First report of Alternaria alternata causing leaf yellow spot on Heteropanax fragrans in China.

Heteropanax fragrans (Roxb.) Seem is a common garden landscape tree in China. In December 2020, a leaf disease on H. fragrans was observed in a 2 ha field in Zhanjiang (20.85° N, 109.28° E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned into necrotic tissues with a clear yellow halo and a white center. The disease incidence on plants was 100%. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed thrice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (HFA-1, HFA-2, and HFA-3). The colonies first produced a light-grayish aerial mycelia, which turned dark grayish upon maturity. Conidiophores were branched. Conidia numbered from two to four in chains, were dark brown, ovoid, or ellipsoid and mostly beakless; had 1-4 transverse and 0-3 longitudinal septa; measured within 7.2-17.8 (average = 10.2) × 2.5-7.5 (average = 4.3) µm (n = 30). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify the large subunit (LSU), internal transcribed spacer (ITS) region, translation elongation factor (TEF) , and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with NL1/LR3, ITS1/ITS4, EF-1α-F/EF-1α-R, and GDF1/GDR1 (Walther et al. 2013;Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (LSU, ON088978-ON088980; ITS, MW629797, ON417005 and ON417006; TEF, MW654167, ON497264,and ON497265;GAPDH, MW654166, ON497262,and ON497263). The obtained sequences were 100% identical with those of Alternaria alternata strain CBS 102600 upon BLAST analysis . The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. alternata (CBS 102600, CBS 102598, CBS 118814, CBS 918.96,CBS 106.24, CBS 119543, CBS 916.96). The fungus associated with leaf yellow spot on H. fragrans was thus identified as A. alternata. Pathogenicity tests were conducted in a greenhouse at 24 â„ƒ-30 â„ƒ with 80% relative humidity. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control). The test was performed thrice. Disease symptoms were found on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch's postulates. To our knowledge, this report is the first one on A. alternata causing leaf yellow spot on H. fragrans. Thus, this work provides an important reference for the control of this disease in the future.

First Report of Fusarium proliferatum Associated with Leaf Yellow on Alternanthera philoxeroides in China.

Alternanthera philoxeroides (Mart.) Griseb is a highly invasive weed commonly found in rice fields in China. In May 2021, leaf yellowing was observed on this weed (about 10 ha) in Zhanjiang (21°19'N, 110°20'E), Guangdong Province, China. Disease incidence was approximately 20% (n = 100 investigated plants). Ten yellow leaves from 10 plants were sampled, surface-sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 5 min. The leaves were rinsed three times in sterile distilled water and four sections of each leaf were placed onto potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Fusarium ssp. (69% of the isolates) were obtained from 55% of the leaf samples. Three representative single-spore isolates (APF-1, APF-2, and APF-3) were used for further study. Colonies were white to pink on PDA. Conidiogenous cells were monophialidic or polyphialidic. Macroconidia were slightly curved, tapering apically with three to five septa, and measured from 32.5-55.8 µm × 2.5-5.1 µm in size (n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006). For molecular identification, a colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) and portions of elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; O'Donnell et al. 2010). The sequences were submitted to GenBank under accession numbers MZ026797-MZ026799 (ITS) and MZ032209-MZ032217 (RPB1, RPB2, EF1-α). The sequences of the three isolates were 100% identical (ITS, 537/537 bp; RPB1, 1606/1606 bp; RPB2, 770/770 bp and EF1-α, 683/683 bp) with those of F. proliferatum (accession nos. MT378328, MN193921, MH582196, and MH582344) through BLAST analysis. Analysis of the sequences revealed a 99.87 - 100% identity with the isolates of the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 30 days old) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The test was performed three times. The plants were grown in pots in a greenhouse at 25 °C to 28 °C, with relative humidity of approximately 80%. Yellowing was observed on the inoculated plants after 7 days, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in terms of morphology and ITS sequences. No fungi were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing yellow symptoms on A. philoxeroides. The fungus has some potential biological control properties, but its host range needs to be further determined.

Pseudocercospora rhododendricola Causing Leaf dark Spot on Rhododendron pulchrum by the first phylogenetic analyses.

Rhododendron pulchrum Sweet is a famous ornamental flower in China. In December 2020, a leaf spot disease was observed on cv. Maojuan in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The spots were irregular and distributed on both sides of the main vein. They were dark to black, and their borders were obvious. The coalescence of the spots eventually led to leaf wilt. The disease incidence was 100% (n = 100, about 50 ha ). Thirty infected leaves were collected from the field, and the margin of the diseased tissues was cut into 2 mm × 2 mm pieces. Samples were surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively. They were rinsed thrice with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 ℃. After 5 days, fungal colonies appeared on the PDA. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates (RSP-1, RSP-2, and RSP-3) were obtained and the colonies of isolates were preserved in glycerol (15%) at -80 °C deposited at the Museum of Guangdong Ocean University. The morphology of these three isolates was consistent, and their sequences showed 100% homology according to ITS, TEF1, and ACT analysis results. The colonies grew to approximately 5 cm in diameter after 10 days. They showed olive green with off-white aerial mycelia. Stromata and conidia were observed on leaf lesions. Stromata were olivaceous brown. Conidia were solitary, cylindrical to narrowly obclavate, mildly curved, obtuse to rounded at the apex, and 1- to 3-septate; they had dimensions of 20 to 60 × 2.0 to 3.0 µm (n = 30). These morphological characteristics were not different from the description of Pseudocercospora rhododendricola (J.M. Yen) Deighton (Liu et al. 1998). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), and actin (ACT) loci of the isolates using primer pairs ITS4/ITS5, EF1/EF2, and ACT-512F/ACT-783R, respectively (White et al., 1990; O'Donnell et al. 1997). The sequences of the isolate RSP-1 were deposited in the GenBank (ITS, MW629798; TEF1, MW654168; and ACT, MW654170). BLAST analysis showed that the sequences of P. rhododendricola were submitted to GenBank for the first time by the author of this paper. A phylogenetic tree was generated based on the concatenated data of ITS, TEF1, and ACT sequences from GenBank by the Maximum Likelihood method. The isolates were closest to Pseudocercospora sp. CPC 14711 (Crous et al., 2013). Phylogenetic and morphological analyses identified the isolates as P. rhododendricola. Pathogenicity tests were conducted in a greenhouse at 24 °C-30 â„ƒ with 80% relative humidity. Healthy cv. Maojuan were grown in pots. Unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control) (5 leaflets per plant, 3 plants, 2-month-old plants). The test was performed thrice. Disease symptoms were found on the leaves after 2 weeks, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. P. rhododendricola was the cause of leaf spot of Rhododendron sp. from Singapore (Liu et al., 1998). For the first time, this pathogen was identified by combining phylogenetic and morphological analyses. The sequences in this study would be used as the reference sequences for further studies.

Dominant-negative Ikaros cooperates with BCR-ABL1 to induce human acute myeloid leukemia in xenografts.

Historically, our understanding of mechanisms underlying human leukemogenesis are inferred from genetically engineered mouse models. Relatively, few models that use primary human cells recapitulate the full leukemic transformation as assayed in xenografts and myeloid transformation is infrequent. We report a humanized experimental leukemia model where xenografts develop aggressive acute myeloid leukemia (AML) with disseminated myeloid sarcomas within 4 weeks following transplantation of cord blood transduced with vectors expressing BCR-ABL1 and a dominant-negative isoform of IKAROS, Ik6. Ik6 induced transcriptional programs in BCR-ABL1-transduced progenitors that contained repressed B-cell progenitor programs, along with strong stemness, proliferation and granulocyte-monocytic progenitor (GMP) signatures-a novel combination not induced in control groups. Thus, wild-type IKAROS restrains stemness properties and has tumor suppressor activity in BCR-ABL1-initiated leukemia. Although IKAROS mutations/deletions are common in lymphoid transformation, they are found also at low frequency in AML that progress from a prior myeloproliferative neoplasm (MPN) state. Our experimental system provides an excellent model to gain insight into these rare cases of AML transformation and the properties conferred by IKAROS loss of function as a secondary mutation. More generally, our data points to the importance of deregulated stemness/lineage commitment programs in human myeloid leukemogenesis.

The occurrence of melioidosis is related to different climatic conditions in distinct topographical areas of Taiwan.

This study assessed the correlations between the incidence of melioidosis and rainfall, wind strength and wind direction in both the flat and hilly regions of Taiwan. Data from the melioidosis and climate databases from 2005 to 2011 were combined and analysed. With the inclusion of a lag time accounting for a possible incubation period for melioidosis, the daily rainfall and wind-speed data were correlated with the number of confirmed melioidosis cases. The incidence of melioidosis in the flat region was related to the wind speed (>19 m/s) and the specific angle (150°, 220°, 280°) of the wind direction. Rainfall is a common environmental factor that contributes to an increase in the incidence of melioidosis in both areas; however, the contribution of wind strength or wind direction to the spread of melioidosis was restricted to areas with specific topographical characteristics, such as hills.

Influenza-associated excess mortality in southern Brazil, 1980-2008.

In order to estimate influenza-associated excess mortality in southern Brazil, we applied Serfling regression models to monthly mortality data from 1980 to 2008 for pneumonia/influenza- and respiratory/circulatory-coded deaths for all ages and for those aged ≥60 years. According to viral data, 73∙5% of influenza viruses were detected between April and August in southern Brazil. There was no clear influenza season for northern Brazil. In southern Brazil, influenza-associated excess mortality was 1∙4/100,000 for all ages and 9∙2/100,000 person-years for persons aged ≥60 years using underlying pneumonia/influenza-coded deaths and 10∙0/100,000 for all ages and 86∙6/100,000 person-years for persons aged ≥60 years using underlying respiratory/circulatory-coded deaths. Influenza-associated excess mortality rates for southern Brazil are similar to those published for other countries. Our data support the need for continued influenza surveillance to guide vaccination campaigns to age groups most affected by this virus in Brazil.

Reactive oxygen species mediate cyclic strain-induced endothelin-1 gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in endothelial cells.

Endothelin-1 (Et-1) is a peptide synthesized by endothelial cells (ECs) both in culture and in vivo. Cyclic strain induces gene expression of Et-1, however, the molecular mechanisms remain unclear. Since cyclic strain induces a sustained increase in intracellular reactive oxygen species (ROS), we hypothesized that the ROS could be a modulator in strain-induced Et-1 gene expression. Human umbilical vein ECs (HUVECs) subjected to cyclic strain had increased Et-1 secretion. Pretreatment of HUVECs with antioxidants, catalase (300 U/ml) or 1,3-dimethyl-2-thiourea (DMTU, 0.1 mm), abolished the strain-induced Et-1 release. ECs strained for 6 h had elevated Et-1 mRNA levels. In contrast, ECs treated with catalase or DMTU did not have increase Et-1 mRNA levels stimulated by cyclic strain. Bovine aortic ECs (BAECs) transfected with fusion plasmid containing Et-1 5'-flanking sequence (4.4 kb) and chloramphenicol acetyltransferase reporter gene produced a maximal Et-1 promoter activity after undergoing strain for 6 h, whereas pretreatment with catalase decreased this activity. BAECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or catalytically inactive mutant of extracellular signal-regulated kinase (mERK2) had inhibited strain-induced Et-1 promoter activity, indicating the Ras/Raf/ERK pathway was involved; moreover, ERK phosphorylation was induced in ECs which were strained. This strain-activated ERK phosphorylation was attenuated in the presence of catalase. Functional analysis of the Et-1 promoter with site-directed mutagenesis indicates that the activator protein-1 (AP-1) binding site had to be within 143 base-pairs upstream of transcription initiation site for strain-induced promoter activity. Pretreatment of ECs with catalase also decreased the strain-induced promoter activity in the minimal construct (-143 bp). Our data demonstrate that strain-induced Et-1 gene expression is modulated by ROS via Ras/Raf/ERK signaling pathway, and indicate the responsiveness of the AP-1 binding site for strain-induced Et-1 expression.

X-ray absorption spectroscopy study of Co structure in the epitaxial Co/Pt multilayers on Al2O3(11-20) substrates.

In this study, five epitaxial [Co(t nm)/Pt(1 nm)]30, multilayer samples (t=0.16-1.07 nm) were studied using polarized X-ray absorption spectroscopy method. These samples were prepared on Mo(110)/ Al2O3(11-20) substrates by MBE technique. The results show that the Co layer is more like an fcc pseudomorphic structure for the Co thickness of less than 0.3 nm. For Co layer thickness of 1 nm, the first shell distance is 0.25 nm, which is very close to the Co-Co distance of bulk hcp Co. On the other hand, for Co layer of less than 0.3 nm, the in plane first shell distance is expanded by 4% and most of the neighboring atoms are Pt atoms. The fitting results of the Co/Pt multilayers seem to support a sharp boundary model rather than an interdiffusion model.

Hepatic cytochrome P450 regulation in disease states.

Hepatic cytochrome P450 (P450) enzyme activities and gene expression can be profoundly altered in disease states. In general the levels of affected hepatic P450 enzymes are depressed by diseases, causing potential and documented impairment of drug clearance and clinical drug toxicity. However, modulation of P450s is enzyme selective and this selectivity differs among different diseases. This review will concentrate on regulation of P450s in diabetes, obesity and infectious and inflammatory disease, conditions that affect millions of people worldwide every day.

Reactive oxygen species modulate angiotensin II-induced beta-myosin heavy chain gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in neonatal rat cardiomyocytes.

Angiotensin II (Ang II) causes cardiomyocytes hypertrophy. Cardiac beta-myosin heavy chain (beta-MyHC) gene expression can be altered by Ang II. The molecular mechanisms are not completely known. Reactive oxygen species (ROS) are involved in signal transduction pathways of Ang II. However, the role of ROS on Ang II-induced beta-MyHC gene expression remains unclear. Here we found that Ang II increased beta-MyHC promoter activity and it was blocked by Ang II type 1 receptor antagonist losartan. Ang II dose-dependently increased the intracellular ROS. Cardiomyocytes cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal regulated kinase (mERK2) inhibited Ang II-induced beta-MyHC promoter activity, indicating Ras/Raf/ERK pathway was involved. Antioxidants such as catalase or N-acetyl-cysteine decreased Ang II-activated ERK phosphorylation and inhibited Ang II-induced beta-MyHC promoter activity. These data indicate that Ang II increases beta-MyHC gene expression in part via the generation of ROS.

Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-kappaB binding at the transcription start site.

Inflammatory cytokines cause the down-regulation of multiple cytochrome P450 mRNAs, but the transcriptional mechanisms involved are not known. We investigated the role of a putative negative NF-kappaB-responsive element, nkappaB-RE1, in the down-regulation of the CYP2C11 gene in rat hepatocytes. This sequence spans the transcription start site of CYP2C11, from positions -2 to +8. Electrophoretic mobility shift assays showed that nuclear extracts from livers of rats treated with bacterial lipopolysaccharide, or from hepatocytes treated with interleukin-1beta, formed a protein complex with an oligonucleotide probe containing the nkappaB-RE1, and that this complex contained predominantly the p50 subunit of NF-kappaB. Binding of NF-kappaB to the nkappaB-RE1 probe was of lower affinity than to a probe containing the prototypic NF-kappaB enhancer of the immunoglobulin kappa chain gene. Mutations in the 5'-end of the nkappaB-RE1, and to a lesser extent the 3'-end, reduced the affinity of NF-kappaB for this element. Introduction of the 5'-mutation into nkappaB-RE1 abolished the response of the -200-CYP2C11-chloramphenicol acetyltransferase reporter construct to interleukin-1 or lipopolysaccharide. We conclude that nkappaB-RE1 is a functional negative regulatory element that participates in the inflammatory suppression of CYP2C11.

Three zinc finger nuclear proteins, Sp1, Sp3, and a ZBP-89 homologue, bind to the cyclic adenosine monophosphate-responsive sequence of the bovine adrenodoxin gene and regulate transcription.

Adrenocorticotropin acting through cyclic adenosine monophosphate (cAMP) regulates transcription of the bovine adrenodoxin (Adx) gene in the adrenal cortex. The bovine Adx cAMP-responsive transcription sequence (CRS) has previously been found to contain two consensus GC boxes. By use of nuclear extracts from adrenocortical cells, Sp1 and Sp3 are shown here to bind to CRS. Mutations designed to enhance the identification of additional CRS binding proteins by reducing Sp protein binding showed the presence of an additional DNA-binding protein (Adx factor). Adx factor binding is inhibited by the zinc-chelating agent, 1,10-o-phenanthroline, suggesting it might be a zinc finger protein. By a fractionation/renaturation technique the Adx factor in mouse Y1 adrenocortical cells was found to be in the size range of 106-115 kDa by gel mobility shift assay. On the basis of size, the CRS sequence to which it binds, and its tentative identification as a zinc finger protein, Adx factor has been identified as a Krüppel-like zinc finger protein (a mouse ZBP-89 homologue). Further mutagenesis of CRS demonstrates that it can further be divided into two similar cAMP-responsive elements, and elimination of ZBP-89 binding does not affect cAMP responsiveness of either. Expression of these three nuclear proteins in Drosophila SL2 cells has been used to decipher the role of Adx CRS binding proteins in regulating transcription. Sp1 and Sp3 confer basal transcriptional activities, yet only Sp1 confers cAMP-responsive activity. ZBP-89 represses basal transcriptional activity.

Dynorphin stimulates corticotropin release from mouse anterior pituitary AtT-20 cells through nonopioid mechanisms.

Dynorphin (Dyn) peptides were previously shown to increase plasma corticotropin (ACTH) in the ovine fetus, but the site of its action remains unclear. In the present study, Dyn A(1-17) was found to stimulate ACTH release from mouse anterior pituitary tumor AtT-20 cells in a dose-dependent manner. Naloxone did not block the effect of Dyn A(1-17) and the selective kappa-opioid receptor agonist U50488H did not stimulate ACTH release. Dyn A(2-17), a degradative peptide fragment that does not bind to opioid receptors, also stimulated ACTH release from AtT-20 cells. Although the nonopioid effects of Dyn have previously been attributed to N-methyl-D-aspartate (NMDA) receptors, the ACTH-releasing effects of Dyn A(1-17) in AtT-20 cells were not affected by co-administration of NMDA receptor antagonist LY235959. The ACTH response to Dyn A(1-17) could not be blocked by alpha-helical CRH (CRH antagonist) and was additive with a maximal stimulatory dose of CRH, suggesting different mechanisms of action. These results show that the release of ACTH by Dyn A(1-17) in AtT-20 cells is not mediated by kappa-opioid receptors or by the NMDA receptor.

Dual ultrastructural localization of mu-opiate receptors and substance p in the dorsal horn.

Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence-specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP-immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR-labeled dendrites that received any afferent input were contacted by at least one SP-containing axon or terminal. Although SP-immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP-containing spinal interneurons may be regulated by MOR ligands. A few SP-labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second-order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post- and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics.

The N-methyl-D-aspartate (NMDA) receptor is postsynaptic to substance P-containing axon terminals in the rat superficial dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor is thought to mediate the postsynaptic effects of excitatory amino acids released from primary afferent terminals in the superficial layers of the dorsal horn of the spinal cord where synergistic associations with substance P (SP) have been implicated in the production of hyperalgesia. We examined the electron microscopic dual immunocytochemical localization of SP and the R1 subunit of the NMDA receptor (NMDAR1) in this region to determine the cellular basis for interactions between SP and NMDA receptor ligands. Of 971 profiles immunolabeled for NMDAR1, 40% were dendrites and the remainder were primarily unmyelinated axons and astrocytic processes. In dendrites, NMDAR1-like immunoreactivity (NMDAR1-LI) was associated with synaptic and non-synaptic portions of the plasma membrane, as well as intracellular membranes including smooth endoplasmic reticulum. These NMDAR1-labeled dendrites received synaptic input from unlabeled terminals and from terminals containing SP and/or NMDAR1-LI and they occasionally (25/389) also contained SP. In contrast, of 540 SP-immunoreactive profiles, 60% were axon terminals and the majority (252/324) of these SP-labeled terminals were presynaptic to NMDAR1-containing dendrites. These results provide anatomical evidence that the synergistic nociceptive effects of SP and NMDA ligands are attributed mainly to dual modulation of the activity of single dendritic targets in the dorsal horn of the spinal cord. They also suggest that activation of NMDA receptors may also play a role in the modulation of SP neurons, presynaptic release of SP or other neurotransmitters, and in glial function in the dorsal horn.

Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.

The delta opioid receptor (DOR) and mu opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2-C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and < 1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.

Immunolabeling of Mu opioid receptors in the rat nucleus of the solitary tract: extrasynaptic plasmalemmal localization and association with Leu5-enkephalin.

Activation of the mu opioid receptor (MOR) by morphine within the caudal nucleus of the solitary tract (NTS) is known to mediate both cardiorespiratory and gastrointestinal responses. Leu5-enkephalin (LE), a potential endogenous ligand for MOR, is also present within neurons in this region. To determine the cellular sites for the visceral effects of MOR ligands, including LE, we used immunogold-silver and immunoperoxidase methods for light and electron microscopic localization of antisera against MOR (carboxyl terminal domain) and LE in the caudal NTS of rat brain. Light microscopy of coronal sections through the NTS at the level of the area postrema showed MOR-like immunoreactivity (MOR-LI) and LE labeling in punctate processes located within the subpostremal, dorsomedial and medial subnuclei. Electron microscopy of sections through the medial NTS at this level showed gold-silver particles identifying MOR-LI prominently distributed to the cytoplasmic side of the plasma membranes of axons and terminals. MOR labeled terminals formed mostly symmetric (inhibitory-type) synapses but sometimes showed multiple asymmetric junctions, characteristic of excitatory visceral afferents. MOR-LI was also present along extrasynaptic plasma membranes of dendrites receiving afferent input from unlabeled and LE-labeled terminals. We conclude that MOR ligands, possibly including LE, can act at extrasynaptic MORs on the plasma membranes of axons and dendrites in the caudal NTS to modulate the presynaptic release and postsynaptic responses of neurons. These are likely to include local inhibitory neurons and both gastric and cardiorespiratory afferents known to terminate in the subnuclei with the most intense MOR-LI.

Ultrastructural localization of mu-opioid receptors in the superficial layers of the rat cervical spinal cord: extrasynaptic localization and proximity to Leu5-enkephalin.

Many of the analgesic effects of opiate drugs and of endogenous opioid ligands, such as Leu5-enkephalin (LE) are thought to be mediated in part by mu-opioid receptors (MOR) in the dorsal horn of the spinal cord. To establish the cellular sites for the spinally mediated analgesic effects of MOR activation and the potential anatomical substrates for interactions with LE, we examined the ultrastructural localization of MOR and LE immunoreactivities in the adult rat cervical spinal cord (C3-C5). Anti-MOR sera recognizing the carboxyl terminal domain of MOR was localized using immunoperoxidase and immunogold-silver methods. mu-opioid receptor-like immunoreactivity (MOR-LI) was observed mainly in the superficial layers of the dorsal horn. Electron microscopy of this region revealed that small unmyelinated axons and axon terminals constituted 48% (91/189) and 15% (28/189), respectively, while dendrites comprised 36% (68/189) of the total population of neuronal profiles containing the MOR. MOR-LI was localized mainly along extrasynaptic portions of the plasma membrane in both axons and dendrites. In sections dually labeled for MOR and LE, 21% (14/68) of the dendrites containing MOR-LI closely apposed or received synaptic contact from axon terminals exhibiting LE reaction product. The results provide the first ultrastructural evidence that within the dorsal horn of the spinal cord, LE, as well as exogenous opiates may alter both axonal release of neurotransmitters and postsynaptic responsiveness of target neurons to afferent input through activation of extrasynaptic MOR.