3D TOCSY-HSQC NMR for Metabolic Flux Analysis Using Non-Uniform Sampling.

(13)C-Metabolic Flux Analysis ((13)C-MFA) is rapidly being recognized as the authoritative method for determining fluxes through metabolic networks. Site-specific (13)C enrichment information obtained using NMR spectroscopy is a valuable input for (13)C-MFA experiments. Chemical shift overlaps in the 1D or 2D NMR experiments typically used for (13)C-MFA frequently hinder assignment and quantitation of site-specific (13)C enrichment. Here we propose the use of a 3D TOCSY-HSQC experiment for (13)C-MFA. We employ Non-Uniform Sampling (NUS) to reduce the acquisition time of the experiment to a few hours, making it practical for use in (13)C-MFA experiments. Our data show that the NUS experiment is linear and quantitative. Identification of metabolites in complex mixtures, such as a biomass hydrolysate, is simplified by virtue of the (13)C chemical shift obtained in the experiment. In addition, the experiment reports (13)C-labeling information that reveals the position specific labeling of subsets of isotopomers. The information provided by this technique will enable more accurate estimation of metabolic fluxes in large metabolic networks.

Hydrogenase activity of mineral-associated and suspended populations of Desulfovibrio desulfuricans Essex 6.

The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell-mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl2 and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.

Role of outer membrane c-type cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell production, accumulation, and detachment during respiration on hematite.

The iron-reducing bacterium Shewanella oneidensis MR-1 has the capacity to contribute to iron cycling over the long term by respiring on crystalline iron oxides such as hematite when poorly crystalline phases are depleted. The ability of outer membrane cytochromes OmcA and MtrC of MR-1 to bind to and transfer electrons to hematite has led to the suggestion that they function as terminal reductases when this mineral is used as a respiratory substrate. Differences in their redox behavior and hematite-binding properties, however, indicate that they play different roles in the electron transfer reaction. Here, we investigated how these differences in cytochrome behavior with respect to hematite affected biofilm development when the mineral served as terminal electron acceptor (TEA). Upon attachment to hematite, cells of the wild-type (WT) strain as well as those of a ΔomcA mutant but not those of a ΔmtrC mutant replicated and accumulated on the mineral surface. The results indicate that MtrC but not OmcA is required for growth when this mineral serves as TEA. While an OmcA deficiency did not impede cell replication and accumulation on hematite prior to achievement of a maximum surface cell density comparable to that established by WT cells, OmcA was required for efficient electron transfer and cell attachment to hematite once maximum surface cell density was achieved. OmcA may therefore play a role in overcoming barriers to electron transfer and cell attachment to hematite imposed by reductive dissolution of the mineral surface from cell respiration associated with achievement of high surface cell densities.

Antibiosis activity of Pantoea agglomerans biocontrol strain E325 against Erwinia amylovora on apple flower stigmas.

Pantoea agglomerans E325, the active ingredient in a commercial product for fire blight control, was previously shown in vitro to produce a unique alkaline- and phosphate-sensitive antibiotic specific to Erwinia amylovora. Antibiosis was evaluated as a mode of antagonism on flower stigmas using two antibiosis-deficient mutants. On King's medium B, mutants E325ad1 and E325ad2 have stable smooth-butyrous or hypermucoid colony morphologies, respectively, and the parental strain E325 exhibits phenotypic plasticity with predominantly hypermucoid colonies accompanied by slower-growing, smooth-butyrous colonies. Mutants were tested against E. amylovora on stigmas of detached flowers of crab apple (Malus mandshurica) in growth chambers and apple (Malus domestica) in the orchard. Epiphytic fitness of the antibiosis-negative mutants was similar or greater than the parental strain as determined by relative area under the population curve (RAUPC). In laboratory and orchard trials, both mutants had significantly lower inhibitory activity against the pathogen (i.e., less reduction of E. amylovora RAUPC) compared with the parental strain. E325 and the mutants caused similar decreases in pH in a broth medium, indicating that acidification, which was previously reported as a possible mechanism of pathogen inhibition on stigmas, is not directly related to antibiosis. In this study we provide the first evidence for E325 antibiosis involved in E. amylovora growth suppression on apple flower stigmas.

Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR-1.

In an effort to improve the understanding of electron transfer mechanisms at the microbe-mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer-membrane cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe(3)(PO(4))(2)x8H(2)O] and a switzerite-like phase [Mn(3),Fe(3)(PO(4))(2)x7H(2)O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.

Effects of dog ownership in early childhood on immune development and atopic diseases.

BACKGROUND: Exposure to pets in childhood has been associated with a reduced risk of wheezing and atopy. OBJECTIVE: Our objective was to determine whether the effects of pet exposure on immune development and atopy in early childhood can be explained by alterations in exposure to innate immune stimuli in settled dust. METHODS: Two hundred and seventy-five children at increased risk of developing allergic diseases were evaluated to age 3 years for pet ownership, blood cell cytokine responses, and atopy. Can f 1, Fel d 1, endotoxin, ergosterol, and muramic acid were measured in settled dust from 101 homes. RESULTS: Dog exposure at birth was associated with decreased atopic dermatitis (AD) (12% vs. 27%; P=0.004) and wheezing (19% vs. 36%; P=0.005) in year 3. The rates of AD (23%) and wheezing (42%) in year 3 were relatively high in children who acquired dogs after birth. The prevalence of dog sensitization (10-12%) did not vary according to dog exposure. Can f 1 levels in bedroom dust were positively associated with IL-10 (r=0.26; P=0.01), IL-5 (r=0.34, P<0.001), and IL-13 (r=0.28; P=0.004) responses at age 1, and IL-5 (r=0.24; P=0.022) and IL-13 (r=0.25; P=0.015) responses at age 3. In contrast, endotoxin was associated with IFN-gamma (r=0.31; P=0.002) and IL-13 (r=0.27; P=0.01) responses at age 3 but not at age 1, and similar relationships were present for muramic acid. Adjustment for levels of innate immune stimuli in house dust did not significantly affect the relationships between Can f 1 and cytokine responses. CONCLUSIONS: Exposure to dogs in infancy, and especially around the time of birth, is associated with changes in immune development and reductions in wheezing and atopy. These findings are not explained by exposure to endotoxin, ergosterol, or muramic acid.

What are subcutaneous adipocytes really good for?

Our acute awareness of the cosmetic, psychosocial and sexual importance of subcutaneous adipose tissue contrasts dramatically with how poorly we have understood the biology of this massive, enigmatic, often ignored and much-abused skin compartment. Therefore, it is timely to recall the exciting, steadily growing, yet underappreciated body of evidence that subcutaneous adipocytes are so much more than just 'fat guys', hanging around passively to conspire, at most, against your desperate attempts to maintain ideal weight. Although the subcutis, quantitatively, tends to represent the dominant architectural component of human skin, conventional wisdom confines its biological key functions to those of energy storage, physical buffer, thermoregulation and thermoinsulation. However, already the distribution of human superficial adipose tissue, by itself, questions how justified the popular belief is that 'skin fat' (which actually may be more diverse than often assumed) serves primarily thermoinsulatory purposes. And although the metabolic complications of obesity are well appreciated, our understanding of how exactly subcutaneous adipocytes contribute to extracutaneous disease - and even influence important immune and brain functions! - is far from complete. The increasing insights recently won into subcutaneous adipose tissue as a cytokine depot that regulates innate immunity and cell growth exemplarily serve to illustrate the vast open research expanses that remain to be fully explored in the subcutis. The following public debate carries you from the evolutionary origins and the key functional purposes of adipose tissue, via adipose-derived stem cells and adipokines straight to the neuroendocrine, immunomodulatory and central nervous effects of signals that originate in the subcutis - perhaps, the most underestimated tissue of the human body. The editors are confident that, at the end, you shall agree: No basic scientist and no doctor with a serious interest in skin, and hardly anyone else in the life sciences, can afford to ignore the subcutaneous adipocyte - beyond its ample impact on beauty, benessence and body mass.

Activation of murine epidermal V gamma 5/V delta 1-TCR(+) T cell lines by Glu-Tyr polypeptides.

The physiologic role of gamma delta-T-cell-receptor-bearing cells and the T cell receptor ligands that they recognize is still poorly understood. Previous studies have suggested that one possible antigen for gamma delta-TCR(+) cells is the random copolymer poly-glutamic acid-tyrosine (poly-Glu-Tyr), because poly-Glu-Tyr-reactive gamma delta-TCR(+) hybridoma cells were produced from poly-Glu-Tyr-immunized mice. We have found, however, that clonal V gamma 5/V delta 1-TCR(+) epidermal T cell lines from nonimmune mice also respond to poly-Glu-Tyr by producing cytokines. Other amino acid homopolymers, copolymers, and tripolymers were not stimulatory for the V gamma 5/V delta 1-TCR(+) epidermal T cells, except for poly-glutamic acid-alanine-tyrosine (poly-Glu-Ala-Tyr). Of the poly-Glu-Tyr and poly-Glu-Ala-Tyr polymers, only those that contained Glu and Tyr in an equimolar ratio were stimulatory. The cytokine interleukin-2 was strictly required for the responses to poly-Glu-Ala-Tyr, whereas the responses to poly-Glu-Tyr were merely enhanced with interleukin-2. The response to poly-Glu-Tyr was also enhanced by crosslinking CD28 molecules with plate-bound anti-CD28 crosslinking antibody. This finding suggests that the poly-Glu-Tyr response has a partial dependence on CD28-mediated costimulation, a characteristic of TCR-dependent responses. Consistent with this observation, V gamma 5/V delta 1-TCR-loss variants of the epidermal T cell line could no longer respond to poly-Glu-Tyr. The unpredicted responses of epidermal gamma delta-TCR(+) T cells to poly-Glu-Tyr and poly-Glu-Ala-Tyr demonstrate that the functions of these cells potentially can be triggered by peptidic ligands, probably through a TCR-mediated process.

Response of murine gamma delta T cells to the synthetic polypeptide poly-Glu50Tyr50.

Random heterocopolymers of glutamic acid and tyrosine (pEY) evoke strong, genetically controlled immune responses in certain mouse strains. We found that pE50Y50 also stimulated polyclonal proliferation of normal gamma delta, but not alpha beta, T cells. Proliferation of gamma delta T cells did not require prior immunization with this Ag nor the presence of alpha beta T cells, but was enhanced by IL-2. The gamma delta T cell response proceeded in the absence of accessory cells, MHC class II, beta 2-microglobulin, or TAP-1, suggesting that Ag presentation by MHC class I/II molecules and peptide processing are not required. Among normal splenocytes, as with gamma delta T cell hybridomas, the response was strongest with V gamma 1+ gamma delta T cells, and in comparison with related polypeptides, pE50Y50 provided the strongest stimulus for these cells. TCR gene transfer into a TCR-deficient alpha beta T cell showed that besides the TCR, no other components unique to gamma delta T cells are needed. Furthermore, interactions between only the T cells and pE50Y50 were sufficient to bring about the response. Thus, pE50Y50 elicited a response distinct from those of T cells to processed/presented peptides or superantigens, consistent with a mechanism of Ig-like ligand recognition of gamma delta T cells. Direct stimulation by ligands resembling pE50Y50 may thus selectively evoke contributions of gamma delta T cells to the host response.

Murine epidermal V gamma 5/V delta 1-T-cell receptor+ T cells respond to B-cell lines and lipopolysaccharides.

The V gamma 5/V delta 1(+)-T-cell receptor (TCR)-bearing T-cell clone, 2CBET-3, was generated from C57BL/6 mice. Upon stimulation, 2CBET-3 cells produce interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, but not IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, macrophage colony-stimulating factor, or interferon-gamma. These cells were evaluated for their ability to be stimulated by a variety of murine cell lines, including fibroblasts, trophoblasts, melanoma cells, embryonic carcinomas, B-cell lymphomas, mastocytoma cells, and keratinocytes. The human B-lymphoma cell line, Daudi, also was included in these studies. We found that 2CBET-3 cells produced cytokines up to several hundredfold above the control levels in response to the B-cell lines, Daudi, and A20/2J, but not to the B-cell line 439.4.2. After fixation with glutaraldehyde, Daudi and A20/2J continued to stimulate this gamma delta T-cell line. 2CBET-3 cells also responded to the keratinocyte line PAM212, but not to another, XB-2. When lipopolysaccharides (LPS) from Escherichia coli or S. typhimurium were added to 2CBET-3 cells in the presence of A20/2J cells, 2CBET-3 cells responded with increased cytokine production compared with the cytokine production in the presence of A20/2J cells alone. 2CBET-3 cells by themselves did not respond to LPS alone or to supernatants from A20/2J cells incubated with LPS. Unlike 2CBET-3, the epidermal T-cell hybridoma 70BET-49, expressing a V gamma 5/V delta 1-TCR identical to that of 2CBET-3, did not respond to A20/2J cells in the presence or absence of LPS, suggesting a requirement for molecules other than the TCR for V gamma 5/V delta 1-TCR+ T-cell stimulation by the B-cell lines and by LPS. This unique reactivity of gamma delta-TCR+ cells is different from that of alpha beta-TCR+ cells and may reflect a functional specialization of gamma delta-TCR+ cells in the response to bacterial infections.

Response of a murine epidermal V gamma 1/V delta 6-TCR+ hybridoma to heat shock protein HSP-60.

In the epidermis, the major population of T lymphocytes expresses a T-cell receptor (TCR) with V gamma 5 and V delta 1 variable regions, which is unique to this tissue. Roberts et al and Ezquerra et al, also describe a minor population of gamma delta-TCR+ cells in the epidermis that expresses a V gamma 1/V delta 6 TCR. These cells are different from other epidermal T cells in that they "spontaneously" produce cytokines, a result thought to be due to autoreactivity. Over the past 5 years, our laboratory has produced V gamma 1/V delta 6+ T-cell hybridomas from many tissue sources. These spontaneously produce cytokines but also are activated by heat shock protein (HSP-60)-derived peptides. Ezquerra et al report that none of their V gamma 1/V delta 6+ epidermal T-cell lines derived from C3H/HeN mice respond to HSP-60. Of the 99 gamma delta-TCR+ hybridomas we have produced from epidermal T cells of C57BL/6 mice, only one expressed the V gamma 1/V delta 6 TCR. This hybridoma, 70BET-2.12, not only spontaneously produces cytokines, but, unlike the V gamma 1/V delta 6-TCR+ epidermal T cells of Ezquerra et al, it also responds to the whole HSP-60 protein and a 17-mer HSP-60 peptide from M. leprae, producing increased levels of interleukin-2 of up to approximately ten-fold above the spontaneous level. This shows that V gamma 1/V delta 6-TCR+ epidermal T cells can respond to HSP-60. To confirm that 70BET-2.12 expresses TCR genes similar to those of cells that have HSP-60 reactivity, V gamma 1-C gamma 4 and V delta 6-C delta cDNA were produced from RNA isolated from this hybridoma, amplified by the polymerase chain reaction, and sequenced. The gamma and delta TCR gene sequences were similar but not identical to previously published sequences of HSP-60-reactive cells from lymphoid and other organs. No explanation can be found for the discrepancy between our findings and those of others at the level of TCR expression such that other strain-specific factors might be responsible for HSP-60 reactivity.

Heavy-metal mitogenesis: Zn++ and Hg++ induce cellular cytotoxicity and interferon production in murine T lymphocytes.

Splenic and lymph node lymphocytes from Balb/c mice were activated in vitro by the heavy-metal cations, Zn++ and Hg++, as noted by the several-fold increases in 3H-thymidine incorporation at 144 h of culture. Optimal mitogenic concentrations of Zn++ and Hg++ were 200 microM and 10 microM, respectively. Data from experiments using T or B splenic lymphocytes enriched by cell passage over nylon wool columns, through use of athymic Nu/Nu mouse spleen cells, or by cell lysis with monoclonal anti-Thy-1 and antibody plus complement, indicated than Zn++ and Hg++ are mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron, followed by cell passage through nylon wool, eliminated the lymphocyte responses to Zn++ and to Hg++. Moreover, addition of macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn++ and Hg++ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce acid-labile interferon.

Heavy-metal mitogenesis: thymocyte activation by Zn++ requires 2-mercaptoethanol and lipopolysaccharide as cofactors.

Thymocytes from Balb/C mice were activated in vitro by Zn++ as noted by the several-fold increases in 3H-thymidine incorporation at 144 h of culture. Thymocyte activation by Zn++ required the presence of 2-mercaptoethanol (2-ME) and bacterial lipopolysaccharide (LPS) in concentrations as low as 1.0 ng/ml. Thymocytes were not stimulated by these agents in the absence of Zn++. Bovine serum products, thought to contain trace amounts of LPS, appeared to satisfy this LPS requirement. Interleukin 1 (IL 1) could not replace LPS as a cofactor. Thymocytes did not respond to Hg++ under the culture conditions used here. Thymocyte subpopulation studies showed that cell preparations enriched for peanut lectin receptor-negative, mature thymocytes were activated by Zn++ and required LPS for the response.