Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Regulation of plasma membrane Ca2 +-ATPase activity by acetylated tubulin: Influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca^2 +-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300 μg/ml) in combination with acidic lipids at concentrations > 10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50 μg/ml tubulin, increased PMCA activity > 12-fold, whereas tubulin alone at high concentration (≥ 300 μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca^2 + transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (< 50 μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

The effects of drying on sediment nitrogen content in a Mediterranean intermittent stream: a microcosms study

Mediterranean climates predispose aquatic systems to both flood and drought periods, therefore, stream sediments may be exposed to desiccation periods. Changes in oxygen concentrations and sediment water content influence the biotic processes implicated in nitrogen dynamics. The objectives of this study were to identify (1) the changes of inorganic nitrogen in stream sediments during the transition from wet to dry conditions, and (2) the underlying processes in N dynamics and its regulation. Extractable sediment NO₃ ⁻-N and NH₄ ⁺-N, organic matter and extractable organic carbon content were assessed during natural desiccation in microcosms with sediments from an intermittent Mediterranean stream. In agreement with our initial hypothesis, our results showed how the NO₃ ⁻-N content of the sediment was enhanced during the first 10 days of sediment drying, whereas NH₄ ⁺-N was lost by 14 days post-drying. During the first 10 days, sediment desiccation seemed to stimulate the net N-mineralization and net nitrification from sediments. Afterwards, the extractable NO₃ ⁻-N concentration sharply dropped, which may be attributed to lower ammonium-oxidation rates as ammonium and organic matter are depleted, and to an increase in NO₃ ⁻-N consumption by microbial populations. Denitrification was inhibited, with a significant decrease as % water-filled pore space lowered. We hypothesize that the sediment inorganic N content enhanced during sediment desiccation could be released as part of the N pulse observed after sediment rewetting. However, the stream N availability after rewetting dried sediments would differ depending on desiccation period duration.     

Effects of cytoprotective antioxidants on lymphocytes from representative mitochondrial neurodegenerative diseases

Two new aza analogues of the neuroprotective agent idebenone have been synthesized and characterized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scavengers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol derivatives were also shown to augment ATP levels in coenzyme Q₁₀-deficient human lymphocytes. The more lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibition of the electron transport chain and improved antioxidant activity.     

The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides.

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.     

Soil water availability and rooting depth as determinants of hydraulic architecture of Patagonian woody species

Adaptations of species to capture limiting resources is central for understanding structure and function of ecosystems. We studied the water economy of nine woody species differing in rooting depth in a Patagonian shrub steppe from southern Argentina to understand how soil water availability and rooting depth determine their hydraulic architecture. Soil water content and potentials, leaf water potentials (Ψ_Leaf), hydraulic conductivity, wood density (ρ_w), rooting depth, and specific leaf area (SLA) were measured during two summers. Water potentials in the upper soil layers during a summer drought ranged from −2.3 to −3.6 MPa, increasing to −0.05 MPa below 150 cm. Predawn Ψ_Leaf was used as a surrogate of weighted mean soil water potential because no statistical differences in Ψ_Leaf were observed between exposed and covered leaves. Species-specific differences in predawn Ψ_Leaf were consistent with rooting depths. Predawn Ψ_Leaf ranged from −4.0 MPa for shallow rooted shrubs to −1.0 MPa for deep-rooted shrubs, suggesting that the roots of the latter have access to abundant moisture, whereas shallow-rooted shrubs are adapted to use water deposited mainly by small rainfall events. Wood density was a good predictor of hydraulic conductivity and SLA. Overall, we found that shallow rooted species had efficient water transport in terms of high specific and leaf specific hydraulic conductivity, low ρ_w, high SLA and a low minimum Ψ_Leaf that exhibited strong seasonal changes, whereas deeply rooted shrubs maintained similar minimum Ψ_Leaf throughout the year, had stems with high ρ_w and low hydraulic conductivity and leaves with low SLA. These two hydraulic syndromes were the extremes of a continuum with several species occupying different portions of a gradient in hydraulic characteristics. It appears that the marginal cost of having an extensive root system (e.g., high ρ_w and root hydraulic resistance) contributes to low growth rates of the deeply rooted species.     

Occurrence of <Emphasis Type="Italic">TRGV-BJ</Emphasis> hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.     

Tubulin must be acetylated in order to form a complex with membrane Na+,K+-ATPase and to inhibit its enzyme activity

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na⁺,K⁺-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na⁺,K⁺-ATPase and consequent inhibition of enzyme activity.     

Triploidy Induction in the Pacific White Shrimp Litopenaeus vannamei: An Assessment of Induction Agents and Parameters, Embryo Viability, and Early Larval Survival

In this study, we trialed 6-dimethylaminopurine (6-DMAP) chemical shocks to induce meiosis I or meiosis II Pacific White shrimp, Litopenaeus vannamei, triploids for the first time, and cold temperature shocks to induce meiosis II L. vannamei triploids as done previously. Inductions were performed on 37 spawnings in total with experiments being progressively designed in a factorial manner to allow optimization of induction parameters. Treatment with a 200-μm 6-DMAP final concentration at 1?min post-spawning detection for a 6 to 8?min duration resulted in the most consistent induction of chemically induced meiosis I triploids while treatment at 7?min 30?s post-spawning detection for a 10-min duration resulted in the most consistent induction of chemically induced meiosis II triploids. A cold temperature shock of 11.7°C to 13.25°C (final treatment temperature; spawning water temperature 28.5°C) applied at 8?min post-spawning detection for a 4 to 10?min duration resulted in the most consistent induction of cold-temperature-induced meiosis II triploids. 6-DMAP shocks resulted in meiosis I induction rates from 29% to 100% in unhatched embryos and 50% in nauplii, and meiosis II induction rates from 65% to 100% in unhatched embryos and 52% to 100% in nauplii. Cold shocks resulted in induction rates from 5% to 100% in unhatched embryos and nauplii. Confocal microscopy analysis of embryos revealed that there are major developmental abnormalities in a large proportion of later stage triploid L. vannamei embryos compared to their diploid sibling controls. Despite this, however, some triploid embryos did appear normal and both shock agents induced small numbers of viable triploid L. vannamei nauplii which were successfully reared to protozoeal stage 3 as confirmed by flow cytometry. Triploids beyond this life-history stage were not observed in the present study as confirmed by flow cytometry at mysis stages. This study adds to our knowledge base of triploid induction in L. vannamei and further highlights the inherent difficulties with triploid embryonic and larval viability in this species.