Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

On the ultrastructure of polypeptide hormone-producing cells in the gut of the ascidian,Ciona intestinalis L. and the Bivalve,Mytilus edulis L.

Summary Ultrastructural evidence has been found for the presence of polypeptide hormone-producing cells in the gut of Ciona intestinalis L. and Mytilus edulis L. which do not appear to have been described before. Due to their localization and ultrastructural characteristics, it is suggested that the cells in Mytilus edulis probably produce an insulin-like substance and that some of these cells in Ciona intestinalis may produce 5-HT (5-Hydroxytryptamine). In each species only one granulated cell type can be observed. The granules, which are electron dense and membrane bound, also show a halo. The average diameter of the granules is 100–200 nm for Ciona and 200–400 nm for Mytilus.I thank Mr. G. Bargsten, M.A., Dept. of Marine Zoology, University of Kiel, for the supply of the animals     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

Increased frequency of micronucleated exfoliated cells among humans exposed in vivo to mobile telephone radiations

The health concerns have been raised following the enormous increase in the use of wireless mobile telephones throughout the world. This investigation had been taken, with the motive to find out whether mobile phone radiations cause any in vivo effects on the frequency of micronucleated exfoliated cells in the exposed subjects. A total of 109 subjects including 85 regular mobile phone users (exposed) and 24 non-users (controls) had participated in this study. Exfoliated cells were obtained by swabbing the buccal-mucosa from exposed as well as sex–age-matched controls. One thousand exfoliated cells were screened from each individual for nuclear anomalies including micronuclei (MN), karyolysis (KL), karyorrhexis (KH), broken egg (BE) and binucleated (BN) cells. The average daily duration of exposure to mobile phone radiations is 61.26 min with an overall average duration of exposure in term of years is 2.35 years in exposed subjects along with the 9.84 ± 0.745 micronucleated cells (MNCs) and 10.72 ± 0.889 total micronuclei (TMN) as compared to zero duration of exposure along with average 3.75 ± 0.774 MNC and 4.00 ± 0.808 TMN in controls. The means are significantly different in case of MNC and TMN at 0.01% level of significance. The mean of KL in controls is 13.17 ± 2.750 and in exposed subjects is 13.06 ± 1.793. The value of means of KH in exposed subjects (1.84 ± 0.432) is slightly higher than in controls (1.42 ± 0.737). Mean frequency of broken egg is found to be more in exposed subjects (0.65 ± 0.276) as compared to controls (0.50 ± 0.217). Frequency of presence of more than one nucleus in a cell (binucleated) is also higher in exposed (2.72 ± 0.374) in comparison to controls (0.67 ± 0.231). Although there is a slight increase in mean frequency of KH, BE and BN in exposed subjects but the difference is not found statistically significant. Correlation between 0–1, 1–2, 2–3 and 3–4 years of exposure and the frequency of MNC and TMN has been calculated and found to be positively correlated.     

Involvement of the adenylyl cyclase signaling system in the action of insulin and mollusk insulin-like peptide

Involvement of the adenylyl cyclase signaling system in the mechanism of action of the mammalian insulin and epidermal growth factor as well as of insulin-like peptide isolated from the bivalve mollusk Anodonta cygnea has been studied. It was shown for the first time that insulin and insulin-like peptide exert in vitro the GTP-dependent stimulating action on the adenylyl cyclase activity. Epidermal growth factor has an analogous effect. Effectiveness of the peptides decreased in the order insulin-like peptide > epidermal growth factor > insulin in the foot smooth muscles of A. cygnea and insulin > epidermal growth factor > insulin-like peptide in the skeletal muscles of rat.     

Different strategies for the chemical synthesis of lignans

This paper summarises the results of three projects. The first is concerned with developing general routes for the synthesis of lignans. In particular, two routes involving tandem conjugate addition reactions and Diels Alder reactions respectively that have been used to synthesise podophyllotoxin derivatives are described. The second project is concerned with the asymmetric synthesis of lignans and involves the application of these reactions, with the introduction of a menthyloxy group as a chiral auxiliary, to achieve the asymmetric synthesis of podophyllotoxin derivatives. The third project is concerned with the attempted biomimetic syntheses of podophyllotoxin derivatives using oxidative coupling reactions. Attention is focussed primarily on the use of hypervalent iodine reagents, which yield stegane and isostegane derivatives rather than podophyllotoxin derivatives. Other examples of biaryl coupling leading to stegane and isostegane derivatives are included, and other examples of lignan synthesis involving hypervalent iodine reagents are also described. Abbreviations: DDQ – 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMAD – dimethyl acetylenedicarboxylate (dimethyl butynedioate); PIDA – phenyliodonium diacetate, iodobenzene diacetate; PIFA – phenyliodonium bis(trifluoroacetate), [bis(trifluoroacetoxy) iodobenzene]; Ra-Ni – Raney nickel; TBAF – tetrabutylammonium fluoride; TBDMS –t-butyldimethylsilyl; TFA – trifluoroacetic acid; TFAA – trifluoroacetic anhydride; TFE – 2,2,2-trifluoroethanol; TTFA – thallium(III) trifluoroacetate.     

Comparison of the average structures,from molecular dynamics,of complexes of GTPase activating protein (GAP) with oncogenic and wild-type ras-p21: identification of potential effector domains

GTPase activating protein (GAP) is a known regulator of ras-p21 activity and is a likely target of ras-induced mitogenic signaling. The domains of GAP that may be involved in this signaling are unknown. In order to infer which domains of GAP may be involved, we have performed molecular dynamics calculations of GAP complexed to wild-type and oncogenic (Val 12–containing) ras-p21, both bound to GTP. We have computed and superimposed the average structures for both complexes and find that there are four domains of GAP that undergo major changes in conformation: residues 821–851, 917–924, 943–953, and 1003–1020. With the exception of the 943–953 domain, none of these domains is involved in making contacts with ras-p21, and all of them occur on the surface of the protein, making them good candidates for effector domains. In addition, three ras-p21 domains undergo major structural changes in the oncogenic p21-GAP complex: 71–76 from the switch 2 domain; 100–108, which interacts with SOS, jun and jun kinase (JNK); and residues 122–138. The change in conformation of the 71–76 domain appears to be induced by changes in conformation in the switch 1 domain (residues 32–40) and in the adjacent domain involving residues 21–31. In an accompanying paper, we present results from microinjection of peptides corresponding to each of these domains into oocytes induced to undergo maturation by oncogenic ras-p21 and by insulin-activated wild-type cellular p21 to determine whether these domain peptides may be involved in ras signaling through GAP.     

Direct actions of cortisol, thyroxine and growth hormone on IGF-I mRNA expression in sea bream hepatocytes

The present study aims to investigate potential regulatory effect of different growth-related hormones including growth hormone (GH), human insulin-like growth factor-I (hIGF-I), thyroxine (T₄), triiodothyronine (T₃) and cortisol, on insulin-like growth factor-I (IGF-I) mRNA expression of hepatocytes isolated from silver sea bream. By using real-time PCR, IGF-I mRNA expression profiles of hepatocytes in response to individual hormones were determined in vitro. Hepatocytes incubated with GH at concentrations of 10–1000 ng/mL showed significantly higher IGF-I expression, but the elevation was attenuated at high concentration of GH (1000 ng/mL). IGF-I expression remained unchanged in hepatocytes after incubation with hIGF-I. Hepatocytes incubated with T₄ at concentration of 1000 ng/mL exhibited a significant elevation in IGF-I expression, whereas no difference in IGF-I expression was demonstrated in hepatocytes after incubation with T₃. Upon incubation with cortisol (1–1000 ng/mL), IGF-I expression was significantly decreased in hepatocytes in a dose-dependent manner. Our study demonstrated that GH, T₄, and cortisol had direct modulatory effects on IGF-I expression in fish hepatocytes in vitro.     

The Distribution of Bacterio- and Mesozooplankton in the Coastal Waters of the White and Barents Seas

The total population density and the biomass of bacterioplankton, mesozooplankton, and phosphate-accumulating bacteria (PAB) were estimated during the 2000–2001 summer–autumn seasons in the coastal waters of the White and Barents Seas, which are subject to the action of tidal and sea currents, the inflow of riverine waters, and anthropogenic impact. In the shallow estuarine waters with salinities of 6.5–32 near the Chernaya, Pesha, and Pechora River mouths, the population of PAB fluctuated from 0.1 to 9.1 million cells/ml (0–36% of the total bacterial population). In pelagic seawaters, which are low in phosphates (12–50 g/l) and are characterized by an increased iron/phosphorus ratio (2.0–3.6), bacterioplankton amounted to 0.1–1.6 million cells/ml and was mainly represented by small organisms with a volume of 0.08–0.15 m³, commonly lacking intracellular polyphosphates. In the pelagic zone of the Barents Sea, the biomass of mesozooplankton (B _z) was comparable with that of bacterioplankton (B _b = 39–175 mg/m³), the B _b/B _z ratio being 1.4–4.6. Off the Varandeiskii, Pechora, and Kolguyev oil terminals, B _b increased to 155–300 mg/m³ and the B _b/B _z ratio rose to 1.4 to 50.3 (with an average value of 20.9), presumably due to the severe anthropogenic impact on these waters. In this case, the dense population of bacterioplankton (0.9–7.6 million cells/ml) was mainly represented by large cells (0.12–0.76 m³ in volume), most of which (3–43% of the total bacterioplankton population) contained polyphosphates. The chemical composition of these waters was characterized by an elevated content of the total phosphorus (65–128 g/l) and by a low iron/phosphorus ratio (0.9–1.2).     

Relevance of the Diastereotopic Ligation of Magnesium Atoms of Chlorophylls in the Major Light-harvesting Complex II (LHC II) of Green Plants

The recent high-resolution crystal structure of LHC II [Liu et al. (2004) Nature 428: 287–292] makes possible an unprecedented insight into the stereochemical features of how chlorophylls (Chl)s are bound. The diastereotopic ligation generates four structurally different pigment types, two Chl a and two Chl b, which are distinguished not only by the groups in the 7-position (methyl in Chl a and formyl in Chl b) but also by the face of the tetrapyrrole to which the fifth magnesium ligand is bound. Within a LHC II monomer, out of the eight Chl a six have a ‚normal’ α-coordination and two are β-coordinated while out of the six Chl b only one has the ‚special’ β-coordination. In Photosystem I where a more meaningful statistical analysis could be made, out of 96 Chl a only 14 are β-coordinated, again indicating a preference for the ‚normal’ α-coordination [Balaban et al. (2002) Biochim Biophys Acta Bioenerget 1556: 197–207; Oba and Tamiaki (2002a) Photosynth Res 74: 1–10]. Astonishingly, all the special β-Chls are part of the stromal ring of Chls within the LHC II trimers and occupy key positions for the excitation energy transfer. Sequential energy traps are engineered with one hetero- and three homo-dimers. A careful pairing of carotenoids with the special β-Chls, which could quench their triplet states efficiently, implies a functional relevance of this diastereotopic ligation.     

Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions

To define sequences in the cruciferin gene cru1 promoter of importance for expression, tobacco (Nicotina tabacum L.) plants were transformed with constructs in which the cru1 promoter, in front of the intact cru1 structural gene, was truncated at –1216, –974, –736, –515, –306, –46 and –17 bp relative to the cap-site. Cru1 expression in tobacco seeds was studied by Northern analysis, Western analysis and in-situ hybridizations. Comparisons of the Northern analysis of RNA from tobacco seeds harvested at 18 d after pollination with the Western analysis of protein from mature seeds showed that the regions between –974 to –736 and –306 to –46 were important for the expression of cru1 at an early developmental stage, whereas the regions –736 to –515 and –515 to –306 were important for expression throughout embryogenesis. By investigating the mRNA levels in transgenic seeds at different stages of development, indications were obtained that the two latter regions exerted their effects during the later stages. The in-situ hybridization showed that cru1 mRNA was distributed in parenchyma cells throughout the embryo in seeds expressing constructs –974 and –736. Constructs –515 and –306 showed an expression restricted to the axis or axis and parts of the cotyledons. Sequence comparisons of the cru1 promoter with other storage-protein gene promoters, identified several motifs implicated in gene regulation. Gel retardation assays with synthetic oligonucleotides showed that a region present in both cru1 and BnC1 promoters, a CANNTG motif, an SEF3 motif, an abscisic-acid-responsive element and an RY-like motif interacted specifically in vitro with DNA-binding proteins present in nuclear extracts from seeds of Brassica napus L. harvested 40 d after pollination.Abbreviations ABA abscisic acid - DAP days after pollination This work was supported by grants from the Swedish Research Council for Forestry and Agriculture and from the Swedish Natural Science Research Council. Ms Ulla Pihlgren, Elisabeth Westergren and Elfi Öhren are acknowledged for expert technical assistance.     

Synthesis of bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by stepwise and selective formation of three disulfide bridges.

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for -amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Chaetosphaeria tortuosa, the newly discovered teleomorph of Menispora tortuosa, with a key to known Menispora species

Chaetosphaeria tortuosa is described as the newly discovered teleomorph of Menispora tortuosa, based on specimens from Canada and the Czech Republic, and single spore isolations from both morphs. The fungus produces superficial, more or less globose, papillate, dark brown to black smooth perithecia (200–)220–250 × (220–)230–260 μm. The asci are unitunicate, 8-spored, cylindrical-fusiform, (110–)120–133(–145) × 12–14 with a distinct apical, nonamyloid annulus 1–1.5 μm high, 3.5–4 μm wide. The ascospores are fusiform, 19–24 × 5–6 μm, hyaline, 3-septate, smooth, and 2-seriate in the ascus. The morphology of the teleomorph and anamorph are similar to that of C. ovoidea (anamorph: M. glauca), differing in dimensions of asci and ascospores, and in the disposition and morphology of the phialides of the anamorphs. The generic concept and phylogeny of Menispora is briefly discussed, and a key to the 11 species currently accepted in the genus is provided.     

Diversification of Lupinus (Leguminosae) in the western New World: derived evolution of perennial life history and colonization of montane habitats

Previous phylogenetic studies of Lupinus (Leguminosae) based on nuclear DNA have shown that the western New World taxa form a monophyletic group representing the majority of species in the genus, with evidence for high rates of recent diversification in South America following final uplift of the Andes 2–4 million years ago (Mya). For this study, three regions of rapidly evolving non-coding chloroplast DNA (trnL intron, trnS–trnG, and trnT–trnL) were examined to estimate the timing and rates of diversification in the western New World, and to infer ancestral states for geographic range, life history, and maximum elevation. The western New World species (5.0–9.3 Mya, 0.6–1.1 spp./My) comprise a basally branching assemblage of annual plants endemic to the lower elevations of western North America, from which two species-rich clades are recently derived: (i) the western North American perennials from the Rocky Mountains, Great Basin, and Pacific Slope (0.7–2.1 Mya, 2.0–5.9 spp./My) and (ii) the predominantly perennial species from the Andes Mountains of South America and highlands of Mexico (0.8–3.4 Mya, 1.4–5.7 spp./My). Bayesian posterior predictive tests for association between life history and maximum elevation demonstrate that perennials are positively correlated with higher elevations. These results are consistent with a series of one or more recent radiations in the western New World, and indicate that rapid diversification of Lupinus coincides with the derived evolution of perennial life history, colonization of montane habitats, and range expansion from North America to South America.     

Immunocytochemical identification of α-endorphin-like material in neurones of the brain and corpus cardiacum of the blowfly,Calliphora vomitoria (Diptera)

Summary A group of the 24–26 paraldehyde fuchsin-positive median neurosecretory cells (MNC) in the pars intercerebralis of the brain of the blowfly, Calliphora vomitoria, has shown immunoreactivity towards three different antibodies to -endorphin, a peptide that corresponds to the amino acid sequence present between residues 61 and 76 of the precursor molecule, -lipotropin (-LPH). The immunoreactive material could be followed in axons within the median bundle, the tract through which neurosecretory material from the MNC is passed down to the corpus cardiacum (CC). The -endorphin-immunoreactive material was observed leaving the CC in the cardiac-recurrent nerve, dorsal to the proventriculus, in the direction of the abdomen. The cells that contain the -endorphin-like material are different from those of the MNC that contain insulin-, pancreatic polypeptide-, and gastrin/CCK-like peptides. This finding demonstrates the considerable complexity and peptidergic nature of the MNC and constitutes further evidence that morphinomimetic-like peptides are present in the nervous system of invertebrates.     

Nitrogen and phosphorus uptake in seedlings of the seagrass Amphibolis antarctica in Western Australia

The uptake of nitrate, ammonium and phosphate was examined in vitro in seedlings of the seagrass Amphibolis antarctica ((Labill.) Sonder ex Aschers.). Uptake of all three nutrients was significantly correlated with external concentration up to 800 µ g l^–1. The uptake of nitrate (0–200 µ g NO₃-N g dry wt^–1 h^–1) was significantly lower than the uptake of ammonium (0–500 µ g NH₄-N g dry wt^–1 h^–1), suggesting that the seedlings have a higher affinity for this form of nitrogen in the water column.Data were in general agreement with uptake rates recorded for other seagrasses, notably Zostera marina. In comparison to the dominant macroalgae for the same region, seedlings had either similar or higher uptake rates in relation to external concentration, lending support to the hypothesis that seedlings, which do not possess roots, behave like macroalgae in terms of nutrient acquisition from the water column.A comparison with literature data on adult seagrass suggests, however, that seagrasses show lower uptake rates than macroalgae suggesting that the macroalgae, which are totally reliant on the water column for nutrients, are more efficient at uptake than seagrasses which may potentially use the sediment for a nutrient source.     

Evaluation of the sample size used for the rapid bioassessment of rivers using macroinvertebrates

Experiments were designed to investigate selective predation by medium (40–55 mm carapace width: CW) and large (55–70 mm CW) Carcinus maenas when feeding on four bivalves of contrasting shell morphology. Size-selection was examined by presenting individual crabs with a wide size range of Mytilus edulis, Ostrea edulis, Crassostrea gigas and Cerastoderma edule. Medium-sized crabs preferred mussels 5–15 mm shell length (maximum shell dimension: SL) and cockles 5–10 mm SL, whereas large crabs preferred mussels 15–25 mm and cockles 10–20 mm SL. Crabs generally showed no preference for any particular size of either oyster species. Species-selection was examined by presenting individual crabs with paired combinations of the four bivalves in various proportions. When offered mussels and oysters simultaneously, both size categories of crabs consistently selected mussels, and food choice was independent of prey relative abundance. By contrast, C. maenas selected mussels and cockles as expected by the frequency in which each size category of crab encountered the preferred size ranges of prey. Crab preference clearly paralleled the rank order of prey profitability, which in turn was mainly determined by prey biomass, suggesting that active selection takes place at some point of the predation cycle. Experiments with epoxy resin models showed that initial reluctance of crabs to attack oysters was not associated with the ultimate energy reward. Moreover, they suggest that foraging decisions are partly based on evaluations of overall prey shape and volume, and that the minimum dimension of the shell constitutes an important feature which crabs recognise and associate with prey value.     

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain

We have previously found that a peptide corresponding to residues 35–47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras–p21 peptide corresponding to residues 96–110 that blocks ras–jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35–47, but not the 96–110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35–47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras–p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35–47 and 96–110, act at two different critical downstream sites, the former at raf, the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.     

Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii

Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m^–3 h^–1 and 55 g m^–3 h^–1 were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial–fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m^–3 h^–1. Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m^–3 h^–1 and 94 g m^–3 h^–1 in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m^–3 h^–1 or increased to 95.6 g m^–3 h^–1, respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m^–3 h^–1 under steady-state conditions, with near-complete toluene degradation.     

Mechanism of Cl− transport at the plasma membrane ofChara corallina: II. Transinhibition and the determination of H+/Cl− binding order from a reaction kinetic model

Summary Internal Cl^– and low internal pH are strong inhibitors of Cl^– influx at the plasma membrane ofChara. The present investigation seeks to understand the mechanism by which this is achieved. Since both Cl^– and H⁺ are transported by the same system, one possible mechanism is simply through a change in the electrochemical gradients of these ions. However, it is found that transport is more sensitive to theinternal concentrations of the two ions than to their respective gradients. It is demonstrated that Cl^– influx, which shows Michaelis-Menten kinetics with respect to external concentration, is affected only in itsV _max by internal Cl^– and pH; the apparentK _m of the transport system for external Cl^– is unchanged. In addition, it is found that there is an apparent interaction between internal Cl^– and pH in their effects on Cl^– influx, both in intact cells and those that have been perfused internally. A kinetic model is proposed which can account quantitatively for all these observations simply through the effects of substrate concentration on the apparent rate constants of a recycling carrier. The model predicts (i) strictly ordered binding of Cl^– and H⁺ to the carrier at both internal and external surfaces, with Cl^– first on and first off (ii) movement of charge through the membrane on the loaded, rather than the unloaded, carrier. The present model is expected to account for similar kinetic observations from a variety of other cotransport systems.