The hmu locus of Yersinia pestis is essential for utilization of free haemin and haem-protein complexes as iron sources

Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by trans-duction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enter-ocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.     

Correlation between limitation of growth ofPachysolen tannophilus on D-xylose with the formation of ethanol and other products

The formation of ethanol, xylitol, ribitol, arabitol and acetic acid from D-xylose byPachysolen tannophilus correlated with the limitation of growth. The correlation was consistent with these products being secondary metabolites.Issued as NRCC Publication Number 24259.     

Predator preference for brightly colored males in the guppy: a viability cost for a sexually selected trait

Although conspicuous visual sexual signals, such as bright colors,in males serve to attract females in numerous species, theymay also attract the attention of potential predators and thusmay be costly in terms of increasing individual risk of mortalityto predation. Most models of the evolution of extravagant malesexual traits and female preferences for them assume that thesexually preferred male trait is costly to produce and maintain.However, there is surprisingly little empirical evidence fordirect fitness costs associated with sexually selected visualtraits that enhance male mating success. In the present study,we report a direct fitness cost for sexually selected, brightbody-color patterns in males in the form of an associated greaterrisk of mortality to predation. By using the guppy (Poeciliareticulata) and the blue acara cichlid fish (Aequidens pulcher)as a model prey–predator system, we demonstrate experimentallythat individual cichlids preferentially and consistently approached,attacked, and captured the more brightly colored of two size-matchedmale guppies presented simultaneously in staged encounters.This resulted in the brightly colored male incurring, on average,a significantly higher risk of mortality given an encounterwith the predator than with the drabber male in matched pairs.Our results constitute strong behavioral evidence for a directviability cost associated with bright coloration in male guppies,and they corroborate the generally accepted paradigm that directionalpredation by visual fish predators against brightly colored,adult male guppies underlies the evolution of the known divergentcolor patterns in natural guppy populations that experiencedifferent intensities of predation. The viability cost associatedwith bright conspicuous coloration in male guppies potentiallyreinforces for females the reliability of this sexually selectedtrait as an indicator trait of male quality.     

Comparative morphometrics of the primate apical tuft

The relationship between the structure and function of the primate apical tuft is poorly understood. This study addresses several hypotheses about apical tuft morphology using a large modern primate comparative sample. Two indices of tuft size are employed: expansion and robusticity. First, comparisons of relative apical tuft size were drawn among extant nonhuman primate groups in terms of locomotion and phylogenetic category. Both of these factors appear to play a role in apical tuft size among nonhuman primates. Suspensory primates and all platyrrhines had the smallest apical tufts, while terrestrial quadrupeds and all strepsirrhines (regardless of locomotor category) had the largest tufts. Similarly, hypotheses regarding the apical tufts of hominins, especially the large tufts of Neandertals were addressed using a comparison of modern warm- and cold-adapted humans. The results showed that cold-adapted populations possessed smaller apical tufts than did warm-adapted groups. Therefore, the cold-adaptation hypothesis for Neandertal distal phalangeal morphology is not supported. Also, early modern and Early Upper Paleolithic humans had apical tufts that were significantly less expanded and less robust than those of Neandertals. The hypothesis that a large apical tuft serves as support for an expanded digital pulp is supported by radiographic analysis of modern humans in that a significant correlation was discovered between the width of the apical tuft and the width of the pulp. The implications of these findings for hypotheses about the association of apical tuft size and tool making in the hominin fossil record are discussed.     

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.     

Optimization of the Water-Insoluble Procedures for <Emphasis Type="Italic">USP</Emphasis> General Chapter <Emphasis Type="Italic">Residual Solvents</Emphasis> <467>

The water-insoluble procedures in US Pharmacopeia (USP) General Chapter Residual Solvents <467>, which are based on European Pharmacopoeia procedures, were optimized and modified before their inclusion in the chapter to improve their scope, performance, and ruggedness. The optimized procedures use a static headspace introduction system with a gas chromatograph equipped with a flame ionization detector. This article describes some of the key changes made to the USP published procedures, including use of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) as the solvent, addition of 5 mL of water and 1 mL of sample (dissolved in DMSO or DMF) to the headspace vial, use of a 3:1 GC split ratio, and use of new matrix-matched system suitability solutions. These procedures were verified with two different active pharmaceutical ingredients—hydroxyzine pamoate and prednisone. In the investigation, the more polar material (hydroxyzine pamoate) showed greater recoveries for the optimized procedures when prepared in DMSO. The less polar material (prednisone) typically had greater recoveries in DMF for the optimized procedures. During experimentation, insights into sample preparation, additional types of headspace instrumentation, solvent purity, and other parameters were also gained.     

A tetrameric allyl complex of sodium, and computational modeling of the Na-allyl chemical shift

Transmetallation of Li[A′] (A′ = [1,3-(SiMe₃)₂C₃H₃]⁻) with sodium tert-butoxide produces the corresponding sodium salt, which crystallizes from THF/toluene in the form of a cyclic tetramer, {Na[A′](thf)}₄. The Na atoms are in a square planar arrangement, bridged with π-bound allyl ligands; the Na-C distances range from 2.591(3)-2.896(3) Å, with an average of 2.70 Å. The geometries of several model organosodium complexes containing cyclopentadienyl and allyl ligands were optimized with density functional theory methods. The optimized structures were used with the gauge-including atomic orbital (GIAO) method to calculate their ²³Na NMR magnetic shielding values. Unlike the case with NaCp, the chemical shift of unsubstituted Na(C₃H₅) is very sensitive to the presence of coordinated THF (causing a 20 ppm upfield shift); silyl substitution has an even larger effect (30 ppm upfield shift). The observed ²³Na shift of δ −3.3 ppm for Na[A′] in THF-d₈, however, cannot be reliably distinguished from that calculated for the [Na(thf)₄]⁺ cation alone.     

Cytochrome c-d regulates developmental apoptosis in the Drosophila retina

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.     

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.     

Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health

Reports that elasmobranchs (sharks, skates, and rays) may havea low incidence of disease have stimulated interest in understandingthe role of their immune system in this apparent resistance.Although research in this area may potentially translate intoapplications for human health, a basic understanding of theelasmobranch immune system components and how they functionis essential. As in higher vertebrates, elasmobranch fishespossess thymus and spleen, but in the absence of bone marrowand lymph nodes, these fish have evolved unique lymphomyeloidtissues, namely epigonal and Leydig organs. As conditions forshort-term culture of elasmobranch immune cells have becomebetter understood, the opportunity to examine functional activityof cytokine-like factors derived from conditioned culture mediumhas resulted in the identification of growth inhibitory activityagainst a variety of tumor cell lines. Specifically, the mediumenriched by short term culture of bonnethead shark (Sphyrnatiburo) epigonal cells (epigonal conditioned medium, ECM) hasbeen shown to inhibit the growth of mammalian tumor cell lines,including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-celllymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer(PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinomacell lines (MCF7, HCC38, Hs578T). Of the cell lines tested,WEHI-164, A375.S2, Daudi, and Jurkat cells were among the mostsensitive to growth inhibitory activity of ECM whereas PANC-1and NIH:OVCAR-3 cells were among the least sensitive. In addition,ECM demonstrated preferential growth inhibition of malignantcells in assays against two different malignant/non-malignantcell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separationof protein components of ECM using SDS-PAGE resulted in a veryreproducible pattern of three major bands corresponding to molecularsizes of approximately 40–42 kD, 24 kD, and 17 kD. Activityis lost after heating at 75°C for 30 min, and can be diminishedby treatment with proteinase K and protease. Activity is notaffected by treating with trypsin, DNase I or RNase A.