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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 6, Issue 6 - Dec 1996
Volume 6, Issue 5 - Oct 1996
Volume 6, Issue 4 - Aug 1996
Volume 6, Issue 3 - Jun 1996
Volume 6, Issue 2 - Apr 1996
Volume 6, Issue 1 - Feb 1996
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-Lactamase Inhibitory Protein from Streptomyces exfoliatus SMF19 and Cloning of the Corresponding Gene
PARK, HYEON-UNG ; KYE JOON LEE ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 369~374
-lactamase inhibitory protein (BLIP) produced by Streptomyces exfoliatus SMF19 was purified(33 kDa) and the N-terminal amino acid sequence was determined as NH2-ATSVVAWGGNND. Genomic DNA library of S. exfoliatus SMF19 was constructed in pWE15 and recombinants harbouring the corresponding gene were selected by colony hybridization to the mixture of 36-mer oligonucleotide designed from the N-terminal amino acid sequence. The corresponding gene (bliX) was isolated on a 4-kb ApaI fragment of S. exfoliatus SMF19 chromosomal DNA and then sequenced. The bliX consisting of 1, 119bp encoded a mature protein with a deduced amino acid sequence of 342 residues and also encoded a 40-amino-acid signal sequence. No significant sequence similarity to bliX was found by pairwise comparison using various protein and nucleotide sequences.
Cloning and Characterization of GL-7-ACA Acylase Gene from Pseudomonas sp. GK16
LEE, YOUNG-SIK ; HAN-CHUL YANG ; SUNG-SOO PARK ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 375~380
The gene coding for glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase was cloned from Pseudomonas sp. GK16 and some of its characteristics were analyzed. The complete nucleotide sequence revealed that the putative open reading frame is 2160 bases long and encodes 720 amino acids. By SDS-PAGE three proteins, approximately corresponding to 70, 54 and 16 kDa of molecular weight, were detected in E. coli cells carrying pGAP18. The largest protein should be a precursor which is not processed yet, while the other two proteins must be derived from the precursor by the proteolytic processing.
A Plasmid of Lactococcus lactis subsp. lactis ML8 Linked with Lactose Metabolism and Extracellular Proteinase
LEE, JONG-HOON ; HYONG JOO LEE ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 381~385
Three distinct plasmids, with approximate molecular weights of 1, 4.5, and 33 megadaltons, were found in Lactococcus lactis subsp. lactis (L. lactis) ML8. Slow acid-producing mutants of L. lactis ML8, isolated by plasmid curing with acriflavine treatment, lacked the 33-megadalton plasmids. The plasmid-cured mutant showed lactose-negative (Lac) characteristics and the alteration of extracellular proteinase pattern. The possible involvement of extracellular proteinase with the 33-megadalton plasmid is highlighted in this research.
Molecular Cloning of a
-D-Galactosidase Gene from Lactococcus lactis subsp. lactis 7962
CHANG, HAE-CHOON ; YANG-DO CHOI ; HYONG-JOO LEE ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 386~390
-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing
-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned
-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the
-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was
, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.
-dependent NADH:quinone Oxidoreductase in the Respiratory Chain of the Marine Bacterium Marinomonas vaga
Kim, Young-Jae ; Park, Yong-Ha ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 391~396
The Gram-negative marine bacterium Marinomonas vaga, which requires 0.5 M NaCl concentration for optimal growth, is slightly halophilic. The growth of M vaga was highly resistant to the proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP) under alkaline pH conditions (pH 8.5) but very sensitive to CCCP under acidic pH conditions (pH 6.5). These results suggest that the respiratory chain-linked NADH oxidase system of M. vaga may lead to generation of a
electrochemical gradient. In order to examine the existence of
-stimulated NADH oxidase in M. vaga, membrane fractions were prepared by the osmotic lysis method. The membrane-bound NADH oxidase oxidized both NADH and deamino-NADH as substrates and required
for maximum activity. The maximum activity of NADH oxidase was obtained at about pH 8.5 in the presence of 0.2 M NaCl. The site of
-dependent activation in the NADH oxidase system was at the NADH:quinone oxidoreductase segment. The NADH oxidase and NADH:quinone oxidoreductase were very sensitive to the respiratory chain inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) in the presence of 0.2 M NaCl but highly resistant to another respiratory inhibitor, rotenone. Based on these findings, we conclude that M. vaga possesses the
-dependent NADH:quinone oxidoreductase that may function as an electrogenic
Production of Cycloinulooligosaccharide Fructanotransferase (CFTase) from Bacillus sp. CFCl
Kim, Hwa-Young ; Park, Jeong-Bok ; Kwon, Young-Man ; Choi, Yong-Jin ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 397~401
A bacterial strain CFCl, which produced an extracellular cycloinulooligosaccharide fructanotransferase(CFTase), was isolated from soil. The isolated strain was identified as a strain of Bacillus sp. The synthesis of CFTase by the bacterium was found to be induced by inulin which was added to the culture medium as a carbon source. The highest activity of CFTase was observed at pH 7.5 and
in the medium containing 4% inulin and 0.5% peptone as a carbon source and a nitrogen source, respectively. Under the optimal conditions, the enzyme activity in the culture supernatant reached the highest level of 85 munits/ml after 96 h cultivation.
Purification and Characterization of Inulin Fructotransferase (Depolymerizing) from Arthrobacter sp. A-6
PARK, JEONG-BOK ; YONG-JIN CHOI ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 402~406
Inulin fructotransferase (depolymerizing) (EC 184.108.40.206) was purified 34-fold from the culture broth of Arthrobacter sp. A-6 by using a combination of ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The purified enzyme converts inulin into di-D-fructofuranose dianhydride III(DFA III) and small quantities of fructo-oligosaccharides. The temperature and pH optima of the enzyme were
and 6.0, respectively. Molecular weight of the enzyme was determined to be 49 kDa by 12
SDS-polyacrylamide gel electrophoresis, and 145 kDa by Sephacryl S-200gel filtration. This indicates that the functional inulin fructotransferase of Arthrobacter sp. A-6 has a homomeric trimer structure. The enzyme had an isoelectric point of pH 4.6. The N-terminal amino acid sequence of the purified enzyme subunit was Ala-Asp-Asn-Pro-Asp-Gly(\ulcorner)-Ser-Asn-Met(or Glu)-Tyr-Asp-Val.
Purification and Biochemical Properties of Extracellular Phospholipase
from Serratia sp. MK1
Kim, Myung-Kee ; Rhee, Joon-Shick ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 407~413
A novel type of extracellular phospholipase
was isolated from Serratia sp. MK1 and purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The purified enzyme was a monomer with a molecular mass of about 43, 000 Da. This enzyme showed the highest lipolytic activity toward phosphatidylserine among the phosphoglycerides tested, and preferentially catalyzed the hydrolysis of the ester bond in phosphatidic acid to lyso-phosphatidic acid. Enzyme activity was completely inhibited by the addition of a chelating agent such as EDTA, and inhibited enzyme activity was fully recovered by the presence of
. This implies that the enzyme requires
for activity. The enzyme was stable up to
when incubated for 1 h at pH 8.5, and the optimal pH and temperature were 8.5 and
Purification and Characterization of Clostridium thermocellum Xylanase from Recombinant Escherichia coli
Koo, Bon-Joon ; Oh, Hwa-Gyun ; Cho, Ki-Haeng ; Yang, Chang-Kun ; Jung, Kyung-Hwa ; Ryu, Dai-Young ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 414~419
The xylnX gene encoding a xylanase from Clostridium thermocellum ATCC27405 was cloned in the plasmid pJH27, an E. coli-Bacillus shuttle vector and the resultant recombinant plasmid, pJX18 was transformed into E. coli HB101. The overexpressed xylanase was found to be secreted into the periplasmic space of the recombinant E. coli cells. The crude enzyme was obtained by treating the E. coli cells with lysozyme, and purified by DEAE-Sepharose column chromatography. Molecular wieght of the xylanase was estimated to be 53 kDa by gel filtration. The pI value was determined to be pH 8.8. The N-terminal sequence of the enzyme protein was Asp-Asp-Asn-Asn-Ala-Asn-Leu-Val-Ser-Asn which was considered to be the sequence of that of the mature form protein. The Km value of the enzyme for oat spelt xylan was calculated to be 2.63 mg/ml and the Vmax value was
. The xylanase had a pH optimum for its activity at pH 5.4 and a temperature optimum at
. The enzyme hydrolyzed xylan into xylooligosaccharides which were composed mainly of xylobiose (40%) and xyloltriose (12%) after 5 hour reaction. This result indicates that the xylanase from C. thermocellum ATCC27405 is an endo-acting enzyme.
Cultivation of Phanerochaete chrysosporium and Lignin Peroxidase Activity
Kim, Yeong-Kwan ; Kim, Gieun ; Jeong, Myoung-Sun ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 420~424
Effects of exogenous veratryl alcohol addition on the growth of basidiomycete Phanerochaete chrysosporium ME-446 and the induction of lignin peroxidase activity were investigated in this study. The organism was grown in ligninolytic (low-nitrogen) culture conditions in which extracellular enzymes are produced. Analyses showed that a statistically significant decrease of cell growth was associated with the veratryl alcohol addition. The effect of veratryl alcohol addition on LiP activity was nearly instantaneous and this effect diminished with culture aging. The extent of this effect was different depending on the time of addition, which led to a speculation that there might be some other effector species which played a role in regulation of lignin peroxidase activity.
Enzymatic Characteristics of Biosynthesis and Degradation of Poly-
-hydroxybutyrate of Alcaligenes latus
Kim, Tae-Woo ; Park, Jin-Seo ; Lee, Yong-Hyun ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 425~431
The enzymatic characteristics of Alcaligenes latus were investigated by measuring the variations of various enzyme activities related to biosynthesis and degradation of poly-
-hydroxybutyrate (PHB) during cultivation. All PHB biosynthetic enzymes,
-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase, were activated gradually at the PHB accumulation stage, and the PHB synthase showed the highest value among three enzymes. This indicates that the rate of PHB biosynthesis is mainly controlled by either
-ketothiolase or acetoacetyl-CoA reductase rather than PHB synthase. The enzymatic activities related to the degradation of PHB were also measured, and the degradation of PHB was controlled by the activity of PHB depolymerase. The effect of supplements of metabolic regulators, citrate and tyrosine, was also investigated, and the activity of glucose-6-phosphate dehydrogenase was increased by metabolic regulators, especially by tyrosine. The activities of
-ketothiolase and acetoacetyl-CoA reductase were also activated by citrate and tyrosine, while the activity of PHB depolymerase was depressed. The increased rate and yield of PHB biosynthesis by metabolic regulators may be due to the increment of acetyl-CoA concentration either by the repression of the TCA cycle by citrate through product inhibition or by the activation of sucrose metabolism by the supplemented tyrosine.
Enzymatic Hydrolysis of Crystalline Chitin in an Agitated Bead Reaction System and Its Reaction Characteristics
Lee, Yong-Hyun ; Bae, Young-Ki ; Jeong, Eui-Jun ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 432~438
Native crystalline chitin was hydrolyzed in an agitated bead reaction system using crude chitinase excreted from Aspergillus fumigatus JC-19. The reaction was enhanced significantly, and the concentration and yield of reducing sugar after 48 hours were measured to be 35.42 g/I (w/v) and 0.64, respectively, around 1.86 times higher than those of the conventional system that was carried out without glass beads. The effect of reaction conditions, such as the amounts of chitin, chitinase and glass beads, and the size of glass bead, were examined. Ball milled chitin was also hydrolyzed in the agitated bead reaction system, the conversion yield and reaction rate of ball milled chitin for 24 hours increased up to 0.87 and 48.02 g/I, respectively. Chitinase showed relatively high stability in the agitated bead reaction system, particularly in the presence of enzyme stabilizer,
, which played a critical role in preventing the deactivation of chitinase by the physical impact of glass beads. The variations of the structural features of chitin during the reaction were followed by SEM and X-ray diffraction, and the enhanced hydrolysis reaction was caused by both the fragmentation of chitin particles and the destruction of the crystalline structure owing to the synergic effects of the attrition of glass beads and the hydrolytic action of chitinase.
Isolation of Chitin-utilizing Bacterium and Production of Its Extracellular Chitinase
Woo, Cheol-Joo ; Yun, Un-Jung ; Park, Heul-Doung ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 439~444
A bacterial strain, designated as WY22, producing extracellular chitinase was isolated from the soil around the Youngduck area, after enrichment culture in a medium containing
(w/v) wet colloidal chitin as a sole carbon source. The isolate was identified as a strain of Bacillus sp. based on its morphological and physiological characteristics. It was observed that Bacillus sp. WY22 could inhibit the growth of Fusarium oxysporum with hyphal extention-inhibition assay on potato dextrose agar plate supplemented with
collidal chitin. Optimum culture conditions of Bacillus sp. WY22 were examined for chitinase production in a chitin medium. High level production of chitinase was observed not only in the chitin medium but in a medium supplemented with
N-glucosamine or lactose instead of chitin. The optimum concentrations of colloidal chitin and yeast extract were 3.0 and
, and the optimum culture conditions for initial pH of medium and temperature were 7.0 and
, respectively, for the production of chitinase.
Pyridoxatin, an Inhibitor of Gelatinase A with Cytotoxic Activity
Lee, Ho-Jae ; Chung, Myung-Chul ; Lee, Choong-Hwan ; Chun, Hyo-Kon ; Kim, Hwan-Mook ; Kho, Yung-Hee ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 445~450
Gelatinase A is a member of the matrix metalloproteinases that play an important role in cancer invasion and metastasis. In the course of screening gelatinase A inhibitors from microbial sources, a fungal strain PT-262 showed a strong inhibitory activity. The strain was identified as Chaunopycnis alba on the basis of its morphological characteristics. The inhibitor was isolated from acetone extract of mycelial cake by sequential chromatographies on MCI-gel, Sephadex LH-20, and a reverse-phase HPLC column. The purified inhibitor was identified as pyridoxatin by its physico-chemical properties and spectroscopic analysis. Pyridoxatin is not a peptide analog and has cyclic hydroxamic acid moiety. It inhibited activated gelatinase A with an
value of 15.2
using fluorescent synthetic peptide. It also had a strong cytotoxicity against human cancer cell lines in vitro. Furthermore, this compound inhibited DNA synthesis with an
value of 2.92
in PC-3 prostate cancer cells by [
]thymidine incorporation assay.
Production of Toxin Protein by Recombinant Escherichia coli with a Thermally Inducible Expression System
Jong, Se-Han ; Chang, Ho-Nam ; Chang, Yong-Keun ; Rhim, Seong-Lyul ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 451~455
Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to
to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at
and then 5-8 hours at
. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.
Protein Aggregation and Adsorption upon In vitro Refolding of Recombinant Pseudomonas Lipase
Lee, Young-Phil ; Rhee, Joon-Shick ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 456~460
Recombinant Pseudomonas lipase was used to study protein aggregation and adsorption upon in vitro refolding. Protein adsorption as well as aggregation was responsible for major side reactions upon in vitro refolding as a function of protein concentration. The optimal range of protein concentration was determined by the relative contribution of protein aggregation and adsorption. Above the optimal range, the yield of active lipase inversely correlated with protein aggregation, showing a competition between folding and aggregation. However, adsorption of protein rather than protein aggregation is thought to contribute as a major side reaction of the refolding process at sub-optimal concentrations at which the formation of aggregates should be more reduced. Protein aggregation was influenced by the amount of guanidine hydrochloride in the refolding solvent. The refolding temperature was a critical factor determining the extent of protein aggregation. The refolding yield was also affected by the dilution fold and dilution mode, which suggests that the refolding process might kinetically compete with the rate of mixing.
Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Antagonistic Effects in Sausage Fermentation
Kim, Wang-June ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 461~467
Four strains of lactic acid bacteria (LAB), that lower the pH of sausage
4.2 within 24 h of incubation at
, were screened from 57 bacteriocin producing LAB which were isolated from kajamie shikhae and natural fermented sausages. The proteinaceous nature of the bacteriocin was confirmed by losing antimicrobial activity after pronase treatment. Inhibitory activity against pathogens, times of bacteriocin production and sensory tests were compared between 4 isolates and 3 commercial starters. Especially, strain NFS #8-1, screened from natural fermented sausage and identified as Pediococcus acidilactici, antagonized a large number of foodborne pathogens including Listeria monocytogenes, Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Salmonella typhimurium and Staphylococcus aureus. Production of bacteriocin by strain NFS #8-1 was early in the growth phase (mid log phase) and its sensory acceptance was high. The feasibility of using strain NFS #8-1 as a starter for the production of microbiologically safe fermented sausage is envisaged.
Functional Analysis of the Tomato Spotted Wilt Virus(TSWV) NSm Protein by Using Immunoblotting and Immunogold Labelling Assay
Choi, Tae-Jin ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 468~473
The genome of tomato spotted wilt virus (TSWV) is composed of three RNA segments, S, M, and L RNA and the 5.0 kb M RNA encodes two glycoproteins Gl, G2 and NSm protein of unknown function. In an effort to investigate the function of the NSm protein, antibody was raised against NSm fusion protein overexpressed in Escherichia coli. This antibody was used to detect the NSm protein by using western blot analysis and electron microscopic observation after immunogold labelling. For the cloning of the NSm gene, total RNA extracted from a TSWV infected plant was used for cDNA synthesis and polymerase chain reaction (PCR) instead of going through time-consuming virus purification. A protein band specifically reacting to the NSm antibody was detected from TSWV inoculated plants. The NSm protein was detected in the cell wall fraction and in pellet from low speed centrifugation when the infected plant tissue was fractionated into 4 fractions. In the immuno-electron microscopic observation, gold particles were found around the plasmodesmata of infected plant tissue. These results suggest that the NSm protein of TSWV plays some role in cell-to-cell movement of this virus.
Rabbit Antibody Raised against Murine Cyclin D3 Protein Overexpressed in Bacterial System
Jun, Do-Youn ; Kim, Mi-Kyung ; Kim, Young-Ho ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 474~481
Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl
-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.
Effect of pH on the Production of Lactic Acid and Secondary Products in Batch Cultures of Lactobacillus casei
Yoo, Ik-Keun ; Chang, Ho-Nam ; Lee, Eun-Gyo ; Chang, Yong-Keun ; Moon, Seung-Hyeon ;
Journal of Microbiology and Biotechnology, volume 6, issue 6, 1996, Pages 482~486
Batch fermentations of lactic acid were performed with Lactobacillus casei to investigate the effect of pH on cell growth and production of lactic acid and by-products. Maximum productivity of lactic acid increased with increasing pH from 5.0 to 6.5, and the extent of D-lactate production was different at each pH. Acetate and D-lactate concentrations increased even after the complete consumption of glucose in the medium. While a pH range of 6.0-6.5 was optimal for cell growth and lactic acid production, superior results were achieved at pH 6.0 when both maximum lactic acid productivity and minimum by-product formation were considered.