Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

Biosynthesis and posttranslational processing of the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line. Effect of dexamethasone.

Neurotensin (NT) and Neuromedin N (NN) are two biologically active peptides present in one copy each in the C-terminal region of a 169-residue precursor. Four basic Lys-Arg doublets occur within the precursor and represent putative processing sites. We investigated the effects of dexamethasone on the biosynthesis and the posttranslational processing of the NT/NN precursor in the rat medullary thyroid carcinoma 6-23 cell line (rMTC 6-23). Western blot analysis and RIA coupled to HPLC and arginine-directed tryptic cleavage of precursor forms were performed with antisera specific for precursor sequences adjacent to the four basic doublets. These studies revealed that rMTC 6-23 cells synthesized the NT/NN precursor in response to dexamethasone and had the capability to process this precursor at the three Lys-Arg doublets that flank and separate NT and NN, thus yielding authentic NT, NN, and several larger products. The most N-terminal Lys-Arg doublet was not processed in this system. Dexamethasone increased in a concentration- and time-dependent manner the levels of all the NT/NN precursor-derived products. This increase did not affect the relative proportion of the different products. We also showed by Northern blot analysis that both the 1.1-kilobase and 1.5-kilobase NT/NN precursor messenger RNAs were present in the rMTC 6-23 cell line and that the time course and dose response of dexamethasone-induced messenger RNA synthesis were in good agreement with those observed for dexamethasone-induced increase in processing products. The rMTC 6-23 cell line represents a good model to elucidate the steps involved in the posttranslational processing of the NT/NN precursor.

Neuromedin-N is not released with neurotensin from rat ileum.

Neuromedin-N (NN) and neurotensin (NT) were shown recently to be encoded in the same precursor molecule. Colocalization and corelease of ileal NT and NN have not yet been demonstrated and were investigated in the rat using antisera that separately recognized intact NT and NN in ileal extracts. Immunofluorescence labeling of full thickness ileal wall revealed that NN-positive fluorescence was only found in the N-cells. However, only 50% of the N-cells also contained NN-like immunoreactivity (NN-LI). This was associated with a level of extractable NN that was 5-fold lower than that of NT. Corelease of NN- and NT-LI was investigated with the isolated, vascularly perfused jejunoileum model by using various substances that were described as potent stimulants of NT release in vivo. Luminal infusion of mixed nutrients, oleic acid (100 mM), glucose (5%), and taurocholic acid (1%) induced a well sustained release of NT, with plateau secretion of about 200%, 120%, 300%, and 700% above basal, respectively. Vascular bombesin (10(-7) M) and carbachol (10(-5) M) provoked a biphasic release of NT, consisting of a transient rise (approximately 600% above basal) followed by a less pronounced but sustained response. HPLC analysis of portal effluent revealed that 70-80% of NT-LI was intact NT. NN-LI was not coreleased with NT even upon vascular coinfusion of phenanthroline, which markedly protected exogenously infused NN. The coexistence but lack of corelease to any significant degree of NN with NT suggests different fates of these two precursor-related peptides within the ileal mucosa.

Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat--II. Immunohistochemical localization of maturation products.

The neurotensin/neuromedin N precursor molecule possesses four lysine-arginine dibasic residues which represent potential sites of cleavage by proteolytic maturation enzymes. As shown in the preceding paper, all of these dibasic residues are cleaved to a variable extent in rat brain. The aim of the present study was to localize immunohistochemically the resulting maturation products using site-specific antibodies directed against neurotensin, as well as against the exposed KLPLVL (K6L) and EKEEVI (E6I) sequences of the precursor. In a first set of experiments, each antigen was singly labelled in serial adjacent sections through the rat brain using a peroxidase-antiperoxidase technique. In a second series of experiments, neurotensin and either E61 or K6L antigens were double labelled in pairs using indirect immunofluorescence and visualized by confocal microscopy. In both types of preparations, immunoreactivity for all three antigens was detected in nerve cell bodies and axon terminals. In the absence of colchicine pretreatment, labelled nerve cell bodies were sparse in both neurotensin- and E6I-immunostained material and virtually undetectable in K6L-immunoreacted sections. By contrast, terminal immunostaining was intense and comparable in distribution for both neurotensin and E6I in most regions examined. K6L axonal labelling showed the same topographic pattern as that of E6I and neurotensin but was consistently weaker, except in the globus pallidus, where both E6I- and K6L-immunoreactive arbors were more widespread than those of neurotensin. These results suggest that the cleavage of the dibasic sites adjacent to the E6I and K6L sequences is more extensive in certain brain regions than in others. Colchicine pretreatment markedly increased the number of neurotensin- and, to a lesser extent. E6I-immunoreactive perikarya throughout the rat brain. However, it only marginally augmented the number of K6L-immunoreactive cell bodies, which remained sparse throughout. These results suggest that the maturation cleavages exposing the E6I and K6L sequences occur further distal to the cell body than the one giving rise to neurotensin. Both E6I- and K6L-immunoreactive perikarya were essentially confined to areas displaying neurotensin immunoreactivity. Furthermore, E6I and K6L antigens were shown in double labeling experiments to be present in the same cells as neurotensin, indicating that even if it is quantitatively different among brain regions, the basic pattern of neurotensin/neuromedin N precursor processing remains qualitatively similar throughout the brain.

Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat--I. Biochemical characterization of maturation products.

Neurotensin and neuromedin N are two biologically active related peptides which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are closest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present studies were aimed at determining the extent to which each of these four dibasic sites is cleaved and at identifying and quantifying the intermediate and mature products to which this cleavage gives rise in extracts from whole rat brain, hippocampus and globus pallidus. This was achieved by means of radioimmunoassays specific for sequences of the neurotensin/neuromedin N precursor that are adjacent to the dibasic processing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all tissue extracts, it was found that the three most C-terminal dibasic processing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neuromedin N-like sequence is processed to a lesser extent. As reported previously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

PC12 cells can be induced to produce, but do not process, the neurotensin/neuromedin N precursor.

Neurotensin and neuromedin N are two biologically active, related peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences, which are thought to represent putative processing sites, occur in the precursor molecule. Studies by others have shown that rat pheochromocytoma PC12 cells produced neurotensin and dramatically increased their neurotensin/neuromedin N precursor mRNA content in response to a combination of nerve growth factor, dexamethasone, forskolin, and Li+. Here, we investigated the effects of this combination of inducers on the posttranslational processing of the neurotensin/neuromedin N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and arginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doublets. Thus, mature neurotensin and neuromedin N represented less than 1% of the total precursor content in PC12 cells. The PC12 cell line may represent an interesting model with which one could transfect the recently cloned prohormone convertases PC1 and PC2, thereby allowing the study of the role of these enzymes in the processing of the neurotensin/neuromedin N precursor.

Calcium-dependent release of neuromedin N and neurotensin from mouse hypothalamus.

Neuromedin N (NN), a hexapeptide, was isolated from porcine spinal cord. Its C-terminal tetrapeptide sequence is identical to that of neurotensin (NT) and it exhibits NT-like effects when injected in the central nervous system. Both peptides were recently shown to be encoded in the same precursor molecule. We have just developed a sensitive and specific radioimmunoassay (RIA) for NN and showed that the peptide central nervous system distribution paralleled that of NT, the highest concentrations being found in the hypothalamus. Using this assay and a specific RIA for NT, we show here that NN and NT were simultaneously released from slices of mouse hypothalamus by K(+)-induced depolarization in a Ca(++)-dependent manner. The ratio of released NN over NT was 0.3 and was identical to the ratio of endogenous NN over NT. For both NN and NT, the releasable peptide pool represented 2% of the endogenous peptide pool. HPLC characterization of the releasable and endogenous immunoreactive material reacting with the NN and NT antisera showed that it coeluted with synthetic NN and NT, respectively. The present data further support the hypothesis that NN acts as a neuromodulator in the central nervous system.

The characterization and regional distribution of neuromedin N-like immunoreactivity in rat brain using a highly sensitive and specific radioimmunoassay. Comparison with the distribution of neurotensin.

Neuromedin N is a hexapeptide that shares a 4 amino acid homology with the C-terminus of neurotensin and exhibits neurotensin-like effects in the central nervous system. Both peptides were recently shown to be encoded in the same precursor molecule. In this study, a radioimmunoassay for neuromedin N was developed using monoiodo [125I-Tyr4]neuromedin N as the tracer and a rabbit antiserum raised against synthetic [Cys6]neuromedin N coupled to ovalbumin through its Cys residue. The antiserum showed strong structural requirement for the N-terminal sequence of neuromedin N and did not cross-react with neurotensin and other related peptides. The limit of detection of the radioimmunoassay was 0.5 fmol/tube and the IC50 was 5 fmol/tube. Neuromedin N-like immunoreactivity was present in 0.1 N HCl extracts of rat brain at a concentration of 9.3 +/- 1.3 pmol/g of tissue and behaved like synthetic neuromedin N on HPLC. Its concentration was significantly lower than that of neurotensin assayed in the same extracts (15.1 +/- 1.4 pmol/g), and this was not the consequence of lower extraction yield or lower post-mortem stability of neuromedin N as compared to neurotensin. The regional rat brain distribution of neuromedin N-like immunoreactivity paralleled that of neurotensin-like immunoreactivity, being highest in the hypothalamus and lowest in the cerebellum. These data support the proposal of a neuromodulator role for neuromedin N. The highly specific and sensitive radioimmunoassay described here will make it possible to investigate in more detail the regional brain distribution of neuromedin N and to study its release from brain tissues.

Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms.

Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS)