Cloning, Expression, and Purification of Insecticidal Protein Pr596 from Locust Pathogen Serratia marcescens HR-3

A novel insecticidal protein (Pr596) produced by Serratia marcescens HR-3 was found be a metalloprotease and responsible for insecticidal activity toward locusts. Two pairs of primers were designed to amplify Pr596, a putative open reading frame (ORF) by similarity search and the N-terminal amino-acid sequence of insecticidal protein. The results revealed that the ORF consisted of 1464 nucleotides encoding a protein of 487 amino-acid residues. Pr596 was cloned into expression vector pET32a(+) and was expressed in Escherichia coli BL21 (DE3)/pLysS strain with isopropyl-β-d-thiogalactopyranoside induction. The Pr596 was found to be highly expressed as inclusion bodies by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Pr596 inclusion bodies were isolated and subjected to Ni-NTA His Bind Resins (Pharmacia, Germany). Pr596 purified and refolded was revealed by SDS-PAGE and had proteolytic activity and insecticidal activity. Results suggested that there is a potential to develop this protein to be used as an alternative locus control agent.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strain A1

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coliXL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M _r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M _r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the -helix domain in Bacillus anthracisthermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50 °C.     

Characteristics of a novel secreted zinc-dependent endopeptidase of <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

A novel zinc-dependent metalloendopeptidase of Bacillus intermedius (MprBi) was purified from the culture medium of a recombinant strain of Bacillus subtilis. The amino acid sequence of the homogeneous protein was determined using MALDI-TOF mass spectrometry. The sequence of the first ten residues from the N-terminus of the mature protein is ASTGSQKVTV. Physicochemical properties of the enzyme and its substrate specificity have been studied. The molecular weight of the metalloproteinase constitutes 19 kDa, the K _m and k _cat values are 0.06 mM and 1210 sec⁻¹, respectively, and the pI value is 5.4. The effect of different inhibitors and metal ions on the enzyme activity has been studied. Based on the analysis of the amino acid sequence of the active site motif and the Met-turn together with the enzyme characteristics, the novel bacterial metalloproteinase MprBi is identified as a metzincin clan adamalysin/reprolysin-like metalloprotease.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus

Several entomopathogenic fungi produce toxins that could be used as bioinsecticides in integrated pest management programs. Paecilomyces fumosoroseus is currently used for the biological control of the whiteflies Bemisia tabaci and B. argentifolii. Supernatants from submerged batch culture, where the fungus produced abundant dispersed mycelium, conidia and blastospores, were toxic to the whitefly nymphs. The most abundant metabolite was purified by HPLC and identified by mass spectrometry and NMR as dipicolinic acid. Both the dipicolinic acid produced by the fungus and the chemically synthesized compound had insecticidal activity against third-instar nymphs of the insect. Dipicolinic acid was toxic to the whitefly nymphs in bioassays involving topical applications. In submerged culture, the specific growth rate of P. fumosoroseus was 0.054 h⁻¹, the specific glucose consumption rate was 0.1195 g g⁻¹ h⁻¹ and the specific dipicolinic acid production rate was 0.00012 g g⁻¹ h⁻¹. Dipicolinic acid was detected after 24 h when the fungus started growing; and dipicolinic acid production was directly correlated with fungal growth. Nevertheless, the yield was low and the maximal concentration was only 0.041 g l⁻¹. The maximal concentrations of conidia and blastospores (per milliliter) were 1.4×10⁸ and 7×10⁷, respectively.     

A Novel β-Agarase with High pH Stability from Marine Agarivorans sp. LQ48

A novel endo-type β-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40°C, the purified recombinant AgaA had a high specific activity of 349.3 μmol min⁻¹ mg⁻¹, a K _m of 3.9 mg ml⁻¹, and a V _max of 909.1 μmol min⁻¹ mg⁻¹ for agarose. The recombinant enzyme hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40°C and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0–11.0 for 1 h, a characteristic much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications.     

Characterization of a recombinant aryl β-glucosidase from Neosartorya fischeri NRRL181

An isolated gene from Neosartorya fischeri NRRL181 encoding a β-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V _max of 886 μmol min⁻¹ mg-protein⁻¹ and a K _m of 68 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.     

A unique β-agarase,AgaA, from a marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. strain PO-303

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg⁻¹, a K _m of 1.10 mg ml⁻¹, and a V _max of 22.5 μmol min⁻¹ mg⁻¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.     

High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l⁻¹ with an activity of 55,300 U ml⁻¹ in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.     

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Summary A metalloprotease from sorghum malt variety KSV8-I was purified by a combination of 4-M sucrose fractionation, ion-exchange chromatography on Q-Sepharose (Fast flow), gel-filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2128.7 U mg⁻¹ protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 35 kDa. The purified enzyme had optimal activity at 60 °C and maximal temperature stability between 40 and 60 °C but retained over 77% of its initial activity after incubation at 70 °C for 30 min. Both pH optimum and maximal stability were at 7.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. Using 0.2 ml of 5 mM solution of each metal ion, the purified protease was slightly (P<0.05) inhibited by Zn²⁺, appreciably (P<0.01) inhibited by Ca²⁺ and Co²⁺ and highly significantly (P<0.001) inhibited by Ag⁺, Ba²⁺, Hg²⁺, Mn²⁺ and Pb²⁺. The enzyme was equally highly significantly (P<0.001) inhibited by EDTA and hydrolysed casein to give the following kinetic constants: K_m = 21.0 mg ml⁻¹; V_max = 8.2 μmol ml¹ min⁻¹ and K_i = 0.390 mM.     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(?)-mandelic acid production

A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His₆-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V _max and K _m parameters were determined to be 27.9 μmol min⁻¹ mg⁻¹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(−)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(−)-mandelic acid.     

Production,purification and partial characterisation of a novel laccase from the white-rot fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> CBS 577.79

Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)⁻¹ and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55°C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60°C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99 × 10⁶ and 3.07 × 10⁶ M⁻¹ s⁻¹, respectively). Catalytic rate constants for typical N–OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s⁻¹), violuric acid (8.4 s⁻¹) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s⁻¹), were found to be higher than those reported for other high redox potential fungal laccases.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Efficient constitutive expression of chitinase in the mother cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and its potential to enhance the toxicity of Cry1Ac protoxin

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry⁻B. The recombinant plasmid was stably maintained over 240 generations in Cry⁻B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l⁻¹ Na₂CO₃–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.     

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.     

Characterization of a Novel Proteinous Toxin from Sea Anemone <Emphasis Type="Italic">Actineria villosa</Emphasis>

The sea anemone Actineria villosa expresses a lethal protein toxin. We isolated a novel 120-kDa protein, Avt120, from partially purified toxin and found it to possess extremely strong lethal activity. The 3,453-bp Avt120 gene translates to a 995-amino acid protein. The 50% lethal dose (LD₅₀) of purified Avt120 in mice was 85.17 ng. Among several tested cell lines, Colo205 cells were most sensitive to Avt120: 50% of them were damaged by 38.4 ng/mL Avt120. Avt120 exerted ATP degradation activity (10 μmol ATP h⁻¹ mg⁻¹), which was strongly inhibited by ganglioside GM1 to decrease the cytotoxicity of Avt120.     

Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties

Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension cultures to a specific activity of 4.68 μmol (mg protein)⁻¹ min⁻¹. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of 5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843–854). There was 62% identity with the eucaryotic FDH consensus sequence. Michaelis constants of approx. 20 μm (formaldehyde), approx. 50 μm (glutathione) and approx. 31 μm (NAD⁺) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and ω-hydroxyfatty acids served as substrates for both FDH preparations. The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation products. Received: 1 April 1998 / Accepted: 18 November 1998