Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

Effects of brain‐derived neurotrophic factor on dopaminergic function and motor behavior during aging

Brain‐derived neurotrophic factor (BDNF) is critical in synaptic plasticity and in the survival and function of midbrain dopamine neurons. In this study, we assessed the effects of a partial genetic deletion of BDNF on motor function and dopamine (DA) neurotransmitter measures by comparing Bdnf^+/? with wildtype mice (WT) at different ages. Bdnf^+/? and WT mice had similar body weights until 12 months of age; however, at 21 months, Bdnf^+/? mice were significantly heavier than WT mice. Horizontal and vertical motor activity was reduced for Bdnf^+/? compared to WT mice, but was not influenced by age. Performance on an accelerating rotarod declined with age for both genotypes and was exacerbated for Bdnf^+/? mice. Body weight did not correlate with any of the three behavioral measures studied. Dopamine neurotransmitter markers indicated no genotypic difference in striatal tyrosine hydroxylase, DA transporter (DAT) or vesicular monoamine transporter 2 (VMAT2) immunoreactivity at any age. However, DA transport via DAT (starting at 12 months) and VMAT2 (starting at 3 months) as well as KCl‐stimulated DA release were reduced in Bdnf^+/? mice and declined with age suggesting an increasingly important role for BDNF in the release and uptake of DA with the aging process. These findings suggest that a BDNF expression deficit becomes more critical to dopaminergic dynamics and related behavioral activities with increasing age.     

Technical enzymes produced in transgenic plants

Technical enzymes are used in many industrial applications. Nowadays technical enzymes are often produced in transgenic host organisms. The use of transgenic plants with respect to high level of expression at low costs as a prerequisite for successful commercial production of technical enzymes is discussed. This review summarises recently published examples for production of technical enzymes in plants. In addition, plastid transformation and viral vectors are discussed as methods which might be useful for obtaining high expression level of recombinant proteins in plants.     

Demonstration of substance P in aortic nerve afferent fibers by combined use of fluorescent retrograde neuronal labeling and immunocytochemistry

Combined use of the intraaxonal retrograde transport of the fluorescent marker ‘true blue’ with substance P (SP) immunocytochemistry has been used to trace the nodose ganglion projections of SP-containing neurons of the aortic depressor nerve. It has been found that (1) SP immunoreactive (SP-I) cell bodies are clearly demonstrable in clusters in the rostral part of the nodose ganglion without the aid of colchicine pretreatment; (2) ‘true blue’ is retrogradely transported to the nodose ganglion following its application to the central cut end of the aortic nerve; (3) ‘true blue’ fluorescence and SP fluorescent immunoreactivity can be visualized in the same tissue section and certain cell bodies in the nodose ganglia contain both SP-I and retrogradely transported ‘true blue’. These results indicate that the aortic nerve which projects from the aortic arch baro- and/or chemoreceptors to brainstem vasomotor centers contains SP-I afferent fibers which emanate from the nodose ganglion.     

Myxidium mackiei (Myxosporea) in Indo-Gangetic flap-shelled turtles Lissemys punctata andersonii: parasite-host interaction and ultrastructure

Myxosporeans are common parasites of fish, and uncommon parasites of amphibians, reptiles and invertebrates, that can cause significant morbidity and mortality. The common genus Myxidium infects the excretory system of turtles, yet knowledge of its pathogenicity in these hosts is limited. We offer new knowledge of morphological and ultrastructural aspects of host-parasite interactions in Myxidium infections from our recent diagnostic investigations on captive freshwater turtles listed in CITES (Appendix II). We investigated the cause of death of 2 adult Indo-Gangetic flap-shelled turtles Lissemys punctata andersonii from a zoo collection. After post-mortem examination, tissues were processed for histopathology, and special stains were used to demonstrate morphology of myxosporean spores. Additional kidney tissue, immersion-fixed in formalin, was processed for transmission electron microscopy. Both turtles were infected with a myxosporidian, Myxidium mackiei, in the kidney, which occluded 5 to 10% of the renal proximal convoluted tubules. The polysporic plasmodia contained pairs of developing and mature spores. Each mature, spindle-shaped spore had 2 asymmetric valves (1 overlapping, 1 overlapped), with 10 to 13 and 10 to 14 longitudinal ridges per valve, and 2 polar capsules each containing a polar filament with 4 to 5 turns. A pair of spores, each surrounded by a membrane-bound electron-lucent matrix, lay in an enclosing cell within the plasmodium. Regions of the parasite-host interface consisted of undulations of the parasite surface, with intense pinocytotic activity beneath, intermingled with the hosts' microvilli, and endocytotic channels at the apex of renal epithelial cells. The microvilli of the renal epithelial cells of infected tubules were frequently sheared or compressed, or occasionally missing; we did not detect other pathology induced by the parasite. Our report of M. mackiei in L. punctata is a new host record. Both individuals also had disseminated pale yellow nodules (bacterial granulomas) present in lung, heart, kidney, and skeletal muscle, and both were infected with coccidia (tentatively identified as Eimeria sp.) in multiple organs. The cause of death for one turtle was septicemia, but remained unknown for the other individual.     

Amino acid residues constituting the agonist binding site of the human P2X3 receptor

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.     

Rho GTPases RhoA and Rac1 Mediate Effects of Dietary Folate on Metastatic Potential of A549 Cancer Cells through the Control of Cofilin Phosphorylation

Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.     

Disseminated visceral coccidiosis in Indo-gangetic flap-shelled turtles, Lissemys punctata andersonii

Eimerioriniid coccidia commonly infect vertebrates and might contribute to morbidity and mortality under captive conditions. The common genus Eimeria typically shows tissue specificity, usually being limited to the epithelium of the gut; disseminated infections are rare in vertebrates. Disseminated visceral coccidiosis was found in two wild-caught adult female Indo-gangetic flap-shelled turtles (Lissemys punctata andersonii) that died while in captivity at a zoo. Sporulated oocysts of Eimeria spp. were found in lung and liver of one turtle and in auditory canal, nasal mucosa, pharynx, lung, liver, kidney, spleen, and intestine of the second. Two distinct species of Eimeria were indicated for the latter case by polymerase chain reaction amplification and sequencing of a portion of the 18S rRNA gene; one species was present in nasal mucosa and liver, with a separate species in lung, spleen, and intestine. Severity of inflammation was correlated with coccidial density. Coccidia were in melanomacrophages in liver and spleen; in the interstitium of auditory canal, nasal mucosa, pharynx, lung, and intestine; and within the interstitium and epithelial cells of the renal tubules in kidney. We suggest these disseminated infections might have been facilitated by a compromised immune system.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.