Application of compact electron cyclotron resonance ion source.

The compact electron cyclotron resonance (ECR) ion source with a permanent magnet configuration (Kei2 source) has been developed at National Institute of Radiological Sciences for a new carbon therapy facility. The Kei2 source was designed for production of C(4+) ions; its performance such as beam intensity and stability has already reached the medical requirements. Therefore, the prototype development of the source for medical use is essentially finished. Recently, we have started a few studies on other applications of the source. One is the production of fullerenes in the ECR plasma and modified fullerenes with various atoms for new materials. A second application is the production of multiply charged ions (not only carbon) for ion implantation. In this paper, some basic experiments for these applications are reported.

Subtype-selective corticotropin-releasing factor receptor agonists exert contrasting, but not opposite, effects on anxiety-related behavior in rats.

The corticotropin-releasing factor (CRF) system mediates stress responses. Extrahypothalamic CRF1 receptor activation has anxiogenic-like properties, but anxiety-related functions of CRF2 receptors remain unclear. The present study determined the effects of intracerebroventricular administration of a CRF2 agonist, urocortin 3, on behavior of male Wistar rats in the shock-probe, social interaction, and defensive withdrawal tests of anxiety-like behavior. Equimolar doses of stressin1-A, a novel CRF1 agonist, were administered to separate rats. The effects of pyrazolo[1,5-a]-1,3,5-triazin-4-amine,8-[4-(bromo)-2-chlorophenyl]-N, N-bis(2-methoxyethyl)-2,7-dimethyl-(9Cl) (MJL-1-109-2), a CRF1 antagonist, on behavior in the shock-probe test also were studied. Stressin1-A increased anxiety-like behavior in the social interaction and shock-probe tests. Stressin1-A elicited behavioral activation and defensive burying at lower doses (0.04 nmol), but it increased freezing, grooming, and mounting at 25-fold higher (1-nmol) doses. Conversely, systemic administration of MJL-1-109-2 (10 mg/kg) had anxiolytic-like effects in the shock-probe test. Unlike stressin1-A or MJL-1-109-2, i.c.v. urocortin 3 infusion did not alter anxiety-like behavior in the shock-probe test across a range of doses that reduced locomotion and rearing and increased grooming. Urocortin 3 also did not decrease social interaction, but it decreased anxiety-like behavior in the defensive withdrawal test at a 2-nmol dose. Thus, i.c.v. administration of CRF1 and CRF2 agonists produced differential, but not opposite, effects on anxiety-like behavior. Urocortin 3 (i.c.v.) did not consistently decrease or increase anxiety-like behavior, the latter unlike effects seen previously after local microinjection of CRF2 agonists into the septum or raphe. With increasing CRF1 activation, however, the behavioral expression of anxiety qualitatively changes from "coping" to "noncoping" and offensive, agonistic behaviors.

Gastrointestinal phenotype of GAD67lacZ transgenic mice with early postnatal lethality.

It has been proposed that gamma-aminobutyric acid (GABA) in the gut may function as a neurotransmitter, hormone and/or paracrine agent. Our aim was to examine transgenic mice of the GAD67-lacZ line with impaired postnatal growth and early postnatal lethality for gastrointestinal abnormalities. The gastrointestinal tract was dissected and processed for histology, immunohistochemistry, electron microscopy, western blotting and measurement of GAD activity. Homozygous mice of both sexes displayed an intestinal phenotype characterized by a fragile and haemorrhagic intestinal wall, a reduced number of villi, epithelial lesions and the occasional appearance of pseudostratified epithelium. The number of GABA-immunoreactive enteroendocrine cells and mucin-secreting goblet cells increased significantly relative to wild-type epithelium. The appearance of GABA-immunopositive neuronal perikarya and the lack of GABA-immunoreactive varicose fibres were observed in the enteric plexuses of transgenic mice. Tissue homogenates of transgenic mice showed higher levels of expression of GAD67 and GAD65 as compared with wild-type mice. Our results suggest that the possible reason underlying the growth impairment and postnatal lethality observed in GAD67 transgenic mice is a functional impairment of GABAergic enteric neurons and disintegration of intestinal epithelium.

Enteric neuromuscular junctions: comparison of ultrastructural features in different phylogenetic groups.

The enteric neuromuscular junctions of snail (Helix pomatia), locust (Locusta migratoria migratorioides), cockroach (Periplaneta americana), carp (Cyprinus carpio) and tench (Tinca tinca) were studied by means of different light and electron microscopic methods. The nitroblue tetrazolium staining revealed that the myenteric plexuses of the above species are composed of nerve cells, a network of varicose nerves and nerve bundles. Instead of highly organized ganglia, single neurons or small groups of 2-4 cells are characteristic of the invertebrates and fish studied. Catecholaminergic fluorescence induced by glyoxylic acid was detected in the muscular layer of the entire alimentary tract in snail and the hindgut of tench. Fluorescent nerves and perikarya were frequent in the snail gut, while only nerves and no perikarya were found in tench. A close contact between enteric muscles and nerves is the most common form of enteric neuromuscular junction in both the smooth (i.e. the molluscan and fish gut) and the striated (i.e. the insect gut) musculature. The striated musculature (i.e. the insect gut, the oesophagus of carp, and the oesophagus, stomach and the midgut of tench) also receives a synaptic input. Cytochemical evidence is provided of the cholinergic character of fish motor endplates. The ultrastructural appearance and vesicle population of certain nerve terminals suggest a universal role of aminergic and peptidergic control in gut motility.

Immunological importance of chimerism in transplantation: new conditioning protocol in BMT and the development of chimeric state.

Chimerism is an exceptional immunogenetic state, characterized by the survival and collaboration of cell populations originated from two different individuals. The prerequisits to induce chimerism are immuno-suppression, myeloablation, or severe immunodeficiency of the recipients on the one side and donor originated immuno-hematopoietic cells in the graft on the other. The pathologic or special immunogenetic conditions to establish chimerism are combined with bone marrow transplantation, transfusion, and various kinds of solid organ grafting. Different types of chimerism are known including complete, mixed and mosaic, or split chimerism. There are various methods used to detect the type of chimera state, depending on the immunogenetic differences between the donor and recipient. The induction of complete or mixed chimerism is first determinated by the effect of myeloablative therapy. The chimera state seems to be one of the leading factors to influence the course of the post-transplant period, the frequency and severity of GVHD, and the rate of relapse. However, the most important contribution of the chimeric state is in development of graft versus leukemia effect. A new conditioning protocol (DBM/Ara-C/Cy) for allogeneic BMT in CML patients and its consequence on chimera state and GVL effect is demonstrated.

Rapid stimulation of type I 5'-deiodinase in rat pituitaries by 3,3',5-triiodo-L-thyronine.

The negative feedback control of thyrotropin production by the anterior pituitary involves local 5'-deiodination of L-thyroxine (L-T4) to the active thyroid hormone 3,3',5-triiodo-L-thyronine (T3) by two 5'-deiodinase isozymes which are distinctly regulated by thyroid hormone. T3 rapidly increased steady-state mRNA levels and activity of type I iodothyronine-5'-deiodinase (5'DI) in rat anterior pituitary and in reaggregate cultures of anterior pituitaries. Type II 5'-deiodinase activity determined in parallel with 3,3',5-triiodo-L-thyronine (rT3) as substrate was markedly lower than that of 5'DI. 5'DI mRNA levels and activity were higher in anterior pituitaries of female compared to male rats. Neither gender differences nor T3 stimulation of 5'DI activity were found in the posterior part. These data demonstrate a T3 dependent expression of 5'DI in euthyroid anterior pituitary and suggest that this isozyme serves a major function within the complex network of thyroid hormone homeostasis.

Tenascin differentiates dermatofibroma from dermatofibrosarcoma protuberans: comparison with CD34 and factor XIIIa.

Differentiation of dermatofibroma (DF) from dermatofibrosarcoma protuberans (DFSP) can be difficult. CD34 and Factor XIIIa have been used to differentiate DF from DFSP. However, there is overlap and lack of specificity of their expression. Tenascin is an extracellular matrix glycoprotein that is involved in embryogenesis, carcinogenesis, and wound healing. The aim of the study was to assess the role of tenascin in DF and DFSP and compare the results with those obtained with CD34 and Factor XIIIa. Immunohistochemical staining was performed on 20 cases each of DFSP and DF, using antibodies to tenascin, CD34 and Factor XIIIa, and the streptavidin biotin technique. Positivity for all 3 antibodies was assessed within the tumors. Tenascin expression was also assessed at the dermal-epidermal junction. Strong tenascin positivity was noted at the dermal-epidermal junction overlying the lesion in 20 of 20 cases of DF (100%) and was negative over the lesion in 20 of 20 cases DFSP (100%). Tenascin was noted within the lesion of 80% of both DF and DFSP (16/20 cases). CD34 was strongly expressed in 16 of 20 (80%) DFSP and 5 of 20 (25%) DF, whereas Factor XIIIa was strongly expressed in 19 of 20 (95%) DF and 3 of 15 (15%) DFSP. Although CD34 was expressed in 80% DFSP and Factor XIIIa in 95% of DF, there was overlap in their expression in the 2 types of tumors. The increased expression of tenascin at the dermal-epidermal junction overlying the lesion in DF but not in DFSP, differentiated these 2 tumors. In contrast, tenascin expression within the lesion did not differentiate DF from DFSP.

Some morphological and histochemical features of the midgut myenteric plexus of the common European frog, Rana esculenta.

The neuron morphology and distribution of four putative transmitters were investigated in the myenteric plexus of frog (Rana esculenta) midgut. The gross morphology was revealed by NADH-diaphorase histochemistry, and the shape of the neurons by silver impregnation. Nerve cells had heterogeneous distribution: they either formed ganglia or placed as solitary neurons in the duodenum, while in the rest of the midgut only solitary neurons were observed. Three morphologically distinct cell types were revealed by silver impregnation: mainly type I and type II neurons cells were seen in the duodenum, while the rest of the intestine contained type II and III cells. Catecholamine fluorescence was revealed in nerve fibres in the duodenum, while few small nerve cells were observed in the small intestinal region. Acetylcholinesterase histochemistry showed strongly reactive nerve cells that were associated with the main fibre bundles in the duodenum. Only longitudinally oriented fibres and occasionally stained neurons were seen in the small intestine. Substance P immunocytochemistry revealed an extensive plexus, which contained a moderate number of stained perikarya in the full length of the midgut. Gamma-aminobutyric acid showed non-uniform distribution in the two parts of the midgut: a stronger and more regular fibre staining was found in the duodenum then in the rest of the intestine. Ultrastructural observations demonstrated that intrinsic neurons received synaptic inputs from the profiles contained agranular vesicles, while "P"-type profiles established close contacts with neurons. Both profile types formed close contacts with the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical and ultrastructural features of the developing enteric nervous system of the human foetal small intestine.

The developing enteric nervous system of the human foetus has been analyzed at the 10th and 18th week of gestation with a special reference to the development of nerve-muscle contacts. The myenteric plexus formation was analyzed by means of electron microscopy and on whole-mounts after NADH diaphorase histochemistry. The development of noradrenergic innervation as an extrinsic inhibitory supply was followed by means of a glyoxylic acid-induced fluorescence method. Differentiated neurons and neuroblasts both occurred in myenteric ganglia of the 10- and 18-week-old foetus although the ganglionic neuropil was almost unidentifiable ultrastructurally at the 10th week of gestation but was mature looking at the 18th week. The nerve plexuses connecting the ganglia frequently formed distant and close myoneural contacts. NADH-diaphorase histochemistry revealed that in the 18-week-old human foetus most of the neural perikarya was within the ganglia. Also, at the 18th week of gestation a well-defined fluorescent network was demonstrated within the ganglia, as well as in the internodal segments. On the basis of these observations we concluded that the time between the 10th and 18th week of gestation has paramount importance for both the morphological and functional maturation of the enteric nervous system.

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).

Computer-aided morphometric analysis of the developing concentric structure of the human fetal intestinal tube.

The Image-Pro Plus 3.0 morphometric program was used to study the region-specific organization of the human fetal intestine across the radial axis of the gut at weeks 12 and 18 of gestation. The thicknesses of the epithelium, the submucosa, the muscular layers and the myenteric ganglia were measured in resin-embedded semithin sections. Statistical analysis of the collected data was performed by using the two-way ANOVA, the SNK test and the Pearson correlation. The structural changes relating to the gut morphogenesis within this developmental period were followed both light and electron microscopically. The various tissues forming the radial axis of the intestinal tube exhibited different trends concerning their individual development. The thickness of the epithelium did not change in the fetal period investigated, although the epithelial surface displayed characteristic ultrastructural changes. The thickness of the submucosal layer increased significantly, but with different dynamics along the longitudinal axis, whereas the increases in size of the muscular layers and the myenteric ganglia did not differ significantly along the longitudinal axis of the embryonic intestine. The Pearson correlation revealed a significant correlation between the development of the circular muscle layer and that of the myenteric plexus along the whole length of the intestinal tube. The epithelium, the submucosa and the longitudinal muscle layers developed independently between weeks 12 and 18 of gestation.

Sequential pattern of nerve-muscle contacts in the small intestine of developing human fetus. An ultrastructural and immunohistochemical study.

The developing enteric nervous system of the human fetus has been studied by means of electron microscopy and neuron-specific enolase immunocytochemistry between the 10th and 26th week of gestation, with special reference to the development of nerve-muscle contacts. In the 10th week of gestation the circular muscle layer is formed, followed by the appearance of a primitive myenteric plexus, and the longitudinal smooth muscle layer in the 12th week of gestation. Adherens-type junctions between the smooth muscle cells and the elements of the myenteric plexus, interdigitation of nerve and muscle processes, and also contacts without any morphological specialization are frequent until the 18th week, when the mechanical points of attachment are relocated from the circular to the longitudinal muscle layer. By this time the developing myenteric plexus becomes ensheathed by non-neuronal cells, disrupting the direct contacts between smooth muscle cells and the primary strands of the myenteric plexus. The possible functional significance of these changing nerve-muscle contacts is discussed in the present paper.

Localization and quantitative distribution of biogenic monoamines in the intestinal tract of locust, snail and carp.

The localization and quantitative distribution of the biogenic monoamines of the intestinal tract has been studied in Locusta migratoria, Helix pomatia and Cyprinus carpio with morphological and biochemical methods. Electron microscopically dense-core vesicles of aminergic character were found in the varicose nerve fibres located in the intestinal muscles of all three animal species. Intensive green fluorescence characteristic of catecholamines was detectable in both the varicose nerve fibres and perikarya. Using a fluorimetric method the quantity of biogenic monoamines was measured in the gastrointestinal tract of all three species. The amount of adrenaline and noradrenaline was rather low: 0.03-0.29 ?g/g. The dopamine content reached the value of 6 ?g/g in the locust gut, and a significant amount of serotonin was present in the intestinal tract of all three species: 1.00-2.59 ?g/g. Compared to the adrenaline and noradrenaline serotonin proved to be higher by more than one order in each case. On the basis of morphological and biochemical results the authors suggest that biogenic monoamines are involved in the regulation of the gut muscle functioning both in the form of transmitters as well as neurohormones.

Bombesin injection into the central amygdala influences feeding behavior in the rat.

The present study was performed to determine whether low doses (10 or 40 ng) of bombesin microinjected into the amygdala could modify solid food intake. Forty ng of bombesin in 24 h deprived rats caused transient inhibition of food intake. This inhibitory effect was eliminated by prior bombesin antagonist treatment. A series of quantitative behavioral tests indicated that low doses of bombesin application specifically reduced food intake without altering the behavioral pattern or influencing the body temperature. The present results suggest, that bombesin-like peptides may act as a satiety signal in the central part of the amygdala.

Bombesin microinjection into the basolateral amygdala influences feeding behavior in the rat.

It has been demonstrated that the basolateral amygdala (ABL) represents a satiety mechanism. Experimental data indicate that peripheral or central applications of neuropeptide bombesin (BN) and BN-like peptides inhibit feeding. Since the amygdala (AMY) is rich in BN-like immunoreactive elements, the present study was performed to determine whether 10 or 40 ng doses of BN microinjected bilaterally into the ABL could modify solid food intake. Twenty nanograms of BN (10 ng per injection site) in 24-h deprived rats caused transient inhibition of food intake and 80 ng resulted in a significant reduction of food consumption for 1 h. This inhibitory effect of BN on feeding was eliminated by prior BN antagonist treatment. Results of behavioral tests showed that BN microinjections into the ABL specifically reduced food intake without altering behavioral patterns or influencing the body temperature. Present results suggest that BN-like peptides may act as a complex satiety signal in the ABL.

Relationship between appearance of GABA, fluorogenic monoamines and cytochrome oxidase activity during prenatal morphogenesis of chick myenteric plexus.

The basic histology of the developing embryonic gut wall of the chick was examined on haematein and eosin-stained paraffin sections. In parallel with this, the ontogenic sequence of myenteric plexus formation was followed on whole mounts after NADH diaphorase histochemistry. The presence of nerve elements was verified also by electron microscopy. The appearance of enteric gamma-aminobutyric acid-containing neurons, as an example of an intrinsic inhibitory neuronal system, was studied by using an antiserum against the gamma-aminobutyric acid glutaraldehyde bovine serum albumin conjugate. The development of noradrenergic innervation as an extrinsic inhibitory supply was followed by means of a glyoxylic acid-induced fluorescence method. Cytochrome oxidase activity was detected histochemically. Three consecutive steps of the morphogenesis of the myenteric plexus were revealed; first the appearance of a cellular crest at the mesenteric border on embryonic day 9; second the migration and clustering of nerve cells between embryonic days 10 and 16; then the elongation of neurites on embryonic days 16 and 21. Immunoreactive and also fluorescent fibres were first detected on the 14th day of incubation, while immunopositive cell bodies appeared only after hatching. In the early stages the cytochrome oxidase activity was restricted to the perikarya, while at the end of embryonic development the activity also appeared in the ganglionic neuropile. On the basis of these observations, we concluded that there is a close time relation between the morphogenesis and the biochemical and functional maturation of the myenteric plexus.

Consecutive diaphorase-acetylcholinesterase histochemistry in the myenteric plexus of frog stomach.

An investigation was made on the frog stomach myenteric plexus with 2 different histochemical techniques. Neuronal perikarya were stained with nicotinamide-adenine-dinucleotide-diaphorase (NADHd), while the acetyl-cholinesterase (AChE) staining showed rather the axoarchitectonic arrangement of the frog myenteric plexus. In double-labelled "whole mounts", NADHd-positive cell bodies and AChE-positive nerve processes were revealed. Some of the nerve cells and neuronal processes did not exhibit AChE activity at all. Since glyoxylic acid-induced fluorescence (GIF) was not detected in the myenteric plexus, the presence of catecholamines can be excluded. As a consequence of these observations, we suggest the presence of a non-cholinergic, non-adrenergic intrinsic neuronal system in the frog stomach myenteric plexus, containing purines or peptides as transmitters.

Glutathione metabolism of Acremonium chrysogenum in relation to cephalosporin C production: is gamma-glutamyltransferase in the center?

Methionine increased the intracellular glutathione (reduced) (GSH) pool and the specific gamma-glutamyltransferase (gamma-GT) activity in the cephalosporin C (CPC) producer Acremonium chrysogenum. The accelerated turnover of GSH might be indicative of the existence of a functioning gamma-glutamate cycle, and might supply the CPC biosynthetic machinery with L-cysteine. The gamma-GT was not subject to nitrogen metabolic repression but was more active in cells exposed to different oxidative-stress-generating agents. Exogenous cysteine hindered both the uptake of methionine and the induction of gamma-GT, and was not beneficial for CPC production. There was no causal relationship between the redox status of the cells and the observed cell morphology.

Urocortin 2 treatment is protective in excitotoxic retinal degeneration.

Urocortin 2 (Ucn 2) is a corticotrop releasing factor paralog peptide with many physiological functions and it has widespread distribution. There are some data on the cytoprotective effects of Ucn 2, but less is known about its neuro- and retinoprotective actions. We have previously shown that Ucn 2 is protective in ischemia-induced retinal degeneration. The aim of the present study was to examine the protective potential of Ucn 2 in monosodium-glutamate (MSG)-induced retinal degeneration by routine histology and to investigate cell-type specific effects by immunohistochemistry. Rat pups received MSG applied on postnatal days 1, 5 and 9 and Ucn 2 was injected intravitreally into one eye. Retinas were processed for histology and immunocytochemistry after 3 weeks. Immunolabeling was determined for glial fibrillary acidic protein, vesicular glutamate transporter 1, protein kinase Cα, calbindin, parvalbumin and calretinin. Retinal tissue from animals treated with MSG showed severe degeneration compared to normal retinas, but intravitreal Ucn 2 treatment resulted in a retained retinal structure both at histological and neurochemical levels: distinct inner retinal layers and rescued inner retinal cells (different types of amacrine and rod bipolar cells) could be observed. These findings support the neuroprotective function of Ucn 2 in MSG-induced retinal degeneration.

Effect of particle size on the surface properties and morphology of ground flax.

Flax fibers were ground with a ball-mill and four fractions with different size ranges were collected by sieving. These were tested for water sorption, degree of polymerization (DP), copper number, hydroxyl number and analyzed by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and inverse gas chromatography (IGC). Significant differences were found between the properties of the flax fiber and those of the ground versions, including fragmentation of fibers, increase of water sorption, copper number, hydroxyl number and surface O/C ratio, and decrease of DP, crystallite size and dispersive component of surface energy (γs(d)). Some parameters depended on the particle size: O/C ratio and hydroxyl number had local maxima at 315-630 µm, while γs(d) increased steadily with the decrease of particle size. These relationships were explained by fiber disintegration, destruction of waxy surface layer, exposure of cellulosic components, increase of surface area and crystalline imperfections.