Nitric oxide synthase and background adaptation in Xenopus laevis

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by α-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). α-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain.In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background.Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3′,5′ guanosine monophosphate (cGMP)-accumulating fibers in the PI.The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.     

Quantification of synapse formation and maintenance in vivo in the absence of synaptic release

Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.     

The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts

Summary An immunoconjugate composed of natural interferon (nIFN) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFN/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFN/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFN on the injected tumors. Further enhancement was obtained when nIFN or nIFN together with Mc5 (at a dose 10 times larger than that present in nIFN/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of¹²⁵I-nIFN/Mc5 by the tumors was greater and its elimination slower than for¹²⁵I-nIFN alone or conjugated to irrelevant mouse IgG₁. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFN alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.     

Informed consent, parental awareness, and reasons for participating in a randomised controlled study

BACKGROUND: The informed consent procedure plays a central role in randomised controlled trials but has only been explored in a few studies on children. AIM: To assess the quality of the informed consent process in a paediatric setting. METHODS: A questionnaire was sent to parents who volunteered their child (230 children) for a randomised, double blind, placebo controlled trial of ibuprofen syrup to prevent recurrent febrile seizures. RESULTS: 181 (79%) parents responded. On average, 73% of parents were aware of the major study characteristics. A few had difficulty understanding the information provided. Major factors in parents granting approval were the contribution to clinical science (51%) and benefit to the child (32%). Sociodemographic status did not influence initial participation but west European origin of the father was associated with willingness to participate in future trials. 89% of participants felt positive about the informed consent procedure; however, 25% stated that they felt obliged to participate. Although their reasons for granting approval and their evaluation of the informed consent procedure did not differ, relatively more were hesitant about participating in future. Parents appreciated the investigator being on call 24 hours a day (38%) and the extra medical care and information provided (37%) as advantages of participation. Disadvantages were mainly the time consuming aspects and the work involved (23%). CONCLUSIONS: Parents' understanding of trial characteristics might be improved by designing less difficult informed consent forms and by the investigator giving extra attention and information to non-west European parents. Adequate measures should be taken to avoid parents feeling obliged to participate, rather than giving true informed consent.     

Intracellular signal transduction by the extracellular calcium-sensing receptor of Xenopus melanotrope cells

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist l-phenylalanine (l-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G_s/PKA nor G_q/PKC pathways are involved. However, pertussis toxin (G_i/o protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaR-evoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of l-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.     

Rearrangement of the T cell receptor gamma-chain gene in childhood acute lymphoblastic leukemia

We analyzed rearrangements of the T cell receptor gamma-chain (T gamma) gene as well as rearrangements of the T cell receptor beta-chain (T beta) gene and immunoglobulin heavy-chain (IgH) gene in 68 children with acute lymphoblastic leukemia (ALL). All 15 patients with T cell ALL showed rearrangements of both T beta and T gamma genes. Twenty-four of 53 non-T, non-B ALL patients (45%) showed T gamma gene rearrangements and only 14 of these also showed T beta gene rearrangements. Only a single patient rearranged the T beta gene in the absence of T gamma gene rearrangement. The rearrangement patterns of the T gamma gene in non-T, non-B ALL were quite different from those observed in T cell ALL, as 20 of 23 patients retained at least one germline band of the T gamma gene. In contrast, all T cell ALL patients showed no retention of germline bands. These data indicate that rearrangement of the T gamma gene is not specific for T cell ALL. Further, the results also suggest that T gamma gene rearrangement precedes T beta gene rearrangement. The combined analysis of rearrangement patterns of IgH, T beta, and T gamma genes provides new criteria for defining the cellular origin of leukemic cells and for further delineation of leukemia cell heterogeneity.     

Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.     

Morphology of neurosecretory cells in basommatophoran snails homologous with egg-laying and growth hormone-producing cells of Lymnaea stagnalis

In a light and electron microscope study, neurosecretory cells morphologically homologous with the egg-laying hormone-producing caudodorsal cells (CDC) and growth hormone-producing dorsal cells (DC, light green cells) of the freshwater basommatophoran snail Lymnaea stagnalis have been found in five genera (seven species) of Basommatophora, viz. in Lymnaea palustris and Lymnaea ovata, in Planorbis planorbis and Planorbis vortex, in Planorbarius corneus, in Bulinus truncatus, and in Biomphalaria glabrata. It is concluded that the functions of these cells are homologous as well. The homologies of the respective neuron types regard their locations in the cerebral ganglia, their clustering in groups, location of their neurohemal area (CDC: cerebral commissure; DC: median lip nerves), and ultrastructural characteristics (e.g., abundance of rough endoplasmic reticulum, well-developed Golgi apparatus, presence of two types of neuron-specific secretory granules, and release of granule contents by exocytosis into the hemolymph). In addition, CDC show large electron-dense granules and DC reveal infoldings of the plasma membrane at the abaxonal side of the soma as well as synaptic input. On the other hand, each neuron type shows species-specific characteristics, particularly with regard to the number of cells and the structure of the neurohemal area. Furthermore, the CDC show marked differences between genera in the morphology (especially the mean diameter) of type 2 and, particularly, type 1 secretory granules. The morphology of the two types of secretory granules in the DC differs strongly between species. The possible relation between the morphology and the chemical contents of secretory granules has been discussed.