(4R)-[(1S)-1-[(tert-Butyldimethylsilyl)oxy]-1-(2-methoxy-4,5-methylenedioxyphenyl)methyl]-3-methylenedihydro-2(3H)-furanone and (4R)-[(1R)-1-[(tert-butyldimethylsilyl)oxy]-1-(2-methoxy-4,5-methylenedioxyphenyl)methyl]-3-methylenedihydro-2(3H)-furanone, which are key intermediates for the synthesis of 1,2-oxidized furofuran lignan, were each stereoselectively synthesized from L-glutamic acid.

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  Cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy analogue (DIMBOA), occur transiently in high amounts in wheat and maize during the juvenile, non-autotrophic stage of growth. To elucidate the biosynthetic enzymes operating for the transient production of these compounds, we examined the hydroxylating activities for 2-hydroxy-1,4-benzoxazin-3-one (HBOA), the immediate precursor of DIBOA, and indole, the first intermediate of the biosynthetic pathway that branches off from the tryptophan pathway, by using microsomes prepared from wheat seedlings. Both hydroxylases occurred soon after germination, reached a maximum 48 h after germination, and decreased to finally disappear as the plants grew into the autotrophic growth stage. The mode of appearance and disappearance similar to that of hydroxamic acids, suggesting that elevated expression of the whole set of enzymes involved in the biosynthesis after indole was responsible for the transient occurrence of hydroxamic acids. The hydroxylating activity was also observed for 1,4-benzoxazin-3-one, a putative precursor of HBOA, but to significantly less extent than that for HBOA and indole.

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  In order to elucidate the enzyme-substrate-cofactor interaction in 3-isopropylmalate dehydrogenase, the coenzyme activity of NAD analogs which have a 3-substituted pyridine ring was examined. Analogs 3-5 showed diminished k_cat values compared with those of NAD⁺, whereas thiocarboxamide 2 was almost as equally active as NAD⁺. This suggests that the NH₂ functionality of NAD⁺ is more important for the catalysis of IPMDH than a carbonyl group.

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  Bioassay-guided isolation of anti-inflammatory compounds from the methanol extract of Myrsine seguinii yielded an anti-inflammatory compound (1). The structure of compound 1 was elucidated to be 3-geranyl-4-hydroxy-5-(3′-methyl-2′-butenyl)benzoic acid on the basis of its spectroscopic data. Compound 1 strongly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 μg (inhibitory effect (IE): 65%). The acetate and the methyl ether of 1 showed moderate activity at a 500-μg application, with IE 38% and 27%, respectively. However, the methyl ester and the dimethyl derivative of 1 did not show activity at the same dose. The related compounds of 1, o-, m- and p-hydroxybenzoic acids also did not exhibit notable activity. These results indicate that the carboxylic acid and lipophilic terpene moieties of 1 were significant structural features for anti-inflammatory activity.

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  The newly established GUS expression bioassay on the callus extracts of 22 species of plants revealed that the methanol extract of Phytolacca americana callus had the most potent inhibitory activity against agrobacterial plant transformation. A triterpene glycoside phytolaccoside B was isolated from the extract as a genuine plant transformation inhibitor having neither antiagrobacterial nor phytotoxic activity. This compound is promising for use as a biochemical probe for studies on the plant transformation mechanism.

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  Eleven taxoids were isolated from needles of the Japanese yew, Taxus cuspidata Sieb. et Zucc., one of them being a new compound whose structure was established as5α-cinnamoyl-9α,10β,13α-triacetoxy-11(15→1)abeotaxa-4(20),11-dien-15-ol (7-deactoxytaxuspine J) on the basis of a spectroscopic analysis. Its relative stereochemistry is proposed from the results of a NOESY experiment.

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  A preparative-scale asymmetric synthesis of (R)-α-fluorophenylacetic acid, a useful chiral derivatizing reagent, is described. Starting from ethyl α-bromophenylacetate, α-fluorophenylmalonic acid dipotassium salt was prepared in three steps (54% yield), including nucleophilic substitution by the fluoride ion as the keystep. Both the purified form and crude preparation of arylmalonate decarboxylase in E. coli worked well on this substrate, and (R)-α-flurophenylacetic acid (>99% e.e.) was prepared in a quantitative yield.

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  A ²H-NMR analysis of 6α-hydroxy-3-oxo-pinguis-5(10)-ene-11,6-olide produced by axenic culture of liverwort Aneura pinguis in the presence of [4-²H₂]-labeled mevalonate clarified the presence of a 1,2-hydride shift and retention of deuterium at the C-4 position in the biosynthesis of pinguisane-type sesquiterpenes from FPP.

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  (-)-Indolactam-V (1) has the fundamental structure of potent tumor-promoting teleocidins. A new teleocidin-related metabolite, (-)-14-O-malonylindolactam-V (2), was isolated from the culture broth of Streptomyces blastmyceticum NA34-17 which produced a large quantity of 1 along with a small amount of (-)-14-O-acetylindolactam-V (3). Heat treatment of 2 in methanol readily produced 1 and 3, suggesting that S. blastmyceticum NA34-17 excreted 1 as a malonic acid conjugate to accumulate 1 and 3 in the culture broth during cultivation.

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  Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 μgl^-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34~40 μm) was observed after division in the medium containing 1~200 μgl^-1 2,4-D; and significant cell elongation (average cell length: 95~130 μm) was observed after cell division in the medium containing 500~2000 μgl^-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.

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  To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1′, 2′-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3′ end ³²P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3′-5′ exonuclease, ribonuclease H, and 3′-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3′-5′ exonuclease activity than that of E. coli exonuclease III.

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  α-Amylase (LAMY) from alkaliphilic Bacillus sp. strain KSM-1378 is a novel semi-alkaline enzyme which has 5-fold higher specific activity than that of a Bacillus licheniformis enzyme. The Arg124 in LAMY was replaced with proline by site-directed mutagenesis to increase thermostability of the enzyme. The wild-type and engineered LAMYs were very similar with respect to specific activity, kinetic values, pH-activity curve, and degree of inhibition by chelating reagents. Thermostability and structure stiffness of LAMYs as measured by fluorescence were increased by the proline substitution. The change of Arg124 to proline is assumed to stabilize the loop region involving aminio acid residues from 122 to 134. This is the first report that thermostability of an α-amylase is improved by proline substitution.

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  Inhibitory activities of 1-deoxynojirimycin and gluconolactone on Aspergillus niger glucoamylase were studied in relation to the subsite structure of the enzyme. Although both of these inhibitors are considered to bind at subsite 1 of the enzyme active site, 1-deoxynojirimycin showed competitive type inhibition but gluconolactone was a mixed type (or noncompetitive type) inhibitor for the hydrolysis of p-nitrophenyl α-D-glucoside. The former type of inhibition suggested that the main binding mode of the substrate was productive, but the latter, nonproductive. A possible way of explaining these apparent inconsistent results is to assume that the main binding mode of the substrate is productive and gluconolactone forms a nonproductive ternary complex with the enzyme and the substrate.

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  It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with β-naphthyl laurate, and β-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of β-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C_10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.

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  The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.

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  The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M_r, 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.

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  Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4′-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s,(2S)-flavanone 2-hydroxylase and isoflavone 2′-hydroxylase, were cloned.

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  His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of “sensor histidine (His)-kinases”, “response regulators”, and “histidine-containing (HPt) phosphotransmitters”. Results from recent intensive studies suggested that, in the higher plant Arabidopsis thaliana, His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we characterized, in vitro, the putative cytokinin-responsive CKI1 His-kinase, in terms of His-Asp phosphorelays. It was demonstrated for the first time that the receiver domain in this sensor exhibits a strong phosphohistidine phosphatase activity toward some Arabidopsis HPt phosphotransmitters (AHP1 and AHP2), suggesting the functional importance of the receiver domain for a presumed interaction of the sensor His-kinase with other His-Asp phosphorelay components.

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  θ-Toxin (perfringolysin O) modified by diethyl pyrocarbonate, a histidine-specific reagent, lost its hemolytic activity. The modified toxin retains the activities of binding to and insertion into cholesterol-containing membranes but lacks the ability to form oligomers. These results suggest that histidine residues of θ-toxin contribute their share to cytolysis, especially the oligomerization process.

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  A chitinase was purified from the culture filtrate of nigeran-degrading Arthrobacter sp. NHB-10 by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50 and Superose 12. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was 30,000 and its isoelectric point was 6.8. The optimum pH and temperature for the enzyme activity were 5.0 and 45°C, respectively. The enzyme was stable from pH 3 to 7 and up to 55°C. The enzyme activity was inhibited by Hg²⁺ and p-chloromercuribenzoic acid. Two internal amino acid sequences of the enzyme were AGPQLLTGYY and IGGVMT.

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  The digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture (IMO-H) was investigated. In an in vitro experiment, the digestibility of IMO-H was examined by models of the digestive system. IMO-H was resistant to two types of α-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa hydrolyzed tri-, tetra- and higher saccharide alcohols to disaccharide alcohol, removing successive glucose units from the non-reducing ends of the chains. The hydrolysis ratio for IMO-H was intermediate between the values for maltose and maltitol. In an in vivo study, growing rats were fed on an experimental diet containing IMO-H, maltitol, or hydrogenated palatinose in the range from 5% to 20%. The growth parameters of the rats fed on the test sugar show that the availability of IMO-H was about 1.2 to 1.25 times that of maltitol or hydrogenated palatinose.

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  Food preference in individual animals is regulated by brain activity. Two murine model systems for investigating food preference were developed by focusing on fruit juices. In a home-cage, two-bottle test, the volume of apple juice consumed was found to be much larger than that of orange juice. In a two-nozzle “Drinkometer” test, by which each mouse was kept in a 38 cm (W)×32 cm (D) cage and each drinking event was recorded by an electronic “Drinkometer” device, it was again found that the mice preferred drinking apple juice to orange juice. To elucidate the role of olfaction in this food preference, mice were subjected to an olfactory bulbectomy to remove the olfaction capability. In the home-cage two-bottle test, the preference for apple juice over orange juice was apparent even after the olfactory bulbectomy, indicating that olfaction was not essential for the formation of food preference behavior. In contrast, in the two-nozzle “Drinkometer” test, the preference for apple juice over orange juice was found to be abrogated by this surgery, implying the involvement of olfaction-based memory on food preference behavior.

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  The effects of a probiotic (a mixture of Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces and Candida) on the lipid metabolism, and caecal flora and metabolites of cocks were studied. The cholesterol level of the liver and serum was significantly decreased in the cocks fed on the cholesterol-enriched diet containing the probiotic. The distribution and frequency of occurrence of flora, and the chemical characteristics of the metabolites in the caecal content of the cocks were also affected by the inclusion of the probiotic in the basal and cholesterol-enriched diets. The Enterobacteriaceae species were significantly decreased in number, while the Bacillus, Streptococcus, Bifidobacterium and Lactobacillus species were significantly increased. The presence of yeast was observed, and the ammonia level was significantly reduced. The pH value, however, was not affected. The concentration of short-chain fatty acids in the caecal content of the cocks fed on the cholesterol-enriched diet supplemented with the probiotic was increased. It is, therefore, suggested that the incorporation of a probiotic in the diet would improve the balance of the intestinal flora and metabolites. Furthermore, it would also suppress the serum and liver cholesterol levels of cocks fed on the cholesterol-enriched diet.

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  (-)-Epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical, and 1,1-diphenyl-2-picrylhydrazyl radical. Examination of the scavenging effects of tea catechins and their glucosides on superoxide anion showed that the presence of at least an ortho-dihydroxyl group in the B ring and a galloyl moiety at the 3 position was important in maintaining the effectiveness of the radical scavenging ability. Stoichiometric factors of tea catechins were estimated to be 2 for (+)-catechin and (-)-epicatechin, 5 for (-)-epigallocatechin, 7 for (-)-epicatechin gallate, and 10 for (-)-epigallocatechin gallate.

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  6-O-β-D-Xylopyranosyl-β-D-glucopyranosides (β-primeverosides) of (Z)-3-hexenol and trans-linalool 3,7-oxide were newly isolated from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita). In addition, the already identified β-primeverosides of benzyl alcohol, methyl salicylate, and trans-linalool 3,6-oxide from an oolong tea cultivar were isolated from the Yabukita cultivar. It was confirmed that all aglycones of the linalool oxide glycosides isolated here were of the optically active S-form by chiral GC after enzymatic hydrolysis with glycosidase.

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  2-Amino-1-methyl-phenylimidazo[4,5b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine produced in cooked meat and fish, is known to be a carcinogen for rats and mice. This study provides the first evidence for hypertriglyceridemia in rats exposed to PhIP, suggesting its potential risk to induce not only carcinogenesis, but also atherosclerosis, and highlighting the potential importance of PhIP for humans.

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  A potent antioxidative 6-hydroxydaidzein (6-OHD) was newly isolated from soybean koji fermented with Aspergillus oryzae. 6-OHD, in addition to 8-hydroxydaidzein and 8-hydroxygenistein, were found to be present in various fermented soybean products, including their koji. Considering that these o-dihydroxyisoflavones had strong antioxidative activities, they may contribute to protecting from oxidative deterioration during the processing of fermented soybean products.

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  The DNA cleavage activities of (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCg) were examined with 16 different metal ions. Cu²⁺ with all the catechins facilitated DNA cleavage, while Ag⁺ with EGC and EC showed a strong repressive effect. The other metal ions examined showed little effect.

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  The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe²⁺ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 μmol O₂ uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-β-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55°C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 μM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 μM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 μmol O₂ uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 μmol O₂ uptake/mg/min) showed a large α-peak of cytochrome a at 602 nm and a smaller α-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an α-peak characteristic of heme a at 587 nm, but not the α-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe²⁺ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.

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  Microbial degradation of carbazole (CA), a model of hard-removal heterocyclic nitrogen compounds contained in petroleum oil, was examined using Sphingomonas sp. CDH-7 isolated from a soil sample by screening for CA-assimilating microorganisms. CDH-7 used CA as a sole source of carbon and nitrogen, and metabolized CA to ammonia via anthranilic acid as an intermediate product. When CDH-7 was cultivated in the medium containing CA at the concentration of 500 mg/l (2.99 mM), CA was completely degraded within 50 h. By the reaction with the resting cells of CDH-7, 500 mg/l of CA was completely degraded within 4 h, with 1.64 mM of ammonia accumulated in the reaction mixture. When CA was added at the concentration of 100 mg/l (0.599 mM) periodically to the reaction mixture ten times, 925 mg/l (5.54 mM) of CA was degraded within 48 h by the resting cells, and 4.50 mM of ammonia was accumulated in the reaction mixture with a 75.1% molar conversion yield based on total CA added. The resting cells could almost completely degrade CA in a two-liquid-phase system which consists of water and organic solvent, even in the presence of 20% (v/v) isooctane, n-hexane, cyclohexane, and kerosene as a model petroleum oil. In the presence of an organic solvent system such as 20% (v/v) p-xylene, toluene, and heptanol, however, CA degradation yields decreased.

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  NAD-dependent D-sorbitol dehydrogenase (EC 1.1.1.14) was crystallized from the cytosolic fraction of Gluconobacter suboxydans IFO 3257. This is the first example of the enzyme crystallized from acetic acid bacteria. The enzyme catalyzed oxidoreduction between D-sorbitol and D-fructose in the presence of NAD or NADH. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 5.1s. Gel filtration on a Sephadex G-200 column gave the molecular mass of 98 kDa for the enzyme, which dissociated into 26-kDa subunits on SDS-PAGE, indicating that the enzyme is composed of four identical subunits. Oxidation of D-sorbitol to D-fructose and xylitol to D-xylulose predominated in the presence of NAD at the optimum pH of 9.5-10.0. Reductions of D-fructose to D-sorbitol and D-xylulose to xylitol were also observed in the presence of NADH with the optimum pH around 6.0. The relative rate of D-fructose reduction was about one-fourth of that of D-sorbitol oxidation. NADP and NADPH were inert for the both reactions. Since the reation rate in D-sorbitol oxidation predominated over D-fructose reduction at some alkaline pH, the enzyme could be available for direct enzymatic measurement of D-sorbitol. Even in the presence of a large excess of D-glucose and other substances, reduction of NAD to NADH was highly specific and stoichiometric to the D-sorbitol oxidized.

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  The nucleotide sequence of the Clostridium stercorarium F-9 xynC gene, encoding a xylanase XynC, consists of 3,093 bp and encodes a 1,031-amino acids with a molecular weight of 115,322. XynC is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family IX cellulose-binding domain, and two S-layer homologous domains. Immunological analysis indicated the presence of XynC in the culture supernatant of C. stercorarium F-9 and in the cells, most likely on the cell surface. XynC purified from a recombinant E. coli was highly active toward xylan and slightly active toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, p-nitrophenyl-β-D-glucopyranoside, and carboxymethylcellulose. XynC hydrolyzed xylan and xylooligosaccharides larger than xylotriose to produce xylose and xylobiose. This enzyme was optimally active at 85°C and was stable up to 75°C at pH 5.0 and over the pH range of 4 to 7 at 25°C.

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  Human erythropoietin (EPO)-producing recombinant BHK cells were cultured in culture medium containing microcarriers, and then microcarriers attached with cells were replenished in the hollow fiber culture cassette. By culture for 14 days, it was possible to produce 450 μg of the recombinant EPO, which corresponded to over two-fold of the recombinant EPO production by control hollow fiber culture without microcarriers.

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