CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

Physiological studies and determination of chromosome number in Maca,Lepidium Meyenii (Brassicaceae)

Experiments based on four accessions of maca (Lepdium meyenii) disclosed higher developmental rates in plants grown in neutral pH (6.6) soil when compared with those grown in acidic soil (5.3). Photoperiod response studies revealed similar growth rate for plants grown under either long day or short day condition. Plants in the field and growth chambers completed their life cycle in 11 months or less, therefore maca can be considered an annual crop. These results suggest that the range of adaptation of maca is not as narrow as previously believed, and therefore it can be successfully produced outside its natural habitat. Chromosome counts and predominance of bivalents in diakinesis and metaphase I disclosed that maca is a disomic octoploid of 2n=8x=64 chromosomes. Field and growth chamber observations and morphological uniformity of the plants within accessions indicate that maca relies mainly on self-fertilization for its reproduction.     

The acidic polysaccharide from Palmaria decipiens (Palmariales,Rhodophyta)

Palmaria decipiens, one of the most abundant red seaweeds of the chilean Antarctic, was collected in King George Island. The hot water extract (26% yield) showed by acid hydrolysis to contain xylose, galactose and traces of glucose. Fractionation with cetrimide gave a soluble neutral xylan and an insoluble fraction. The insoluble fraction afforded an acidic polysaccharide that contained 4.8% of uronic acids, 2.8% of sulfate and 18.9% of protein. Polyacrylamide gel electrophoresis showed that it was homogeneous. The GLC and HPLC analysis of the total acidic hydrolysis products showed that the acidic polysaccharide was composed of the neutral sugars galactose and xylose in the molar ratio 8.2:1.0 and of galacturonic and glucuronic acid in the ratio 1.5:1.0. The second-derivative FT-IR spectrum showed the characteristic amide I, II and III bands of proteins. Alkaline cleavage with 0.1 M NaOH indicated the presence of a glycoprotein with O-glycosidic linkage.Results found in this work suggest that the acidic polysaccharide extracted from Palmaria decipiens is an acidic xylogalactan-protein complex.     

Vertical distribution of benthic nematodes in an oligotrophic lake: seasonality, species and age segregation

The vertical distribution of nematodes up to 20 cm sediment depth was studied over a one-year period in the alpine, oligotrophic lake Königssee. Ten water depths were examined, four of which correspond to the littoral (1 m, 2 m, 5 m, 10 m), three to the littoriprofundal (15 m, 20 m, 30 m) and three to the profundal (60 m, 120 m, 190 m). The sediment was devided into four layers (0–2 cm, 2–5 cm, 5–10 cm and 10–20 cm). (1) The highest proportion of nematodes was found in the top layer. Out of 45 263 nematodes, 89% were found in the first 5 cm of the sediment and only 1 % deeper than 10 cm. (2) The proportion of nematodes in the top layer increased along with water depth. Water depth was a better predictor of percentage of nematodes in the top layer than particle size or content of organic carbon in the sediment. (3) There were considerable differences among species in their vertical distribution in the sediment. (4) There was a significant trend for juveniles to occupy more superficial layers than adults across various species. (5) There are time related vertical preferences among adults and juveniles of several nematode species across the year, suggesting vertical migration in the sediment (e.g. Aphanolaimus aquaticus Daday, Eumonhystera longicaudatula Gerlach & Riemann, Tobrilus gracilis Bastian, Monhystera paludicola de Man, Ethmolaimus pratensis de Man and Ironus tenuicaudatus de Man). The factors determining the vertical variation in nematode abundance in freshwater systems across space and time are still unknown.     

Modeling competition between Laurencia obtusa (Ceramiales,Rhodophyta) and Hypnea spinella (Gigartinales,Rhodophyta) at Cabo Frio Island,Rio de Janeiro,Brazil

The Lotka-Volterra competition model was used to represent the interaction between Laurencia obtusa and Hypnea spinella. A new model that considers effects of competition on algal carrying capacity is suggested. To test the models, data from field experiments conducted in an intertidal region at Cabo Frio Island, Rio de Janeiro, Brazil, were used. Both models showed that Hypnea was a stronger competitor than Laurencia. The model of interaction through the carrying capacity showed a stable coexistence between the algal populations and better represented the experimental data.     

Measurement of nutrient spiralling during a period of continuous surface flow in a Mediterranean temporary stream (Arroyo de La Montesina, Spain)

Uptake rate of calcium, potassium, nitrate-N and phosphorus were measured in a second order Mediterranean temporary stream, in February and March 1992. This study analyzed a period of continuous surface flow between two hydrologic disturbance events (flood and drought) of an annual hydrological cycle (1991–92).The lowest values of uptake length were recorded for nitrate-N in February 92 and calcium in March 92. Nitrate had the highest uptake rate in both release performances, and potassium showed the lowest uptake rate values. The increase of calcium and nitrate uptake rate between February 92 and March 92 suggested a higher ecosystem efficiency in nutrient retention with a higher temperature and light intensity and slower water velocity, discharge and water depth. These results obtained were similar to those reported in permanent streams, indicating that in periods of continuous surface flow (without extreme hydrologic disturbance), abiotic factors can influence nutrient retention in temporary streams.     

Neurotrophin-4: The odd one out in the neurotrophin family

Neurotrophin-4 (NT-4) is a member of a family of neurotrophic factors, the neurotrophins, that control survival and differentiation of vertebrate neurons (2–4). Besides being the most recently discovered neurotrophin in mammals, and the least well understood, several aspects distinguish NT-4 from other members of the neurotrophin family. It is the most divergent member and, in contrast to the other neurotrophins, its expression is ubiquitous and appears to be less influenced by environmental signals. NT-4 seems to have the unique requirement of binding to the lowaffinity neurotrophin receptor (p75^LNGFR) for efficient signalling and retrograde transport in neurons. Moreover, while all other neurotrophin knock-outs have proven lethal during early postnatal development, mice deficient in NT-4 have so far only shown minor cellular deficits and develop normally to adulthood. Is NT-4 a recent addition to the neurotrophic factor repertoire in search of a crucial function, or is it an evolutionary relic, a kind of wisdom tooth of the neurotrophin family?     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

NMDA Receptor antagonists prevent acute ammonia toxicity in mice

We proposed that acute ammonia toxicity is mediated by activation of NMDA receptors. To confirm this hypothesis we have tested whether different NMDA receptor antagonists, acting on different sites of NMDA receptors, prevent death of mice induced by injection of 14 mmol/Kg of ammonium acetate, a dose that induces death of 95% of mice. MK-801, phencyclidine and ketamine, which block the ion channel of NMDA receptors, prevent death of at least 75% of mice. CPP, AP-5, CGS 19755, and CGP 40116, competitive antagonists acting on the binding site for NMDA, also prevent death of at least 75% of mice. Butanol, ethanol and methanol which block NMDA receptors, also prevent death of mice. There is an excellent correlation between the EC₅₀ for preventing ammonia-induced death and the IC₅₀ for inhibiting NMDA-induced currents. Acute ammonia toxicity is not prevented by antagonists of kainate/AMPA receptors, of muscarinic or nicotinic acetylcholine receptors or of GABA receptors. Inhibitors of nitric oxide synthase afford partial protection against ammonia toxicity while inhibitors of calcineurin, of glutamine synthetase or antioxidants did not prevent ammonia-induced death of mice. These results strongly support the idea that acute ammonia toxicity is mediated by activation of NMDA receptors.