A novel serine/proline-rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts

AtTic40 is part of the chloroplastic protein import apparatus that is anchored in the inner envelope membrane by a single N-terminal transmembrane domain, and has a topology in which the bulk of the C-terminal domain is oriented toward the stroma. The targeting of AtTic40 to the inner envelope membrane involves two steps. Using an in vitro import assay, we showed that the sorting of AtTic40 requires a bipartite transit peptide, which was first cleaved by the stromal processing peptidase (SPP), thus generating a soluble AtTic40 stromal intermediate (iAtTic40). iAtTic40 was further processed by a second unknown peptidase, which generates its mature form (mAtTic40). Using deletion mutants, we identified a sequence motif N-terminal of the transmembrane domain that was essential for reinsertion of iAtTic40 into the inner envelope membrane. We have designated this region a serine/proline-rich (S/P-rich) domain and present a model describing its role in the targeting of AtTic40 to the inner envelope membrane.     

Unique glycine-activated riboswitch linked to glycine–serine auxotrophy in SAR11

The genome sequence of the marine bacterium ' Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that ' Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 μM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 × 10⁶ cells ml⁻¹ to 8.16 × 10⁶ cells ml⁻¹ ( R ² =  0.992). Serine was capable of substituting for glycine at 1.5 μM. ' Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that ' Cand. P. ubique' is therefore a glycine–serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.     

Detection of Mycoplasma pneumoniae in simulated and true clinical throat swab specimens by nanorod array-surface-enhanced Raman spectroscopy

The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%-100% specificity and 94-100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application.     

Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase

The regulation of arachidonic acid conversion by the 5-lipoxygenase and the cyclooxygenase pathways in mouse peritoneal macrophages has been studied using particulate and soluble agonists. Particulate agonists, zymosan and latex, stimulated the production of cyclooxygenase metabolites as well as the 5-lipoxygenase product, leukotriene C4. In contrast, incubation with the soluble agonist phorbol myristate acetate or exogenous arachidonic acid led to the production of cyclooxygenase metabolites but not leukotriene C4. We tested the hypothesis that the 5-lipoxygenase, unlike the cyclooxygenase, requires activation by calcium before arachidonic acid can be utilized as a substrate. Addition of phorbol myristate acetate to macrophages in the presence of calcium ionophore (A23187) at a concentration which alone did not stimulate arachidonate metabolism resulted in a synergistic increase (50-fold) in leukotriene C4 synthesis compared to phorbol ester or A23187 alone. No such effect on the cyclooxygenase pathway metabolism was observed. Exogenous arachidonic acid in the presence of A23187 produced similar results yielding a 10-fold greater synthesis of leukotriene C4 over either substance alone without any effects on the cyclooxygenase metabolites. Presumably, calcium ionophore unmasked the synthesis of leukotriene C4 from phorbol myristate acetate-released and exogenous arachidonate by elevating intracellular calcium levels enough for 5-lipoxygenase activation. These data indicate that once arachidonic acid is released from phospholipid by an agonist, it is available for conversion by both enzymatic pathways. However, leukotriene synthesis may not occur unless intracellular calcium levels are elevated either by phagocytosis of particulate agonists or with calcium ionophore.     

Evidence for an intracellular pool of a migration inhibitory factor-associated, activation antigen of human mononuclear phagocytes, Mo3e

Mo3e is a protease-sensitive Ag (p75,50) selectively expressed by human monocytic cells stimulated in vitro by exposure to various activating factors including PMA. Here, we report the existence of a large intracellular pool of Mo3e Ag in addition to that expressed on the surface of activated U-937 cells. As detected by quantitative immunofluorescence analysis, permeabilization of unstimulated and PMA-stimulated U-937 cells revealed a latent pool of Mo3e Ag that was 75-fold and 9-fold greater, respectively, than the magnitude of Mo3e Ag expressed on the surface. PMA stimulation not only induced an increase in the relative proportion of Mo3e antigen expressed on the surface membrane, but also stimulated a 1.8-fold increase in "total" Mo3e detectable in permeabilized cells. Trypsin treatment of intact PMA-stimulated U-937 cells eliminated surface Mo3e expression but had little measureable effect on the total Mo3e pool. Permeabilization also uncovered a sequestered compartment of Mo3e Ag in I-937 cells, a variant of U-937 that is surface Mo3e negative. Although the PMA-induced surface Mo3e expression of U-937 was abrogated by cycloheximide, the total pool of Mo3e detectable in permeabilized PMA-stimulated cells was only partially reduced; cycloheximide treatment caused no reduction in the intracellular Mo3e compartment of unstimulated U-937 cells. Detergent lysates of PMA-stimulated U-937 cells exhibited undiminished quantities (relative to untrypsinized cells) of p75 and p50 proteins immunoreactive with anti-Mo3e mAb as detected by Western blotting. This trypsin-sequestered intracellular Mo3e Ag may serve as a reservoir for the up-regulated surface expression of Mo3e that occurs as a result of mononuclear phagocyte activation.     

Influence of silver,mercury, lead,cadmium, and selenium on glutathione peroxidase and transferase activities in rats

At the levels used in the experiments, mercury and silver significantly depressed the activity of glutathione peroxidase (assayed with either H₂O₂ or cumene-OOH) in rat tissues, whereas cadmium or lead had no effect on this activity. The most pronounced effects of mercury and silver on glutathione peroxidase were found in the liver and kidneys, with much less effect in the testes and erythrocytes. Similar trends for the effects of these metals were noted for tissue selenium levels. Silver and mercury significantly depressed the selenium concentrations, but cadmium and lead had no effect upon the selenium levels. Mercury and silver had no effect upon the activity of glutathione transferase in liver and testes, but mercury caused a significant initial increase of its activity in the kidneys. At no time did silver have any significant effect on its activity in this organ.     

Influence of prostaglandins (PG) E2 and F2α on the inflammatory process

PGF_2^α, but not PGE₂, induces a slight pedal edema when given alone. Both compounds were equipotent in the carrageenin-induced rat paw edema. Locally administered, PGE₂ and PGF_2^α did not exacerbate, but rather inhibited inflammations induced by various agents such as 1% carrageenin or 1% egg white. The administration of PGE₂ directly into cotton pellets or into the rat's hind paw in combination with M. butyricum significantly inhibited, respectively, granuloma formation and the polyarthritis. Subcutaneously, both prostaglandins inhibited the adjuvant induced polyarthritis. Neither PGE₂ nor PGF_2^α inhibited the anti-edema properties of non-steroidal or steroidal anti-inflammatory standards. A greater anti-edema activity was observed with the combination treatment than with the anti-inflammatory standards alone. We were unable to decrease the anti-inflammatory activity of the steroidal and non-steroidal standards or increase the inflammatory potential of the phlogistic agents.     

Cytotoxic T-Lymphocyte Precursor Frequencies in BALB/c Mice after Acute Respiratory Syncytial Virus (RSV) Infection or Immunization with a Formalin-Inactivated RSV Vaccine

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.Respiratory syncytial virus (RSV) infection in infants and young children often results in lower respiratory tract disease and is a high priority for vaccine development (1, 2). Attempts to develop an effective live, inactivated, or subunit vaccine have been unsuccessful (24, 25, 28). Early efforts at vaccinating young children with a formalin-inactivated RSV (FI-RSV) vaccine failed to protect the children from naturally acquired infection and actually enhanced lower respiratory tract disease upon later virus infection (2, 15, 24, 25). This enhanced disease has created concern about the safety of any nonlive RSV vaccine and, consequently, understanding the pathogenesis of FI-RSV-induced enhanced disease is critically important to vaccine development. Studies with BALB/c mice suggest that induction of memory T cells producing Th2-like cytokines, as a result of FI-RSV vaccination, may be key to the pathogenesis of enhanced disease (6, 16, 28, 32, 40). Th2-like cytokine mRNA has been demonstrated in cells from lung tissue or bronchoalveolar lavage (BAL) specimens after RSV challenge of FI-RSV-immunized mice (17, 32, 40). In addition, in vivo studies using antibody (Ab) blockade showed that the enhanced histopathology in FI-RSV-immune mice challenged with live virus could be eliminated by using anti-interleukin-4 (IL-4) and anti-IL-10 Abs but not anti-IL-12 Abs (6). Recent evidence suggests that CD8⁺ T lymphocytes may be important in directing the type of inflammatory response to RSV in challenge of G glycoprotein-sensitized mice (21, 31).One aspect of the FI-RSV immune response that has not been well characterized is the cytotoxic T-lymphocyte (CTL) response. There is limited information on major histocompatibility complex (MHC) class I-restricted CTLs after FI-RSV immunization (29), while the information about the CTL response after live-RSV infection has been well documented. Several studies have shown class I-restricted CTLs to kill predominantly target cells expressing the M, N, or F RSV protein (5, 7, 9, 26, 29, 41). The role of CTLs in the immune response to RSV is well illustrated by in vivo depletion studies with BALB/c mice (8, 18, 30). These studies suggest that both CD4⁺ (class II) and CD8⁺ lymphocytes are important for clearing RSV and that both contribute to the inflammatory response associated with infection. A vaccinia virus construct expressing RSV membrane-associated, nonglycosylated protein M2 has been affiliated with short-term protection in the BALB/c mouse (7). This protein does not induce neutralizing Abs, and therefore, protection likely is mediated by CTLs. Passive transfer of CD8⁺ T lymphocytes has been associated with both clearance of the virus and enhanced histopathology (1).In this report, we describe studies of CTL precursor (CTLp) frequencies in both live-RSV-infected and FI-RSV-immunized mice for MHC class I- and class II-restricted target cells. These studies demonstrate clear differences in the CTLp response between RSV and FI-RSV immunizations and provide additional approaches to identifying potential FI-RSV-induced enhanced disease mechanisms.     

IS FLORAL SPECIALIZATION AN EVOLUTIONARY DEAD‐END? POLLINATION SYSTEM TRANSITIONS IN RUELLIA (ACANTHACEAE)

Pollination systems frequently reflect adaptations to particular groups of pollinators. Such systems are indicative of evolutionary specialization and have been important in angiosperm diversification. We studied the evolution of pollination systems in the large genus Ruellia. Phylogenetic analyses, morphological ordinations, ancestral state reconstructions, and a character mapping simulation were conducted to reveal key patterns in the direction and lability of floral characters associated with pollination. We found significant floral morphological differences among species that were generally associated with different groups of floral visitors. Floral evolution has been highly labile and also directional. Some specialized systems such as hawkmoth or bat pollination are likely evolutionary dead-ends. In contrast, specialized pollination by hummingbirds is clearly not a dead-end. We found evidence for multiple reverse transitions from presumed ancestral hummingbird pollination to more derived bee or insect pollination. These repeated origins of insect pollination from hummingbird-pollinated ancestors have not evolved without historical baggage. Flowers of insect-pollinated species derived from hummingbird-pollinated ancestors are morphologically more similar to hummingbird flowers than they are to other more distantly related insect-pollinated flowers. Finally, some pollinator switches were concomitant with changes in floral morphology that are associated with those pollinators. These observations are consistent with the hypothesis that some transitions have been adaptive in the evolution of Ruellia.     

Survey of antibodies to Trypanosoma cruzi and Leishmania spp. in gray and red fox populations from North Carolina and Virginia

American trypanosomiasis and leishmaniasis are caused by related hemoflagellate parasites, Trypanosoma cruzi and Leishmania spp., which share several common host species. Both zoonotic protozoans are endemic in the United States. Canines, including domestic and wild canids, are reservoir hosts for human infections with T. cruzi and Leishmania spp. The present study examined the seroprevalence of T. cruzi and Leishmania spp. in wild canids from North Carolina and Virginia. Wild canine species tested in this work included 49 gray foxes (Urocyon cinereoargenteus) and 5 red foxes (Vulpes vulpes). Overall, sera samples from 54 foxes (North Carolina = 43; Virginia = 11) were tested by immunochromatographic strip assays (ICT). Antibodies to T. cruzi were found in 4 (9%) gray foxes from North Carolina and 2 (18%) gray foxes from Virginia. Antibodies to Leishmania spp. were detected in 1 (2%) gray fox from North Carolina. Our results indicate that wild canids are exposed more frequently to T. cruzi in North Carolina than Leishmania spp. and only T. cruzi in Virginia.