Search

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10–90% (v/v), and was tolerant to organic solvents whose log P ow (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.

The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding "P6C reductase" that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%.

Biotransformation of L-lysine (L-Lys) to L-pipecolic acid (L-PA) using lat-expressing Escherichia coli has been reported (Fujii et al., Biosci. Biotechnol. Biochem., 66, 622-627 (2002)). The rate-limiting step of this biotransformation seemes to be the transport of L-Lys into cells. To improve the L-PA production rate, we attempted to increase the rate of L-Lys uptake. E. coli BL21 carrying a plasmid with lat and lysP (pRH125) caused a 5-fold increase in the rate of L-PA production above the level of cells carrying a plasmid with lat (pRH124). Moreover, E. coli BL21 carrying a plasmid with lat, lysP, and yeiE (pRH127) caused a 6.4-fold increase in the rate of L-PA production above the level of cells carrying pRH124. Our results from RT-PCR experiments and the sequence similarity of YeiE to LysR transcriptional regulators suggest the possibility that yeiE expression induces lysP expression. The amplification of lysP, or rather both lysP and yeiE, increases the rate of L-PA production using lat-expressing E. coli.

An optimal feed rate profile of a substrate (tylosin) for a novel antibiotic, acetyl-isovaleryl tylosin (AIV) production process was investigated. In the first step of optimization, a kinetic model for production of AIV from tylosin by Streptomyces thermotolerans was established properly using the least square method, followed by the confirmation that the proposed model could be used to predict the production process of AIV from tylosin. An objective function, state equations and an inequality constraint with respect to the tylosin feeding rate profile were applied to maximize the amount of AIV produced from tylosin in a fed-batch culture. The optimized tylosin feeding rate profile was determined using a direct iterative search algorithm based on the modified complex method. The simulation of AIV production at the optimal tylosin feeding profile indicates that the final amount of AIV is expected to be about 30% higher than that at the conventional constant tylosin feeding rate, which was also confirmed experimentally using a 30-l jar fermentor.

The red-pigmented yeast, Phaffia rhodozyma, containing high concentration of astaxanthin (Ax) was evaluated as a dietary pigment source for egg yolk of laying hens fed on a low-carotenoid diet. Diets supplemented with the yeast to provide 0 to 16ppm Ax were fed for 4 weeks, while canthaxanthin (Cx), a synthetic pigment source, was added to diets at 0 to 1.5ppm as the comparing counterpart in 2 experiments. Roche color fan score and a* value on Chroma Meter increased linearly with the increase of logarithmic concentration of dietary Ax and Cx. Cx (Carophyll Red(R)) was 4 to 5 times as potent as Ax in the yeast for the pigmentation of egg yolk probably because of low digestibility of the yeast. Ax concentrations in plasma and egg yolk increased with dietary Ax concentration and the increases paralleled with the pigmentation of egg yolk. These results suggest that Phaffia yeast containing high concentration of Ax has the potential as natural pigment source for egg yolk of laying hens.

Two experiments were conducted if the meat color-modifying potential of Phaffia rhodozyma, a yeast containing high levels of astaxanthin (Ax), is influenced by the feeding duration and the dietary concentration and if the potential is enhanced by an addition of lecithin+α-tocopherol in broiler chickens. Experimental diets supplemented with cell wall-fractured yeast to provide 10, 20 and 30ppm Ax were fed to 3, 5 and 7 week-old chickens for 5, 3 and 1 weeks, respectively, prior to market in Experiment 1. In Experiment 2, Phaffia yeast-supplemented diets (20ppm Ax), which were supplemented with or without lecithin (5g/kg diet)+α-tocopherol (0.1g/kg diet), were fed to 5 week-old chickens for 3 weeks prior to market. Feeding of Phaffia yeast influenced neither performance of broilers nor edible meat yields, irrespective of either the feeding duration, the dietary concentration or the addition of lecithin+α-tocopherol. Concern-ing the color analysis of edible meats using with Minolta reflectance colorimeter, light-ness (L* value) was not essentially influenced by feeding of the yeast-supplemented diets, while yellowness (b* value) was slightly increased by the yeast feeding. On the other hand, the redness (a* value) of edible meats and abdominal adipose tissues was significantly or numerically increased by feeding of the yeast-supplemented diets, in which the increase was less extent in chickens received the yeast-supplemented diet (30ppm Ax) for 1 week prior to market than chickens received the yeast-supplemented diets (20ppm Ax) for 3 weeks. The extent of increase in a* values of edible meats was relatively dependent on the total Ax intake. Addition of lecithin+α-tocopherol did not intensify the coloration (redness) of edible meats by the feeding of yeast-supplemented diets. These results show that cell wall-fractured Phaffia yeast containing high concen-trations of Ax can be a useful source for modification of meat color and the effect is briefly due to the total Ax intake in broiler chickens.

トウモロコシ•大豆粕主体の慣用飼料を給与した産卵鶏を供試し,カロチノイドの一種であるアスタキサンチン(Ax)を高濃度に含む赤色酵母の卵黄色強化効果を検討した。飼料中のアスタキサンチン濃度が0から6ppmになるように赤色酵母を段階的に添加した飼料,赤色酵母からアセトンで抽出したAx 3ppm添加飼料,市販Ax製剤3ppm添加飼料,市販カンタキサンチン(Cx)製剤1ppm添加飼料,試薬用Cx 1ppm添加飼料を2-4週間給与した。赤色酵母,AxおよびCx給与では産卵鶏の増体量,飼料摂取量,産卵率および卵重に変化は見られなかった。ロッシュカラーファンスコアおよびChroma Meterによる赤色度(a*値)はいずれのAxおよびCx添加でも有意に上昇した。また,カラーファンスコア,a*値および卵黄中のAx濃度のいずれもが飼料中Axの添加濃度にしたがって有意に上昇した。抽出Axは赤色酵母中Axに比較して,卵黄色強化効果では8倍以上,卵黄へのAx移行率では約3倍高く,赤色酵母中のAxの利用性を高めるためには酵母の細胞壁破砕などの処理により消化性(酵母の分解性)を高めることが有効であると推定された。試薬用Cxの卵黄着色効果は市販Cx製剤よりも小さいことが示された。以上のことから,産卵鶏の実用的飼育条件下でも,赤色酵母は天然の卵黄色強化用の飼料原料として有用であること,そして,その利用性は酵母細胞壁の破砕処理などにより大きく改善可能であることが示唆された。

We have isolated a 2.5-kb DNA fragment from plasmid pST5R7 encoding a sucrose utilization system from Escherichia coli B-62 which confers a sucrose-fermenting phenotype to transformed E. coli K-12 strains. DNA-sequence determination revealed one full-length open reading frame 98% identical to cscA, the sucrose-hydrolase (invertase) gene of the csc regulon from E. coli EC3132. Functional characterization indicates that high-level expression and limited periplasmic release of invertase is responsible for the sucrose-fermenting capacity of transformed E. coli K-12 strains carrying cscA.Key words: sucrose utilization, sucrose hydrolase, invertase, recombinant protein production.

A tylosin-producer, Streptomyces fradiae, was transformed with plasmids carrying genes from Streptomyces thermotolerans that are involved in acyl modification of macrolide antibiotics. A transformant with pMAB3, in which macrolide 4"-O-acyltransferase gene (acyB1) and its regulatory gene (acyB2) are subcloned, produced several types of 4"-O-acyltylosins. A transformant with pAB11 delta EH containing macrolide 3-O-acyltransferase gene (acyA) in addition to the above two genes produced 3-O-acetyltylosin and 3-O-acetyl-4"-O-acyltylosins. Among the products of the latter transformant, 3-O-acetyl-4"-O-isovaleryltylosin (AIV) was detected as a minor component. When L-leucine, a precursor of isovaleryl-CoA, was added to the medium at the late stage of the fermentation, AIV content among the total macrolides increased ten-fold and AIV became a main product. This fact suggests that a high level of endogenous isovaleryl-CoA may be essential for the selective production of AIV by S. fradiae carrying pAB11 delta EH.

A gene encoding the macrolide modification enzyme 3-O-acyltransferase (acyA) was cloned by chromosome walking onto the carbomycin biosynthetic region in Streptomyces thermotolerans TH475, with the 3' region of the gene encoding the macrolide modification enzyme 4"-O-acyltransferase (acyB1) as a probe. A shortened fragment (1.8 kb) containing acyA was subcloned with pIJ350. A high-level tylosin producer, Streptomyces fradiae MBBF, transformed with the plasmid could produce a hybrid macrolide, 3-O-acetyltylosin, most efficiently.

To elucidate functional diversity of cytochrome P450 monooxygenases from the white-rot basidiomycete Phanerochaete chrysosporium (PcCYPs), we conducted a comprehensive functional screening using a wide variety of compounds. A functionomic survey resulted in characterization of novel PcCYP functions and discovery of versatile PcCYPs that exhibit broad substrate profiles. These results suggested that multifunctional properties of the versatile PcCYPs would play crucial roles in diversification of fungal metabolic systems involved in xenobiotic detoxification. To our knowledge, this is the first report describing multifunctional properties of versatile P450s from the fungal kingdom. An increased compilation of PcCYP functions will facilitate a thorough understanding of metabolic diversity in basidiomycetes and provide new insights that could also expedite practical applications in the biotechnology sector.

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid.

A DNA fragment responsible for the 4''-O-acylation of macrolide antibiotics was cloned from a mutant strain of the carbomycin producer Streptomyces thermotolerans. The gene encoding the macrolide 4''-O-acyltransferase was within a 2.7-kb region of the cloned fragment (15-kb). Streptomyces lividans carrying the region converted exogenously added tylosin to 4''-O-acyltylosins. Nucleotide sequencing of the region showed two open reading frames (ORFs). Expression assay using deleted plasmids showed that both ORFs were essential for optimal expression of the acyltransferase activity. One of them (acyB1) was identical with carE reported previously as a gene encoding 4''-mycarosyl isovaleryl-CoA transferase. The other (acyB2) was assumed to encode a novel regulatory protein that could active acyB1 expression. acyB1 and acyB2 were highly conserved among streptomycetes with macrolide 4''-O-acyl transferase activity.

We examined the effect of conflicting vestibular and smooth pursuit information on the vestibulo-ocular reflex (VOR) in alert monkeys. Sinusoidal whole body rotation was applied either in the pitch or yaw plane while presenting a target spot that moved orthogonally to the rotation plane. The monkeys were rewarded for tracking the spot and eye movements induced by rotation alone were examined every 15–30 min in complete darkness. Orthogonal eye movement responses to rotation were not observed before training but appeared after about 30 min of training. The gains (eye/chair) of the orthogonal component increased up to 0.2 after 1–2 h and were largest at the training frequency and approximately in phase with the stimulus; phase advanced at lower frequencies and lagged at higher frequencies. Amplitude tuning was also demonstrated when examined using different amplitudes at a constant frequency after training. Two hours of training to fixate an earth-stationary spot projected onto a patterned visual background that moved orthogonally to the rotation plane during rotation, did not induce a cross axis response. These results indicate that pursuit training during VOR is effective in inducing cross-axis eye movement responses that are tuned to the metrics of the training stimulus.

A 3.2-kb DNA fragment of the carbomycin biosynthetic region including the 3-O-acyltransferase gene (acyA) from Streptomyces thermotolerans was sequenced, and four ORFs were found in the fragment. The second ORF, designated ORF-A, was transcribed in the opposite direction to the other three ORFs. The first ORF was identified as carA, a gene for carbomycin resistance. The amino acid sequence of ORF-A was homologous to proteins of the cytochrome P-450 family. Streptomyces lividans transformed with pCB20, in which ORF-A was subcloned, epoxidized carbomycin B at its C-12, 13 positions, thus producing carbomycin A. The third ORF, the amino acid sequence of which showed a homology to macrolide antibiotics O-acyltransferases was identified as acyA. The last ORF (ORF-B), which starts just 3 bp downstream from the TGA termination codon of acyA, was thought to be a carbomycin 4-O-methyltransferase gene, because the amino acid sequence deduced from ORF-B showed high homology to a putative midecamycin 4-O-methyltransferase encoded on mdmC.

The purpose of this research is to study methods of constructing a system which gives intelligent support to plant operators by applying artificial intelligence and fuzzy theory. The research also aims at construction of a “phenomenon explanation system with designers’ knowledge” as one form of intelligent support system.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.

In 198k we developed a method of hyperthermia using Nd:YAG laser (laser thermia) a system for transmitting continuous low power energy (2 W) through an artificial sapphire interstitial probe for local hyperthermia (43-0.5°C). Our experimental and clinical evidence indicated that this method is safe and effective. The process of tumor destruction induced by laser thermia is, however, poorly understood. We performed in vivo histopathological studies and obtained the following results; 1) At the tissue level, the initial change after laser thermia was the damage of tumor feeding vessels. 2) At the cell level, immediately after laser thermia the mitochondria in the intracellular organella were completely destroyed.

Investigations were made on the survivals of 81 patients with advanced gastric cancer. Of these, 55 patients were treated with a combination chemotherapy of mitomycin C, 5-fluorouracil, and cytosine arabinoside (i.e., MFC therapy). Another 26 cases were treated with immunochemotherapy of MFC plus OK-432, an immunotherapeutic agent. Fifty percent survival times were 5.4 months for the MFC group, and 7.5 months for the MFC plus OK-432 group. One year survival rates of each group were 13 and 35 percent, respectively. The two year survival was null for the MFC group, and 13 percent for the MFC plus OK-432 group. Statistical analysis revealed that the survival curve of the MFC plus OK-432 decreased more slowly as compared with that of the MFC (p less than 0.05). In other words, the patients treated with MFC plus OK-432 showed a significantly longer survivals compared to the patients treated with MFC alone. Some cases were also presented for the better understanding of the immunochemotherapy of MFC plus OK-432.

The precipitation behaviour of the a phase in a Ti-27 at. %Nb-6 at. %Ta-6 at. %Zralloy has been investigated mainly by means of small-angle X-ray scattering measurements, by which the average size and number density of a precipitates were determined. The alloy was isothermally aged at 643 K after cold-rolling to various thicknesses. During ageing the average size of α precipitates increased but the number density decreased. The effect of cold-rolling was to increase the volume fraction of a precipitates. The superconducting critical current density was measured for the same specimens after the metallographical investigation. The specific pinning force produced by α precipitates, which corresponds to the global pinning force density divided by the number density of precipitates, was found to be proportional to the cube of the particle radius. It was found that the global pinning force density can be described using a scaling rule in terms of the volume fraction of α precipitates and the reduced ma...

A wide band chopper was designed to provide polychromatic pulsed neutrons in the range 2–10 A for small angle neutron scattering (SANS), and has been installed at Kyoto University Research Reactor. In the SANS by means of the time-of-flight technique with this chopper, scattering patterns are provided in the range of the scattering vector K from 0.01 to 0.1 A −1 . The shape of the slit of the rotor was determinedafter computer calculations of neutron burst shapes and transmission probabilities for various slit shapes. The experimental tests for the rotor have satisfactorily been performed. A practical application of the chopper to SANS was carried out to examine theprecipitation in a superconducting composite wire of TiNb alloy during aging at 643 K.

The ester enolate Claisen rearrangement of (E)-1-alkyl-3-trimethylsilyl-2-propenyl glycolates gave (E)-erythro-2-hydroxy-3-trimethylsilyl-4-alkenoates with high diastereoselectivity, which were stereoselectively converted into (2E,4E)- and (2Z,4E)-2,4-alkadienoates by the Peterson reaction.

Magnification radiography was applied for the diagnosis of the biliary tract diseases including cancer. Our X-ray unit was consisting of HITACHI DR-155-23 U-6M-55P and 0.1 mm focal-spot tube, FFD 80 cm giving a magnification of x2 with the conditions of 60 kVp, 20 mA, 0.8 sec. Intensifying screens such as Kyokko LH II (for a standard sized subject) or DuPont Q II (for an obese subject) were used in combination with Fuji RX films. We confirmed a high diagnostic value in revealing early lesions of this sytem. The skin dose test indicated that this technique was routinely applicable with safety for clinical use.

A feedback resistant trp operon plasmid that transformed a multiple mutant (trpR tnaA) of Escherichia coli was found to enhance remarkably the production of tryptophan in a bench-scale fermentor. 5.5 g of tryptophan was accumulated per litre of culture medium at 24th hr in batch. The productivity was 0.229 g/l/hr. This productivity is the highest among those ever reported by other workers. The recombinant plasmid (Tcr Trp+ I-) used was completely stable in each run when tetracycline was added by 10 μg/l into the medium.

The percentage of cells of different T-cell subsets and their functions in tuberculous pleural effusion were investigated. The percentage of OKT4-positive cells was 65 +/- 2% (mean +/- SEM, n = 8) and that of OKT8-positive cells was 19 +/- 3% (n = 8). Pleural T lymphocytes of patients with tuberculous pleurisy responded well to stimulation with purified protein derivative of tuberculin, and deoxyribonucleic acid synthesis was observed along with gamma interferon (IFN-gamma) production. When pleural T lymphocytes of patients with tuberculous pleurisy were treated with OKT4 monoclonal antibody and complement, a significant decrease in IFN-gamma production was observed in all eight patients (P less than 0.005), whereas no definite decrease in IFN-gamma production was found after treatment with OKT8 monoclonal antibody and complement. These results suggest that at least the OKT4+/OKT8- T-cell subset is responsible for the antigen-specific IFN-gamma production in pleural T lymphocytes of patients with tuberculous pleurisy.

Effect of adhesion strength on the Bendability was studied in a compositetype vibration-damping steel sheet which consists of two steel skins bonded with a viscoelastic resin. The adhesion strength of the damping sheet was evaluated in tensile shear strength (TSS); TSS values of the damping sheets used for the test were 6, 61, 126 and 148kgf/cm2. U-and 90 deg-bend tests were adopted as bend formability tests.It was found that the bendability of the damping sheet is closely related to their TSS, and it is improved with an increase of TSS. When the TSS is higher than 126kgf/cm2, the bendability of the damping sheet is quite similar to that of conventional steels. On the other hand, when the TSS is as low as 6kgf/cm2, the slip and peel-off between two skins were observed in the bend tests.In the 90 deg-bend test, the damping sheet with TSS of lower than 61 kgf/cm2 bend properties are different from the conventional steel. In large size of bend radius, bend angles of the damping sheet were larger than those of the conventional steel. However, in small size of bend radius, bend angles of the damping sheet were smaller than those of the conventional steel. These behaviors of the damping sheet are based by elastic property of the core resin.

It is rather seldom to detect a cancer of the common bile duct at the non-icteric stage of the disease. This report presents a non-icteric case of carcinoma of the choledochus. A 70-year-old woman complained of r-hypochondralgia with occasional fever. Laboratory examination revealed an increased serum alkaline phosphatase activity without any elevation of serum bilirubin content. The oral cholecystography failed to visualize the gallbladder. By endoscopic retrograde cholangiopancreatography (ERCP), a bilateral filling defect was demonstrated at the common bile duct. Pancreatoduodenectomy was performed and the resected speicmen showed a 1 × 2 cm tumor (adenocarcinoma) at the choledochus. She is now in good health over 13 months after operation.

The ester enolate Claisen rearrangement of (E)-1-alkyl-3-trimethylsilyl-2-propenyl glycolates gave (E)-erythro-2-hydroxy-3-trimethylsilyl-4-alkenoates with high diastereoselectivity, which were stereoselectively converted into (2E,4E)- and (2Z,4E)-2,4-alkadienoates by the Peterson reaction.

The recombinant plasmids RSF2124-trp and pSC101-trp were examined for their phenotypic stability in Escherichia coli W3110 and its derivatives under various culture conditions. RSF2124-trp and pSC101-trp were stable in a trpAE1 strain. In an amber mutant of the tryptophan repressor gene, RSF2124-trp was fairly stable, whereas pSC101-trp was unstable. All Trp- segregants from the pSC101-trp carrier had lost the entire plasmid. In a mutant carrying the tnaA mutation, RSF2124-trp was unstable in rich media. Most Trp- segregants that appeared under these conditions were deleted in trp genes as well as in the cI gene on the recombinant plasmid. pSC101-trp in this tnaA mutant was also unstable. All Trp- segregants had lost the plasmid. Studies of enzyme activities revealed that the greater the activity of anthranilate synthase and tryptophan synthase in bacteria, the more segregants tended to appear in the stability test. RSF2124 and pSC101 without the trp gene were completely stable in the same bacteria. The apparent instability of bacteria carrying the recombinant plasmid could be explained by the lower growth rate compared with bacteria carrying only the vector plasmid, resulting in the enrichment of Trp- bacteria during culture.

The elevation of lipid peroxide level has been considered as a cause of the degeneration of organs or tissues (1–6). It was also considered that the lipid peroxide formed in the primary site would be transferred via blood to other organs or tissues where the damage would be provoked by the propagation of lipid peroxidation (7,8). Recently, it was reported from our laboratory that plasma lipid peroxide levels of diabetic patients were significantly higher than those of control subjects and that the levels in diabetics with angiopathy were markedly higher than those in diabetics without angiopathy (9). From these results, we suspected that a high level of lipid peroxide in plasma would be, at least partly, the cause of angiopathy in patients suffering from diabetes, and that it would be worthwhile to obtain further information on the lipid peroxides of blood plasma of diabetic patients. The present paper deals with the lipid peroxide levels of serum lipoprotein fractions of diabetic patients examined in relation to some abnormal features of lipid metabolism.

The stability test of RSF2124-trp and pSC101-trp in E. coli Trp− strains revealed that the higher the activities of trp operon enzymes in cells, the more frequently Trp− segregants tended to appear. The apparent instability of the recombinant-plasmid-carrying cells could be explained by their lower growth rate than that of cells carrying only the vector plasmid and thus, by the enrichment of Trp− cells during the culture. Phenotypic expression of Trp− was due to either the disappearance of entire plasmid from host cells or the deletion of trp operon from the recombinant plasmid. A specific plasmid named pMT-trp was constructed by digestion of RSF2124-trp with restriction endonuclease PstI and ligation with T4 ligase. In pMT-trp, transposon TnA which is inherent in RSF2124-trp was deleted, resulting in the loss of ampicillin resistance. All of Trp− segregants from pMT-trp-carriers in E. coli, W3110 and its derivatives were found to have lost the entire plasmid. The experimental fact that deletion occurred exclusively in RSF2124-trp suggests that the presence of TnA in the plasmid RSF2124-trp was responsible for the deletion.