Substance P-like immunoreactivity in the central nervous system of the blattarian insect Periplaneta americana L. revealed by a monoclonal antibody

Summary Brains, retrocerebral complexes and frontal and suboesophageal ganglia of adult American cockroaches, Periplameta americana, were immunohistochemically investigated with a specific monoclonal antibody (McAb) directed against a well characterized antigenic determinant, namely the COOH terminus of the endecapeptide substance P (SP). This resulted in the detection of several neurons and nerve fibres containing a substance antigenically closely related to this typically vertebrate neuropeptide.No difference in staining pattern could be observed between male and female insects. Related to the age of the adult specimens, however, a slight quantitative difference in SP immunoreactivity seems to occur, which probably might have functional implications. The SP-like peptide demonstrated in this study appears to be located in different neuronal structures than the ones that we earlier described as containing ACTH-, CRF-, OT-, AVP-, NP I-, NP II-, BPP-, FMRFamide-, AKH-, met-ENK-, FSH-, LH- and LHRF-like material (Verhaert et al. 1984a, b, 1985; Verhaert and De Loof 1985a, b).     

Peptide-containing pathways from the parabrachial complex to the central nucleus of the amygdala

The present investigation was undertaken to examine the organization of peptidergic projections that exist between the parabrachial nuclear complex (PB) and the central nucleus of the amygdala (CNA). The retrograde tracer True Blue was injected into the CNA of adult rats. The brain tissue was then reacted immunocytochemically to localize neurotensin (NT), substance P (SP), methionine enkephalin (ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SS), and cholecystokinin octapeptide (CCK). Following microinjection of True Blue in the CNA, retrogradely-labeled neurons were located primarily in the external lateral subnucleus, abutting the brachium conjunctivum. In animals that received colchicine pretreatment, two populations of neurons, containing either SP or NT, were found to project to the CNA. In addition, cells containing CCK, ENK, VIP, or SS were not found to be a part of this projection system. These data suggest that neurons in the PB project to the CNA and are, in part peptide-containing.     

Ecology and Translocation-Aided Recovery of an Endangered Badger Population

Abstract Southern British Columbia, Canada, is the northwestern range limit of the American badger (Taxidea taxus) and supports a nationally endangered subspecies. We initially investigated space-use, diet, and demography in southeastern British Columbia to characterize range-limit ecology. Resident badgers in the northern part (NP) of our study area were extirpated or nearly so during our study (λ = 0.7), whereas the southern (SP) badger population remained viable (λ = 1.2). This apparent difference in viability between NP and SP may have been confounded by timing because research occurred later in SP; litter size, number of Columbian ground squirrels (Spermophilus columbianus) consumed, and home range size were each correlated nearly equally to latitude and date of research, and survivorship was greater later in the study for both the NP and SP. Collectively, these factors indicated temporal, not just spatial, variability. Therefore, we translocated badgers into the NP to 1) determine whether the NP had lost its capacity to support badgers or had merely experienced the variability expected at a range limit and, if the latter, 2) initiate recovery. Translocated animals and their offspring had kit production equivalent to that of SP residents, adult survivorship intermediate between the NP and SP residents, and no confirmed kit mortality, with population growth projected (λ = 1.3). Diet of translocated individuals was similar to that of residents. Home ranges of translocated females were intermediate between the 2 resident groups, and home ranges of translocated males were not different than either resident group. Juvenile dispersal dates and distances were similar to those of residents for each sex. Our results were consistent with the extirpation of the NP being driven by temporally variable conditions or the effect of random events expected at range limits. The extirpation of NP did not appear to have been primarily due to any permanent loss of the NP's capacity to support badgers. At 3.5 years after starting translocations, badgers remained in the NP within an apparently growing population. We found translocation to be a useful diagnostic and conservation tool for badgers at their northern limit. Its utility may extend to countering the fluctuations typical of other rare, range-limit species.     

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

Introduction

The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.

Methods

Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.

Results

Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.

Conclusions

The release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.     

Immunohistochemistry of the guinea-pig trachea using an anti-idiotypic antibody recognizing substance P receptors

The airways receive a dense innervation from sensory neurons containing substance P (SP). An anti-SP anti-idiotypic antibody (anti-Id ab) recognizing SP receptors was previously characterized pharmacologically and proved to be useful in immunohistochemistry of the central nervous system. This antibody was used to localize SP binding sites in the guinea-pig trachea by immunohistochemistry. Immunolabelling was considered as specific when it could be prevented by a) preabsorption of the anti-Id ab with a C-terminal specific monoclonal anti-SP antibody, and b) preincubation of the tissue sections with either of the tachykinins, substance P and neurokinin A, in the presence of the inhibitor of neutral endopeptidase, phosphoramidon, and addition of these compounds into the antibody incubation medium. Moreover, immunofluorescence was absent when the acetone-fixed of fresh frozen sections were exposed to the detergent Tween 20 prior to immunohistochemistry, which points to a membrane localization of the detected tissue antigen, as expected for SP receptors. Compared with previous reports on autoradiographic localization of SP receptors in the guinea-pig trachea, the present immunohistochemical approach proved to be superior in enabling discrimination of labelled elements: Trachealis muscle, cylindrical epithelial cells and some roundish, singly lying cells in the epithelium and subepithelial lamina propria displayed specific immunofluorescence. These morphological findings match well with the known pharmacological actions of SP on the guinea-pig trachea.     

Nitric Oxide Regulates Substance P Release from Rat Spinal Cord Synaptosomes

Abstract: In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 ± 2.50 fmol/min/mg of protein and 26.2 ± 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S -nitroso- N -acetyl-penicillamine or membrane-permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration-dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.     

Peptidergic inputs to sympathetic preganglionic neurons

This paper presents data showing that the sympathetic autonomic areas of the cat thoracolumbar spinal cord contain nerve terminals and fibres with immunoreactivity for at least seven neuropeptides. The distribution in the intermediolateral cell column of the terminals and fibres which contain enkephalin-, neuropeptide Y-, neurotensin-, substance P-, and neurophysin II-like immunoreactivity (ENK, NPY, NT, SP, and NP2, respectively) suggests that these peptides are involved in more generalized functions of the autonomic nervous system. On the other hand, peaks in density of immunoreactivity at certain levels suggest that different levels of influence of sympathetic preganglionic neurons by the various peptides may occur along the length of the thoracolumbar cord. The distribution of terminals and fibres containing somatostatin- and oxytocin-like immunoreactivity (SS and OXY) suggests that these peptides may be part of specific pathways to particular sympathetic preganglionic neurons. The possible sources of the terminals and fibres containing ENK, NPY, NT, SS, and SP include the spinal cord and supraspinal areas, whereas the source of these structures with OXY and NP2 is most likely supraspinal. The data suggest that coexistence of peptides and interactions between structures containing different neuropeptides occur in the spinal autonomic areas. It is speculated that neuropeptides have an important role to play in the regulation of the cardiovascular division of the autonomic nervous system.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Regional distribution of neuropeptide gamma and other tachykinin peptides derived from the substance P gene in the rat.

Substance P (SP) and multiple neurokinin A (NKA)-related peptides can be derived from alpha-, beta- and/or gamma-preprotachykinin (PPT) mRNAs. In this study, the relative concentrations of the tachykinin peptides derived from the SP gene in rat brain, duodenum, jejunum, submandibular gland, parotid gland, urinary bladder and vas deferens was determined using high-performance liquid chromatography (HPLC) and radioimmunoassays (RIAs). In all tissues, SP levels were the highest. The relative abundance of NKA-related peptides was NKA greater than neuropeptide gamma (NP gamma) = neuropeptide K (NPK) greater than NKA(3-10). These results demonstrate that multiple tachykinin peptides are present in tissues where the SP gene is expressed, and that the NKA portion of the beta- and gamma-PPT precursors can be differentially processed posttranslationally in rat tissues into NKA, NPK, NP gamma and/or NKA(3-10).     

Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.     

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.     

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

Summary The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.     

Evidence for the existence of substance P in the prepubertal rat ovary. II. Immunocytochemical localization

Nerve fibers containing substance P (SP) were localized in ovaries from juvenile and peripubertal rats by immunofluorescence. These fibers were closely associated with the theca externa of antral follicles, as well as being in the interstitial tissue and within the tunica adventitia of small blood vessels, mostly arterioles. Consistently, the greatest amount of SP immunoreactivity was observed surrounding the ovarian vasculature. Substance P was not detected in cells or within the corpora lutea (CL). Additionally, the peripubertal animals seemed to have a greater concentration of ovarian SP than the juvenile animals. Possible functional roles for this peptide in the ovary are discussed.     

Structural similarity between the bone marrow extracellular matrix protein and neurokinin 1 could be the limiting factor in the hematopoietic effects of substance P

In the adult bone marrow (BM), immune cells are replenished through the process of definitive hematopoiesis, which is regulated by a complex process of cellular and humoral interactions. The latter include substance P (SP), a neurotransmitter that is produced by neural and nonneural cells. Neurokinin-1 (NK-1), the high-affinity SP receptor, shares structural similarity with fibronectin, a component of the BM extracellular matrix proteins. This study examines how such similarity could alter the effects of SP on the proliferation of the immature BM progenitors. In vitro studies show that 1 ng fibronectin/mL enhanced the stimulatory effect of SP on the proliferation of primitive BM progenitors. This finding was studied by computational studies: proteomics and three-dimensional molecular modeling. Use of surface-enhanced laser desorption/ionization ProteinChip technology showed that despite the induction of neutral endopeptidase, exogenous fibronectin hindered the degradation of SP to SP(1-4). These findings support a protective role for fibronectin in the digestion of SP. Since SP(1-4) is a negative regulator of hematopoiesis, this report indicates that the structural similarity between fibronectin and NK-1 could be important for maintaining hematopoietic stimulation. These studies could be extrapolated to hematological disorders that are associated with SP-fibronectin complexes.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Resorptive clearance and transepithelial potential difference in capsaicin-treated F344 rats.

The long-term effects of substance P (SP) depletion on epithelial integrity were assessed by measurements of airway transepithelial potential difference (PD) and resorptive clearance. Both techniques are highly sensitive to differences in epithelial permeability. PD (mV) was assayed with a KCl-saturated agar bridge technique in the lower trachea of Fischer 344 rats depleted of SP by neonatal capsaicin treatment. Capsaicin treatment is known to ablate a subpopulation of primary afferent fibers containing SP. Resorptive clearance (clearance in 1 h) was measured with 99mTc-labeled diethylenetriaminepentaacetic acid administered as a 100-microliters (100 microCi) bolus. Depletion of SP by neonatal treatment with capsaicin resulted in a highly significant increase in resorptive clearance (P less than 0.001). There was also a significant difference in PD in the lower trachea, the values being less negative for the capsaicin-treated rats (P less than 0.005). The increase in resorptive clearance may reflect the leakiness of the epithelial membrane, as also suggested by the decrease in PD. Aside from its known involvement as a neurotransmitter in neurogenic inflammation, SP may, under normal physiological conditions, have an additional role in the maintenance of epithelial barrier function.     

Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca²⁺]_i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons G_αq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.     

福建南北泥蚶种内分化的RAPD分析

采用随机扩增多态性DNA(RAPD)技术,对泥蚶在福建以南(广东汕头和湛江)和福建以北(浙江温岭和韩国)种群(分别合称南方类群和北方类群)做了遗传分化研究。由筛选出的20个随机引物共获得103个清晰可辨的RAPD标记,扩增片段长度在250—2500bp。汕头种群与湛江种群,韩国种群与温岭种群之间的最小遗传距离分别为0．0612和0．0692,而南、北类群间的遗传距离却在0．3261-0．4511。类群间近交系数也大于类群内。NJ和UPGMA法构建的系统树均显示汕头种群、湛江种群首先聚在一起,再与温岭种群和韩国种群聚合,说明两个类群发生了较明显的遗传分化,估计与地理隔离有关。     

Preference for sucralose predicts behavioral responses to sweet and bittersweet tastants

Rats can be classified as either sucralose avoiders (SA) or sucralose preferrers (SP) based on their behavioral responses in 2-bottle preference, 1-bottle intake, and brief-access licking tests. The present study demonstrates that this robust phenotypic variation in the preference for sucralose predicts acceptance of saccharin, an artificial sweetener with a purported concentration-dependent "bitter" side taste and a 0.25 M sucrose solution adulterated with increasing concentrations of quinine hydrochloride (QHCl). Specifically, SA displayed decreased preference for and intakes of saccharin (≥41.5 mM) and sucrose-QHCl (>0.5 mM QHCl) solutions, relative to SP. In a second experiment involving brief-access (30-s) tests, SP and SA did not differ in their unconditioned licking responses across a range of sodium chloride or QHCl solutions (0.03-1 mM). However, the acceptability threshold for sucrose was lower in SA, relative to SP (0.06 and 0.13 M, respectively). Our findings suggest that phenotypic differences in sucralose preference are indicative of a more general difference in the hedonic processing of stimuli containing "bittersweet" or "sweet" taste qualities.