Characteristics and distribution of endogenous RFamide-related peptide-1

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.     

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.     

Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family. Insights from novel lamprey RFamide peptides

Among the RFamide peptide groups, PQRFamide peptides, such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF), share a common C-terminal Pro-Gln-Arg-Phe-NH(2) motif. LPXRFamide (X = L or Q) peptides, such as gonadotropin-inhibitory hormone (GnIH), frog growth hormone-releasing peptide (fGRP), goldfish LPXRFamide peptide and mammalian RFamide-related peptides (RFRPs), also share a C-terminal Leu-Pro-Leu/Gln-Arg-Phe-NH(2) motif. Such a similar C-terminal structure suggests that these two groups may have diverged from a common ancestral gene. In this study, we sought to clarify the evolutionary origin and divergence of these two groups, by identifying novel RFamide peptides from the brain of sea lamprey, one of only two extant groups of the oldest lineage of vertebrates, Agnatha. A novel lamprey RFamide peptide was identified by immunoaffinity purification using the antiserum against LPXRFamide peptide. The lamprey RFamide peptide did not contain a C-terminal LPXRFamide motif, but had the sequence SWGAPAEKFWMRAMPQRFamide (lamprey PQRFa). A cDNA of the precursor encoded one lamprey PQRFa and two related peptides. These related peptides, which also had the C-terminal PQRFamide motif, were further identified as mature endogenous ligands. Phylogenetic analysis revealed that lamprey PQRFamide peptide precursor belongs to the PQRFamide peptide group. In situ hybridization demonstrated that lamprey PQRFamide peptide mRNA is expressed in the regions predicted to be involved in neuroendocrine and behavioral functions. This is the first demonstration of the presence of RFamide peptides in the agnathan brain. Lamprey PQRFamide peptides are considered to have retained the most ancestral features of PQRFamide peptides.     

Adenylate deaminase. A multigene family in humans and rats

Multiple AMP deaminase (AMP-D) isoforms have been found in vertebrates, and tissue-specific inherited deficiencies of AMP-D have been described in two unrelated clinical syndromes suggesting there may be more than one AMP-D gene in higher eukaryotes. Using a newly isolated cDNA cloned from an adult rat brain library and a previously reported cDNA cloned from adult rat skeletal muscle, two linked AMP-D genes have been identified in rat and man. ampd1 is expressed at high levels in skeletal muscle of the adult rat. ampd2 is the predominant gene expressed in non-muscle tissues and smooth muscle of the adult rat, and it is also the predominant gene expressed in embryonic muscle and undifferentiated myoblasts. Both genes are expressed in cardiac muscle of the adult rat. The peptides encoded by these two genes have distinct immunological properties. The conservation of nucleotide sequence and exon/intron boundaries in these two genes suggests they arose by duplication of a common primordial gene around 150 million years ago.     

Effect of RFRP-3 on reproduction is sex- and developmental status-dependent in the striped hamster (Cricetulus barabensis)

RFamide-related peptides (RFRPs) are orthologous to gonadotropin-inhibitory hormone (GnIH) inhibiting gonadotropin release. There are only two RFRP sequences (RFRP-1 and RFRP-3) encoded in rodents. RFRP-3, which was considered as a hypothetical inhibitor on GnRH, shows a stimulatory effect on the male Syrian and male Siberian hamster in short days. As a dominant rodent pest in northern China farmland, the striped hamster (Cricetulus barabensis) has higher reproductive activities and could act as a model to study the mechanism of reproduction. However, the effect of RFRP-3 on the reproductive activity for the striped hamster is less understood. In the study, we cloned 643 bp RFRP cDNA from the striped hamster hypothalamus, which contained an ORF of 570 bp encoding two RFamide-related peptide (RFRP) sequences: SPAPANKVPHSAANLPLRF-NH₂ (C. barabensis RFRP-1) and TLSRVPSLPQRF-NH₂ (C. barabensis RFRP-3). We also investigated the expression variation of RFRP mRNA and GnRH mRNA in the hypothalamus from hamsters with different developmental statuses (7-week-, 13-week- and 1.5-year-olds) using FQ-PCR, in which the 13-week-old female individuals were in estrous. The striped hamsters that are 7 weeks and 1.5 years old are non-breeding individuals, and those that are 13-week hamsters have breeding phenomena. The highest hypothalamus RFRP mRNA level was found in breeding males as compared to non-breeding males. Conversely, the lowest RFRP mRNA level in the hypothalamus was observed in breeding females, with no significant level when the breeding females were compared to the 7-week-old individuals. Additionally, the investigation of GnRH expression level showed a declining expression trend across the developmental stages (7-week-, 13-week- and 1.5-year-olds) in both sexes. Significant negative and positive relationships were detected in the 13-week estrous female (r = − 0.997, P = 0.035) and the 13-week male (r = 0.998, P = 0.029) striped hamsters respectively, which suggest that RFRP-3 has inhibitory and stimulatory effects on female and male adults respectively. Our results suggest that the effects of RFRP-3 on reproduction are sex- and developmental status-dependent in the striped hamster.     

Transglutaminase Catalyzes the Formation of Sodium Dodecyl Sulfate-Insoluble, Alz-50-Reactive Polymers of τ

Abstract: The gene for tryptophan 2,3-dioxygenase (TDO) heretofore was believed to be expressed only in liver. The data presented here demonstrate that RNA encoding TDO is present in rodent brain. Oligonucleotide primers based on the rat liver TDO cDNA sequence were synthesized and used to amplify RNA derived from mouse whole brain and liver and rat brain regions by the RNA-PCR. Reaction products were purified and subjected to DNA sequencing. Identical sequences were obtained when mouse whole brain and liver RNAs were amplified, and these sequences were shown to be 96% identical to the published rat liver tryptophan TDO cDNA sequence. In addition, TDO sequences were found in RNA derived from rat brainstem, cerebellum, cortex, hypothalamus, and the remainder of the brain.     

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.     

MAP2a, an Alternatively Spliced Variant of Microtubule-Associated Protein 2

Abstract: MAP2, a dendritically localized microtubule-associated protein (MAP), consists of a pair of high molecular mass (280 kDa) polypeptides, MAP2a and MAP2b, and several low molecular mass (70 kDa) proteins called MAP2c. Although MAP2b and MAP2c have been shown to arise via alternative splicing, it was not clear whether MAP2a is also created by alternative splicing or by posttranslational modification. Using epitope peptide mapping, we have demonstrated that an element specific to MAP2a is situated at its N-terminal end. A cDNA clone from an adult rat brain library was found to contain an additional 246 nucleotides situated at the 5' end of the 9-kb MAP2 mRNA. Antibodies generated against the encoded protein sequence recognize specifically MAP2a in rat brain homogenates. Moreover, although MAP2a, like MAP2b, is found in dendrites and cell bodies, its temporal appearance and cell type-specific distribution in rat brain differs from MAP2b.     

Human and rodent sequence analogs of Alzheimer's amyloid beta A4 share similar properties and can be solubilized in buffers of pH 7.4

The filamentous amyloid protein aggregates found in the brain of patients affected with Alzheimer's disease principally consist of a peptide termed beta A4, according to its secondary structure of beta-pleated sheets and its molecular mass of about 4 kDa. It has a length of up to 42 or 43 residues. By chemical means, we have synthesized peptide analogs corresponding to the human and rodent beta A4 sequences. We describe structural and functional properties of peptides spanning residues 1-43, 10-23, 1-27 and 4-27 of beta A4. The peptides have been tested for their ability to form filaments in vitro. Their solubilities and secondary structures in solution and in the solid state have been used to detect differences between the properties of human and rodent beta A4 sequences. We show that mouse and rat beta A4 homologs are as amyloidogenic as the human sequence. The absence of amyloid deposits in the brain of aged rats and mice is therefore not due to the three amino acid substitutions identified within the sequence which is homologous to beta A4 of humans. Moreover, peptides corresponding to residues 1-27 of human and rodent beta A4 are solubilized under physiological conditions; thus they are very unlikely to form stable filaments in vivo.     

Identification of Cortexin: A Novel, Neuron-Specific, 82-Residue Membrane Protein Enriched in Rodent Cerebral Cortex

Abstract: Nucleotide sequence analysis of a cDNA clone of a rat cortex-enriched mRNA identifies a novel integral membrane protein of 82 amino acids. The encoded protein is named cortexin to reflect its enriched expression in cortex. The amino acid sequence of rat cortexin and its mouse homologue are nearly identical (98% similarity), and both contain a conserved single membrane-spanning region in the middle of each sequence. Northern blot analysis shows that cortexin mRNA is brain-specific, cortexenriched, and present at significant levels in fetal brain, with peak expression in postnatal rodent brain. In situ hybridization studies detect cortexin mRNA primarily in neurons of rodent cerebral cortex, but not in cells of the hindbrain or white matter regions. The function of cortexin may be particularly important to neurons of both the developing and adult cerebral cortex.     

Cloning and Characterization of a Soluble Kynurenine Aminotransferase from Rat Brain: Identity with Kidney Cysteine Conjugate β-Lyase

Abstract: In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate β-lyase, indicating that rat kidney KAT and β-lyase represent the same molecular entity. Oligonucleotide probes derived from the β-lyase cDNA were then used as primers for PCR of reverse-transcribed rat brain poly(A)⁺ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the β-lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK-293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 m M pyruvate and 2-oxoglutarate, the K _m values for l -kynurenine were 1.2 m M and 86.3 µ M , respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/β-lyase mRNA of ∼2.1 kb.     

Mass spectrometry analyses of rat 2b myosin heavy chain isoform

We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified.     

Roles of prolactin-releasing peptide and RFamide related peptides in the control of stress and food intake

Subsequent to the isolation of the first recognized RFamide neuropeptide, FMRFamide, from the clam, a large number of these peptides have been identified. There are now five groups of RFamide peptides identified in mammals. RFamide peptides show diversity with respect to their N-terminal sequence and biological activity. RFamide peptides have been implicated in a variety of roles, including energy metabolism, stress and pain modulation, as well as effects in the neuroendocrine and cardiovascular systems. In the present minireview, we focus on prolactin-releasing peptide (PrRP) and RFamide related peptide (RFRP) with respect to their roles in the control of energy metabolism and stress responses. Both food intake and stressful stimuli activate PrRP neurons. The administration of PrRP affects energy metabolism and neuroendocrine systems. PrRP-deficient or PrRP receptor-deficient mice show abnormal energy metabolism and/or stress responses. On the other hand, RFRP neurons are activated by stressful stimuli and the administration of RFRP induces neuroendocrine and behavioral stress responses. Taken together, these data suggests that PrRP and RFRP neurons play a role in the control of energy metabolism and/or stress responses.     

Molecular cloning of the cDNA encoding the third polypeptide (gamma) of brain calmodulin-dependent protein kinase II

cDNA clones for three distinct types of rat brain calmodulin-dependent protein kinase II have been isolated. Two of them were identified as cDNA clones for the alpha and beta subunits of this kinase. The other showed a nucleotide sequence similar but, not identical, to that encoding either the alpha or beta subunit. The cDNA sequence encoded a polypeptide, designated gamma, consisting of 527 amino acid residues with a molecular weight of 59,038. The deduced amino acid sequence of gamma was 84 and 87% homologous to those of alpha and beta, respectively. Higher homologies of the sequences were found in the amino-terminal halves of the three species, alpha, beta, and gamma. RNA blot analysis revealed that the mRNAs for alpha, beta, and gamma were expressed in rat brain with different regional specificities.     

Identification, characterization and localization of thyrotropin-releasing hormone precursor peptides in perinatal rat pancreas

The sequence of rat hypothalamic prepro TRH, deduced from its complementary DNA, contains five TRH progenitor sequences and six cryptic sequences separated by paired basic amino acid residues. We have utilised antisera against two synthetic peptides corresponding to sequences within proTRH, [Tyr53] preproTRH (53-74), part of the amino terminal leader sequence of proTRH and [Cys 74,83] preproTRH-(75-82), representing a TRH progenitor sequence flanked by cysteine residues (pCC10) in radioimmunoassays (RIA) to identify and chromatographically characterize proTRH derived peptides in extracts of rat perinatal pancreas and to localize these peptides immunohistochemically. Two forms of immunoreactive pYT22 (ipYT22) were observed, similar in size to ipYT22 seen in extracts of adult rat brain. By RIA immunoreactive pCC10 was detectable in neonatal but not fetal pancreas. However, immunohistochemical double staining of both fetal and neonatal rat pancreas colocalized both ipYT22 and ipCC10 with immunoreactive insulin in the B-cell of the developing Islets of Langerhans. These findings indicate that the B-cell of the perinatal pancreas synthesizes TRH from a prohormone encoded by a mRNA similar to that present in adult rat hypothalamus.     

Tissue-specific transcription initiation and effects of growth hormone (GH) deficiency on the regulation of mouse and rat GH-releasing hormone gene in hypothalamus and placenta

Hypothalamic GRH gene expression has been shown to be negatively regulated by GH in both rat and mouse. The recent reports of different 5' untranslated sequences in mouse GRH cDNA from hypothalamus and placenta have raised the possibility of tissue-specific regulation of the GRH gene. To provide support for this possibility, we have studied rodent models with GH deficiency due to genetic defects in the pituitary. Complementary DNA probes for the hypothalamic and placental 5' regions were used to determine the tissue specificity of each mRNA. Although the hypothalamic form of GRH mRNA was detected in placenta, it constituted less than 0.7% of total placental GRH mRNA. A placental 5' probe (based on the previously reported sequence) hybridized only with a larger mRNA species and was not tissue specific, indicating that it was not related to GRH and was derived possibly from a cloning artifact. The correct 5' sequence of mouse placental GRH cDNA was determined and shown to be distinct from both that previously reported and the hypothalamic sequence. Although the placental form of GRH mRNA was detected in hypothalamus using the polymerase chain reaction, its levels were undetectable by Northern blotting. The 5' end of rat placental GRH cDNA was similarly sequenced and shown to exhibit no homology with the rat 5' hypothalamic sequence, but a high degree of homology with the corresponding mouse placental sequence. In GH-deficient dwarf (dw/dw) rats, hypothalamic GRH mRNA levels were significantly increased above control levels in both females and males, and pregnancy did not alter the levels in either (dw) or control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a cDNA encoding rat cationic trypsinogen

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.