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.     

Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.     

The structure of d(TpA), the major photoproduct of thymidylyl-(3'5')-deoxyadenosine.

Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929-7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight-membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA.     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position.

We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.     

Changes in Metabotropic Glutamate Receptor mRNA Levels Following Global Ischemia: Increase of a Putative Presynaptic Subtype (mGluR4) in Highly Vulnerable Rat Brain Areas

Abstract: Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent l -2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1–mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Pollen mitosis in the slipper orchid Cypripedium fasciculatum

Pollen mitosis in the slipper orchid Cypripedium fasciculatum was studied using correlated methods of immunofluorescence and transmission electron microscopy. Unlike the more highly evolved orchids, the cypripedioid orchids shed pollen as monosulcate monads. Prior to pollen mitosis, the microspore nucleus migrates to a proximal position opposite the aperture, as is typical of monocotyledons. There is no distinct generative pole microtubule system (GPMS) like that recently reported in development of pollen polarity in the vandoid moth orchid Phalaenopsis. Instead, microtubules in early prophase are concentrated around the nucleus and extend into the cytoplasm toward the future generative pole. Once the nucleus has migrated to the continuous surface opposite the aperture, microtubules surround the nucleus evenly and show no tendency to be more concentrated in the generative domain. The mitotic spindle, which develops from the perinuclear microtubules, is asymmetrically placed in the microspore and is cone-shaped. The generative pole is broad and closely appressed to the continuous spore surface, while the vegetative pole is pointed and located in the interior of the microspore. As the chromosomes move poleward, microtubules proliferate in the interzone and a phragmoplast develops. The phragmoplast expands in a hemispherical path beyond the interzone following an array of microtubules that radiates from the generative nucleus. Data from this study indicate that evolution of pollen in orchids includes a shift in location of the generative cell from proximal to distal and the evolution of a GPMS, in addition in the well-known trend toward increased pollen aggregation and loss of exine.     

Isolation and Refined Regional Mapping of Expressed Sequences from Human Chromosome 21

To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3.     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid