Expression of chimeric P450 genes encoding flavonoid-3', 5'-hydroxylase in transgenic tobacco and petunia plants(1).

Flavonoid-3',5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3', 5'-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3'5'H. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3',5'-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3',5'-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3'5'H activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3'5'H activity between tobacco and petunia is discussed.

Evidence that irradiation of far-infrared rays inhibits mammary tumour growth in SHN mice.

To evaluate the effect of irradiation of far infrared rays (FIR), the growth of spontaneous mammary tumours of SHN mice was compared among 3 groups: the control was kept until the end of experiment on the normal rack in the absence of FIR and Experimental group I was constantly exposed to FIR. Experimental group Il was raised as the control followed by movement to the FIR rack after mammary tumour appearance. While there was little difference between the control and Experimental group I in mammary tumour growth for 16 days, Experimental group II was significantly lower than the control in this parameter. Furthermore, the percentage of rapidly growing tumours showing greater than 200% of growth rate was apparently lower in Experimental group II. Associated with this, epidermal growth factor receptor expression in mammary tumours, anterior pituitary weight and serum leptin level were significantly decreased in Experimental group II. The findings suggest that whole-body FIR irradiation at ambient temperature could be a possible way of a hyperthermic therapy for tumours.

Nucleus-electron model for states changing from a liquid metal to a plasma and the Saha equation.

We extend the quantal hypernetted-chain (QHNC) method, which has been proved to yield accurate results for liquid metals, to treat a partially ionized plasma. In a plasma, the electrons change from a quantum to a classical fluid gradually with increasing temperature; the QHNC method applied to the electron gas is in fact able to provide the electron-electron correlation at an arbitrary temperature. As an illustrating example of this approach, we investigate how liquid rubidium becomes a plasma by increasing the temperature from 0 to 30 eV at a fixed normal ion density 1.03x10(22)/cm(3). The electron-ion radial distribution function (RDF) in liquid Rb has distinct inner-core and outer-core parts. Even at a temperature of 1 eV, this clear distinction remains as a characteristic of a liquid metal. At a temperature of 3 eV, this distinction disappears, and rubidium becomes a plasma with the ionization 1.21. The temperature variations of bound levels in each ion and the average ionization are calculated in Rb plasmas at the same time. Using the density-functional theory, we also derive the Saha equation applicable even to a high-density plasma at low temperatures. The QHNC method provides a procedure to solve this Saha equation with ease by using a recursive formula; the charge population of differently ionized species are obtained in Rb plasmas at several temperatures. In this way, it is shown that, with the atomic number as the only input, the QHNC method produces the average ionization, the electron-ion and ion-ion RDF's, and the charge population that are consistent with the atomic structure of each ion for a partially ionized plasma.

Inhibition by whole-body hyperthermia with far-infrared rays of the growth of spontaneous mammary tumours in mice.

To evaluate possible therapeutic benefits of irradiation with far-infrared rays (FIR) on breast cancer, we examined combined effects of the chronic exposure to FIR at ambient temperature (26.5-27.5 degrees C) and the whole-body hyperthermia induced by FIR (WBH) (35-41 degrees C) on the growth of spontaneous mammary tumours of mice. A high mammary tumour strain of SHN virgin mice born on the normal rack or FIR rack were maintained on the respective racks until mammary tumour appearance. When the mammary tumour size reached approximately 7 mm, some mice in each group received no further treatment (Control and FIR groups, respectively) and the remaining mice received 3 hours of WBH each of 5 consecutive days (C + WBH and FIR + WBH groups, respectively). There was little difference between the control and FIR groups in the tumour growth over 10 days of examination. On the other hand, the tumour growth was inhibited significantly in both C + WBH and FIR + WBH groups and the degree of inhibition was similar. The data confirmed that the chronic exposure to FIR at ambient temperature has little effect on the growth of spontaneous mammary tumours in mice. WBH with FIR, however, strongly inhibited the tumour growth without deleterious side-effects, while chronic FIR irradiation itself again had little effect in this process. This WBH regimen may serve as a useful animal model for long-term studies of a noninvasive treatment of breast cancer.

Evidence that far-infrared radiation promotes growth of Xenopus laevis.

In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment.

Evidence that far infrared radiation promotes growth of Xenopus laevis.

In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment.

[Efficacy and safety of intramuscular imipenem/cilastatin (IPM/CS) for complicated urinary tract infections].

An intramuscular preparation of imipenem/cilastatin (IPM/CS, 500 mg/500 mg) was administered to 59 patients with complicated urinary tract infections (UTI; cystitis and pyelonephritis) to evaluate its efficacy and safety. The obtained results are summarized as follows: In patients with cystitis, evaluations based on daily frequencies of administration were also performed. 1) According to the treating doctors, the drug showed an overall efficacy rate of 80% (45/56 patients). The efficacy rate was 89% in patients with cystitis treated by a u.i.d. regimen. Among patients treated by a b.i.d. regimen, the efficacy rate was 67% for cystitis cases and 84% for pyelonephritis cases. 2) When clinical efficacy was assessed according to the criteria for UTI drug efficacy evaluation, the drug was 'markedly effective' in 14 patients, 'effective' in 23, and ineffective in 11 patients, for an efficacy rate of 77% (37/48 patients). 3) The microbiological eradication rate was 88% (59/67 strains). The rate was 95% (20/21 strains) for Gram-positive bacteria and 85% (39/46 strains) for Gram-negative bacteria. The efficacy for Enterobacter faecalis and Pseudomonas aeruginosa was 100% and 73%, respectively. 4) As side effects, pain at the injection site was reported by one patient and abnormal laboratory test values were observed in 2 patients. All of these reactions were mild and resolved shortly after the completion of treatment. Based on these findings, it is concluded that this intramuscular preparation of IPM/CS is effective for treating complicated urinary tract infections.

Genetic transformation of Begonia tuberhybrida by Ri rol genes.

We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals.

Distribution of lipid microspheres incorporating prostaglandin E1 to vascular lesions.

The intravascular distribution of 0.2 mu lipid microspheres (LM) containing prostaglandin E1 (lipo-PGE1) injected intravenously in spontaneously hypertensive rats (SHR) and arteriosclerotic rabbits was investigated by electron microscopic observation and quantification of radiolabelled compounds. LM were observed under an electron microscope to concentrate in subendothelial space of vascular walls, particularly in vascular lesions associated with hypertension or arteriosclerosis. Radiolabelled lipo-PGE1 accumulated more densely in the vascular walls than did free PGE1, and the difference was more conspicuous in vascular lesions. This indicates that lipo-PGE1 penetrates vascular endothelium and then accumulates in blood vessels to result in augmentation of the pharmacological action of prostaglandin. These findings suggest the usefulness of LM as a carrier of prostaglandin to vascular lesions.

Prostaglandin E1 incorporated in lipid microspheres in the treatment of peripheral vascular diseases and diabetic neuropathy.

Lipo-PGE1 is a drug preparation of prostaglandin E1 (PGE1) incorporated in lipid microspheres similar in properties to liposomes. A randomized, single-blind, cross-over study comparing free PGE1 (PGE1 cyclodextrine, PGE1CD) with lipo-PGE1 was performed. Twenty patients with peripheral vascular diseases and diabetic neuropathy entered the trial. The first seven days' treatment was either 5 micrograms/day of lipo-PGE1 or 40 micrograms/day of PGE1CD, followed by a seven-day wash-out period; then cross-over was performed for another week's administration. Improvements were achieved by both PGE1 preparations. The comparison between lipo-PGE1 and PGE1CD showed that the former was significantly superior, both in final global improvement (p less than 0.01) and in terms of patients' preference (p less than 0.01); lipo-PGE1 also produced fewer side-effects. This study suggests that lipo-PGE1 is a very valuable agent for the treatment of peripheral vascular disorders and diabetic neuropathy.

Nucleotide sequences and stability of a Nicotiana nuclear DNA segment possessing autonomously replicating ability in yeast.

The nucleotide sequence of a tobacco (Nicotiana tabacum) chromosomal DNA segment(t3-ars) capable of replication in yeast (ars: autonomously replicating sequences) is presented. The subcloned region (618 bp) contained 11 bp consensus (5' A/TTTTATPuTTTA/T 3') essential for several yeast ars, and 73% A and T. Unique 70 bp repetitive sequences resided next to this sequence. Thirty-two bp AT repeats were also seen in the neighbourhood of the repetitive sequence. The hybrid plasmid containing t3-ars was mitotically stabilized by the help of yeast centromere (CEN4).

Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid.

The octopine tumor-inducing (Ti) plasmid pTiA66 has an insertion mutation in its T region (the DNA region incorporated into the plant genome) that results in the slow growth of crown gall tumors. These tumors exhibit hormonal autonomy different from that of the crown gall tumors caused by wild-type Ti plasmids. In the present study, the nucleotide sequences of both the DNA segment inserted into pTiA66 and its target site have been determined. The inserted segment is 2548 base pairs long and has 20-base-pair terminal inverted repeats. An 8-base-pair sequence at the target site is duplicated at both integration junctions. These structural features of the insert suggest that it is a bacterial insertion sequence (IS) element, which we have named IS66. Blot-hybridization analyses using IS66 probes revealed that genomes of octopine Ti plasmids contain at least three sequences homologous to IS66: two homologues are located in the virulence region and one is located between the left-hand (TL-DNA) and right-hand (TR-DNA) portions of T-DNA. The chromosome of Agrobacterium tumefaciens A66 also contains two sequences highly homologous to IS66. These results suggest that the mutant pTiA66 plasmid was generated by translocation of one of the sequences showing homology with IS66 into the T region. The fact that a sequence homologous to IS66 is present between TL-DNA and TR-DNA also suggests that the octopine T region was split into two portions, TL-DNA and TR-DNA, by translocation of IS66 or its relatives. Thus, IS66 may cause genetic and structural variations of the T region and the vir region of the octopine Ti plasmids.