A phylogenetic approach to the identification of phosphoglucomutase genes

The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes. We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM). Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms. In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3. Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database. Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium-- and sugar-binding sites, were conserved in all 47 sequences. The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified. Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2. Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2. A third lineage may contain homologs to human PGM3. On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.      

Short chain oligogalacturonides induce ethylene production and expression of the gene encoding aminocyclopropane 1-carboxylic acid oxidase in tomato plants

Oligogalacturonic acids (OGAs), derived from plant cell wall pectin, have been implicated in a number of signal transduction pathways involved in growth, development and defense responses of higher plants. This study investigates the size range of OGAs capable of inducing ethylene synthesis in tomato plants, and demonstrates that in contrast with many other effects, only short chain OGAs are active. Oligomers across a range of DP from 2-15 were separated and purified to homogeneity by QAE-Sephadex anion exchange chromatography using a novel elution system. The OGAs were applied to tomato plants and assayed for their ability to induce ethylene gas release and changes in steady state levels of mRNA encoding the ethylene forming enzyme aminocyclopropane-1-carboxylic acid oxidase (ACO). The study demonstrated that only OGAs in the size range of DP4-6 were active both in eliciting ACO expression and in the production of ethylene.      

Release of Membrane-Bound Trails by Trypanosoma cruzi Amastigotes onto Modified Surfaces and Mammalian Cells

ABSTRACT. Upon incubation at 37° C onto glass coverslips coated with Concanavalin A, poly-L-lysine, or a monoclonal antibody (1D9) directed to the parasite major surface glycoprotein Ssp-4, extracellular Trypanosoma cruzi amastigotes release trails of material barely visible by light microscopy. This release is not associated with parasite movements. Immunolabeling studies confirmed that the material is derived from the parasite's membrane since thin section through samples labeled with 1D9 revealed that the trails are membrane-bound structures. Scanning electron microscopy showed that the ∼0.1-μm thick trails of material emerging from the amastigotes can be uniform or beaded, indicating a tendency to vesiculation. The trails are preferentially released from the flagellar pocket region and/or at the opposite posterior end of the parasite body, and seem to be devoid of microtubules. The release is time and temperature-dependent and fixed parasites do not form trails. All attempts to inhibit trail release using drugs (antimycin A, sodium azide, cytochalasin D, nocodazole, genistein, staurosporine, EGTA) failed. The observation of trails associated with intracellular parasites and amastigotes invading Vero cells suggests that this is probably a physiological process.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures

Baculoviruses are a group of viruses that infect invertebrates and that have been used worldwide as a biopesticide against several insect pests of the Order Lepidoptera. In Brazil, the baculovirus Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV; Baculoviridae) has been used experimentally to control S. frugiperda (Lepidoptera: Noctuidae), an important insect pest of corn (maize) fields and other crops. Baculoviruses can be produced either in insect larvae or in cell culture bioreactors. A major limitation to the in vitro production of baculoviruses is the rapid generation of mutants when the virus undergoes passages in cell culture. In order to evaluate the potential of in vitro methods of producing SfMNPV on a large-scale, we have multiplied a Brazilian isolate of this virus in cell culture. Extensive formation of few polyhedra mutants was observed after only two passages in Sf9 cells.     

Photosynthesis of oak trees [Quercus petraea (Matt.) Liebl.] during drought under field conditions: diurnal course of net CO2 assimilation and photochemical efficiency of photosystem II

Adult trees of Quercus petraea were submitted to controlled water shortage in a natural stand near Nancy, France. Diurnal course of net CO₂ assimilation rate (A) was measured in situ together with chlorophyll a fluorescence determined on dark adapted leaves. In 1990, trees experienced a strong water stress, with predawn and midday leaf water potentials below –2·0 and –3·0 MPa, respectively. Diurnal course of A of well-watered trees exhibited sometimes important midday decreases in A related to high temperature and vapour pressure deficit. Decreases in initial (F_o) and maximal (F_m) fluorescence and sometimes in photochemical efficiency of photosystem II (F_v/F_m) were observed and probably revealed the onset of mechanisms for thermal de-excitation. These mechanisms were shown to be sensitive to dithiothreitol. All these effects were reversible and vanished almost completely overnight. Therefore, they may be considered as protective mechanisms adjusting activity of photosystem II to the electron requirement for photosynthesis. Water stress amplified these reactions: A was strongly decreased, showing important midday depression; diurnal reductions in F_m and F_v/F_m were enhanced. The same trends were observed during summer 1991, despite a less marked drought. These protective mechanisms seemed very effective, as no photoinhibitory damage to PS II could be detected in either water stressed or control trees.     

Hydrogen and Oxygen Isotopic Fractionation During Heterotrophic Cellulose Synthesis

Hydrogen and oxygen isotopic fractionation relative to mediumwater for two different carbohydrate metabolic pathways leadingto cellulose synthesis were measured. This was accomplishedby analysing stable hydrogen and oxygen isotope ratios of waterand cellulose for seedlings. The seedlings had been germinatedand heterotrophically grown in closed vessels from species havingstarch (Triticum aestivum L. and Hordeum vulgare L.) and lipids(Ricinus communisL. and Arachis hypogaea L.) as the primarysubstrate. Isotopic fractionation factors occurring during enzyme-mediatedexchange of carbon-bound hydrogen with water or the additionof carbon-bound hydrogens from water during the synthesis ofcellulose from either starch or lipids were similar (rangingfrom +144 to +166%). About 34% and 67% of carbon-bound hydrogenswere derived from water during the synthesis of cellulose fromstarch and lipid, respectively. Thus, the greater deuteriumenrichment in cellulose from oil seed species associated withgluconeogenesis was caused by a greater proportion of water-derivedcarbon-bound hydrogens and not because of differences in fractionationfactors. The proportion of carbon-bound hydrogens derived fromwater during these metabolic pathways was similar to that ofoxygen derived from water. These results may explain the variabilityin D/H ratios of cellulose nitrate from terrestrial and aquaticplants. Key words:     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Biomass of primary and secondary vegetation in Rondônia, Western Brazilian Amazon

Biomass estimates of primary and different ages of secondary vegetation are reported for a tropical forest region in Rondônia, Western Brazilian Amazon. The estimates are based on published allometric equations, and on vegetation composition and allometric data collected in areas of primary forest and secondary vegetation of ages 2, 3, 5, 9, 11, 16 and 18 years. Primary forest biomass estimates varied from 290 to 495 t ha^–1. Secondary vegetation biomass estimates accounted for 40–60% of the primary forest biomass after 18 years of abandonment. Secondary growth rates in lightly used areas are estimated to have varied from 6.6 to 8.7 t ha^–1 y^–1 between the third and the eighteenth years after abandonment. CO₂ sequestration by regrowing vegetation is discussed for two scenarios of land abandonment.