Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Influences of habitat features and human disturbance on use of breeding sites by a declining population of southern fur seals (Arctocephalus australis)

Southern fur seals Arctocephalus australis in Peru have declined gradually over the past decade, and declined dramatically (72%) as a result of low food availability during the severe El Niño in 1997–98. In 1999, seals abandoned some historically important breeding sites. This is particularly alarming because new sites were not colonized. Our objective was to examine how habitat features and human disturbance influenced whether sites were currently used, abandoned or apparently not used in the past by fur seals for breeding. Data were collected on 14 variables at 70 potential breeding sites at three guano reserves in Peru. Discriminant analysis revealed significant multivariate differences among sites currently used for breeding, abandoned sites and unused sites ( F =5.97, P <0.00001), and the model classified 74% of sites correctly. Currently used sites were less likely to have human disturbance and more likely to have offshore islands, stacked rocks, tide pools and abundant shade. Separate discriminant analyses for each reserve produced similar results. Habitat associated with thermoregulation (e.g. shade or pools) may be more important to fur seals in Peru, which breed at lower latitudes and are at greater risk of overheating on land than other populations. Habitat with minimized human access may be especially important to seals in small populations in which individuals may perceive themselves as more vulnerable because of decreased vigilance and dilution effects. Seals in our study selected breeding habitat with stacked rocks, which create shade and tide pools for thermoregulation and make human access difficult; but pups might suffer higher mortality in this habitat. We hypothesize that fur seals in Peru may exhibit an Allee effect, whereby suitability of habitat varies with population abundance.     

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×10⁷/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO₄ (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.     

Glyptal as a support for enzyme immobilisation

Glyptal, a polyester obtained from phthalic anhydride and glycerol, was used as a support for protein immobilisation. Hydrazide groups were introduced in the polymer and then converted to azide groups, through which protein was covalently immobilised. Amyloglucosidase was used as a model and an insoluble water derivative was synthesised retaining 24 % of the specific activity of the native enzyme. Some properties of this immobilised enzyme were studied: Km (4.54 g.l^–1 using starch as substrate), optimal temperature (55°C) and half life (8 days). Furthermore, ferromagnetic-azide-glyptal derivative showed to be useful for the amyloglucosidase immobilisation.     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

Sequence analysis of an actin isoform in the flatworm Diphyllobothrium dendriticum (Cestoda)

We have examined actin cDNA of the flatworm Diphyllobothrium dendriticum (Cestoda). Actin is a contractile protein that has been implicated in a variety of developmental and cellular processes. It is highly conserved and present in all eukaryotic cells. It is of particular interest to analyze evolutionary preserved genes in flatworms, because ancestral flatworms are regarded to play a central role in the evolution of the metazoans (Barnes et al., 1998). Screening a cDNA library of D. dendriticum (UniZap XR, Stratagene) with a human -actin probe resulted in several positive clones. One of the cDNA inserts, Didactl, consisting of 1392 bp was completely sequenced. The established nucleotide sequence revealed a 5 untranslated region of 33 bp, the entire open reading frame of 1128 bp and a 3 untranslated region of 231 bp which ends in a stretch of 21 A residues. The potential polyadenylation signal (AATAAA) is located 14 bp upstream of the poly (A) tail. The deduced amino acid sequence of Didactl is 376 amino acids long. It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins. Didactl is for example 96% homologous to human cytoplasmic -actin but only 92.6% identical with human smooth muscle -actin. The actin proteins are generally encoded by a multigene family which differs in size from species to species. Most organisms have four to eight genes coding for actin in their genome, but the number of actin genes can also be over 20 (Hamelin et al., 1988). Sequence comparisons of Didactl and the partly sequenced cDNA clones indicate that D. dendriticum has at least four different genes coding for actin in its genome.     

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m₃G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hibernating dormice, were present in much higher amouts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.     

Spatio-temporal distribution of the microphytobenthic biomass in intertidal flats of Tagus Estuary (Portugal)

A study on spatio-temporal distribution of microphytobhethos in intertidal zones of Tagus Estuary was carried out from 1990 to 1992. Near Lisbon, Portugal, Tagus Estuary is a shallow mesotidal estuary, covering an area of 320 km². The intertidal area ranges from 20 to 40% off the total area and it is constituted mainly by mudflats. Intertidal flats are richly populated by microalgae, diatoms being the most important and ubiquitos group.Spatial variation of microphytobethos was studied in spring 1990, 21 different sites were sampled. Microphytobenthos biomass was evaluated as chlorophyll a content of the surface centimeter, ranging from 10 to 240 mg m^–2. A Principal Component Analysis showed that 62% of the total variability found in intertidal flats of Tagus estuary could be attributed to two major factors: sediment type and tidal height. A hierarchical grouping defined 3 major groups of similar stations, each one representing a different strata of the ecosystem.One station from each group was chosen for the study of the temporal variation. A sampling, rogram took place from April 1991 to April 1992, with fortnightly sampling, the Chl a ranged from 20–300 mg m^–2. No clear seasonal variation was found, and our results indicated that tidal height of sampledsite played an essential role in temporal biomass evolution, thus upper littoral sites were influenced by climatic parameters, whereas in lower sites action of tides mainly controlled microphytic biomass.

---

Résumé Une étude sur l'hétérogénéité spatio-temporelle du microphytobenthos dans les sédiments intertidaux de l'Estuaire du Tage a été accompli de 1990 á 1992.L'Estuaire du Tage, prés de Lisbonne (Portugal) est un estuaire peu profond, mesotidal, avec une aire total de 320 km². L'aire intertidale est comprise entre 20 et 40% du total, et constituéé surtout par des vasiéres. Ces slikkes sont peuplées par une communauté assez riche de microalgues, ou les diatomées sont les plus abundantes.La variation spatialle du microphytobenthos était évalué au Printemps 1990, ou 21 différentes stations étaient échantillonnées. La biomasse était évalué par la concentration enchlorophylle a du premier centimétre de sédiment, qui a varié de 10 á 240 mg Chl a m^–2. Une Analyse en Composants Principales a montré que 62% de la variabilité de la biomasse était lié á deux facteurs: le sédiment et l'hauteur vis-á-vis la marée. Une classification hiérarchique des stations par similitude a établi 3 groupes principaux, représantantles différents strates de écecosytéme.Une station de chaque groupement a été choisie pour l'étude de la variation temporelle, qui s'est deroulé d'avril 1991 á avril 1992, avec des prélévements deux fois par mois. Les valeurs de Chl a obtenus vont de 20 á 300 mg m^–2. Les variations saisonniéres observées ne sont pas claires: nos résultats indiquent que l'hauteur de la station (m) joue un rôle essentiel dans l'évolution temporel de la biomasse, c'est á dire, la biomasse microalgal des sites du supra-littoral est influencié par les paramétres climatiques, tandis que dans l'infra-littoral c'est l'action des marées le facteur principal.

     

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.