Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

Molecular cloning and expression analysis of the ethylene insensitive3 (EIN3) gene in cucumber (Cucumis sativus).

The plant gaseous hormone ethylene regulates many aspects of plant growth, development, and responses to the environment. Ethylene insensitive3 (EIN3) is a key transcription factor involved in the ethylene signal transduction pathway. To gain a better understanding of this particular pathway in cucumber, the full-length cDNA encoding EIN3 (designated as CsEIN3) was cloned from cucumber for the first time by rapid amplification of cDNA ends. The full length of CsEIN3 was 2560 bp, with an open reading frame of 1908 bp encoding 635 amino acids. Sequence alignment and phylogenetic analyses revealed that CsEIN3 has high homology with other plant EIN3/EIL proteins that were derived from a common ancestor during evolution, and CsEIN3 was grouped into a cluster along with melon. Homology modeling demonstrated that CsEIN3 has a highly similar structure to the specific DNA-binding domain contained in EIN3/EIL proteins. Based on quantitative reverse transcription-polymerase chain reaction analysis, we found that CsEIN3 was constitutively expressed in all organs examined, and was increased during flower development and maturation in both male and female flowers. Our results suggest that CsEIN3 is involved in processes of flower development. In conclusion, this study will provide the basis for further study on the role of EIN3 in relevant biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

Kinetics of Ge diffusion, desorption and pit formation dynamics during annealing of Si(0.8)Ge(0.2)/Si(001) virtual substrates.

Thermal stability of Si(0.8)Ge(0.2)/Si(001) virtual substrates (VS) is studied as a function of annealing temperature and time by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Two regimes describing different Ge behavior are observed when the Si(0.8)Ge(0.2) VS is annealed. Heating the substrate from room temperature to 500 degrees C results in some degree of Ge segregation. The surface morphology however remains relatively smooth and there is no formation of 3D islands on the surface. Above 500 degrees C, Ge is preferentially lost from the surface and microscopic pits with edges aligned along 110 azimuth are formed. As temperature increases, Ge% decreases and the size of pits also increases. The decrease in Ge% and the formation of holes at the surface are attributed to Ge desorption from the surface. A kinetic model involving diffusion and desorption processes is proposed to describe the Ge behavior and pits formation in this regime.

Laser-fluoride effect on root demineralization.

Although the individual cariostatic effects of laser and fluoride have been shown, the combined effect of CO(2) laser and fluoride on root demineralization remains uncertain and was the main aim of this study. By using a pH-cycling system and Polarized Light Microscopy, we demonstrated the synergistic effect of fluoride combined with CO(2) laser treatment on reducing root demineralization. The mean lesion depths (in microm) for each group were 160 +/- 14 (Control), 113 +/- 8 (Laser treatment alone), 111 +/- 6 (Fluoride treatment alone), and 25 +/- 7 (Fluoride followed by laser treatment). A significant laser-enhanced fluoride uptake, characterized by the ToF-SIMS analysis, was revealed by the 37% and 400% increments in loosely and firmly bound fluorides (both p < 0.002) in laser-irradiated areas, compared with the non-irradiated controls. In conclusion, there is a significant synergistic effect of combined CO(2) laser and fluoride treatment on the inhibition of root demineralization, possibly due to laser-enhanced fluoride uptake in the root.

Nucleation and growth of cobalt nanostructures on highly oriented pyrolytic graphite.

Cobalt in the form of three-dimensional (3D) hemispherical clusters (size approximately 10-30 nm) were observed to grow on pristine graphite surfaces via a Volmer-Weber growth mode. X-Ray photoelectron spectroscopy (XPS) reveals that these clusters are physisorbed on the surface. In the presence of minute surface contamination, the morphology of Co changes into a mixture of irregular and hemispherical three-dimensional islands. The formation of irregular islands appears to be mediated by the chemical interactions between Co and the surface contaminants as evidenced from analysis of the carbon pi-pi* transitions. Further analysis of size distribution of Co nanoclusters grown on pristine surfaces shows a critical nucleus size of i* = 1, i.e. a Co dimer forms the smallest stable cluster on a pristine graphite surface.

Inhibitive effect of genistein on interleukin-8 expression in cultured human retinal pigment epithelial cells.

Recent studies showed that interleukin-8 (IL-8) played an important role in retinal neovascularization. In this study, the effects of genistein on the expression of IL-8 in the arising retinal pigment epithelia-19 cells were studied. The levels of IL-8 protein expression in supernatants were punctually detected by ELISA. When the cells were treated with hypoxia (5% CO2, 95% N2), IL-8 secretion increased from 0.29 +/- 0.04 to 2.59 +/- 0.42 ng/ml. To study calcium-dependent IL-8 expression, cells were treated with KCl at 25 mM, norepinephrine (NE) at 10 nM, and glutamate (Glu) at 1 microM for 8 h. As a result, the levels of IL-8 protein in supernatants were significantly increased compared with that in the controls. When the cells are treated with genistein (50, 100, 200 microM) for 30 min before hypoxia or stimulations by KCl, NE, and Glu, the elevated expression of IL-8 protein was all suppressed in a concentration-dependent manner. These results suggested that suppression of IL-8 expression in retinal pigment epithelial cells might partly account for the inhibitive effect of genistein on retinal neovascularization.

Effects of chemotherapy and/or radiotherapy on survivin expression in ovarian cancer.

Survivin, a member of apoptosis protein inhibitor family, is upregulated in various malignancies, especially in chemotherapy- and/or radiation-resistant cell lines. In this study, the correlation between the level of survivin expression and degree of apoptosis was investigated in three ovarian cancer lines (two chemoresistant cell lines SKOV-3 and OVCAR-3, as well as one chemosensitive cell line OV2008) treated with 5 microg/ml of cisdiamminedichloroplatinum (cisplatin, CDDP) for 24 h, 2 Gy of (60)Co irradiation, or 5 microg/ml CDDP for 3 h plus 2 Gy of (60)Co, respectively. We also evaluated the survivin mRNA abundance in patients with advanced ovarian cancers during CDDP treatment. In the ovarian cancer cell lines, survivin mRNA abundance and protein contents were significantly increased after the treatments while the apoptotic rates did not change in SKOV-3 and OVCAR-3. Moreover, in OVA2008 cells the expression of survivin decreased and the apoptotic rate significantly increased after CDDP and combined treatments. Survivin mRNA was not detectable in normal ovarian tissues and benign ovarian tumors. However, it was observed in the resected tumor specimens from 20 patients with advanced ovarian cancer. These results suggested that survivin may play an important role in the resistance to chemotherapy and radiotherapy in ovarian cancer cell lines and in the progression of ovarian tumors. Survivin may also provide a pivotal prognostic implication for epithelial ovarian carcinomas.

The molecular mechanism of actinomycin D in preventing neointimal formation in rat carotid arteries after balloon injury.

The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (SMC). Actinomycin D has been regarded as a potential candidate to prevent balloon injury-induced neointimal formation. To explore its molecular mechanism in regulating cell proliferation, we first showed that actinomycin D markedly reduced the SMC proliferation via the inhibition of BrdU incorporation at 80 nM. This was further supported by the G1-phase arrest using a flowcytometric analysis. Actinomycin D was extremely potent with an inhibitory concentration IC50 at 0.4 nM, whereas the lethal dose LD50 was at 260 microM. In an in vivo study, the pluronic gel containing 80 nM and 80 microM actinomycin D was applied topically to surround the rat carotid adventitia; the thickness of neointima was substantially reduced (45 and 55%, respectively). The protein expression levels of proliferating cell nuclear antigen (PCNA), focal adhesion kinase (FAK), and Raf were all suppressed by actinomycin D. Extracellular signal-regulated kinases (Erk) involved in cell-cycle arrest were found to increase by actinomycin D. These observations provide a detailed mechanism of actinomycin D in preventing cell proliferation thus as a potential intervention for restenosis.

[Production of herbicide-resistant rice with transforming heterogene].

Using pAHC20 (containing Bar gene), pWRG1515 (containing GUS gene and hygromycin phosphotransferase gene), and pCAMBIA3300 RG with Bar gene and snowdrop lectin (GNA) gene as donor DNA, the micro-adventitious shoots and the calli induced from mature embryos of Oryza sativa 87203, Eyi105, Shangnong aromatic glutinous rice as recipients were transformed with particle bombardment and Agrobacterium tumefaciens strain LBA4404 containing pAL4404, respectively. After chosen with phosphinothricin and antibiotic, GUS detection and PCR analysis, The results showed that the foreign genes had been transformed microprojectile-mediated to Oryza sativa Eyi105, the regeneration plants were obtained, and, 5 transgenic calli of Oryza sativa Eyi105 were obtained with Agrobacterium-mediated transformation.

Protein crystal growth in microgravity using a liquid/liquid diffusion method.

Crystallization of proteins by liquid liquid diffusion method was performed in microgravity using the MDA Minilab aboard the US Space Shuttle. Three proteins, namely lysozyme, trichosanthin, and a new lechin, were crystallized in the space experiment. In contrast to the results of space experiments with a tube-like vapor diffusion method, the crystallization conditions for growing better crystals in space are remarkably different from the conditions optimized on earth. This may be due to difficulties in ground optimization, which are caused by gravity-dependent phenomena, in particular the specific convective flow occurring with liquid liquid diffusion.

Glucose turnover in lean and obese rats of the SHR/N-cp and LA/N-cp strains.

1. The relationship between hypertension, obesity, non-insulin-dependent diabetes mellitus and various parameters of glucose metabolism was studied. Lean and obese rats of the SHR/N-cp and LA/N-cp congenic strains were studied at four months of age. 2. Tritium and 14C-labeled glucoses were infused in one set of rats while tritiated water and 14C-labeled alanine were infused in a second group. 3. Glucose oxidation, turnover, conversion to glycogen, fatty acid synthesis, and alanine conversion to glucose were determined, as were blood pressure, pulse pressure and heart rate. 4. The presence of obesity influenced body weight, body fat, de novo fatty acid synthesis, organ weights, glucose mass, glucose oxidation, glucose synthesis, glucose carbon turnover and pulse pressure. 5. It had no effect on glycogen synthesis, tissue glycogen levels, blood glucose, glucose space, or blood pressure. 6. Strain differences were observed in final body weight, organ weights, blood pressure, pulse pressure, hepatic fatty acid synthesis, glucose mass, glucose space, glucose synthesis, liver glycogen levels and glucose conversion to muscle glycogen. 7. Strain-phenotype interaction effects were observed on glucose incorporation into hepatic glycogen, Cori cycle activity, hepatic de novo fatty acid synthesis, final body weight, fat pad weight, heart weight, and mean arterial pressure. 8. These results suggest that although obesity and hypertension are genetic traits in these rats, these traits are independent in their influence on the metabolism of glucose and the development of non-insulin-dependent diabetes mellitus.

In vitro studies on the effects of dehydroepiandrosterone and corticosterone on hepatic steroid receptor binding and mitochondrial respiration.

1. The in vitro effects of dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S) and related steroids on glucocorticoid corticosterone (GC) binding in hepatic cytosol and hepatic mitochondrial respiration were examined in male BHE/cdb and Sprague-Dawley (SD) rats. 2. The Kd and the IC50 for GC binding in SD rats were 5- and 2-fold higher, respectively, than in BHE/cdb rats. 3. Hepatic cytosol from BHE/cdb rats bound six times more [3H]-GC to receptors than that from SD rats. 4. The percentage displacement and Bmax of GC for its receptors were similar for the two strains of rats. 5. DHEA and DHEA-S did not displace GC from its receptors. 6. DHEA and related non-sulfated steroids decreased state 3 mitochondrial respiration, respiratory control and oxidative phosphorylation capacity in a dose-dependent manner with malate + pyruvate as substrate.

A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice.

A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.

Dietary fat saturation affects glucose metabolism without affecting insulin receptor number and affinity in adipocytes from BHE rats.

The effects of dietary fat source on epididymal fat cell insulin receptor binding and affinity and on glucose transport and use by genetically diabetic rats were studied. Male BHE rats were fed 6% fat/64% sucrose diets. The fat consisted of 1% corn oil plus 5% beef tallow, menhaden oil or corn oil. Glucose tolerance was assessed at 100, 300 and 600 d of age. At 100 d of age the fat pads were excised, isolated adipocytes prepared and insulin receptor number, receptor affinity, 3-O-methyl glucose uptake and glucose use determined. Insulin receptor number and binding affinity were not affected by dietary fat type. The transport and subsequent use of glucose were greater in fat cells from rats fed beef tallow compared with those from rats fed corn oil or menhaden oil. All three groups exhibited a deterioration in glucose tolerance with age. Although we observed greater glucose transport, oxidation and conversion to fatty acids in beef tallow-fed rats, we saw no differences in these measurements between cells from corn or menhaden oil-fed rats. Thus, we conclude that the effects of these dietary lipids are attributable to effects of saturated fatty acids on intracellular events rather than on the insulin receptor per se, and that the type of unsaturated fatty acid [(n-3) vs. (n-6)] is of little importance to the regulation of glucose metabolism by isolated adipocytes.

Dietary fat saturation affects hepatocyte insulin binding and glucose metabolism in BHE rats.

The influence of feeding 6% hydrogenated coconut oil, corn oil or menhaden oil on hepatocyte insulin binding, receptor number and glucose use was studied. Hepatocytes isolated from rats fed menhaden oil had a significantly greater affinity for insulin than hepatocytes from rats fed hydrogenated coconut oil. Glucose use was not influenced by diet; uniformly labeled glucose was metabolized to CO2 or to lipid similarly in cells isolated from rats fed the three oils. Thus, dietary fat type in a low fat diet influenced events at the plasma membrane without influencing intracellular events.

[Diagnosis and treatment of rapidly progressive glomerulonephritis. An analysis of 20 cases].

In order to know the incidence and character of rapidly progressive glomerulonephritis (RPGN) in China, 20 patients with RPGN were analysed and 10 of them followed. Their diagnosis were confirmed by biopsy showing extensive crescent formation. Five patients were rebiopsied. RPGN was found in about 3% of the patients with glomerulonephritis in our center. Only one case was mediated by anti-GBM antibody, while the other 19 by immune complex. Prodromal infection was common (12/20). Hematuria, nephrotic syndrome and hypertension were found in 20, 15 and 12 patients respectively. Five patients without special treatment died of renal cause within 6 months. Intensive treatment (pulse methylprednisone or plasmapheresis) was effective in all of the four cases with cellular crescents. After the treatment, their renal function improved with decrease of proliferation and crescent formation as shown by rebiopsy. However, deterioration of renal function reappeared after 3 months to 3 years in three of the cases. It is shown that in China, RPGN was predominantly mediated by immune complex and is associated with a high incidence of prodromal infection. Clinically, nephrotic syndrome was more frequently seen here than in western countries. For improvement of the prognosis, it is necessary to pay attention not only to earlier diagnosis but also to protection of renal function after intensive therapy.

[Renal involvement in amyloidosis: analysis of 14 cases].

UNLABELLED: 14 cases were investigated between 1963 to 1988. Age ranging from 25-66 (mean 48.2). 9 cases (64%) were over age 50. Diagnosis was established by renal biopsy or autopsy. 11 cases (79%) were primary. 3 cases (21%) were secondary with chronic infection. Besides renal involvement, primary amyloidosis principally affected the heart, gastrointestinal tract and tongue. Hepatosplenomegaly and anemia were found in secondary cases. Proteinuria was found in 14 cases. Nephrotic Syndrome (NS) found in 8 cases, both renal insufficiency and hypertension were 21% respectively. In 3 cases performed renal venography, 2 cases showed renal vein thrombosis. 9 cases (64%) showed kidneys were enlarged. Congo red test showed positive reaction were 100% in secondary and 50% in primary. By light microscopy stained with congo red there was a specific protein deposited in glomeruli and mesangium, by electron microscopy. There was fine amyloid fibrils. These findings are characteristic and to have diagnostic value. CONCLUSION: (1) Renal amyloidosis is not very rare in China. (2) Proteinuria and NS were major clinical manifestations. (3) Renal biopsy and autopsy have diagnostic value, congo red test does not prove the absence of primary amyloidosis.

Thyroxine effects on parameters of glucose turnover in BHE rats fed menhaden oil.

The effect of hyperthyroidism on glucose turnover in BHE rats fed menhaden oil was studied. Thyroxine-treated rats had a greater glucose mass, a greater absolute glucose synthesis rate, less hepatic and muscle glycogen levels, and greater hepatic and peripheral fat cell lipogenic rates than nontreated rats. No differences in body weight gain were observed, nor were there differences in blood glucose levels, glucose space, or fractional reversible or irreversible glucose use. These observations suggest that thyroxine and menhaden oil were additive in their effects on glucose metabolism in BHE rats, which are genetically programmed to develop non-insulin-dependent diabetes mellitus.

Glucose turnover in BHE rats fed EFA deficient hydrogenated coconut oil.

The effects of feeding corn or hydrogenated coconut oil on various parameters of glucose metabolism in prediabetic BHE rats was studied. Weanling rats were fed a 6% fat-64% sucrose diet. At seven weeks of age, the rats were weight matched within diet treatments. Half of the rats were injected with 6(3)H/U14C glucose while their weight matched counterparts were injected with U14C alanine and 3HOH. Diet had no effect on glucose mass, glucose space, hepatic glycogen or blood glucose levels. However, diet did affect other parameters. HCO fed rats had higher fractional irreversible glucose turnover rates, fractional glucose carbon recycling, hepatic fatty acid synthesis rates, adipose fatty acid synthesis rate, lower muscle glycogen and lower rates of incorporation of glucose into muscle glycogen than corn oil fed rats. These differences in glucose flux explain the maintenance of glucose homeostasis in these prediabetic coconut oil fed rats in the face of increased fatty acid and glucose synthesis.