Sulfation of fucoidin in focus embryos: III. Required for localization in the rhizoid wall

Zygotes of the brown alga Fucus distichus L. Powell accumulate a sulfated polysaccharide (fucoidin) in the cell wall at the site of rhizoid formation. Previous work indicated that zygotes grown in seawater minus sulfate do not sulfate the preformed fucan (an unsulfated fucoidin) but form rhizoids. Under these conditions, we determined whether sulfation of the fucan is required for its localization in the rhizoid wall. This was accomplished by developing a specific stain for both the fucan and fucoidin. Using a precipitin assay, we demonstrated in vitro that the lectin ricin (RCA(I)) specifically complexes with both the sulfated and desulfated polysaccharide. No precipitate is observed when either is incubated in 0.1 M D-galactose or when RCA(I) is mixed with laminarin or alginic acid, the other major polysaccharides in Fucus. RCA(I) conjugated with fluorescein isothiocyanate (FITC) is also shown to bind specifically to fucoidin using a filter paper (DE81) assay. When added to zygotes, RCA(I)-FITC binds only to the site of fucoidin localization, i.e., the rhizoid cell wall. However, RCA(I)-FITC is not observed in the rhizoid wall of zygotes grown in the absence of sulfate. This observation is not due to inability of RCA(I)-FITC to bind to the fucan in vivo. Chemically desulfated cell walls that contained fucoidin in the rhizoid wall bind RCA(I)-FITC only in the rhizoid region. Also, the concentration of fucose-containing polymers and polysaccharides that form precipitates with RCA(I) is the same in embryos grown in the presence or absence of sulfate. If sulfate is added back to cultures of zygotes grown without sulfate, fucoidin is detected at the rhizoid tip by RCA(I)-FITC several hours later. These results support the conclusion that the enzymatic sulfation of the fucan is a modification of the polysaccharide required for its localization and/or assembly into a specific region of the cell wall.     

Inhibitory systems sensitive to catecholamines and GABA in the inner plexiform layer of the frog retina

Local electroretinography was used to study the effect of pharmacological agents injected into the eye on lateral inhibitory interaction in the inner plexiform layer of the frog retina. Adrenoblocking agents, unlike cholinoblocking agents, were found to abolish dynamic lateral inhibition. It is postulated on the basis of the results that the inhibitory mediator in the system of dynamic lateral inhibition is a catecholamine. Yet another type of inhibition at the level of the amacrine cells, differing from dynamic inhibition (steady inhibition of adaptive type), sensitive to picrotoxin, was revealed with the aid of GABA and analeptics (strychnine and picrotoxin). It is postulated that GABA acts in this inhibitory system as an inhibitory mediator. On the basis of the results and analysis of the morphological picture of the plexiform layer, a model is proposed for the synaptic organization of lateral inhibition at the level of the amacrine cells.     

New Thioureas and Related Substances Intended for Melanoma Targeting

Thiouracil and a few related drugs are known to be melanoma-seeking agents owing to specific incorporation into nascent melanin. The melanin-affinic properties are apparently due to binding to intermediates, preferably dopaquinone, produced in the melanin synthetic pathway by tyrosinase-catalysed oxidation of tyrosine. In the present paper, in vitro screening methods have been used for the identification of possible melanoma seekers according to the above principle. The binding of test substance to dopaquinone suppresses dopachrome formation by the withdrawal of dopaquinone from the reaction mixture, and the decrease in dopachrome concentration was monitored spectrophotometrically at 475 nm. In order to eliminate false results caused by tyrosinase inhibition, which also will decrease the dopachrome concentration, the oxygen consumption was followed potentiometrically. To avoid the effect of tyrosinase inhibition on dopachrome formation, additional experiments with autoxidation of L-dopa in the presence of test substance were performed. Of the 22 substances (mainly thioureylenes and thioamides) studied, 4,5,6-triamino-2(H)-pyrimidinehtionsulfate, trithiocyanuric acid, 2-thiouracil, 6-methyl-2-thiouracil, and 4-amino-2-mercaptopyrimidine most effectively decreased the dopachrome formation with no or little inhibition of tyrosinase activity. They should therefore be regarded as potential melanoma seekers. In a complementary autoradiographic study on the uptake of the potent tyrosinase inhibitor mercaptobenzothiazole (MBT) in B 16 melanoma, transplanted to mice, it was found that strong tyrosinase inhibition seems to decrease incorporation into melanin in vivo. MBT was partly accumulated in restricted areas of the tumor, which may be explained by the small molar dose injected.     

Effect of different sera on amniotic cell growth in culture

The growth properties of the FCS (calf foetus serum), obtained from the blood of died fetals have been investigated, the "Gibco" FCS and human serum IV being used as a control. It was shown, that FCS prepared in our laboratory provided the growth and karyotyping of amniotic cells in vitro. This FCS was equally effective for cell planting as was human serum IV and somewhat less effective than the "Gibco" FCS. The FCS obtained in our laboratory is not deficient, and cheap. It may be recommended for a wide use in prenatal detection of genetic disorders.     

Characteristic of sorption properties of skeletal muscle nuclei in the normal state and with E-avitaminotic muscular dystrophy

Sorption properties of skeletal muscles nuclei in rabbits in normal state and with E-avitaminosis were studied using organic dyes: neutral red (cationic) and turquoise direct light-fast "K" (anionic) and the influence of calcium-modified membrane of nuclei on their sorption. The nuclear surface is established to have both positive and negative charged groups sorbing turquioise direct and neutral red, respectively. The maximum volume of the dyes binding and the dissociation constants of the membrane-dye complex are estimated. It is shown that with muscular dystrophy the number of charged groups of both signs on the nuclear surface decreases. Calcium ions decrease the cationic dye sorption both in the normal state and with dystrophy and insignificantly decrease the anionic dye with dystrophy.     

The effect of acid rain on ultrastructure and functional parameters of photosynthetic apparatus in pea leaves

The ultrastructure and functional parameters of the photosynthetic apparatus in leaves of 14-day-old pea seedlings were studied in conditions of laboratory simulated acid rain (SAR). Pea seedlings were sprayed with an aqueous solution containing NaNO₃ (0.2 mM) and Na₂SO₄ (0.2 mM) (pH 5.6, a control variant), or with the same solution, which was acidified to pH 2.5 (acid variant). Functional characteristics were determined by chlorophyll fluorescence analysis. There was reduction in the efficiency of the photosynthetic electron transport by 25% accompanied by an increase in the quantum yield of thermal dissipation of excess light quanta by 85% without significant change in maximum quantum yield of PSII photochemistry (F_v/F_m). Ultrastructural changes in chloroplasts were revealed by transmission electron microscopy (TEM) 2 days after the SAR treatment of pea leaves. In this case, changes in the structure of the grana and heterogeneity of the thylakoids packing in the granum, namely, an increase in thylakoid intraspace widths and thickness of granal thylakoids compared to the control, were found. It was shown also that carbonic anhydrase activity was significantly inhibited in chloroplast preparations isolated from SAR-treated pea leaves. We hypothesize possible involvement of chloroplast carbonic anhydrase in thylakoid granal structure maintenance. The structural disturbances and the inhibition of photochemical activity of chloroplasts are possible consequences of the carbonic anhydrase inactivation by SAR treatment leading to violation of HCO₃ ^?–CO₂ equilibrium. The data obtained suggest that acid rains negatively affect the photosynthetic apparatus by disrupting the membrane system of the chloroplast.     

Akap1 Deficiency Promotes Mitochondrial Aberrations and Exacerbates Cardiac Injury Following Permanent Coronary Ligation via Enhanced Mitophagy and Apoptosis

A-kinase anchoring proteins (AKAPs) transmit signals cues from seven-transmembrane receptors to specific sub-cellular locations. Mitochondrial AKAPs encoded by the Akap1 gene have been shown to modulate mitochondrial function and reactive oxygen species (ROS) production in the heart. Under conditions of hypoxia, mitochondrial AKAP121 undergoes proteolytic degradation mediated, at least in part, by the E3 ubiquitin ligase Seven In-Absentia Homolog 2 (Siah2). In the present study we hypothesized that Akap1 might be crucial to preserve mitochondrial function and structure, and cardiac responses to myocardial ischemia. To test this, eight-week-old Akap1 knockout mice (Akap1^-/-), Siah2 knockout mice (Siah2^-/-) or their wild-type (wt) littermates underwent myocardial infarction (MI) by permanent left coronary artery ligation. Age and gender matched mice of either genotype underwent a left thoracotomy without coronary ligation and were used as controls (sham). Twenty-four hours after coronary ligation, Akap1^-/- mice displayed larger infarct size compared to Siah2^-/- or wt mice. One week after MI, cardiac function and survival were also significantly reduced in Akap1^-/- mice, while cardiac fibrosis was significantly increased. Akap1 deletion was associated with remarkable mitochondrial structural abnormalities at electron microscopy, increased ROS production and reduced mitochondrial function after MI. These alterations were associated with enhanced cardiac mitophagy and apoptosis. Autophagy inhibition by 3-methyladenine significantly reduced apoptosis and ameliorated cardiac dysfunction following MI in Akap1^-/- mice. These results demonstrate that Akap1 deficiency promotes cardiac mitochondrial aberrations and mitophagy, enhancing infarct size, pathological cardiac remodeling and mortality under ischemic conditions. Thus, mitochondrial AKAPs might represent important players in the development of post-ischemic cardiac remodeling and novel therapeutic targets.     

Golgi membranes are absorbed into and reemerge from the ER during mitosis

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.     

Botryoid odontogenic cyst. Exploration of proliferative activity,apoptosis and expression of TP53 and BCL2 compared to the histologically identical lateral periodontal and gingival cysts

The botryoid odontogenic cyst (BOC) is a rare, locally more aggressive variant of the usually indolent lateral periodontal cyst (LPC) and gingival cyst (GC). A recent case of BOC provided an opportunity for an exploratory study on the causes of its more aggressive behavior. The limited objective was to see if the BOC was sufficiently different from the other cysts to warrant an investment in a large study. Sections of neutral buffered formalin fixed, paraffin-embedded tissues from the BOC and archival specimens of four GCs, four LPCs and three odontogenic keratocysts (OKCs) were stained using immunohistochemistry for Ki-67, a marker of proliferating cells, caspase-3, a marker of cells undergoing apoptosis, tumor suppressor p53, and the apoptosis inhibitor BCL2. The mean labeling index (LI) of immunoreactive cyst epithelial cells was computed for each antibody and type of cyst. Compared to the LPCs and GCs, the BOC exhibited a moderately larger Ki-67/caspase-3 LI difference, which indicates that the BOC had a net higher rate of growth. We found a much higher level of LI, therefore likely dysregulation of p53. We also found a much higher LI of BCL2. The LIs of p53 and BCL2 in the BOC were similar to and more than twice that of the OKCs, respectively. Although meaningful statistical analysis was precluded by our use of only one case of BOC and a small number of the other cysts, the high p53 and very high BCL2 labeling indices of the BOC offer a potential explanation for its reportedly more aggressive behavior that clearly is worthy of further investigation.