De novo transcriptome analysis of tobacco seedlings and identification of the early response gene network under low-potassium stress.

Tobacco is an economically important crop, and its potassium content can greatly affect the quality of tobacco leaves. However, the molecular mechanism involved in potassium starvation in tobacco has not been elucidated to date. In this study, Illumina (Solexa) sequencing technology was used to analyze the transcriptome of tobacco seedlings under low-potassium stress for 6, 12, and 24 h. After analysis, 107,824 assembled unigenes were categorized into 57 GO functional groups, and 31,379 unigenes (29.08%) were clustered into 25 COG categories. A total of 9945 genes were classified into 233 KEGG pathways, and 15,209 SSRs were found among the 107,824 unigenes. Between the two samples, 1034 genes were differentially expressed. Twelve randomly selected gene expression levels were analyzed by quantitative RT-PCR, and the results were highly consistent with those obtained by Solexa sequencing. Our results provide a comprehensive analysis of the gene-regulatory network of tobacco seedlings under low-potassium stress.

Identification of low potassium stress-responsive proteins in tobacco (Nicotiana tabacum) seedling roots using an iTRAQ-based analysis.

Potassium is one of the three main mineral nutrients, and is vital for leaf growth and the quality of tobacco (Nicotiana tabacum) plants. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has been one of the most popular techniques for quantitative proteomic analysis. In this study, we used iTRAQ to compare protein abundances in the roots of control and low potassium-treated tobacco seedlings, and found that 108 proteins were differentially expressed between the two treatments. Of these, 34 were upregulated and 74 were downregulated, and 39 (36%) were in the chloroplasts. Kyoto Encyclopedia of Genes and Genomes pathway enrichment results suggested that metabolic pathways were the dominant pathways (10 upregulated and 14 downregulated proteins). Ten proteins involved in the pyruvate metabolism pathway increased their expression levels, and 17 upregulated proteins were enriched in the ribosomes category. To evaluate correlations between protein and gene transcript abundances, the expression patterns of 12 randomly chosen genes were examined. A quantitative real-time polymerase chain reaction revealed that the 12 genes were induced after low potassium treatment for 3, 6, 12, and 24 h. Our results demonstrate that low potassium levels affect protein profiles in tobacco roots.

Proteome analysis of tobacco leaves reveals dynamic changes in protein expression among different cultivation areas.

The leaves of tobacco plants were used to analyze differences in protein content of tobacco grown in the four main flue-cured tobacco-producing areas of Sichuan Province, China. An improved protein extraction method, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gel electrophoretic separation, was used to extract and separate total protein from tobacco leaves. Proteomic maps with relatively high resolution and repeatability were produced. At isoelectric points 4 to 7 and molecular weight ranging from 20-100 kDa, we detected 1032, 1030, 1019, and 1011 clearly visible protein spots in tobacco leaves from the four study areas. Proteome comparison between these protein spots showed that 119 spots with a greater than 2-fold change in expression quantity contributed to the variation in expression. Of which, 115 were successfully identified and annotated. According to the annotation results, these proteins participate in photosynthesis, energy metabolism, mineral nutrition, terpene metabolism, defensive reaction, and other physiological and biochemical processes. This study preliminarily explains the effects of ecological conditions on the physiological metabolism of tobacco leaves and how such effects directly or indirectly contribute to tobacco leaf quality.

First Report of Foliar Blight Caused by Phytophthora capsici on Citrus reticulata Blanco cv. Nian Ju in Guangdong, China.

Citrus reticulata Blanco cv. Nian Ju, an important ornamental plant, is traditionally displayed during the Chinese Spring Festival because its golden fruits are a symbol of auspiciousness. In the spring of 2012, foliar blight was observed on 10 to 30% of the Nian Ju plants at four nurseries in Yangjiang, Guangdong Province, China. Initial symptoms appeared as brown to black foliar lesions, followed by expansion of spots into blight. Some young branches also had necrosis. During frequent rainfall and prolonged wet periods at 22°C to 30°C, white and dense mycelia and sporangia were observed on the infected seedlings. To isolate the causal organism, leaves and stems were cut into sections. Each section included some partial lesion and adjacent asymptomatic tissues. They were surface-disinfested in 1% sodium hypochlorite for 60 s, rinsed three times with sterile water, and placed on V8 juice agar (V8A) at 25°C. After 3 days, 10 isolates were obtained and purified by single-zoospore method. These isolates were identified to species level by sequencing the rRNA internal transcribed spacer (ITS) region. Four representative isolates had an identical ITS sequence (GenBank Accession No. KF750568), which had 99% homology with Phytophthora capsici sequences in GenBank. In addition, all recovered isolates were identical in morphological characteristics. They produced caducous, papillate, and ovoid to ellipsoid sporangia (Length × width = 46.2 ± 7.7 × 23.6 ± 11.3 µm), often with a tapered base. The average length of pedicels was 33.3 ± 4.5 µm. All isolates are A2 mating type. They produced gametangia when paired with an A1 tester of P. capsici isolated from pepper on V8A. Plerotic oospores were 25.3 ± 2.1 µm in diameter. Amphigynous antheridia were 13.6 ± 2.8 µm long and 11.2 ± 0.9 µm wide. Oogonia were 27.4 ± 3.2 µm in diameter. To determine the pathogenicity, three 3-year-old potted C. reticulata cv. Nian Ju plants were sprayed with 20 ml of zoospore suspension from one representative isolate at 105 per ml. Two control plants were sprayed with 20 ml distilled water. All plants were then maintained at 90% relative humidity at 25°C with a 12-h photoperiod. Symptoms similar to those observed in the nurseries developed on all inoculated plants but not on any control plants after 10 days. The pathogenicity test was repeated once and similar results were obtained. P. capsici was recovered from all inoculated plants and resultant isolates had identical morphology to that of the isolates used for inoculation. P. capsici has a relatively broad host range including pumpkins, cucumbers, peppers, beans, squashes, and spinach (1,2). To our knowledge, this is the first report of foliar blight of C. reticulata cv. Nian Ju caused by P. capsici. This study indicates that P. capsici is potentially an important pathogen of C. reticulata cv. Nian Ju plants and further investigations into its epidemiology and development of site-specific integrated management programs for this new disease are warranted. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN, 1996. (2) D. Tian and M. Babadoost. Plant Dis. 88:485. 2004.

Variation of genomic DNA methylation in the nitrate reductase gene of sibling tobacco (Nicotiana tabacum) cultivars.

To better understand genomic DNA methylation in sibling plant cultivars, methylation-sensitive amplification polymorphism analysis was used to investigate two sibling tobacco cultivars, Yunyan85 and Yunyan87, and their two parents, K326 and Yunyan No. 2. Differences in the degree of genomic DNA methylation were found among the four tobacco cultivars. Compared with parents, the two sibling cultivars had fewer methylated sites. Twenty-nine methylation-sensitive amplification polymorphism fragments that exhibited methylation alteration in the four tobacco cultivars were recovered and sequenced. BLAST (nucleotide BLAST) searches showed that two of the 29 sequences have 99% similarity with nucleotides 1442-1694 of the nia-1 gene and the other 27 sequences contain GC, CAAT or TATA box. The nitrate reductase genes from Yunyan87, K326 and Yunyan No. 2 were found to be identical; however, the third intron of the nitrate reductase gene from Yunyan85 was different compared to the third introns of Yunyan87, K326 and Yunyan No. 2. We conclude that methylation alteration of promoter regions could be responsible for the different phenotypes in tobacco and that introns of the nitrate reductase gene can vary as a result of intra-species crossing in tobacco.

First Report of Pestalotiopsis mangiferae and P. vismiae Causing Twig Dieback of Myrica rubra in China.

Chinese bayberry (Myrica rubra Siebold & Zucc.), an evergreen fruit tree, is widely grown in southern China. In 1999, severe twig dieback was observed on M. rubra in Taizhou and it spread to several major M. Rubra-producing areas of Zhejiang covering more than 6,000 ha by 2011. Symptoms were usually observed from June to November and first appeared as chlorosis of leaves and leaf drop, followed by the formation of dark brown lesions covered with white mycelia surrounding leaf scars. The lesions can extend to the whole twig and tree causing discoloration of the xylem. In most cases, infected trees die within 1 to 4 years. Two distinct fungi totaling 46 isolates were isolated from the surface-disinfested diseased twigs and cultured on potato dextrose agar (PDA) at 28°C. An isolate of each fungus, designated as C1 and B1, was characterized further following 10 days of growth on PDA at 28°C. C1 formed zonate, white colonies and black, acervular conidiomata with the conidia aggregated on acervuli as a creamy mass. Isolate B1 formed nonzonate, white colonies and black, acervular conidiomata with the conidia aggregated on acervuli as droplets. Conidia for each isolate were fusiform with five cells; one hyaline apical cell, one hyaline basal cell, and three, dark brown median cells. Conidia ranged from 17.8 to 25.2 × 6.7 to 9.2 µm for C1 and 21.2 to 27.8 × 4.3 to 7.5 µm for B1. There were two to three hyaline, filamentous appendages (9.8 to 23.5 µm long for C1 and 10.5 to 25.5 µm long for B1) attached to each apical cell, and one hyaline appendage (3.5 to 7.2 µm long for C1 and 3.0 to 6.8 µm long for B1) attached to each basal cell. The cultural and morphological characteristics of C1 (16 isolates) matched the description for Pestalotiopsis mangiferae while B1 (27 isolates) matched the description for P. vismiae (2). The PCR-amplified and sequenced internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) for isolate C1 (GenBank Accession No. JQ281542) and B1 (GenBank Accession No. JQ281543) were 99 and 100% homologous to that of the P. mangiferae isolate MM 102 (GenBank Accession No. GU722595) and P. vismiae isolate xsd08116 (GenBank Accession No. FJ481027), respectively. For pathogenicity tests, nine healthy detached leaves and 12 potted plants of M. rubra were wound inoculated with sterile water (control) or conidial suspensions (105 conidia per ml; 20 µl on each site) of C1 and B1, respectively, and maintained with relative humidity of more than 90% under fluorescent light at 28°C. Tests were performed twice. Necrotic lesions, resembling those that occurred in the field, were observed on all inoculated detached leaves and 33.3% of C1 and 25% of B1 inoculated potted plants 10 and 30 days following inoculation, respectively, while the controls remained healthy. Two fungi were reisolated from the lesions with identical morphology to the initial C1 and B1 inoculums. Therefore, P. mangiferae and P. vismiae were determined to be the causal agent for twig dieback of M. rubra in China. Pestalotiopsis spp. were previously reported as pathogens of loquat (4), mango (3), and blueberry (1) causing economic loss. To our knowledge, this is the first report of twig dieback disease of M. rubra caused by P. mangiferae and P. vismiae. References: (1) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (2) Q. X. Ge et al. Flora Fungorum Sinicorum. Vol. 38, Pestalotiopsis. Science Press, Beijing, 2009. (3). Y. Ko et al. Plant Dis. 91:1684, 2007. (4). A. E. Perelló and S. Larran. Plant Dis. 83:695, 1999.

Clinical effect of unilateral balloon infusion of low dose bone cement in PKP for osteoporotic thoracolumbar compression fractures in the elderly.

OBJECTIVE: The study was undertaken to determine the clinical effectiveness of percutaneous kyphoplasty (PKP) with unilateral balloon infusion of low dose of bone cement for treatment of osteoporotic vertebral compression fractures (OVCFs) in the elderly. PATIENTS AND METHODS: A retrospective study was carried out. A total of 36 patients with OVCFs treated by PKP from August 2019 and August 2020 were included. Patients were divided into two groups according to the amount of bone cement infused into the vertebral body. The amount of cement in conventional-dose group was 3.5-6.0 mL and the amount of cement in small-dose group was 1.8-3.0 mL. Pain relief before and after the operation were evaluated, and the leakage of bone cement in the two groups was also observed. RESULTS: Two groups of patients have obtained a good clinical efficacy. Pain has significant differences before and after the operation (p < 0.05). More importantly, compared with conventional-dose group, small-dose group has lower bone cement leakage rate (p < 0.05). CONCLUSIONS: PKP with small-dose bone cement infusion can obtain the same clinical effects of conventional-dose, but the incidence of bone cement leakage is lower and safe.

[A descriptive analysis of tea consumption in adult twins in China].

Objective: To describe the distribution characteristics of tea consumption in adult twins recruited in the Chinese National Twin Registry (CNTR) and provide clues to genetic and environmental influences on tea consumption. Methods: Enrolled in CNTR during 2010-2018, 25 264 twin pairs aged 18 years and above were included in subsequent analysis. Random effect models were used to estimate tea consumption in the population and regional distribution characteristics. The concordance rate of the behavior and difference in consumption volume of tea within pairs were also described. Results: The mean age of all subjects was (35.38±12.45) years old. The weekly tea consumers accounted for 17.0%, with an average tea consumption of (3.36±2.44) cups per day. The proportion of weekly tea consumers was higher among males, 50-59 years old, southern, urban, educated, and the first-born in the twin pair (P<0.05), and lower among unmarried individuals (P<0.001). Within-pair analysis showed that the concordance rate of tea consumption of monozygotic (MZ) twins was higher than that of dizygotic (DZ) twins and the overall heritability of tea consumption was 13.45% (11.38%-15.51%). Stratified by the characteristics mentioned above, only in males, the concordance rate of MZ showed a tendency to be greater than that of DZ (all P<0.05). The differences in consumption volume of tea within twin pairs were minor in MZ among males (P<0.05), while the differences were not significant in female twins. Conclusion: There were discrepancies in the distribution of tea consumption among twins of different demographic and regional characteristics. Tea consumption was mainly influenced by environmental factors and slightly influenced by genetic factors. The size of genetic factors varied with gender, age, and region, and gender was a potential modified factor.

Mifepristone acts as progesterone antagonist of non-genomic responses but inhibits phytohemagglutinin-induced proliferation in human T cells.

BACKGROUND: Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. The stimulation of membrane progesterone receptors (mPRs) would seem to be the cause of rapid non-genomic responses in human peripheral T cells, such as an elevation of intracellular calcium ([Ca(2+)](i)) and decreased intracellular pH (pH(i)). Mifepristone (RU486) produces mixed agonist/antagonist effects on immune cells compared with progesterone. We explored whether RU486 is an antagonist to mPRs and can block rapid non-genomic responses and the induction by phytohemagglutinin (PHA) of cell proliferation. METHODS: Human male peripheral T cell responses in terms of pH(i) and [Ca(2+)](i) changes were measured using the fluorescent dyes, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and fura-2, respectively. Expression of mPR mRNA was determined by RT-PCR analysis. Cell proliferation and cell toxicity were determined by [(3)H]-thymidine incorporation and MTT assay, respectively. RESULTS: The mRNAs of mPRalpha, mPRbeta and mPRgamma were expressed in T cells. RU486 blocked progesterone-mediated rapid responses including, the [Ca(2+)](i) increase and pH(i) decrease, in a dose related manner. RU486 did not block, but enhanced, the inhibitory effect of progesterone on PHA induced cell proliferation. RU486 alone inhibited proliferation induced by PHA and at >25 microM seems to be cytotoxic against resting T cells (P < 0.01). CONCLUSIONS: RU486 is antagonistic to the rapid mPR-mediated non-genomic responses, but is synergistic with progesterone with respect to the inhibition of PHA-induced cell proliferation. Our findings shine new light on RU486's clinical application and how this relates to the non-genomic rapid physiological responses caused by progesterone.

Two dominant host resistance genes to pre-B lymphoma in wild-derived inbred mouse strain MSM/Ms.

To explore possible host genes suppressing spontaneous B-lymphomagenesis in the mouse, expression of ecotropic murine leukemia virus (E-MuLV) and lymphoma development were observed in crosses between the pre-B lymphoma-prone SL/Kh and low-lymphoma strains of mice. E-MuLV expression was intensely inhibited in F1 hybrids with the strains either with the Fv-1b allele (BALB/C, C57BL/10, and A/J) or with the Fv-1nr allele (NZB). In these F1 mice, no lymphoma developed by 18 months of age. On the other hand, F1 hybrids with the strains with the Fv-1n allele [C3H/He, CBA/N, SJL, DBA/2, and MSM/Ms (hereafter referred to as MSM)], high or intermediate levels of E-MuLV expression were observed. Lymphoma incidence in these F1 hybrids, however, was low. This observation suggests the presence of non-Fv-1 dominant resistance genes in these strains. In an attempt to characterize such host genes, we analyzed crosses between SL/Kh mice and a wild mouse-derived inbred strain, MSM/Ms. The latter was susceptible to N-tropic virus expression, but (SL/Kh x MSM)F1 hybrids, did not develop and lymphomas. Of 60 SL/Kh x (SL/Kh x MSM)F1 hybrids, 14 B-lineage lymphomas, including 13 pre-B and 1 follicular center cell lymphoma, developed by 18 months of age. This was compatible with the hypothesis of two independently segregating dominant genes of MSM suppressing lymphomagenesis. By scanning all chromosomes for linkage of lymphoma susceptibility with polymorphic microsatellite loci, one significant linkage disequilibrium was found in the proximal segment of chromosome 17, containing D17MIT44 (map position 15.0) to D17MIT150 (position 33.3), and another linkage disequilibrium, in the midproximal segment of chromosome 18, containing D18MIT90 (map position 28.0) and D18MIT140 (37.0). All 13 pre-B lymphoma-bearing backcross mice were homozygous for SL/Kh-derived alleles at these loci. We named the gene on chromosome 17 Msmr1 (for MSM resistance 1) and that on chromosome 18 Msmr 2 (for MSM resistance 2).

Bone marrow pre-B-1 (Bomb1): a quantitative trait locus inducing bone marrow pre-B-cell expansion in lymphoma-prone SL/Kh mice.

Abnormalities of regulatory genes in early B-cell development often lead to lymphomagenesis. Our previous study showed that there is an abnormal transient expansion of bone marrow (BM) pre-B cells in lymphoma-prone SL/Kh strain mice. Such expansion is a genetic property of SL/Kh stem cells rather than BM microenvironments. Using the percentage of BP1+ B220+ pre-B cells in total BM lymphoid cells as a quantitative parameter, we studied the genetic control of BM pre-B cells in 159 F2 offspring of crosses between SL/Kh and NFS/N mice and 334 back-crosses to SL/Kh mice. A highly significant quantitative trait locus was identified on the distal segment of chromosome 3, showing logarithm of odds scores of 22.7 in the F2 cohort and 10.7 in back-cross mice. This quantitative trait locus, named bone marrow pre-B-1, colocalized with lymphoid enhancer factor-1, which encodes a high mobility group DNA-binding protein that is expressed in T and pre-B cells.

To be or not to be a T-lymphomas, that is determined by a dominant gene Tlsm-1 in mouse models.

A single dominant gene Tlsm-1 was found to determine the type of spontaneous lymphomas to be T in the cross between AKR/Ms and SL/Kh. Microsatellite analysis mapped Tlsm-1 at the position 61 cM from centromere of Chr. 7. Study of this segment of T-lymphoma prone AKXD Rl strains also showed association of Tlsm-1 with T-lymphomas. On the other hand, lymphoma latency was significantly shorten by a recessive gene lla of SL/Kh. By a quantitative trait analysis, lla was located in class II gene in MHC.

Expression of LECAM-1 and LFA-1 on pre-B lymphoma cells but not on preneoplastic pre-B cells in SL/KH mice.

The pre-B lymphoma-prone inbred strain SL/Kh mice showed a polyclonal expansion of BP-1+ pre-B cells in bone marrow early in life. Preneoplastic pre-B cells did not express adhesion molecules LECAM-1 and LFA-1, whereas neoplastic pre-B cells consistently expressed both molecules. There were two types of pre-B lymphomas in SL/Kh with distinct in vivo behavior. One infiltrated lymph nodes and spleen and another, predominantly bone marrow. However, lymphoma cells of both types expressed BP-1, LECAM-1 and LFA-1. Expression of these adhesion molecules on BP-1+ cells, therefore, may represent an important consequence of pre-B lymphomagenesis in SL/Kh strain, but is not sufficient to explain the in vivo behavior of the pre-B lymphoma cells.

[Angiotensin II participates in stress-induced high blood pressure via stimulating hypothalamic vasopressin synthesis and release].

Experiments were carried out in male Sprague-Dawley rats. The animals were randomly divided into three groups: control, stressed and stress + captopril. Stress stimulations were composed of repeated electric foot-shock combined with noise, twice one day (2 h each session) for 15 consecutive days. Animals in the stress+captopril group were administered with captopril (50 mg/kg.d) intraperitoneally. The results showed that at the end of the 15-day experiment the systolic pressure of the tail artery in stressed rats was significantly higher than that of the control rats, i.e., 19.75+/ C1.0 kPa (n=8, P<0.05) versus 16.32+/ C0.55 kPa (n=7); the vasopressin (AVP) mRNA level in the hypothalamus of the stressed rats also increased significantly compared with that of the control rats, i.e., 12990.33+/ C1533.58 (n=6, P<0.001) versus 7332.66+/ C522.65 (n=6). However, in the stress + captopril rats, both the tail artery systolic pressure and hypothalamic AVP mRNA level were significantly higher than those of the control rats, but lower than those of the stressed rats. In the control rats, no significant change in mean blood pressure (MBP) was observed after intracerebroventricular (icv) injection of 0.3 microgram of d(CH(2))(5)Tyr(Me)AVP, a selective AVP V(1) receptor antagonist; however, a decrease in MBP was observed in both stressed and stress+captopril rats (P<0.05), but the decrease in stress+captopril rats was more obvious than that of the stressed rats after icv a same dose of d(CH(2))(5)Tyr(Me)AVP. These results indicate that the endogenous renin-angiotensin system participates in the mechanism of the stress-induced high blood pressure in rats, and that the effect of Ang II is mediated mainly by stimulating hypothalamic AVP synthesis and release, which in turn result in an increase in blood pressure by acting on the central V (1) receptors.

[Effects of gonadal steroid hormones on hypothalamic vasopressin mRNA level in male and female rats].

Experiments were carried out in 10-11-week old gonadectomized male and female Sprague-Dawley rats. Dot-blot analysis and 3'-end digoxigenin-labeled 26 meroligonucleotide probe was used in detecting the mRNA level hypothalamic vasopressin (AVP). The basal hypothalamic AVP-mRNA level in the sham-operated intact males was 45% higher than that in the sham-operated intact females (P < 0.05). Plasma osmolality was also higher in the sham-operated intact males than in the sham-operated intact females (P < 0.05). The hypothalamic AVP-mRNA level in ovariectomized rats was 30% higher than that in sham-operated intact females (P < 0.05). Although the hypothalamic AVP mRNA level tended to be lower in castrated males than in sham-operated intact males, the difference was not statistically significant. The difference in plasma osmolality between gonadectomized males and females was statistically insignificant. In castrated males, hypothalamic AVP-mRNA level was decreased following sc injection of estradiol (P < 0.05), but testosterone, progesterone or a combination of estradiol and progesterone were without effect. In ovariectomized rats, sc injection of estradiol or a combination of estradiol and progesterone resulted in a decrease in hypothalamic AVP-mRNA level (P < 0.01), but progesterone or testosterone had no effect. The difference in plasma osmolality between gonadal steriod hormones-treated rats and vehicle-treated rats was not statistically significant. These findings indicate that gonadal steriod hormones can affect hypothalamic AVP-mRNA level in rats, through some central mechanism.

[Effect of angiotensin II on vasopressin gene transcription in the hypothalamus of rats].

Experiments were carried out in male Sprague-Dawley rats. 3'-end digoxigenin-labeled 26 bp oligonucliotide probe was used to detect the vasopressin (AVP) mRNA in the hypothalamus. Dot blotting technique was used in the investigation of AVP gene transcription level. The results showed that continuous intracerebroventricular administration (i.c.v.) of angiotensin II at a rate of 0.2 nmol/h for 2 days by using a miniosmotic pump resulted in a significant increase in daily water intake. An increase of AVP gene transcription level in the hypothalamus was also observed, but statistically insignificant. When daily water intake was limited (25 ml/d), continuous i.c.v. infusion of ANG II caused a significant increase in hypothalamic AVP gene transcription. It was also observed that hypothalamic AVP gene transcription level increased after salt loading and dehydration. However, intraperitoneal application of angiotensin converting enzyme inhibitor captopril (5 mg/(kg.d)) or i.c.v. nonpeptide angiotensin II receptor antagonist Dup753 (0.9 nmol/h) did not attenuate the increase of AVP gene transcription level induced by salt loading or dehydration. It is therefore suggested that the administration of ANG II enhances AVP gene transcription in the hypothalamus, especially when water intake is limited. However, this increase does not involve the participation of endogenous ANG II.

[Changes in vasopressin V1 receptor mRNA level in rats with high blood pressure induced by chronic stress].

Using reverse transcription polymerase chain reaction (RT-PCR) combined with Southern blotting hybridization technique, the AVP V1 receptor mRNA was found to be widely distributed in central neuronal system and other tissues of rats, such as cerebral cortex, hypothalamus, medulla, liver and kidney. The receptor mRNA levels in the cortex, hypothalamus and medulla were decreased significantly in chronically stressed rats, as compared with normal controls (cortex: P < 0.05; hypothalamus: P < 0.01; medulla: P < 0.001). But no significant change was observed in tissues of heart, liver and kidney (heart: P > 0.05; liver: P > 0.05; kidney: P > 0.05). These results suggest that chronic stress may lead to a decrease of AVP V1 receptor density in the CNS as a result of decreased synthesis.

[Quantitative analysis of mRNA level by PCR method using single basemutated template as inner standard].

Using simple polymerase chain reaction (PCR) method, a single base changed mutant was generated, to which an EcoR I restriction site was added at a specific position of the primer template. The copies of the mutant could be quantified after amplification by PCR. Then the diluted mutant used as an inner standard was added into the samples to be detected. In the same PCR reaction, the mutated and primer template DNA were amplified. After digestion by EcoR I, the PCR products were electrophoresed in 2% agarose gel. The DNAs of different size were separated by electrophoresis and the copies of the primer template in the samples was quantified. The experimental results showed that the cDNA copies of AVP V1 receptor in reverse transcription products from 1 microgram of total RNA isolated from liver tissue of Sprague Dawley rats was about 1.25 x 10(-20) mol.

[Effect of stressful stimulations on hypothalamic vasopressin mRNA level of rats].

Experiments were carried out in stressful foot shock and noise stimulated male Sprague Dawlay rats. 3'-end digoxigenin-labeled 26 mer oligonucliotide probe was used to detect the vasopressin (AVP) mRNA in hypothalamic tissues. Dot blotting technique was used in the assessment of AVP mRNA level. The results showed that the tail systolic arterial pressure of rats increased gradually after stressful stimulations of foot shock in combination with noise. The difference in tail systolic blood pressure between control and stimulated rats was statistically significant after 5-day stimulation. By the ninth day, the systolic blood pressure of the stressed rats reached the highest level and maintained thereafter for several days. No significant difference was observed in the AVP-mRNA level between control and stressed rats by the time when stimulated for 4 days, but it significantly increases when stimulated for 6 days or longer (by the 6th day: P < 0.005; by the 15th day: P < 0.001). No difference was found in plasma osmolalities between control and stressed animals. These results indicate that the stressful foot shocks in combination with noise stimulations may cause an increase in AVP-mRNA level in the hypothalamus, which was accompanied by an increase in systolic arterial pressure. The mechanism of increase in AVP-mRNA level upon stress stimulation remains to be elucidated.