Regulatory peptides in fruit fly midgut

Regulatory peptides were immunolocalized in the midgut of the fruit fly Drosophila melanogaster. Endocrine cells were found to produce six different peptides: allatostatins A, B and C, neuropeptide F, diuretic hormone 31, and the tachykinins. Small neuropeptide-F (sNPF) was found in neurons in the hypocerebral ganglion innervating the anterior midgut, whereas pigment-dispersing factor was found in nerves on the most posterior part of the posterior midgut. Neuropeptide-F (NPF)-producing endocrine cells were located in the anterior and middle midgut and in the very first part of the posterior midgut. All NPF endocrine cells also produced tachykinins. Endocrine cells containing diuretic hormone 31 were found in the caudal half of the posterior midgut; these cells also produced tachykinins. Other endocrine cells produced exclusively tachykinins in the anterior and posterior extemities of the midgut. Allatostatin-immunoreactive endocrine cells were present throughout the midgut. Those in the caudal half of the posterior midgut produced allatostatins A, whereas those in the anterior, middle, and first half of the posterior midgut produced allatostatin C. In the middle of the posterior midgut, some endocrine cells produced both allatostatins A and C. Allatostatin-C-immunoreactive endocrine cells were particularly prominent in the first half of the posterior midgut. Allatostatin B/MIP-immunoreactive cells were not consistently found and, when present, were only weakly immunoreactive, forming a subgroup of the allatostatin-C-immunoreactive cells in the posterior midgut. Previous work on Drosophila and other insect species suggested that (FM)RFamide-immunoreactive endocrine cells in the insect midgut could produce NPF, sNPF, myosuppressin, and/or sulfakinins. Using a combination of specific antisera to these peptides and transgenic fly models, we showed that the endocrine cells in the adult Drosophila midgut produced exclusively NPF. Although the Drosophila insulin gene Ilp3 was abundantly expressed in the midgut, Ilp3 was not expressed in endocrine cells, but in midgut muscle.     

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.     

Immunohistological localization of regulatory peptides in the midgut of the female mosquitoAedes aegypti

The midgut of the female mosquitoAedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatotropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.     

Peptidomics and peptide hormone processing in the Drosophila midgut

Peptide hormones are key messengers in the signaling network between the nervous system, endocrine glands, energy stores and the gastrointestinal tract that regulates feeding and metabolism. Studies on the Drosophila nervous system have uncovered parallels and homologies in homeostatic peptidergic signaling between fruit flies and vertebrates. Yet, the role of enteroendocrine peptides in the regulation of feeding and metabolism has not been explored, with research hampered by the unknown identity of peptides produced by the fly's intestinal tract. We performed a peptidomic LC/MS analysis of the fruit fly midgut containing the enteroendocrine cells. By MS/MS fragmentation, we found 24 peptides from 9 different preprohormones in midgut extracts, including MIP-4 and 2 forms of AST-C. DH(31), CCHamide1 and CCHamide2 are biochemically characterized for the first time. All enteroendocrine peptides represent brain-gut peptides, and apparently are processed by Drosophila prohormone convertase 2 (AMON) as suggested by impaired peptide detectability in amon mutants and localization of amon-driven GFP to enteroendocrine cells. Because of its genetic amenability and peptide diversity, Drosophila provides a good model system to study peptide signaling. The identification of enteroendocrine peptides in the fruit fly provides a platform to address functions of gut peptide hormones in the regulation of feeding and metabolism.     

Beyond chemotherapy and targeted therapy: adoptive cellular therapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is an intractable disease for which effective treatment approaches are urgently needed. The ability to induce antigen-specific immune responses in patients with lung cancer has led to the development of immunotherapy as a novel concept for the treatment of NSCLC. Adoptive cellular therapy (ACT) represents an important advancement in cancer immunotherapy with the utilization of tumor infiltrating lymphocytes, cytokine-induced killer cells, natural killer cells and γδ T cells. In this study, we review recent advances in ACT for NSCLC in clinical trials and provide a perspective on the improvement in ACT and potential therapeutic approaches using engineered T cell therapy for NSCLC.     

Cockroach allatostatin-immunoreactive neurons and effects of cockroach allatostatin in earwigs

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to demonstrate the presence of allatostatin-immunoreactive cells and fiber tracts in the neuroendocrine system of the earwig Euborellia annulipes. The corpora cardiaca cells were not immunoreactive, nor were the neurosecretory endings of fiber tracts from the brain to the corpora cardiaca. No immunoreactive material was detected in the corpus allatum, although the corpus allatum contained neurosecretory endings, and some cells of the brain, including medial and lateral protocerebral cells, showed immunoreactivity. In addition, the recurrent and esophageal nerves were allatostatin-positive. The last abdominal ganglion contained immunoreactive somata, and immunoreactive axons of the proctodeal nerve innervated the rectum, anterior intestine, and posterior midgut. We did not detect reactive endocrine cells in the midgut. Allatostatin I at concentrations of 10^–5 and 10^–7 M did not inhibit juvenile hormone biosynthesis by E. annulipes corpora allata in vitro. This was true for glands of low activity from 2-day females and brooding females, as well as for relatively high activity glands from 10-day females. In contrast, 10^–7 M allatostatin I significantly and reversibly decreased hindgut motility. Motility was decreased in hindguts of high endogenous motility from 2-day females and in those of relatively low activity from brooding females. These results support the notion that a primary function of allatostatin might be to reduce gut motility. Arch. Insect Biochem. Physiol. 38:155–165, 1998. © 1998 Wiley-Liss, Inc.     

The neurobiological link between OCD and ADHD

Obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD) are two of the most common neuropsychiatric diseases in paediatric populations. The high comorbidity of ADHD and OCD with each other, especially of ADHD in paediatric OCD, is well described. OCD and ADHD often follow a chronic course with persistent rates of at least 40–50 %. Family studies showed high heritability in ADHD and OCD, and some genetic findings showed similar variants for both disorders of the same pathogenetic mechanisms, whereas other genetic findings may differentiate between ADHD and OCD. Neuropsychological and neuroimaging studies suggest that partly similar executive functions are affected in both disorders. The deficits in the corresponding brain networks may be responsible for the perseverative, compulsive symptoms in OCD but also for the disinhibited and impulsive symptoms characterizing ADHD. This article reviews the current literature of neuroimaging, neurochemical circuitry, neuropsychological and genetic findings considering similarities as well as differences between OCD and ADHD.     

The Neuroprotective Effects of α-Iso-cubebene on Dopaminergic Cell Death: Involvement of CREB/Nrf2 Signaling

As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.     

Urokinase-type plasminogen activator receptor regulates apoptotic sensitivity of colon cancer HCT116 cell line to TRAIL via JNK-p53 pathway

The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved.     

A comparative study of PKH67, DiI,and BrdU labeling techniques for tracing rat mesenchymal stem cells

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1–3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3?±?1.6% PKH67+ and 98.4?±?1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2?±?4.6% 10 μM BrdU+ and 77.6?±?4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91?±?3.8% PKH67+, 90?±?1.5% DiI+, 91?±?0.8% 5 μM BrdU+, and 76.9?±?0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.     

Carvedilol Attenuates 6-Hydroxydopamine-Induced Cell Death in PC12 Cells: Involvement of Akt and Nrf2/ARE Pathways

Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson’s disease. Carvedilol, a nonselective β-adrenergic receptor blocker with pleiotropic activity has been shown to exert neuroprotective effect due to its antioxidant property. However, the neuroprotective mechanism of carvedilol is still not fully uncovered. The phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway plays key role in cell survival and the nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is the major cellular defense mechanism against oxidative stress. Here we investigated the effects of carvedilol on 6-hydroxydopamine (6-OHDA)-induced cell death as well as the Akt and Nrf2/ARE pathways in PC12 cells. We found that carvedilol significantly increased cell viability and decreased reactive oxygen species in PC12 cells exposed to 6-OHDA. Furthermore, carvedilol activated the Akt and Nrf2/ARE pathways in a concentration-dependent manner, and increased the protein levels of heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1(NQO-1), two downstream factors of the Nrf2/ARE pathway. In summary, our results indicate that carvedilol protects PC12 cells against 6-OHDA-induced neurotoxicity possibly through activating the Akt and Nrf2/ARE signaling pathways.     

Brain-derived neurotrofic factor (BDNF) secretion of human mesenchymal stem cells isolated from bone marrow,endometrium and adipose tissue

The ability of mesenchymal stem cells (MSCs) to differentiate into neuronal lineage determines the potential of these cells as a substrate for a cell replacement therapy. In this paper we compare the neurogenic potential of the MSCs from different donors, isolated from the bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was established that the native eMCSs, BMSCs and AD MSCs express neuronal marker β-III-tubulin with a frequency of 90, 50 and 14%, respectively. Also we showed that the eMSCs have a high endogenous level of brain-derived neurotrophic factor (BDNF), whereas the BMSCs and the AD MSCs are characterized by low basal BDNF levels. An induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMP induced changes in the cells morphology, the increase of β-III-tubulin expression, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. During the differentiation the BDNF secretion was significantly enhanced in the BMSCs and decreased in the eMSCs cultures. However, no correlation between the basal and induced levels of the neuronal markers expression in the studied MSCs has been established.     

Ag and Sn Nanoparticles to Enhance the Near-Infrared Absorbance of a-Si:H Thin Films

Silver (Ag) and tin (Sn) nanoparticles (NPs) were deposited by thermal evaporation onto heated glass substrates with a good control of size, shape and surface coverage. This process has the advantage of allowing the fabrication of thin-film solar cells with incorporated NPs without vacuum break, since it does not require chemical processes or post-deposition annealing. The X-ray diffraction, TEM and SEM properties are correlated with optical measurements and amorphous silicon hydrogenated (a-Si:H) films deposited on top of both types of NPs show enhanced absorbance in the near-infrared. The results are interpreted with electromagnetic modelling performed with Mie theory. A broad emission in the near-infrared region is considerably increased after covering the Ag nanoparticles with an a-Si:H layer. Such effect may be of interest for possible down-conversion mechanisms in novel photovoltaic devices.