Bacteriovorax stolpii proliferation and predation without sphingophosphonolipids

Bacteriovorax stolpii strain UKi2, a facultative predator-parasite of larger Gram-negative bacteria, synthesizes distinct sphingophosphonolipids. These lipids are characterized by a direct P-C bond, the novel head group 1-hydroxy-2-aminoethylphosphonate, iso-branched long chain bases and fatty acids, and fatty acids dominated by those with α-hydroxy groups. Myriocin, an inhibitor of serine:fatty acyl CoA transferase, reversibly blocked sphingophosphonolipid synthesis in B. stolpii UKi2. However, the inhibitor did not block cell proliferation indicating that these lipids are not vital for B. stolpii UKi2 viability and growth. When mixed with Escherichia coli prey cells, control predator-parasite bacteria were effective in forming large E. coli bdelloplasts and cleared the suspension of the prey cells. Although myriocin-treated cells could attack prey cells and form bdelloplasts, their locomotory behavior was altered and fewer and smaller bdelloplasts were produced. These observations open up the possibility for a role of sphingophosphonolipids in B. stolpii UKi2 complex behavior.     

Effects of hydrophobic helix length and side chain chemistry on biomimicry in peptoid analogues of SP-C

The hydrophobic proteins of lung surfactant (LS), SP-B and SP-C, are critical constituents of an effective surfactant replacement therapy for the treatment of respiratory distress syndrome. Because of concerns and difficulties associated with animal-derived surfactants, recent investigations have focused on the creation of synthetic analogues of the LS proteins. However, creating an accurate mimic of SP-C that retains its biophysical surface activity is extraordinarily challenging given the lipopeptide's extreme hydrophobicity and propensity to misfold and aggregate. One successful approach that overcomes these difficulties is the use of poly-N-substituted glycines, or peptoids, to mimic SP-C. To develop a non-natural, bioactive mimic of SP-C and to investigate the effects of side chain chemistry and length of the helical hydrophobic region, we synthesized, purified, and performed in vitro testing of two classes of peptoid SP-C mimics: those having a rigid alpha-chiral aromatic helix and those having a biomimetic alpha-chiral aliphatic helix. The length of the two classes of mimics was also systematically altered. Circular dichroism spectroscopy gave evidence that all of the peptoid-based mimics studied here emulated SP-C's secondary structure, forming stable helical structures in solution. Langmuir-Wilhelmy surface balance, fluorescence microscopy, and pulsating bubble surfactometry experiments provide evidence that the aromatic-based SP-C peptoid mimics, in conjunction with a synthetic lipid mixture, have superior surface activity and biomimetic film morphology in comparison to the aliphatic-based mimics and that there is an increase in surface activity corresponding to increasing helical length.     

Short-term temporal dynamics of yeast abundance on the tall fescue phylloplane

Six replicate trials were conducted to determine the short-term temporal dynamics and the effects of foliar applications of nutrients on the phylloplane yeast community of tall fescue (Festuca arundinacea Schreb.). In each trial, 2% sucrose + 0.5% yeast extract solution or sterile deionized water (control) was applied to the experiment plots. Twelve hours post-treatment (at 0600 hours), leaf samples were collected and yeast colony-forming units (cfu) were enumerated by dilution plating. This process was repeated at 1200, 1800, and 2400 hours in each trial. Significant differences were observed between the number of yeast cfu and the time at which the samples were collected. On average, the number of yeast cfu recovered was significantly less at 1800 hours and significantly greatest at 2400 hours when compared with all other sampling times. Averaged over all time intervals, we observed a trend of increased yeast abundance in turf treated with the nutrient solution compared with control treatments. In a separate investigation, atmospheric yeast abundance above the canopy of tall fescue was assessed in the morning (0900) and in the afternoon (1500) using a Thermo Andersen single stage viable particle sampler. In 5 of the 6 trials of this experiment, atmospheric yeast abundance was significantly greater in the morning than in the afternoon. Results suggest the following colonization model: phylloplane yeasts on tall fescue reproduce during the late evening and early morning, stabilize during the late morning and early afternoon through exchange of immigrants and emigrants, and decline during the late afternoon and (or) early evening.     

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.     

Systematic comparison of gene expression between murine memory and naive B cells demonstrates that memory B cells have unique signaling capabilities

Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naive precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of Ag-specific memory and naive cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. We identified genes with differential expression and confirmed the differential expression of many of these by quantitative RT-PCR and of some of these at the protein level. Our initial analysis revealed differential expression patterns of genes that regulate signaling. Memory B cells have increased expression of genes important in regulating adenosine signaling and in modulating cAMP responses. Furthermore, memory B cells up-regulate receptors that are essential for embryonic stem cell self-renewal. We further demonstrate that one of these, leukemia inhibitory factor receptor, can initiate functional signaling in memory B cells whereas it does not in naive B cells. Thus, memory and naive B cells are intrinsically wired to signal differently from one another and express a functional signaling pathway that is known to maintain stem cells in other lineages.     

Unique receptor repertoire in mouse uterine NK cells

Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3(-)CD122(+) NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.     

Molecular Characterization of a Variant of Bacillus anthracis-Specific Phage AP50 with Improved Bacteriolytic Activity

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.     

A role for Acp29AB, a predicted seminal fluid lectin, in female sperm storage in Drosophila melanogaster

Females of many animal species store sperm for taxon-specific periods of time, ranging from a few hours to years. Female sperm storage has important reproductive and evolutionary consequences, yet relatively little is known of its molecular basis. Here, we report the isolation of a loss-of-function mutation of the Drosophila melanogaster Acp29AB gene, which encodes a seminal fluid protein that is transferred from males to females during mating. Using this mutant, we show that Acp29AB is required for the normal maintenance of sperm in storage. Consistent with this role, Acp29AB localizes to female sperm storage organs following mating, although it does not appear to associate tightly with sperm. Acp29AB is a predicted lectin, suggesting that sugar–protein interactions may be important for D. melanogaster sperm storage, much as they are in many mammals. Previous association studies have found an effect of Acp29AB genotype on a male's sperm competitive ability; our findings suggest that effects on sperm storage may underlie these differences in sperm competition. Moreover, Acp29AB's effects on sperm storage and sperm competition may explain previously documented evidence for positive selection on the Acp29AB locus.     

Examination of the cellular mechanisms by which marinobufagenin inhibits cytotrophoblast function

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in the inhibition of Na(+)/K(+)-ATPase. Increased plasma levels have been reported in patients with volume expansion-related hypertension. We have recently demonstrated that MBG impairs first trimester cytotrophoblast (CTB) cell proliferation, migration, and invasion, which may play a role in the development of preeclampsia. However, whether apoptosis contributes to altered CTB cell function by MBG remains unknown. Using the human extravillous CTB cell line SGHPL-4, we examined the effect of MBG and a similar Na(+)/K(+)-ATPase inhibitor, ouabain, on the phosphorylation status of Jnk, p38, and Src. Additionally, we measured apoptosis by caspase 9 and 3/7 activity and by annexin-V staining. We also investigated interleukin-6 (IL-6) secretion with or without p38 and Jnk inhibition. MBG significantly increased the phosphorylation of Jnk, p38, and Src and increased the expression of caspase 9 and 3/7 indicating the activation of apoptosis. MBG treatment also stimulated the expression of the early apoptosis marker, annexin-V, which was prevented by Jnk and p38 inhibition. MBG also stimulated the secretion of IL-6, which was attenuated by p38 inhibition. Ouabain had similar effects to those of MBG, suggesting that the apoptotic effects on CTB cells may be mediated by inhibition of Na(+)/K(+)-ATPase. In conclusion, the MBG-induced impairment of CTB function occurs via activation of Jnk, p38, and Src leading to increased apoptosis and IL-6 secretion. These observations may have clinical applicability with respect to the therapy of preeclampsia.