bluuebunny/crossref_metadata_2025 huggingface dataset:
Laccase‐mediated formation of the phenoxazinone derivative, cinnabarinic acid
Authors: Eggert Claudia , Temp Ulrike , Dean Jeffrey F.D. , Eriksson Karl-Erik L.
Published: April 2003

The phenoxazinone chromophore occurs in a variety of biological systems, including numerous pigments and certain antibiotics. It also appears to form as part of a mechanism to protect mammalian tissue from oxidative damage. During cultivation of the basidiomycete, Pycnoporus cinnabarinus, a red pigment was observed to accumulate in the culture medium. It was identified as the phenoxazinone derivative, cinnabarinic acid (CA). Laccase was the predominant extracellular phenoloxidase activity in P. cinnabarinus cultures. In vitro studies showed that CA was formed after oxidation of the precursor, 3‐hydroxyanthranilic acid (3‐HAA), by laccases. Moreover, oxidative coupling of 3‐HAA to form CA was also demonstrated for the mammalian counterpart of laccase, the blue copper oxidase, ceruloplasmin.
Poly(U) binding activity of hepatitis C virus NS3 protein, a putative RNA helicase
Authors: Kanai Akio , Tanabe Kyoko , Kohara Michinori
Published: April 2003

A non‐structural protein of the hepatitis C virus (HCV), NS3, contains amino acid sequence motifs characteristic of serine‐proteinases and RNA helicases. RNA binding activity of the NS3 protein with an apparent dissociation constant of 2 × 10−7 M was detected using a poly(U)‐Sepharose resin. Competitive RNA binding analysis suggested that the NS3 protein binds preferentially to the poly(U) sequence, which is located at the 3′ end of HCV RNA. Mutational analysis of NS3 protein revealed the possibility that both the RNA helicase region and the serine‐proteinase region were necessary for full RNA binding activity.
The K+ channel inward rectifier subunits form a channel similar to neuronal G protein‐gated K+ channel
Authors: Velimirovic Bratislav M. , Gordon Eric A. , Lim Nancy F. , Navarro Betsy , Clapham David E.
Published: April 2003

G protein‐activated inwardly rectifying K+ channel subunits GIRK1 (Kir 3.1), GIRK2 (Kir 3.2), and CIR (Kir 3.4) were expressed individually or in combination in Xenopus oocytes and CHO cells. GIRK1 coexpressed with CIR or GIRK2, produced currents up to 10‐fold larger than any of the subunits expressed alone. No such clear synergistic effects were observed upon coexpression of CIR/GIRK2 under the same conditions. Coexpression of G protein βγ (mGβ1γ2) increased the current through GIRK1/GIRK2 and GIRK2 channels. Gβγ subunits purified from bovine brain, increased channel activity 50–1000‐fold in patches from cells expressing GIRK1/GIRK2 or GIRK2 alone. The single GIRK1/GIRK2 channels resembled previously described neuronal G protein‐gated K+ channels. In contrast, single GIRK2 channels were short‐lived and unlike any previously described neuronal K+ channel. We propose that some neuronal G protein‐activated inward rectifier K+ channels may be formed by a GIRK1/GIRK2 heteromultimer and that Gβγ activation may involve both subunits.
Murine and human mast cell express acetylcholinesterase
Authors: Nechushtan Hovav , Soreq Hermona , Kuperstein Valentina , Tshori Sagi , Razin Ehud
Published: April 2003

Expression of catalytically active protein was detected in a murine mast cell line. The primary type of AChE mRNA produced by these cells was found to be the brain and muscle type by PCR amplification of alternative exons from the 3′ of mast cells AChE cDNA. AChE was further found to be expressed in the HMC‐1 the human mast cell precursor line. Furthermore, utilizing the single cell RT‐PCR method we detected AChE mRNA expression in FcϵRI‐positive single cells derived from human colonic mucosal biopsies. Our findings predict the involvement of mast cell AChE in neuronal‐mast cell interactions.
Differential effects of endogenous and exogenous nitric oxide on the release of endothelin‐1 from the intact perfused rat adrenal gland in situ
Authors: Hinson J.P. , Kapas S. , Cameron L.A.
Published: April 2003

Studies using an inhibitor of nitric oxide (NO) synthesis have suggested that endogenous NO may have a role in regulating endothelin release. We investigated the effect of endogenous and exogenous nitric oxide (NO) on the release of irET‐1. l‐NAME stimulated, but l‐arginine inhibited irET‐1 release. Perfusing sodium nitroprusside (SNP), however, did not inhibit irET‐1 secretion. CyclicGMP, the second messenger for NO action, was stimulated by SNP but not by l‐arginine. These data demonstrate that endogenous NO inhibits of irET‐1, in a manner which is independent of cGMP, and suggest that this action may contribute to the vasodilatory effect of NO.
Interaction between human amphipathic apolipoproteins and amyloid β‐peptide: surface plasmon resonance studies
Authors: Shuvaev Vladimir V. , Siest Gérard
Published: April 2003

Several apolipoproteins including apoE and apoA‐I are known to be associated with amyloid β‐peptide, a major component of senile plaques in Alzheimer's disease. In the present study the interaction between three human amphipathic apolipoproteins apoE3, apoA‐I and apoA‐II and immobilized amyloid β‐peptide (1–40) was quantified by plasmon resonance. The interactions were saturable and reversible. The results demonstrated a high affinity of the binding of amphipathic apolipoproteins to amyloid β‐peptide. On the other hand, only a small population of synthetic amyloid β‐peptide participated in the interaction. The apparent equilibrium dissociation constants K
D were 10 nM for apoE3, 25 nM for apoA‐I and 80 nM for apoA‐II under physiological conditions. The affinity of the apoE3‐amyloid β‐peptide binding was not affected by pH in the range 6.0–8.0 but was significantly increased by high salt concentration. ApoA‐I mainly followed similar patterns. A major participation of hydrophobic forces in the binding of apoE3 and apoA‐I to amyloid β‐peptide was suggested.
Calcium dependence of ryanodine‐sensitive calcium channels from brain cortex endoplasmic reticulum
Authors: Marengo Juan José , Bull Ricardo , Hidalgo Cecilia
Published: April 2003

Endoplasmic reticulum vesicles isolated from rat brain cortex and fused with lipid bilayers displayed ryanodine‐sensitive calcium channels, with three cytoplasmic calcium dependences. A: Channels (n = 5) stimulated by Ca2+ (K
0.5 = 1.2 μM and n
Hill = 1.9) and not inhibited up to 0.5 mM Ca2+. B: Channels (n = 14) cooperatively activated (K
0.5 = 6.9 μM and n
Hill = 1.8), and inhibited by Ca2+ (K
0.5 = 152 μM and n
Hill = 1.8). C: Low P
o (< 0.1) channels (n = 22), non‐cooperatively activated and inhibited with the same K
0.5 = 26.3 μM Ca2+. These three types of responses to cytoplasmic [Ca2+] may underlie separate calcium release pathways in neurons of rat brain cortex.
Cloning and sequencing of the 5′ region of the human glucose‐6‐phosphatase gene: transcriptional regulation by cAMP, insulin and glucocorticoids in H4IIE hepatoma cells
Authors: Schmoll Dieter , Allan Bernard B. , Burchell Ann
Published: April 2003

We have cloned and sequenced the first 1.2 kb of the 5′ region of the human glucose‐6‐phosphatase gene. Transfection of H4IIE hepatoma cells with the 1.2 kb fragment fused to a luciferase reporter gene demonstrated both basal and hormone responsive luciferase activity. Dexamethasone increased and insulin decreased luciferase activity. Insulin and dibutyryl cyclic AMP both significantly decreased activity in the presence of dexamethasone.
H2O2 renders cells accessible to lysis by exogenous phospholipase A2: a novel mechanism for cell damage in inflammatory processes
Authors: Dan Phyllis , Nitzan Dorrit W. , Dagan Arie , Ginsburg Isaac , Yedgar Saul
Published: April 2003

Phospholipase A2 (PLA2) and H2O2, secreted from activated inflammatory cells, play a central role in the tissue damage occurring in inflammatory processes. However, while exogenous PLA2 alone does not cause cell lysis, it readily does so when acting with H2O2. We have found that H2O2 degrades cell surface proteoglycans, thus rendering the membrane PL accessible to hydrolysis by exogenous PLA2. This novel mechanism introduces a role for cell surface proteoglycans in protection of cells from damage by pro‐inflammatory agents, and may assign a central role for the combined action of H2O2 and PLA2 in inflammatory and bacteriocidal processes.
Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c‐Src in human platelets
Authors: Falet Hervé , Ramos-Morales Francisco , Bachelot Christilla , Fischer Siegmund , Rendu Francine
Published: April 2003

Protein tyrosine phosphatase 1C (PTP1C), highly expressed in hematopoietic cells, is a soluble protein tyrosine phosphatase containing two Src homology 2 (SH2) domains at the N‐terminus and two putative sites of tyrosine phosphorylation at the C‐terminus. This paper reports that PTP1C and c‐Src could be coimmunoprecipitated during thrombin‐induced platelet activation. Moreover, association between the two signalling proteins occurred only after PTP1C had been tyrosine phosphorylated. In in vitro experiments, PTP1C bound to the SH2 domain of c‐Src, suggesting that association between tyrosine phosphorylated PTP1C and c‐Src was mediated by the SH2 domain of c‐Src. Finally, in resting platelets, PTP1C was mainly found in the Nonidet P‐40 soluble fraction whereas following thrombin‐induced activation, around 17% of PTP1C was associated with the insoluble fraction.
Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: Enzyme purification, characterisation and N‐terminal sequence
Authors: Bonete María-José , Pire Carmen , Llorca Francisco I. , Camacho Mónica L.
Published: April 2003

An NAD(P)‐glucose dehydrogenase from the extremely halophilic Archaeon, Haloferax mediterranei, has been purified to electrophoretic homogeneity. The purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability. The N‐terminal amino acid sequence has been determined and N‐terminus alignment with sequences of other glucose dehydrogenases shows that the halophilic enzyme most closely resembles the NAD(P)‐linked glucose dehydrogenase from the thermophilic Archaeon Thermoplasma acidophilum. However, the halophilic glucose dehydrogenase appears to be a dimeric protein, in contrast to the tetrameric enzyme from the thermophile.
Kinetic modelling of the proton translocating CF0CF1‐ATP synthase from spinach
Authors: Pänke Oliver , Rumberg Bernd
Published: April 2003

The rate of both ATP synthesis and hydrolysis catalysed by the thiol‐modulated and activated ATP synthase from spinach is measured as a function of all substrates including the protons inside the thylakoid lumen. The most important findings are: (1) sigmoid kinetics with respect to Hin
+, (2) hyperbolic kinetics with respect to ADP, ATP and phosphate, with K
m for phosphate and ADP decreasing upon increasing Hin
+, (3) binding of ADP and phosphate in random order and competitive to ATP. Simulation of the complete set of experimental data is obtained by a kinetic model featuring Boyer's binding‐change mechanism.
Tetrameric triosephosphate isomerase from hyperthermophilic Archaea
Authors: Kohlhoff Michael , Dahm Anke , Hensel Reinhard
Published: April 2003

Triosephosphate isomerase (TIM) of the hyperthermophilic Archaea Pyrococcus woesei and Methanothermus fervidus have been purified to homogeneity. The enzymes from the two hyperthermophiles represent homo‐tetramers of 100 kDa, contrary to all known bacterial and eukaryotic TIMs, which are dimers of 48–60 kDa. Molecular size determination of the TIM from the mesophilic methanogen Methanobacterium bryantii yielded the usual molecular mass of only 57 kDa, indicating that the tetrameric aggregation state does not represent an archaeal feature but rather correlates with thermoadaptation. A similar preference for higher protein aggregates in hyperthermophilic Archaea has previously been demonstrated for 3‐phosphoglycerate kinases. The gene of the P. woesei TIM was cloned and sequenced. The archaeal TIM proved to be homologous to its bacterial and eukaryotic pendants. Most strikingly, the deduced protein sequence comprises only 224 residues and thus represents the shortest TIM sequence known as yet. Taking the three‐dimensional structure of the eucaryal TIM as a basis, from the shortenings of the chain considerable rearrangements at the bottom of the α/β barrel and at its functionally inactive flank are expected, which are interpreted in terms of the formation of new subunit contacts.
SAP kinase‐3, a new member of the family of mammalian stress‐activated protein kinases
Authors: Mertens Stephan , Craxton Molly , Goedert Michel
Published: April 2003

Stress‐activated protein kinases are MAP kinase homologues that are activated by cellular stresses, bacterial endotoxin and inflammatory cytokines. They are activated by a dual threonine/tyrosine phosphorylation within a TPY sequence in the case of stress‐activated protein kinase‐1 and its isoforms (also called JNKs) or a TGY sequence in the case of stress‐activated protein kinase‐2 and its isoforms (also called p38, p40, RK, CSBPs, XMpk2 and Mxi2). Here we report the cloning and sequencing of a new protein kinase from rat with a TGY sequence in the activation domain. This stress‐activated protein kinase‐3 is 60% identical to mouse stress‐activated protein kinase‐2 and 45% identical to HOG1 from Saccharomyces cerevisiae. Transcripts encoding stress‐activated protein kinase‐3 are widely expressed, with high levels in skeletal muscle.
Benzophenone synthase from cultured cells of Centaurium erythraea
Authors: Beerhues Ludger
Published: April 2003

A central step in xanthone biosynthesis is the formation of the C13 skeleton, i.e. an intermediate benzophenone. Biosynthesis of 2,3′,4,6‐tetrahydroxybenzophenone from m‐hydroxybenzoyl‐CoA and malonyl‐CoA was shown in cell‐free extracts from cultured cells of Centaurium erythraea. The enzyme catalyzing this reaction was named benzophenone synthase.
Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity
Authors: Yoshihara Eisaku , Gotoh Naomasa , Nishino Takeshi , Nakae Taiji
Published: April 2003

We report here our discovery that protein D2 of the outer membrane of Pseudomonas aeruginosa is a novel porin bearing protease activity. Homogeneously purified protein D2 hydrolyzed several synthetic peptides according to the Michaelis‐Menten kinetics. A specific serine protease inhibitor, diisopropyl fluorophosphate (DFP), inactivated the protease activity and [3H]DFP covalently labeled protein D2. We tested the effect of two monoclonal antibodies raised against protein D2 on the protease activity. One antibody lowered the protease activity to about 20%, while the other enhanced it to about 300% of that without antibody. In addition, the fractions derived from the outer membrane of the protein D2‐deficient mutants showed negligible protease activity, whereas similarly fractionated outer membrane proteins of the protein D2‐positive parent strain showed strong protease activity.
Subcellular localization of annexin V in human foreskin fibroblasts: nuclear localization depends on growth state
Authors: Barwise Jennifer L. , Walker John H.
Published: April 2003

Annexin V is a major intracellular calcium‐binding protein in human foreskin fibroblasts. Immunocytochemistry revealed that annexin V was localized in the nucleus and throughout the cytoplasm in human foreskin fibroblasts. The presence of annexin V in the nucleus was variable depending on the growth state. Nuclear staining was strongest in proliferating cells immediately after sub‐culture, and decreased on prolonged culture without changing the culture medium. The cytoplasmic location of annexin V was not greatly affected by the same conditions. Refeeding cells with fresh serum restored annexin V to the nuclei of all cells within in 24 h indicating that nuclear localization of annexin V is dependent on serum factors.
Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1
Authors: Terada Tomohiro , Saito Hideyuki , Mukai Mayumi , Inui Ken-ichi
Published: April 2003

The LLC‐PK1 cells stably transfected with a rat PEPT1 cDNA transported ceftibuten (anion) and cephradine (zwitterion), both oral β‐lactam antibiotics, in a H+‐gradient‐dependent manner. Diethylpyrocarbonate, a histidine residue modifier, abolished ceftibuten uptake. This inhibition was prevented in the presence of glycylsarcosine or cephradine. When expressed in Xenopus oocytes, replacement of either histidine 57 or histidine 121 of the rat PEPT1 with glutamine by site‐directed mutagenesis eliminated ceftibuten and [14C]glycylsarcosine transport activities. Immunostaining of oocyte sections indicated that insertion of the mutant transporters in the plasma membranes was not impaired. These findings suggest that both histidine 57 and histidine 121, which are conserved in the rat, rabbit and human PEPT1, are involved in substrate recognition of this molecule.
Structural modifications of the Ω loop in human acetylcholinesterase
Authors: Velan Baruch , Barak Dov , Ariel Naomi , Leitner Moshe , Bino Tamar , Ordentlich Arie , Shafferman Avigdor
Published: April 2003

Conformational mobility of the surface Ω loop (Cys‐69‐Cys‐96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen‐bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the Ω loop in the HuAChE is not involved in large lid‐like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp‐86 supporting the proposed coupling between the structure of the Ω loop and the positioning of the Trp‐86 indole moiety, in catalytic activity and in allosterism.
Vitamin D3 elicits calcium response and activates blood monocyte‐derived macrophages from patients with vitamin D dependent rickets type II
Authors: Takeda Eiji , Takata Kazumi , Yamanaka Hisami , Taketani Yutaka , Morita Kyoto , Miyamoto Ken-ichi , Shono Masayuki , Teshima Shigetada , Rokutan Kazuhito
Published: April 2003

We studied the effects of vitamin D3 metabolites on intracellular free Ca2+ concentration ([Ca2+]i) and the respiratory burst of monocyte‐derived macrophages (MDM) from patients with vitamin D dependent rickets type II. Treatment of MDM from the patients and healthy donors with 1 nM 1,25(OH)2D3 produced a rapid elevation of [Ca2+]i and similarly primed both types of cells for enhanced capacity for O2
− release with phorbol diester. These results suggest that macrophages may have distinct non‐genomic pathways of vitamin D3, which partly explain the absence of immunodeficiency and the disappearance of rickets after treatment with vitamin D3 in the patients.
Two‐dimensional crystals of the Kdp‐ATPase of Escherichia coli
Authors: Iwane Atsuko Hikikoshi , Ikeda Ikuko , Kimura Yoshiaki , Fujiyoshi Yoshinori , Altendorf Karlheinz , Epstein Wolfgang
Published: April 2003

A variant form of the Kdp‐ATPase of Escherichia coli was overproduced to a level approaching 37% of the protein in the inner membrane of this organism. Membranes from overproducing cells were prepared with an inside‐out orientation. Incubation of the membranes on ice for 1–2 weeks in the presence of sodium vanadate resulted in the formation of two‐dimensional crystals of the Kdp‐ATPase. The calculated projection map of the p1 crystal form showed three prominent density peaks at a resolution of 22 Å. This technique is a useful and simple method to obtain low‐resolution structures of membrane proteins.
Specific blockade of slowly activating IsK channels by chromanols — impact on the role of IsK channels in epithelia
Authors: Suessbrich H. , Bleich M. , Ecke D. , Rizzo M. , Waldegger S. , Lang F. , Szabo I. , Lang H.-J. , Kunzelmann K. , Greger R. , Busch A.E.
Published: April 2003

Chromanols, which were recently shown to inhibit cAMP‐mediated Cl− secretion in colon crypts via a blockade of a cAMP‐activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I
sK
channels with an IC50 of 7 μmol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I
sK
inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I
sK
blocker azimilide on cAMP mediated Cl− secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl− secretion. These data suggest that I
sK
protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.
Localization of desmin in cultured astrocytes and its distribution in relation to glial fibrillary acidic (GFA) protein
Authors: Raju T. R. , Dahl D.
Published: April 2003

In dissociated cultures from newborn rat brain, it is shown that astrocytes which are positive with GFA antisera also stain with desmin antisera. However, the localization of these two proteins within the same cell seems to be different as revealed by double immunofluorescence employing both these antisera.
The adrenocortical response to stress from the pre‐hatching period to the adult state in the pigeon: Thalamic participation
Authors: Ramade Fred , Baylé Jean‐Dominique
Published: April 2003

The stress‐induced adrenocortical response was kinetically evaluated for 100 min after ether vapor exposure in late embryos (30 h before hatching) and in 1, 2, 3 and 6‐wk‐old squabs. An‐adult‐pattern of corticosterone profile was seen in intact 6‐wk‐old pigeons, i.e. a polyphasic increase of corticosterone including successive peaks at 12–15, 35, 60 and 90 min after stress application. In 1‐wk‐old squabs, a small adrenocortical response to stress included only a single peak of corticosterone at 12–15 min. The early peak was found again, with the same magnitude, 2 wk after hatching but it was followed by two later and smaller peaks 35 and 60 min after stress. The adult pattern was visible in 3‐wk‐old stressed pigeons, although the magnitude of the response was still smaller than in adult controls. A single, high peak of corticosterone occurred 12–15 min after stress application, without any later peak, in embryos and a similar monophasic response was seen in thalamic lesioned adult birds. Therefore, it can be suggested that the polyphasic reverberating adrenocortical response to stress depends upon relationships between the adrenocorticotrophic hypothalamus and the anterior mediodorsal thalamus. Such thalamic‐hypothalamic interrelations appear to mature during the early post‐hatching weeks.
Lipid composition of gray and white matter in developing rat brain
Authors: Reddy T. Sanjeeva , Rajalakshmi R. , Ramakrishnan C. V.
Published: April 2003

The lipid composition of gray and white matter was studied in the developing rat brain. For this purpose, gray and white matter were separated from the brains of rats from different age groups and were used for the estimation of moisture and lipids. The moisture content of both gray and white matter decreased with age, but the decrease was more significant in the latter. The concentration of different lipids in gray matter reached adult values at 4 weeks of age, whereas the lipids in white matter showed a slower increase even after this period. Similarly, the content of gray matter reached adult values much earlier (by 3 weeks) than white matter, which continued to increase throughout the period of study. Myelin represented 40% of the dry weight of whitematter. The non‐myelin portion of white matter had a different lipid composition than gray matter, white matter, or myelin.
Some electrophysiological properties of developing rat skeletal myotubes grown in serum‐free, chemically defined medium
Authors: Yoles Esther , Bak Asia , Sampson S.R.
Published: April 2003

Serum‐free, chemically defined media have been reported to provide suitable conditions for growth and proliferation of mammalian skeletal muscle, but there is no information regarding the ability of myotubes to develop normal electrophysiological properties in these media. We have recorded transmembrane resting and action potentials from rat skeletal myotubes grown in both serum‐containing (GM) and serum‐free chemically defined (CDM) growth media. Muscle cells in CDM do not develop as high resting Em as their counterparts in conventional growth media. CDM myotubes also had a lower incidence and frequency of spontaneously occurring action potentials. Treatment with ouabain or decrease in temperature of the recording medium reduced resting Em of both GM and CDM cells to the same level. We found that the sensitivity of CDM cells to ouabain was about 10‐fold higher than that of GM cells. An increase in temperature of the recording medium increased Em of GM myotubes but not of CDM myotubes. The change in resting Em in response to a 10‐fold change in extracellular K+‐ion concentration was the same for both groups of cells thus indicating that there was no difference in membrane permeability to K+‐ion. We conclude that the difference in Em can be accounted for largely, if not entirely, by differences in activity or amount of electrogenic Na+‐K+ ATPase.
Ontogeny of glutamine synthetase in rat brain
Authors: Chatterjee D. , Sarkar P. K.
Published: April 2003

The developmental pattern of glutamine synthetase (GS) in rat brain has been studied with respect to that of the cells involved in the synthesis of the enzyme. GS activity is very low until day 13 after birth, rises sharply between days 13 and 15, and slowly thereafter. In contrast, more than half of the total number of protoplasmic astrocytes—the cells involved in synthesis of GS—are formed by day 12, following which the rate of proliferation declines considerably. GS is precociously inducible by cortisol in purified protoplasmic astrocytes, in organ cultures of 6–13 day rat brain and in the brains of cortisoladministered 12 day rats. These results and the temporal coincidence of the period of increase of GS with the onset of the function of adrenal cortex suggest that GS activity in the developing rat brain is under steroidal control.
Chemically defined medium for rat astroglial cells in primary culture
Authors: Weibel M. , Pettmann B. , Daune G. , Labourdette G. , Sensenbrenner M.
Published: April 2003

We have developed a serum‐free defined medium that supports the growth in primary culture of rat astroglial cells. Cells dissociated from cerebral hemispheres of newborn rats were maintained for 4 days in a basal medium (Waymouth's medium) containing 10% fetal calf serum, which was substituted by a serum‐free medium. The basal medium was supplemented with insulin (5 μg/ml) and fatty acid free bovine serum albumin (0.5 mg/ml). Under these conditions the cells proliferate as estimated by cell counts and DNA content; however, growth was less than in Waymouth's medium supplemented with 10% fetal calf serum. In contrast, a very similar morphology was observed between cultures grown in the serum‐free or serum‐containing media. The serum‐free medium allows some maturation of the astroglial cells as shown by the presence of glial fibrillary acidic (GFA) protein, S‐100 protein and glutamine synthetase (GS) activity. The astroglial cells can survive and grow in this chemically defined medium for up to 5 weeks. The ability to culture astroglial cells in such a minimal defined medium should facilitate investigations concerning the effects of growth factors on their proliferation and maturation.
Long term reduction in eight arm maze performance after early exposure to phenobarbital
Authors: Pick Chaim G. , Yanai Joseph
Published: April 2003

Performance in the hippocampal eight arm maze was studied in mice after early exposure to phenobarbital (PhB). since previous studies suggested that these animals suffered neural deficits in the hippocampus. For prenatal exposure pregnant mothers were fed 3 g PhB/kg milled food on gestation days 9–18. Neonates were injected daily with 50 mg PhB/kg. on postnatal days 2–21. After a week of water deprivation, the animals were tested at age 50 days for 5 days preceded by 1 day of habituation. Deficits in eight arm maze performance were demonstrated in early treated mice on every testing day. For example, on day 5 of testing the number of correct entries during the first eight attempts in the prenatally treated group were 12% below control level (P<0.01), the respective reduction in the neonatal group was 10% (P< 0.001). The number of trials needed to enter all arms on day 5 was 27% above control level among prenatally treated mice (P< 0.001), and 13% in neonatally treated mice (P< 0.05). It took prenatal PhB animals twice the time to reach criterion than their controls (P< 0.001) and four times as long for neonatally treated mice (P< 0.001).
Development of neuronal markers in aggregating fetal rat telencephalon cells cultured in the presence of triiodothyronine
Authors: Jørgensen Ole Steen , Honegger Paul
Published: April 2003

The concentrations of the general neuronal markers D2‐protein (N‐CAM), D3‐protein and neuron specific enolase (NSE) in reaggregating cultures of fetal rat telencephalon cells were affected by the presence of 30 nM triiodothyronine in the defined culture medium. The extent of normal developmental changes were enhanced by triiodothyronine, as demonstrated by crossed immunoelectrophoresis. From 13 to 19 days in culture, the concentration of D2‐protein decreased, and the concentrations of both D3‐protein and NSE increased. Nerve growth factor (NGF) was without effect on the development of these general neuronal markers. However, as shown previously both triiodothyronine and NGF increased the activity of choline acetyltransferase, a marker for cholinergic neurons. The results suggest an enhanced overall differentiation of several types of telencephalon neurons in the presence of triiodothyronine, and a specific stimulation of cholinergic telencephalon neurons by NGF.
Development of gaba binding sites in chick embryo optic lobe: Effect of triton X‐100
Authors: Flores Vladimir , Rios Hugo , de Plazas Sara Fiszer
Published: April 2003

The temporal course of the development of GABA receptor sites in chick optic lohe was studied as a parameter of neuronal differentiation in the central nervous system. At 10 days of incubation, specific [3H]GABA binding was of 0.08 pmol/optic lobe and increased 7–8 fold between 12 and 16 days of incubation, reaching at 16 days a value of 0.60 pmol/optic lobe. This coincides with the period of arrival of the retinal fibers to the optic lobe. After this stage, the number of GABA binding sites decreased to a value of 0.35 pmol/optic lobe at hatching. After hatching a new increase appeared which reached at 5 days post‐hatching a value of 0.87 pmol/optic lobe. Scatchard analysis of the saturation binding data obtained at 16 days of incubation and at hatching revealed the presence of two binding sites: one with high affinity and the other with low affinity, while at 12 days of incubation, the earliest stage examined, only the low‐affinity binding site appeared. The high‐affinity binding site for [3H]GABA was inhibited by muscimol, GABA, and bicuculline (ic50: 0.006, 0.002 and 10 μM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X‐100) showed a marked increase in the amount of specific [3H]GABA binding after 16 days of incubation reaching a 3‐fold increase at hatching. These results suggest that endogenous inhibitors of the higher affinity binding site, probably appear during this period.
The development of serotonergic raphespinal projections in Xenopus laevis
Authors: van Mier P. , Joosten H.W.J. , van Rheden R. , ten Donkelaar H. J.
Published: April 2003

The development of serotonin‐immunoreactive neurons in the central nervous system of Xenopus laevis larvae has been studied with special emphasis on the development of the raphe nuclei and raphespinal projections. The first serotonergic neurons were observed in the rostral part of the brain stem at stage 25, only 28 hr after fertilization. By stage 28 some 20 serotonin‐immunoreactive neurons were found in the rostral part of the brain stem, bearing small protrusions on the ventromedial side of the soma. These initial axonal outgrowths reach the rostral part of the spinal cord at stage 32. By stage 35/36 the growth cones of the descending serotonergic axons in the spinal cord have reached the level of the anus (10th to 15th myotome). Up to stage 45 the majority of the descending serotonergic axons was found in the dorsolateral part of the marginal zone of the spinal cord. After stage 45 some serotonergic axons were also found scattered over other parts of the spinal marginal zone. Collateral oranches were first observed in the caudal part of the brain stem at stage 35/36. Later they occurred also in the rostral (stage 43) and caudal (stage 50) spinal cord, usually on fibers in the ventral half of the spinal cord. The number of serotonergic neurons in the central nervous system (brain stem and hypothalamus) increased steadily throughout development until stage 45. After that the total number of serotonergic neurons in the central nervous system increased about two times faster than the number of serotonergic neurons in the raphe nuclei, due to a massive increase of serotonergic neurons in the hypothalamus.

The present study shows that young, just differentiated raphe neurons already contain serotonin. The generation of these neurons appears to take place in the ventricular zone (matrix) of the brain stem between the caudal border of the mesencephalon and the entrance of the nervus octavus. From here these neurons seem to migrate to their final destination. The distribution of serotonin‐immunoreactive neurons in the brain stem suggests that a superior (not described so far in Anura) and an inferior raphe nucleus can be distinguished in Xenopus. A rostrocaudal gradient seems to be present in the production of serotonergic neurons which project to the spinal cord. Spinal projections from the raphe nuclei are particularly extensive from the nucleus raphes inferior and gradually decrease rostralwards. In the rostral part of the nucleus raphes superior almost no neurons projecting to the spinal cord are found.
Hypothalamic dopamine may play a role in inducing prolactin in pituitary cells
Authors: Hagino N. , Inoue Kinji
Published: April 2003

The effects of hypothalamic dopamine on the functional differentiation of prolactin cells was investigated in the developing rat brain. The treatment of pregnant rats with α‐methyl‐p‐tyrosine (tyrosine hydroxylase inhibitor) resulted in a noticeable decrease of dopamine concentration in the hypothalamus at birth and at 5 days of age of offspring. Moreover, treatment with this regimen caused a marked decrease in the population of prolactin cells and in the production of prolactin at birth and at 5 days of age of offspring. It seems, then, that hypothalamic dopaminergic neurons precede the functional differentiation of prolactin cells in the developing brains and that hypothalamic dopamine may play an important role in producing prolactin in the anterior pituitary during development.
Pharmacological evidence that effects of vasotocin on brain maturation are mediated by GABA mechanisms
Authors: Goldstein R. , Rodica Bartok
Published: April 2003

Active sleep, brain weight as well as total lipids and galactolipids were assayed in 8‐day‐old rat pups, after daily administration (between 2 and 8 days of postnatal age) of saline, arginine vasotocin (AVT) (10−7 mg), picrotoxin (5 × 10−5 mg), valproic acid (10−5 and 10−6 mg) or of AVT+picrotoxin and AVT+valproic acid. AVT increases active sleep and decreases the weight, total lipids and the galactolipids of the brain. Although picrotoxin alone was without any apparent effects on maturational parameters, when administered together with AVT, it completely prevented AVT from producing its effects. Valproic acid had AVT‐like effects when administered in a higher dose (10−5 mg), but was without any apparent effects when administered in the lower dose (10−6 mg). However, both doses intensely potentiated the effects of AVT. Since both drugs specifically interfere with the GABAergic neurotransmission, it is concluded that AVT retards the accumulation of brain lipids by activating a GABAergic pathway.
A small subset of cortical astrocytes in culture accumulates glycogen
Authors: Rosenberg Paul A. , Dichter Marc A.
Published: April 2003

We are interested in identifying the target cells for norepinephrine in cerebral cortex and in characterizing the effects of norepinephrine on these target cells. Norepinephrine inhibits the incorporation of tritiated glucose into glycogen in rat cerebral cortex in dissociated cell culture. To identify which cells store glycogen in these cultures we combined glycogen cytochemistry with glial flbrillary acidic protein immunocytochemistry. Using this technique we show that cytochemically detectable glycogen is restricted to a small subset of astrocytes as well as an unidentified cell type which does not contain glial fibrillary acidic protein.

These results demonstrate that only a minority of astrocytes in cortical cultures accumulate glycogen. Therefore cortical astrocytes are differentiated with respect to glycogen accumulation, an important metabolic function. We do not know if glycogen accumulation in astrocytes is a constitutive or facultative property. In either case the subset of astrocytes which accumulates glycogen might be one of the major cellular targets for norepinephrine in cerebral cortex.
Fibronectin and collagens modulate the proliferation and morphology of astroglial cells in culture
Authors: Goetschy Jean‐François , Ulrich Gabrielle , Aunis Dominique , Ciesielski‐Treska Jaroslava
Published: April 2003

The proliferation and morphology of astroglia derived from neonatal rat cortex and cultured in serum‐free medium on either untreated, or fibronectin‐, or collagen I‐, or collagen IV‐treated substrates were investigated using tritiated thymidine autoradiography and immunocytochemical staining of glial fibrillary acidic protein (GFAP) and actin. Modification of culture substratum with fibronectin enhanced the rate of proliferation of astroglial cells and increased the proportion of process‐bearing astroglial cells. The distribution of actin and patterns of adhesion observed were typical for motile cells. Both types of collagen decreased the proportion of astroglial cells undergoing mitosis. Many of the astroglial cells exhibited a flat morphology and displayed prominent stress fibres in the cell body and processes. The data suggest that specific interactions with the substratum modulate the proliferation and morphological behaviour of astroglial cells.
Acquisition of acoustic startle response in relation to growth and thyroid function in rats
Authors: Schneider Bernard F. , Golden Wendy L.
Published: April 2003

Thiourea was administered by gavage to pregnant and lactating rats from gestational day 18 until postnatal day 10, at doses of 100 or 250 mg per day (approximately 350 or 900 mg/kg/day). From the day after birth until postnatal day 14 the neonates were weighed, and beginning on postnatal day 10 acquisition of acoustic startle reflex was assessed. Serum thyroxine and thyrotropin were measured on postnatal day 14. High dose thiourea severely inhibited thyroid function in pups, significantly depressed body growth, and retarded development of the acoustic startle response. At the low dose there was a significant reduction in growth, but no effect on acquisition of acoustic startle reflex. Low dose pups appeared to be functionally euthyroid (free T4 and thyrotropin were not significantly different from control). In conjunction with other studies, these results strongly suggest that, while development of acoustic startle is dependent upon thyroid hormone, it is not readily affected by retarded body growth.
Ontogeny of the dopamine and cyclic adenosine‐3′: 5′ ‐monophosphate‐regulated phosphoprotein (DARPP‐32) in the pre‐ and postnatal mouse central nervous system
Authors: Foster G. A. , Schultzberg M. , Kökfelt T. , Goldstein M. , Hemmings H. C. , Ouimet C. C. , Walaas S. I. , Greengard P.
Published: April 2003

The ontogeny of a dopamine and cyclic adenosine‐3′:5′‐monophosphate‐regulated phosphoprotein with an apparent molecular weight of 32 kilodaltons (DARPP‐32) has been studied in the central nervous system of the prenatal, newborn and adult mouse.

DARPP‐32‐immunoreactive somata were first identified at day 12 of gestation, in the primary olfactory cortex and in the ventrolateral medulla oblongata. On day 14 of gestation, neurons containing DARPP‐32‐like immunoreactivity became apparent in the caudate nucleus, olfactory tubercle, nucleus accumbens, frontoparietal cortex and the ventral medulla oblongata. During the period up to and including birth, the number of cell bodies and fibres in all these areas increased markedly. In addition, DARPP‐32‐positive neurons became visible in the olfactory nucleus, the arcuate nucleus, and DARPP‐32‐positive cells appeared in the choroid plexus of the lateral, third and fourth ventricles. DARPP‐32‐containing fibres could be seen in the median eminence, the ventrolateral thalamus, and in the striatonigral projection, descending in the internal capsule to ramify extensively in the substantia nigra. Only in the cerebellum and suprachiasmatic nucleus did the development of DARPP‐32‐like immunoreactivity occur postnatally.

The development of tyrosine hydroxylase, the rate‐limiting enzyme for catecholamine synthesis, was simultaneously examined. The arrival of the tyrosine hydroxylase‐containing projection to the caudate nucleus, the olfactory tubercle and the nucleus accumbens apparently occurred 1–2 days after the appearance of DARPP‐32‐immunoreactive cells within these regions. In the ventral and ventrolateral medulla oblongata, and the primary olfactory cortex, no tyrosine hydroxylase innervation was seen near the DARPP‐32‐positive neurons at days 12–14. The organization of the DARPP‐32‐containing somata of the caudate nucleus into aggregates of 5–15 neurons was partly paralleled spatially by an increased density of tyrosine hydroxylase‐positive fibres.

Many DARPP‐32‐immunoreactive cells in the immature mouse brain are present by the day of birth, particularly in the areas known to receive a dopaminergic innervation. The development of these presumptive dopaminoceptive DARPP‐32‐containing neurons does not seem to be dependent on the presence, however, of a dopaminergic input, since in all regions examined DARPP‐32‐LI preceded the appearance of tyrosine hydroxylase‐like immunoreactivity by at least 1–2 days. Indeed, the results suggest that the existence of DARPP‐32‐like immunoreactivity in cell bodies and dendrites may be a pre‐requisite for the formation or subsequent stabilization of dopaminergic synapses.
DNA content in neurons of Auerbach's plexus under experimental conditions in adult rats
Authors: Robecchi M. G. Giacobini , Poncino A. , Geuna S. , Giacometti S. , Filogamo G.
Published: April 2003

Some nerve cells of the Auerbach's myenteric plexus of the intestine of the adult rat, which hypertrophied following a surgically induced stenosis, began DNA synthesis unrelated to mitotic division.

The cytophotometric analysis confirmed and quantified the amount of synthesis revealed by autoradiography with tritiated thymidine uptake.

Numerous nerve cells show a DNA content exceeding the diploid level. Only a few of these show twice the diploid content.

The significance of the DNA synthesis is discussed.
GABA agonist promoted formation of low affinity GABA receptors on cerebellar granule cells is restricted to early development
Authors: Belhage B. , Hansen G. H. , Schousboe A. , Meier E.
Published: April 2003

The ability of the GABA receptor agonist 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP) to promote formation of low affinity GABA receptors on cerebellar granule cells was tested using primary cultures of these neurons. Granule cells were exposed to THIP (150 μM) for 6 hr after, respectively, 4, 7, 10 and 14 days in culture. It was found that THIP treatment of 4‐ and 7‐day‐old cultures led to formation of low affinity GABA receptors, whereas such receptors could not be detected after THIP treatment in the older cultures (10 and 14 days) in spite of the fact that these cultured granule cells expressed a high density of high affinity GABA receptors. It is concluded that the ability of THIP to promote formation of low affinity GABA receptors on cerebellar granule cells is restricted to an early developmental period.
Role of glucocorticoids in the chromaffin‐neuron developmental decision
Authors: Anderson David J. , Michelsohn Arie
Published: April 2003

Chromaffin cells and sympathetic neurons develop from a common neural crest‐derived progenitor cell. The developmental fate of this cell differs depending upon whether it migrates to the sympathetic ganglion or to the adrenal gland primordium, suggesting that local environmental signals control its differentiation. Glucocorticoid (GC) is a good candidate for an important adrenal environmental signal. These steroids are known to regulate PNMT, an adrenal‐specific enzyme. However, in vivo observations suggest that the adrenal microenvironment influences the phenotype of sympathoadrenal progenitor cells as early as E14.5, 2 days before PNMT is first expressed by developing chromaffin cells. Using cDNA probes, we find that GC receptor mRNA can be detected in the embryonic adrenal at least one full day before the initial appearance of PNMT mRNA. This observation is compatible with the idea that the apparent early influence of the adrenal microenvironment reflects the action of GC on progenitors which have migrated into this environment. In support of this, we show that similar influences can be exerted by GC on PC12 cells, which contain GC receptor mRNA but do not express or induce PNMT mRNA. Taken together, these data suggest that other factors in addition to the presence of the GC receptor may be necessary for the developmental appearance of PNMT expression.
In vitro evidence for a neurite growth‐promoting activity in trembler mouse serum
Authors: Koenig J. , Hantaz‐Ambroise Dj. , De La Porte S. , Thi N. A. Do , Bourre J. M. , La Chapelle F. , Koenig H. L.
Published: April 2003

Basal lamina components, such as heparan sulfate proteoglycan (HSPG) and laminin play an important role in neuritic outgrowth for CNS and PNS neurons in culture. The mutant mouse ‘Trembler’ is characterized by hypomyelinization and production of an excess of basal lamina layers around Schwann cells in peripheral nerves.

In order to analyse whether or not the serum of the mutant animals contains neurite outgrowthpromoting factors, we cultured rat spinal cord neurons in the presence of Trembler serum. Under these conditions, the outgrowth of neurites was increased approx, 2 times as compared to control serum. Trembler serum induces neuritic outgrowth characterized both by an increase in number of primary neurites emerging from the nerve cell body as well as by an increase in peripheral branching of neurites.

To characterize the factors implicated in this increase we added antibodies directed against HSPG or laminin to the mutant serum. As a result, the increase in neuritic outgrowth was reduced or abolished in both cases.

Trembler effect on neurite growth disappeared when the number of the non‐neuronal cells was reduced, suggesting that the mutant serum did not act directly on neurons but by the intermediary action of non‐neuronal cells.