Growth of Corynebacterium glutamicum in ammonium- and potassium-limited continuous cultures under high osmotic pressure

 In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower than previously reported in glucose-limited continuous cultures (50 mg/g and 10 mg/g dry weight respectively). On the other hand, the internal trehalose pool was much higher at 40 mg/g dry weight. When the medium osmolality was increased to 1.2 osmol/kg by NaCl addition, under ammonia limitation, the proline content rose from 5 mg/g to 20 mg/g dry weight and the trehalose content from 40 mg/g to 70 mg/g dry weight, whereas the intracellular pool of glutamate remained essentially constant. An increase in the internal sodium content was also observed. Similar results were found for the internal pool of glutamate, proline and trehalose when C. glutamicum was grown under potassium limitations at an osmolality of 1.2 osmol/kg. There were also higher levels of sodium ions, glutamine and alanine. According to the present results, whereas proline was previously reported to be the dominantly accumulated osmoprotectant in C. glutamicum grown under glucose limitations, under ammonia and potassium limitations trehalose represented the dominantly synthesized metabolite. Received: 19 December 1995/Received revision: 9 April 1996/Accepted: 15 April 1996     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996     

The ubiquity of natural adsorbable organic halogen production among basidiomycetes

 Recently, several species of basidiomycetes were shown to produce de novo high concentrations of chloroaromatic metabolites. Since these lignocellulose-degrading fungi play a major role in the ecosphere, the purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes. A total of 191 fungal strains were monitored for adsorbable organic halogen (AOX) production when grown on defined liquid media. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. A low production of 0.1–0.5 mg AOX/l was observed among 25% of the strains, a moderate production of 0.5–5.0 mg AOX/l was observed among 16% of the strains and 9% of the strains produced high levels (5–67 mg AOX/l). The latter group was dominated by species belonging to the genera Hypholoma, Mycena and Bjerkandera, showing specific AOX productions in the range 1074–30893 mg AOX/kg dry weight of mycelial biomass. Many highly ecologically significant fungal species were identified among the moderate to high producers. These species were also able to produce AOX when cultivated on natural lignocellulosic substrates. Hypholoma fasciculare and Mycena metata respectively produced up to 132 mg and 193 mg AOX/kg dry weight of forest litter substrate in 6 weeks. Received: 5 October 1995/Received revision: 28 December 1995/Accepted: 12 February 1996     

A second polymorphic dinucleotide repeat in the 5′ flanking region of the human IL10 gene

Received: 1 July 1996 / Revised: 10 July 1996     

Effect of feed composition and upflow velocity on aggregate characteristics in anaerobic upflow reactors

Two upflow anaerobic hybrid reactors treated lactose and a mixture of ethanol, propionate and butyrate, respectively, at a volumetric loading rate of 3.7 kg chemical oxygen demand (COD) m⁻³day⁻¹, a hydraulic retention time of 5 days and a liquid upflow velocity of 0.01 m/h. Under steady-state conditions, the lactose-fed sludge had much higher (20%–100%) specific methanogenic conversion rates than the volatile-fatty acid␣(VFA)/ethanol-fed sludge for all substrates tested, including VFA. In both reactors, a flocculant sludge developed, although a much higher content of extracellular polysaccharide was measured in the lactose-fed sludge [1900 μg compared to 305 μg uronic acid/g volatile suspended solids (VSS)]. When the liquid upflow velocity of a third, VFA/ethanol-fed reactor was increased to 0.5 m/h, granulation of the sludge occurred, accompanied by a large increase (200%–500%) in the specific methanogenic conversion rates for the syntrophic and methanogenic substrates studied. Granulation reduced the susceptibility of the sludge to flotation. Glucose was degraded at a high rate (100 mg glucose gVSS⁻¹h⁻¹) by the sludge from the third reactor, despite not having been exposed to a sugar-containing influent for 563␣days. Received: 7 June 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

Purification and some properties of a novel microbial lactate oxidase

 Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability. Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996     

Effects of electron donor and microbial concentration on the enhanced dechlorination of carbon tetrachloride by anaerobic consortia

 Reductive dechlorination of carbon tetra-chloride (CCl₄) by anaerobic bacterial communities from anaerobic digester sludge with the amendment of low concentrations of electron donors and microorganisms was undertaken to evaluate the influence of electron donors and microbial concentration on the rate of dechlorination of CCl₄. Humic acid, acetate, and glucose were selected to examine the feasibility of the electron donor with respect to the remediation of a contaminated subsurface. The addition of an electron donor and microorganisms significantly enhanced the dechlorination rate of carbon tetrachloride. The addition of an electron donor increased the cell numbers of anaerobic consortia, thereby increasing the rate of dechlorination. Glucose was a better electron donor than acetate and humic acid under reducing environments. The pseudo-first-order degradation rate constants of CCl₄ ranged from 0.0057 day^-1 to 0.135 day^-1, depending on the conditions of the electron donor and biomass supplemented. Furthermore, the addition of the electron donor in the batches amended with 0.56 mg volatile suspended solids (VSS)/l biomass had a higher enhanced efficiency than those with 1.7 mg VSS/l biomass. These results suggest that there is a potential for stimulating the dechlorinating capability of anaerobic consortia to remedy the chlorinated hydrocarbons in the oligotrophic environment if the conditions of the supplementing electron donor are properly selected. Received: 14 August 1995/Received last revision: 15 March 1996/Accepted: 15 April 1996     

Induction of gene expression for nitric oxide synthase by immunomodulating drugs in the RAW264.7 murine macrophage cell line

 We have elucidated the direct effects of PSK (a protein-bound polysaccharide) and OK-432 (a streptococcal preparation), both immunomodulating drugs, on the gene expression for an inducible nitric oxide synthase and on the production of nitric oxide (NO) in the RAW264.7 murine macrophage cell line. As determined by northern blot analysis, both immunomodulating drugs were potent inducers of gene expression for inducible NO synthase when cells were costimulated with interferon-γ (IFNγ). Expression of mRNA for the enzyme occurred in a dose-dependent manner after 3 h, when 10 – 50 μg/ml PSK or 0.001 – 1 KE/ml OK-432 was used. Furthermore, NO was also produced in response to these drugs, as detected by the Griess reagent reaction. The enhancement of NO synthesis was thought to be mediated, in part, through tumor necrosis factor α (TNFα) induction by these agents, since a neutralizing antibody to TNFα significantly suppressed NO production in RAW264.7 cells stimulated with PSK or OK432 in combination with IFNγ. We speculate that NO production may play a role in tumoricidal and microbicidal activities of PSK or OK-432 in vivo. Received: 9 August 1995 / Accepted: 1 April 1996     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996     

The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier,OK-432

 Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432. Received: 29 April 1996 / Accepted: 27 July 1996     

Simultaneous utilization of pyridine and fructose by Rhodococcus opacus UFZ B 408 without an external nitrogen source

 A bacterium classified as Rhodococcus opacus, which is able to use pyridine (a potentially growth-inhibiting substrate) as its sole source of carbon, energy and nitrogen, was isolated. In a carbon-limited chemostat culture, the kinetics was determined for growth on both pyridine and a mixture of pyridine and fructose (9 mM/22.15 mM). With growth on pyridine, stable steady states were achieved up to dilution rates of about 0.1 h^-1. A further increase in the dilution rate resulted in the progressive accumulation of pyridine in the culture liquid and the cells were washed out. The maximum specific growth rate (μ_max = 0.23 h^-1) and the K _S value (0.22 mM) for growth on pyridine were determined from the residual pyridine concentrations measured within the range of stable steady states. With growth on the substrate mixture, the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone, and stable steady states were established at dilution rates of up to 0.13 h^-1. The maximum pyridine degradation rate was enhanced to 270 mg pyridine l^-1 h^-1 compared to 210 mg pyridine l^-1 h^-1with growth on pyridine as a single substrate. An external nitrogen source did not need to be added in the case of growth on the substrate mixture. Fructose was assimilated by means of ammonium released from pyridine. Analysis of the nitrogen balance furnished proof that pyridine is an energy-deficient substrate; pyridine was assimilated and dissimilated at a ratio of 1 mol/0.67 mol respectively. The resulting yield coefficient was about 0.55 g dry weight/g pyridine. Moreover, it was demonstrated that, in regard to the biologically usable energy, 1 mol pyridine corresponds to 0.43 mol fructose. Received: 3 July 1995/Received revision: 19 October 1995/Accepted: 24 October 1995     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

Analysis of anchor residues in a naturally processed HLA-DR53 ligand

 The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. Received: 29 April 1996 / Revised: 16 June 1996     

Suppressive effects of Lactobacillus casei cells,a bacterial immunostimulant,on the incidence of spontaneous thymic lymphoma in AKR mice

 The mean survival age of female AKR/J mice was significantly prolonged, the enlargement of thymus was markedly suppressed, and the proliferation of ecotropic and recombinant murine leukemia viruses (MuLV) was markedly inhibited when 8-week-old female AKR/J mice were injected intraperitoneally (i. p.) with heat-killed Lactobacillus casei cells twice weekly for 8 weeks. In contrast, such actions of heat-killed L. casei cells were not seen in 20-week-old female AKR/J mice. The leukemogenic activity of the cell-free extract of thymus from adult female AKR/J mice in newborn female AKR/J mice was drastically reduced by i. p. treatment with heat-killed L. casei cells. The difference in adjuvant effectiveness of heat-killed L. casei cells on 8- and 20-week-old animals may be dependent on the difference in the enhancing activity of the cell-mediated immune systems between the groups induced by heat-killed L. casei cells, and, as a result, on the difference in the degree of proliferation of ecotropic and recombinant MuLV in thymus, which consequently causes thymic lymphoma. Received: 13 February 1996 / Accepted: 18 April 1996     

Enrichment culture and isolation of slow-growing bacteria

 Enrichment containing large numbers of slow-growing bacteria was developed by repeated batch culture under high biomass concentrations (more than 10 000 mg biomass/l). The characteristics of slow-growing bacterial populations were elucidated by application of colony-forming-curve (CFC) analysis. The CFC were obtained by counting the number of visible colonies on agar plates at successive intervals. The enrichment consisted of several groups with different colony-forming rates and the slow-growing bacteria appeared on cell extract/agar plates after 7 days of incubation. It was found that large numbers of slow-growing bacteria survived under starvation conditions. One of the slow colony-forming bacteria, strain TI-X7, was tentatively identified as being of the genus Micrococcus. The enrichment contained a large amount of Micrococcus-like tetrad cells. The dialysate fractions in excess cell extract, permeable through dialysis tubing, were extremely effective for growth of strain TI-X7. Received: 15 December 1995/Accepted: 20 February 1996     

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae

 β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis. Received: 18 January 1995/Received last revision: 23 August 1995/Accepted: 13 September 1995     

Stable carbon isotopes in tree rings of beech: climatic versus site-related influences

 Stable carbon isotopes in tree rings are a promising tool in palaeoclimate research, provided attempts are made to disentangle climatic from local effects (e.g. soil properties, competition, light). The ¹³C/¹²C variations in cellulose of tree rings of beech (Fagus sylvatica) were determined at several sites in the Swiss Central Plateau covering the last 50 years. We chose sites which differ in moisture conditions and sampled cores from four to six trees per site. The mean ¹³C/¹²C series from the different dry sites (distant by up to 40 km) are closely interrelated suggesting a common external cause. Correlation analysis with climate data proved the total precipitation in the months May, June and July to have the strongest effect on the carbon isotopes (r =  – 0.73). This result is in agreement with the commonly used model which relates the isotope discrimination to the water use efficiency. On the other hand, the isotope series of the wet sites are not as well correlated to the climate. At two of the sites (a dry and a humid) tree ring width suddenly increased. We used this effect as a test-case to study the influence of local growth conditions on the climate-isotope relationship. Received: 17 April 1996 / Accepted: 2 September 1996     

MHCDB: database of the human MHC (release 2)

 The second release of the human major histocompatibility complex (MHC) database is now publicly available. It contains an updated physical map and considerably more genomic sequence. cDNA sequences of all current alleles are accessible as individual sequence entries. The variability of different genes is displayed graphically as static and dynamic images accessible from the database. Known disease-serotype associations have also been incorporated, together with data from the MHCPEP database of eluted peptides. Received: 13 February 1996 / Revised: 12 August 1996