Proline-specific dipeptidyl peptidase activity in the cockroach brain and intestine: partial characterization, distribution, and inactivation of tachykinin-related peptides.

Proline-specific dipeptidyl peptidase (DPP IV) is an established enzyme known to degrade neuropeptides and peptide hormones in vertebrate tissues. DPP IV cleaves peptides at the Pro2 residue. Because several neuropeptides of the cockroach Leucophaea maderae, such as LemTRP-1 (APSGFLGVRamide), are potential substrates for this peptidase, we investigated the occurrence of proline-specific DPP activity in cockroach tissues. Partly purified DPP activity was characterized from the brain and midgut of L. maderae by using Gly-Pro-4-nitroanilide as a substrate. The highest activity was obtained from the membrane fraction of intestine; about 10 times less activity (per milligram protein) was obtained from brain membranes. A smaller amount of soluble DPP activity could also be identified in both tissues. Gel chromatography of the solubilized intestinal DPP activity revealed a molecular mass of about 75 kDa. The enzyme had a pH optimum of 8.5. Diprotin A (Ile-Pro-Ile) was an efficient competitive inhibitor of the cockroach DPP, whereas other known DPP inhibitors were found to be less potent. When incubated with human and cockroach DPP IV, the cleavage products of LemTRP-1 were AP and SGFLGVRamide (des-AP-LemTRP-1) as determined by mass spectrometry of high-performance liquid chromatography (HPLC)-purified peptide fragments. The AP fragment was biologically inactive and the des-AP fragment had a drastically reduced myostimulatory activity on the hindgut of L. maderae. The blowfly TRP callitachykinin-I (CavTK-I; APTAFYGVRamide) was cleaved in two steps to des-AP-CavTK-I and desAPTA-CavTK-I, showing that cockroach DPP does not only liberate Xaa-Pro, but also Xaa-Ala dipeptides. The fragment desAPTA-CavTK-I was completely inactive on the cockroach hindgut. To compare, LemTRP-3 and CavTK-II, which lack a Pro2, were not cleaved by DPP IV. Enzyme histochemistry for DPP IV was performed on cryostat sections of brain and intestine with Gly-Pro-4-methoxy-2-naphthylamide as the substrate and Fast Blue B as the chromogen. Strong histochemical labeling was seen in specific neuropils of the brain such as the calyces of the mushroom bodies, the antennal glomeruli, and the central body. Also, the inner lining of the midgut (the peritrophic membrane) and the malpighian tubules were strongly labeled by reaction product. In both the brain and intestine, the enzyme-histochemical reaction was inhibited by diprotin A.

Preliminary observations on ascidian and echinoderm neurons and neural explants in vitro.

As part of a study on echinoderm and ascidian neural regeneration, attempts were made to develop a system for the maintenance of their neurons in vitro. It was found that neurons and neural tissue explants from the starfish, Asterias rubens, and the brittlestar, Ophiura ophiura, and explants from the brain of the ascidian, Ciona intestinalis, could be cultured for up to 6 weeks in a modified L15-based medium. Some cells extended axonal projections and produced growth cones under certain conditions. Attempts were made to stimulate neuron survival and outgrowth of echinoderm cultures with conditioned media containing growth factors or tissue extracts and with various substrates including extracellular matrix extracts from native tissue. Ascidian brain explants from both normal and regenerating animals were cultured in the standard conditions established for echinoderm tissue, with outgrowth being observed in 25% of explants. In these cultures labelling with bromodeoxyuridine suggested that regeneration continues in vitro, although results using substance P immunocytochemistry indicate neuronal differentiation may be impeded. These preliminary studies suggest it is possible to maintain adult echinoderm and ascidian neurons in vitro.

Regeneration and post-metamorphic development of the central nervous system in the protochordate Ciona intestinalis: a study with monoclonal antibodies.

In this study, we use three monoclonal antibodies that recognise antigens present in the central nervous system of the ascidian Ciona intestinalis to study regeneration and post-metamorphic development of the neural ganglion. We have also used bromodeoxyuridine labelling to study generation of the neuronal precursor cells. The first antibody, CiN 1, recognises all neurones in the ganglion, whereas the second, CiN 2, recognises only a subpopulation of the large cortical neurones. Western blotting studies show that CiN 2 recognises two membrane-bound glycoproteins of apparent Mr 129 and 100 kDa. CiN 1 is not reactive on Western blots. Immunocytochemical studies with these antibodies show that CiN 1-immunoreactive neurone-like cells are present at the site of regeneration as early as 5-7 days post-ablation, a sub-population of CiN 2-immunoreactive cells being detected by 9-12 days post-ablation. The third antibody, ECM 1, stains extracellular matrix components and recognises two diffuse bands on Western blots of whole-body and ganglion homogenates. The temporal and spatial pattern of appearance of CiN 1 and CiN 2 immunoreactivity both during post-metamorphic development and in regeneration occurs in the same sequence in both processes. Studies with bromodeoxyuridine show labelled nuclei in some neurones in the regenerating ganglion. Plausibly these originate from the dorsal strand, an epithelial tube that reforms by cell proliferation during the initial phases of regeneration. A second population of cells, the large cortical neurones, do not incorporate bromodeoxyuridine and thus must have been born prior to the onset of regeneration. This latter finding indicates a mechanism involving trans-differentiation of other cell types or differentiation of long-lived totipotent stem cells.

The Patterns of Bromodeoxyuridine Incorporation in the Nervous System of a Larval Ascidian, Ciona intestinalis.

The fates of cells from the anterior region of the ascidian neural plate are described either as neural or as mixed neural and non-neural. In Ciona intestinalis, all cellular progeny are accounted for until a time 60% between the onset of embryonic development and larval To resolve the issue of their fates in this species, we have examined the later mitotic history of neural-plate cells. Because cessation of cell division in the neural plate has been claimed to occur at 70% of embryonic development, we need to account for cell production from 60% onward, to determine whether more cells are produced than populate the larval CNS, allowing some to adopt non-neural fates. The embryonic incorporation of bromodeoxyuridine (BrdU), 500 {mu}M in seawater, was monitored in 1 -h larvae by anti-BrdU immunocytochemistry. The pattern of incorporations indicates that all larval neurons are born before 70% of embryonic development, but that cell division unexpectedly continues to generate ependymal cells until at least 95%. Divisions in the neurohypophysis continue throughout embryonic development. The total number of cells produced appears sufficient only to complete the complement of larval CNS cells, denying non-neural fates for anteriorly migrating neural plate cells, and indicating a general absence of cell death. Consistent numbers of incorporations after the same exposure in different larvae provide evidence for determinacy of neural plate lineages. The last three conclusions confirm those reached previously (Nicol and Meinertzhagen, 1988b).

Pattern of substance P- and cholecystokinin-like immunoreactivity during regeneration of the neural complex in the ascidian Ciona intestinalis.

The neural ganglion of ascidians exhibits a novel and rapid pattern of regeneration whereby within approximately 28-35 days of total ablation an entirely new neural complex is formed. In normal adults, neuronal cell bodies expressing substance P- (SP-Li), neurokinin A-(NKA-Li), CCK/gastrin- (CCK-Li), and insulin-like immunoreactivity exhibit a clearly defined pattern of localization in the cortical rind of the ganglion with characteristic long processes arising from the perikarya running throughout the neuropile. CCK-Li cell bodies are particularly concentrated close to the points of exit of the main nerve trunks. We have used antisera raised against these peptides to monitor the process of regeneration up to postoperative (pa) day 35. Only SP and CCK antisera produced positive staining in the regenerating tissue. Immunoreactive cell bodies first appear following 14 days pa. At this time CCK-Li neurons are more abundant than SP-Li neurons and in contrast to the pattern found in the normal adult ganglion, immunoreactive cell bodies are located both peripherally and centrally in the core of the ganglion and processes were rarely seen. Later stages exhibited an increasing number of SP-Li neurons and at 35 days pa SP-Li cell bodies clearly predominate. CCK-Li neurons typically become clustered close to the points of emergence of the anterior nerve roots. The early expression of CCK-Li and SP-Li molecules during regeneration is considered in terms of their potential role in development and cell proliferation in the newly forming ganglion.

GABA-Like Immunoreactivity in the Nervous System of Oikopleura dioica (Appendicularia).

The cellular localization of γ-aminobutyric acid (GABA) has been visualized immunocytochemically in the nervous system of Oikopleura dioica by using an antiserum to glutaraldehyde fixation complexes of GABA. The results show GABA-like immunoreactivity in neurons of the brain, in cells of the sensory vesicle, in the caudal ganglion, and in the nerve cord. Positive reactions were also found at the neuromuscular terminals in the tail.