Evolution of a novel lysine decarboxylase in siderophore biosynthesis

Structural backbones of iron‐scavenging siderophore molecules include polyamines 1,3‐diaminopropane and 1,5‐diaminopentane (cadaverine). For the cadaverine‐based desferroxiamine E siderophore in Streptomyces coelicolor, the corresponding biosynthetic gene cluster contains an ORF encoded by desA that was suspected of producing the cadaverine (decarboxylated lysine) backbone. However, desA encodes an l ‐2,4‐diaminobutyrate decarboxylase (DABA DC) homologue and not any known form of lysine decarboxylase (LDC). The only known function of DABA DC is, together with l ‐2,4‐aminobutyrate aminotransferase (DABA AT), to synthesize 1,3‐diaminopropane. We show here that S. coelicolor desA encodes a novel LDC and we hypothesized that DABA DC homologues present in siderophore biosynthetic clusters in the absence of DABA AT ORFs would be novel LDCs. We confirmed this by correctly predicting the LDC activity of a DABA DC homologue from a Yersinia pestis siderophore biosynthetic pathway. The corollary was confirmed for a DABA DC homologue, adjacent to a DABA AT ORF in a siderophore pathway in the cyanobacterium Anabaena variabilis, which was shown to be a bona fide DABA DC. These findings enable prediction of whether a siderophore pathway will utilize 1,3‐diaminopropane or cadaverine, and suggest that the majority of bacteria use DABA AT and DABA DC for siderophore, rather than norspermidine/polyamine biosynthesis.     

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma. The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.     

Mutations in the carboxyl-terminal domain of phospholipase C-beta 1 delineate the dimer interface and a potential Galphaq interaction site.

The carboxyl-terminal domain of phospholipase C-beta is required for its stimulation by Galpha(q) and for its Galpha(q)-specific GTPase-activating protein (GAP) activity. We subjected this domain to a combination of deletion and alanine/glycine scanning mutagenesis to detect mutations that would inhibit either responsiveness to G(q) or G(q) GAP activity. Most mutations that altered either response or GAP activity diminished both in parallel. Many of these mutations map at the interface at which the carboxyl-terminal domain was recently shown to form a dimer (Singer, A. U., et al. (2001) Nat. Struct. Biol., 9, 32-36). Most others clustered in an area that is a plausible Galpha(q) binding site. In addition, one mutation that differentially inhibited GAP activity relative to responsiveness to Galpha(q) mapped in this region at a location modeled to be in close contact with the switch II region of Galpha(q). This is the site at which RGS proteins are thought to exert their GAP activity. Last, a deletion mutation differentially inhibited the response of phospholipase C-beta1 to Galpha(q) without blocking GAP activity. Its location in the molecule suggests that moving the attachment point of the catalytic domain can disrupt its ability to be activated by Galpha(q).     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.      

Geographical distribution and the origin of life: The development of early nineteenth-century British explanations

Conclusions By the 1840s and 1850s biogeographical theory had polarized into two opposing views — both of which had their origins in the sixteenth or seventeenth centuries. At issue in this polarization was the question of God's involvement with His creation. At one end of the spectrum were Sclater, Agassiz, Kirby, and others who saw a neatly designed world in which geographical distributions were planned and executed by the hand of God at creation. For most of these naturalists, organisms were created en masse within the regions they now occupy. Disjunct distributions were proof to them that God had indeed created species in situ as many individuals. These naturalists hoped to reveal God's biogeographical plan by discovering His regions of creation. They had hoped to demonstrate a neatly devised set of regions of creation which might be applicable to all creatures, but in attempting to do so, they arrived at conflicting sets of delineations — thus helping to undermine their conceptions of nature in which design (both idealist and utilitarian) played an important part.⁹³ At the other end of the biogeographical spectrum were the theoretical ideas of Prichard and Lyell, who viewed a more remote God — one who allowed His creation to be shaped and modified by secondary laws. Lyell in particular wished to leave considerations of design aside, hoping to demonstrate that the shape of the present creation is due to natural laws. Prichard and Lyell saw God's role in the creation of species (and distributions) as being extremely limited. In fact, the regions of creation seen today are in actuality only natural artifacts produced by migrations and barriers. They saw distributions being in constant flux, as was the rest of nature.Those supporting the views of Prichard and Lyell spent a great deal of effort in attempting to remove a major obstacle in their paths — disjunct distributions. If disjunct distributions were indeed the products of separate creative acts, as Sclater and others claimed, then the arguments of Prichard and Lyell would be negated. For if the creation of a species was shown to be the product of multiple creations, then what was the need of migrations and dispersal mechanisms? Also at stake, of course, was the concept of species based upon generation. Darwin was well aware that if the supernatural implications of disjunct distributions could not be refuted, then his evolutionary system — founded upon a species concept based on descent — would be in peril.⁹⁴ A further barrier to the acceptance of the Prichard/Lyell view was the fact that those sympathetic to a nonsupernatural explanation of disjunct distributions could not agree upon a natural explanation for those anomalies, and an internal debate between naturalists within this group raged for decades.⁹⁵ By 1859 a biogeographical stalemate had occurred. Sclater and others, supporting their static view of nature, continued to look for regions of creation, pointing to disjunct distributions in support of their arguments, while those favorable to the views of Prichard and Lyell continued to search for natural explanations for such biogeographical anomalies.The key needed to resolve the biogeographical debate was a credible theory for species origins. By 1858 there were essentially three options for British naturalists: supernatural creation, Lamarckian transmutation, or natural creation. A few British naturalists grasped at these straws, but most workers preferred the option of remaining silent until a more viable explanation for the origin and distribution of species could be advanced.⁹⁶ And not until the publication of Darwin's theory did that explanation become available.     

E-Cadherin Engagement Stimulates Tyrosine Phosphorylation

Cadherins are cell adhesion molecules concentrated at intercellular adherens junctions, where they form a multiprotein complex with cytoplasmic catenins. Although cell-cell interactions affect many aspects of cell behavior, little is known about signaling pathways triggered by cadherin engagement. We show here that E-cadherin-mediated cell-cell adhesion leads to a rapid increase in tyrosine phosphorylation at sites of cell-cell contact and that this stimulation of tyrosine phosphorylation can be mimicked by aggregation of E-cadherin with antibodies. The proteins that become phosphorylated are distinct from those previously shown to be tyrosine phosphorylated in response to integrin-mediated adhesion and include ras-GAP. We also find that E-cadherin-mediated tyrosine phosphorylation is not required for the assembly of adherens-type junctions.     

Two levels of resting potential in cardiac purkinje fibers

In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was “N-shaped,” and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride.