Functional characterization of the corticular photosynthetic apparatus in grapevine.

A comparative analysis of functional characteristics of the grapevine leaf photosynthetic apparatus (LPA) and corticular photosynthetic apparatus (CPA) in chlorenchyma tissues of first-year lignified vine was performed. Obtained results demonstrate significant differences between the functional properties of the CPA and the LPA. CPA contains an increased proportion (about 2/3) of QB-non-reducing centers of photosystem II (PSII) that is confirmed by elevated O-J phase in fluorescence kinetics, high PSIIß content, and slower QA-• reoxidation. CPA and LPA use different strategies to utilize absorbed light energy and to protect itself against excessive light. CPA dissipates a significant proportion of absorbed light energy as heat (regulated and non-regulated dissipation), and only a smaller part of the excitation energy is used in the dark stages of photosynthesis. The rate constant of photoinhibition and fluorescence quenching due to photoinhibition in CPA is almost three times higher than in LPA, while high-energy state fluorescence quenching value is twice lower. The saturation of vine chlorenchyma tissue with water increases the CPA tolerance to photoinhibition and promotes the ability to restore the photosynthetic activity after photoinhibition. The electron microscopy analysis confirmed the presence of intact plastids in vine chlorenchyma tissue, the interior space of plastids is filled with large starch grains while bands of stacked thylakoid membranes are mainly localized on the periphery. Analyzes showed that corticular plastids are specialized organelles combining features of chloroplasts, amyloplasts and gerontoplasts. Distinct structural organization of photosynthetic membranes and microenvironment predetermine distinctive functional properties of CPA.

Suppression of RAC1-driven malignant melanoma by group A PAK inhibitors.

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma. Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1. Activating mutations in RAC1 are of special interest, as small-molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1. In this work, we explore the effects of PAK inhibition on RAC1P29S signaling in zebrafish embryonic development, in the proliferation, survival and motility of RAC1P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice. We report that RAC1P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK. We also found and that RAC1-mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors. These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

Targeting group I p21-activated kinases to control malignant peripheral nerve sheath tumor growth and metastasis.

Malignant peripheral nerve sheath tumors (MPNSTs) are devastating sarcomas for which no effective medical therapies are available. Over 50% of MPSNTs are associated with mutations in NF1 tumor suppressor gene, resulting in activation of Ras and its effectors, including the Raf/Mek/Erk and PI3K/Akt/mTORC1 signaling cascades, and also the WNT/ß-catenin pathway. As Group I p21-activated kinases (Group I Paks, PAK1/2/3) have been shown to modulate Ras-driven oncogenesis, we asked if these enzymes might regulate signaling in MPNSTs. In this study we found a strong positive correlation between the activity of PAK1/2/3 and the stage of human MPNSTs. We determined that reducing Group I Pak activity diminished MPNST cell proliferation and motility, and that these effects were not accompanied by significant blockade of the Raf/Mek/Erk pathway, but rather by reductions in Akt and ß-catenin activity. Using the small molecule PAK1/2/3 inhibitor Frax1036 and the MEK1/2 inhibitor PD0325901, we showed that the combination of these two agents synergistically inhibited MPNST cell growth in vitro and dramatically decreased local and metastatic MPNST growth in animal models. Taken together, these data provide new insights into MPNST signaling deregulation and suggest that co-targeting of PAK1/2/3 and MEK1/2 may be effective in the treatment of patients with MPNSTs.

[LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed.

[Valve-like structures in motor cells of the leaf of Distichlis spicata "Yesen 4A" (Poaceae)].

The salt gland of the leaf of Distichlis directly contacts the cells of interfascicular aquiferous parenchyma (motor cells). The cytoplasmic strand of motor cells produce deep invaginations, which form with the participation of mitochondria. The constriction of the cytoplasmic strand at the site of the localization of mitochondria leads to the fusion of the tonoplast and plasmalemma with mitochondrial membranes and the formation of a thin one-layer plate, a valve. At this locus, vacuolar and apoplast spaces are separated only by a valve. The cytoplasm of motor cells is filled with electron dense granules, which are considered as contractile elements. It is assumed that the cytoplasmic strand is involved in the reduction of the volume, which results in the generation of pressure on the valve. This leads to the direct throw-in of water into the apoplast space adjacent to the salt gland.

[POSSIBILITIES FOR THE USE OF THE ANTI-LEUKOTRIENE DRUG MONTELUKAST IN COMBINED THERAPY OF PATIENTS WITH BRONCHIAL ASTHMA AND COLD-INDUCED BRONCHIAL HYPERACTIVITY].

AIM: To study effectiveness of the use of the anti-leukotriene drug montelukast in combination with inhalation glucocorticoid and long-acting beta-agonist in patients with bronchial asthma (BA) and cold-induced bronchial hyperactivity (CBHA) with a view to optimizing control of the disease. MATERIALS AND METHODS: We carried out an open comparative prospective study of patients with persistent BA in a cold season under conditions of real clinical practice. The patients were divided into 2 groups. Group 1 included patients with CBHA, group 2 consisted of subjects with constant bronchial reactivity in response to cold in the standard provocative test. The patients were followed up for 24 weeks. During the first 12 weeks of the treatment, the patients of group I were given sodium montelukast with budesonide/formoterol. In the next 12 weeks they received only budesonide/formoterol at the same dose. Patients of group 2 were treated with budesonide/formoterol during the entire study period. Efficacy of therapy was assessed by asthma control test (ACT). RESULTS: Control of BA in group 1 (20-25 scores in A CT) was achieved in 83% of the patients within the first 12 week period The result was comparable (87%) with that in group 2. Impairment of control (to 52%) was documented in group I during the last 12 weeks although it was preserved (20-25 scores) in group 2 (81%). CONCLUSION: The use of sodium montelukast in combination with budesonide/formoterol for the treatment of BA with CBHA in winter season ensured control of the disease in most patients during 12 weeks. Withdrawal of montelukast in the subsequent period leads to the loss of control and a rise in the frequency of exacerbation.

[Ultrastructural organization and composition of carotenoids in the eyespot in the mutant Chlamydomonas reinhardtii].

Biogenesis of the ultrastructure of the eyespot in the chloroplasts of unicellular green algae Chlamydomonas reinhardtii has been studied. We have found that the development of the structure of the eyespot correlates with the accumulation of carotenoids. Depending on their accumulation, the eyespots form from 1 to 4 lines of lipid-carotenoid globules. It has been shown that only carotenes are accumulated in the globules of the eyespots. We first have found that the composition of carotenes in the eyespots of the mutants may vary due to the changes in their composition in the membranes of chloroplasts.

[Mitochondrial dysfunction in children with hepatic forms of glycogen storage disease].

AIM: The purpose of the study was to assess mitochondrial dysfunction severity in patients with hepatic forms of glycogen storage disease (GSD). PATIENTS AND METHODS: We examined 53 children with GSD in the dynamics. Distribution of children by disease types was: 1st group--children with GSD type I, 2nd group--children with GSD type III, 3rd group--children with GSD type VI and IX; comparison group consisted of 34 healthy children. Intracellular dehydrogenases activity: succinate dehydrogenase (SDH), glycerol-3-phosphate-dehydrogenase (GPDH). nicotinamideadenin-H-dehydrogenase (NADH-D) and lactatdehydrogenase (LDH) was measured using the quantitative cytochemical method in the peripheral lymphocytes. RESULTS: It was revealed decrease of SDH- (p < 0.001) and GPDH-activities (p < 0.001), along with increase of the NADH-D activity (p < 0.05) in all patients with GSD, (SDH/ NADH-D) index was decreased (p < 0.001). LDH activity was increased in groups 1 (p < 0.05) and 3 (p < 0.01), compared with comparison group. The most pronounced intracellular enzymes activity deviations were observed in children with GSD type I, that correspond to more severe clinical form of GSD. It was found strong correlation between intracellular enzymes activity and both hepatomegaly level (R = 0.867) and metabolic acidosis severity (R = 0.987). CONCLUSION: Our investigation revealed features of mitochondrial dysfunction in children with GSD, depending on the GSD type. Activities of lymphocytes enzymes correlates with the main disease severity parameters and can be used as an additional diagnostic criteria in children with hepatic form of GSD.

[Structural-functional organization of the cells of Brc-1 mutant Chlamydomonas reinhardtii, supplying protoporphyrin IX in the dark].

The structural-functional characteristics of the cells of wild type CC-124 and Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii while growing in the dark and light were studied. It has been shown that the cells of the wild type in heterotrophic and mixotrophic growth conditions had a well developed structure and high functional activity due to the ability of the cells to synthesize chlorophyll both in the light and in the dark. The cells of Brc-1 mutant lost their ability to synthesize chlorophyll in the dark and the cells' color was orange due to brc-1 mutation in the nuclear gene LTS3 that regulated the activity of Mg-chelatase enzyme. In the dark the mutant cells accumulated protoporphyrin IX and had a weakly developed structure with low functional activity. It has been ascertained that due to high content of protoporphyrin IX even a short-term exposure of the cells of Brc-1 mutant to the light was accompanied by very strong destructive changes in all the membranes in a cell: plasmalemma, chloroplast, mitochondrion, shells of the nucleus and vacuoles. The reasons of these significant damages of the membrane components and O2-gas exchange in the cells of Brc-1 mutant are discussed.

[Comparative analysis of the exogenous ammonium influence on soybean tissue and Chlamydomonas cells].

In this work we studied the influence of exogenous ammonium on the total protein and chlorophyll contents, on the number of ribosomes and on the expression of ribosomal genes encoding the small subunit 18S rRNA and rpS6 protein in unicellular green alga Chlamydomonas reinhardtii and in callus tissue of Glycine max. Comparative analysis of two sets of data showed that although the lack of ammonium resulted in reduction of the number of ribosomes in alga and plant cells, this effect was not caused by decreasing of the expression level of the ribosomal genes. Possible mechanisms of the ammonium regulatory role in the ribosome biogenesis are discussed.

[The "crystals" in the sugar beet (Beta vulgaris L.) leaves].

Crystal containing cells widely distributed in plant tissues, though the origin of the crystals and their functions are still opened to question. Membrane vesicles in beet leaves are visible in electronic microscope. They originate in cytoplasm and penetrate into vacuole by pinocytosis with participation of tonoplast. In light microscope, vesicles are luminous likewise crystals in crystal cells. Such vesicles-"crystals" fulfill crystal cells also. The content of vesicles-"crystals" are electronic transparent at every path of leaf development. It was proposed that distinct vesicles-"crystals" in cytoplasm and vacuole and mass of them in crystal cells, vein bundles, and epidermal cells--all of them are lytic compartments. Later, obviously, true crystals are formed.

[Secondary autoimmune hemolytic anemia as a result of B19 parvovirus persistence in immunodeficient patients].

AIM: To ascertain informative value of immunological diagnosis of B19 parvovirus in combination with polymerase chain reaction (PCR); to analyse frequency of development of secondary autoimmune hemolytic anemia (AIHA) in immunodeficient patients as a result of virus persistence--persistent infection eliminated only by treatment causing suppression of erythropoiesis. MATERIAL AND METHODS: B19 parvovirus detection was performed in blood serum of 207 subjects: 144 patients with anemia (Hb < 100 g/1) and 500 blood donors. DNA of parvovirus B19 was detected in the sera by PCR, antibodies to this virus--by enzyme immunoassay (EIA). IgG, IgM, IgA and components of compliment Clq, C3 on the surface of erythrocytes were detected by EIA in anemic patients. RESULTS: Parvovirus infection was registered in 30% patients, in 70% the infection was persistent. The latter were diagnosed to have secondary AIHA. CONCLUSION: Combined application of PCR and EIA extends potentialities of diagnosis of infection caused by B19 parvovirus. Persistence of the parvovirus provokes onset of symptomatic hemolytic anemia in immunodeficient patients.

[Structural-functional organization of chloroplasts in leaves of xantha-702 mutant of Gossypium hirsutum L].

For cotton mutant xantha (Gossypium hirsutum L.), it has been established that synthesis of 5-aminolevulinic acid was blocked in the light. In the light this mutant accumulates chlorophyll by 30 times lower as compared to the parent type. In mutant xantha, a very few pigment-protein complexes of PS-I and PS-II are formed in chloroplasts, and formation of membrane system in these is blocked at the early stages, in most cases, at the stage of bubbles and single short thylakoids. Functional activity of reaction centers of PS-I and PS-II is close to zero. Only light-harvesting chlorophyll-a/b protein complexes of the two photosystems are formed in mutant xantha plastid membranes with maximum chlorophyll fluorescence at 728 and 681 nm, respectively. It has been concluded that in mutant xantha genetic block of 5-aminolevulinic acid biosynthesis in the light disturbs the formation and functioning of the complexes of reaction centers of PS-I and PS-II, hindering the development of the whole membrane system in chloroplasts, causing a sharp decrease in productivity.

[Electron dense substance of thylakoids in chloroplasts of tansy Tanacetum vulgare L].

An intrathylakoid electron opaque substance, further referred to as loculin, is found in 80-90 % of thylakoids of tansy leaf mesophyll chloroplasts at the stage of flower bud formation and flowering. Upon conventional isolation of chloroplasts in aqueous solution, and fixation in osmium solution alone, loculin is not retained in thylakoids. Preliminary fixation of leaves in glutaraldehyde makes it possible to isolate chloroplasts without injuring the envelope and stroma (glutar chloroplasts), and loculin is retained in thylakoids under these conditions. Upon prolonged incubation of glutar chloroplasts (for 24 h), loculin leaves thylakoids in the form of drops concentrating on the chloroplast envelope. Upon crossing the thylakoid membrane and chloroplast, loculin properties remain unchanged. It is assumed that loculin is an important metabolite necessary for active growth.

[The influence of ammonium and nitrate of nutrient medium on ultrastructural organization and photosynthesis of callus cells in Glycine max L]. / Vliianie ammoniia i nitrata pitatel'noi sredy na ul'trastrukturnuiu organizatsiiu i fotosintez kletok kallusa Glycine max L.

We studied the influence of exogenic ammonium on the functional activity and ultrastructural organization of cells of the mixotrophic soybean callus (Glycine max L.). Ammonium available in the nutrient medium increased the chlorophyll content, accelerating the rate of photosynthetic O2 evolution per unit of biomass. The presence of ammonium in the medium promoted formation of the protein-synthesizing system, which manifested itself as increased numbers of ribosomes, and thylakoids of chloroplasts, and higher electron density of the stroma in mitochondria and cytoplasm of mixotrophic cells. It has been concluded that the use of ammonium may lead to activation of protein synthesis, thus rising photosynthetic activity and favouring formation and development of membrane structures in chloroplasts.

[Ultrastructure organization of chloroplasts in chlorotica mutants of Pisum sativum L]. / Ul'trastrukturnaia organizatsiia khloroplastov chlorotica-mutantov Pisum sativum L.

A study was made of chlorophyll-protein complexes of photosystems, and of ultrastructural organization of chloroplasts in pea leaves of the primary cultivar Torsdag and of its mutants, chlorotica 2004 and 2014. It has been shown that mutants accumulated 80 and 55% chlorophyll, respectively, and were able to synthesize all four types of photosystem complexes. The value of the light-harvesting antenna in mutant 2014 was close to the control one, and in mutant 2004 it increased significantly (by 30%). These changes were caused by a proportional decrease (40-50%) in any complexes in mutant 2014, whereas the number of PS-I reaction centre complexes, decreased by 50% in mutant 2004 at nearly complete storage of PS-I reaction centre complexes, decreased by 50% in mutant 2004 at nearly complete storage of PS-II complexes. The proportional decrease of PS-I and PS-II complexes in mutant chlorotica 2014 was followed by partial reduction of the entire membrane system in chloroplasts, but with a normal development of both granal and intergranal thylakoids. On the contrary, the loss of PS-I reaction centre complexes in mutant chlorotica 2004 leads to reduction of unstacked sites of thylakoids in chloroplasts. It is concluded that this effect may be associated with localization of PS-I complexes mainly in unstacked sites of thylakoids.

[Structural and functional organization of chloroplasts in leaves of Pisum sativum L. under conditions of root hypoxia and iron deficiency]. / Strukturno-funktsional'naia organizatsiia khloroplastov v list'iakh Pisum sativum L. v usloviiakh kornevoi gipoksii i defitsita zheleza.

A combined effect of iron deficiency and root hypoxia on the biochemical composition activity and structure of chloroplasts in pea leaves have been studied. Both factors are shown to affect the accumulation of chlorophyll causing leaf chlorosis. At iron deficiency chlorosis occurs from the top of plant leaves. At root hypoxia chlorosis starts from the lower strata. At a combined action of both factors the destructive effects are summarized. It was established that light-harvesting complexes of photosystems were reduced stronger at iron deficiency, while complexes of reaction centers of photosystem I and photosystem II are lessened at root hypoxia. Nevertheless, even at a combined effect of both factors yellow leaves preserved small amounts of any pigment-protein complexes and their functional activities. The ultrastructure of chloroplasts during leaf chlorosis was gradually reduced. At first, intergranal sites of thylakoids and then granal ones were destroyed, that was typical of iron deficiency. However, even yellow and almost white leaves kept small thylakoids, capable of forming stacking and small grana made of 2-3 thylakoids. It has been concluded that the destructive effects are summarized due to different kinds of action of iron deficiency and root hypoxia on the structure and functioning of leaves at their combined action.

[Choline chloride effect on chloroplast ultrastructure and cell growth in Chlamydomonas reinhardtii]. / Ul'trastruktura khloroplastov i rost kletok Chlamydomonas reinhardtii pri deistvii kholinkhlorida.

The influence of growth retardant choline chloride (0.02, 0.2 and 2 g/l) on cell size and division as well as chlorophyll accumulation and chloroplast ultrastructure of unicellular green algae Chlamydomonas was studied. It was shown that at any concentration used (0.02, 0.2, and 2 g/l) choline chloride decreased the rate of cell division. The content of chlorophyll and carotenoid per cell decreased and the sizes of cells increased at all concentrations of choline chloride. On the basis of electron microscopy data, the conclusion was made that an increase in the concentration of choline chloride intensified destruction processes in membranes of chloroplasts and other cell organelles.