Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Objective

To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system.

Methods

Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using ³⁵SO₄²⁻-labelling and the dimethylmethylene blue assay. ³H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured.

Results

The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days. ³H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the ³⁵SO₄²⁻ incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts.

Conclusion

The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.     

β-Lactamase activity and resistance to penicillins in Myxococcus xanthus

Several mutants and other variants of Myxococcus xanthus HP100 were obtained with differences in their sensitivity to carbenicillin and other penicillin derivatives. The specific activities of -lactamase in different resistant organisms varied from strain to strain but were consistently higher than in HP100. The relative molecular mass (M _r ) of the enzyme in M. xanthus HP100 was found to be 22,300. In certain carbenicillin resistant strains a second fraction of -lactamase activity of molecular weight 186,000 presumed to be an octamer of the other form was present. The enzyme was found in cell free extracts and also in culture supernatants of all carbenicillin resistant mutants but not in culture supernatants of strain HP100. In all the carbenicillin resistant mutants a part of the intracellular enzyme activity was released by osmotic shock and this activity may be periplasmic. The forms of the enzyme present in the culture supernatants and released by osmotic shock were monomeric. Carbenicillin resistance was not transferable between strains by conjugation. One resistance allele inhibited the transfer of the R factor Sa between myxococci.Non-standard abbreviations C^S C^R sensitivity and resistance to carbenicillin - C _u ^R C _S ^R unstable and stable resistance to carbenicillin     

Sizes of isotopically labeled molecules released during lysis of tumor cells labeled with 51Cr and [14C]nicotinamide.

Previous studies of mechanisms of immune lysis have utilized the release of isotopically labeled molecules from cells labeled with Na₂⁵¹Cr0₄ and [¹⁴C]nicotinamide, among others. The interpretation of specific isotope release from such cells was hampered by the lack of knowledge about the molecular sizes of the released radioactive molecules. In the present study, P815 tumor cells were labeled with the above two isotopic compounds. The cells were then lysed by freezing and thawing, antibody and complement, or immune T-lymphocyte-mediated killing. Soluble supernatants from the lysates were chromatographed in the cold on Sephadex G-10, G-25, and G-200 using several elution media. No differences were detected due to different kinds of lysis or different eluting media. Eighty-five percent of ¹⁴C-labeled molecules released from the cells were indistinguishable from nicotinamide and clearly distinct from nicotinamide adenine dinucleotide. The results suggest that [¹⁴C]nicotinamide taken up by the tumor cells during overnight culture is released unaltered during lysis. Ninety percent of ⁵¹Cr-labeled lysate molecules had an apparent molecular weight less than 4000. Most of this material was distinct from inorganic chromate, but its chemical form is not known.     

Photomixotrophic cultures of blueberries (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis>) accumulate or release phenylpropanoids via inductive treatments

The in vitro production of phenylpropanoids has been studied in temporary immersion bioreactors (TIBs) cultures of blueberries (Vaccinum corymbosum) supplemented with 0.4 MPa CO₂, light intensity of 150 μM/m²/s, and induced with ABA (4 mM) and H₂O₂ (5 mM). Results demonstrated differences in the accumulation and/or the release of patterns of metabolites. In TIBs plus ABA, a reddish-maroon color was consistent with the accumulation of phenolics, where shoots formed clusters without internodal segments. While in TIBs plus H₂O₂, the phenylpropanoids were released into the culture medium. In both cases, experimental treatments with sucrose-reduced medium displayed the highest phenylpropanoids values, in contrast with the controls (conventional culture). Altogether, differential expressions of genes, linked to key pathways as photosynthesis, phenylpropanoids, and oxidative burst, could corroborate for the increase in the phenolics production in parallel with an improvement of photosynthesis in photomixotrophic culture.     

Glial Metabolism of Isoleucine

Isoleucine, together with leucine and valine, constitutes the group of branched-chain amino acids (BCAAs). BCAAs are transported from the blood into the brain parenchyma, where they can serve several distinct functions. Since brain tissue is known to oxidatively metabolize BCAAs to CO₂, they are considered as fuel material in brain energy metabolism. Also, in the case of leucine, cultured astrocytes have been reported to be able to completely oxidize BCAA. While the metabolism of leucine by astroglia-rich primary culture (APC) has already been studied in detail, the metabolic fates of isoleucine and valine in these cells remained to be identified. Therefore, in the present study an NMR analysis was performed of ¹³C-labelled metabolites generated in the catabolism of [U-¹³C]Ile by astrocytes and released by them into the incubation medium. APC potently removed isoleucine from the medium and metabolized it. The major isoleucine metabolites released from APC are 2-oxo-3-methylvalerate, 2-methylbutyrate, 3-hydroxy-2-methylbutyrate and propionate. To a lesser extent, APC generate and release also [2,3-¹³C]glutamine, [4,5-¹³C]glutamine and ¹³C-labelled isotopomers of lactate and citrate. These results show that APC can release into the extracellular milieu catabolites and several TCA cycle dependent metabolites resulting from the degradation of isoleucine. Special issue article in honor of Dr. George DeVries.     

Role of planktonic and sessile extracellular metabolic byproducts on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> intra and interspecies relationships

Bacterial species are found primarily as residents of complex surface-associated communities, known as biofilms. Although these structures prevail in nature, bacteria still exist in planktonic lifestyle and differ from those in morphology, physiology, and metabolism. This study aimed to investigate the influence of physiological states of Pseudomonas aeruginosa and Escherichia coli in cell-to-cell interactions. Filtered supernatants obtained under planktonic and biofilm cultures of each single species were supplemented with tryptic soy broth (TSB) and used as the growth media (conditioned media) to planktonic and sessile growth of both single- and two-species cultures. Planktonic bacterial growth was examined through OD₆₄₀ measurement. One-day-old biofilms were evaluated in terms of biofilm biomass (CV), respiratory activity (XTT), and CFU number. Conditioned media obtained either in biofilm or in planktonic mode of life triggered a synergistic effect on planktonic growth, mainly for E. coli single cultures growing in P. aeruginosa supernatants. Biofilms grown in the presence of P. aeruginosa biofilms-derived metabolites presented less mass and activity. These events highlight that, when developed in biofilm, P. aeruginosa release signals or metabolites able to prejudice single and binary biofilm growth of others species and of their own species. However, products released by their planktonic counterparts did not impair biofilm growth or activity. E. coli, living as planktonic or sessile cultures, released signals and metabolites or removed un-beneficial compounds which promoted the growth and activity of all the species. Our findings revealed that inter and intraspecies behaviors depend on the involved bacteria and their adopted mode of life.     

Field decomposition of transgenic Bt maize residue and the impact on non-target soil invertebrates

The release of chemical compounds from plant roots that suppress soil nitrification is termed biological nitrification inhibition (BNI). Determining the environmental factors that control the synthesis and release of BNI-compounds from Brachiaria humidicola (Rendle) Schweick, a tropical pasture grass that thrives on acid soils, is the focus of this investigation. Because the BNI trait is related to the N status of the plant, we investigated the possibility that the expression of this trait would be related to the forms of N found in the root environment. Plants were grown with two sources of N, NH₄⁺ or NO₃⁻ for 60 days and the release of BNI-compounds monitored. Only plants grown with NH₄⁺ released BNI-compounds from roots. The presence of NH₄⁺ and possibly the secondary effect of its uptake (i.e., acidic pH) in the root environment significantly enhanced the release of BNI-compounds. Both the NH₄⁺ and NO₃⁻ grown plants responded to the stimulus from NH₄⁺ in the root environment. BNI-compounds found in root tissue and their release were nearly three times greater in NH₄⁺ grown than from NO₃⁻ grown plants. The BNI-compounds released from roots composed of at least three active components—Type-I (stable to pH changes from 3.0 to 10), Type-II (temporarily loses its inhibitory effect at a pH higher than a threshold pH of 4.5 and the inhibitory effect is reestablished when the root exudate pH is adjusted to <4.5) and Type-III (inhibitory effect is irreversibly lost if the pH of the root exudate reaches 10.0 or above). A major portion of BNI-compounds released in the presence of NH₄⁺ is of Type-I. In the absence of NH₄⁺, mostly Type-II and Type-III BNI-compounds were released. The BNI-compounds inhibited the function of Nitrosomonas europaea through the blocking of both ammonia monooxygenase and hydroxylamino oxidoreductase pathways. These results indicate that the release of BNI-compounds from B. humidicola roots is a regulated function and that presence of NH₄⁺ in the root environment is necessary for the sustained synthesis and release of BNI.     

Release of fatty acid-binding protein and long chain fatty acids from isolated rat heart after ischemia and subsequent calcium paradox

To obtain insight into the relation between the release of heart-type fatty acid-binding protein (H-FABP_c) and of long-chain fatty acids (FA) from injured cardiac tissue, rat hearts were Langendorff perfused according to the following scheme: 30 min normoxia, 60 min ischemia, 30 min reperfusion, 10 min Ca²⁺ free perfusion and finally 10 min Ca²⁺ repletion. During this protocol right ventricular (Q _rv ) and interstitial effluent samples (Q _i ) were collected at regular intervals. During reperfusion a total of 0.8±0.1 nmol H-FABP_c but no FA were detected in the effluents. However, during Ca²⁺ readmission, 45±4 nmol H-FABP_c (80–90% of total tissue content) was released with an initial (first 3 min) simultaneous release of FA (FA/H-FABP_c ratio 0.90±0.07 mol/mol). Thereafter, FA release continued at 10–15 nmol per min mainly inQ _rv while the rate of H-FABP_c release decreased. During Ca²⁺ repletion, tissue FA content raised rapidly from 168±20 to 1918±107 nmol/g dry weight. These findings suggest that after severe cardiac damage initially FA is released bound to H-FABP_c, whereas further FA release occurs in a non-protein bound manner.     

Mass Balance Studies with 14C-Labeled 2,4,6-Trinitrotoluene (TNT) Mediated by an Anaerobic Desulfovibrio Species and an Aerobic Serratia Species

Investigations were carried out to evaluate the level of incorporation of radiolabeled 2,4,6-trinitrotoluene (TNT) and metabolites into the bacterial biomass of two different bacterial species after cometabolically mediated TNT transformation. Biotransformation experiments with ¹⁴C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with the cells. It was found that more than 42% of the initially applied radiolabel was associated with the cell biomass after cometabolic ¹⁴C-TNT transformation with the strictly anerobic Desulfovibrio species strain SHV, whereas with the strictly aerobic Serratia plymuthica species strain B7, 32% of cell-associated ¹⁴C activity was measured. The remainder of the radiolabel was present in the supernatants of the liquid cultures in the form of different TNT metabolites. Under anoxic conditions with the Desulfovibrio species, TNT was ultimately transformed to 2,4,6-triaminotoluene (TAT) and both diaminonitrotoluene isomers, whereas under oxic conditions with the Serratia species, TNT was converted to hydroxylaminodinitrotoluenes and aminodinitrotoluenes, with 4-amino-2,6-dinitrotoluene (4ADNT) being the major end product. In both culture supernatants, small amounts of very polar, radiolabeled, but unidentified metabolites were detected. At the end of the experiments approximately 92% and 96% of the originally applied radioactivity was recovered in the studies with the Serratia and Desulfovibrio species, respectively. Received: 21 May 1998 / Accepted: 6 July 1998     

Antigen-Induced leukotriene relaese from rat lung

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

Methane release from stands of water horsetail ( Equisetum fluviatile ) in a boreal lake

1. Annual and diel variations in methane (CH₄) release in stands of Equisetum fluviatile were measured from June to November in Lake Pääjärvi, southern Finland, where E. fluviatile is the dominant emergent macrophyte. An estimate of total annual release of CH₄ from stands of E. fluviatile in this lake was also made. Diel variation was measured twice (June and August), whereas measurements for annual variation were performed monthly. The hypothesis that a relationship exists between the productivity of stands and CH₄ release was also tested, whereupon net ecosystem exchange (NEE) of CO₂ as well as standing stock of E. fluviatile were determined, in addition to simultaneous recordings of air temperature and solar radiation. 2. Seasonal variations in CH₄ release were pronounced, with the highest release rate of 813 mg m^–2 day^–1 measured in July and the lowest 6.5 mg m^–2 day^–1 in November, when the shoreline was already frozen. 3. Methane release rates were strongly correlated with mean air temperature in the measuring chambers and with total solar radiation. There was no significant correlation between the instantaneous radiation and CH₄ release rates. 4. The seasonal patterns of CH₄ release and NEE of CO₂ resembled each other, except in July when NEE suddenly dropped. The decrease in NEE coincided with the highest CH₄ release rate measured and the highest temperature during the measuring period, i.e. 32 °C outside and 37 °C inside the chamber. Excluding this date, daily CH₄ release was strongly correlated with NEE (r² = 0.971). 5. No diel changes in CH₄ release rates were detected. In June and August the maximum release rates were 11.4 and 16.8 mg CH₄ m^–2 h^–1, respectively. 6. The standing stock of E. fluviatile at different times of the growing season was not correlated with CH₄ efflux; the CH₄ release rates could be related neither to the number of shoots, i.e. sufficient conduits for gas transport were always present, nor to the shoot biomass in the measuring chambers. 7. For an estimate of the annual release, the monthly values measured at noon were integrated over the entire growing season; this resulted in 43.7 g CH₄ m^–2 for the annual emission. The total annual emission of CH₄ from the area covered with E. fluviatile in Lake Pääjärvi was calculated to be ≈ 5000 kg. 8. Significant amounts of CH₄ are released from stands of E. fluviatile in boreal lakes. The CH₄ release rate follows a seasonal pattern but there is no diel pattern. Methane release rate can be related to temperature, solar radiation and NEE of CO₂, but not to the standing stock of E. fluviatile or the number of shoots.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (T_c) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB₄ can induce human T_c to exert suppressor cell activity, and incubation of lymphocytes with LTB₄ for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40–90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O₂^.−) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 ± AA for icosanoid release; phorbol myristate acetate for O₂^.− production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca²⁺-dependent K⁺ permeability in PMNL was measured by the cell K⁺ release induced by A 23187. T_c and T_c subsets were determined using monoclonal antibodies (OKT₃₊, OKT₄₊ and OKT₈₊). A biphasic sequential release of the different substances (leukocytic icosanoids and O₂^.− was monitored: increase ( 36–48 h after Tl) and decrease ( 72 h after Tl). The increase in AA stimulation was more transient than that of O₂^.−. The decline in the release of AA metabolites and O₂^.− production was associated with the anergic phase (decrease OKT₄₊/OKT₈₊ ratio) and with the clinical outcome of the patients. The decrease in LTB₄ and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca²⁺-dependent K⁺ permeability increased early up to 2 or 3 times normal.In order to go further with the mechanism of inhibition of LTB₄ and O₂^.− release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB₄ secretion. A less important inhibition was observed with O₂^.− release (−32%) and Ca²⁺-dependent K⁺ permeability (−30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): “suppressive factor(s) of membrane activation ”SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromotography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O₂^.− release.     

Salmonella typhi Ty2 OmpC Porin Induces Bactericidal Activity on U937 Monocytes

The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid-encoded S. typhi Ty2 OmpC. The mononuclear cell supernatants, immunized in vitro with OmpC antigen, derived from 10 out of 17 patients activated U937 bactericidal capacity. In contrast, the supernatants from the immunization with outer membrane preparation lacking S. typhi Ty2 OmpC induced a significantly reduced bactericidal capacity of U937 cells. This procedure should prove useful for in vitro characterization of cellular immunogens from exclusive human pathogens.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Stabilization and functional studies of high-molecular-weight murine lymphotoxins

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2–4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (α_H 130,000–160,000 daltons). It was found that the Cx and α_H LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr ⁷⁵Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell.     

Antigen-Induced leukotriene relaese from rat lung in vitro

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

ORGANIC CARBON RELEASE BY DUNALIELLA SALINA (CHLOROPHYTA) UNDER DIFFERENT GROWTH CONDITIONS OF CO2, NITROGEN,AND SALINITY1

Two strains of Dunaliella salina (Dunal) Teod., UTEX 1644 and UTEX 200, were cultured under different growth regimes, including 10 mM NO₃^? or NH₄⁺, 1.5 or 3.0 M NaCl, and low (0.035%) or high (5%) CO₂ in air. The release of ¹⁴C-labeled dissolved organic carbon (DOC), expressed as a rate and as a percentage of photosynthetic ¹⁴CO₂ assimilation, was subsequently determined. The percentage of DOC released was inversely related to cell density in the assay medium, but photosynthesis on a per-cell basis was not. Release of DOC was low, in the range of 1–5% of photosynthesis, but during acclimation to growth on NH₄⁺, it rose to 11%. The presence of NH₄⁺ rather than NO₃^? in the growth medium increased the rate of release by both strains, but the percentage release was stimulated only in UTEX 200 cells, because their photosynthetic rate was depressed by NH₄⁺. For UTEX 1644, high, as compared to low, CO₂-grown cells, had somewhat higher rates and percentages of DOC release, but release from UTEX 200 cells was unaffected by the growth-CO₂. The rate of DOC release by high CO₂-grown cells was not enhanced at a low concentration of dissolved inorganic carbon, indicating that the released material did not originate from the photorespiratory pathway. The effects of NaCl on DOC release varied with strain and growth conditions. For UTEX 200, the cells in NO₃^?, but not NH₄⁺, exhibited a doubling or more in percentage of release with a doubling in NaCl concentration, irrespective of growth-CO₂. With UTEX 1644 the low CO₂-grown cells showed the greatest enhancement in 3.0 M NaCl. Organic matter accumulated on the external surface of the cell membrane and constituted a well-defined cell-coat, which was more dense in NH₄⁺ than in NO₃^?-grown cells. Microtubules, which may play a role in maintaining cell shape, were observed just below the plasma membrane. From a practical viewpoint, the presence of organic material in the hypersaline ponds of salt-works is detrimental to salt production. When D. salina cells become abundant in such ponds, the attendant, continuous release of DOC may make a significant contribution to the problem.     

And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Shewanella putrefaciens CN32对粘土附着态铵氮释放的影响

【目的】研究产胞外分泌物微生物Shewanella putrefaciens CN32对土壤中常见粘土矿物附着态NH_4~+的释放效果及影响机制。【方法】以吸附NH_4~+的蒙脱石、蛭石、伊蒙混层矿物和黑云母为对象,通过监测S. putrefaciens CN32作用下不同粘土释放的NH_4~+含量及过程,以及监测微生物量及释放的胞外聚合物(Extracellular Polymeric Substances,EPS)的含量变化,研究S. putrefaciens CN32作用下不同粘土矿物类型附着态NH_4~+释放的差异性。【结果】粘土矿物附着态NH_4~+含量从高到低依次为蒙脱石蛭石伊蒙混层矿物黑云母(黑云母NH_4~+吸附量极低,会在非生物作用下几乎完全释放),CN32作用下粘土附着态NH_4~+相对释放量依次为蒙脱石伊蒙混层矿物蛭石;然而,尽管CN32有效促进了粘土附着态NH_4~+释放,但释放的NH_4~+并未在溶液中大量累积,而是多被微生物同化吸收转化为生物有机氮(EPS为主)并吸附在粘土表面,且粘土对EPS的吸附能力表现为蒙脱石伊蒙混层矿物蛭石黑云母;由于粘土吸附NH_4~+及EPS都与矿物中的羟基(结构水或层间水)关系密切,推测EPS对矿物羟基的竞争吸附可能是CN32促进NH_4~+释放的重要原因之一。【结论】以上结果表明,产EPS微生物S. putrefaciens CN32能够促进各类粘土矿物的附着态NH_4~+释放,但释放的NH_4~+可以通过微生物作用转化为有机氮,从而在减少NH_4~+流失的同时增加土壤氮肥的生物可利用性,因此微生物在降低土壤氮肥流失、转化土壤氮肥污染过程中可能起到了重要作用,也揭示了深入系统地分析不同类型土壤(粘土类型不同)中粘土附着态NH_4~+在不同功能微生物作用下的迁移转化过程,是精准评估土壤氮肥施用效率及流失风险的前提之一。     

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×10⁷/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO₄ (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.