Effects of Metals on Methanogenesis, Sulfate Reduction, Carbon Dioxide Evolution, and Microbial Biomass in Anoxic Salt Marsh Sediments

The effects of several metals on microbial methane, carbon dioxide, and sulfide production and microbial ATP were examined in sediments from Spartina alterniflora communities. Anaerobically homogenized sediments were amended with 1,000 ppm (ratio of weight of metal to dry weight of sediment) of various metals. Time courses in controls were similar for CH₄, H₂S, and CO₂, with short initial lags (0 to 4 h) followed by periods of constant gas production (1 to 2 days) and declining rates thereafter. Comparisons were made between control and experimental assays with respect to initial rates of production (after lag) and overall production. Methane evolution was inhibited both initially and overall by CH₃HgCl, HgS, and NaAsO₂. A period of initial inhibition was followed by a period of overall stimulation with Hg, Pb, Ni, Cd, and Cu, all as chlorides, and with ZnSO₄, K₂CrO₄, and K₂Cr₂O₇. Production of CO₂ was generally less affected by the addition of metals. Inhibition was noted with NaAsO₂, CH₃HgCl, and Na₂MoO₄. Minor stimulation of CO₂ production occurred over the long term with chlorides of Hg, Pb, and Fe. Sulfate reduction was inhibited in the short term by all metals tested and over the long term by all but FeCl₂ and NiCl₂. Microbial biomass was decreased by FeCl₂, K₂Cr₂O₇, ZnSO₄, CdCl₂, and CuCl₂ but remained generally unaffected by PbCl₂, HgCl₂, and NiCl₂. Although the majority of metals produced an immediate inhibition of methanogenesis, for several metals this was only a transient phenomenon followed by an overall stimulation. The initial suppression of methanogenesis may be relieved by precipitation, complexation, or transformation of the metal (possibly by methylation), with the subsequent stimulation resulting from a sustained inhibition of competing organisms (e.g., sulfate-reducing bacteria). For several environmentally significant metals, severe metal pollution may substantially alter the flow of carbon in sediments.     

Bacterial Activity in South Pole Snow

Large populations (200 to 5,000 cells ml⁻¹ in snowmelt) of bacteria were present in surface snow and firn from the south pole sampled in January 1999 and 2000. DNA isolated from this snow yielded ribosomal DNA sequences similar to those of several psychrophilic bacteria and a bacterium which aligns closely with members of the genus Deinococcus, an ionizing-radiation- and desiccation-resistant genus. We also obtained evidence of low rates of bacterial DNA and protein synthesis which indicates that the organisms were metabolizing at ambient subzero temperatures (−12 to −17°C).     

Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments.

The metabolism of D-[U-14C]glucose and the incorporation of [methyl-3H]thymidine by aerobic and anaerobic marine sediment microbes exposed to 1 to 1,000 ppm anthracene, naphthalene, p,p'-dichlorodiphenyltrichloroethane, and pentachlorophenol were examined. Cell-specific rates of [14C]glucose metabolism averaged 1.7 X 10(-21) and 0.5 X 10(-21) mol/min per cell for aerobic and anaerobic sediment slurries, respectively; [3H]thymidine incorporation rates averaged 43 X 10(-24) and 9 X 10(-24) mol/min per cell for aerobic and anaerobic slurries, respectively. Aerobic sediments exposed to three of the organic pollutants for 2 to 7 days showed recovery of both activities. Anaerobic sediments showed little recovery after 2 days of pre-exposure to the pollutants. We conclude that (i) anaerobic sediments are more sensitive than aerobic sediments to pollutant additions; (ii) [3H]thymidine incorporation is more sensitive to pollutant additions than is [14C]glucose metabolism; and (iii) the toxicity of the pollutants increased in the following order: anthracene, p,p'-dichlorodiphenyltrichloroethane, naphthalene, and pentachlorophenol.     

A Simple, High-Precision, High-Sensitivity Tracer Assay for N(inf2) Fixation

We describe a simple, precise, and sensitive experimental protocol for direct measurement of N(inf2) fixation using the conversion of (sup15)N(inf2) to organic N. Our protocol greatly reduces the limit of detection for N(inf2) fixation by taking advantage of the high sensitivity of a modern, multiple-collector isotope ratio mass spectrometer. This instrument allowed measurement of N(inf2) fixation by natural assemblages of plankton in incubations lasting several hours in the presence of relatively low-level (ca. 10 atom%) tracer additions of (sup15)N(inf2) to the ambient pool of N(inf2). The sensitivity and precision of this tracer method are comparable to or better than those associated with the C(inf2)H(inf2) reduction assay. Data obtained in a series of experiments in the Gotland Basin of the Baltic Sea showed excellent agreement between (sup15)N(inf2) tracer and C(inf2)H(inf2) reduction measurements, with the largest discrepancies between the methods occurring at very low fixation rates. The ratio of C(inf2)H(inf2) reduced to N(inf2) fixed was 4.68 (plusmn) 0.11 (mean (plusmn) standard error, n = 39). In these experiments, the rate of C(inf2)H(inf2) reduction was relatively insensitive to assay volume. Our results, the first for planktonic diazotroph populations of the Baltic, confirm the validity of the C(inf2)H(inf2) reduction method as a quantitative measure of N(inf2) fixation in this system. Our (sup15)N(inf2) protocols are comparable to standard C(inf2)H(inf2) reduction procedures, which should promote use of direct (sup15)N(inf2) fixation measurements in other systems.     

Relationships between cytoplasmic microtubular complex,DNA synthesis and cell morphology in mouse embryonic fibroblasts (effects of age,serum deprivation,aphidicolin, cytochalasin B and colchicine)

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an “aging effect” upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).     

Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.     

Nitrate requirement for acetylene inhibition of nitrous oxide reduction in marine sediments

The inhibition of nitrous oxide (N₂O) reduction by acetylene (C₂H₂) in saltmarsh sediment was temporary; we investigated this phenomenon and possible causes. The reduction of N₂O in the presence of C₂H₂ was biological. N₂O consumption in the presence of C₂H₂ began when nitrate concentration became very low. The time course of N₂O consumption after periods of N₂O accumulation was unaffected by initial nitrate concentrations between 16 and 200M, or C₂H₂ concentrations between 10 and 100% of the gas phase. Sulfide had no effect on the kinetics of N₂O reduction in the presence of C₂H₂. In more dilute slurries of saltmarsh sediments and in estuarine sediment, N₂O persisted in the presence of C₂H₂ unless sufficient organic carbon was added to deplete nitrate. In saltmarsh sediments, the rate of N₂O consumption in the presence of C₂H₂ was not changed by preincubation with C₂H₂. Initial positive rates of N₂O production in the presence of C₂H₂ occurred only when the block was apparently effective (i.e., at nitrate concentrations greater than about 5–10M) and appeared to represent a valid estimate of denitrification. Conversely, and in agreement with previous studies, concentrations of NO₃ ^– below these levels resulted in reduced efficiency of C₂H₂ blockage of N₂O reductase.     

A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with Oct-1.

The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1. Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.     

An inducible mammalian amber suppressor: propagation of a poliovirus mutant

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.