Search

Rooted cuttings from two carnation (Dianthus caryophyllus L.) cultivars showing contrasting responses to the vascular wilt caused by Fusarium oxysporum f. sp. dianthi (Fod) were inoculated with this phytopathogen, and some of the biochemical responses associated with flavonoid biosynthesis were investigated in the roots. The resistant cultivar (' Golem ') showed a significant increase in the levels of phenolic and flavonoid compounds at 48–96 h post-inoculation (hpi) (α = 0.05). LC-MS-based analysis indicated that the flavonoids mainly included flavanol-type glycosides, especially quercetin and kaempferol aglycones. Quantification of the mRNA levels of genes encoding CHS (Chalcone Synthase), CHI (Chalcone Isomerase), FLS (Flavonol Synthase), and the transcription factor MYB11 by using reverse transcription quantitative polymerase chain reaction (RT-qPCR) indicated that the resistant cultivar exhibited higher expression levels of these genes and, therefore, showed more flavonoid accumulation at 96 hpi. The differences in the temporal regulation of the assessed variables during infection support the idea that the early expression of enzymes of the flavonoid biosynthesis pathway in carnation roots is linked to a resistance response to the hemibiotrophic pathogen Fod race 2. The present experimental approach is the first report describing the molecular mechanisms underlying flavonoid biosynthesis in carnation roots during their interaction with Fod. [Display omitted] • Glycosylated flavonols are the most common flavonoids in carnation cultivars. • Fod infection causes an early upregulation of flavonoid biosynthesis in carnation roots. • Targeted metabolite profiling allowed discrimination of carnation cultivars. • mRNA levels of chi , chs and myb1 genes are associated with carnation resistance to Fod. [ABSTRACT FROM AUTHOR]

Abstract: Juvenile hormone (JH) plays key roles in the regulation of growth, development, diapause and reproduction in insects, and juvenile hormone esterase (JHE) plays an important role in regulating JH titers. We obtained a full-length cDNA encoding JHE in Nilaparvata lugens (NlJHE), the first JHE gene cloned from the hemipteran insects. The deduced protein sequence of Nljhe contains the five conserved motifs identified in JHEs of other insect species, including a consensus GQSAG motif that is required for the enzymatic activity of JHE proteins. Nljhe showed high amino acid similarities with Athalia rosae JHE (40%) and Apis mellifera JHE (39%). Recombinant NlJHE protein expressed in the baculovirus expression system hydrolyzed [3H] JH III at high activity and yielded the specificity constants (k cat/K M =4.28×106 M−1 s−1) close to those of the validated JHEs from other insect species, indicating that Nljhe cDNA encodes a functional JH esterase. The Nljhe transcript was expressed mainly in the fat body and the expression level reached a peak at 48h after ecdysis of the 5th instar nymphs. In the 5th instar, macropterous insects showed significantly higher Nljhe mRNA levels and JHE activities, but much lower JH III levels, than those detected in the brachypterous insects soon after ecdysis and at 48h after ecdysis. These data suggest that NlJHE might play important roles in regulation of JH levels and wing form differentiation. [Copyright &y& Elsevier]

Previous work has shown that recombinant protein yield can be improved from in vitro-cultured mammalian cells by culturing at sub-physiological temperatures, although this effect is cell line and product dependent. The mechanism(s) by which low temperature leads to enhanced product yield are currently unknown; however, recent reports suggest that increased mRNA levels at sub-physiological temperatures may be largely responsible for this. Here, we have investigated whether low-temperature cultivation of cell lines selected for antibody production at 37°C leads to changes in heavy- and light-chain mRNA levels and if this is reflected in antibody yields. Low-temperature in vitro culturing resulted in reduced viable cell concentration, prolonged cell viability, a reduction in metabolite consumption and production, cell cycle arrest in both CHO and NS0 cells, and changes in the levels of heavy- and light-chain mRNA. Despite increases in the level of heavy- and light-chain mRNA upon culturing at 32°C in our model CHO cell line, this did not result in increased total product yield; however, changes in cell-specific yields were observed that reflected the metabolic rate of glucose utilization and changes in mRNA levels. [ABSTRACT FROM AUTHOR]

Using a rabbit model of flexor tendon injury, mRNA levels for a subset of relevant molecules involved in inflammatory and fibrotic processes were assessed by reverse transcriptase-polymerase chain reaction 3, 6, 12 and 24 days after injury. Increased levels of COX-2, IL-1β, MMP-13 and TIMP-1 mRNA were detected in both tendon and tendon sheath following injury, with each molecule exhibiting tissue and time-dependent changes. MMP-13 and TIMP-1 mRNA levels were markedly upregulated in both tissues, whereas COX-2 and IL-1β predominantly increased in tendon. Both hyaluronan synthase (HAS) 2 and 3 exhibited increases in mRNA levels in tendon tissue after injury, HAS 2 being more pronounced. These findings support the concept that healing in the flexor tendon and the sheath involve different molecular events and that each tissue may require unique modifications if healing is to be enhanced and adhesions reduced. [Copyright &y& Elsevier]

Abstract: Recent studies have shown that dietary Ca2+ supplementation strongly inhibits uptake of Ca2+ and Cd at the fish gill. To better understand the influence of dietary Ca2+ on branchial Ca2+ transport, we examined the expression of two trout gill calcium transporters during waterborne and dietary Cd exposure, at two different levels of dietary Ca2+. Quantitative polymerase chain reaction (PCR) was used to monitor epithelial calcium channel (ECaC) and sodium–calcium exchange (NCX) mRNA levels following 7–28 days of exposure to these treatments. In brief, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to control, 3μg/L waterborne Cd, 500mg/kg dietary Cd, or a combined 3μg/L waterborne plus 500mg/kg dietary Cd exposure, supplemented with either 20mg/g or 60mg/g dietary calcium (Ca2+). Two-way analysis of variance was used to discern the main effects of Cd exposure and dietary Ca2+ supplementation on ECaC and NCX mRNA levels. We found that dietary Ca2+ supplementation decreased significantly ECaC mRNA expression on days 14 and 21. In comparison, NCX mRNA levels were not influenced by dietary Ca2+ supplementation, but rather were significantly inhibited in the combined waterborne and dietary Cd exposure on day 7 alone. Statistical analysis found no interactive effects between Cd exposure and dietary Ca2+ exposure at any time point, except for day 28.This study provides evidence of the importance of nutritional status on the transcriptional regulation of ion transport at the fish gill. We discuss the importance of diet and nutritional status to the development of new regulatory approaches, such as the biotic ligand model, which currently do not account for the significance of diet on metal bioavailability in aquatic organisms. [Copyright &y& Elsevier]

The article cites a study that aims to determine if larval whitefish Coregonus lavaretus showing the signs of blue sac disease (BSD) had altered mRNA levels. The mRNA levels of three genes, encoding CYP1A, ERα, and p53, which might be differentially expressed in larval fish are investigated. Whitefish larvae used in the study were from farmed broodstock, at the Salmonid Research Laboratory in Rutki, Poland. The species showing the signs of BSD were recognized on the day of hatching.

Synucleins are proteins known for their malfunction in a group of illnesses called synucleopathies, which includes Alzheimer's and Parkinson's disease. To learn more about the role of synucleins in the CNS, we have studied levels of message coding for α-, β-, and γ-synuclein using quantitative RT-PCR. Levels of synuclein mRNAs were studied in the cerebral cortex (left and right, anterior and posterior), hippocampus, striatum, and cerebellum, obtained from 5-d-old (newborn), 1-mo (juvenile)-, and 6-, and 9-mo (adult)-old rats. The mRNA levels for all synucleins varied significantly among structures. The rank order of mRNA levels in different structures was cortex=hippocampus>striatum>cerebellum for α-synuclein; cortex>hippocampus=cerebellum >striatum for β-synuclein; and hippocampus=striatum>cortex=cerebellum for γ-synuclein. There was significant effect of age for mRNA levels for all synucleins. The dynamics of these changes were different depending on type of synuclein and brain structure. Levels of mRNA for α-synuclein were significantly reduced with age in all structures except hippocampus. For β- and γ-synuclein, levels increased significantly only in the cerebral cortex and only from 5 d to 1 mo of age. In contrast, γ-synuclein levels in the cerebellum were very high at 5 d and significantly reduced at 1 mo of age. The revealed pattern and dynamics of changes in the levels of mRNA coding for synucleins would support the conclusion for an important role of these molecules during development and the aging process. [ABSTRACT FROM AUTHOR]

It is well documented that over-expression of the c-myc proto-oncogene occurs in the vast majority of mouse thymic lymphomas induced by γ-irradiation, evidencing the importance of this gene in T-cell lymphomagenesis. However, it remains unknown whether elevated levels of c-myc expression are driven by extra c-myc copy numbers. Here we use a quantitative test on the basis of real-time PCR to determine the cellular copy number of c-myc in a set of 14 g-radiation-induced thymic lymphomas obtained from (C57BL/6J x BALB/cJ) F1 hybrid mice with increased mRNA c-myc expression. Since 5 out of 14 (35.7%) cases had no extra copy numbers of c-myc, gene amplification was obviously not the cause of c-myc over-expression in these tumours. In the remaining 9 tumours, c-myc over-expression was also accompanied with extra DNA copy numbers. Therefore, c-myc amplification might be a consequence of the genomic instability subsequent to the up-regulation of c-myc. However, linear regression analysis showed a lack of correlation between increasing DNA copy numbers and mRNA over expression of c-myc in these tumours (r =0.029, p=0.94). De-regulation of c-myc does not necessarity imply amplification of this gene in these tumours. This report is, to our knowledge, the first one comparing c-myc amplification with expression in lymphomas of the T-cell lineage. [ABSTRACT FROM AUTHOR]

Cytokines, viral load and opportunistic infections play an important role in HIV-disease progression. Hundred children vertically infected with HIV were enrolled to determine mRNA levels of TNF-α, IL-10, IL-4 and IFN-γ. These levels were estimated by amplifying cytokine mRNA from peripheral blood mononuclear cells. Severity of HIV was staged by the reduction in CD4 T cells and the onset of opportunistic infections. IL-10 mRNA levels were observed to increase with the severity. Despite the rising IL-10 mRNA levels, TNF-α mRNA levels increased with severity of HIV and decrease in CD+ T cell counts. IL-4 mRNA levels increased with the reduction in CD4 T cell numbers. Depleting mRNA levels of IFN-γ contributed to the worsening of HIV disease. Increase in TNF-α and IL-4 levels appended to the disease severity by upregulation of the viral replication. Increased IL-10 levels and decreased IFN-γ levels predisposed the children to HIV associated opportunistic infections, which in return contributed to cytokine disarray. [ABSTRACT FROM AUTHOR]+ T cell counts. IL-4 mRNA levels increased with the reduction in CD4 + T cell numbers. Depleting mRNA levels of IFN-γ contributed to the worsening of HIV disease. Increase in TNF-α and IL-4 levels appended to the disease severity by upregulation of the viral replication. Increased IL-10 levels and decreased IFN-γ levels predisposed the children to HIV associated opportunistic infections, which in return contributed to cytokine disarray. [ABSTRACT FROM AUTHOR]

Abstract: A high density lipoprotein-beta glucan binding protein (HDL-BGBP) is synthesized in the hepatopancreas of the white shrimp Litopenaeus vannamei and secreted to the hemolymph. Recently, we reported the HDL-BGBP full length cDNA sequence and found that the predicted polypeptide is larger than the mature protein and also, that it contains a long 5′- and 3′-UTRs that may be involved in transcript level regulation. To test whether starvation and feeding may play a role in regulating HDL-BGBP mRNA levels, two different stimuli were evaluated: starvation and composition of diets. After 24 h, the steady state HDL-BGBP mRNA levels of starved shrimp decreased, suggesting that synthesis of the lipoprotein is less required in the absence of food. When shrimp were fed with diets containing different concentrations of protein and lipids, changes in HDL-BGBP mRNA levels were also detected. Shrimp fed the lower concentration of protein and lipid feed accumulated higher levels of HDL-BGBP mRNA. These results indicate that feeding influences HDL-BGBP transcript levels in the hepatopancreas. [Copyright &y& Elsevier]

Abstract: The overall composition upstream of start codons in Escherichia coli was evaluated and viewed in connection with global transcriptome data. Genes starting with AUG as initiation codon tended to be expressed at higher levels than the non-AUG genes, and the upstream region of the non-AUG genes showed negligible signs of Shine–Dalgarno sequences. The latter is in sharp contrast to the AUG genes. Viewing these findings in connection with the current literature, it is proposed that a distinct mechanism for initiation of translation might exist for non-AUG genes that are not preceded by a Shine–Dalgarno sequence. A survey covering a range of other eubacteria (Firmicutes, Proteoacteria, and Actinobacteria) reveals that it is mainly among the Proteobacteria that non-AUG genes do not display clear signs of Shine–Dalgarno regions. [Copyright &y& Elsevier]

Abstract: Thymidylate synthase (TS) intratumoural expression may be a prognostic marker and predict outcome of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer patients. The TS gene promoter enhancer region contains two different polymorphisms which can influence TS mRNA transcriptional and translational efficiency: a polymorphic tandem repeat sequence (2 or 3 repeats; 2R and 3R) and a single nucleotide polymorphism (SNP), G>C, within the second repeat of the 3R alleles. We studied the relationship between tumoural TS mRNA expression levels and TS gene polymorphisms in the colonic mucosa of 48 colorectal cancer patients. The 3R/3R genotype was characterised by higher TS mRNA levels in the tumour than the 2R/2R–2R/3R genotypes (P =0.071). Regarding the relationship with the SNP polymorphism, a statistically significant difference in TS gene expression between the 3RG/3RG genotype and 2R/2R–2R/3RC–2R/3RG genotype subset was observed (P =0.017). No statistically significant correlation was observed between experimental data and baseline clinical-pathological characteristics as well as clinical outcome in the relatively small patient series investigated. This is the first study reporting an association between the TS intra-repeat SNP and gene expression levels in colorectal cancer patients. These results suggest that in 3R/3R patients, the G>C polymorphism may be an important factor in determining TS mRNA expression levels, and warrant further investigation of the role of TS promoter polymorphisms as predictors of sensitivity to 5-FU-based chemotherapy in larger case series. [Copyright &y& Elsevier]

Exercise is known to upregulate mRNA synthesis for carnitine palmitoyl transferase1 (CPT1) and possibly also other mitochondrial carnitine acyltransferases in muscle tissue. The aim of this study was to test whether such an adaptation of oxidative metabolism in skeletal muscle is a systemic process and consequently, also affects other cells. Messenger RNA levels of five genes [carnitine palmitoyl transferases 1 and 2 (CPT1 and CPT2), carnitine acetyltransferase (CRAT), carnitine palmitoyltransferase 2 (CPT2), microsomal carnitine palmitoyltransferase (GRP58) and organic cation transporter (OCTN2)] were determined with quantitative real time polymerase chain reaction (PCR) in blood cells and in muscle biopsy samples from six cross country skiers before and 6 months after a high volume/low intensity exercise training, when training had elicited a significantly slower rate of lactate accumulation. Quantitative real time PCR showed that levels of mRNA in blood cells correlated significantly (CPT1B: P< 0.001) with those in muscle tissue from the same donors. After 6-months training, there was a 15-fold upregulation of CPT1B mRNA, a six to ninefold increase of CRAT mRNA, of CPT2 mRNA, GRP58 mRNA, and of OCTN2 mRNA. The observation of a concordant stimulation of CPT1, CPT2, CRAT, GRP58 and OCTN2 transcription in blood cells and muscle tissue after 6-month-endurance training leads the hypothesis of a common stimulation mechanism other than direct mechanical stress or local chemical environment, but rather humoral factors. [ABSTRACT FROM AUTHOR]

Adiponectin is an adipocyte-derived hormone that plays an important role in lipid metabolism and glucose homeostasis. Objectives of this study were 1) to determine the presence and distribution of adiponectin and its receptors 1 and 2 (adipoR1 and adipoR2) in porcine tissues; 2) to characterize pig adiponectin, adipoR1, and adipoR2 mRNA levels in various fiat depots from three different breeds of pigs; and 3) to study, in stromal-vascular cell culture, the effects of leptin and tumor necrosis factor-α (TNFα) on pig adiponectin, adipoR1, and adipoR2 gene expression. To this end, fat Chinese Upton Meishan (UM, n = 10), lean Ham Line (HL, n = 10), and Large White (LW, n = 10) gilts were used. We report the isolation of partial cDNA sequences of pig adipoR1 and adipoR2. Porcine deduced AA sequences share 97 to 100% homology with human and murine sequences. Pig adipoR1 mRNA is abundant in skeletal muscle, visceral fat, and s.c. fat tissues, whereas adipoR2 mRNA is predominantly expressed in liver, heart, skeletal muscle, and visceral and s.c. fat tissues. Pig adiponectin mRNA levels in s.c. and visceral fat tissues were not associated with plasma insulin and glucose in fasting animals. Subcutaneous (r = -0.44, P < 0.05), visceral (r = 0.43, P < 0.05), and total body fat (r = -0.42, P < 0.05) weights were negatively correlated with adiponectin mRNA levels measured in visceral, but not s.c., fat. Pig adipoR1 and adipoR2 mRNA levels, in visceral fat, were less expressed in fat UM gilts than in the lean HL gilts (P < 0.05). Inverse associations were found between s.c. (r = -0.57, P < 0.01), visceral (r = -0.46, P < 0.05), and total body fat (r = -0.56, P < 0.01) weights and adipoR2 mRNA levels in visceral fat only. We were unable to find such associations for adipoR1 mRNA levels in the overall gilt population. The current study demonstrated that TNFα downregulates adiponectin and adipoR2, but not adipurl, mRNA levels in stromal-vascular cell culture. Moreover, leptin significantly decreased adiponectin mRNA levels, whereas there was no effect on adiponectin receptors. We conclude that adiponectin and adipoR2 mRNA levels, but not adipoR1, are modulated in pig visceral fat tissues. Furthermore, our results indicate that TNFα interferes with adiponectin function by downregulation of adipoR2 but not of adipoR1 mRNA levels in pigs. [ABSTRACT FROM AUTHOR]

Biological mediators can influence the activity and differentiation of bone cells. 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) is known to induce differentiation of precursors into mature osteoblasts, and transforming growth factorβ1 (TGF-β1) can modulate the activity of bone cells leading to alterations in proliferation and gene expression patterns. Bone-derived cells were loaded via intermittent cyclic hydrostatic pressure (icHP) on cells under basal conditions and in the presence of 1,25-(OH)2D3 or TGF-β1. Evaluating the effects of loading on the cells allowed for a comparison to be made between responsiveness to biomechanical and biochemical stimuli and their potential interplay. The effects of icHP on mRNA levels for the specific genes involved in bone remodelling and differentiation were measured in MG-63 cells using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels for matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) were significantly, and uniquely, increased (p < 0.001) in cells exposed to icHP under serum-free conditions for 4–12 h. However, mRNA levels for MMP-3, but not MMP-1, were significantly enhanced in cells subjected to static hydrostatic pressure (HP). Treatment of cells with 1,25-(OH)2D3 resulted in increased (p < 0.001) mRNA levels for osteocalcin and decreased (p < 0.001) mRNA levels for both MMP-1 and MMP-3. In cells exposed to icHP and 1,25-(OH)2D3, the mRNA levels for both MMP-1 and MMP-3 were elevated (p < 0.001) compared with hormone alone, but not to the same degree (p < 0.01) as cells subjected to icHP alone. Addition of TGF-β1 to cells led to increases in cell proliferation and expression of collagen I, as well as decreases in expression of osteocalcin and MMP-1 and MMP-3. Exposure of cells to icHP and TGF-β1 again led to unique and significant increases in expression of MMP-1 and MMP-3. No changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or any of the other 9 genes assessed, including those for MMP-2 and MMP-13, were detected under any of the conditions described. Therefore, icHP can induce alterations in mRNA levels for a specific subset of genes in both premature and mature osteoblasts. Such stimuli can modulate the impact of potent biological mediators in defining patterns of gene expression by bone cells and potentially modify function in vivo. [ABSTRACT FROM AUTHOR]

Abstract: The amount of peroxisome proliferator-activated receptor-α (PPARα) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARα abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARα and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARα and ACO was seen to increase when the level of FFA in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARα gene in adult rats seems to be less sensitive to nutritional changes than in neonates. [Copyright &y& Elsevier]

The enzyme 5α-reductase (5α-R) is present in many mammalian tissues, including the brain. The physiological importance of 5α-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone to its 5α-reduced derivative, precursors of allopregnanolone, potent allosteric modulator of the γ-aminobutyric acid receptor (GABAA-R). 5α-R occurs as two isoforms, 5α-R type 1 (5α-R1) and 5α-R type 2 (5α-R2). We present an accurate, rapid, and modestly labor-intensive method to precisely quantitate 5α-R mRNA species in the cerebral cortex of the rat. This approach combines the high specificity of “one-step” reverse transcription-polymerase chain reaction (RT-PCR) with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were co-amplified in a single reaction in which the same primers are used. The method presented in this paper enables a more efficient quantitative determination of 5α-R mRNA isozymes, and may lead to a better understanding of the role of 5α-R isozymes in the physiology of the central nervous system. [Copyright &y& Elsevier]

The liver is a multifunctional organ capable of steroid hormone catabolism. The steroid metabolizing enzyme activities have been identified in the liver and it has been demonstrated that neonatal testicular androgens irreversibly program several hepatic enzyme activities in the rat including 5α-reductase (5α-R). In this paper we have applied a quantitative RT-PCR method coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE) to measure the mRNA levels of 5α-R type 1 isozyme (5α-R1) in the liver of female intact and ovariectomized rats. Both groups were treated with testosterone (T) to study the possible regulation of 5α-R1 by androgens in females and to allow comparison with our previous findings in the liver of male rats. Our results demonstrate that the 5α-R1 mRNA isozyme is higher in the liver of female rats than in males and that the regulation of 5α-R1 mRNA by T is different in the liver of female than in male rats. Finally, our results could point to hepatic 5α-Reductase participation in the female not only in the catabolism of steroids with Δ4,5 double bond, but also in other physiological functions such as the production of 3α,5α-reduced metabolites of progesterone and deoxycorticosterone (DOC), potent allosteric modulators of GABAA receptors. [ABSTRACT FROM AUTHOR]

Background: Inhibition of cholesteryl ester transfer protein (CETP) is an effective way to increase HDL levels in animals and humans. The effects of a CETP inhibitor, JTT-705, on the in vivo kinetics of apolipoprotein (apo) A-I and apo A-I gene expression in the liver and intestine were investigated. Methods: Japanese White rabbits were randomly fed normal rabbit chow LRC-4 (n=10, control) or a food admixture of LRC-4 and 0.75% JTT-705 (n=10, treated) for 7 months. An in vivo kinetics study of apo A-I was performed by injecting rabbit 125I-apo A-I, and apo A-I mRNA levels were quantified by RT-PCR. Results: JTT-705 significantly inhibited CETP activities, increased serum levels of HDL-cholesterol (C), HDL2-C, HDL-phospholipid, and apo A-I, and decreased HDL-triglyceride levels. The synthetic rate of apo A-I was higher in the treated rabbits than in control rabbits (13.7±2.6 versus 9.5±1.3 mg/kg per day, P<0.05), while the fractional catabolic rate was similar in the two groups. JTT-705 increased apo A-I mRNA levels in the liver without affecting those in the intestine. Conclusion: Inhibition of CETP activity by JTT-705 increases HDL levels by increasing the synthesis of apo A-I, suggesting that it could be a promising therapeutic approach for atherosclerosis. [Copyright &y& Elsevier]

A novel, accurate, rapid and modestly labor-intensive method has been developed to quantitate specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR). This strategy combines the high degree of specificity of competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). The specific target mRNA and a mimic DNA fragment, used as an internal standard (IS), were co-amplified in a single reaction in which the same primers are used. The amount of mRNA was then quantitated by extrapolation from the standard curve generated with the internal standard. PCR primers were designed to amplify both a 185 bp fragment of the target cDNA for steroid 5α-reductase 1 (5α-R1) and a 192 bp fragment of the target cDNA for steroid 5α-reductase type 2 (5α-R2). The 5′ forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). Two synthetic internal standard DNAs of 300 bp were synthesized from the sequence of plasmid pEGFP-C1. The ratio of fluorescence intensity between amplified products of the target cDNA (185 or 192 bp fragments) and the competitive DNA (300 bp fragment) was determined quantitatively after separation by capillary electrophoresis and fluorescence analysis. The accurate quantitation of low-abundance mRNAs by the present method allows low-level gene expression to be characterized. [Copyright &y& Elsevier]

Bipolar affective disorder (BPD) is a multifactorial, severe, chronic and disabling illness with 50% heritability that affects 1–2% of the population. Lithium ions (Li) are the drug of choice for BPD. Yet, 20–40% of patients fail to respond to Li. Although numerous biochemical and cellular effects have been attributed to Li, its therapeutic mechanism of action has not been elucidated. This review presents the possible involvement of 3′(2′)-phosphoadenosine-5′-phosphate (PAP) phosphatase in the etiology of bipolar disorder and the mechanism of action of Li. Of the enzymes inhibited by Li, PAP phosphatase is inhibited with the lowest Ki (0.3 mM). At therapeutic concentrations of Li (0.5–1.5 mM), inhibition is greater than 80%. Therefore, PAP phosphatase is a strong candidate for Li''s therapeutic mechanism of action. In yeast, a PAP phosphatase knockout mutation leads to the accumulation of PAP, which affects ribosomal-, transfer- and small nucleolar-RNA processing. PAP accumulation in the mammalian brain following Li inhibition of PAP phosphatase may very well account for the observed effects of Li on gene expression and behavior. Furthermore, we have reported significant changes in PAP phosphatase levels in postmortem frontal cortex of bipolar patients. [Copyright &y& Elsevier]

In the de novo synthesis of inositol, the conversion of d-glucose-6-phosphate to l-myo-inositol-1-phosphate (MIP) is catalyzed by MIP synthase. Little is known about mammalian MIP synthase and nothing is known about its regulation. The second step in inositol biosynthesis is the conversion of MIP to inositol by inositol-monophosphatase (IMPase), a common step to inositol production via the de novo pathway and its recycling from inositol phosphates. Because lithium inhibits IMPase both in yeast and in mammals, and the drug upregulates yeast MIP synthase (INO1) and downregulates IMPase (INM1), the present study was undertaken to determine whether chronic in vivo therapeutic lithium concentrations affect MIP synthase and IMPase expression in mouse frontal cortex and hippocampus. Mice were treated with food containing LiCl (2.5 g/kg) for 10 days. RNA was purified from the brain areas and mRNA amplified using RT-PCR. Expression of MIP synthase and IMPA1 (one of the genes coding for IMPase) but not IMPA2 was upregulated in mouse hippocampus. None of the genes were affected in the frontal cortex. In yeast, when inositol is limiting, the heterodimeric transcriptional activator Ino2p/Ino4p derepresses expression of INO1 by binding to the upstream activation sequence UASINO. Using the TFSEARCH program, we found that the promoter of the virtual human MIP synthase gene contains upstream stimulating factor (USF) elements with a similar core binding sequence. The fact that lithium treatment upregulates both MIP synthase and IMPA1 mRNA levels in mouse hippocampus may reflect a compensatory response of both genes to inositol depletion. [Copyright &y& Elsevier]

During peak smoltification, the interactive effects of cortisol and ambient seawater challenge were compared in two strains of Atlantic salmon (Salmo salar) smolts: a domesticated strain, AquaGen and a native River Imsa strain. Tissue and blood samples were taken from untreated fish on 20 May. Fish were then transferred to experimental tanks, allowed to recover for 24 h and cortisol (50 mg kg body mass−1), dissolved in vegetable oil, or vegetable oil alone (sham) was implanted. Samples were taken 5 days post implantation. Fish were then exposed to 24 h ambient seawater challenge (FW–SW) or freshwater to freshwater replacement (FW–FW) and sampled as before. Sham implantation had no significant impact on any of the measured parameters. Cortisol implantation significantly elevated plasma cortisol in FW–FW Imsa smolts, while no effect was observed in FW–SW Imsa smolts. Cortisol implantation had no effect on the plasma cortisol levels of AquaGen smolts regardless of FW–SW challenge. Increased plasma cortisol corresponded with significantly higher plasma glucose levels in FW–FW Imsa smolts. Plasma Na+ and Cl− levels were not affected by cortisol implantation but were significantly increased in FW–SW smolts of both strains. Gill Na+,K+-ATPase activity increased in response to cortisol implantation in only FW–FW smolts but not FW–SW smolts. Gill Na+,K+-ATPase α-subunit mRNA levels were not affected by strain, cortisol injection or transfer protocol, while both CFTR I and CFTR II mRNA levels were significantly higher in AquaGen versus Imsa smolts regardless of treatment. CFTR I mRNA was elevated following cortisol implantation in FW–FW smolts from both strains suggesting CFTR I expression is under the control of cortisol. These findings also suggest that plasma cortisol levels are regulated differently between strains and that cortisol implantation and ambient FW–SW challenge interact, interfering with the individual effects of each of these factors. [Copyright &y& Elsevier]

This study identified two potential novel biomarkers of peroxisome proliferation in the rat. Three peroxisome proliferator-activated receptor (PPAR) ligands, chosen for their high selectivity towards the PPARα, -δ and -γ subtypes, were given to rats twice daily for 7 days at doses known to cause a pharmacological effect or peroxisome proliferation. Fenofibrate was used as a positive control. Daily treatment with the PPARα and -δ agonists produced peroxisome proliferation and liver hypertrophy. 1 H nuclear magnetic resonance spectroscopy and multivariate statistical data analysis of urinary spectra from animals given the PPARα and -δ agonists identified two new potential biomarkers of peroxisome proliferation - N -methylnicotinamide (NMN) and N -methyl-4-pyridone-3-carboxamide (4PY) - both endproducts of the tryptophan-nicotinamide adenine dinucleotide (NAD + ) pathway. After 7 days, excretion of NMN and 4PY increased 24- and three-fold, respectively, following high doses of fenofibrate. The correlation between total NMN excretion over 7 days and the peroxisome count was r =0.87 ( r 2 =0.76). Plasma NMN, measured using a sensitive high performance liquid chromatography method, was increased up to 61-fold after 7 days' treatment with high doses of fenofibrate. Hepatic gene expression of aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) was downregulated following treatment with the PPARα and -δ agonists. The decrease was up to 11-fold compared with controls in the groups treated with high doses of fenofibrate. This supports the link between increased NMN and 4PY excretion and regulation of the tryptophan-NAD + pathway in the liver. In conclusion, NMN, and possibly other metabolites in the pathway, are potential non-invasive surrogate biomarkers of peroxisome proliferation in the rat. [ABSTRACT FROM AUTHOR]

Many hypothalamic neuropeptides are involved in the regulation of energy homeostasis and feeding behavior. We have investigated whether and to what extent neuropeptide Y (NPY), agouti-related protein (AGRP), melanin-concentrating hormone (MCH), and prepro-orexin (prepro-OX) as well as pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) mRNA levels are affected in rat hypothalamus. An experimental model of long-term fasting rat characterized by three metabolic phases from changes in lipid and protein utilization was used. Except for prepro-OX and compared to fed group, starvation induced an increase in the orexigenic gene expressions that was much more marked in phase 3 (by 2.5-, 8.1-, and 13.5-fold for MCH, AGRP, and NPY, respectively) than in phase 2 (by about 1.5–2.2-fold as an average) of fasting. AGRP and NPY mRNA levels were inversely related to body fat content. Anorexigenic gene expression was only slightly affected at both fasting stages. We conclude that the regulation of NPY and AGRP gene expression is primarily involved during late fasting and could mediate the concomitant enhanced drive for refeeding. [Copyright &y& Elsevier]

This study investigated the effects of the natural polyphenol mangiferin (MA) on superoxide anion (O2−) production, xanthine oxidase (XO) activity, vascular contractility, inducible nitric oxide synthase (iNOS) mRNA levels, tumour necrosis factor-alpha (TNF-α) mRNA levels, and tumour growth factor-beta (TGF-β) mRNA levels. O2− was generated by the hypoxanthine–xanthine oxidase (HX–XO) and phenazine methosulphate (PMS)–NADH systems. XO activity was determined by measurement of uric acid production with xanthine as substrate. Vascular contraction experiments were performed with intact rat aortic rings. iNOS, TNF-α and TGF-β gene expression in rat macrophages stimulated in vivo with 3% thioglycollate and in vitro with 100 ng/mL lipopolysaccharide and 10 U/mL of interferon-gamma were evaluated semiquantitatively by the retrotranscriptase–polymerase chain reaction. MA at 10–100 μM, like the known O2− scavenger superoxide dismutase (1 U/mL), scavenged O2− produced by the HX/XO and PMS–NADH systems. By contrast MA at 1–100 μM, unlike allopurinol (10 μM), was unable to inhibit XO activity. MA at 1–100 μM did not modify resting tone or the contractile responses elicited by 1 μM phenylephrine or 1 μM phorbol 12-myristate 13-acetate in rat aorta. MA at 1–100 μM, like dexamethasone (100 μM), decreased iNOS mRNA levels in activated macrophages. At 100 μM, MA also reduced TNF-α mRNA levels, but increased TGF-β mRNA levels. These results thus indicate that MA is an O2− scavenger and that it inhibits expression of the iNOS and TNF-α genes, suggesting that it may be of potential value in the treatment of inflammatory and/or neurodegenerative disorders. In addition, the finding that MA enhances TGF-β gene expression suggests that this polyphenol might also be of value in the prevention of cancer, autoimmune disorders, atherosclerosis and coronary heart disease. [Copyright &y& Elsevier]

Abstract Background The contractile response of human splanchnic vessels to different vasoconstrictors is attenuated in cirrhosis. Functional studies indicate a cellular signalling defect upstream of the G-protein level. The aim of the present study was to analyze expression and mRNA levels of the following most relevant vasopressor receptors in the smooth musculature of human hepatic arteries: α1 adrenoceptor (AR) subtypes a, b and d, angiotensin II type 1 receptor (AT1), arginine vasopressin receptor type 1a (V1a), endothelin receptor type A (ETA ) and B (ETB ). Materials and methods Hepatic arteries were collected from 10 donors (noncirrhotic) and 14 recipients (cirrhotic) at liver transplantations. Real-time-PCR was performed to quantify steady-state levels of receptor mRNAs. Results α1aAR mRNA levels showed no significant difference between the cirrhotic arteries and the controls while the mRNA levels of the other vasoactive receptors were significantly higher in the cirrhotic hepatic arteries (α1bAR: 4-fold, P = 0·013; AT1: 16-fold, P = 0·024; V1a: 23-fold, P = 0·001; ETA: 4-fold, P = 0·02; ETB: 8-fold, P = 0·008). No mRNA for the α1dAR was detected either in the donor or recipient hepatic arteries. Conclusion We conclude that vascular hyporeactivity to the most relevant endogenous vasoconstrictors of cirrhotic hepatic arteries is not caused by a receptor down-regulation at mRNA levels. In contrast they were up-regulated. [ABSTRACT FROM AUTHOR]

Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobilization of this compound have been proposed, including a novel mechanism that involves the release of arachidonic acid from arachidonoyl-CoA by a thioesterase with substrate specificity for very-long-chain fatty acids. In rat heart, the acyl-CoA thioesterase activity can be regulated by a mechanism that involves β-adrenoceptors. In this paper we demonstrate that β-adrenergic agonists also regulate the acyl-CoA thioesterase mRNA levels. Isoproterenol (10−7 M)—a concentration known to exert physiological responses—increases in a time-dependent manner the acyl-CoA thioesterase mRNA levels, an effect blocked by a specific β-adrenoceptor antagonist. In addition, our results show that cAMP is involved in this process. The acyl-CoA thioesterase mRNA levels are also increased by fasting, but not by di(2-ethylhexyl)phthalate, a peroxisome proliferator. These results may suggest the existence of a β-adrenoceptor-activated regulatory pathway for arachidonic acid release in cardiac tissue. [Copyright &y& Elsevier]

Objective: 3'(2')-Phosphoadenosine 5'-phosphate (PAP) phosphatase is a novel lithium (Li) inhibitable enzyme. Thus the enzyme seemed an important candidate for studies of the molecular etiology of bipolar disorder. Methods: RT-PCR, Western-blot analysis and Pi liberation were used to measure PAP phosphatase mRNA levels, protein levels and enzyme activity (respectively) in postmortem frontal cortex specimens of bipolar patients versus normal subjects. Results: The PAP phosphatase protein levels were 24% significantly lower in bipolar patients than in normal subjects. PAP phosphatase mRNA levels and enzymatic activity did not differ between normal controls and bipolar patients. Conclusions: Abnormality of PAP phosphatase in bipolar patients offers a new direction for study of bipolar disorder etiology. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan, biglycan, decorin, fibromodulin, MMP-1,-3,-13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent. [Copyright &y& Elsevier]

The secretion of gonadotropins, the key reproductive hormones in vertebrates, is controlled from the brain by the gonadotropin-releasing hormone (GnRH), but also by complex steroid feedback mechanisms. In this study, after the recent cloning of the three gonadotropin subunits of sea bass (Dicentrarchus labrax), we aimed at investigating the effects of GnRH and sexual steroids on pituitary gonadotropin mRNA levels, in this valuable aquaculture fish species. Implantation of sea bass, in the period of sexual resting, for 12 days with estradiol (E2), testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT), almost suppressed basal expression of FSHβ (four to 15-fold inhibition from control levels), while slightly increasing that of α (1.5-fold) and LHβ (approx. twofold) subunits. Further injection with a GnRH analogue (15 μg/kg BW; [d-Ala6, Pro9-Net]-mGnRH), had no effect on FSHβ mRNA levels, but stimulated (twofold) pituitary α and LHβ mRNA levels in sham- and T-implanted fish, and slightly in E2- and DHT-implanted fish (approx. 1.5-fold). The GnRHa injection, as expected, elevated plasma LH levels with a parallel decrease on LH pituitary content, with no differences between implanted fish. In conclusion, high circulating steroid levels seems to exert different action on gonadotropin secretion, inhibiting FSH while stimulating LH synthesis. In these experimental conditions, the GnRHa stimulate LH synthesis and release, but have no effect on FSH synthesis. [Copyright &y& Elsevier]

BackgroundProtein synthesis in skeletal muscle is reduced following starvation and restored by feeding. The mediators and mechanisms are incompletely understood. The aim of this study was to evaluate whether prolongation of undernutrition induced changes in muscle gene expression at the level of mRNA and protein. Materials and methodsThe changes in myosin heavy-chain 2X mRNA in adult partially starved (50% of ad libitum standard rodent chow intake for 4 or 7 days) C57BL mice or subsequently refed mice were studied. Ad libitum-fed mice were used as controls. Protein synthesis, total RNA and myosin heavy-chain 2X mRNA concentrations were determined. Plasma concentrations of amino acids were measured by high-performance liquid chromatography. ResultsPartial starvation of 4 and 7 days reduced body weight by 15.6 ± 1% and 17.1 ± 2.1% (P ≤ 0.05) vs. ad libitum fed controls. Protein synthesis was reduced by 32 ± 9% and protein content by 20 ± 4% (P ≤ 0.05) following 7 days of partial starvation. Plasma amino acid concentrations were increased (6297 ± 853) in refed animals vs. ad libitum-fed controls (3057 ± 141, P ≤ 0.05). Total RNA concentration (μg RNA μg-1 DNA) in skeletal muscle was unchanged. Myosin heavy-chain 2X mRNA concentration did not change following 4 days of partial starvation but increased by 24 ± 5% (P ≤ 0.05) following 7 days of partial starvation, hence suggesting that expression of myosin mRNA was nutritionally altered. ConclusionPostprandial stimulation of protein synthesis following starvation may thus be a combination of increased mRNA availability and increased translation. This effect may be activated by peak concentrations of amino acids in plasma following feeding. [ABSTRACT FROM AUTHOR]

Expression of the mdr1 (multidrug resistance), mrp (multidrug resistance associated protein), and lrp (lung resistance related protein) genes is associated with transport related MDR (multidrug resistance). We quantified mRNA levels of these genes using competitive reverse transcription polymerase chain reaction (RT-PCR) in 128 samples of leukaemic cells from 92 patients with acute myelogenous leukaemia (AML). There was a wide variation between the samples in mRNA levels of all three genes. The mean mdr1 mRNA level was 1.3 transcripts per cell (range undetectable to 15.8), the mean mrp level was 7.9 (range 0.1–36.2) and mean lrp 3.9 (range 0.1–29). Lrp mRNA levels were higher in samples drawn at diagnosis from the 15 patients with resistant disease than from the 37 with chemosensitive disease (4.9 SD 3.1 v 2.9 SD 2.3, P = 0.016). Neither mdr1 nor mrp mRNA levels were predictive for response to chemotherapy. In samples from patients who had received chemotherapy, those that had received mitoxantrone (n = 24) had higher lrp mRNA levels (mean 4.8, SD 2.5) than those that had not (n = 20, mean 2.8, SD 2.4, P = 0.012). In conclusion, the results indicate that lrp expression is associated with inferior response to chemotherapy in AML and that lrp expression increases after exposure to mitoxantrone. [ABSTRACT FROM AUTHOR]

Comparisons were made between the changes in mRNA levels induced by low night temperatures in the cold-sensitive tomato and two altitudinal ecotypes of the wild species L. hirsutum. Changes in mRNA levels were detected by resolving in vitro translation products of poly (A)+ RNA by 2-D PAGE. The treatment was applied by first growing plants in a thermoperiod of 25/18°C and then switching to 25/6°C. All tomatoes displayed a diurnal cycling in which a set of mRNAs accumulated at the end of the 18°C nights, whereas another accumulated at the end of the 25°C days. The accumulation of night specific mRNAs was inhibited by 6°C nights in the cold sensitive tomatoes while that of the tolerant one was only marginally affected. All tomatoes showed a similar reduction in the apparent turnover rate of the day specific mRNAs during the 6°C nights. Finally, low night temperatures induced the accumulations of six to eigh mRNAs in all genotypes. This number increased by 15 in L. esculentum after the seventh night and are likely involved in stress response rather than acclimation/tolerance. The tomato is proposed as a genetic model to discriminate genes involved in acclimation/tolerance from those involved in stress response. [ABSTRACT FROM AUTHOR]

We have isolated and sequenced several overlapping cDNA clones from a bovine λgt10 library which encode all but the first five amino acids of the entire mature 24 kDa subunit of NADH : ubiquinone oxidoreductase (EC 1.6.99.3), the first enzyme of the respiratory chain. The derived amino acid sequence agrees with that determined by direct sequencing of the purified protein, filling in a gap in the published sequence. A comparison of the nucleotide and amino acid sequences of the bovine 24 kDa subunit with those recently determined for the rat homologue has shown that this nuclear-encoded subunit of an OXPHOS complex has diverged in these two species much less than the mitochondrial DNA-encoded subunits of the same enzyme complex, and also less than a set of available non-mitochondrial nuclear DNA-coded proteins. The sequence analysis of the clones has revealed the expression in the brain of two mRNAs differing in the length of the 3′-untranslated region. Furthermore, two polyadenylated RNA species, 930 and 1080 nucleotides in length, probably corresponding to the above mRNAs, have been detected in bovine brain and other tissues by RNA gel blot hybridization. The level of expression of the 24 kDa subunit gene varies by more than an order of magnitude among different tissues. A cross-hybridizing mRNA species of 930 nucleotides has also been observed in HeLa cells and found to be strongly growth regulated. [ABSTRACT FROM AUTHOR]

The effects of antipsychotic administration (1-32 days, twice per day) on the levels of mRNA coding for dopa decarboxylase (DDC) and tyrosine hydroxylase (TH) in rat brain has been assessed by a procedure utilising solution hybridisation with oligonucleotides. Saline and sulpiride (20 mg/kg/day) had no apparent effect on DDC mRNA levels. Haloperidol (3 mg/kg/day) elicited increases in DDC mRNA levels of 240% after 32 days and loxapine (4 mg/kg/day) elicited increases of 180% in DDC mRNA levels. None of the drugs affected TH mRNA levels. These results indicate that DDC may be more important than TH in the long term regulation of dopamine production. [ABSTRACT FROM AUTHOR]

The effects of administration of antipsychotic drugs (1-32 days, twice per day) on the rat brain mRNA levels of dopamine D, D and D receptors has been assessed by a novel procedure utilising solution hybridisation with oligonucleotides. Saline and sulpiride (10 mg/kg/injection) had no effect on D, D and D receptor mRNA levels. Haloperidol (1.5 mg/kg/injection) elicited increases in D, D and D receptor mRNA levels of 100%, 100% and 300%, respectively, after 32 days and loxapine (2 mg/kg/injection) elicited increases of 450%, 150% and 550%, respectively. These results indicate that the up-regulation of dopamine receptors may be associated with the occurrence of tardive dyskinesia but not the clinical mode of action of antipsychotics. [ABSTRACT FROM AUTHOR]

The photosynthetic apparatus is rich in iron-containing cofactors and iron deficiency causes severe impairment of photosynthesis in plants, algae, and cyanobacteria. Synechocystis sp. PCC 6803 serves as a model system to investigate the complex assembly and integration of the multi-subunit protein complexes of oxygenic photosynthetic electron transport; particularly when coupled to developmental cues due to nutrient limitation or requirements. We study Fe-deficient and Fe-supplemented cultures of Synechocystis sp. PCC 6803. The autotrophic growth rate of Fe-deficient cultures is slower than Fe-supplemented cultures. Whole cell spectral analysis reveals differences in both the quantity and the peak absorbance of chlorophyll. Fe deficiency decreases rates of photosynthetic electron transport and the mRNA and corresponding protein levels as observed using specific probes. mRNA levels of psaB increased 20-fold during recovery from Fe deficiency, as compared to the control. psaD transcript levels increased to 160% during recovery as compared to the control. PsaA/B heterodimer formation and turnover is dependent on Fe and the complete assembly on the reducing side of photosystem I (PS I) is PsaD-dependent. Recovery from Fe deficiency suggests that regulation occurs at both the mRNA and protein level. [ABSTRACT FROM AUTHOR]

Oat polysomes direct the synthesis in vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased amount of the other products synthesized in vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments using oat poly A mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is partially responsible for the levels of globulin in the mature grain. [ABSTRACT FROM AUTHOR]

At least three classes of legumin, encoded by the gene families legA, legJ and legS, and a lectin, encoded by a single gene, accumulate in the developing cotyledons of Pisum sativum L. Transcription rates for the genes encoding these proteins were measured in nuclei isolated from cotyledons at 12 and 16 days after flowering (DAF). The steady-state levels of the corresponding mRNA species were also measured in absolute terms throughout cotyledon development, from 8-9 to 28 DAF. When transcription rates and steady-state mRNA levels of the different gene families are compared, there is little correlation. This indicates a posttranscriptional regulation of the level of expression of these storage proteins in the developing cotyledons. Expression of the legumin genes is known to be seed-specific, whereas expression of the lectin gene is found in both seed and root. When transcription rates were measured in leaf nuclei the levels of legumin and lectin transcripts detected approached background levels, indicating that these genes are either inactive or transcribed at very low levels in leaves; however, the rate of transcription of the chlorophyll a/b-binding protein gene was high. This points to transcriptional control as the major factor in the organ-specificity of legumin and lectin expression. [ABSTRACT FROM AUTHOR]

Mesembryanthemum crystallinum responds to high salinity in the soil by shifting the mode of carbon assimilation from the C3 mode to Crassulacean acid metabolism (CAM). Several enzymes of carbon metabolism have increased apparent activities in the CAM mode, including phosphoenolpyruvate carboxylase (PEPcase) and pyruvate orthophosphate dikinase (PPDK). We have identified cDNA clones for PEPcase and PPDK by immunological screening of a cDNA library constructed in the protein expression vector lambda gt11. The clones were characterized by immunoblotting and RNA blotting techniques. RNA blotting showed that during CAM induction the steady-state level of mRNAs for both PEP case and PPDK increased. [ABSTRACT FROM AUTHOR]