Neuropeptides with similarities to vertebrate tachykinins, designated tachykinin-related peptides (TRPs), have been identified in several insect species. In this investigation we have utilized an antiserum raised to one of the locust TRPs, locustatachykinin-I (LomTK-I), to determine the distribution pattern of LomTK-like immunoreactive (LTKLI) neurons in the developing nervous system of the moth Spodoptera litura. A number of LTKLI neurons could be followed from the larval to the adult nervous system: a set of median neurosecretory cells (MNCs) in the brain, a pair of brain descending neurons and a few sets on neurons in the ventral nerve cord. The distribution of LTKLI neurons in the adult brain is very similar to that seen in other insect species with prominent arborizations in the central body, antennal lobes, mushroom body calyces, optic lobe neuropils and other distinct neuropil areas in the protocerebrum and tritocerebrum. A new finding is the presence of LTKLI neurosecretory cells with axon terminals in the anterior aorta and corpora cardiaca, suggesting for the first time a neurohormonal role of tachykinin-related peptide(s) in insects. During postembryonic development the number of LTKLI neurons in the ventral nerve cord decreases somewhat, whereas the number increases in the brain. Thus the functional roles of TRPs may change to some extent during development.
We have examined the distribution of two tachykinin-related neuropeptides, callitachykinin I and II (CavTK-I and CavTK-II), isolated from whole-animal extracts of the blowfly Calliphora vomitoria. Extracts of dissected brains, thoracic-abdominal ganglia and midguts of adult blowflies and the entire central nervous system of larval flies were analysed by high performance liquid chromatography (HPLC) combined with enzyme-linked immunosorbent assay (ELISA) for the presence of CavTKs. To identify the two neuropeptides by HPLC, we used the retention times of synthetic CavTK-I and II as reference and detection with an antiserum raised to locustatachykinin II (shown here to recognise both CavTK-I and II). The brain contains only two immunoreactive components, and these have exactly the same retention times as CavTK-I and II. The thoracic-abdominal ganglia and midgut contain immunoreactive material eluting like CavTK-I and II as well as additional material eluting later. The larval central nervous system (CNS) contains material eluting like CavTK-I and II as well as a component that elutes earlier. We conclude that CavTK-I and II are present in all assayed tissues and that additional, hitherto uncharacterised, forms of tachykinin-immunoreactive material may be present in the body ganglia and midgut as well as in the larval CNS. An antiserum was raised to CavTK-II for immunocytochemistry. This antiserum, which was found to be specific for CavTK-II in ELISA, labelled all the neurones and midgut endocrine cells previously shown to react with the less selective locustatachykinin antisera. It is not clear, however, whether CavTK-I and II are colocalised in all LomTK-immunoreactive cells since there is no unambiguous probe for CavTK-I.
We employed human SK-MEL-28 cells as a model system to identify cellular proteins that accompany N-(4-methyl)phenyl-O-(4-methoxy)phenyl-thionocarbamate (MMTC)-induced apoptosis based on a proteomic approach. Cell viability tests revealed that SK-MEL-28 skin cancer cells underwent more cell death than normal HaCaT cells in a dose-dependent manner after treatment with MMTC. Two-dimensional electrophoresis in conjunction with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis or computer matching with a protein database further revealed that the MMTC-induced apoptosis is accompanied by increased levels of caspase-1, checkpoint suppressor-1, caspase-4, NF-kappaB inhibitor, AP-2, c-Jun-N-terminal kinase, melanoma inhibitor, granzyme K, G1/S specific cyclin D3, cystein rich protein, Ras-related protein Rab-37 or Ras-related protein Rab-13, and reduced levels of EMS (oncogene), ATP synthase, tyrosine-phosphatase, Cdc25c, 14-3-3 protein or specific structure of nuclear receptor. The migration suppressing effect of MMTC on SK-MEL-28 cell was tested. MMTC suppressed the metastasis of SK-MEL-8 cells. It was also identified that MMTC had little angiogenic effect because it did not suppress the proliferation of HUVEC cell line. These results suggest that MMTC is a novel chemotherapeutic and metastatic agents against the SK-MEL-28 human melanoma cell line.
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