bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of supercritical carbon dioxide on the diffusion coefficient of phenol in poly(bisphenol A carbonate)
Authors: Shi Chunmei , Roberts George W. , Kiserow Douglas J.
Published: April 2003

For some polymers, the rate of solid‐state polymerization (SSP) is higher with supercritical carbon dioxide (scCO2) as the sweep gas than with atmospheric N2. One explanation for this higher rate is that the diffusion coefficient of the condensate molecule is higher in the CO2‐swollen polymer. To investigate this hypothesis, we measured the diffusion coefficient of phenol in poly(bisphenol A carbonate) (BPA‐PC) by carrying out SSP of this polymer under diffusion‐limited conditions. Under these conditions, the diffusion coefficient of the condensate molecule could be calculated from the profile of the molecular weight versus time. The phenol diffusivity was determined between 135 and 180 °C in the presence of N2 at about 1 bar and in the presence of scCO2 at about 138, 207, and 345 bar. The diffusion coefficient of phenol was up to 200% higher in scCO2 than in N2, depending on the temperature and CO2 pressure. With both N2 and scCO2, the activation energy for phenol diffusion in BPA‐PC was larger than the activation energy for the reaction between hydroxyl and phenyl end groups that occurred during SSP of BPA‐PC. As a result, the overall SSP reaction shifted from diffusion control at low temperatures toward chemical‐reaction control at high temperatures. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1143–1156, 2003
Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast Saccharomyces cerevisiae
Authors: Chen Ching‐Nen , Porubleva Larysa , Shearer Georgia , Svrakic Maja , Holden Lauren G. , Dover James L. , Johnston Mark , Chitnis Parag R. , Kohl Daniel H.
Published: April 2003

Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH‐dependent) is encoded by GRE2 (YOL151w). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI–TOF the proteins in candidate bands on SDS–PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione‐S‐transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae, by our estimate). Copyright © 2003 John Wiley & Sons, Ltd.
Characterization of a protein tyrosine phosphatase (RIP) expressed at a very early stage of differentiation in both mouse erythroleukemia and embryonal carcinoma cells
Authors: Chida Dai , Kume Tsutomu , Mukouyama Yousuke , Tabata Satoshi , Nomura Nobuo , Thomas Matthew L. , Watanabe Toshio , Oishi Michio
Published: April 2003

From our previous studies, several protein tyrosine phosphatases (PTPase) are implicated in the early events leading to in vitro differentiation of both mouse erythroleukemia (MEL) and embryonal carcinoma (F9) cells. Among the PTPases, recent experiments suggest that a new PTPase (RIP) plays a critical role in differentiation processes, particularly at their early stages. We isolated cDNA clones for RIP from a RNA preparation isolated from differentiating MEL cells, and determined the total 7932 bp base sequence for RIP cDNA. The cDNA codes for a putative 269.8 kDa (2450 amino acids) protein with a PTPase catalytic domain. We have demonstrated that the transcripts exist in multiple forms, and among mouse tissues they were found predominantly in kidney and, to a lesser extent, in lung, heart, brain and testis. The RIP gene was mapped between D5Mit90 and D5Mit25 on mouse chromosome 5.
Multi‐step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents
Authors: Cain Kelvin , Inayat-Hussain Salmaan H. , Kokileva Ludmilla , Cohen Gerald M.
Published: April 2003

DNA fragmentation in isolated rat liver nuclei is a Mg2+‐dependent, multi‐step process which is potentiated by Ca2+ and cleaves the DNA into ≥700, 200–300 and 30–50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca2+/Mg2+‐dependent endonuclease(s). We now show that Cd2+, Hg2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N‐ethylmaleimide (NEM) block both Mg2+ and Ca2+/Mg2+‐dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono‐/oligonucleosomes to 30–50, 200–300, ≥700 kbp and finally to intact DNA. NEM and DCI inhibition was blocked by dithiothreitol, and it is proposed that a critical thiol(s) is involved in the DNA cleavage reactions which are a feature of the apoptotic process.
Purification and characterization of leukotriene A4 hydrolase from human epidermis
Authors: Iversen Lars , Kristensen Peter , Nissen Judith B. , Merrick William C. , Kragballe Knud
Published: April 2003

The leukotriene A4 hydrolase is a central enzyme in leukotriene B4, formation. Unlike 5‐lipoxygenase, leukotriene A4 hydrolase activity is present in normal human epidermis, where it is likely to be involved in transcellular leukotriene formation. In this study the leukotriene A4 hydrolase was purified from human epidermis and human cultured keratinocytes and compared with leukotriene A4 hydrolase from human neutrophils. To purify leukotriene A4 hydrolase from human epidermis a new non‐specific affinity chromatography column, with the leukotriene A4 hydrolase inhibitor bestatin coupled to AH‐Sepharose, was introduced. The epidermal leukotriene A4 hydrolase was purified to apparent homogeneity and the molecular weight was determined to be approximately 70,000 Da by SDS‐PAGE. The pI was 5.1–5.4 for the epidermal as well as the keratinocyte and neutrophil leukotriene A4 hydrolase, as determined by chromatofocusing. Only minor differences in the amino acid composition were seen between the three enzyme sources. The optimal pH for the hydrolase activity was 7.5–8.5 for the epidermal and neutrophil leukotriene A4 hydrolases. Finally, it was also shown that the epidermal leukotriene A4 hydrolase undergoes suicide inactivation when transforming leukotriene A4 into leukotriene B4. It was concluded that there is a close resemblance between the epidermal leukotriene A4, hydrolase and the hydrolase found in other cell types. Therefore, the human epidermis may be a good model for the in vivo study of transcellular leukotriene formation.
Internal actin filament dynamics in the presence of vinculin: a dynamic light scattering study
Authors: Götter R. , Goldmann W.H. , Isenberg G.
Published: April 2003

Analyses of dynamic light scattering data by stretched exponential fit show that vinculin has a negligible influence on internal actin filament dynamics and actin bending stiffness which contrasts with our previous observations with talin, another actin and vinculin‐binding protein from focal adhesions. The results here agree with kinetic and rheologic measurements.
Electrogenic Na+ transport by Enterococcus hirae Na+‐ATPase
Authors: Kakinuma Yoshimi , Igarashi Kazuei
Published: April 2003

Energy‐dependent generation of a membrane potential (Δψ) (−45 mV, interior negative) was observed in the F0F1, H+‐ATPase‐defective mutant of Enterococcus hirae. The generation of Δψ was found at high pH (but not at low pH), for which intracellular Na+ was required but not extracellular K+. The Δψ‐generating activity was induced in cells cultured in media containing high concentrations of Na+, and was not observed in the Na+‐ATPase mutants. These results suggest that E. hirae Na+‐ATPase is responsible for the electrogenic sodium pump.
Synaptotagmin I‐ and II‐deficient PC12 cells exhibit calcium‐independent, depolarization‐induced neurotransmitter release from synaptic‐like microvesicles
Authors: Bauerfeind Rudolf , Jelinek Ruth , Huttner Wieland B.
Published: April 2003

Synaptotagmin I‐ and II‐deficient PC12 cells (ShojiKasai et al. [1]) were used to compare the role of this protein in the calcium‐dependent exocytosis of secretory granules and synaptic‐like microvesicles (SLMVs). While neither catecholamine nor protein secretion from secretory granules were altered, the depolarization‐induced acetylcholine release from SLMVs was no longer calcium‐dependent. We propose that within the exocytotic process of SLMVs, there exist two depolarization‐induced steps. One is calcium‐dependent and no longer present in synaptotagmin I‐ and II‐deficient cells. The other is induced by depolarization, does not require calcium, and suffices to trigger neurotransmitter release from SLMVs in synaptotagmin I‐ and II‐deficient PC12 cells.
Identification of a phosphatidylinositol‐4,5‐bisphosphate‐binding domain in the N‐terminal region of ezrin
Authors: Niggli Verena , Andréoli Christophe , Roy Christian , Mangeat Paul
Published: April 2003

Purified human recombinant ezrin cosediments with large liposomes containing phosphatidylserine (PS). This interaction is optimal at low ionic strength. At physiological ionic strength (130 mM KCl) ezrin interacts strongly with liposomes containing ≥5% phosphatidylinositol‐4,5‐bisphosphate (PIP2), the residual being phosphatidylcholine (PC). When PIP2 is replaced by phosphatidylinositol‐4‐monophosphate (PIP), phosphatidylinositol (PI) or PS, the interaction is markedly reduced. Furthermore we show, that a purified N‐terminal glutathione S‐transferase (GST) fusion protein of ezrin (1–309) still has retained the capacity to interact with PIP2‐containing liposomes, whereas a C‐terminal fusion protein (310–586) has lost this ability.
The osmo‐inducible gpdl+ gene is a target of the signaling pathway involving Wis1 MAP‐kinase kinase in fission yeast
Authors: Aiba Hirofumi , Yamada Hisami , Ohmiya Ryusuke , Mizuno Takeshi
Published: April 2003

The gpd1
+ gene of Schizosaccharomyces pombe encodes an isozyme of NADH‐dependent glycerol‐3‐phosphate dehydrogenases that is involved in glycerol synthesis, whose expression is induced upon an upshift of the medium osmolarity. We provide evidence that this osmotic induction of gpd1
+ in S. pombe is under the control of a MAP‐signaling pathway involving the wis1
+ gene‐product, which is a homologue of MAP‐kinase kinases. The results suggested that the gpd1
+ gene is a downstream target of the osmosensing signaling that is transmitted through Wis1, thereby defects of either of these genes result in the similar phenotype, namely, osmosensitive for growth, because of the failure in accumulation of the intracellular osmoprotectant, glycerol.
Amino‐terminal sequence determinants for substrate recognition by platelet‐derived growth factor receptor tyrosine kinase
Authors: Chan Perry M. , Keller Paul R. , Connors Richard W. , Leopold Wilbur R. , Miller W.Todd
Published: April 2003

The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determinants for platelet‐derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N‐terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti‐phosphotyrosine antibody. Further separation of bound phosphopeptides with reverse‐phase HPLC led to the identification of three optimal substrates for PDGFR: Ala‐Ala‐‐Tyr‐Ala‐Ala‐Arg‐Arg‐Gly, Ala‐Ala‐‐Tyr‐Ala‐Ala‐Arg‐Arg‐Gly and Ala‐Ala‐‐Tyr‐Ala‐Ala‐Arg‐Arg‐Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.
Expression of monocyte chemoattractant protein‐1 mRNA and protein in cultured human thyrocytes
Authors: Kasai Kikuo , Banba Nobuyuki , Motohashi Satoshi , Hattori Yoshiyuki , Manaka Kenichi , Shimoda Shin-Ichi
Published: April 2003

Monocytes as well as lymphocytes infiltrate in the stroma of thyroid tissue in autoimmune and destructive thyroiditis. Monocyte chemoattractant protein‐1 (MCP‐1) is a cytokine that attracts T‐lymphocytes as well as monocytes. Using human thyrocytes in primary cultures, we show that expression of MCP‐1 mRNA and protein is remarkably stimulated by both interleukin‐1 (IL‐1) and tumor necrosis factor‐α (TNF‐α), and also that interferon‐γ (IFN‐γ) by itself is a weak stimulant but has a synergistic activity with either IL‐1 or TNF‐α. The finding indicates that MCP‐1 can be produced by thyrocytes themselves, suggesting a possible role of thyrocytes on accumulation of monocytes and T‐lymphocytes to the tissue from the blood in autoimmune and destructive thyroiditis.
Inhibition of coagulation factors by recombinant barley serpin BSZx
Authors: Dahl Søren W. , Rasmussen Søren K. , Petersen Lars C. , Hejgaard Jørn
Published: April 2003

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k
ass) of 4.5 × 103−1.3 × 105 M−1 s−1 at 22°C. Only factor Xa tunred a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k
ass = 1−2 × 102 M−1 s−1) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
Expression of photoreceptor cyclic nucleotide‐gated cation channel α subunit (CNGCα) in the liver and skeletal muscle
Authors: Feng Li , Subbaraya Iswari , Yamamoto Nair , Baehr Wolfgang , Kraus-Friedmann Naomi
Published: April 2003

Glucagon and β‐adrenergic agents increase cAMP levels and stimulate Ca2+ influx in liver cells. There is no consensus as to the mechanism by which these hormones stimulate the influx of Ca2+. Using mouse retinal rod CNGCα cDNA probes, we cloned rat liver and skeletal muscle, and human hepatic CNGCα subunit sequences showing 97–100% identity with the human rod channel. In order to assess channel activity, the effect of cyclic nucleotides on free intracellular Ca2+ levels of isolated hepatocytes was measured. Dibutyryl‐cAMP was more effective in increasing free Ca2+ levels than dibutyryl‐cGMP. These data indicate that the CNGCα subunit is expressed in the both liver and skeletal muscle possibly mediating hormonal effects on ion fluxes.
Cathepsin D is involved in the clearance of Alzheimer's β‐amyloid protein
Authors: Hamazaki Hideaki
Published: April 2003

The cerebral deposition of 39–42 residue amyloid β‐protein (Aβ) is a histopathological characteristic of Alzheimer's disease. The present study is aimed at finding proteinases responsible for the intracellular clearance of Aβ. The Aβ‐degrading proteinase was purified from rat brain. Amino‐terminal sequence analysis indicated the Aβ‐degrading proteinase was cathepsin D. Purified cathepsin D hydrolyzed Aβ between Phe19 and Phe20. Cathepsin D is likely to be involved in the intracellular clearance of aggregatable Aβ, since Aβ fragments with Phe20 at the amino‐terminus have been reported to be secreted from several lines of cultured cells.
Phosphorylation of the eIF4E‐binding protein PHAS‐I after exposure of PC12 cells to EGF and NGF
Authors: Kleijn Miranda , Korthout Marsha M.R. , Voorma Harry O. , Thomas Adri A.M.
Published: April 2003

PHAS‐I or the eIF4E‐binding protein 1 regulates the cap‐binding activity of eIF4E by sequestering eIF4E. Binding of eIF4E to PHAS‐I is regulated by phosphorylation of PHAS‐I. PC12 cells were used to study the signal transduction pathway leading to phosphorylation of PHAS‐I. Both EGF and NGF induced phosphorylation of PHAS‐I. Wortmannin, a PI‐3 kinase inhibitor, staurosporine, a PKC inhibitor, and rapamycin, a FRAP inhibitor all blocked the phosphorylation of PHAS‐I. Of the three inhibitors, only wortmannin was able to inhibit MAPK phosphorylation. This excludes a role for MAPK in NGF‐ and EGF‐induced PHAS‐I phosphorylation in PC12 cells. Apparently, PHAS‐I was phosphorylated in a PI‐3 kinase‐, PKC‐, and FRAP‐dependent manner after EGF or NGF stimulation. Only PI‐3 kinase and FRAP are involved in the regulation of the basal level of PHAS‐I phosphorylation.
Bicarbonate/CO2 induces rapid activation of phospholipase A2 and renders boar spermatozoa capable of undergoing acrosomal exocytosis in response to progesterone
Authors: Roldan E.R.S. , Vazquez J.M.
Published: April 2003

We tested whether capacitation, a phenomenon that renders spermatozoa capable of undergoing acrosomal exocytosis, may be completed rapidly after a short exposure to bicarbonate/CO2. We found that, in the presence of Ca2+, a 10‐min exposure of boar spermatozoa to bicarbonate led to a partial activation of phospholipase A2, primed spermatozoa for a major subsequent activation of this enzyme upon stimulation with progesterone and furthermore rendered spermatozoa capable of undergoing exocytosis in response to this steroid. These results suggest that capacitation may be completed in a relatively short period of time and open up new possibilities for unravelling molecular mechanisms underlying this process.
DNA topoisomerase I changes the mode of interaction between camptothecin drugs and DNA as probed by UV‐resonance Raman spectroscopy
Authors: Feofanov Alexei V. , Baranov Alexandre V. , Fleury Fabrice , Riou Jean-François , Nabiev Igor R. , Manfait Michel
Published: April 2003

Pronounced differences of interactions of camptothecin (CPT) and its derivative 7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloycamptothecin (CPT11), inhibitors of DNA topoisomerase I, with oligonucleotides were found using UV resonance Raman spectroscopy. 30‐mer oligonucleotides were derived from the sequences of the topoisomerase I‐induced and CPT‐enhanced cleavage sites in SV40 DNA. CPT induces well‐defined alterations of the oligo structure, whereas CPT11 interacts with oligonucleotides more weakly and in another manner than CPT. Formation of cleavable ternary complexes between CPT11, topoisomerase I and oligonucleotides causes CPT11 to interact with oligonucleotides in the same fashion as was found for its parent compound CPT, and enhances this interaction as compared to CPT‐oligonucleotide complexes. The data present evidence of molecular interactions of CPT11 with both other partners (topoisomerase I and oligonucleotide) of the ternary cleavable complex at the oligonucleotide‐enzyme interface.
AZT‐induced hypermethylation of human thymidine kinase gene in the absence of total DNA hypermethylation
Authors: Lucarelli Marco , Palitti Franco , Carotti Daniela , Cianfriglia Maurizio , Signoretti Claudia , Bozzi Argante , Strom Roberto
Published: April 2003

Genome‐wide DNA hypermethylation induced by 3′‐azido‐3′‐deoxythymidine (AZT) has been suggested to be involved in the development of AZT resistance. We used a CD4 T‐lymphoblastoid CEM line and its AZT‐resistant MT500 variant with reduced thymidine kinase activity. Evaluation of total DNA methylation, after AZT treatment, failed to show an increase in the 5‐methylcytosine level in both parental and AZT‐resistant cells. The effect was instead observed at a more specific gene level, on the three HpaII sites present in exon 1 of the human thymidine kinase gene. These results suggest that AZT treatment can induce site‐specific hypermethylation, even in the absence of a more general DNA hypermethylating effect.
NAD+‐dependent internalization of the transmembrane glycoprotein CD38 in human Namalwa B cells
Authors: Zocchi Elena , Franco Luisa , Guida Lucrezia , Piccini Daniele , Tacchetti Carlo , De Flora Antonio
Published: April 2003

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD+ (cyclase) and the hydrolysis (hydrolase) of the calcium‐mobilizing metabolite cyclic ADP‐ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium‐releasing activity of its intermediate product cADPR. Incubation of CD38+ human Namalwa B cells with external NAD+ elicited extensive membrane down‐regulation of CD38 and its internalization in non‐clathrin‐coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD+‐dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.
Binding and uptake of putative neurotransmitters in mutant mouse cerebellum and cerebellar reaggregat
Authors: Collins Janet M. , Seeds Nicholas W.
Published: April 2003

γ‐Amino butyric acid (GABA) appears to be the inhibitory transmitter of various cerebellar interneurons, while glutamate has been suggested as the excitatory transmitter of granule cells. In this study we have determined the developmental profile from day 2 to day 60 for the Naa‐independent (presumably post‐synaptic receptor) binding of [3H]GABA (Kd = 34 nM) and [3H]kainic acid, a glutamate analog (Kd = 53 nM. Both binding activities increase steadily to adult levels with kainic acid achieving this more rapidly. Cerebellar cells (7 day) cultured as reaggregates for 14 days showed an increase in GABA binding and glutamate uptake compared to the starting tissue but did not achieve the levels seen in vivo, while kainic acid binding and GABA uptake remained low. Binding and uptake measurements were performed in neurological mutants. Agranular mutants, weaver, reeler and staggerer demonstrated as expected, marked decreases in total GABA binding and glutamate uptake into synaptosomes, and to a lesser extent GABA uptake. In addition total kainate binding was depressed. In comparison, Purkinje cell degeneration mutants showed only a small decrease in kainic acid binding. These results suggest that kainic acid binding does not identify the post‐synaptic glutamate receptor.
Resistance to barbiturate is changed by developmental alteration of dopamine receptor sensitivity
Authors: Yanai Joseph , Fishman Rachelle H.B.
Published: April 2003

Adult rats whose dopamine receptor number was reduced via prenatal haloperidol exposure were less resistant than controls to pentobarbital narcosis. This altered resistance was functional (CNS) and not metabolic, as shown by pentobarbital brain levels. These results are consistent with our previous experiments, indicating that changes in the number of dopamine receptors may underlie relative susceptibility to barbiturates.
Sex difference in dendritic development of the sexually dimorphic nucleus of the preoptic area in the rat
Authors: Hammer Ronald P. , Jacobson Carol D.
Published: April 2003

Sex differences in the growth and dendritic development of neurons in the sexually dimorphic nucleus of the preoptic area were examined with quantitative Golgi techniques during early postnatal life in rats. Neuronal size and dendritic extent were found to increase more in males than in females during the first 10 postnatal days, while the numbers of primary and terminal dendrites were similar in the two sexes. The onset of greater dendritic growth in males occurs just after the volume of the nucleus begins to exhibit sexual dimorphism, between 24 and 26 days after fertilization. Growth of dendrites in this region may be related to the presence of sex hormones during the critical period of sexual brain differentiation.
Phenylethanolamine‐N‐methyltransferase‐immunoreactive cells in developing rat superior cervical ganglion and the effect of hydrocortisone on their number
Authors: Päivärinta H. , Soinila S. , Eränkö O. , Joh T.H.
Published: April 2003

Rats were subcutaneously injected with hydrocortisone acetate. Control rats were similarly injected with saline. PNMT‐immunoreactive cells in the superior cervical ganglia were immunohistochemically demonstrated, and the cell densities (number/mm3) of the PNMT‐immunoreactive cells were counted.

PNMT‐immunoreactive cells were detected in ganglia of all age groups of the saline‐treated control rats. With increasing age, there was a tendency to cluster formation. The number of the PNMT immunoreactive cells dramatically increased if hydrocortisone administration was initiated during the first two postnatal weeks. Hydrocortisone also caused appearance of numerous PNMT‐immunoreactive fibres, which were not seen in controls. The cell density of the PNMT‐immunoreactive cells in the hydrocortisone‐treated rats was still increased, although less, if the hydrocortisone injections were started during the third postnatal week, as compared with the saline‐injected control rats of the same age.

After discontinuation of the first, early postnatal hydrocortisone treatment on days 2–6, the number of the PNMT‐immunoreactive cells decreased during the following 2 weeks, but a second treatment with hydrocortisone daily for 7 days initiated as late as on postnatal day 63 caused a new significant increase in cell density and the appearance of immunoreactive fibres from them.

It is concluded that PNMT‐immunoreactive cells are present in the normal rat superior cervical ganglion during the whole postnatal developmental period. Their number can be increased with hydro‐cortisone, as assessed on the basis of counted cell densities, during the first three postnatal weeks. Early postnatal hydrocortisone treatment preserves some cells in a stage in which they, on second exposure to glucocorticoids, can express PNMT immunoreactivity and increase in cell density.
Neurofilament immunoreactivity in developing rat autonomic and sensory ganglia
Authors: Ayer‐Lelievre C. , Dahl D. , Björklund H. , Seiger Å.
Published: April 2003

Immunoreactivity to neurofilament (NF) antiserum appears early in the development of both the central and peripheral nervous systems of the rat fetus. In 10 somite embryos, positive cell bodies are present in the ventromedial part of anterior rhombencephalic and mesencephalic neural tube. From there the appearance of immunoreactivity spreads cranially to the prosencephalic anlage before closure of the anterior neuropore and caudally following the sequence of neural tube closure. Immunoreactivity increases rapidly in axon bundles of central and peripheral systems, but in immature cell bodies of sensory ganglia the NF material only forms a ring around the nucleus. At 16 days of gestation, some cell bodies are progressively loaded with NF‐immunoreactive material as a thick perinuclear network first and then in more excentrically located aggregates. This category of neurons is mainly observed in the distal part of the trigeminal ganglion, in petrous and nodose ganglia and in cervical dorsal root ganglia. In adult ganglia large cell bodies and some small ones present high NF immunoreactivity. In autonomic cell bodies (in superior cervical ganglion and in parasympathetic cranial ganglia) the immunoreactive material only forms a perinuclear ring slowly transformed into a loose perinuciear meshwork at the end of gestation. Intensely reactive nerve fibers are observed in cranial sensory as well as in sympathetic and parasympathetic ganglia and nerves. No positive cell bodies and only a few NF‐immunoreactive nerves are observed in the carotid bodies. The NF immunoreactivity is better visualized on sections of fresh frozen material, treated with acetone, than in fixed specimens.

These results are compared to previous observations reported for other species and for developing dorsal root ganglia. This immunostaining may be used to detect differentiation of peripheral sensory and autonomic neurons under experimental conditions. The uneven distribution of NF immunoreactivity in sensory neurons from stage 16 days of gestation as specific for precise subpopulations of neurons is discussed.
Postnatal development of γ‐GT activity in rat brain microvessels corresponds to capillary growth and differentiation
Authors: Santoso A. W. Budi , Bär Th.
Published: April 2003

It has been demonstrated histochemically that endothelial cells of the cerebral capillaries show a high activity in γ‐glutamyl transpeptidase (γ‐GT), an enzyme which takes part in the transfer of large neutral amino acids across the blood‐brain barrier. Reports of the disappearance of enzyme activity in endothelial cells (EC) grown in culture12 suggest that the presence of astroglial cells (AG) is required for the expression of γ‐GT in these cells. The present study deals with the developmental changes in γ‐GT activity in capillaries of rat cerebral cortex during ontogenesis (i.e. on days 2, 7, 11, 14, 21 and 60 after birth).

γ‐GT activity is determined by measuring the enzyme kinetics of the histochemical reaction on isolated brain capillaries using a flying spot microscope densitometer. Enzyme activity is expressed as an increase in relative optical density (at 500 nm) in arbitrary units/min/μm2 during the 2 min immediately after initiating incubation and corresponds to the γ‐GT active area (in μm2) of the capillary segment. During early postnatal development, a biphasic change of γ‐GT activity in capillaries of rat cerebral cortex is observed. The first phase (i.e. the postnatal period between the 2nd and 12th day) is characterized by a significant decrease in γ‐GT activity, which coincides with the onset of the rapid mitotic proliferation of cortical endothelial cells. During the second phase (i.e. the postnatal period between the 12th and 21st day), a fast increase in the γ‐GT activity can be measured. Then enzyme activity reaches the adult level between the 21st and 60th postnatal day.

With regard to changes in capillary length density and in astroglial proliferation the results correspond to the differentiation and maturation of the blood‐brain barrier and confirm evidence stressing the important role of astroglial cells for the expression of γ‐GT activity.
The role of neuron death in the development of the gender difference in the number of neurons in the rat superior cervical ganglion
Authors: Wright L. L. , Smolen A. J.
Published: April 2003

Adult male rats have more neurons in their superior cervical ganglia than do adult females. This difference arises over the first two postnatal weeks and is apparently related to perinatal levels of circulating testosterone.

Exposure of neonatal rats to testosterone or estradiol during the first postnatal weeks results in an increase in the number of neurons in the superior cervical ganglion seen at 15, 30 or 60 days postnatally. The present studies were undertaken to determine whether this observed increase in neurons is due to an increase in neuronal proliferation or to a decrease in neuronal death. Results of autoradiographic studies show no evidence of enhanced neuronal proliferation following postnatal exposure to estradiol, but do show an increased survival of a prenatally labeled population of cells.

Counts of degenerating cells in the superior cervical ganglion show that during the peak period of normal neuronal degeneration, on postnatal day 5, 17‐β‐estradiol or testosterone propionate treated animals have significantly fewer degenerating superior cervical ganglion cells than do vehicle‐injected littermate controls. In addition, vehicle‐injected females have more degenerating cells on day 5 than do vehicle‐injected males. Taken together these results provide strong evidence that the increase in superior cervical ganglion neurons seen after neonatal exposure to estradiol results from a reduction in developmental neuron death.
Intraocular herpes simplex virus injection in neonatal rats induces sympathetic nerve cell destruction: Effect of nerve growth factor
Authors: Aloe Luigi
Published: April 2003

The effect of herpes simplex virus (HSV) injection on the sympathetic nerve system of newborn rats was studied at structural, ultrastructural and immunohistochemical levels. It was found that HSV injected into the anterior eye chamber is retrogradely transported and reaches the nerve cell bodies of the ipsilateral superior cervical ganglion (SCG) after 18–24 hr, causing complete cell destruction within 3–4 days. In subsequent days, nerve cells of the contralateral SCG, spinal sensory ganglia, chromaffin cells and brain cells also become infected and are eventually killed by the virus. Pretreatment with nerve growth factor (NGF) produces an initial protection from viral cell destruction, but does not block the final, lethal effect of the virus. These investigations demonstrate that sympathetic nerve cell destruction can be induced in newborn rodents by HSV and that NGF treatment renders the cells, for a time‐limited period, more resistant to the virus.
Effects of gonadal steroids on nerve growth factor receptors in sympathetic and sensory ganglia of neonatal rats
Authors: Wright L. L. , Marchetti D. , Perez‐Polo J. R.
Published: April 2003

The numbers of neurons in the rat superior cervical sympathetic ganglion (SCG) differ in males and females, with the males having 30% more SCG neurons than females at 60 days of age. This sex difference arises during the early postnatal period, when testosterone administration increases the numbers of neurons and alters the nerve growth factor (NGF) content of the rat SCG. In contrast, there is no gender difference in number of neurons in the L1 dorsal root ganglion.

In both males and females, the amount of NGF bound per ganglion increased between postnatal days 5 and 15 (P5 and P15) in both dorsal root ganglia (DRGs) and the SCG. There is also a gender difference in NGF binding: SCGs and DRGs of female rats at both P5 and P15 bind more NGF per ganglion than do those of males. This effect was more marked in DRGs than in the SCG. Treatment of neonatal females with testosterone reduced NGF binding in both SCGs and DRGs to levels comparable to males at P5, and in DRGs at P15. In contrast, treatment of males with testosterone from birth resulted in a 2–3 fold increase of NGF binding in both SCGs and DRGs as compared to controls at P15. At P15, testosterone treatment of females increased NGF binding in the SCG. Males and females had opposing responses to neonatal exposure to estradiol. Treatment with estradiol from birth increased NGF binding in SCGs and DRGs of females, but had no effect on NGF binding of SCGs, and reduced NGF binding in DRGs of males.

These data suggest that there are more NGF receptors in sensory and sympathetic ganglia in developing females as compared to males, and that neonatal exposure to gonadal steroids affects the expression of NGF receptors.
Cytoarchitecture in cultured rat neocortex explants
Authors: de Jong B. M. , Ruijter J. M. , Romijn H. J.
Published: April 2003

Neocortex explants obtained from 6‐day‐old rat pups and cultured in a serum‐free medium from 5 hr to 13 days in vitro (DIV) show preservation of cytoarchitectural characteristics. Major changes in the size of the explants and their layers occur during the first 2 DIV. A radial arrangement of neurons within layer 2‐3‐4, which becomes apparent between 2 and 10 DIV, suggests an advance in maturation in culture. In contrast to the situation in vivo, a distinct layer 4 cannot be consistently identified. During the first 2 DIV, a transposition of cells into the pial direction can be seen. Individual degenerated cells are spotted especially in layer 5. The presence of these cells amidst healthy neurons suggests that their death has a functional cause. Neurons at the ventricular border of layer 6 become relatively large in comparison with other neurons. Within the cultured tissue there is a marked increase in GFA reactivity compared to the situation in vivo. The described results clearly indicate that in these cultured expiants, both similarities and differences are of interest for studies on the formation of neuronal circuitry within the cerebral cortex.
Histochemical localization of Ca2+, Mg2+‐ATPase of the rat cerebellar cortex during postnatal development
Authors: Yoshioka Takafumi , Tanaka Osamu
Published: April 2003

In order to investigate the membrane activities underlying development of neural cells, a histochemical localization of Ca2+‐ATPase, Mg2+‐ATPase and alkaline phosphatase (AlPase) activities in the rat cerebellar cortex during postnatal development was carried out. In the developing cerebellar cortex, ATPase activity was mainly associated with the plasma membranes of Purkinje and granular cells. This activity appeared in the immature Purkinje cells at birth and was proportionally increased throughout postnatal development. It was observed that the ATPase activity of migratory granular cells during a critical period from 3 and 15 postnatal days was increased in a funicular pattern in the developing cerebellar cortex. Conversely, peak AlPase activity in the developing cerebellar cortex was localized in the proliferative external granular cells until 7 postnatal days. Apparently, these phosphatase activities were not present in Bergmann glial fibers during the course of granular cell migration. The present findings were taken to indicate that neuronal cells in the cerebellar cortex have acquired a membrane‐bound ATPase which can participate in Ca2+ transport or ATP metabolism during the course of early postnatal development.
Neuronal localization of the nerve growth factor precursor‐like immunoreactivity in the rat brain
Authors: Senut Marie‐Claude , Lamour Yvon , Lee James , Brachet Philippe , Dicou Eleni
Published: April 2003

The distribution of the nerve growth factor precursor(proNGF)‐like immunoreactivity was examined in the adult rat brain with affinity‐purified antisera directed against synthetic peptides that reproduce sequences of the precursor protein. Immunoreactivity was localized in defined areas of the neocortex. hippocampal formation, thalamus/hypothalamus, striatum, olfactory bulb, pons and spinal cord, which are regions previously reported to contain NGF mRNA. Interestingly, immunoreactivity was also observed in the septum and diagonal band of Broca known to contain very low NGF mRNA levels. Using immunohistochemical methods combined with the retrograde transport of a protein gold complex we demonstrate that proNGF‐like immunoreactivity is localized within neuronal cell bodies, in the cortex, hippocampus and septum. These results suggest that the immunological approach may not only identify NGF‐synthesizing cells, but also cells which may accumulate proNGF or some cleavage products by an uptake mechanism.
Pre‐ and post‐synaptic cholinergic dysfunction in aged rodent brain regions: New findings and an interpretative review
Authors: Sherman Kathleen A. , Friedman Eitan
Published: April 2003

Age‐related impairment of dynamic aspects of central cholinergic neurotransmission has been indicated by many studies of aged rodents, but the regional distribution of cholinergic deficits and the relative contribution of presynaptic hypofunction and reduced acetylcholine release, loss of synaptic integrity or loss of muscarinic receptors remains unclear. This study therefore compared choline acetyltransferase activity (as a structural marker) and sodium‐dependent high affinity choline uptake (which reflects both ongoing cholinergic neuronal activity and structural integrity) in the hippocampus, cortex and striatum of male C57BL mice at 3–4, 10–12 or 28–32 months of age. To evaluate the relationship of changes in muscarinic receptors to presynaptic alterations, binding of the antagonist 3H‐quinuclidinyl benzilate was compared in membranes prepared from each of these brain regions. High affinity choline uptake was significantly reduced in all three brain regions by 28–32 months of age. This trend was already evident by 10–12 months of age, especially in hippocampus and cortex. By contrast, choline acetyltransferase activity was unchanged in striatum and actually increased in hippocampus and cortex of aged mice. Muscarinic binding was reduced significantly only in striatum and this effect was significant by 10–12 months of age. This decrease in antagonist binding was accompanied by a small but significant reduction in the relative proportion of high affinity agonist sites as defined by carbachol displacement.

The impairment of high affinity choline uptake in the absence of a parallel reduction of choline acetyltransferase activity suggests a decline of ongoing cholinergic activity rather than loss of terminal integrity as the basis of presynaptic deficits in aging. This functional decline may be exacerbated by reduction of muscarinic receptors in striatum. Despite considerable literature support for the hypothesis that cholinergic mechanisms are impaired with age, several controversies leave important issues unresolved. Therefore, the present results are discussed in the context of a critical review with emphasis on dynamic properties of presynaptic function which require analysis in experimental animal models. The impact of normal aging on brain cholinergic systems is distinguished from the neurodegenerative changes in Alzheimer disease in that presynaptic function is compromised with a relative preservation of the integrity of innervation. Nonetheless, in addition to a role in the behavioral changes associated with normal aging, age‐related cholinergic hypoactivity may contribute to the vulnerability of brain cholinergic neurons to degenerative insult and alter the efficacy of drug therapies for this age‐dependent disease.
Persistent protein damage despite reduced oxygen radical formation in the aging rat brain
Authors: LeBel Carl P. , Bondy Stephen C.
Published: April 2003

The relation between cerebral oxygen radicals and the aging process was investigated in crude synaptosomal (P2) fractions from rats. The rate of formation of oxygen radicals was measured using the probe 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA), which is de‐esterified and subsequently oxidized by oxygen radicals to a fluorescent product 2′,7′‐dichlorofluorescein (DCF). There was a significant agedependent decrease in the formation rate of oxygen radicals, observed by decreased formation of DCF. No difference in oxygen radical formation was apparent between age groups following an in vitro challenge with an ascorbate/FeSO4 mixture. This age‐dependent decrease in cerebral oxygen radical generation coincided with age‐dependent increases in Superoxide dismutase. No age‐related alterations in lipid order in either the hydrophilic or lipophilic membrane regions were observed using fluorescence polarization analysis. Age‐dependent losses in cerebral P2 tryptophan fluorescence (a measure of protein degradation), and increased liberation of [14C]protein fragments into the acid‐soluble fraction (a measure of overall proteolytic activity) were observed. Results suggest that aging does not proceed as a result of elevated rates of generation of oxygen radicals, a finding that does not support the proposed free radical theory of aging. The observed age‐dependent decrease in the formation of oxygen radicals does not effect membrane lipid order. These findings implicate modifications in proteins and activated protein catabolic pathways as major contributing factors in the normal physiological process of senescence.
Effect of chronic exposure to high magnesium on neuron survival in long‐term neocortical explants of neonatal rats in vitro
Authors: Baker R.E. , Ruijter J.M. , Bingmann D.
Published: April 2003

In order to assess the effect of elevated magnesium, neuronal morphology and physiology was studied in chronically cultured organotypic neonatal rat occipital neocortex. Explants grown in 10 mM magnesium were found to experience an approximate 30% cell loss (as shown by cell count and DNA‐protein analysis), while 12.5 and 15 mM magnesium showed ca. 47 and 60% cell losses, respectively. Intracellular recording from 10 mM magnesium explants revealed that measurable postsynaptic potentials and action potentials could occur, apparently depending on the type of cell examined. All postsynaptic activities ceased in 12.5 mM magnesium cultures, though action potentials could be elicited by current stimulation. The effects of known depolarizing agents, viz. potassium and N‐methyl‐D‐aspartate, on 12.5 mM magnesium‐grown explants were also examined. Explants grown in the presence of 12.5 mM magnesium plus 10 mM potassium showed a dramatic increase in the loss of neurons. The simultaneous addition of 6,7‐dinitro‐quinoxaline‐2,3‐dione showed this to be due to an increase in non‐N‐methyl‐D‐aspartate mediated cell death in response to glutamate release brought about by the depolarizing effects of the potassium. The addition of 10 μM N‐methyl‐D‐aspartate to 12.5 mM magnesium‐grown cultures, on the other hand, improved cell survival to control levels. The mechanism of this reciprocal neuroprotective effect of N‐methyl‐D‐aspartate against magnesium has yet to be elucidated. We conclude that these findings are consistent with regard to the opposing actions of N‐methyl‐D‐aspartate and magnesium on calcium influx and various metabolic processes within the explants.
Distribution of taurine‐like immunoreactivity in cerebellum of kittens from taurine‐supplemented and taurine‐deficient mothers
Authors: Lu Peimin , Schuller‐Levis Georgia , Sturman John A.
Published: April 2003

Using an antibody prepared against taurine conjugated to bovine serum albumin with glutaraldehyde, the distribution of taurine in cerebellum of newborn and 8‐week‐old kittens from mothers fed 0, 0.02, 0.05, or 1% dietary taurine has been determined. In general, taurine‐like immunoreactivity was greater in kittens from mothers fed the greatest amounts of taurine, as was the total cerebellar taurine concentration. The most notable feature in newborn kitten cerebellum was a dense band of staining in the inner molecular layer adjacent to the Purkinje cell layer, which corresponds to the short Purkinje cell dendrites. In cerebellum of 8‐week‐old kittens, taurine‐like immunoreactivity was present in Purkinje cells and their dendrites, most granule cells, and a few interneurons in the molecular layer of the 0.02, 0.05, and 1% groups. The cerebellum of the 0% group was distinctive in that virtually no neurons were reactive, appearing as ‘ghosts’ against the background, and both white matter and the granule cell layer contained large numbers of reactive astrocytes. The presence of such large numbers of reactive astrocytes and the immunoglobulin within the brain suggests an impairment of the blood‐brain barrier in such taurine‐deficient kittens.