauma of calf-bones as well as in the rats exposed to a head-down suspension (HDS) have been studied. In microgravity-exposed animals there were no significant changes in fluid-electrolyte composition of the liver, kidney, myocardium, skin, bone (calf bone). Specific "functional load" on the system of fluid-electrolyte homeostasis in the form of operation and subsequent healing of bone fracture did not result in additional changes in fluid and electrolyte contents of body tissues. In the HDS animals there was a significant change in fluid-electrolyte composition of skin, kidney and bone tissue.