Effects of isatin on atrial natriuretic peptide-mediated accumulation of cGMP and guanylyl cyclase activity of PC12 cells

We have previously demonstrated that isatin (indole-2,3 dione), an endogenous compound widely distributed in mammalian tissues and body fluids, effectively inhibits atrial natriuretic peptide (ANP) receptor binding and ANP-stimulated guanylyl cyclase activity of rat membrane preparations. In the present study the effects of isatin on ANP-mediated accumulation of cGMP and guanylyl cyclase (GC) activity of PC12 cells were studied. Isatin (0.1 mM) effectively inhibited ANP-stimulated GC-activity of broken cells but was nearly inactive in attenuating ANP-dependent accumulation of cGMP in intact PC12 cells. The ATP-analogue adenylylimidodiphosphate (AMP-PNP) slightly potentiated the ANP effect on GC activity in broken cell preparations and significantly reduced GC sensitivity to isatin. Isatin caused a more pronounced reduction of ANP-dependent cGMP accumulation in cells grown in the presence of 10% embryonal calf serum (ECS) than in 0.5% ECS. The data obtained suggest that, in intact cells, the manifestation of the isatin effect on ANP-mediated signal transduction may depend on intracellular factor(s), possibly interacting at the kinase domain.     

Tooth replacement rates in early chondrichthyans: a qualitative approach

The continuous replacement of teeth throughout their lifetime is a common characteristic of most chondrichthyans. This process was already present in the earliest representatives of the group. It has been well established that different species of extant sharks show rapid tooth replacement rates; however, some authors have suggested that in early chondrichthyans this rate might have been much slower. Here we present a qualitative approach to analyse tooth replacement rates in the Early Devonian shark Leonodus carlsi , the earliest tooth-bearing shark known to date. For this, we have examined 1,103 isolated teeth from Celtiberia, Spain. Our study provides strong evidences of an extremely slow dental replacement in this primitive chondrichthyan based on three independents analyses: (1) statistical analysis of the wear degree, demonstrating that teeth remain functional for a long period of time; (2) analysis of both the histological and the morphological features of the teeth cusps suggests that this chondrichthyan used a maturation process that optimizes its function, thus worn teeth show an efficient working shape that implies their teeth remained functional for a long time after being modelled by use; and (3) estimations of size increments between teeth (Δs) of the same dental family for some recent sharks whose rates of replacement were known prove that Δs is inversely proportional to the rate of replacement ( R ² = 0.8327). The estimated values of tooth replacement rates obtained from Δs for L. carlsi and for some Late Devonian cladoselachian sharks are significatively slower than those observed in current sharks.     

Ligands of boron in Pisum sativum nodules are involved in regulation of oxygen concentration and rhizobial infection

Boron (B) is an essential nutrient for N₂‐fixing legume–rhizobia symbioses, and the capacity of borate ions to bind and stabilize biomolecules is the basis of any B function. We used a borate‐binding‐specific resin and immunostaining techniques to identify B ligands important for the development of Pisum sativum–Rhizobium leguminosarum 3841 symbiotic nodules. arabinogalactan–extensin (AGPE), recognized by MAC 265 antibody, appeared heavily bound to the resin in extracts derived from B‐sufficient, but not from B‐deficient nodules. MAC 265 stained the infection threads and the extracellular matrix of cortical cells involved in the oxygen diffusion barrier. In B‐deprived nodules, immunolocalization of MAC 265 antigens was significantly reduced. Leghaemoglobin (Lb) concentration largely decreased in B‐deficient nodules. The absence of MAC 203 antigens in B‐deficient nodules suggests a high internal oxygen concentration, as this antibody detects an epitope on the lipopolysaccharide (LPS) of bacteroids typically expressed in micro‐aerobically grown R. leguminosarum 3841. However, B‐deprived nodules did not accumulate oxidized lipids and proteins, and revealed a decrease in the activity of the major antioxidant enzyme ascorbate peroxidase (APX). Therefore, B deficiency reduced the stability of nodule macromolecules important for rhizobial infection, and for regulation of oxygen concentration, resulting in non‐functional nodules, but did not appear to induce oxidative damage in low‐B nodules.     

Long-term exposure of Boeckellagibbosa (Copepoda, Calanoida) to in situ levels of solar UVB radiation

1. The freshwater calanoid copepod Boeckella gibbosa is typical of high elevation lakes and ponds in Patagonia (Argentina). Previous studies have shown that this species is highly tolerant to short-term exposure to natural and artificial UVB radiation, and that its tolerance is due to photoreactivation by longer wavelength radiation. In this study, we investigate the potential sublethal effects of solar radiation after prolonged exposure.
2. We incubated B. gibbosa at 1 m depth in oligotrophic Lake Toncek for 24 days. The incubation chambers were 1.2 l acrylic cylinders covered with appropriate filters in order to obtain three radiation treatments: visible radiation only, visible radiation + UVA and visible radiation + UVA + UVB.
3. The three treatments did not differ significantly in variables considered as indicators of survival (number of individuals), reproduction (proportion of ovigerous females, clutch size) and development (instar composition). Although resistance to solar UVB radiation is certainly a requisite to live in transparent high elevation habitats, the fact of being effectively exposed to natural levels of UVB radiation does not seem to have measurable consequences on an already adapted species, such as B. gibbosa     

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H₂O₂) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H₂O₂ derives from superoxide (O₂^˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O₂^˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O₂^˙̄ and H₂O₂ may provide feedback signals to modulate mitochondrial metabolism.     

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines

The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser??s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.     

Bird community composition across an Andean tree‐line ecotone

Few studies have found strong evidence to suggest that ecotones promote species richness and diversity. In this study we examine the responses of a high‐Andean bird community to changes in vegetation and topographical characteristics across an Andean tree‐line ecotone and adjacent cloud forest and puna grassland vegetation in southern Peru. Over a 6‐month period, birds and vegetation were surveyed using a 100 m fixed‐width Distance Sampling point count method. Vegetation analyses revealed that the tree‐line ecotone represented a distinctive high‐Andean vegetation community that was easily differentiated from the adjacent cloud forest and puna grassland based on changes in tree‐size characteristics and vegetation cover. Bird community composition was strongly seasonal and influenced by a pool of bird species from a wider elevational gradient. There were also clear differences in bird community measures between tree‐line vegetation, cloud forest and puna grassland with species turnover (β‐diversity) most pronounced at the tree‐line. Canonical Correspondence Analysis revealed that the majority of the 81 bird species were associated with tree‐line vegetation. Categorizing patterns of relative abundance of the 42 most common species revealed that the tree‐line ecotone was composed primarily of cloud forest specialists and habitat generalists, with very few species from the puna grassland. Only two species, Thlypopsis ruficeps and Anairetes parulus, both widespread Andean species more typical of montane woodland vegetation edges, were categorized as ecotone specialists. However, our findings were influenced by significant differences in species detectability between all three vegetation communities. Our study highlights the importance of examining ecotones at an appropriate spatial and temporal scale. Selecting a suitable distance between sampling points based on the detection probabilities of the target bird species is essential to obtain an unbiased picture of how ecotones influence avian richness and diversity.     

Protein, RNA and DNA synthesis in skin fibroblast cultures from healthy donors and patients with rheumatic diseases

Synthesis of protein, RNA and DNA was studied in skin fibroblast cultures of healthy donors and patients with systemic scleroderma (SSD) and in those with rheumatoid arthritis (RA) with the use of 14C-protein hydrolyzate, 14C-uridine and 14C-thymidine, respectively. A study was also made of the stimulation of 14C-proline incorporation in protein fibroblasts upon addition to serum-free media of 5% bovine embryonic serum. The stability of RNA in fibroblasts was tested. It was shown that the rate of protein synthesis was 11 times higher in fibroblasts of RA patients and 6 times higher in those of SSD patients as compared to the rate of protein synthesis in fibroblasts of normal subjects. The rate of DNA synthesis in skin fibroblasts of RA patients was 15 times higher and in those of SSD patients 4 times higher than normal. In both RA and SSD patients, the synthesis of short-labeled RNA was 2-3 times higher than normal. The addition of embryonic serum increased 2-3 times the incorporation of 14C-proline in protein skin fibroblasts of SSD patients. It was found that all RNA in skin fibroblasts was represented by long-living molecules and that 30-40% of short-labeled RNA in skin fibroblasts of healthy donors and SSD patients underwent degradation within 1-2 hours. The data obtained indicate that fibroblasts of the two pathologies under study are characterized by considerable differences in the synthesis of DNA and the activity of the protein-synthesizing system.