Evaluation of CuO oxidation parameters for determining the source and stage of lignin degradation in soil

The composition of phenols and other aromatic compounds in organic and mineral soil horizons and their respective source vegetation from different climatic zones of the Canadian Prairies were analyzed using CuO oxidation and gas chromatography-mass spectrometry (GC-MS) to investigate the stage of lignin degradation. Parameters based on the CuO oxidation products were calculated for the soils and corresponding vegetation to determine the lignin sources and to monitor the lignin degradation. In addition to the widely used lignin monomer parameters, parameters resulting from lignin-derived phenolic dimers are used for the first time to assess lignin degradation in soils. The composition of lignin-derived phenols (S/V, C/V) in soil closely matches the composition observed in their respective source plants (grass, Aspen, Pine) reflecting the preservation of characteristic lignin patterns in soils. Degradation parameters based on lignin phenols and benzenes derived from tannins or other phenolic biomolecules indicate a progressive degradation from the vegetation to the soil horizons. In addition to commonly used lignin monomer indicators, parameters based on the lignin dimers are applied. Lignin degradation is found to be lowest in the Pine forest, intermediate in the grassland soils and highest in the Aspen-grassland transition soil. Degradation parameters based on non-lignin aromatic derivatives (3,5-dihydroxybenzoic acid, benzenepolycarboxylic acids) demonstrate a similar trend. The lignin from samples in the cooler climate (Black Chernozems) is observed to be more oxidized than in the soils from the warmer climate (Brown Chernozems) suggesting that abiotic processes may be in involved in the alteration of lignin and other phenolic biomolecules in soils. The results indicate that the comparative analysis of CuO oxidation products of soils and source vegetation is a valuable tool to assess the sources and degradation of lignin in soils.     

Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Effects of growth hormone overexpression and growth hormone resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice

Transgenic mice overexpressing growth hormone (GH) exhibit alterations in the function of the hypothalamic-pituitary-gonadal (HPG) axis and the H-P-adrenal axis. Alterations in the turnover of hypothalamic neurotransmitters, in plasma hormone levels, and in regulation of their release are associated with reproductive deficits, particularly in females. Results reported after publication of our minireview on this subject provided evidence that GH-transgenic mice have increased binding of GH to GH binding proteins in plasma, are hyperinsulinemic and insulin resistant, and have major alterations in energy budgets with increased allocation to growth. Reduced life span and fertility of these animals may be related to insufficient allocation of energy to reproduction and maintenance. Growth hormone resistance induced by transgenic expression of an antagonistic bGH analog or by targeted disruption (knock-out, KO) of the GH receptor (GH-R) gene leads to dramatic suppression of plasma levels of insulin-like growth factor-1 (IGF-1), and dwarf phenotype due to reduced growth and increased adiposity. In both models of GH resistance, there are marked reproductive deficits in females, decline of breeding performance of males, and alterations in the function of the HPG axis. In GH-R-KO females, puberty is delayed, and litter size is reduced. Fetal weights are reduced whereas placental weights are increased, and the weight of newborn pups is reduced despite an increase in the length of gestation. In GH-R-KO males, copulatory behavior and fertility are reduced, plasma PRL is elevated, and responses to luteinizing hormone releasing hormone (LHRH) in vivo and to LH in vitro are suppressed. However, reproductive deficits in GH-R-KO mice are very mild when compared to those described previously in IGF-KO animals. Apparently, the amounts of IGF-1 that may be produced locally in the absence of GH stimulation are sufficient for sexual maturation and fertility in both sexes, whereas quantitative deficits in reproductive function reflect absence of GH-dependent IGF-1 production and other consequences of eliminating GH signaling. The reproduction phenotype of the GH-R-KO mice is also mild when compared to dwarf mice that lack GH, prolactin (PRL), and thyroid stimulating hormone (TSH). This is presumably related to the presence of redundant mechanisms in the stimulatory control of the gonads by the pituitary and the ability of animals capable of producing PRL and TSH to compensate partially for the absence of GH signaling.     

Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.     

The Cytochrome b5 Tail Anchors and Stabilizes Subdomains of Human DNA Topoisomerase IIα in the Cytoplasm of Retrovirally Infected Mammalian Cells

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIα are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIα and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b₅ anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1–266) is weakly and diffusely expressed, addition of the cytb₅ anchor (1–266-b₅) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1–426). A C-terminal domain (aa 1030–1504) of human topo IIα containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb₅ anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.