bluuebunny/crossref_metadata_2025 huggingface dataset:
Search for virus nucleic acid sequences in postmortem human brain tissue using in situ hybridization technology with cloned probes: Some solutions and results on progressive multifocal leukoencephalopathy and subacute sclerosing panencephalitis tissue
Authors: Shapshak P. , Tourtellotte W.W. , Wolman M. , Verity N. , Verity M.A. , Schmid P. , Syndulko K. , Bedows E. , Boostanfar R. , Darvish M. , Nakamura S. , Tomiyasu U. , Steiner R.C. , Hawkins S. , Hoffman D. , Adhami F. , Martinez S.
Published: January 2005

In order to obtain a useful and readily applicable in situ hybridization (ISH) protocol for progressive central nervous system (CNS) diseases of unknown etiology that are possibly due to persistent viral infection, known and well described diseases were studied, namely, progressive multifocal leukoencephalopathy (PML) and subacute sclerosing panencephalitis (SSPE). The procedures described were validated by confirming results obtained by other investigators using histology, immunocytochemistry, electron microscopy, and ISH. A number of frequently encountered problems of tissue preparation are addressed as well as techniques to reduce autoradiography exposure times. A multi‐staged specific, sensitive, reliable, and valid procedure for detection of viral genomes, mRNA and proteins is approached.

Formalin‐fixed and paraffin‐embedded (FFPE) brain material from six patients who died with PML and one patient who died from SSPE were studied using ISH with a tritium‐labeled cloned JC virus DNA probe and a measles‐cloned nucleocapsid (NC) gene cDNA probe, respectively. This report constitutes a methodological framework as well as a detailed neuropathological analysis of identified brain cell populations within which in situ hybridization was detected. In early PML lesions, swollen nuclei or oligodendrocytes were the predominant cells labeled, whereas older lesions revealed increased numbers of reactive and bizarre hypertrophic astrocytes hybridized at the outer periphery of the demyelinated lesions. The hybridization varied greatly in intensity in different cells. Intense hybridization was noted very rarely in microglial cells, including rod cells and rarely in venular pericytes, intravascular mononuclear cells, or in vascular endothelial cells. These results, considered together with previous findings, indicate that in PML the viral infection runs different courses in the various cells: in astrocytes the viral genome persists for a long time inducing pathological changes in some cells. In oligodendrocytes the infection rapidly lyses the cells. There was a good correlation between chromatic changes observable in routinely stained sections and virus presence.

In addition, in situ hybridization using a measles‐NP‐cloned probe in white matter from FFPE SSPE brain is presented confirming earlier results in SSPE cryopreserved brain.
Defining the lower limits of blood‐brain barrier permeability: Factors affecting the magnitude and interpretation of permeability‐area products
Authors: Preston E. , Haas N.
Published: January 2005

Experimental alteration in the restricted permeability of the blood‐brain barrier to polar, blood‐borne molecules is often quantitated in the rat with use of 14C‐sucrose or 3H‐mannitol delivered as a test substance into the circulation. The underlying principle is to relate the quantity of saccharide that has permeated into brain parenchyma, after an arbitary time period, to some index of the circulating tracer level. This study indicates that to correct the radioactivity level in the brain tissue for intravascular tracer, it is an erroneous practive to estimae the latter as the poduct of tissue blood volume and the tracer concentration measured in a systematic blood sample. Dissected brain tissue was found to have a lower hematocrit and thereby larger plasma/tracer compartment per unit blood volume than femoral arterial blood. It is further shown that, although commercially supplied stocks of 14C‐sucrose or 3H‐mannitol may contain only small quantities of radioactive impurities, their inclusion in injectates and preferential uptake into brain may cause significant overestimation of permeability to the parent tracer. It is also confirmed that magnitude of permeability‐area (PA) products for permeation of purified sucrose or mannitol into brain varies inversely with the length of time allotted for tracer circulation in the bloodstream. This finding is at variance with the assumptions of a two‐compartment (plasma/brain) diffusion model underlying such measurements and supports a recently published model for blood‐to‐brain transfer based on multiple uptake compartments in brain parenchyma. The factors compromising PA values published from several laboratories that had been attributed to genetic differences in laboratory rats.
Neuritic growth from a new subline of PC12 pheochromocytoma cells: Cyclic AMP mimics the action of nerve growth factor
Authors: Katoh‐Semba R. , Kitajima S. , Yamazaki Y. , Sano M.
Published: January 2005

We have identified a new subline of PC12 pheochromocytoma cells (PC12D cells) in which neurites are extended within 24 hr in response to cAMP‐enhancing reagents as well as in response to nerve growth factor (NGF), but not in response to epidermal growth factor or phorbol diester. Anti‐NGF antiserum did not affect forskolin (FRK)‐induced neuritic recruitment. FRK‐induced neurites exhibited growth cones and contained secretion granules and many parallel arrays of microtubules as was the case with NGF‐induced neurites. FRK, but not NGF, increased the levels of intracellular cAMP and activated adenylate cyclase in the membrane fraction. Both NGF and FRK enhanced the activities of tyrosine hydroxylase (TH), acetylcholinesterase (AchE), and ornithine decarboxylase (ODC), but not the levels of neuron‐specific enolase. Enhanced levels of intracellular cAMP mimicked the effects of NGF on neuritic growth, TH, AchE, and ODC activities in PC12D cells, even though NGF does not act through elevation of levels of cAMP.
Evidence for the expression of four myelin basic protein variants in the developing human spinal cord through cDNA cloning
Authors: Roth H. J. , Kronquist K. E. , de Rosbo N. Kerlero , Crandall B. F. , Campagnoni A. T.
Published: January 2005

Four human myelin basic protein (MBP) variants with molecular masses of 21.5, 20.2, 18.5, and 17.3 kilodaltons (kDa) have been identified in the developing human spinal cord and their structures determined through an analysis of cDNA clones of their mRNAs. The 20.2‐kDa MBP mRNA encoded a novel MBP variant, the structure of which has not been reported in any species. Its amino acid sequence was identical with that of the 21.5‐kDa MBP except for a deletion of 11 amino acid residues encoded by exon 5 of the MBP gene. All four human MBP variants were identical except for the insertion or deletion of two peptide fragments corresponding to those encoded by exons 2 and 5 of the MBP gene. In this study, no mature human MBP cDNAs missing exon 6 sequences were identified. This suggests that, unlike the mouse, the four human MBP mRNAs encoding these MBP variants arise by the alternative splicing of only exons 2 and 5 from the primary MBP gene transcript. This indicates that the predominant MBP splicing pathways in human and mouse are different.

Immunoblots of human fetal spinal cords (11–21 weeks) indicated that MBP expression turned on abruptly between 14 and 16 weeks. Expression of the 20.2‐kDa MBP variant was most evident at 16 weeks and its relative proportion declined thereafter, suggesting that its expression was developmentally regulated.
Psychoneuroimmunology: Interactions between central nervous system and immune system
Authors: Solomon G. F.
Published: January 2005

Psychoneuroimmunology, a rapidly developing field, has to do with the complex bidirectional interactions between the central nervous system and the immune system. Neuroendocrine influences modulate immune function, and there is feedback from the immune system to the brain. CNS‐immune interaction appears to play a role in psychosocial influences on immunologically resisted and mediated diseases. With the growing evidence now at hand, over 30 “postulates” can be proposed for specific implication of CNS‐immune interaction.
Noradrenergic sympathetic innervation of the spleen: II. Tyrosine hydroxylase (TH)‐positive nerve terminals form synapticlike contacts on lymphocytes in the splenic white pulp
Authors: Felten S. Y. , Olschowka J.
Published: January 2005

Immunocytochemistry was used to demonstrate tyrosine hydroxylase (TH)‐positive profiles in the spleen of the adult Fischer 344 rat. At the light microscopic level, numerous varicose nerve profiles were seen in the white pulp, particularly surrounding the central arteries and their arteriole branches. At the electron microscopic level, varicosities were seen in close proximity to smooth muscle cells of the arteries, and directly abutting lymphocytes (presumable T‐lymphocytes) of the nearby periarteriolar lymphatic sheath. There were no intervening cell processes between the TH‐positive terminals and the lymphocyte. The opposing membranes were smooth and evenly spaced approximately 6 nm apart. Additional TH‐positive nerve profiles were seen in the inner marginal zone and within trabeculae. The correlation between this immunocytochemical staining and previously demonstrated histofluorescence for norepinephrine leads to the conclusion that lymphocytes in the splenic white pulp have direct associations with noradrenergic fibers of the sympathetic nervous system. This association provides a route by which the autonomic nervous system could directly influence specific immune system effector cells.
Noradrenergic sympathetic innervation of the spleen: III. Development of innervation in the rat spleen
Authors: Ackerman K. D. , Felten S. Y. , Bellinger D. L. , Felten D. L.
Published: January 2005

The ontogeny of noradrenergic innervation and its compartmental devlopment were studied in the rat spleen using glyoxylic acid histofluorescence and high‐performance liquid chromatography (HPLC). Noradrenergic nerves were present at birth in bundles adjacent to the splenic artery and vein. On days 1‐3, fluorescent profiles largely were associated with the vasculature and with the perivascular zone. By day 6, these fibers formed increasingly elaborate and tortuous plexuses around the central arteries and their branches. By day 10, fibers were present along the marginal sinus and extended into the developing marginal zone. Between day 10 and day 13 the largest increase in norepinephrine (NE) levels (per mg protein) were noted, and the periarteriolar lymphatic sheath (PALS) achieved its adult form, with increased innervation of the parenchyma. In contrast, the venous/trabecular system developed relatively late. The first trabecular fibers were evident at day 10, and the capsule was not innervated until day 13. From 13 days to adulthood, there was a gradual refinement and extension of existing patterns with no change in NE levels as measured by HPLC (per mg protein), suggesting that the innervation was keeping pace with rapid increases in spleen growth.

The pattern of growth and development for noradrenergic nerves in the PALS remarkably parallels changes in T cell compartmentation during this period. We propose that norepinephrine is available for interaction with T cells at the earliest stages of development and could play a role in such precesses as lymphocyte packing and the onset of immunocompetence.
Interleukin‐2 inhibition of oligodendrocyte progenitor cell proliferation depends on expression of the TAC receptor
Authors: Saneto R. P. , Chiappelli F. , de Vellis J.
Published: January 2005

Interleukin‐2 (IL‐2) has been shown to inhibit oligodendroctye progenitor cell proliferation. Within the immune system, IL‐2 biological action is dependent strictly on the expression of the IL‐2 receptor. The antibody TAC, whcih specifically binds the lymphocyte IL‐2 receptor, has been shown to also bind oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium. The expression of the TAC antigen was found necessary for IL‐2 inhibition of oligodendrocyte progenitor cell proliferation. After IL‐2 induced down‐regulation of the TAC antigen, the progenitor cell was unresponsive to IL‐2, even 72 hr after IL‐2 withdrawal. During this unresponsiv period, the oligodendrocyte progenitor cell was immunocytochemically negative for the TAC antigen. Thus, in contrast to IL‐2 receptors on T‐cells, IL‐2 does not up‐regulate its receptor on oligodendrocyte progenitor cells. However, upon interleukin 1 (IL‐1) addition both IL‐2 responsiveness and TAC immunocytochemical staining reappeared. These data suggest that IL‐2 inhibition of progenitor cell proliferation depends on the expression of the TAC antigen, which can be regulated by IL‐1.
Pharmacological characteristics of diazepam receptors in neurons and astrocytes in primary cultures
Authors: Bender A. S. , Hertz L.
Published: January 2005

Benzodiazepine binding sites in mouse astrocytes and neurons in primary cultures were labeled with [3H]diazepam (1.8nM), and 3 benzodiazekpine antagonists was studied. RO 5‐4864, RO 7‐3351, and, especially, the antagonist PK 11195 were much more potent in astrocytes than in neurons, whereas the opposite was true for the agonists alprazolam, clonazepam, flurazepam, RO 11‐3128, and chlordiazepoxide, and, especially, the antagonists CGS‐8216 and RO 15‐1788. Flunitrazepam, diazepam, midazolam, RO 11‐6893, and RO 5‐2181 were about equipotent in the two cell types. The neuronal, but not the astrocytic, binding site showed stereospecificity. In astrocytes most of the drugs had pseudo‐Hill coefficients close to one, whereas thepseudo‐Hill coefficients in neurons, except for RO 5‐4864 and PK 11195, were distintly lower than one. Thus, the benzodiazepine binding sites had profoundly different pharmacological characteristics in neurons and in astrocytes.
Nerve growth factor immunoreactivity in mouse kidney: An immunoelectron microscopic study
Authors: Barajas L. , Salido E. C. , Laborde N. P. , Fisher D. A.
Published: January 2005

We have recently reported the immunolocalization of nerve growth factor (NGF) in mouse kidney by light microscopy. In the present study, we have investigated the ultrastructural localization of NGF by the preembedding immunoperoxidase method for electron microscopy. NGF immunoreactivity was present in the connecting tubule cells of the distal nephron. These cells showed immunostaining associated with the Golgi complex, vesicles, rough endoplasmic reticulum (RER), and polyribosomes. The intercalated cells, in contrast, lacked immunoreactivity.
Ganglioside interactions with the dopaminergic system of rats
Authors: Tilson H. A. , Harry G. J. , Nanry K. , Hudson P. M. , Hong J. S.
Published: January 2005

The effects of ganglioside treatment on changes in dopaminergic funciton following 6‐hydroxydopamine (6‐OHDA) or repeated exposure to haloperidol were studied in male Fischer‐344 rats.Rats were injected sc with 30 mg/kg of a mixed ganglioside preparation (GM) at the time of the surgery and for 13 days after receiving an intranigral injection of 6‐OHDA. GM treatment attenuated 6‐OHDA depletion of striatal dopameine (DA) and DOPAC. Another group of rats was implanted with chronic indwelling cannulas in the lateral cerebroventricles at the time of 6‐OHDA administration into the substantia nigra. Daily intraventricular injection of 25 or 50 μg GM attenuated depletions of striatal DA and DOPAC. A separate experiment sought to determine the effects of GMI on the development of receptor supersensitivity produced by repeated exposure to a dopamine receptor antagonist, rats were injected sc with 1 mg/kg haloperidol for 8 or for 16 days; some rats were coadministered 2 mg/kg GMI sc. Four days after the last dose, the rats were challenged with 1 mg/kg apomorphine, and activity was counted for 6 min. Treatment with GMI decreased the behavioral supersensitivity to apomorphine induced by repeated exposure to haloperido. Theses experiments suggest that treatment with GM can have a protective effect against 6‐OHDA‐induced depletion of dopamine if treatment with GM begins at the time of lesioning. These studies also support receptor binding data from other laboraories indicating that treatment with GM1 can affect up‐regulation of dopamine receptors.
Kinetic and pharmacologic characterization of dopamine binding in the mouse cerebellum and the effects of the reeler mutation
Authors: Panagopoulos N. , Matsokis N. A. , Valcana T.
Published: January 2005

The purpose of this study was to characterize the dopaminergic system in the mouse cerebellum and to determine whether the dyskinesia of the reeler mutant is accompanied by alterations in cerebellar and/or striatal dopamine binding. From the analysis of (3H) dopamine ((3H) DA) and (3H)spiperone ((3H)Sp) binding, and the comparison of these parameters between the cerebellum and striatum, we conclude that an dopaminergic system exists in the cerebellum with properties common to the striatal system but also with some differences. That is, (1) with (3H)DA as ligand, we find two binding sites in cerebellum with similar Kd to those of striatum but of lower density, (2) with (3H)Sp as ligand we observe two binding sites in cerebellum and one in striatum, and (3) the competition of (3H)DA binding by various drugs shows that among the cerebellar sites, relative to striatum, there is a higher proportion that corresponds to high affinity D3 and D4(D2 high) binding sites. In cerebellum and striatum of reeler mice, (3H) binding increases 125–174% and 14%, respectively.
Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures
Authors: Avola R. , Condorelli D. F. , Surrentino S. , Turpeenoja L. , Costa A. , Stella A. M. Giuffrida
Published: January 2005

The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15–30 days in vitro in 10% fetal calf serum (FCS)‐supplemented medium and then maintained in serum‐free basal medium (DMEM) supplemented with fatty acid‐free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl‐3H]thymidine or [5, 6 ‐3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37°C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl‐3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr.

The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4–10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied.

Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum‐supplemented (DMEM + BSA + FCS) cultures. The addition of EGF to DMEM + BSA significantly increased cell number, while addition of insulin had no effect. The highest values for cell number were observed when insulin and transferrin were added together with EGF. Moreover, EGF treatment in the presence of serum significantly increased the incorporation of labeled amino acids into vimentin, glial fibrillary acidic protein, and actin.
Glutamate and kainate effects on the noradrenergic neurons innervating the rat vas deferens
Authors: Lara H. , Bastos‐Ramos W.
Published: January 2005

The effects of glutamate on the noradrenergic innervation of the rat vas deferens have been investigated. Administration of a single dose of kainate (50μg) to the peripheral noradrenergic ganglia innervating the vas deferens induced a time‐dependent decrease of norepinephrine in the organ; after 10 days the norepinephrine concentration had fallen to 35% of control values. This effect was accompanied by a 70% decrease in the potassium‐induced release of recently incorporated 3H‐norepinephrine. Concomitantly, post‐synaptic hypersensitivity to both norepinephrine and dopamine appeared. The finding that adrenergic ganglia possess high‐affinity glutamate binding sites suggests that the effect of kainate may be ascribed to glutamate receptors present on the perikarya of the noradrenergic neurons. It is concluded that noradrenergic neurons of the vas deferens are under glutamatergic control and that this might be important in the motor control of the organ.
Purification and partial characterization of brain adenosine deaminase: Inhibition by purine compounds and by drugs
Authors: Centelles J. J. , Franco R. , Bozal J.
Published: January 2005

Rat brain adenosine deaminase (E. C. 3.5.4.4.) was purified 667‐fold from the supernatant fraction by the following techniques: heat treatment (60°C), fraction‐ation with ammonium sulfate, column chromatography on DEAE‐Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 Å) using a calibrated Sephacryl S‐300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and ΔH (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme‐substrate complex. The enzyme had a Km (adenosine) of 4.5 × 10−5 M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 μM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.
Rat interferon enhances the expression of acetycholine receptors in rat myotubes in culture
Authors: André P. , Braun S. , Passaquin A. C. , Coupin G. , Bartholeyns J. , Warter J. M. , Poindron P.
Published: January 2005

Interferon enhances α‐bungarotoxin binding on 5‐day‐old rat myotubes. The increase is time‐ and dose‐dependent. KD for bungarotoxin remains unchanged in the presence and in the absence of interferon, while the maximum number of binding sites is dramatically affected by interferon. Cycloheximide inhibits the effect of interferon, which suggests that the enhancement of α‐bungarotoxin binding depends on protein synthesis.
Enhanced expression of a protein antigen (JI‐31 antigen, 30 kilodaltons) by reactive astrocytes in lacerated spinal cord
Authors: Predy R. , Malhotra S. K. , Das G. D.
Published: January 2005

A mouse monoclonal antibody IMAb JI‐31, isotype IgG 2b was raised against an autopsy sample of cerebral white matter from a multiple sclerosis (MS) patient. MAb JI‐31 recognizes a protein (JI‐31 antigen) in human brain which has a molecular weight of approximately 30,000 daltons (30 kD) as determined by immunoprecipitaion followed by SDS‐gel electrophoresis (reducing conditions) and autoradiography (Singh et al.: Biosci Rep 6:73–79,1986). By immunofluorescence micoroscopy,. Mab JI‐31 stains glial fibrillary acidic protein (GFAP)‐positive cells, namely astrocytes from GFAP (Predy et al.:Biosci Rep 7:491–502,1987).

In this paper we report we report that greatly enhanced staining for JI‐31 antigen is exhibited by reactive astrocytes which arise following CNS injury. (Laceration‐tape surgical lesion of the rat spinal cord served as the expermental modle.) Enhanced expression of JI‐31 antigen reveals some new aspect of the astrocyte response to injury, involving transformation to the reactive state. Consequently, MAb Jl‐31 may be a useful marker for studies or reactive astrocytes.
Differential outgrowth of retinal neurites on purified extracellular matrix molecules
Authors: Carri N. G. , Perris R. , Johansson S. , Ebendal T.
Published: January 2005

Organotgpic cultures of the embryonic retina were system. Microexplants from the chick retina (embryonic day 6) were grown in medium matrix molecules adsorbed to plastic at various concentrations. The maximum neurite length obtained on each type of substratum was measured on day 4 of vitronectin or a hyaluronate‐binding chondroitin sulmolecules On neurite during containing appropriate trophic On purified fate proteoglycan. In contrast, neurite was strongly promoted on laminin in a dose‐dependent manner. Fibronectin elicited a neurite outgrowth corresponding to about one‐third the length Of the Outgrowth On laminin. A 319000‐dalton fibronectin moted by the intact fibronectin molecule. Other isolated domains Of fibronectin, including the 1059000‐ dalton “cell‐binding” domain, did not allow neurite outgrowth. Furthermore, preincubation of fibronec tin substratum with antibodies to the heparin‐binding to fragment elicited neurite elongation the heParin‐binding fibronectin fragment entirely Prevented outgrowth. Fiber Outgrowth was evoked On substrata Of Platelet factor 4, a protein binding heparan Adding increasing concentrations of heparin Progressively inhibited the neurite extension on laminin, whereas similar addition of soluble chondroitin sulfate Proteogbcan had no effect. The results indicate that growing retinal neurites show strong preference for indicate that laminin versus fibronectin. Moreover, the outgrowth‐ to be localized to their heparin‐binding regions. It is tern, elongating retinal neurites can actively discriminate between different extracellular molecules by a mechanism that may involve participation of cell surface heparan sulfate proteoglycans.
Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: Comparative analysis of tyrosine hydroxylase gene products
Authors: D'Mello S. R. , Weisberg E. P. , Stachowiak M. K. , Turzai L. M. , Gioio A. E. , Kaplan B. B.
Published: January 2005

Investigations into the structure and mechanisms regulating the expression of the genes involved in cate cholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3′ untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N‐terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3′ untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine‐rich region in the N‐terminal portion of the rat and human proteins (amino acids 51‐68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.
Human γ enolase: Isolation of a cDNA clone and expression in normal and tumor tissues of human origin
Authors: Van Obberghen E. , Kamholz J. , Bishop J. G. , Zomzely‐Neurath C. , Lazzarini R. A.
Published: January 2005

We have isolated and sequenced a cDNA clone encod‐ ing the human γ enolase. Comparison of our cDNA sequence and the rat γ enolase sequence revealed 97% homology at the level of amino acid sequence. The two coding regions were 91% homologous on the nu‐ cleotide level, whereas the 3′ noncoding regions were much less homologous (32%). Further comparison of our cDNA sequence with the human a enolase re‐ vealed an 82% homology at the amino acid level and a 75% homology at the nucleotide level for the two coding regions, whereas the 3′ nontranslated regions were only 30% homologous. Using a portion of the 3′ nontranslated region of our cDNA, shown to be spe‐ cific for human γ enolase, a single 2.5 kb mRNA was detected in human brain tissue. This same γ enolase message was also found in a number of human normal nonneuronal tissues, and in several human tumor‐ derived cell lines. Expression of the mRNA for the γ enolase subunit should thus be used with caution when identifying the cells of neuronal or neuroendocrine origin.
Inhibition of K+ permeability diminishes alpha2‐adrenoceptor mediated effects on norepinephrine release
Authors: Zimanyi I. , Folly G. , Vizi E. S.
Published: January 2005

The effect of two different potassium channel blockers, 4‐aminopyridine (4‐AP) and quinine, on the alpha2‐adrenoceptor mediated modulation of norepinephrine (NE) release was investigated. Pairs of mouse vasa deferentia were loaded with 3H‐norepinephrine (3H‐NE), superfused continuously, and stimulated electrically. 4‐AP (5.3 × 10−4 M), and quinine (10−5 M) enhanced the stimulation‐evoked release of tritium significantly. The electrically induced release of radioactivity was reduced by alpha2‐adrenoceptor agonists (1‐NE and xylazine) and enhanced by the alpha2‐adrenoceptor antagonist yohimbine. Both effects were affected markedly by 4‐AP or quinine: the depressant action of 1‐NA and xylazine was partially antagonized and the facilitatory effect of yohimbine was completely abolished during the blockade of the potassium channels. It is suggested that the blockade of potassium permeability counteracts negative feedback modulation; therefore, it seems likely that the stimulation of alpha2‐adrenoceptors leads to an enhanced potassium permeability and hyperpolarization of varicose axon terminals.
Developing rod photoreceptors from normal and mutant Rd mouse retinas: Altered fatty acid composition early in development of the mutant
Authors: Scott B. L. , Racz E. , Lolley R. N. , Bazan Nicolas G.
Published: January 2005

The phospholipid and fatty acid contents of developing rod photoreceptor cells were determind in dissociated photoreceptor cells obtained from normal mice and from rd mice exhibiting an inherited retinal degeneration. Photoreceptors were dissociated from retinas by mechanical agitation after mild protease treatment and characterized by light and electron microscopy. Phospholipid classes were isolated by thin‐layer chromatography, and fatty acyl groups separated and quantitated by capillary gas‐liquid chromatography. Developing photoreceptor cells of normal retinas accumulated all phospholipid classes, but in proportions which shifted with age. The mole % contents of phosphatidylcholine (PC) and phosphatidylinositol (PI) decreased with age, whereas phosphatidylethanolamine (PE) and phosphatidylserine (PS) increased. The content of the polyunsaturated fatty acid docosahexaenoate (22:6), expressed as nmol/μg lipid phosphorus, increased rapidly during development, whereas arachidonate (20:4) content tended to decline. Mono‐unsaturated fatty acid levels (palmitoleate, 16:1; oleate, 18:1) declined with age. Among saturated fatty acids, palmitate (16:0) decreased during normal development, whereas stearate (18:0) increased. The total mass of phospholipid/photoreceptor cell in the normal, adult mouse retina was estimated to be approximately 14 pg.

The total phospholipid content and mole % distribution of individual phospholipid classes in immature rd photoreceptors were similar to values for normal cells. In contrast, significant changes in fatty acid composition were detected between immature rd cells and normal cells. Rd cells generally had higher levels of saturated (myristate, 14:0; palmitate, 16:0) and monounsaturated fatty acids (oleate, 18:1) and lower levels of polyunsaturated fatty acids (arachidonate, 20:4; docosahexaenoate, 22:6), suggesting that fatty acid metabolism is altered by expression of the rd gene and/or by the associated impairment of photoreceptor cell differentiation.
Tyrosine hydroxylase and serotonin containing cells in embryonic rat rhombencephalon: A whole‐mount immunocytochemical study
Authors: König Norbert , Wilkie M. B. , Lauder J. M.
Published: January 2005

Rhombencephala from rat embryos were processed as whole‐mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5‐HT). Specific advantages of the whole‐mount technique over the classical serial‐section method were that even isolated immunoreactive (IR) cells could be detected easily, and three‐dimensional relationships could be ascertained without the need for serial reconstruction.

Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5‐HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown‐rump length: 6mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4‐7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1‐2 and C1‐3), whereas 5‐HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4‐9 complex). Although the three‐dimensional distribution of the TH‐IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5‐HT‐IR cell groups and their spatial transformations during this period.

Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5‐HT‐IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5‐HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term “rostral rhombencephalic raphe nuclei” for the rostral 5‐HT cell groups instead of “mesencephalic raphe nuclei,” which is a misnomer.

Close spatial association between TH and 5‐HT‐IR elements was observed mainly in the caudal rhombencephalon, where 5‐HT‐IR fibres coursed through an area containing numerous TH‐IR cell bodies (the presumed anlagen of groups A1‐2 and C1‐3).
Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development: In vivo and in vitro study with monoclonal antibodies
Authors: Dahl D.
Published: January 2005

Neurofilament phosphorylation in rat nervous system development was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes in tissue sections and in primary dissociated cultures. The antibodies either decorated neurofilaments shortly after their appearance or after a considerable delay (from 4 to 9 days in vivo and from 12 to 27 days in vitro), thus suggesting the existence of at least two classes of phosphorylated epitopes. With most antibodies there was a good correlation between in vivo and in vitro findings as to the early or late appearance of phosphorylated epitopes. Monoclonal NE14 was the main exception in that immunoreactivity with this antibody was present in 1‐day cultures, while it only occurred 4 days after the first appearance of neurofilaments in vivo. The effect of phosphorylation on neurofilament structure and function remains to be determined. Neurofilament expression is an early phenomenon in ontogeny coinciding with neuronal differentiation. It is possible that late phosphorylation events may stabilize the axonal cytoskeleton following the massive loss of axons that occurs in several fiber tracts during late fetal and neonatal life.
Lipids of the developing human retina. I. Total fatty acids, plasmalogens, and fatty acid composition of ethanolamine and choline phosphoglycerides
Authors: Martinez M. , Ballabriga A. , Gil‐Gibernau J. J.
Published: January 2005

The total fatty acid composition of the human retina was studied during early normal development and compared to that found in infancy and in adulthood. The retina of an infant undernourished prenatally and of two malnourished postnatally were also studied and compared to the normal values for the age. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) and choline phosphoglycerides (CPG) were also studied. Total and ethanolamine plasmalogens (EP) were estimated by the aldehyde dimethyl acetal (DMA) content of total lipids and of EPG, respectively. After acid methanolysis, analyses of fatty acid methyl esters (FAME) and of DMA were effected by capillary GLC on a single 30 m long, SP‐2330, capillary column. The main developmental fatty acid changes were an increase in 22:6ω3, 22:5ω3 and 20:3ω6 and a decrease in 20:4ω6. The 22:6ω3/20:4ω6 ratio increased in a very significant, parabolical way throughout development. In contrast to the brain, the proportion of ethanolamine plasmalogens decreased with maturation, whereas the ratio 18DMA/16DMA increased. The two postnatally malnourished infants had a very significant increase in retinal 22:5ω6, but only the child that had been fed on a very unbalanced ω3/ω6 diet since 25 weeks of gestation showed an important decrease in retinal 22:6ω3.
Acylation in vitro of the myelin proteolipid protein and comparison with acylation in vivo: Acylation of a cysteine occurs nonenzymatically
Authors: Ross N. W. , Braun P. E.
Published: January 2005

Characteristics of fatty acylation of myelin proteolipid protein (PLP) in vitro were compared with the corresponding process in vivo. Rapid and efficient separation of labelled PLP from other proteins and lipids was effected by extraction into chloroform/methanol/0.1 N HCl (10/10/1) and chromatography on Sephadex LH‐60 in the same solvent. Covalent linkage of [3H]‐palmitate to PLP was demonstrated by repetitive chromatography on LH‐60, thin layer chromatography, and polyacrylamide gel electrophoresis. Reductive cleavage with sodium borohydride of PLP acylated in vitro or in vivo yielded [3H]‐hexadecanol, identifying at least one of the acyl linkages as a thiolester bond. When PLP was acylated with acyl‐CoA as the fatty acid donor, the reaction occurred non‐enzymatically as supported by the following observations: (1) acylation activity increased with increasing pH above pH 7.5, (2) acylation activity was heat stable, (3) acylation activity was not removed from PLP during purification in organic solvents or in Triton X‐100–containing buffers, and (4) acylation of tryptic fragments occurred in the absence of an exogenously added enzyme source. The relevance of in vitro fatty acylation of PLP to that in vivo was confirmed by comparison of proteolytically derived peptide maps that showed that likely the same domain of PLP was acylated in vitro and in vivo.
Monoclonal antibodies made to chick mesencephalic neural crest cells and to ciliary ganglion neurons identify a common antigen on the neurons and a neural crest subpopulation
Authors: Barald Kate F.
Published: January 2005

We previously reported the production of monoclonal antibodies (Mabs) that identified cell surface components of cultured chick and quail ciliary ganglion (CG) neurons and of a subpopulation of neural crest (NC) cells from 31‐hr chick embryos (stage 9). Here we demonstrate that another Mab, CG‐14, which was prepared to nitrocellulose‐immobilized, lightly fixed (0.125% paraformaldehyde) mesencephalic NC cells from 31‐hr (stage 9) chick embryos, labels the same antigen(s) recognized by CG‐1 and CG‐4 on both the CG neurons and the subpopulation of NC cells. All three Mabs label a polypeptide of 75 kD on Western blots of one‐dimensional SDS‐polyacrylamide gels. CG‐14 blocked the binding of CG‐1 and/or CG‐4 to the 75 kD band on Western blots and blocked the binding of CG‐1 and CG‐4 to CG and NC cells. CG‐1 and/or CG‐4 antibodies, in turn, blocked the binding of CG‐14 to Western blots, as well as NC and CG cells. We had previously shown that antibodies CG‐1 and CG‐4 were synergistically cytotoxic for the majority (95%) of cultured CG neurons in vitro in the presence of guinea pig complement. Here we show that the antibodies, which are both of the γ2a subclass, are also cytotoxic for the NC cells that they label in vitro. After the cells are ablated in culture, no other cells bearing the antigen(s) recognized by any of the three Mabs appear over a 2.5‐week period. CG‐14, however, is not cytotoxic for either the CG or NC cell populations alone or in combinations with CG‐1 or CG‐4.

These results confirm our original observation that cultured CG neurons and NC cells share cell‐surface antigen(s). The antigen recognized by all three Mabs appears to be the same whether the immunogen used to produce the antibodies was CG neurons or NC cells. This finding encourages us to continue tests of the hypothesis that the subpopulation of mesencephalic neural crest cells contributes to the formation of the ciliary ganglion in the embryo. Further characterization of the antigen appears in the accompanying paper.
Myelin basic protein and transferrin characterize different subpopulations of oligodendrocytes in rat primary glial cultures
Authors: de los Monteros A. Espinosa , de Vellis Jean
Published: January 2005

The iron transport glycoprotein, transferrin (Tf), localizes exclusively in oligodendrocytes in brain tissue sections. Previously, we showed that Tf is also expressed in oligodendrocytes in primary cultures established from newborn rat brains. Its developmental appearance precedes that of galactocerebroside (GC). In this study, Tf expression in primary brain cell cultures was investigated over a 4‐week period in relation to GC and myelin basic protein (MBP), respectively, early and late markers of oligodendrocyte development. From 9 days in vitro and thereafter, all Tf+ cells were also found to be GC+. With increasing age the number of Tf+ cells decreased while the number of MBP+ cells increased. However, less than 10% of oligodendrocytes co‐expressed Tf and MBP at any age. MBP+ cells were largely found in cell clusters which increased in size and number with age in culture. Interestingly, Tf+ cells were located around the clusters of MBP+ cells which displayed elaborate branched processes. The transient expression of Tf in oligodendrocytes which become MBP+, suggests a role for Tf in the early stages of myelinogenesis. The results also demonstrate the existence of three phenotypically distinct populations of oligodendrocytes. A new model of developmental and functional subpopulations of oligodendrocytes is proposed.
Nerve growth factor rapidly stimulates arachidonate metabolism in PC12 cells: Potential involvement in nerve fiber growth
Authors: DeGeorge J. J. , Walenga R. , Carbonetto S.
Published: January 2005

Homogenates prepared from pheochromocytoma (PC12) cells that are extending nerve fibers in response to nerve growth factor (NGF) have an increased capacity to metabolize exogenous arachidonate compared with homogenates prepared from cells untreated with NGF. These changes are not a consequence of cell attachment, since they are also seen in NGF‐treated PC12 cells grown in suspension and are not found in attached cells grown in the absence of NGF. This NGF‐stimulated increase in arachidonate metabolic capacity occurs rapidly and before the extension of nerve fibers. In contrast to NGF, epidermal growth factor does not alter the metabolism of exogenous arachidonate by PC12 cells. Radioimmunoassay of medium from PC12 cultures indicates that intact cells produce and release increased amounts of prostaglandin (PGE) in response to NGF.

Drugs that inhibit arachidonate liberation from membrane phospholipids (mepacrine or 4‐bromphenacyl bromide) block NGF‐stimulated nerve fiber growth by PC12 cells. Selective inhibitors of cyclooxygenase metabolism of arachidonate (indomethacin and aspirin) fail to block growth, but inhibitors of lipoxygenase metabolism (baicalein, BW755, and eicosatetraynoic acid) are potent blockers. In cultures of dorsal root ganglion neurons, inhibitors of arachidonate release (mepacrine, 4‐bromphenacyl bromide) or its subsequent metabolism by lipoxygenases (nordihydroquaiaretic acid, eicosatetraynoic acid) also prevent the early morphological events of nerve fiber growth.

Our data suggest that NGF rapidly and specifically increases the capacity of PC12 cells to synthesize arachidonate metabolites, and that arachidonate metabolism may be important in nerve fiber growth by both PC12 cells and dorsal root ganglion neurons.
Brain macrophages synthesize interleukin‐1 and interleukin‐1 mRNAs in vitro
Authors: Hetier E. , Ayala J. , Denèfle P. , Bousseau A. , Rouget P. , Mallat M. , Prochiantz A.
Published: January 2005

Amoeboid microglial cells (brain macrophages) were purified from early post‐natal mouse brain cultures. The percentage of cells stained with an anti‐Mac‐1 antibody was > 95%. Stimulation of these brain macrophages by lipopolysaccharides induced the synthesis of interleukin‐1 (IL‐1), which, in part, remained associated with the cell surface and, in part, was released into the culture medium. In contrast, pure primary astrocyte cultures and cell lines of transformed or immortalised astrocytes did not synthesise significant amounts of IL‐1, demonstrating that amoeboid microglia and not astrocytes synthesise IL‐1 in vitro. These physiological data were confirmed by RNA hybridisation studies showing that, on LPS treatment, brain macrophages synthesise significant amounts of IL‐1α and IL‐1 β mRNAs.