Isolation and characterization of Calcineurin B-like gene (PbCBL1) and its promoter in birch-leaf pear (Pyrus betulifolia Bunge).

Calcium plays a critical role in regulating abiotic stress responses in plants. Calcineurin B-like (CBL) proteins are calcium sensors in calcium signaling pathways. However, the molecular mechanisms underlying calcium signaling remain to be elucidated. In this study, the CBL1 gene, which codes for the CBL protein, was isolated from the birch-leaf pear. One 2,969-bp sequence was cloned using PCR, and using the cloned 2,027-bp sequence was isolated from pear genomic DNA via genome walking. Sequencing analysis revealed that the 4,996-bp sequence was a PbCBL1 gene consisting of eight exons and seven introns, and the 2,027-bp sequence was identified as the promoter of the PbCBL1 gene, which contains the basic promoter elements TATA and CAAT boxes. In addition, some other cis-acting elements including heat, cold, drought, and hormone responsive elements were also present. To further investigate the activity of this promoter, the sequence was used to drive a GUS fusion gene into leaf discs of tobacco (Nicotiana benthamiana) with Agrobacterium-mediated transformation method. GUS gene expression could be regulated by the PbCBL1 promoter following induction by GA, ABA, SA, and MeJA. Furthermore, the results of real-time RT-qPCR indicate that the PbCBL1 gene can respond to changes in the intracellular calcium concentration, and that it can be induced by cold, heat, drought, and stress by several hormones including GA, ABA, SA, and MeJA. PbCBL1 gene may be involved in several signal transduction pathways, and play an important role in the condition of adversity stress in pear.

Cloning and expression analysis of PpSUT2 encoding a sucrose transporter in pear.

A 1794-bp cDNA fragment was amplified from mRNA isolated from pear (Pyrus pyrifolia NaKai. Cuiguan) leaves by using primers based on the sequences generated during the analysis of the pear transcriptome. The 597-amino acid sequence encoded by the cDNA was compared with the sequences in GenBank, and it was found to be similar to that of members of the sucrose-proton co-transporter family. The hydrophobic protein, which was predicted to have 11 transmembrane domains, was designated as PpSUT2. Real-time fluorescent quantitative polymerase chain reaction analysis indicated the accumulation of PpSUT2 mRNA throughout the plant, with the highest levels in the buds. Analysis of the expression of PpSUT2 during fruit development showed that the abundance of its transcripts increased at the end of April and then decreased to the lowest level at the end of July. Subcellular localization studies with the pCXDG vector as a probe demonstrated that PpSUT2 localized to cell membranes. An expression vector was constructed by inserting the PpSUT2 cDNA into pET32(a), and the vector was expressed in Escherichia coli (strain BL21) after induction with 1 mM isopropyl b-d-1-thiogalactopyranoside at 25°C. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the induction of a 71-kDa protein. Further analysis indicated that PpSUT2 might be not directly involved in sucrose transport, instead, functioning as a sucrose sensor on the cytoplasmic membrane.

Development of a novel and efficient strategy for practical identification of Pyrus spp (Rosaceae) cultivars using RAPD fingerprints.

Accurate and reliable cultivar identification of crop species is essential to guarantee plant material identity for purposes of registration, cultivar protection and production. To facilitate identification of plant cultivars, we developed a novel strategy for efficient recording of DNA molecular fingerprints in genotyped plant individuals. These fingerprints can be used as efficient referential information for quick plant identification. We made a random amplified polymorphic DNA (RAPD) marker analysis of 68 pear cultivars. All pear genotypes could be distinguished by a combination of eight 11-mer primers. The efficiency of the method was further verified by correct identification of four cultivars randomly chosen from the initial 68. The advantages of this identification include use of fewer primers and ease of cultivar separation by the corresponding primers marked on the cultivar identification diagram. The cultivar identification diagram can efficiently serve for pear cultivar identification by readily providing the information needed to separate cultivars. To the best of our knowledge, this is the most efficient strategy for identification of plant varieties using DNA markers; it could be employed for the development of the pear industry and for the utilization of DNA markers to identify other plant species.

Characterization of cholesterol oxidation products formed by oxidative modification of low density lipoprotein.

Oxidative modification of LDL is evidenced by alterations in both the protein and lipid components of the particle. Progressive oxidation of the apoprotein is associated with loss of specific amino acids and a gradual increase in electronegativity. Electronegative LDL has been isolated from human plasma (LDL-) by several groups using liquid chromatographic techniques and appears to be oxidized based on increased lipid peroxide levels and cholesterol oxidation products (ChOx). Formation of LDL- also takes place following Cu(2+)-induced oxidation. Cu(2+)-induced oxidation caused a small fraction of the normal unoxidized LDL (n-LDL) to convert to LDL-during the oxidative lag phase while minimal increases in conjugated dienes were apparent. After the lag phase, there was a further increase in LDL-, a rapid accumulation of conjugated dienes, and another more electronegative particle was formed (LDL2-). By the end of the lag phase, approximately 30% and 12% of the total LDL converted to LDL- and LDL2-, respectively. Nearly 40% of the total ChOx formed was present by the end of the lag period, accompanied by small increases in conjugated dienes. The major products accumulating during this time were 7-ketocholesterol, cholesterol-beta-epoxide and 7-alpha-hydroxycholesterol. Accumulation of predominated during the subsequent propagation phase. At the end of propagation phase there was a six fold increase in conjugated dienes and total ChOx increased eight-fold. It appears that a subpopulation of LDL rapidly converts to LDL-, representing a mildly oxidized but oxidant sensitive LDL population. Oxidation of cholesterol accompanies these early events in LDL oxidation with formation of specific ChOx.

Sialic acid and oxidizability of low density lipoprotein subfractions of hyperlipidemic patients.

OBJECTIVES: Low density lipoprotein (LDL) does not constitute an homogenous fraction and it is known that the heavy LDL subfraction is potentially more atherogenic than the light one. Because concentration of LDL subfractions tend to be different in hyperlipidemias, it was verified whether these subfractions can also differ in sialic acid and neutral sugar content, as well as their resistance to oxidation. DESIGN AND METHODS: Two subfractions of low density lipoprotein (light LDL, density 1.019-1.034 g/mL and heavy LDL, density 1.034-1.063 g/mL) were isolated from the plasma of 17 patients with hypercholesterolemia, 11 with combined hyperlipidemia, 7 with hypertriglyceridemia, and 19 normolipidemic subjects. The content of sialic acids and neutral sugars of apo B was determined, respectively, by the periodate-thiobarbituric acid method and by reaction with phenol. The oxidation of LDL subfractions was determined by exposure to 5 microM copper (II) followed by the measurement of lipid hydroperoxides production by high performance liquid chromatography with electrochemical detection. RESULTS: The study groups did not differ in the neutral sugar content of LDL subfractions. However, compared to normolipidemic subjects, the sialic acid concentration of both LDL subfractions was lower in patients with hypercholesterolemia and hypertriglyceridemia and higher in those with combined hyperlipidemia (p < 0.05). In the hypercholesterolemia and combined hyperlipidemia groups, the lipid hydroperoxide content (microM) of heavy LDL was higher than in normolipidemic subjects (p < 0.05). CONCLUSIONS: The heavy LDL subfraction was more susceptible to oxidation in the patients with combined hyperlipidemia compared to controls and the other hyperlipidemic groups. The effect of sialic acids on heavy LDL oxidizability seems to vary according to the type of hyperlipidemia.

Plasma antioxidant enzymes and oxidized lipoproteins in hypercholesterolemic rabbits.

The activities of superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPx, EC 1.11.1.9) and the levels of alpha-tocopherol and oxidized lipoproteins were investigated in the plasma of New Zealand rabbits either before or after cholesterol-diet induced hypercholesterolemia. Plasma SOD activity increased while GPx activity decreased after 60 days of cholesterol feeding. However, in the cholesterol-fed rabbits the release of superoxide dismutase fraction C from vasculature by heparin was lower than that in control rabbits. The levels of triglyceride hydroperoxides increased in low density and high density lipoproteins after feeding rabbits with the cholesterol-rich diet during 60 days. Also, a trend for increasing cholesteryl ester hydroperoxides was observed in beta-very low density and high density lipoproteins. An increase in alpha-tocopherol concentration (microM) was observed in very low density and low density lipoprotein fractions, but after normalization of these results to the cholesterol content of lipoprotein particles only the alpha-tocopherol content of low density lipoprotein remained higher after 60 days of cholesterol feeding. The data suggest that low glutathione peroxidase and superoxide dismutase fraction C activities may facilitate intravascular lipoprotein oxidation by oxidant species generated by the endothelium or blood cells.

Effect of withdrawal from long-term nifedipine administration on open-field habituation in the rat.

The effects of withdrawal from long-term administration of nifedipine (2.5 mg/kg, ip, twice daily for 30 days) on open-field habituation were evaluated in 3-month old male Wistar rats (13-14 animals per group). Habituation was evaluated by the ratio between locomotion or rearing frequencies obtained in the second and the first open-field session for each animal. Nifedipine treatment did not modify the locomotion ratio (with a mean +/- SEM ratio of 0.66 +/- 0.12 for control and 0.45 +/- 0.08 for nifedipine-treated group) nor the rearing ratio (with a mean +/- SEM ratio of 0.51 +/- 0.12 for control and 0.62 +/- 0.18 for nifedipine-treated group). The possible factors underlying the discrepancy between the present results and the commonly reported positive effects of calcium channel blockers on memory are discussed.

Effect of withdrawal from long-term nifedipine administration on open-field habituation in the rat

The effects of withdrawal from long-term administration of nifedipine (2.5 mg/kg, ip, twice daily for 30 days) on open-field habituation were evaluated in 3-month old male Wistar rats (13-14 animals per group). Habituation was evaluated by the ratio between locomotion or rearing frequencies obtained in the second and the first open-field session for each animal. Nifedipine treatment did not modify the locomotion ratio (with a mean +/- SEM ratio of 0.66 +/- 0.12 for control and 0.45 +/- 0.08 for nifedipine-treated group) nor the rearing ratio (with a mean +/- SEM ratio of 0.51 +/- 0.12 for control and 0.62 +/- 0.18 for nifedipine-treated group). The possible factors underlying the discrepancy between the present results and the commonly reported positive effects of calcium channel blockers on memory are discussed.

Effects of single and long-term administration of nifedipine on dopamine-related behaviors.

The effects of single (2.5 and 5.0 mg/kg) and long-term (2.5 mg/kg, twice daily, for 30 days) ip administration of nifedipine on open-field and apomorphine-induced stereotyped behavior were evaluated in young male Wistar rats (12-16 animals per group for the open-field studies and 7 animals per group for the stereotypy experiments). Administration of a single dose of nifedipine produced no changes in ambulation or rearing frequencies or in immobility duration in the open-field compared to controls. Similarly, treatment with a single dose of nifedipine did not modify apomorphine-induced stereotypy. Withdrawal from long-term nifedipine administration caused a significant increase only in rearing frequency 24 h after the last drug injection (with a mean +/- SEM frequency of 23.2 +/- 2.8 for the nifedipine group and of 14.7 +/- 2.0 for control rats, after 6-min observation). This enhancement of rearing frequency was no longer observed 48 h after abrupt nifedipine withdrawal (means +/- SEM: 15.0 +/- 2.2 and 19.6 +/- 2.7 for nifedipine-treated and control rats, respectively). The other open-field behavioral parameters and apomorphine-induced stereotypy (which was observed 96 h after nifedipine withdrawal) were not affected by long-term nifedipine treatment; for example, the sum of stereotypy scores (mean +/- SEM) was 26.9 +/- 3.0 for nifedipine-treated rats and 25.5 +/- 2.2 for vehicle-treated animals. The possible mechanisms underlying these results are discussed in light of the changes in dopaminergic neurotransmission induced by dihydropyridine calcium channel blockers.

Effects of single and long-term administration of nifedipine on nociception and stereotyped behavior of rats.

In the present investigation, nociception and stereotyped behavior were evaluated in 3-month old male Wistar rats after a single nifedipine dose (2.5 and 5.0 mg/kg, ip, 1 h before testing, 6-7 rats per group for stereotypy studies and 15 animals per group for nociception experiments) or after long-term nifedipine treatment (2.5 mg/kg, ip, twice daily for 30 days, with testing performed 72 or 96 h after the last injection, 7 rats per group for stereotypy studies and 14-16 animals per group for nociception experiments). Stereotypy was induced with 2.5 mg/kg amphetamine, ip, and nociception was measured by the tail-immersion test. Administration of a single nifedipine dose did not modify nociception or amphetamine-induced stereotypy (with a mean +/- SEM tail-withdrawal latency of 4.5 +/- 0.5 s for control, 4.4 +/- 0.3 s for 2.5 mg/kg nifedipine and 4.7 +/- 0.7 s for 5.0 mg/kg nifedipine and with mean +/- SEM sum of stereotypy scores of 32.5 +/- 1.6 for control, 29.1 +/- 1.0 for 2.5 mg/kg nifedipine and 29.1 +/- 1.6 for 5.0 mg/kg nifedipine). Withdrawal from long-term nifedipine treatment did not affect stereotyped behavior (with mean +/- SEM sum of stereotypy scores of 28.7 +/- 1.6 for control and 30.7 +/- 1.3 for nifedipine-treated rats) but significantly increased tail-withdrawal latencies (with a mean +/- SEM tail-withdrawal latency of 4.1 +/- 0.3 s for control and 6.4 +/- 0.6 s for nifedipine-treated rats). Therefore, long-term nifedipine treatment induced plastic modifications in nociception but not in stereotyped behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of single and long-term administration of nifedipine on nociception and stereotyped behavior of rats

In the present investigation, nociception and stereotyped behavior were evaluated in 3-month old male Wistar rats after a single nifedipine dose (2.5 and 5.0 mg/kg, ip, 1 h before testing, 6-7 rats per group for stereotypy studies and 15 animals per group for nociception experiments) or after long-term nifedipine treatment (2.5 mg/kg, ip, twice daily for 30 days, with testing performed 72 or 96 h after the last injection, 7 rats per group for stereotypy studies and 14-16 animals per group for nociception experiments). Stereotypy was induced with 2.5 mg/kg amphetamine, ip, and nociception was measured by the tail-immersion test. Administration of a single nifedipine dose did not modify nociception or amphetamine-induced stereotypy (with a mean +/- SEM tail-withdrawal latency of 4.5 +/- 0.5 s for control, 4.4 +/- 0.3 s for 2.5 mg/kg nifedipine and 4.7 +/- 0.7 s for 5.0 mg/kg nifedipine and with mean +/- SEM sum of stereotypy scores of 32.5 +/- 1.6 for control, 29.1 +/- 1.0 for 2.5 mg/kg nifedipine and 29.1 +/- 1.6 for 5.0 mg/kg nifedipine). Withdrawal from long-term nifedipine treatment did not affect stereotyped behavior (with mean +/- SEM sum of stereotypy scores of 28.7 +/- 1.6 for control and 30.7 +/- 1.3 for nifedipine-treated rats) but significantly increased tail-withdrawal latencies (with a mean +/- SEM tail-withdrawal latency of 4.1 +/- 0.3 s for control and 6.4 +/- 0.6 s for nifedipine-treated rats). Therefore, long-term nifedipine treatment induced plastic modifications in nociception but not in stereotyped behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of single and long-term administration of nifedipine on dopamine-related behaviors

The effects of single (2.5 and 5.0 mg/kg) and long-term (2.5 mg/kg, twice daily, for 30 days) ip administration of nifedipine on open-field and apomorphine-induced stereotyped behavior were evaluated in young male Wistar rats (12-16 animals per group for the open-field studies and 7 animals per group for the stereotypy experiments). Administration of a single dose of nifedipine produced no changes in ambulation or rearing frequencies or in immobility duration in the open-field compared to controls. Similarly, treatment with a single dose of nifedipine did not modify apomorphine-induced stereotypy. Withdrawal from long-term nifedipine administration caused a significant increase only in rearing frequency 24 h after the last drug injection (with a mean +/- SEM frequency of 23.2 +/- 2.8 for the nifedipine group and of 14.7 +/- 2.0 for control rats, after 6-min observation). This enhancement of rearing frequency was no longer observed 48 h after abrupt nifedipine withdrawal (means +/- SEM: 15.0 +/- 2.2 and 19.6 +/- 2.7 for nifedipine-treated and control rats, respectively). The other open-field behavioral parameters and apomorphine-induced stereotypy (which was observed 96 h after nifedipine withdrawal) were not affected by long-term nifedipine treatment; for example, the sum of stereotypy scores (mean +/- SEM) was 26.9 +/- 3.0 for nifedipine-treated rats and 25.5 +/- 2.2 for vehicle-treated animals. The possible mechanisms underlying these results are discussed in light of the changes in dopaminergic neurotransmission induced by dihydropyridine calcium channel blockers