Existence of two D-alanine:D-alanine ligases in Escherichia coli: cloning and sequencing of the ddlA gene and purification and characterization of the DdlA and DdlB enzymes

Two distinct genes encoding D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) activity in Escherichia coli have been cloned by complementation of E. coli strain ST640(lambda 112) deficient in D-Ala-D-Ala ligase activity with a lambda library of E. coli DNA. One of the two genes, designated as ddlB, is identical with the ddl gene already sequenced [Robinson, A.C., Kenan, D.L., Sweeney, J., & Donachie, W.D. (1986) J. Bacteriol. 167, 809-817]. We describe the subcloning and DNA sequencing of the other gene, designated as ddlA on the basis of similarities with the Salmonella typhimurium ddlA gene [Daub, E., Zawadzke, L.E., Botstein, D., & Walsh, C.T. (1988) Biochemistry 27, 3701-3708]. The predicted amino acid sequence of the E. coli DdlA enzyme shows 90% homology with the S. typhimurium DdlA sequence. The ddlB gene was subcloned by use of the polymerase chain reaction into an expression vector containing an optimized ribosome binding site, which expressed the DdlB enzyme to greater than 50% soluble cell protein. Both DdlA and DdlB enzymes were purified to greater than 90% homogeneity and characterized kinetically.     

Calcium-carbohydrate bridges composed of uncharged sugars. Structure of a hydrated calcium bromide complex of α-fucose

X-ray diffraction data were used to determine the crystal structure of a hydrated CaBr₂ complex of α-fucose, a common terminal sugar of oligosaccharide chains on glycoproteins. Crystals of C₆H₁₂O₅ · CaBr₂ · 3H₂O are orthorhombic, space group P2₁2₁2₁, with a = 14.360(2), b = 12.896(3), and c = 8.043(1)Å. Intensity data for 1442 independent reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by leastsquares to R = 0.052. Ca²⁺ is chelated by a pair of hydroxyl groups from each of two symmetry-related fucose molecules and is coordinated to three water molecules. Thus the structure consists of hydrated fucose-calcium-fucose bridges. The bridge geometry, which is dictated by the coordination requirements of Ca²⁺, is like that of other calcium-carbohydrate complexes. Our results indicate that calcium-fucose interactions can provide an effective, stereospecific mechanism for cross-linking carbohydrate chains. Similar calcium-carbohydrate bridges may be involved in a variety of Ca²⁺-dependent agglutination and adhesion processes.     

Maternal Smoking during Pregnancy and Fetal Organ Growth: A Magnetic Resonance Imaging Study

Objective

To study whether maternal cigarette smoking during pregnancy is associated with alterations in the growth of fetal lungs, kidneys, liver, brain, and placenta.

Design

A case-control study, with operators performing the image analysis blinded.

Setting

Study performed on a research-dedicated magnetic resonance imaging (MRI) scanner (1.5 T) with participants recruited from a large teaching hospital in the United Kingdom.

Participants

A total of 26 pregnant women (13 current smokers, 13 non smokers) were recruited; 18 women (10 current smokers, 8 nonsmokers) returned for the second scan later in their pregnancy.

Methods

Each fetus was scanned with MRI at 22–27 weeks and 33–38 weeks gestational age (GA).

Main outcome measures

Images obtained with MRI were used to measure volumes of the fetal brain, kidneys, lungs, liver and overall fetal size, as well as placental volumes.

Results

Exposed fetuses showed lower brain volumes, kidney volumes, and total fetal volumes, with this effect being greater at visit 2 than at visit 1 for brain and kidney volumes, and greater at visit 1 than at visit 2 for total fetal volume. Exposed fetuses also demonstrated lower lung volume and placental volume, and this effect was similar at both visits. No difference was found between the exposed and nonexposed fetuses with regards to liver volume.

Conclusion

Magnetic resonance imaging has been used to show that maternal smoking is associated with reduced growth of fetal brain, lung and kidney; this effect persists even when the volumes are corrected for maternal education, gestational age, and fetal sex. As expected, the fetuses exposed to maternal smoking are smaller in size. Similarly, placental volumes are smaller in smoking versus nonsmoking pregnant women.     

Isoform switching facilitates period control in the Neurospora crassa circadian clock

A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q₁₀ of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild‐type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic.     

Direct and pleiotropic effects of the Masou Salmon Delta-5 Desaturase transgene in F1 channel catfish (Ictalurus punctatus)

Transgenic Research - Channel catfish (Ictalurus punctatus) is the primary culture species in the US along with its hybrid made with male blue catfish, I. furcatus. In an effort to improve the...     

Regulation and Manipulation of the Biosynthesis of Abscisic Acid, Including the Supply of Xanthophyll Precursors

Mutant plants deficient in the phytohormone abscisic acid (ABA) are typically unable to control their stomatal behavior appropriately in response to water stress, leading to a “wilty” phenotype. In plant species showing strong seed dormancy, ABA deficiency of the seed results in a second clearly recognizable phenotype, that is, early germination. Mutants selected by means of this latter character are often collectively termed “viviparous.” These two broad classes include mutants that are defective in their ability to synthesize ABA. A number of these genetic lesions have been assigned to specific steps in ABA biosynthesis and have been invaluable in elucidating many important features of the pathway. Most of the genes encoding ABA biosynthetic enzymes have now been cloned and their expression has been studied and manipulated. Genetically modified plants constitutively overexpressing ABA biosynthesis genes have been produced and analyzed over the last 6 years. In some cases these plants have been found to have elevated ABA concentrations, leading to altered stomatal behavior and increased seed dormancy. Genetic manipulation of ABA synthesis in photosynthetic tissues has been most effectively achieved through overexpression of the key rate-limiting biosynthetic enzyme 9-cis-epoxycarotenoid dioxygenase, and downregulation of the major catabolic enzyme ABA 8′-hydroxylase. However in non-photosynthetic tissue manipulation of ABA synthesis is a more complex task because of the limiting supply of xanthophyll precursors. The recent cloning of genes encoding enzymes controlling important pathways of ABA catabolism has been reviewed elsewhere, and so only information relevant to the regulation and manipulation of ABA synthesis, including supply of xanthophyll precursors, is discussed in this review.     

Detection of microspheres in vivo using multispectral optoacoustic tomography

We introduce a new approach to detect individual microparticles that contain NIR fluorescent dye by multispectral optoacoustic tomography in the context of the hemoglobin-rich environment within murine liver. We encapsulated a near infrared (NIR) fluorescent dye within polystyrene microspheres, then injected them into the ileocolic vein, which drains to the liver. NIR absorption was determined using multispectral optoacoustic tomography. To quantitate the minimum diameter of microspheres, we used both colorimetric and spatial information to segment the regions in which the microspheres appear. Regional diameter was estimated by doubling the maximum regional distance. We found that the minimum microsphere size threshold for detection by multispectral optoacoustic tomography images is 78.9 µm.     

Attractiveness of common insectary and harvestable floral resources to beneficial insects

Resources for natural enemies are often lacking in agricultural fields. The provisioning of floral resources in crop fields can ameliorate this problem by providing nectar and pollen to natural enemies. To select an appropriate floral resource, plants must be screened for their flowering times and attractiveness to natural enemies and pests. We tested the attractiveness of nine species of annual flowering plants to hoverflies, which are important predators of aphids in California lettuce fields. We also sampled arthropods in the foliage of tested plants, to assess the abundances of other natural enemy and pest species that were present. Tested plants included three commonly-used insectary plants and six flowering plant species that showed potential as either harvestable herbs or cut flowers. Harvestable insectary plants may provide additional economic incentive for growers to set aside land for floral resources. The commonly-used insectary plant sweet alyssum consistently attracted the most hoverflies and the least bees, while potentially harvestable plants attracted few hoverflies. Competition with bees may have reduced hoverfly visits to several of the tested plant species. Sweet alyssum also stayed in bloom the longest, and contained the highest numbers of predatory hemipterans. Results suggest that plants should be screened for their attractiveness to not only the target biological control agent, but also to other potential competitors for floral resources. While this initial study focused on a limited selection of harvestable annual plants, a wide variety of other marketable plant species, particularly perennials, remain to be tested for their attractiveness to hoverflies or other beneficial arthropods.     

RNA-Catalyzed CoA, NAD, and FAD synthesis from phosphopantetheine, NMN, and FMN

A novel in vitro selection method was developed to isolate RNA sequences with coenzyme-synthesizing activities. We used size-heterogeneous libraries containing randomized ribonucleotide sequences of four different lengths (30N, 60N, 100N, and 140N), all with 5'-ATP initiation. Two RNAs, CoES7 (30N) and CoES21 (60N), are able to catalyze the synthesis of three common coenzymes, CoA, NAD, and FAD, from their precursors, 4'-phosphopantetheine, NMN, and FMN, respectively. Both ribozymes require divalent manganese for activities. The results support the availability of these coenzymes in an RNA world, and point to a chemical explanation for the complex bipartite structures of many coenzymes.