bluuebunny/crossref_metadata_2025 huggingface dataset:
The 3′ untranslated region of bovine preprolactin contains a transferable non‐poly(A) mRNA sequence that prolongs translation
Authors: Vassilakos Aikaterini , Hughes Martin J.G. , Andrews David W.
Published: April 2003

Preprolactin transcripts, synthesized in vitro, were actively translated for a prolonged period when injected into Xenopus oocytes. As a result, prolactin continued to be secreted into the media for up to 6 days after injection of the transcript. To investigate the role of the preprolactin 3′ untranslated sequence in stabilizing transcripts, it was fused to coding regions derived from signal recognition particle receptor α‐subunit or preproinsulin receptor. The translational half‐life of the chimeric RNA was increased for both coding regions, suggesting that a sequence within the preprolactin 3′ untranslated region that prolongs translation is transferable. Deletion mutagenesis of this untranslated region demonstrated that a sequence of 98 nucleotides immediately following the prolactin stop codon was sufficient to prolong translation of RNAs injected into Xenopus oocytes.
Steroid hormones and temperature induce changes of binding parameters of their receptors in intact cells
Authors: Rossini Gian Paolo , Malaguti Claudia
Published: April 2003

When MCF‐7 cells were treated with 17β‐estradiol, dexamethasone, or promegestone at 37°C, the K
d of receptors for their cognate ligands was found to decrease as compared to that measured at 2°C. Cell incubation with hormone at 37°C did not affect the B
max of glucocorticoid and progesterone receptors, but caused a 40% increase of that of estrogen receptor. This increase required the presence of ligand, was insensitive to cycloheximide, and was completed within 10 min of cell incubation at physiological temperature. We conclude that an early step of estrogen action is the recruitment of pre‐existing receptor molecules through activation of their binding capacity.
Formation of 8‐nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination
Authors: Yermilov Vladimir , Rubio Julieta , Ohshima Hiroshi
Published: April 2003

Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide in inflamed tissues. We have demonstrated that 8‐nitroguanine is formed dose‐dependently in calf thymus DNA incubated with low concentrations of peroxynitrite in vitro. 8‐Nitroguanine in acid‐hydrolyzed DNA was chemically reduced into 8‐aminoguanine, which was analyzed using high performance liquid chromatography with electrochemical detection. Only peroxynitrite, but not nitrite, tetranitromethane nor NO‐releasing compounds, formed 8‐nitroguanine. Antioxidants and desferrioxamine inhibited the reaction. 8‐Nitroguanine was depurinated from DNA incubated at pH 7.4, 37°C (). Peroxynitrite did not increase 8‐oxoguanine levels in DNA.
cDNA cloning of a human 100 kDa de‐ubiquitinating enzyme: the 100 kDa human de‐ubiquitinase belongs to the ubiquitin C‐terminal hydrolase family 2 (UCH2)
Authors: Falquet Laurent , Paquet Nicole , Frutiger Séverine , Hughes Graham J. , Hoang-Van Khan , Jaton Jean-Claude
Published: April 2003

The full length cDNA encoding a 100 kDa human de‐ubiquitinating enzyme, referred to as de‐ubiquitinase was obtained using one clone selected from a randomly sequenced human brain cDNA library and specific primers. The sequence of 18 peptides generated from the de‐ubiquitinase isolated from outdated human erythrocytes matched perfectly with the predicted amino acid sequence, which would encode a protein containing 858 amino acids (calculated M
r = 95,743 Da). Homology search disclosed that the protein is a member of a large family of ubiquitin C‐terminal hydrolases (UCH2), that was defined on the basis of the presence of two specific patterns, ‘the Cys‐ and His‐domains’, which are likely to be involved in the de‐ubiquitinating activity [7]. An additional conserved region, ‘the aspartic acid domain’, was also identified, the functional role of which is unknown.
Intercellular Ca2+ waves sustain coordinate insulin secretion in pig islets of Langerhans
Authors: Bertuzzi F. , Zacchetti D. , Berra C. , Socci C. , Pozza G. , Pontiroli A.E. , Grohovaz F.
Published: April 2003

Insulin release was investigated in parallel with changes in cytosolic calcium concentration, [Ca2+]i, in pig islets stimulated by glucose. After two days in culture, glucose stimulation failed to induce insulin release, and caused limited [Ca2+]i changes in few cells. After ten days, insulin response was partially restored and [Ca2+]i recordings revealed a slow oscillatory activity of the whole islet. Slow oscillations appeared to be due to the average [Ca2+]i variations resulting from the spreading of waves throughout the islet. These waves demonstrate the reestablishment of functional cell coupling, which appears to play a critical role in insulin release.
Resistance against cucumber mosaic virus in plants expressing the viral replicon
Authors: Suzuki Masashi , Masuta Chikara , Takanami Yoichi , Kuwata Shigeru
Published: April 2003

CMV RNAs 1 and 2 are considered to constitute the viral replicon. Tobacco plants were transformed with either RNA1 or RNA2 to produce plant lines V1 and V2, respectively. Plants homozygous for each of the RNAs were generated and crossed to produce V1V2 (V2V1) lines that expressed both RNA1 and RNA2. An RNase protection assay indicated that RNA1 and RNA2 multiplied in V1V2 (V2V1) plants. Surprisingly, V1V2 (V2V1) plants, unlike their parent lines, showed a remarkably high level of resistance to CMV; this resistance was more effective against RNA inoculation than against virion inoculation. Experiments using protoplasts showed that the resistance was expressed at the single cell level. All the data together suggested that the observed resistance does not fit the criteria for either ‘RNA‐mediated’ or ‘replicase‐mediated’ resistance.
Evidence for farnesol‐mediated isoprenoid synthesis regulation in a halophilic archaeon, Haloferax volcanii
Authors: Tachibana Akira , Tanaka Toshio , Taniguchi Makoto , Oi Susumu
Published: April 2003

Farnesol strongly inhibited growth of a halophilic archaeon, Haloferax volcanii, with an IC50 value of only 2 μM (0.4 μg/ml) in rich medium and 50 nM (0.01 μg/ml) in minimal medium without lysis. Other isoprenoid alcohols such as isopentenol, dimethylallyl alcohol, geraniol, and geranylgeraniol at 500 μM did not affect its growth. Mevalonate, which is the precursor of all isoprenoid membrane lipids in archaea, led to recovery of the growth inhibition of H. volcanii, but acetate had no such effect. Farnesol inhibited incorporation of acetate, but not mevalonate, into the lipid fraction. These results suggest that farnesol inhibited the biosynthetic pathway from acetate (acetyl‐CoA) to mevalonate. Farnesol is known to be derived from the important intermediate of isoprenoids, farnesyl diphosphate (FPP), and found in neutral lipid fraction from this archaeon. Moreover, the cell‐free extracts from H. volcanii could phosphorylate farnesol with ATP to generate farnesyl monophosphate and FPP. We conclude that farnesol‐mediated isoprenoid synthesis regulation system by controlling farnesol concentration is present in H. volcanii.
Novel effects of dendrotoxin homologues on subtypes of mammalian Kv1 potassium channels expressed in Xenopus oocytes
Authors: Robertson Brian , Owen David , Stow John , Butler Carolynn , Newland Claire
Published: April 2003

We have examined the effects of two DTX homologues, toxin I and toxin K, on Kv1.1, Kv1.2 and Kv1.6 channels expressed in Xenopus oocytes. Toxin I blocked all three channels; in contrast, toxin K was selective for Kv1.1. Both toxins slowed channel activation and inactivation kinetics with 10 nM toxin I approximately doubling activation and inactivation time constants of Kv1.1. For the first time, we have demonstrated the selectivity of a DTX homologue for a single cloned Kv1 channel and suggest that these toxins may sterically hinder the conformational changes that occur during channel gating.
Dependence of rat liver CMP‐N‐acetylneuraminate:GM1 sialyltransferase (SAT IV) activity on the ceramide composition of GM1 ganglioside
Authors: Pitto Marina , Palestini Paola , Masserini Massimo
Published: April 2003

The dependence of CMP‐N‐acetylneuraminate:GM1 sialyltransferase (SAT IV) activity of rat liver Golgi apparatus on GM1 ganglioside ceramide composition was evaluated. SAT IV activity was assayed on GM1 molecular species carrying homogeneous ceramide moieties containing long chain bases of different length (18 or 20 C atoms) unsaturated or not, linked to 14:0, 16:0, 18:0 or 22:0 fatty acids. The results obtained in the presence of the detergent Triton CF‐54, when enzyme and substrate are presumably part of the same supramolecular structure, show that either the long chain base or the fatty acid composition can affect enzyme activity. This feature was not displayed when GM1 was embedded in dipalmitoylphosphatidylcholine vesicles in the absence of detergent. Under the latter conditions, the enzyme was not sensitive to the lipid composition of GM1 but to the ganglioside/phospholipid ratio in the vesicles. These results indicate for the first time that SAT IV is affected by the lipid composition of the substrate and strengthen the hypothesis that glycosyltransferases may contribute to control the cellular glycosphingolipid ceramide pattern.
Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter
Authors: Braibant Martine , Lefèvre Philippe , de Wit Lucas , Ooms Josette , Peirs Priska , Huygen Kris , Wattiez Rudy , Content Jean
Published: April 2003

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS‐3 and pstC‐2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS‐3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H‐2b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding‐dependent permeases is proposed.
Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans
Authors: Biely P. , Côté G.L. , Kremnický L. , Greene R.V. , Dupont C. , Kluepfel D.
Published: April 2003

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside and methyl 2,3,4,6‐tetra‐O‐acetyl‐β‐d‐glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside and methyl 2,3‐di‐O‐acetyl‐β‐d‐xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl β‐d‐xylo‐ and ‐glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate‐specific AcXE.
Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts
Authors: Maulik Nilanjana , Watanabe Masazumi , Zu You-Li , Huang Chi-Kuang , Cordis Gerald A. , Schley James A. , Das Dipak K.
Published: April 2003

While much is known about the beneficial effects of myocardial stress adaptation, relatively less information is available about the adaptive mechanisms. To explore the signaling pathways of stress adaptation, isolated working rat hearts were divided into three groups. Group I was adapted to stress by conventional technique of repeated ischemia and reperfusion consisting of 5 min of ischemia followed by 10 min of reperfusion, repeated four times. Group II was treated with 100 μM of genistein, a tyrosine kinase inhibitor, followed by preconditioning as described for group I. The third group, perfused with buffer only for 60 min, served as control. All hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. The results of our study demonstrated better postischemic myocardial functions in the preconditioned hearts as evidenced by increased aortic flow, coronary flow, developed pressure and lesser amount of tissue injury as evidenced by the decreased creatine kinase release. The preconditioning effects were associated with enhancement of phospholipase D activity in the heart. The preconditioning effect was almost abolished by the genistein treatment which also prevented the enhancement of phospholipase D activities. Additionally, preconditioning of the rat hearts stimulated protein kinase C, MAP kinase, and MAPKAP kinase 2 activities which were inhibited by genistein. The results identifies for the first time tyrosine kinase‐phospholipase D as potential signaling pathway for ischemic preconditioning, and implicates the involvement of multiple protein kinases in myocardial adaptation to ischemia.
Distribution of the neural antigen BSP‐2 in the cerebellum during development
Authors: Langley O. K. , Gombos G. , Hirn M. , Goridis C.
Published: April 2003

The monoclonal antibody anti‐BSP‐2 recognizes three glycosylated peptide chains of 180,000, 140,000 and 120,000 daltons in extracts from adult mouse forebrain and cerebellum. In extracts of embryonic or neonatal brain, it recognizes a different form, migrating as a broad band of higher molecular weight on SDS polyacrylamide gels. This report describes the distribution of the antigen BSP‐2 in developing mouse cerebella using a sensitive immunoperoxidase technique at the electron microscope level. As early as 3 days after birth the antigen can be detected on the surface of all cerebellar neurons, including neuroblasts in the external granular layer, basket and stellate neurons and Purkinje cells. In addition, radial glial fibres (astrocytes) terminating on the pial surface contain BSP‐2. At later stages a pronounced surface labelling of parallel fibres is observed. Migrating granule cell perikaryal membranes possess the glycoprotein. The results are discussed in relation to the apparent identity of BSP‐2 and the cell adhesion molecule N‐CAM and its physiological properties.
Cultured embryonic non‐innervated mouse muzzle is capable of generating a whisker pattern
Authors: Andrés Filipe L. , Van der Loos Hendrik
Published: April 2003

The whisker pattern on the muzzle of the mouse is mapped in the contralateral parietal neocortex, each whisker follicle projecting to its own multineuronal unit (‘barrel’). To determine the role, if any, of the peripheral innervation in the establishment of the vibrissal array, we cultured non‐innervated prospective whiskerpads from 9‐ and 10‐day‐old embryos, mostly on chorioallantoic membrane. The results show that skin, alone, is capable of generating the whisker pattern, thus adducing a strong argument for the hypothesis that the central brain maps have their origin in the periphery.
Prenatal and postnatal development of synapses and acetylcholinesterase staining in the dentate gyrus of the rhesus monkey
Authors: Vijayan Vijaya K.
Published: April 2003

Morphogenesis, distribution of cholinergic enzyme acetylcholinesterase and synaptogenesis in the dentate gyrus of the rhesus monkey during the pre‐ and postnatal periods of development were examined using histological, histochemical and ultrastructural methods. The pattern of neuronal differentiation in the dentate gyrus demonstrated distinct superficial‐to‐deep and lateral‐to‐medial gradients. The histochemical reaction for acetylcholinesterase was present on gestation day 120 as minimal staining in the supragranular band and in the inner one‐third of the dentate molecular layer. At term, the laminar distribution of the enzyme assumed mature pattern although considerable enhancement in staining intensity was achieved postnatally. At term and at 9 months of postnatal age, the most pronounced enzyme activity was found in the supragranular band and in the inner one‐third of the molecular layer. Synaptogenesis in the dentate molecular layer was characterized by the early formation of axo‐dendritic contacts on dendritic trunks and branches followed by the appearance of synapses on simple and complex spines. Spines were detected infrequently on gestation day 132. On day 148, they ranged in morphology from short stubby protrusions to pedunculated, triangular processes. The majority of the spines exhibited flat postsynaptic surfaces. Complex, synapse‐bearing U‐ and W‐shaped spines were observed rarely at this age but appeared more frequently at term and at 15 months of postnatal age. However, at all ages, including 15 months postnatally, synapses on flat‐surfaced simple spines predominated. Most synapses were of the asymmetric variety.

With certain exceptions, these features of development of the rhesus dentate gyrus resemble the reported patterns of postnatal ontogenesis of this structure in the rat. However, the ingrowth of cholinergic afferents and the major modifications in synapse structure occur prenatally in the rhesus monkey during the second half of the gestation period. This temporal difference between the two species should receive consideration in the planning of neuroplasticity experiments designed to explore lesion‐induced adaptations in afferent growth and synaptogenesis in the rhesus dentate gyrus.
Ontogenetic changes of the soluble and membrane‐bound D2 glycoprotein in rat forebrain
Authors: Gennarini Gianfranco , Corsi Patrizia , Pavia Rosalia , Vitiello Francesco , Di Benedetta Carlo
Published: April 2003

A soluble form of the D2 glycoprotein, detected in the rat brain hypotonic extract, is described. Its specific relative concentration did not differ significantly in the three examined cerebral regions (forebrain, brainstem and cerebellum), while in the cerebellum the membrane‐bound form was about three and four times more concentrated than in the forebrain and brainstem, respectively. No sizeable developmental variations of the soluble D2 concentration could be detected in forebrain, whereas the amount of the membrane‐bound protein rose from birth to postnatal day 6 and then decreased to the adult value (about 40% of the newborn concentration). Ontogenetic modifications of the membranous D2 glycans (studied through the binding of the molecule to several lectins) occur around postnatal day 18 when the binding to Ricinus communis lectin, specific for galactose, becomes evident. At all ages both soluble and membrane‐bound forms bind to Concanavalin A, specific for mannose and glucose, and to wheat germ agglutinin, specific for N‐acetylglucosamine, while the lack of binding to Ulex europeus lectin suggests the absence of discrete amount of fucose. The results are discussed in relation to the possible involvement of D2 glycoprotein in cell‐to‐cell adhesion.
Influence of brain extract and dibutyryl cyclic AMP on the amount of carbonic anhydrase in primary glial cell cultures from newborn rat brain
Authors: Delaunoy J. P. , Hog F. , Sensenbrenner M.
Published: April 2003

The effect of brain extracts from rat and beef, and of 2′,5′‐dibutyryl cyclic AMP on the CAII content was investigated in rat primary glial cultures maintained in serum‐containing or serum‐free medium. All cultures contained a mixed population of oligodendroglial and astroglial cells, but under certain conditions the cultures were highly enriched in oligodendroglial cells. An immunocytochemical positive reaction to CAII was observed in oligodendrocytes, while astrocytes were not stained. The content of CAII per culture and per oligodendroglial cell was higher after treatment of the culture with soluble brain extract and a partially purified fraction. A combined autoradiographic‐ immunohistochemical study, after [3H]thymidine incorporation and staining for CAII, showed that under the influence of rat brain extract the number of mitotic CAII positive cells was greater at day 11 compared to control cultures. But the proliferation rate decreased with time in culture after brain extract treatment and the number of mitotic CAII positive cells became far below that of controls. Since at the same time CAII quantity per cell was higher in the treated ones it is suggested that brain extract not only influences the proliferation of oligodendroglial cells, but also their maturation. The addition of 2′,5′‐dibutyryl cyclic AMP had no effect on oligodendroglial cells and the amount of CAII in the cultures was not affected. It is discussed that agents, which stimulate 3′,5′‐cyclic AMP content, may not influence the CAII content, but may stimulate the enzymatic activity.
Properties of γ‐glutamyltransferase in developing rat brain
Authors: Pajari M.
Published: April 2003

The activity and properties of brain γ‐glutamyltransferase (EC 2.3,2.2) were studied in 7‐, 14‐and 90‐day‐old rats. The enzyme activity was highest in the pons‐medulla and lowest in the cerebellum in each age group. The activity of glycylglycine, 10 protein amino acids, GABA and taurine as acceptor of the γ‐glutamyl group was studied with 7‐day‐old and adult rats. The best acceptors were glycylglycine, lysine and methionine and the poorest taurine, valine and isoleucine. The relative acceptor activity of lysine changed most during development. Km for the γ‐glutamyl donor, γ‐glutamyl‐p‐nitroanilide, with glycylglycine was about 3 mM in all experimental groups. It did not change during development but V increased about fivefold in all brain areas studied. A mixture of serine and borate strongly inhibited γ‐glutamyltransferase in each age group. Potassium and magnesium ions had no measurable effect on the enzyme activity but sodium ions were stimulatory.
Some properties of uridine‐5′‐diphospho‐N‐acetylgalactosamine:Hematoside N‐acetylgalactosaminyltransferase at early and late stages of embryonic development of chicken retina
Authors: Maccioni H.J.F. , Panzetta P. , Arrieta D.
Published: April 2003

Some properties of the uridine‐5′‐diphospho‐N‐acetylgalactosamine:hematoside N‐acetylgalactosaminyltransferase were studied in retina tissue from chick embryos at 7 and 14 days of development. The Vmax was about 6‐fold higher in retinas from 14 day embryos than in retinas from 7 day embryos. No differences were found either in the apparent Michaelis constant for both donor nucleotide and acceptor glycolipid, or in the optimal detergent concentration, or in the stability upon storage at −14°C or heating at 50°C. Mixtures of homogenates of retinas from 7 day and from 14 day embryos gave the activity values expected for samples free of effectors diffusible and in excess. From experiments of partial delipidation of retina homogenates and reconstitution with lipid from retina homogenates from one or the other age, no indications were found that the activity was modulated by developmental changes in the lipid environment of the enzyme. Taken together, the results suggest that the increase of activity during development was not due to qualitative changes in the catalytic characteristics of the enzyme.
In Purkinje cell dendrites of the young rat, thyroid hormone controls the resistance of microtubules to fixation at low temperature
Authors: Faivre Catherine , Legrand Christiane , Rabié Alain
Published: April 2003

Microtubules were studied in parallel fibres and Purkinje cell dendrites of 14‐day‐old normal and propylthiouracil‐treated rats after fixation of the cerebellum at room and low temperature (4°C). In both control and propylthiouracil‐treated animals, nearly all the microtubules of parallel fibres disappeared after fixation at low temperature. In Purkinje cell dendrites of control animals, 60% of the microtubules persisted at low temperature. Propylthiouracil treatment led to a 78% decrease in the density of these cold‐stable microtubules. In contrast, the density of cold‐labile microtubules remained normal. Administration of low doses of thyroxine to the propylthiouracil‐treated rats produced a rapid increase in the density of cold‐labile microtubules, but long‐term treatment with the hormone was necessary to obtain normal density of cold‐stable microtubules. Thyroid hormone thus seems to regulate some properties of microtubules involved in their resistance to fixation at low temperature. These properties could be of importance in growth and branching of dendrites.
Development of mouse spinal cord in tissue culture—III. Quantification of neuron development in neural tube microfragment cultures
Authors: Houle John , Fedoroff Sergey
Published: April 2003

Mouse neural tube development in vitro was examined following the isolation and culturing of specific regions of the neural tube. Developmental characteristics of neuron formation and differentiation were assessed both quantitatively and qualitatively.

A 1 mm length of embryonic day 10 mouse neural tube was cut into 32 microfragments of equal size and cultured on collagen coated cover‐slips. Neuronal cells were observed to emerge after 3 days in culture and to migrate away from the fragment upon an immature astroglial precursor cell layer that had begun to form within the first 24 h of culturing. The extent of neuronal migration, the density and total number of neurons per outgrowth zone, and the size distribution of neurons was quantitated after 21 days in culture. Three distinct patterns of neuronal outgrowth (Types II, III and IV) could be observed with a fourth pattern (Type I) best described as being neuron free. Neuron free outgrowth zones (Type 1) were comprized totally of glial cells and represented approximately 10% of the outgrowth zones examined. Characteristics of Type II outgrowth zones were a relatively restricted neuronal migration and a predominance (85%) of small neurons (< 10μm diameter) scattered around the outgrowth zone. In contrast the area of Type III outgrowth zones was twice that of the Type II, with a neuronal density 2.5 times greater, composed of a near equal proportion of small to medium sized (10–18 μm) neurons. The neuronal density and extent of migration of Type IV zones was similar to that of Type II zones; however the neurons of this fragment type were primarily medium and large sized neurons.

In a second experiment the brachial region of E10 neural tube was sectioned into dorsal and ventral halves and microfragments from each half were cultured separately. Dorsal fragments produced primarily Type I and Type II outgrowth zones, and never a Type IV. In contrast, ventral fragments produced primarily Type III and Type IV zones with a small proportion of Type II zones.

The results indicate that neuronal precursor cells in vitro maintain an adherence to patterns of neurogenesis programmed in vivo during early stages of neural tube development. The present culture system thus provides a means for the study of the expression in vitro of specific patterns of neuronal development and of the possible environmental influences (epigenetic factors) upon these patterns.
Alterations in the development of the main olfactory bulb of the mouse after ethanol exposure
Authors: Nyouist‐Battie Cynthia , Gochee Angel
Published: April 2003

The effect of ethanol on the development of the main olfactory bulb (OB) was examined by light and electron microscopy in 21‐day‐old Swiss mice (CD‐1), exposed to ethanol from embryonic day 13 to postnatal day 21. Two control groups were included in the study to rule out changes due to malnourishment: a normal control group, whose dams were fed rodent lab chow throughout the study and a pairfed group, whose dams were fed the same amount of liquid diet as the dams in the ethanol group but with sucrose replacement of ethanol. Somatic growth, measured by length and body weight, was retarded to the same degree in ethanol and pairfed mice, indicating a similar level of malnourishment in both groups without an additional ethanol effect. The most striking change in olfactory bulb development was the stunted growth of this structure in the ethanol‐exposed animals with reductions in the volume of the glomerular. external plexiform and granular cell layers. The laminar organization and cellular cytoarchitecture were not substantially altered by ethanol or malnourishment except for a retarded migration of the periglomerular cells in the ethanol‐treated group. Examination of synaptogenesis in the external plexiform layer (EPL) of olfactory bulb from 21‐day‐old mice in the three groups revealed no treatment‐related changes in synaptic structure, appositional length or ratios of the two dominant type of EPL synapses, which are formed between dendrites of the output neurons (mitral and tufted cells) and granule cells. However, morphometric analysis revealed elevated densities of both synaptic types in the ethanol‐exposed group but with no change in the total synaptic number per olfactory bulb. The results suggest that ethanol exposure of mice after the latter half of gestation and during the postnatal period primarily affects the growth of the olfactory bulb probably by decreasing neuronal cell growth, and/or proliferation but has little affect on other aspects of maturation such as formation of cortical structure, differentiation and synaptogenesis.
Microtubules and neurofilaments in the sciatic nerve fibres of the developing rat: Effects of thyroid deficiency
Authors: Marc Christine , Rabié Alain
Published: April 2003

Microtubules and neurofilaments were counted in cross‐sections of the sciatic nerve fibres of young rats at various stages of development. A linear relationship was found between the numbers of microtubules and neurofilaments and the size of the fibre. The number of microtubules progressively increased during the first two postnatal weeks. Neurofilaments appeared slightly later than microtubules. In thyroid deficiency, the number of microtubules was diminished. The number of neurofilaments was not markedly affected. The ratio of neurofilaments to microtubules was thus increased. The severe retardation of sciatic nerve growth observed in thyroid deficiency might be related to the reduced number of microtubules and to a diminution in fibre plasticity induced by the higher neurofilament to microtubule ratio.
Effects of forskolin on growth and morphology of cultured glial and cerebrovascular endothelial and smooth muscle cells
Authors: Kempski O. , Wroblewska B. , Spatz M.
Published: April 2003

The present experiments were designed to evaluate the effectiveness of forskolin on cAMP production, growth and morphology on cell cultures of glia, endothelium and smooth muscle derived from brain microvessels.

Forskolin significantly increased formation of cAMP and decreased incorporation of thymidine in all three cell types. The thymidine incorporation was reduced dose‐dependently with maximal growth inhibition at 100 μM forskolin. A 1 hr preincubation with forskolin abolished thymidine incorporation by cells grown in fetal calf serum (FCS)‐containing media over the following 24 hr.

In cerebromicrovascular endothelium and smooth muscle, forskolin caused drastic and immediate changes of cell morphology and F‐actin composition that were reversible. In glial cells, morphological changes were visible only after exposure to forskolin for more than 24 hr. These changes were accompanied by increased staining with antibodies against glial fibrillary acidic protein (GFAP).

These findings support the contention of cAMP involvement in growth regulation of these cells and indicate that forskolin might be used as a tool to induce growth arrest and possible differentiation in cell cultures from mammalian brain.
Effects of nerve growth factor on sensory neurons in the chick embryo: A stereological study
Authors: Dimberg Yvonne , Hedlund Kjell‐Olof , Ebendal Ted
Published: April 2003

Chicken embryos on days 6–13 of incubation received injections of purified βNGF (80 μ/ day) for 3 or 4 days and were then killed. Sensory ganglia were fixed and taken for embedding and sectioning. A stereological method based on unfolding of cell‐diameter frequencies was used to determine the number of neurons of different size in the spinal, trigeminal and nodose ganglia. The total volume of the ganglia was also determined. NGF induced increases in diameter of the neural crest‐derived dorsomedial (DM) neurons in spinal and trigeminal ganglia. Injected NGF did not influence ventrolateral (VL) neurons of neural crest origin in the spinal ganglia nor the ventrolateral neurons of placodal origin in the trigeminal ganglion. The volumes of spinal and trigeminal ganglia increased by 50 and 100%, respectively. The volume of the nodose ganglion and the total number and size of the placodal nodose neurons were unaffected by NGF. The results demonstrate a clear difference in the response to NGF in vivo between smaller and larger sensory neurons.
Calcium‐dependent regulation of phospholipase A2 and its inhibitors, including tetracaine, for acetylcholine receptor cluster formation in mouse myotubes co‐cultured with spinal cord explant
Authors: Kimura M. , Kimura I. , Shikada A. , Takahashi K.
Published: April 2003

Regulatory effects of phospholipase A2 (PLA2) on acetylcholine receptor (AChR) cluster formation were investigated in developing mouse myotubes co‐cultured with spinal cord explant, using quinacrine, cortisone, tetracaine and related agents. AChR was visualized using fluorescence‐conjugated α‐bungarotoxin. Peak fluorescence intensity and total fluorescence within the fluorescence stain were measured as indices of AChR cluster formation and AChR content, respectively. Both indices were gradually increased from day 9 to 13 in culture. PLA2 (0.2–1.0 μg/ml), melittin (10 μg/ml) and arachidonic acid (100 μM), added to the culture medium from the second day, clearly inhibited both indices at days 11 and 13, whereas the addition of phospholipase C (1 μg/ml) inhibited peak fluorescence but did not affect total fluorescence. The co‐existence of PLA2 with its inhibitors—quinacrine (3 μM), cortisone (0.01 μM) and tetracaine (30 μM)—significantly overcame the PLA2‐induced inhibition of both indices.

The elevation of calcium ion concentrations from 2.9 to 10 mM abolished the increase of both indices. Ouinacrine (10 μM), cortisone (0.1 μM) and tetracaine (100 μM) alone similarly inhibited both fluorescence indices. The addition of EGTA (2 mM) from day 8 overcame tetracaine‐induced inhibition but not quinacrine‐ or cortisone‐induced inhibition. These results suggest that the formation of AChR clusters in developing myotubes is negatively controlled by endogenous PLA2 activity. This overcoming of PLA2‐induced inhibition by tetracaine may be dependent on calcium ion mobilization, whereas that by quinacrine and cortisone may not.
Transplantation immunobiology in relation to neural grafting: Lessons learned from immunologic privilege in the eye
Authors: Streilein J. Wayne
Published: April 2003

The principles of transplantation immunobiology are described and discussed in terms of their applicability to neural grafting, a newly emerging field dedicated to the ultimate goal of reconstituting central nervous system deficits with normally functioning tissue replacements. Unique anatomic and physiologic features of the eye, which are responsible for the phenomenon of immunologic privilege, are compared with the brain and considered in terms of their relationship to the principles of transplantation. The existence of immune privilege in the brain and the newly acquired understanding of immunologic privilege in the eye may offer strategies by which neural grafters can achieve significantly greater graft acceptance.
Effects of injecting antibodies to mouse nerve growth factor into the chick embryo
Authors: Dimberg Yvonne , Ebendal Ted
Published: April 2003

The function of NGF in chick embryos was studied by injecting antibodies to mouse nerve growth factor (NGF). The uptake of mammalian antibodies into the 8‐ to 15‐day‐old chick embryo was followed by an enzyme‐linked immunoassay. Normal rabbit antibodies (250 μg) were administered to the yolk, of which less than 5% was found in the embryo (300 ng of IgG per g wet wt of embryo). The concentration was proportionally lower when 100 μg anti‐NGF antibodies were injected (40 ng/g). The concentration of anti‐NGF antibodies was 1.5 times higher following injection directly into the body of the embryos. The effects of injecting antibodies at days 3–7 were studied in 10‐day‐old embryos by measuring the diameter frequencies of neurons in sympathetic and sensory ganglia. In comparison with controls, significantly smaller neurons were found in the sympathetic ganglia in embryos directly injected with anti‐NGF. In the spinal ganglia, distribution of neuron diameters did not differ between anti‐NGF‐treated embryos and controls. Finally, the ability of different antibodies to mouse NGF to inhibit the in vitro activity of recombinant chick NGF was investigated. Total block was found at 1000–2000 ng of IgG per ml for most of the antibodies tested, levels not reached when injecting the embryo with antibodies to NGF. We conclude that the main reason for the limited effects in chick embryos by injection of NGF antibodies is due to the low levels of penetration of the anti‐NGF IgG into the embryo.
Influence of GM1 gangliosides on the growth of cultured rat embryonic serotonergic neurons
Authors: Marlier L. , Poulat P. , König N. , Drian M. J. , Privat A.
Published: April 2003

GM1 gangliosides were added to the medium of cultured raphe neurons enriched in the serotonergic phenotype in order to study their influence on biochemical and morphological growth parameters of serotonergic neurons. After 2 days of culture in the presence of GM1, specific uptake of serotonin measured by scintillation counting exhibited a moderate but significant increase for a GM1 concentration of 5 × 10−8M. Morphological parameters of 5‐HT neurons were measured after immunocytochemical staining with specific serotonin antiserum and digitalization of immunoreactive cells. Eight parameters were studied; for concentrations of 5 × 10−8 and 10−7M of GM1, the absolute neuritic field area and the total length of the segments were significantly increased, whereas the number of neuritic segments and their mean length were not modified. We conclude that GM1 ganglioside has a significant influence on the growth of serotonergic neurons. Moreover, electron microscopy showed, on treated cultures, a dramatic increase of the number of spicules all along the neuron's process, suggesting that GM1 could act by modifying the attachment of cells to their substrate. The possible molecular mechanisms of the action of GM1 are discussed.
Serotonin‐immunoreactive neurons in the brain of Manduca sexta during larval development and larval‐pupal metamorphosis
Authors: Granger Noelle A. , Homberg Uwe , Henderson Pamela , Towle Andrew , Lauder Jean M.
Published: April 2003

The developing serotonergic system of the tobacco hornworm, Manduca sexta, has been studied immunocytochemically in whole mount preparations of brain‐retrocerebral complexes. The distribution of serotonin‐immunoreactive cell bodies, fibers and terminal fields has been analysed during larval and larval‐pupal development using a specific rabbit antiserum against serotonin‐hemocyanin conjugates. The serotonergic system was conserved from the fourth to the fifth larval stadium, with minimal changes occurring until the onset of pupal development. At this time, alterations in the distribution of serotonin‐immunoreactive cells and processes were observed, including the apparent disappearance of some cell bodies and terminals. Nevertheless, the overall appearance of this system in the pupal brain was not significantly different from that in the larva. The larval pattern was characterized by eight bilateral groups of cell bodies which sent thick bridges of fibers across the midline, a feature strikingly similar to the serotonergic system in vertebrate embryos. In addition, three bilateral immunoreactive fields of arborization were observed around and ventral to these cell groups, together with regions of serotonin immunoreactivity in the medial and lateral protocerebral lobes. The central body, larval antennal centers, larval accessory lobes and the tritocerebrum were also immunoreactive. Fibrous networks of serotonergic processes were usually observed around nerves emanating from the brain, including the connectives from the brain to the corpus cardiacum and corpus allatum. Smaller varicosities were observed in the interior of these neurohemal and glandular organs and a network of 5‐HT fibers was occasionally found around the corpus cardiacum and corpus allatum. The possible relationship of serotonin to cerebral neuroendocrine functions during the postembryonic development of M. sexta is discussed.
Effects of acidic and basic fibroblast growth factors on proliferation and maturation of cultured rat oligodendrocytes
Authors: Besnard François , Perraud Frédéric , Sensenbrenner Monique , Labourdette Gérard
Published: April 2003

A pure culture of oligodendrocytes has been developed starting from brain hemispheres of newborn rats. Various effects of acidic and basic fibroblast growth factors (FGFs) on the development of oligodendrocytes have been examined and compared. Both factors elicited similar effects, i.e. stimulation of the proliferation, inhibition of the specific activity of the marker enzyme 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase and decrease of the ratio of myelin basic protein positive cells. These results indicate that FGFs are very potent mitogens for oligodendrocytes, even in the absence of other cell types, but that they elicit a negative effect on the cell maturation, possibly related to their strong effect on proliferation.
Non‐specific cholinesterase activity of the developing peripheral nerves and its possible function in cells in intimate contact with growing axons of chick embryo
Authors: Dubovy Petr , Haninec Pavel
Published: April 2003

The results presented here demonstrate non‐specific cholinesterase (nChE) activity in the developing peripheral nerves of chick embryos at stages 25–26 according to Hamburger and Hamilton (1951, J. Morphol. 88, 49–92). Under the light microscope the use of simultaneous staining for nChE activity and silver proteinate impregnation revealed the axons to be surrounded by cells exhibiting nChE activity in the main nerve trunks and in the growing tips of nerves. Nerve branches arising from the main nerve trunks contained cells with positive reaction for nChE activity, too. Electron‐dense particles of the reaction product indicating nChE activity were found in the rough endoplasmic reticulum and in the perinuclear envelope of cells in close contact with growing nerve fibers and their growth cones. The same distribution of nChE activity was found in cells which were located near to nerve fasciculi but without direct contact with axons. Surprisingly, the cells in close contact with axons and their growth cones exhibited the end product of nChE activity in the outer part of their plasma membrane. The cells enveloping axons within the nerve trunks were apparently Schwann cells, while those around the growth cones at nerve tips could be identified as Schwann cells and/or mesenchymal cells of the hindlimb. The nChE reaction product was also detected in the axolemma of nerve fibers and their growth cones. The distribution of nChE activity in the developing peripheral nerves of chick embryos suggests that these molecules may influence the process of axonal elongation and locomotion. Several possible mechanisms of nChE action on growing axons can be presumed: (i) intracellular Ca2+ level regulation; (ii) providing an adhesive substrate; (iii) butyrate production affecting the cell metabolism and the distribution of neurotubules and neurofilaments. It is also assumed that nChE molecules are involved in the interactions of nerve fibers with Schwann cells and/or mesenchymal cells as well as in interneuronal interactions.
Factors affecting denervation‐like changes at the neuromuscular junction during aging
Authors: Rosenheimer Julie L.
Published: April 2003

The ability to sustain synaptic transmission diminishes with aging. To determine whether this is accompanied by alterations in the structure of the synapse, end‐plate architecture was examined in EDL muscles from 10‐ to 25‐month rats. There was a significant age‐related increase in end‐plate area and a decrease in the number of nerve terminals per end plate. Furthermore, the percentage of end plates with ultraterminal sprouts increased dramatically with age. To compare the morphological changes associated with aging to the changes in response to denervation, EDL muscles from 10‐ and 25‐month animals were partially denervated. Both end‐plate area and ultraterminal sprouting increased, while terminal number decreased following denervation in the 10‐month muscles. To determine whether the age‐ and denervation‐associated changes were accompanied by alterations in muscle‐derived nerve‐outgrowth factors, in vitro assays were performed. Neurite outgrowth was quantified from embryonic motoneurons incubated with muscle extract, or grown on cryostat‐cut muscle cross sections from 10‐ and 25‐month innervated and denervated EDL muscles. Both aged and denervated muscles induced greater degrees of neurite outgrowth compared with younger innervated muscles. Innervation to the EDL was then examined, and signs of axonal degeneration were observed. It is suggested that aging is associated with alterations in the motor axon to the EDL muscle. These changes are manifest at the neuromuscular junction. In turn, the muscle responds as if it were in a state of functional denervation.
Effects of MPTP on the cerebrovasculature
Authors: Adams James D. , Klaidman Lori K. , Odunze Ifeoma N. , Johannessen Jan N.
Published: April 2003

The neurotoxin, l‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine, has been shown to cause pooling of blood in the brain microvasculature and decrease the permeability of the blood‐brain barrier. All areas of the brain examined in this study were affected. This study points out the possibility that reduced nutrient uptake, hypoxia and ensuing free radical damage are involved in the mechanism of toxicity of this neurotoxin.