Using alternative forage species to reduce emissions of the greenhouse gas nitrous oxide from cattle urine deposited onto soil.

Grazed pastures are a major contributor to emissions of the greenhouse gas nitrous oxide (N2O), and urine deposition from grazing animals is the main source of the emissions. Incorporating alternative forages into grazing systems could be an approach for reducing N2O emissions through mechanisms such as release of biological nitrification inhibitors from roots and increased root depth. Field plot and lysimeter (intact soil column) trials were conducted in a free draining Horotiu silt loam soil to test whether two alternative forage species, plantain (Plantago lanceolate L.) and lucerne (Medicago sativa L.), could reduce N2O emissions relative to traditional pasture species, white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.). The amounts of N2O emitted from the soil below each forage species, which all received the same cow urine at the same rates, was measured using an established static chamber method. Total N2O emissions from the plantain, lucerne and perennial ryegrass controls (without urine application) were generally very low, but emissions from the white clover control were significantly higher. When urine was applied in autumn or winter N2O emissions from plantain were lower compared with those from perennial ryegrass or white clover, but this difference was not found when urine was applied in summer. Lucerne had lower emissions in winter but not in other seasons. Incorporation of plantain into grazed pasture could be an approach to reduce N2O emissions. However, further work is required to understand the mechanisms for the reduced emissions and the effects of environmental conditions in different seasons.

A role for Rac1 activity in malignant progression of sebaceous skin tumors.

The small GTPase Rac1 is crucial for maintaining stem cells (SCs) in mammalian epidermis, and Rac1 activation leads to SC expansion. Loss or inhibition of Rac1 correlates with decreased frequency of skin cancer formation in a chemical carcinogenesis model. Here, we have addressed whether Rac1 activation would enhance carcinogenesis and result in tumor progression. We used K14ΔNLef1 mice, a model for differentiated sebaceous adenomas (SAs), and activated Rac1 in an epidermis-specific manner (K14L61Rac1). Surprisingly, Rac1 activation did not change the incidence and frequency of sebaceous tumors. However, tumors, which occurred exclusively in K14ΔNLef1/K14L61Rac1 double-transgenic mice, were poorly differentiated resembling malignant sebaceous tumors and were termed sebaceous carcinoma-like tumors (SCLTs). Compared with SAs, SCLTs showed an aberrant pattern of cell proliferation, invasive growth and less abundant expression of sebocyte differentiation markers, including stearoyl-CoA desaturase-1 and adipophilin. Interestingly, the adnexal SC marker Lrig1 was upregulated in SCLTs, showing that active Rac1 leads to the accumulation of sebocyte precursors in the context of K14ΔNLef1-induced skin tumors. In a search for targets of Rac1, we found cancer progression-related proteins, Dhcr24/Seladin1 and Nuclear protein 1/P8, to be strongly regulated in SCLTs. At last, Rac1 and Dhcr24/Seladin1 were detected in human sebaceous tumors demonstrating a potential high impact of our findings for human skin disease. This is the first study showing that Rac1 activity can lead to malignant progression of skin tumors.

Losses at chromosome 4q are associated with poor survival in operable ductal pancreatic adenocarcinoma.

Here we tested the prognostic impact of genomic alterations in operable localized pancreatic ductal adenocarcinoma (PDAC). Fifty-two formalin-fixed and paraffin-embedded primary PDAC were laser micro-dissected and were investigated by comparative genomic hybridization after whole genome amplification using an adapter-linker PCR. Chromosomal gains and losses were correlated to clinico-pathological parameters and clinical follow-up data. The most frequent aberration was loss on chromosome 17p (65%) while the most frequent gains were detected at 2q (41%) and 8q (41%), respectively. The concomitant occurrence of losses at 9p and 17p was found to be statistically significant. Higher rates of chromosomal losses were associated with a more advanced primary tumor stage and losses at 9p and 18q were significantly associated with presence of lymphatic metastasis (chi-square: p = 0.03, p = 0.05, respectively). Deletions on chromosome 4 were of prognostic significance for overall survival and tumor recurrence (Cox-multivariate analysis: p = 0.026 and p = 0.021, respectively). In conclusion our data suggest the common alterations at chromosome 8q, 9p, 17p and 18q as well as the prognostic relevant deletions on chromosome 4q as relevant for PDAC progression. Our comprehensive data from 52 PDAC should provide a basis for future studies with a higher resolution to discover the relevant genes located within the chromosomal aberrations identified.

[From single disseminated tumor cells to metastasis insights from molecular genetic analyses of single cells]. / Von der einzelnen disseminierten Tumorzelle zur Metastase: Erkenntnisse aus der molekulargenetischen Einzelzellanalyse.

The prevailing models of cancer metastasis postulate that, after a series of accumulating genetic and epigenetic changes during transformation and invasive growth, the most advanced clone within a primary tumour acquires the critical cellular phenotype enabling dissemination and metastasis. This postulate is particularly based on observations that metastases usually display more genetic changes than the primary tumour. The development of several novel techniques is now enabling experimental testing of the model: The detection of single disseminated cancer cells by epithelial specific antibodies directed against cytokeratin in mesenchymal tissues has become possible even before manifestation of metastasis. After their isolation by micromanipulation comprehensive amplification of the single cell genomes allows the application of several molecular genetic methods for further characterisation. Our first data from single cytokeratin-positive cells that were isolated from the bone marrow of breast cancer patients indicate that the model of cancer progression should be revised. Cancer cells already disseminate in an early stage of genomic development and still have to acquire the critical chromosomal aberrations needed for metastatic outgrowth and full malignant potential. These observations apparently imply consequences for the development of novel adjuvant therapies. The early diversification of primary tumours and disseminated cancer cells precludes a simple extrapolation from local to systemic disease eventually necessitating increasing efforts for the direct analysis of single disseminated cancer cells as the cellular targets of adjuvant therapies.

Seven-fluorochrome mouse M-FISH for high-resolution analysis of interchromosomal rearrangements.

The mouse has evolved to be the primary mammalian genetic model organism. Important applications include the modeling of human cancer and cloning experiments. In both settings, a detailed analysis of the mouse genome is essential. Multicolor karyotyping technologies have emerged to be invaluable tools for the identification of mouse chromosomes and for the deciphering of complex rearrangements. With the increasing use of these multicolor technologies resolution limits are critical. However, the traditionally used probe sets, which employ 5 different fluorochromes, have significant limitations. Here, we introduce an improved labeling strategy. Using 7 fluorochromes we increased the sensitivity for the detection of small interchromosomal rearrangements (700 kb or less) to virtually 100%. Our approach should be important to unravel small interchromosomal rearrangements in mouse models for DNA repair defects and chromosomal instability.

[Direct molecular analysis of single disseminated cancer cells: a prerequisite for the development of adjuvant therapies?]. / Die direkte molekulare Analyse einzelner disseminierter Tumorzellen: Eine Voraussetzung für die Entwicklung adjuvanter Therapien?

Cancer mortality has only marginally decreased in the last decades despite huge diagnostic and therapeutic efforts. Early dissemination of cancer cells has to be blamed for this finding, because ectopically residing cells are necessarily left behind by the surgeon. Consequently, this cell population has been designated as minimal residual disease that may eventually lead to systemic relapse months or years after presumed curative surgery. Paradoxically, systemic adjuvant treatments are currently administered without any precise knowledge about the target population of such drugs. Here it is argued that the direct analysis of disseminated cells may be a prerequiste for the development of future therapies since first molecular genetic data of single disseminated cancer cells suggest an excessive intercellular heterogeneity and distant relationship to the primary tumor.

Early cancer cell dissemination and late metastatic relapse: clinical reflections and biological approaches to the dormancy problem in patients.

Two clinical entities, unknown-primary cancer and inadvertent transmission of cancer with organ transplants are reviewed and discussed in the context of early and occult tumor cell dissemination. Both entities are taken as chief witnesses for cell dissemination being an early event in tumor progression. The involuntary transmission of tumor by organ grafts directly supports the notion that very few quiescent cells lodging at improbable sites such as kidney or heart suffice to generate de novo metastatic disease in the organ recipient. As to the nature of the cells and their biological and clinical significance a short review is given on the detection of disseminated cells in bone marrow and their prognostic significance for a metastatic relapse in patients with resected primary tumors. A novel single-cell genomic analysis is described, that allows the detection of multiple chromosomal aberration in single tumor cells.

The biology and analysis of single disseminated tumour cells.

Despite an increasing molecular-genetic understanding of the development of malignant epithelial neoplasias, the frontline therapy for patients with carcinomas is still surgery. Systemic adjuvant treatments such as chemotherapy or immunotherapy have had limited success perhaps because they are based on analysis of the primary tumour or on cell lines derived from metastasis. However, the characteristics of systemically disseminated tumour cells can be very different from that of the primary tumour or end-stage metastasis. Consequently, there is a need to study the evolution and nature of systemic cancer directly in order to identify new target structures for therapy present on the potential precursors of metastasis--the disseminated tumour cells.

Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.

A PCR strategy is described for global amplification of DNA from a single eukaryotic cell that enables the comprehensive analysis of the whole genome. By comparative genomic hybridization, not only gross DNA copy number variations, such as monosomic X and trisomic 21 in single male cells and cells from Down's syndrome patients, respectively, but multiple deletions and amplifications characteristic for human tumor cells are reliably retrieved. As a model of heterogeneous cell populations exposed to selective pressure, we have studied single micrometastatic cells isolated from bone marrow of cancer patients. The observed congruent pattern of comparative genomic hybridization data, loss of heterozygosity, and mutations as detected by sequencing attests to the technique's fidelity and demonstrates its usefulness for assessing clonal evolution of genetic variants in complex populations.

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

TNF-alpha and cyclooxygenase metabolites do not modulate C. albicans septic shock with disseminated candidiasis.

We analyzed differences in host regulation of tumor necrosis factor-alpha (TNF-alpha) production and pathophysiological responses in conscious rats after infection with two strains of pathogenic Candida albicans spp. (CA-1 and CA-2) compared with Escherichia coli serotype 055:B5 (EC). The hypothesis was tested that, in contrast to EC, hypotension, organ injury, and mortality after candidemia are not obligatorily dependent on TNF-alpha or TNF-alpha-induced cyclooxygenase pathway metabolites. Dose, viability, and strain-specific dependencies were established after intravenous 10(6) or 10(9) viable CA, as well as heat-killed (HK) or Formalin-inactivated (FI) CA blastospores, compared with live EC at the 24-h LD25 [10(9) colony-forming units (CFU)] and LD100 (10(10) CFU). Shock without endotoxemia developed 4-8 h after 10(9) live CA-1 or CA-2 (LD100 at 24 h) with disseminated yeast-mycelial transformation and increased microvascular permeability in multiple organs but not after HK or FI CA-1. Peak serum TNF-alpha after an LD100 of CA-1 or CA-2 was < 3% of LD25 EC values and was < 1% of peak values during lethal bacteremia. Similar pathogen-specific differences were found in liver- and lung-associated TNF. Production of functionally inactive TNF-alpha during candidemia was excluded by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. Passive immunization against TNF-alpha 2 h before microbial challenge was not protective against CA but prevented otherwise lethal EC sepsis. Cyclooxygenase inhibition also failed to attenuate candidemic shock. We conclude that the magnitude and kinetics of TNF-alpha production and TNF-alpha-dependent immunophysiological responses are differentially regulated after lethal fungal vs. gram-negative bacterial infection. Thus TNF-alpha is not a pivotal mediator of the acute Candida septic shock syndrome with disseminated candidiasis.

Differential systemic and intrapulmonary TNF-alpha production in Candida sepsis during immunosuppression.

Candida albicans (CA) increasingly causes septic shock, acute lung injury, and multiple organ damage during immunosuppression-related neutropenia. However, the effects of neutrophil (PMN) depletion on induction of tumor necrosis factor-alpha (TNF) by CA and its potential mediation of Candida septic shock are unknown. We hypothesized that reduced CA uptake by circulating PMNs during cyclophosphamide (CY)-related neutropenia sensitizes to TNF-mediated shock from enhanced cytokine production after phagocytosis by tissue macrophages. Absolute or relative neutropenia (PMNs < or = 500/microliters or 2,500/microliters) was modeled in rats by intraperitoneal CY 4-8 days before 10(9) yeast-phase CA (acute studies < or = 24 h, n = 81 animals) or 10(6) CA (subacute studies < or = 72 h, n = 25). Compared with neutrophil-sufficient rats, absolute neutropenia accelerated hemodynamic collapse and respiratory distress after 10(9) CA, and pulmonary microvascular permeability was amplified. These changes evolved without increased candidemia or elevations in bioactive or antigenic serum TNF, which remained low even at death (42.3 +/- 14.8 U/ml vs. 12.6 +/- 2.9 U/ml for CY + saline, means +/- SE, P = NS). In contrast, significant TNF in lung tissue and bronchoalveolar lavage fluid (BALF) was evident within 6 h in CY + 10(9) CA rats. Electron microscopy confirmed hyphal proliferation into alveoli from yeast within mononuclear cells in lung capillaries. Subacute disseminated candidiasis after 10(6) CA was not associated with elevated serum, lung, or BALF TNF. We conclude that differential systemic and intrapulmonary TNF production occur in CA septic shock during preexisting neutropenia, with compartmentalized TNF production in the lower respiratory tract accompanying yeast-mycelial transformation. Thus TNF is not an obligate mediator of acute candidemic shock or subacute disseminated candidiasis during CY-induced immunosuppression but may initiate pulmonary injury accompanying high-grade candidemia.

Intraerythrocytic parasitosis in humans with Entopolypoides species (family Babesiidae). Association with hepatic dysfunction and serum factors inhibiting lymphocyte response to phytohemagglutinin.

We have observed two cases of human infection with intraerythrocytic protozoa. The organisms appeared to be in the Entopolypoides group, which had not previously been associated with human infection. One patient was asplenic. Both patients had hepatic dysfunction, and their serum samples contained blocking factors that interfered in vitro with the stimulation of normal lymphocytes by phytohemagglutinin. It appears that in humans, as well as in experimental animals, host factors are important in resistance to infection by intraerythrocytic parasites. These factors include the presence of a spleen and cell-mediated and humoral immunities. Possibly similar infections will be observed in patients with other impairments of T-cell function, such as those induced by malignancy, thymic dysfunction, or immunosuppressive drugs.