MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake

The Staphylococcus aureus DtxR-like protein, MntR, controls expression of the mntABC and mntH genes, which encode putative manganese transporters. Mutation of mntABC produced a growth defect in metal-depleted medium and increased sensitivity to intracellularly generated superoxide radicals. These phenotypes resulted from diminished uptake of manganese and were rescued by the addition of excess Mn(II). Resistance to superoxide was incompletely rescued by Mn(II) for STE035 (mntA mntH), and the strain had reduced virulence in a murine abscess model of infection. Expression of mntABC was repressed by Mn(II) in an MntR-dependent manner, which contrasts with the expression of mntH that was not repressed in elevated Mn(II) and was decreased in an mntR mutant. This demonstrates that MntR acts as a negative and positive regulator of these loci respectively. PerR, the peroxide resistance regulon repressor, acts with MntR to control the expression of mntABC and manganese uptake. The expression of the PerR-regulated genes, katA (catalase), ftn (ferritin) and fur (ferric uptake regulator), was diminished in STE031 (mntR) when grown in excess Mn(II). Therefore, the control of Mn(II)-regulated members of the PerR regulon and the Fur protein is modulated by MntR through its control of Mn(II) uptake. The co-ordinated regulation of metal ion homeostasis and oxidative stress resistance via the regulators MntR, PerR and Fur of S. aureus is discussed.     

Detection of conformational changes in Complex III of the respiratory chain by a maleimido spin label

Changes in the conformation of Complex III (CoQH₂-cytochromec reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochromec. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-³H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.     

Molecular diagnostics of economically important Ceratitis fruit fly species (Diptera: Tephritidae) in Africa using PCR and RFLP analyses

The predominantly Afrotropical fruit fly genus Ceratitis contains many species of agricultural importance. Consequently, quarantine of Ceratitis species is a major concern for governmental regulatory agencies. Although diagnostic keys exist for identification of all described Ceratitis species, these tools are based on adult characters. Flies intercepted at ports of entry are usually immatures, and Ceratitis species cannot be diagnosed based on larval morphology. To facilitate identification of Ceratitis pests at ports of entry, this study explores the utility of DNA-based diagnostic tools for a select group of Ceratitis species and related tephritids, some of which infest agriculturally important crops in Africa. The application of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyse three mitochondrial genes (12S ribosomal RNA, 16S ribosomal RNA, and NADH-dehydrogenase subunit 6) is sufficient to diagnose 25 species and two species clusters. PCR analysis of the internal transcribed spacer region 1 (ITS-1) is able to distinguish three of the five species left unresolved by mitochondrial DNA analysis.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Ascaris sp.: water loss during desiccation of embryonating eggs

The ability of embryonating eggs of Ascaris lumbricoides to avoid desiccation by reducing the loss of water through the egg shell was investigated. When exposed to desiccation the eggs lost water at a rate dependent upon the relative humidity and ambient temperature, eventually resulting in the collapse of the eggs and the death of the enclosed embryo. The eggs are small with a large surface to volume ratio. A low permeability to gaseous exchange thus restricts water loss while still ensuring an adequate supply of oxygen for embryonic development. Relative humidity did not appear to affect the rate of development. In eggs exposed to desiccation at various constant temperatures, the rate of water loss increased as an exponential function of increasing temperature. When eggs were exposed to various temperatures before exposure to desiccation at 22 C, the rate of water loss increased as a function of increasing pretreatment temperature. After exposure to 63–65 C, the ability of the egg shell to slow down the loss of water was destroyed. These phenomena suggest that there is not a simple “critical” or “transition” temperature, but a gradual melting of the complex mixture of components forming the lipid layer.     

Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.     

Autoradiographic discrimination of brain and atrial natriuretic peptide-binding sites in the rat kidney.

Brain (BNP) and atrial natriuretic peptides (ANP) have been identified which may represent endogenous agonists of kidney receptor subtypes. Quantitative in vitro autoradiography was used to investigate the regional distribution of receptor subpopulations and the competitive inhibition of 125I porcine BNP1-26 (pBNP1-26) and 125I rat alpha-ANP1-28 (rANP1-28) renal binding sites. Specific, high affinity binding (Kd 0.2-1.37 nM range) was localized to glomeruli, inner medulla, interlobar and arcuate arteries, vasa recta bundles, and smooth muscle in the renal pelvis. pBNP1-26 competed for the same sites as rANP1-28 but displayed a lower potency and was less selective for nonclearance sites. Clearance binding sites were discriminated by competitive inhibition with C-ANP4-23 and comprised some 65% of glomerular sites as well as the vast majority of sites in the renal pelvis. Nonclearance sites predominated in the inner medulla and intrarenal arteries. C-terminal changes in amino acid sequence induced a significant loss of inhibitory potency. Immunohistochemical studies identified a distinct population of BNP-like immunoreactive renal nerve fibers, associated with intra-renal arteries. Circulating natriuretic peptides and BNP sequences derived from renal nerves may influence renal function by interacting with specific receptor subpopulations in the kidney.     

Chlorophylla and adenosine triphosphate levels in Antarctic and temperate lake sediments

Analysis of adenosine triphosphate (ATP) from surficial sediment layers in two antarctic lakes and two temperate lakes showed a high degree of similarity in spite of differences between trophic state, mictic state, or geographic location. Adenosine triphosphate was found at all levels sampled in temperate lake sediment cores but occasionally was present only in surficial layers of antarctic cores. Surficial sediment layers from antarctic lakes contained high chlorophylla (Chla) levels due to the extensive benthic algal mats which occur there. In some antarctic cores, Chla was detectable in deep, old mat layers, whereas Chla was not found in any of the temperate lake cores. Antarctic lake sediments appear to be unique environments where Chla molecules can remain intact for long periods of time due to low light, temperature, and microbial activity. As such, these lakes are important natural laboratories where a long history of microbial interactions can be studied without metazoan perturbation effects. Although there was much variability in concentration of Chla and ATP between samples, there appears to be no relationship between Chla or ATP levels to mictic or trophic states of the lakes. These data suggest that sediment microbial communities may be independent of environmental and biological properties of the overlying water masses.     

PHYLOGENY OF THE SUBFAMILIES OF THE FAMILY BRACONIDAE (HYMENOPTERA: ICHNEUMONOIDEA): A REASSESSMENT

Abstract— The recently published phylogeny of Braconidae by Quicke and van Achterberg is reassessed. Character-state definitions and character polarities are evaluated, and more rigorous methods are suggested. Our results indicate that there are many more parsimonious solutions to their data set, the consensus of which differs substantially from their results. Based on our reassessment, little can be said about the relationships among braconid subfamilies. Consensus trees show the cyclostomes as a largely unresolved basal grade. The two other major lineages which have been proposed, the helconoids and microgastroids, are somewhat better resolved, but not consistently so. Relationships among the helconoids vary considerably depending on the parameters used for parsimony analysis.