Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Circadian and seasonal variation of the body temperature of sheep in a tropical environment

Nychthemeral and annual rhythms of the rectal temperature were determined for Corriedale sheep in a tropical climate. The minimum rectal temperature averaged 39.55° C at 0500 hours in summer, and 38.87° C at 0600 hours in winter. The maximum was 40.03° C in summer (1700 hours) and 39.33° C in winter (1830 hours). Annual cycle of the rectal temperature showed a minimum in July and maximum in December.     

The paracellular channel for water secretion in the upper segment of the Malpighian Tubule of Rhodnius prolixus

Lumen to bath J ₁₂/C ₁ and bath to lumen J ₂₁/ C ₂ fluxes per unit concentration of 19 probes with diameters (d ^m) ranging from 3.0–30.0 Å (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with d ^m 9.1–30.0 Å) were measured during volume secretion (J _v ) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with ¹⁴C or ³H-labeled probes. J _net=(J ₁₂/C ₁ – J ₂₁/C ₂) was studied as a function of J _v · J _v was varied by using different concentrations of 5-hydroxy tryptamine. J _net for ³H-water was not different from J _v We found: (i) A strong correlation between J _net and J _v for 8 probes d ^m =3.0–11.8 Å (group a probes), indicating that the convective component of J _net is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium, (ii) There is no correlation between J _net and J _v for 11 probes with d ^m=11.8–30 Å (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag, (iii) In a plot of J _net/J _v vs. d ^m group a probes show a steep linear relation with a slope = –0.111, while for group b probes the slope is –0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 Å-wide slit, (ii) Most of J _v must follow an extracellular noncytosolic pathway, (iii) Group b probes must diffuse through a 42 Å-wide slit, (iv) A cylindrical pathway does not fit the data.E.G. is a Visiting Scientist at IVIC. It is a pleasure to thank Drs. A.E. Hill and Bruria Shachar-Hill for their suggestion of the use of dextrans, their instruction and help with the dextran separation technique, and their extensive discussions. Dr. R. Apitz, Mr H. Rojas and Mrs. Fulvia Bartoli were most helpful with suggestions during the course of the experimental work. Mr. Jose Mora was fundamental help with the equipment. Mrs. Lelis Ochoa and Mr. Luis F. Alvarez helped with some of the drawings. This work was partially supported by CONICIT, Fundación Polar and CDCH of UCV. It is a pleasure to thank Dr. H. Passow and Dr. K.J. Ullrich at the Max Planck Institut für Biophysik (Frankfurt/Main) where this work was initiated.     

A method for determining the position and size of optimal sequence regions for phylogenetic analysis

The availability of fast and accurate sequencing procedures along with the use of PCR has led to a proliferation of studies of variability at the molecular level in populations. Nevertheless, it is often impractical to examine long genomic stretches and a large number of individuals at the same time. In order to optimize this kind of study, we suggest a heuristic procedure for detection of the shortest region whose informational content can be considered sufficient for significant phylogenetic reconstruction. The method is based on the comparison of the pairwise genetic distances obtained from a set of sequences of reference to those obtained for different windows of variable size and position by means of a simple index. We also present an approach for testing whether the informative content in the stretches selected in this way is significantly different from the corresponding content shown by the larger genomic regions used as reference. Application of this test to the analysis of the VP1 protein gene of foot-and-mouth-disease type C virus allowed us to define optimal stretches whose informative content is not significantly different from that displayed by the complete VP1 sequence. We showed that the predictions made for type C sequences are valid for type O sequences, indicating that the results of the procedure are consistent. Correspondence to: J. Dopazo     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

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.

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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.

     

Linkage Analysis with an Alternative Formulation for the Mixed Model of Inheritance: The Finite Polygenic Mixed Model

This paper presents an extension of the finite polygenic mixed model of FERNANDO et al. (1994) to linkage analysis. The finite polygenic mixed model, extended for linkage analysis, leads to a likelihood that can be calculated using efficient algorithms developed for oligogenic models. For comparison, linkage analysis of 5 simulated 4021-member pedigrees was performed using the usual mixed model of inheritance, approximated by HASSTEDT (1982), and the finite polygenic mixed model extended for linkage analysis presented here. Maximum likelihood estimates of the finite polygenic mixed model could be inferred to be closer to the simulated values in these pedigrees.     

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions

In order to elucidate the relationship between hypertension and hypertrophy in the production of heat shock proteins, we studied the induction of the HSP72 synthesis by the heart and gracilis muscles of normo (WKY) and hypertensive (SHR) rats subjected to hyperthermia (42°C±0.5 for 15 min). Two age groups were investigated in each strain: young (2 months, with developing cardiac hypertrophy) and old (18 months, with fully developed chronic cardiac hypertrophy). The gracilis muscle never developed hypertrophy, independently of hypertension or aging. 72 kDa inducible protein was determined by Western blot analysis using a specific monoclonal antibody. We also used a commercial standard, loaded on each blot, to quantitate densitometrically the signal.The heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8±24.7% vs 88.3 ±8.5%, p<0.001). This response is not maintained during aging as we did not find any significant difference between normo-and hypertensive old rats after exposure to hyperthermia (43.6±5.3% vs 65.3±10.4%).Unlike the heart, the gracilis muscle shows a basal spontaneous HSP72 synthesis in both the SHR (71.4±10.8%) and WKY (40.6±11.7%) animals. There was a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to their control (186.2±18.7% vs 115.8±9.9%, p<0.02) which was maintained also during aging (171.9±17.3% vs 95.2±10.5%, p<0.01).In conclusion, these data show that hypertension results in an increased synthesis of HSP72 both in cardiac and gracilis muscle in response to heat shock. This abnormal response is attenuated by aging in the heart but not in the gracilis muscle. Thus, the abnormality seems to be independent from hypertrophy and linked to genetic determination of the disease.     

Hyperbolic Modeling of Starburst Dendrimers

Starburst dendrimers are highly branched oligomers. A rigid dendritic hydrocarbon, C₁₁₃₄H₁₁₄₆, has recently been synthesized. It consists of 94 phenylacetylene units displayed in a self-similar two-dimensional skeleton isomorphous to the three-coordinated Bethe lattice. The three-dimensional representation of phenylacetylene dendrimer shows a globular architecture with large voids and niches in its interior, characteristic of hyperbolic surfaces. This work investigates the geometrical scaling behavior of this starburst dendrimer using the symmetry properties of a Bethe lattice embedded in the hyperbolic plane. The results for C₁₁₃₄H₁₁₄₆ provide its density profile and an upper bound for its macromolecular size. This revised version was published online in June 2006 with corrections to the Cover Date.