Fluorimetric determination of 5-hydroxytryptamine and catecholamines in the central nervous system and heart of Locusta migratoria migratorioides

5-Hydroxytryptamine (5HT) was measured in the nervous and heart tissues of Locusta migratoria migratorioides in concentrations of 2·34 and 2·69 μg/g wet weight respectively. Among catecholamines, dopamine was found in the nervous tissue (1·31 μg/g wet weight) as well as in the heart (2·42 μg/g wet weight). Noradrenaline was present only in the brain, but in smaller amounts (0·24 μg/g wet weight) than dopamine. Adrenaline was found neither in the ganglia nor in the heart.On the basis of their occurrence in the CNS, 5HT, dopamine, and noradrenaline, and in the heart 5HT and dopamine can be regarded as transmitter or modulator substances.     

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.     

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 μM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 μM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 μM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 μM D-saccharolactone to inhibit β-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.     

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)₂D₃ decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.     

Platelet serotonin transport is altered in streptozotocin-induced diabetic rats

The present work was conducted to examine whether experimental diabetes (streptozotocin-induced) promotes changes in mean platelet volume, and platelet serotonin (5HT) uptake and content. These variables were measured in from four experimental groups: control, diabetic, diabetic+insulin, and non-diabetic+insulin. Animals treated fifteen days before with streptozotocin had platelets with higher 5HT uptake affinity, 5HT content, and volume. The insulin therapy reestablished the control values of all of these three variables. Non-diabetic animals treated one week with insulin did not show any variations. The effects of in vitro application of insulin, hyperglycaemic incubation medium, and streptozotocin on platelet amine uptake and release were also examined. Only those platelets incubated with streptozotocin showed an altered platelet 5HT uptake. No changes were observed for spontaneous 5HT release. The results are consistent with: a) an increase of platelet uptake capacity, as a consequence of an increase in platelet turnover, for explaining alterations of intraplatelet 5HT contents in experimental diabetes; b) a non-direct effect of insulin and glucose levels on platelet 5HT uptake -for explaining its dysfunctions in experimental diabetes-; c) the contribution of alterations in platelet 5HT transport for explaining the higher incidence of vascular complications in diabetic patients; d) the suitability of platelet as a model for investigating neuronal 5HT reuptake.     

Lysophosphatidic acid receptor-5 negatively regulates cell motile and invasive activities of human sarcoma cell lines

LPA signaling via LPA receptors [LPA receptor-1 (LPA₁)–LPA₆] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA₅ in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA₅ negatively regulates the cell motile and invasive activities of human sarcoma cells.     

Modulation of swimming in Tritonia: excitatory and inhibitory effects of serotonin

1. In the mollusc Tritonia escape swimming is produced by a network of central pattern generator (CPG) neurons. The purpose of this study was to determine which neurotransmitters might be involved in the swim system.
2. Injection of serotonin (5HT) into whole animals elicited swimming followed by a long-lasting inhibition of swimming. In isolated brain preparations, bath-applied 5HT elicited a swim pattern at short latency and also caused a long-lasting inhibition of the swim pattern. The activation of swimming by 5HT was associated with a tonic depolarization of cerebral cell 2 (C2) and the dorsal swim interneurons (DSI) which form part of the swim CPG network.
3. In isolated brain preparations, bath applied glycine, histamine, proctolin, and FMFRamide had no effect on the swim motor pattern elicited by electrical stimulation of a peripheral nerve. Aspartate, carbacol, dopamine, glutamate, octopamine, pilocarpine, and small cardioactive peptide-B (SCP_B) inhibited the activation of swimming by nerve stimulation.
4. The 5HT antagonists cyproheptidine, tryptamine, and 7-methyltryptamine had no effect on swimming, but methysergide and fenfluramine inhibited swimming to both normal sensory stimuli and exogenously applied 5HT.
5. Staining with a polyclonal antibody indicated that one class of CPG neurons, the dorsal swim interneurons (DSI), was immunoreactive for 5HT.
6. Taken together, the data suggest that pattern generator interneurons, particularly the DSIs, use 5HT as a neurotransmitter.

     

Behavioral, Cellular, and Molecular Analysis of Memory in Aplysia II: Long-Term Facilitation

Long-term facilitation (LTF) of Aplysia tail sensory neuron–motorneuron (SN–MN) synapses provides a synaptic correlateof memory for long-term behavioral sensitization of the tail-siphonwithdrawal reflex. LTF can be induced by repeated exposuresof serotonin (5HT) in the isolated pleural-pedal ganglion preparation.In addition, we have shown previously (Sherff and Carew, 1999)that LTF can also be induced by coincident 5HT exposure comprisedof a single 25-min exposure of 5HT at the SN cell body and a5 min pulse of 5HT at the SN-MN synapses. If synaptic 5HT isapplied either 15 min before or after somatic 5HT, LTF is significantlyreduced or is not induced at all. These results show that twoanatomically remote cellular compartments can functionally interactwithin a surprisingly short time period. In this chapter, wediscuss some of the mechanistic implications of this temporalconstraint. We also find that coincident LTF and LTF inducedby repeated pulses of 5HT differ (1) in whether they induceanother temporal phase of facilitation (intermediate-term facilitation,ITF, expressed up to 1.5 hr after 5HT), and (2) in their requirementsfor protein synthesis. The results described both in this paperand in the preceding companion paper show that there are multipleforms of both ITF and LTF that differ in their induction andexpression requirements, and at least in some instances, thedifferent temporal phases of facilitation, and perhaps comparablephases of memory, can be induced independently of each other.     

Transcriptome and toxin family analysis of the paralysis tick, Ixodes holocyclus

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100?bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2–HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods.     

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia

Background: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. Aims:The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cord-derived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. Methods:A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1–13 h after HI (n = 7), (ii) HT+IV huMSCs (30 × 10⁶ cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 × 10⁶ cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and oligodendrocytes quantified. In two further piglets, 30 × 10⁶ IN PKH-labeled huMSCs were administered. Results:HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P ≤ 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. Conclusions:After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 × 10⁶ cells/kg total dose) based on more rapid aEEG recovery, improved ³¹P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.     

In Vitro Selection and Identification of Potential Probiotics Isolated from the Gastrointestinal Tract of Nile Tilapia, <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

Fish gut bacteria can be used as probiotics for aquaculture. The aim of this study is to screen and identify beneficial probiotic bacteria from the gut of Nile tilapia, Oreochromis niloticus. Nine out of one hundred thirty-five isolates were non-pathogenic through intraperitoneal injection and had antibacterial activities with at least a strain from the five isolated fish pathogens, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas putida, and Staphylococcus aureus. Further tests showed that such isolates can survive in the presence of high bile concentration (10%) and at different acidic pH values. A strains (14HT) was sensitive to all selected antibiotics, two strains were (9HT and 11HT) resistant to streptomycin and three strains (9HT, 11HT and 38HT) had resistance to two antibiotics. Four isolates (11HT, 33HT, 38HT and 41HT) had an amylase and a protease activities and one strain (47HT) showed only amylase activity. Based on 16S rRNA gene analysis, the isolated strains were identified as follows: Lactococcus lactis (8HT, 9HT, 11HT and 33HT); Enterococcus faecalis (14HT), Lysinibacillus sp. (38HT) and Citrobacter freundii (39HT, 41HT and 47HT).     

Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritordeums and their respective parents

Hexaploid tritordeum (H^chH^chAABB; 2n?=?42) results from the cross between Hordeum chilense (H^chH^ch; 2n?=?14) and cultivated durum wheat (Triticum turgidum ssp. durum (AABB; 2n?=?28). Morphologically, tritordeum resembles the wheat parent, showing promise for agriculture and wheat breeding. Start Codon Targeted (SCoT) polymorphism is a recently developed technique that generates gene-targeted markers. Thus, we considered it interesting to evaluate its potential for the DNA fingerprinting of newly synthesized hexaploid tritordeums and their respective parents. In this study, 60 SCoT primers were tested, and 18 and 19 of them revealed SCoT polymorphisms in the newly synthesized tritordeum lines HT27 and HT22, respectively, and their parents. An analysis of the presence/absence of bands among tritordeums and their parents revealed three types of polymorphic markers: (i) shared by tritordeums and one of their parents, (ii) exclusively amplified in tritordeums, and (iii) exclusively amplified in the parents. No polymorphism was detected among individuals of each parental species. Three SCoT markers were exclusively amplified in tritordeums of lines HT22 and HT27, being considered as polyploidization-induced rearrangements. About 70 % of the SCoT markers of H. chilense origin were not transmitted to the allopolyploids of both lines, and most of the SCoTs scored in the newly synthesized allopolyploids originated from wheat, reinforcing the potential use of tritordeum as an alternative crop.     

Alterations and Mechanism of Gut Microbiota in Graves’ Disease and Hashimoto’s Thyroiditis

To explore the role of gut microbiota in Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). Seventy fecal samples were collected, including 27 patients with GD, 27 with HT, and 16 samples from healthy volunteers. Chemiluminescence was used to detect thyroid function and autoantibodies (FT3, FT4, TSH, TRAb, TGAb, and TPOAb); thyroid ultrasound and 16S sequencing were used to analyze the bacteria in fecal samples; KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Groups) were used to analyze the functional prediction and pathogenesis. The overall structure of gut microbiota in the GD and HT groups was significantly different from the healthy control group. Proteobacteria and Actinobacteria contents were the highest in the HT group. Compared to the control group, the GD and HT groups had a higher abundance of Erysipelotrichia, Cyanobacteria, and Ruminococcus_2 and lower levels of Bacillaceae and Megamonas. Further analysis of KEGG found that the “ABC transporter” metabolic pathway was highly correlated with the occurrence of GD and HT. COG analysis showed that the GD and HT groups were enriched in carbohydrate transport and metabolism compared to the healthy control group but not in amino acid transport and metabolism. Our data suggested that Bacillus, Blautia, and Ornithinimicrobium could be used as potential markers to distinguish GD and HT from the healthy population and that “ABC transporter” metabolic pathway may be involved in the pathogenesis of GD and HT.     

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-l-tryptophan (5HTP, 25 mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2 mg/kg), during the period of the most intensive development of 5HT neurons - from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.     

Killer Cell Immunoglobulin-Like Receptor Genes and their HLA-C Ligands in Hashimoto Thyroiditis in a Chinese Population

Objective: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen-C (HLA-C) ligands. Alterations in NK cell activity have been associated with Hashimoto thyroiditis (HT). The aim of this study was to determine whether certain KIR/HLA-C genotype combinations play a role in HT pathogenesis.Methods: The present study enrolled 107 unrelated HT patients and 108 random healthy individuals in a case-control study. Blood was collected for DNA extraction; typing of KIR genes and HLA-C alleles was performed by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gels.Results: Among a panel of KIR2D/HLA-C genotype combinations, the frequency of KIR2DS2/HLA-C1 was significantly increased in HT patients compared to controls (33.64% vs. 12.96%, P<.001). To further analyze the precise genotype, we investigated inhibitory or activating KIR/HLA-C gene pairs when their corresponding activating or inhibitory KIR genes were absent in the 2 groups. Only the frequency of KIR2DS2(-)2DL2/3(+)HLA-C1(+) was significantly decreased in HT patients compared to controls (48.60% vs. 70.37%, P = .001).Conclusion: Our data suggest that KIR2DS2/HLA-C1 may correlate with HT pathogenesis. On the contrary, the predominance of KIR2DL2/3/HLA-C1 in the absence of KIR2DS2 suggests a potential inhibitory role in HT pathogenesis. In conclusion, our findings may further elucidate the mechanisms underlying the pathogenesis of HT and other autoimmune diseases.Abbreviations:HLA-C = human leukocyte antigen-CHT = Hashimoto thyroiditisKIR = killer immunoglobulin-like receptorNK = natural killerPCR = polymerase chain reaction     

Plasma membrane depolarization and activation of receptors for endogenous vasoconstrictors as possible mechanisms of potentiation of vasoconstrictive response to serotonin in traumatic shock in rats

The goal of this work was to study possible mechanisms underlying the potentiation of vasopressor response to serotonin observed in traumatic shock. Experiments with isolated aorta and mesenteric artery of the rat showed that vasoconstriction is caused by the activation of 5HT2A receptors. Agonists of 5HT1B, 5HT1D, 5HT2B, and 5HT4 receptors induced vasodilation. Agonists of 5HT1A receptors had a dual effect determined by interaction with α₁-adrenergic receptors and 5HT1A receptors. Plasma membrane depolarization with 15 mM KCl increased the vasoconstrictive force in response to serotonin. This effect was determined by the ability of KCl to activate voltage-gated calcium channels, as a result of which the intracellular calcium stores are replenished. Inhibition of the response to serotonin by ketanserin, a 5HT2A receptor blocker, did not depend on the presence of 15 mM KCl. Constriction in response to serotonin was potentiated after its addition to vessels preconstricted with noradrenaline or endothelin-1. The constriction response partially retained in the presence of 2 × 10^?7 M ketanserin, which completely suppressed the serotonin-induced constriction of dilated vessels both at normal membrane potential and after plasma membrane depolarization. It can be assumed that noradrenalin and endothelin-1 alter the characteristics of 5HT2A receptors and possibly 5HT1A receptors as a result of their heterodimerization with the receptors for these vasoconstrictive hormones or receptor-receptor interaction at the level of signaling systems. Along with the potentiating effect of KCl, this mechanism may underlie the enhancement of vasopressor response to serotonin in shock.