Heat-induced gelation in a mixed system of α-lactalbumin (La) and β-lactoglobulin (Lg) was studied to elucidate the gelling properties of whey protein. An Lg concentration of 4% (w/v) was required for the formation of a self-supporting gel following heating at 80°C for 30 min in a 100 mM potassium phosphate buffer (pH 6.8). Solutions of La, even up to a protein concentration of 8% (w/v), did not gel under the same conditions. The addition of 6% La to 2% Lg caused a significant increase in the gel hardness, although each protein did not individually form a gel at these concentrations. By adding La to Lg, firmer gels were formed at a lower heating temperature, compared to that from Lg alone. La and Lg interacted to form a soluble aggregate through a thiol-disulfide interchange reaction during gel formation, and such an interaction was critical in the formation and stabilization of the gel network structure. We conclude that the enhancing effect of La on the gel hardness of Lg was due to the formation of a specific soluble aggregate, and that such an interaction between these proteins contributes to the properties of whey protein gels.

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This study investigates the relationship between the dietary components and quantitative changes in fecal concanavalin A-binding glycoprotein (CBGP) derived from small intestinal epithelia in rats. Although fecal CBGP increased with increasing protein content in the diet, the increase in fecal CBGP due to dietary protein was much smaller than that resulting from the addition of pectin, cellulose or Gobo dietary fiber (GDF) to the diet. The increasing effect of 10% GDF by weight added to a 20% casein high-sucrose diet (HSD) on fecal CBGP was the most pronounced without producing any gastrointestinal disorders, whereas that from the addition of pectin to HSD at a level of 5% significantly decreased the intestinal sucrase activity. A similar increase in fecal CBGP was observed when rice bran dietary fiber, carrot dietary fiber, Hiziki or Wakame was added to HSD, but these increasing effects on fecal CBGP were independent of their insoluble-soluble ratio. This and our previous observations suggest that such dietary fiber may prompt the renewal and exfoliation of small intestinal epithelia.

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Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malns pumila cv.Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65, 000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The K_m value for the enzyme was found to be 122 μM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyldithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.

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A stereochemically pure mimic of the sex pheromone components of the pine sawfly was synthesized. Acetates of (2S, 3S)- and (2S, 3R)-3-methylpentadecan-2-ol were prepared, and the pheromone activity of their mixture was compared with that of a true pheromone mixture of the acetates of (2S, 3S, 7S)- and (2S, 3R, 7R)-3, 7-dimethylpentadecan-2-ol. Although the potency of the true pheromone system was 50 times as high as that of the pheromone mimic, the mimic could attract a sufficient number of male sawflies in the field test.

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During an early stage of cold acclimation, prominent changes in protein-synthetic activities were found to occur in the crown, which is the part where the stem joins the root of winter wheat (Triticum aestivum L. cv. Horoshirikomugi). This stage was complete within a week of cold treatment, and from the protein-synthetic activities, this stage of cold acclimation could be divided into four phases. First, when the plant seedlings were placed at 0°C, there was a lag period of 1 d and no newly inducible proteins were formed during this time. During the second phase (1 to 2 d), as the first response to cold, 16 new proteins were synthesized and the active synthesis of 6 preexisting proteins was reinitiated, while syntheses of at least 5 preexisting proteins were depressed. During the third phase (2 to 5 d), the levels of most of the cold-inducible proteins reached a maximum, but synthesis of at least 6 preexisting proteins started to decrease. During the fourth phase (after 5 d), the synthetic activities of the 6 proteins returned to the original levels and synthesis of another set of 3 new proteins started. During this phase, the synthesis of both protein fractions, the cold-inducible and the preexisting proteins, reached a steady state. After this period, no major changes in the protein profile could be detected. During the third phase, the most active synthesis of the cold-inducible proteins, in particular, proteins designated C10 (M_r 53 k), C12a (M_r 46 k), and C12b (M_r 46 k), occurred, concurrent with the abrupt and transient decrease in the synthetic activities of a set of 6 preexisting proteins. These results suggest that, in addition to the induction of a set of new proteins, the preferential or selective synthesis of proteins required for accommodation to the cold environment takes place at an early stage of acclimation.

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A direct stereospecific conversion of acyclic vicinal diols into olefins was developed, and a study on the reaction mechanism elucidated that the reaction was s-cis with respect to the hydroxy groups and s-trans with respect to the iodo and hydroxy groups of the intermediate iodo alcohols.

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An enzyme linked immunosorbent assay system using a monoclonal antibody, 15E11, specific for a major allergen Der f II in house dust mite, was developed. This system detected only Der f II in the presence of Der p II and other allergens. The Der f II contents in several house dust samples significantly correlated with the numbers of the mites in the same house dust samples (n=14, r=0.88, p<0.001). These data showed that this system was useful for specific measurement of Der f II in house dusts.

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(2E, 4E)-geometry was assigned to 1233A [(7R, 2'R, 3'R)-11-[3'-(hydroxymethyl)-4'-oxo-2'-oxetanoyl]-3, 5, 7-trimethyl-2, 4-undecadienoic acid (1a)], an inhibitor of cholesterol biosynthesis. Both the ¹H- and ¹³C-NMR spectra of 1a and methyl ester 1b were compared with those of the four geometrical isomers of methyl 3, 5-dimethyl-2, 4-heptadienoate (2). NOE experiments on 1b revealed the presence of a remarkable NOE between the proton at C-2 and those of the C-17 methyl group. Similar NOE was also observed with (2E, 4E)-2. This fact suggests the predominant existence of stable s-cis-rotamers at the single bond between C-3 and C-4 of 1b and of (2E, 4E)-2. Some MM2 calculations were attempted to show the presence of two types of stable conformers in the case of the 3, 5-dimethyl-2, 4-dienoate system.

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Callus cultures were derived from flesh sections of young fruit of Gardenia jasminoides ELLIS on Murashige-Skoog agar medium supplemented with auxin and kinetin in the dark. Colored sections having deep yellow or orange color cells have been maintained for four years (32 generations) by subculturing on Linsmaier-Skoog Gelrite medium containing 1.0mg/liter of indole-3-acetic acid and 0.1 mg/liter of kinetin in the dark. High-performance liquid chromatography of the yellow pigment showed the presence of crocin as a main component, but at low levels. An excess of gentiobiose, which was tentatively identified by high-performance liquid chromatography, was liberated from the yellow pigment by ammoniacal hydrolysis, but its origin other than crocin remains unknown.

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The effects of ascorbic acid (AsA) on the formation process for a heat-induced get of fish meat (kamaboko) were examined. An investigation of the bonds influenced by adding AsA indicates that the aggregation of protein by noncovalent binding decreased and that by cross-linking, except for disulfide bonding, significantly increased in comparison with the control during a 30- min incubation at 40°C (suwari process). The results from the same investigation on a heat-induced gel incubated at 90°C for 30 min without using the suwariprocess, and the effects of AsA on the activity of transglutaminase indicate that this difference was derived not from activation of the enzyme by AsA but from the direct effect of AsA on the proteins. No effect of AsA on the increase in surface hydrophobicity of crude actomyosin at 40 and 90°C was found. Moreover, when the surimi with modified sulfhydryl groups was used, the disappearance of aggregation influenced by adding AsA and an accumulation of aggregates by noncovalent bonding during the formation of a heat-induced gel occured. These results suggest that polymerization during the formation of a heat-induced gel proceeded as follows : native proteins were first aggregated by noncovalent bonding, next by disulfide bonding, and finally by cross-linking apart from disulfide bonding, and that AsA improved the quality of a heat-induced gel by accelerating the formation of disulfide bonds.

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The influence on the insecticidal activity of haedoxan A of its 3-(3, 4-methylenedioxyphenyl) group in the 1, 4-benzodioxanyl moiety was examined with two (±)-(1S^*, 2R^*, 5R^*, 6S^*)-6-[(2R^*, 3R^*)-3-alkyl-6-methoxy-2-methoxymethyl-1, 4-benzodioxan-7-yl]-2-(2, 6-dimethoxyphenoxy)-1-hydroxy-3, 7-dioxabicyclo-[3.3.0] octanes. Replacement of the methylenedioxyphenyl group of haedoxan by methyl and n-butyl group resulted in a large decrease in the activity, indicating the importance of the 3-aryl group for the potent insecticidal activity of haedoxan.

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The methoxymethyl group of the 6-methoxy-2-methoxymethyl-3-(3, 4-methylenedioxyphenyl)-1, 4-benzodioxan-7-yl moiety of insecticidal lignans of Phryma was modified with several alkyl groups to evaluate the effect on activity of the substituents. The assay results make it evident that this activity was significantly modified by the chain length or bulkiness of the alkyl groups and that the oxygen atom of the methoxymethyl group was fairly important for enhancing the activity.

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Five analogs of the insecticidal lignan of the Phryma were synthesized by modifying its 3, 4-methyl-enedioxyphenyl group with a phenyl, 4-methoxyphenyl, 3-methoxyphenyl, 3, 4-dimethoxyphenyl, or 4-chlorophenyl group to evaluate the contribution of the methylenedioxy function to the activity. The assay results revealed that the 4-oxymethylene part of the 3, 4-methylenedioxyphenyl group collaborated more for strengthening the insecticidal activity than the 3-oxymethylene part did. Unexpectedly, the 3, 4-dimethoxyphenyl analog was totally inactive, even at a high dose level.

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The antiviral activity of natural phloroglucinols and of synthesized mono- and diacylphloroglucinols, and 2, 6-diacyl-4, 4-dialkylcyclohexa-1, 3, 5-triones was investigated. A correlation between the acyl chain length and inhibitory activity against vesicular stomatitis virus (VSV) was observed. Potent antiviral activity was found in di-isovalerylphoroglucinol. 2, 6-Diacyl-4, 4-dialkylcyclohexa-1, 3, 5-triones inhibited replication of the virus with low cytotoxicity.

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A new bio-antimutagen, petasiphenol [3-(3, 4-dihydroxyphenyl)-2-oxopropyl caffeate](1) was isolated from scapes of Petasites japonicum (AD₅₀=95 μg/ml against UV-induced mutagenic E. coli WP2 B/r Trp^-. Petasiphenol (1) and its isomer (2) were synthesized. The activity of 1 was observed in the presence of soybean oil (glyceride), although the isomer (2) did not show any activity in doses up to 300 μg/ml.

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The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.

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We constructed the plasmid pTTB151 in which the E. coli bio B gene was expressed under the control of the tac promoter. Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E. coli carrying this plasmid. The requirements for this biotin-forming reaction included fructose-1, 6-bisphosphate, Fe²⁺, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate. The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation. Our results suggest that an unidentified enzyme (s) besides the bio B gene product is obligatory for the conversion of dethiobiotin to biotin.

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We examined the expression of cho B, encoding cholesterol oxidase of Brevibacterium sterolicum ATCC 21387, in Eschrichia coli JM105 and Streptomyces lividans TK23 using various deletion DNA fragments within the 5'-flanking region. The enzyme activity could be detected intracellularly in E. coli only when the 5'-flanking region was reduced to less than 256-bp and cho B was transcribed by the lac promoter. A large amount of the enzyme were produced as inactive inclusion bodies when Cho B protein was fused with the NH₂-terminal portion of LacZ protein. In contrast, cho B with more than 256-bp of the 5'-flanking region was efficiently expressed in S.lividans TK23, and about 85 times as much of the active enzyme (170 U/ml) was secreted into the culture filtrate as with B. sterolicum in flask culture. These results suggest that the promoter of cho B exist within 256-bp of the 5'-flanking region and can be efficiently recognized by the RNA polymerase of S. lividans. The characteristics of the enzyme purified from the culture filtrate of the S. lividans transformant and that of B. sterolicum were identical although the NH₂-terminal amino acid sequence of the enzyme from the S. lividans transformant was 6 amino acids shorter than that from B. sterolicum.

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Newley isolated Bacillus sp. No. 195 produced an extracellular α-amylase sensitive to Haim which was found to inhibit specifically animal α-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60, 000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1 : 1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among cry-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.

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DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida. The enzyme had the typical composition of β', β, α, and σ subunits of eubacterial RNA polymerases. The molecular masses of the subunits were 156, 000 Da, 151, 000 Da, 87, 000 Da, and 42, 000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The NH₂-terminal amino acid residues of the α subunit had a marked homology with those of the α subunit of Escherichia coli RNA polymerase. The enzyme activity was dependent on ribonucleoside triphosphates, Mg²⁺, and a DNA template, and was inhibited in vitro by rifampicin. The enzyme activity was maximal in the presence of 10 mM MgCl₂. In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P. putida initiated transcription at the same site as that of E. coli.

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Epiderstatin is a unique glutarimide antibiotic which was found by screening for inhibitors of the signal transduction of epidermal growth factor (EGF). The antibiotic (0.01 μM) was found to reverse the morphology of NRK cells that were infected with a temperature-sensitive mutant of Rous sarcoma virus (src^ts-NRK) from the transformed phenotype to the normal phenotype at the permissive temperature (32°C). Epiderstatin did not inhibit the protein kinase activity of p60^v-src. The cell cycle progression of src^ts-NRK cells was blocked at G0/G1 phase, which was caused by the inhibition of biosynthesis of p60^v-src but not the transcription of v-src mRNA.

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(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxobicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.

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Bovine serum albumin was reduced by incubating with various concentrations (0-200 mM) of 2-mercaptoethanol, and its emulsifying properties were examined for an oil-in-water emulsion system. A particle size analysis revealed that albumin reduced at 30 mM of the thiol yielded smaller oil particles than either native protein, or the protein reduced at 70 or 200 mM of the thiol. Furthermore, the particle size was almost constant during 35 days of storage with albumin reduced at 30 mM of the thiol, while an emulsion prepared using the native protein, or the protein reduced at 70 or 200 mM of the thiol was unstable during the same storage period. Gel filhation chromatography and transmission electron micrography show that serum albumin made aggregates with high molecular size by its disulfide reduction with 70 or 200 mM, but not with 30 mM of 2-mercaptoethanol. It was, therefore, concluded that time emulsifying property of serum albumin can be improved by a mild disufide reduction.

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The effect of the oxidation of linoleic acid on the interfacial adsorptivity of lysophosphatidylcholine (LPC)/free fatty acid (FFA)/ovalbumin (OA) complexes was invesdgated by ³¹P-NMR. The interfacial adsorptivity of the complexes was evaluated by the mean droplet size, phosphorus signal and relaxation time of an emulsion composed of each complex. The interfacial adsorptivity of the LPC/FFA/OA complexes became lower with the oxidation of linoleic acid, which formed a complex with LPC and OA. Reduction of the T₂ relaxation time and peak broadening of Ser-P⁶⁸ for OA correlated well with the formation of fine emulsions from an LPC/linoleic acid/OA complex. The bilayer vesicles composed of LPC and linoleic acid with a low POV value were destroyed by coupling with protein and show high interfacial adsorptivity. On the other hand, the vesicles composed of LPC and linoleic acid with a high POV value remained in liposome and show low interfacial adsorptivity. These results suggest that the affinity of bilayer vesicles composed of LPC and FFA mainly promoted the interfacial adsorption of an LPC/FFA/OA complex, and that the region of Ser-P⁶⁸ in OA was adsorbed at the interface when the complex formed a fine emulsion.

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The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1 : 1, while that of chymotrypsin inhibitor with chymotrypsin was 1 : 2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M_r 19, 242 and M_r 19, 783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.

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A synthesis of unnatural stereoisomers of LL-P880β (2) and subsequent transformation of 2 into LL-P880γ (3) were achieved by starting from diacetoneglucose (4). A chiral building block, 5-deoxy-α-D-ribofuranose derivative 9, was derived from 4 by two synthetic routes. A chiral aldehyde (20) was prepared through a six-step sequence from 9, and treated with the dianion of methyl acetoacetate to complete the synthesis of (6S, 1'R, 2'R)-LL-P880β (2a) and its C₆-epimer (2b). After converting 2a to its diacetate (23), this was transformed into (1'R, 2'R)-LL-P880γ (3a) via a 5-bromopyran derivative (24).

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Three fucoidanases were purified from Vibrio sp. N-5 by ammonium sulfate fractionation and chromatography with DEAE-Toyopearl 650 M, Sephacryl S-300 HR, and chromatofocusing. The purified enzymes gave a single band on disc polyacrylamide gel electrophoresis. The molecular weights of the enzymes, E-1, E-2, and E-3 were 39, 500, 68, 000, and 68, 000, respectively, by SDS polyacrylamide gel electrophoresis and 158, 000, 68, 500, and 67, 500 by gel filtration. The enzymes hydrolyzed gagome-fucoidan to give small oligosaccharides containing sulfate as main product.

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A methanol extract of neem oil indicated antifeedant activity at 200 μg/disc by a no-choice bioassay against Reticulitermes speratus Kolbe. The extract was purified by recycling HPLC to isolate 11 compounds of variable termite antifeedant potency. Deacetylgedunin was the most active compound (95% protective concentration or PC₉₅=113.7 μg/disc). This was followed by salannin, gedunin, 17-hydroxyazadiradione, nimbandiol, azadiradione, deacetylsalannin, and deacetvlnimbin, with PC₉₅ estimates of 203.3, 218.4, 235.6, 245.4, 827.5, 1373.1, and 1581.2 μg/disc, respectively. Epoxyazadiradione, 17-epiazadiradione, and nimbin were not active (PC₉₅ estimates were beyond the bioassay limits). It could be presumed, by comparing their structures and activities, that the furan ring, αβ-unsaturated ketone and hydroxyl group each played an important role in determining the antifeedant activity.

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A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56, 000 and 58, 000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80°C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80°C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.

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The hypocholesterolemic activity of the undigested fraction (UDF) from soybean protein was significantly greater than that of the parent protein even when rats were fed no cholesterol. This fraction prevented the rise of serum cholesterol during a feeding period of 4 months. There was a gradual decrease in serum cholesterol as the proportion of UDF in the casein diet was increased, and 50% replacement produced much reduction. Liver cholesterol and triglyceride also decreased. Compared with the results obtained with soybean protein, UDF increased fecal excretion of both neutral and acidic steroids, suggesting a possible mechanism for its hypocholesterolemic effect. UDF seemed to interfere with the metabolism of linoleic acid to arachidonic acid more than soyben protein, which reduced desaturation more than casein did. The results showed that UDF was hypocholesterolemic when the diet was cholesterol-free, and that the effect on linoleic acid metabolism was specific.

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The Taka-amylase A gene (amy B) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amy B promoter and the Escherichia coli uid A gene encoding β-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amy B promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amy B gene.

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Partially Sau3AI-digested fragments of chromosomal DNA from Bacillus circulans IAM1165, a high producer of β-1, 3-glucanases able to lyse fungal cell walls, were inserted into a BamHI site of the plasmid vector pHSG399. A gene for the glucanase was cloned in Escherichia coli K-12 by the shotgun method. An 8-kb inserted DNA directed synthesis of an 87-kDa endo-β-1, 3-D-glucanase in E. coli. The β-glucanase gene was in a 2.6-kb EcoRI-SmaI segment within the insert DNA. The enzyme activity was found mainly in the periplasmic fraction of E. coli carrying the gene.

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The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca²⁺. Sr²⁺ increased the transfection rate as well, but Ba²⁺, Mn²⁺, and Mg²⁺ did not. Co²⁺ and Zn²⁺ inhibited transfection. The simultaneous use of Ca²⁺ and Mg²⁺ increased the transfection efficiency. Impairment of transfection caused by lack of Ca²⁺ could not be reversed by the addition of Ca²⁺ later. A decrease in the Ca²⁺ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.

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To discover the role of propionate produced by colonic bacteria, this study examined the secretion of bile acids and cholesterol 7α-hydroxylase activity in the primary cultured hepatocytes. Addition of propionate (2 mM) to the medium for 48 h caused an increase in the bile acid secretion and enzyme activity, while acetate and butyrate had no significant influence. Bile acid secretion vvas increased by the addition of succinyl CoA and its precursor substances (α-ketoglutarate, valine, isoleucine, and methionine), but not malate and oxaloacetate, which are the metabolites of succinyl CoA. α-Ketoglutarate and valine also increased the activity of cholesterol 7α-hydroxylase. Since cholesterol 7α-hydroxylase is a microsomal cytochrome P-450 enzyme and the formation of δ-aminolevulinate from succinyl CoA in the mitochondria is the rate-controlling step for the subsequent synthesis of heme proteins, propionate may affect bile acid synthesis via elevation of mitochondrial succinyl CoA.

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