Electronmicroscopical detection of iota-Carrageenan as its ruthenium red complex

Summary Specific detection of iota-Carrageenan (i-CAR) at the ultrastructural level has been obtained by coupling with ruthenium red (RR) — an electron microscopic stain. The i-CAR-RR complex showed electron density on carbon layers. Peritoneal macrophages were treated with the complex and after 3 h it caused the same morphological changes in macrophages as iota-Carrageenan alone. On the surfaces of macrophages, fine filamentous electron dense material — the i-CAR-RR complex — was detected.     

Effects of elevated carbon dioxide and ozone on the phytochemistry of aspen and performance of an herbivore

The purpose of this study was to assess the independent and interactive effects of CO(2), O(3), and plant genotype on the foliar quality of a deciduous tree and the performance of a herbivorous insect. Two trembling aspen (Populus tremuloides Michaux) genotypes differing in response to CO(2) and O(3) were grown at the Aspen FACE (Free Air CO(2) Enrichment) site located in northern Wisconsin, USA. Trees were exposed to one of four atmospheric treatments: ambient air (control), elevated carbon dioxide (+CO(2); 560 microl/l), elevated ozone (+O(3); ambient x1.5), and elevated CO(2)+O(3). We measured the effects of CO(2) and O(3) on aspen phytochemistry and on performance of forest tent caterpillar (Malacosoma disstria Hübner) larvae. CO(2) and O(3) treatments influenced foliar quality for both genotypes, with the most notable effects being that elevated CO(2) reduced nitrogen and increased tremulacin levels, whereas elevated O(3) increased early season nitrogen and reduced tremulacin levels, relative to controls. With respect to insects, the +CO(2) treatment had little or no effect on larval performance. Larval performance improved in the +O(3) treatment, but this response was negated by the addition of elevated CO(2) (i.e., +CO(2)+O(3) treatment). We conclude that tent caterpillars will have the greatest impact on aspen under current CO(2) and high O(3) levels, due to increases in insect performance and decreases in tree growth, whereas tent caterpillars will have the least impact on aspen under high CO(2) and low O(3) levels, due to moderate changes in insect performance and increases in tree growth.     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation

In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.     

Molecular pathology and targeted therapy of clear cell renal cancer

The golden standard of care for advanced renal cell cancer (RCC) was until now the cytokine therapy with relatively low response rates. Advances of molecular genetics in RCC revealed several molecular targets such as VHL and angiogenic genotype or EGFR in the clear cell variant. Among the novel targeted agents, multiple tyrosine kinase inhibitors were proved to be clinically effective against advanced clear cell renal cancer, changing the standard of care. It is a further question how molecular diagnostics can improve these results by the detection of these targets or gene defects in individual tumors.     

Adhesion dynamics and cytoskeletal structure of gliding human fibrosarcoma cells: a hypothetical model of cell migration

During motility of fibroblast type cells on planar surfaces, adhesions are formed at the anterior of the protruding lamella, which remain stationary relative to the substrate and undergo a maturation process as the cell passes over them. Through these adhesions force is exerted, the orientation of which is parallel to the direction of the movement. Here we show that, during gliding-type motility of human tumor cells, characterized by a semicircular shape, adhesions were found at the outer rim of the cells, along the semicircle. Time-lapse microscopy of GFP-vinculin-expressing cells showed that these adhesions were constantly renewed at the cell edge and followed a curved trajectory according to the graded radial extension model. Eventually, the adhesions reached the long axis of the cell where they were retracted into the cell body. Actin cables formed arcs, with the concave face at the anterior of the lamella found to be oriented in the direction of movement. Since adhesions moved backward with respect to the cell, actin cables connected to these adhesions must continuously grow, reaching maximal size at the long axis of the cell. Contraction of the arcs is responsible for the forward movement of the cell body.     

Stability and separation of aminoacyl-transfer RNAs on chromatography columns

In order to demonstrate that the apparent amount of a tRNA isoacceptor depends on the column matrix employed, chromatographic separations of lysyl tRNAs on several polystyrene anion exchangers and on a reversed-phase matrix (RPC-5) have been studied. Experiments were carried out to distinguish between the actual yield of isoacceptors (aminoacylated and free species) and the apparent yield of isoacceptors (aminoacylated species only). The results indicate that several polystyrene anion exchangers with similar physical properties resolve lysyl tRNAs differently. The differences are noted in the apparent yields and in the degree of chromatographic resolution. When fire column matrices are incubated separately with [³H]lysyl tRNAs and the deacylations measured, the results indicate chemical deacylation by two polystyrene anion exchange matrices but not by two other polystyrene matrices or by RPC-5. Further study of two major isoacceptors of lysyl-tRNA on these column matrices confirm that different matrices cause chemical deacylation of the aminoacyl tRNA bond at different rates. Therefore, the apparent yield of an isoacceptor depends upon the column matrix. The dissociation of the ester bond of the aminoacyl tRNA appears to be catalysed by the quaternary ammonium groups of the matrix. Enhanced deesterification of the aminoacyl tRNA bond is noted in slightly alkaline eluants, suggesting a nucleophilic attack by the hydroxyl anion of the medium. The major conclusion to be drawn is that both the yield and relative amounts of isoacceptors are dependent not only upon the resolving power of the column matrix, but also upon the physical and chemical nature of the matrix and upon the experimental conditions employed. This catalytic effect is in addition to the base-catalysed de-esterification promoted by the residual primary, secondary or tertiary amine groups present in the polystyrene anion exchangers.