Phylogeography of microbial phototrophs in the dry valleys of the high Himalayas and Antarctica.

High-elevation valleys in dry areas of the Himalayas are among the most extreme, yet least explored environments on Earth. These barren, rocky valleys are subjected to year-round temperature fluctuations across the freezing point and very low availability of water and nutrients, causing previous workers to hypothesize that no photoautotrophic life (primary producers) exists in these locations. However, there has been no work using modern biogeochemical or culture-independent molecular methods to test the hypothesis that photoautotrophs are absent from high Himalayan soil systems. Here, we show that although microbial biomass levels are as low as those of the Dry Valleys of Antarctica, there are abundant microbial photoautotrophs, displaying unexpected phylogenetic diversity, in barren soils from just below the permanent ice line of the central Himalayas. Furthermore, we discovered that one of the dominant algal clades from the high Himalayas also contains the dominant algae in culture-independent surveys of both soil and ice samples from the Dry Valleys of Antarctica, revealing an unexpected link between these environmentally similar but geographically very distant systems. Phylogenetic and biogeographic analyses demonstrated that although this algal clade is globally distributed to other high-altitude and high-latitude soils, it shows significant genetic isolation by geographical distance patterns, indicating local adaptation and perhaps speciation in each region. Our results are the first to demonstrate the remarkable similarities of microbial life of arid soils of Antarctica and the high Himalayas. Our findings are a starting point for future comparative studies of the dry valleys of the Himalayas and Antarctica that will yield new insights into the cold and dry limits to life on Earth.

Evidence that chytrids dominate fungal communities in high-elevation soils.

Periglacial soils are one of the least studied ecosystems on Earth, yet they are widespread and are increasing in area due to retreat of glaciers worldwide. Soils in these environments are cold and during the brief summer are exposed to high levels of UV radiation and dramatic fluctuations in moisture and temperature. Recent research suggests that these environments harbor immense microbial diversity. Here we use sequencing of environmental DNA, culturing of isolates, and analysis of environmental variables to show that members of the Chytridiomycota (chytrids) dominate fungal biodiversity and perhaps decomposition processes in plant-free, high-elevation soils from the highest mountain ranges on Earth. The zoosporic reproduction of chytrids requires free water, yet we found that chytrids constituted over 70% of the ribosomal gene sequences of clone libraries from barren soils of the Himalayas and Rockies; by contrast, they are rare in other soil environments. Very few chytrids have been cultured, although we were successful at culturing chytrids from high-elevation sites throughout the world. In a more focused study of our sites in Colorado, we show that carbon sources that support chytrid growth (eolian deposited pollen and microbial phototrophs) are abundant and that soils are saturated with water for several months under the snow, thus creating ideal conditions for the development of a chytrid-dominated ecosystem. Our work broadens the known biodiversity of the Chytridomycota, and describes previously unsuspected links between aquatic and terrestrial ecosystems in alpine regions.

Interferon-gamma, but not interferon-alpha beta, synergizes with tumor necrosis factor-alpha and lipid A in the induction of nitric oxide production by murine L929 cells.

Recently, we have demonstrated that tumor necrosis factor (TNF)-sensitive tumor cells produce nitric oxide (NO) in response to TNF whereas TNF-resistant cells do not. Because the addition of interferon-gamma (IFN-gamma) augmented NO production, we were interested in investigating this phenomenon further and comparing the effects of IFN-gamma with those of IFN-alpha beta. We found that cell lines that are sensitive to TNF-mediated cytotoxicity (TMC) produced NO in response to TNF and IFN-gamma, but not in response to IFN-alpha beta. The effect of IFN-gamma on NO production was dose dependent, but IFN-gamma by itself did not induce NO production. A TNF-resistant cell line (MCA) did not produce NO under any of the conditions tested. Different results were obtained when the effect of IFNs on TMC was assayed. TNF-sensitive L929 cells were rendered less sensitive to TNF after treatment with both types of IFN. In contrast, another TNF-sensitive cell, WEHI 164, was rendered more sensitive to TMC after treatment with both types of IFN. The effect of IFNs on WEHI cells was dose dependent. Neither IFN had any effect on TNF sensitivity of TNF-resistant MCA cells. The addition of lipid A (LA) had no effect on TMC under any condition. However, L929 cells treated with LA, TNF, and IFN-gamma produced twice as much NO as cells treated with TNF and IFN-gamma only. Northern analysis for cytokine-inducible NO synthase (NOS) mRNA steady-state levels indicated that TNF synergized with IFN-gamma to induce increased accumulation of NOS mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin A inhibits nitric oxide production by L929 cells in response to tumor necrosis factor and interferon-gamma.

We recently reported that tumor necrosis factor (TNF) induced the production of nitric oxide (NO) by TNF-sensitive, but not-resistant, tumor cells. Paradoxically, NO thus produced does not appear to be involved in the mechanism of TNF-mediated cytotoxicity as inhibitors of NO production and NO scavengers did not block cytotoxicity. Because the immunosuppressive drug cyclosporin A (CsA) inhibits several types of immune-mediated killing, we were interested in what effect CsA would have on TNF-mediated cytotoxicity as well as NO production. Treatment with CsA had no effect on the sensitivity L929 cells to TNF-mediated cytotoxicity, either in the presence or absence of interferon-gamma (IFN-gamma). In the presence of IFN-gamma alone, L929 cells were slightly less sensitive to the cytotoxic effects of TNF. In contrast to the effect on TNF-mediated cytotoxicity, CsA treatment had a profound effect on the ability of these cells to produce NO in response to TNF and IFN-gamma. Cells treated with CsA produced 75% less NO than did their untreated controls. Inhibition of calmodulin-dependent calcineurin-like phosphatases is one mechanism by which CsA may exert its effects. Therefore, we tested the effect of EGTA, which inhibits calcineurin by chelating calcium, on NO production and found that EGTA treatment resulted in a 15% decrease in the amount of NO produced. In addition, cells treated with the calmodulin antagonist W-13 produced 79% less NO than their untreated controls. Therefore, these results provide further evidence that NO produced by TNF-sensitive cells is not involved in the mechanism of TNF-mediated cytotoxicity because reduction of NO production by CsA has no effect on TNF-mediated killing of these same cells.

Nitric oxide production by tumor targets in response to TNF: paradoxical correlation with susceptibility to TNF-mediated cytotoxicity without direct involvement in the cytotoxic mechanism.

Tumor necrosis factor (TNF) is selectively cytotoxic for some tumor cells in vivo and in vitro. We determined whether TNF-mediated cytotoxicity for TNF-sensitive tumor targets was related to TNF-stimulated production of NO by the tumor cell itself. We found that a cell line that was sensitive to TNF-mediated cytotoxicity produced NO in response to TNF as measured by the accumulation of nitrite in the supernatants of TNF-stimulated cells. Production of NO in response to TNF was inhibited by the competitive substrate inhibitor, NG-monomethyl-L-arginine (NMMA). The kinetics of NO production in response to TNF indicated that most of the NO was produced during the first 24 h and peaked after 48 h of culture and that TNF-stimulated NO production was dose dependent. TNF-resistant cell lines produced less NO than a TNF-sensitive cell line, and the amount of nitrite produced correlated with the relative sensitivity of each cell line to TNF-mediated cytotoxicity. In addition, recombinant interferon-gamma augmented the amount of NO produced in response to TNF by both sensitive and resistant cells and correspondingly enhanced the susceptibility of resistant cells to TNF cytotoxicity. Both sensitive and resistant cells were sensitive to NO, however, in that NO generated exogenously by culture in the presence of sodium nitroprusside was cytotoxic for both sensitive and resistant cells in a dose-dependent manner. We were unable, however, to demonstrate directly a role for NO in TNF-mediated cytotoxicity as NMMA- and arginine-free media provided little protection from TNF-mediated cytotoxicity. We tentatively conclude that the ability of adherent murine tumor cells to produce nitric oxide in response to TNF correlates directly with their level of sensitivity to TNF-mediated cytotoxicity, although NO thus produced appears not to be directly involved in the cytotoxic mechanism.

Malignant fibrous histiocytoma.

Two patients are presented who had malignant fibrous histiocytomas of the skin and soft tissue. These tumors characteristically consist of a solitary mass, 0.5 cm to 12.0 cm, which has skin fixation, is circumscribed and firm. Microscopically, there is a pattern of histiocytes and bizarre histiocytic giant cells intermingled in a fibrous stroma with fibroblasts arranged in a whirling pattern. The lesion is treated with wide excision and skin grafting. Local recurrence is common. Metastasis is less common, but with metastasis the prognosis is poor.