bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased expression of pp60c‐src protein‐tyrosine kinase during peripheral nerve regeneration
Authors: Le Beau J. M. , Tedeschi B. , Walter G.
Published: January 2005

Since little is known about the intracellular changes that take place in response to Schwann cell‐neuron interactions that occur during neurite outgrowth and myelination, we investigated the expression of protein‐tyrosine kinase, pp60c‐src, nerve regeneration through a silicone tube. Segments of regenerated nerve, extracted at various times following nerve‐transection, showed an induction of in vitro c‐src kinase activity as measured by autophosphorylation of immunoprecipitated pp60c‐src. This activity occurred at 7 days following nerve transaction coincident with the onset of neurite outgrowth in vivo. This kinase activity, which peaked out between 21 and 35 days and decreased thereafter, appeared to be associated with axonal growth and myelination, but not mitogenesis in the tube. Analysis of c‐src proteins levels by Western blot showed a similar expression profile as that of the kinase activity. Qualitatively, the expression of an immunoreactive c‐src band, migrating slightly slower than pp60, was detected in extracts of regenerating nerve segments as well as in the corresponding L4 and L5 dorsal root ganglia. This protein may be the CNS neuronal‐specificform (pp60 + ) of the c‐src protein. In situ hybridization revealed that Schwann cells and sensory and motor neurons associated with the regenerated sciatic nerve were positive for c‐src mRNA during regeneration possibly accounting for the increased src protein expression during regeneration, Since the in creased expression of pp60c‐src in regenerated nerve segments coincides with both axonal sprouting and myelination, our findings suggest that the c‐src protein may play a role in Schwann cell‐neuron interactions which facilitate the occurrence of these events during regeneration. In addition, although pp60 + generally not detectable in the mature PNS, our findings show that this protein may be induced during conditions of PNS differentiation which promote neurite outgrowth.
Effect of Na+, K+‐ATPase modifiers on high‐affinity ouabain binding determined by quantitative autoradiography
Authors: Antonelli M. , Casillas T. , Rodríguez de Lores Arnaiz G.
Published: January 2005

There is growing evidence on the existence of endog enous ouabain‐like factors that modulate Na+, K+‐ATPase activity. In this laboratory, two soluble sub‐fractions (peaks I and II) were previously separated from rat cerebral cortex, which, had opposite effects on Na,+K+ ‐ATPase activity. Peak I stimulated and peak II inhibited the enzyme (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem Res 11:933‐947, 1986). The same effects are now re ported for K+‐p‐nitrophenyphosphatase activity. Lo calization of high‐affinityouabain binding in rat brain was done by quantitative atitoradiography us ing amicrocomputer digital imaging system. Peak I did not modify, whereas peak II blocked ouabain binding in areas 3–4 of cerebral cortex, dentate gyrus, stria terminalis, thalamic nuclei, and basal gan glia. Similar results were obtained when ouabain binding was determined in rabbit cerebral cortex and by a conventional filtration assay in nerve ending membranes obtained from rat cerebral cortex. These results favour the idea that the factor present in peak II fraction might behave as an ouabain‐like substance.
Extracellular matrix of central nervous system white matter: Demonstration of an hyaluronate‐protein complex
Authors: Asher R. , Perides G. , Vanderhaeghen J.‐J. , Bignami A.
Published: January 2005

Monoclonal antibodies were raised against human glial hyaluronate‐binding protein (GHAP), a major CNS‐specific glycoprotein known to bind hyaluronate in vitro. Frozen sections of dog and human spinal cord were digested with Streptomyces hyaluronidase in order to ascertain whether GHAP is bound to hyaluronate in vivo. Digestion with hyaluronidase, prior to staining of the sections by conventional indirect immunofluorescence, led to a drastic reduction in the intensity of the staining reaction. Chondroitinase ABC (protease‐free) was also effective in bringing about the release of GHAP from tissue sections. This enzyme also degrades hyaluronate. The effects of the chondroitinase were completely reversed by the addition of 1 mM Zn2+ a known inhibitor of this enzyme.

The intact protein was released into the soluble fraction of human brain homogenates by testicular hyaluronidase. An immunoreactive species of 70 kD was released into the soluble fraction of dog spinal cord homogenates by Streptomyces hyaluronidase. Dog GHAP was isolated from spinal cord by means of ion exchange and affinity chromatography. This protein bound efficiently to hyaluronate in vitro. Dog and human GHAP had identical isoelectric points and similar peptide maps but different molecular weights. Dog GHAP (70 kD) was larger than its human counterpart (60 kD).

These findings imply that GHAP exists in association with hyaluronate in CNS white matter. Immunoelectron microscopy revealed that GHAP fills the space between myelin sheaths in dog spinal cord white matter. One is led to conclude therefore that an hyaluronate based extracellular matrix exists in CNS white matter.
Isolation and structural characterization of the murine tryptophan hydroxylase gene
Authors: Stoll J. , Goldman D.
Published: January 2005

The mouse tryptophan hydroxylase gene was isolated and its intron/exon boundaries and putative regulatory sequences identified. To isolate the gene a mouse mastocytoma cDNA clone encoding tryptophan hydroxylase was used to identify and isolate ten overlapping DNA fragments from a mouse genomic library. Restriction mapping and sequence analysis of the clones revealed that the gene contains 11 exons and covers a region of DNA of approximately 21 kb. The transcription initiation site was mapped and the major site of initiation yields an untranslated leader sequence of 124 nucleotides. A minor initiation site is located 9 nucleotides 3′ of the major site. The 5′ untranslated sequence is interrupted by the first intron. Analysis of the sequence upstream of the initiation site showed the presence of several putativepromoter and regulatory sequences. Nine of the ten intron/exon boundaries of tryptophan hydroxylase are conserved with tyrosine hydroxylase and phenylalanine hydroxylase, further delineating the evolutionary relationship of these three genes.
Cytochrome P‐45011β rat brain
Authors: Ozaki H. S. , Iwahashi K. , Tsubaki M. , Fukui Y. , Ichikawa Y. , Takeuchi Y.
Published: January 2005

The presence of cytochrome P‐45011β in rat brain was studied by immunohistochemistry using polyclonal rabbit antibodies raised against purified bovine adrenocortical P‐45011β, which is involvedin the steroid 11β‐hydroxylation and glucocorticoid formation. The results showed that cytochrome P‐45011β immunoreactivity is selectively localized to the tracts of myelinated fibers throughout the brain. The specificity of immunohistochemical stainings with P‐45011β antibodies was established by control tests including nonimmune rabbit immunoglobulin Gs and P‐45011β antibodies absorbed with purified antigen.

Western immunoblots of homogenates from different brain areas with P‐45011β antibodies, together with biochemical enzymatic assays for cytochrome P‐45011β}monooxygenase activity in these homogenates, confirmed the selective localization of this enzyme observed with immunohistochemistry. Cytochrome P‐45011β and 11β‐hydroxylase activity were detected in a homogenate from the cortical white matter (brain area rich in myelinated fibers) as in that from the rat adrenal, but were not detectable in a homogenate from the cerebral cortex (brain area poor in myelinated fibers). Furthermore, quantitation of the P‐45011β bands on the immunoblots by the areal density revealed that the cortical white matter contains approximately 1.4 pmol of cytochrome P‐45011β/mg of tissue protein, the value of which was about one sixth ofthe corresponding value estimated in the rat adrenal. This relatively highcontent of cytochrome P‐45011β was also reflected in a relatively high level of 11β‐hydroxylase activity measured in a homogenate ofthis brain area by biochemical enzymatic assays using [4‐14C]‐11‐deoxycorticosterone.

Thus, from the present data it can be concluded that rat brain containscytochrome P‐45011β in myelinated tracts, in which the 11β‐hydroxylation mechanism (corticosterone synthesis) occurs as in adrenal cortex. This enzyme was detected in the same brain structures in which cytochrome P‐450scc (an enzyme involved in cholesterol side‐chain cleavage and preg‐nenolone synthesis) has been previously shown to be present, which suggests an important role of myelinated tracts for the biosynthesis of steroid hormones within the brain.
Stereotaxic injection of GD1a ganglioside induces limited recovery of striatal dopaminergic system in MPTP‐treated aging mice
Authors: Date I. , Notter M. F. D. , Felten S. Y. , Felten D. L.
Published: January 2005

The systemic administration of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) to young (2 months old) and aging (12 months old) C57BL/6 mice (4 × 20 mg/kg i.p. given 12 hr apart) reduced tyrosine hydroxylase (TH)‐immunoreactive (IR) fibers in the striatum and reduced dopamine (DA) concentration to 35% of controls in young and 22% of controls in aging mouse brain 5 weeks after administration. Stereotaxic injection of GDla ganglioside (3 × 100 μg, 5 days apart) into the striatum of MPTP‐treated young mice restored striatal DA concentration to 52% of the control concentration 5 weeks after MPTP injection. Similar injections of GDla ganglioside restored striatal DA concentration of MPTP‐treated aging mice to only 31% of the control concentration. Immunocytochemical analysis showed significant recovery of TH‐IR fibers in the striatum of MPTP‐depleted young mice treatedwith GDla ganglioside, while TH‐IR fibers in the striatum of MPTP‐depleted aging mice treated with GDla ganglioside showed less recovery. We conclude that treatment of MPTP‐deleted again mice with GD1a ganglioside results in more limited recovery in the nigrostriatal DA system than in young mice.
O‐2A glial progenitors from mature brain respond to CNS neuronal cell line‐derived growth factors
Authors: Hunter S. F. , Bottenstein J. E.
Published: January 2005

During development, myelin‐forming oligodendrocytes and type 2 astrocytes are believed to arise from bipotential (O‐2A) glial progenitors. Previously we found that conditioned medium (CM) from the B 104 rat CNS neuronal cell line promotes growth of neonatal rat O‐2A progenitors in scrum‐free culture conditions with subsequent increases in differentiated progeny. We now report that O‐2A progenitors are present in mature rat brains and that this CM promotes the growth, motility, and bipolar morphology of these cells from 30‐ and 65‐day‐old rat brains, as shown by quantitative studies using double immunostaining and [3H]thymidine‐autoradiography. dition, the growth‐promoting action of B104 CM is not neutralized by antibodies to platelet‐derived growth factor, a proposed progenitor mitogen. Subsequent to the proliferation of these O‐2A progenitors, increases in oligodendrocytes and type 2 astrocytes occur. These data suggest a novel therapeutic strategy for some demyelinating diseases, e.g., multiple sclerosis, where there isa deficit in oligodendrocytes. Although it has been proposed by others thatmature brain O‐2A progenitors are less proliferative and thereby incapable of adequately replenishing lost oligodendrocytes in these diseases, we present in vitro evidence for continued response of mature brain O‐2A progenitors to this neuronal cell line‐derived mitogen.
Selective extraction, solubilization, and reversed‐phase high‐performance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures
Authors: Díaz R. S. , Regueiro P. , Monreal J. , Tandler C. J.
Published: January 2005

The number of solvents capable of dissolving myelin and proteolipid protein (PLP) and of being used as a mobile phase for the separation of myelin proteins by reversed‐phase high‐performance liquid Chromatography (RP‐HPLC) is very limited. In a thorough study, we found that aqueous tetrahydrofuran (THF) fulfilled such a requirement. The maximal amount of protein extracted corresponded to a THF/water ratio of 4:1 v/v and a polarity index of 5.16. This mixture dissolved a purified PLP preparation completely, 60% of proteins from fresh myelin, and 20% of white matter total proteins. Sodium dodecyl sulphate‐poly‐acrylamide gel electrophoresis (SDS‐PAGE) of those extracts, followed by densitometric analysis, showed that the amount and type of proteins dissolved depended on the polarity (i.e., the content of water) of the solvent mixture used. This selective effect was greater for basic protein in myelin preparations. Crude extracts highly enriched in basic protein can be prepared. In addition, the solvent system THF/ water proved to be very useful as a mobile phase in RP‐HPLC for separating myelin proteins. Using a C3 column and a linear gradient from 30% to 100% THF in water, both containing 0.1% trifluoroacetic acid (TFA), we separated completely the three main protein fractions of central nervous system (CNS) myelin in a short period of time. The high solubility power of THF/water mixtures prolonged greatly the life of the column.
Effects of L‐β‐N‐methylamino‐L‐alanine (L‐BMAA) on the cortical cholinergic and glutamatergic systems of the rat
Authors: Rakonczay Z. , Matsuoka Y. , Giacobini E.
Published: January 2005

Neurotoxic properties of L‐β‐methylamino‐alanine (L‐BMAA) after chronic intracerebroventricular (i.c.v.) (500 μg/day) administration up to 60 clays were investigated in the cerebral cortex of the rat. At day 16, there was a significant decrease in acetylcho‐linesterase (AChE) activity, 3H‐QNB binding, 3H‐glu‐tamate (GLU) binding, and 3H‐glutamate binding in the presence of quisqualate (QA). Choline acetyl‐transferase (ChAT) activity and 3H‐nicotine binding were increased at day 16; however, ChAT activity decreased below control levels, at days 40 and 60. 3H‐Nicotine and 3H‐AMPA binding were significantly lower than controls at both days 40 and 60. These significant neurochemical differences from unoper‐ated controls were seen in both drug‐injected and non‐injected sides of the cortex suggesting a generalized cortical damage to glutamatergic and cholinergic systems. In the presence of bicarbonate, L‐BMAA inhibited in vitro both glutamate and AMPA binding sites. L‐BMAA treatment elicited behavioral changes such as splay, jerking movements, and rigidity. These symptoms were present for a period of at least 6 days after daily administration. After this period, symptoms were gradually attenuated and at day 10 the behavior of the L‐BMAA‐treated animals was not different from that of Na‐bicarbonate injected controls. Our results are interpreted as an activation of quisqualate (AMPA) receptors by L‐BMAA involving NMDA as well as non‐NMDA receptors.
Differential expression of MAG isoforms during development
Authors: Pedraza L. , Frey A. B. , Hempstead B. L. , Colman D. R. , Salzer J. L.
Published: January 2005

The myelin‐associated glycoproteins (MAG) mediate the cell interactions of oligodendrocytes and Schwann cells with axons that are myelinated. MAG exists in two developmentally regulated isoforms: large MAG (L‐MAG) and small MAG (S‐MAG). In this paper, we have studied the tissue‐specific and developmentally regulated alternative splicing of these isoforms using monospecific antibodies that recognize epitopes common to both isoforms or that are present only on L‐MAG. In the central nervous system (CNS), L‐MAG is the major form synthesized early in development, and it persists as a significant proportion of the MAG present in the adult. In the peripheral nervous system (PNS), L‐MAG is expressed at modest levels during development; it is virtually absent in the adult. Thus, the expression of L‐MAG is not limited to the CNS, as was formerly believed, suggesting that it plays a common role during the early stages of myelin formation by both oligodendrocytes and Schwann cells. In both the CNS and PNS, S‐MAG is the predominant isoform in the adult. A higher‐molecular‐weight form of MAG is present in the PNS at low abundance, that is developmentally regulated, and appears to be a glycosylation variant. An analysis of the carbohydrate residues on MAG demonstrates that it contains both N‐linked and O‐linked sugars that could be modulated during development. These results suggest a possible mechanism for the regulation of MAG function during myelinogenesis via the expression of alternative isoforms and carbohydrate modifications.
Differential regulation of S100β and mRNAS coding for S100‐like proteins (42A and 42C) during development and after lesion of rat sciatic nerve
Authors: León M. De , Eldik Linda J. Van , Shooter E. M.
Published: January 2005

The changes in the levels of S1007beta; (a protein that stimulates neurite extension and neuronal survival) and 42A and 42C (S100‐like proteins whose mRNAs are induced in PC 12 cells by nerve growth factor) during development and after rat sciatic nerve lesions were analyzed. S100β, 42A, and 42C mRNAs showed differential regulation during development. S100β mRNA was present both in sciatic nerve and brain, and increased more than 11‐fold during the first 3 wk of nerve postnatal development. 42A and 42C mRNAs were essentially restricted to sciatic nerve, with little found in either embryonic or adult brain. The levels of 42C and 42A mRNAs in sciatic nerve increased 4 ‐and 14 ‐fold, respectively, by postnatal day 23 compared to postnatal day 2. 42A, 42C, and S100β mRNAs also showed a differential regulation during sciatic nerve degeneration and regeneration. Axotomized and control sciatic nerves were examined by Northern blots at various times after a crush or cut injury. 42A and 42C mRNA levels increased rapidly in the distal segment of axotomized nerve, remained two‐ to five‐fold higher than controls at day 14 after injury but returned to control levels by 40 days. In contrast, S100β mRNA showed a three‐fold decrease in the axotomized nerve between days 1 and 3 after injury, and slowly returned towards control levels over the next few weeks. The decrease in S100β mRNA was reflected by a corresponding decrease in S100β protein levels. The induction of 42A and 42C mRNAs and repression of S100β mRNA remained if nerve regeneration was prevented. These data suggest that, in spite of the high degree of sequence homology, S100β and its two homologues 42A and 42C play different roles in neuronal development and regeneration.
Chondroitin sulfate proteoglycan surrounds a subset of human and rat CNS neurons
Authors: Bertolotto Antonio , Rocca G. , Canavese G. , Migheli A. , Schiffer D.
Published: January 2005

Chondroitin sulfate proteoglycan (CS‐PG) bearing glycosaminoglycan (GAG) chains containing unsulfate (COS) and 6‐sulfate (C6S) disaccharides was immunolocalized in rat and human CNS by using monoclonal antibodies (MAb) specific for the two disaccharides. The immunostaining with both MAb was restricted to the periphery of a neuronal subset in rat and human CNS. Double immunofluorescence showed codistribution of the antigens around the same neuronal population. The staining with anti‐COS MAb was stronger than with anti‐C6S MAb, suggesting that the proteoglycan (PG) contains mainly COS disaccharjdes. In different rat cortical areas, 40–;60/mm2 positive interneurons were found, the visual cortex showing the highest value. In human cortex, positivity was also observed around the soma of some pyramidal cells. In the rat, positive neurons were also localized in deep cerebellar nuclei, reticular nucleus of the thalamus, and other structures of the midbrain and hindbrain. CA3 region of hippocampus and the external layer of pyriform cortex were characterized by positivity of the neuropil.

Immunoelectronmicroscopy showed the antigens in the extracellular space around the neuronal soma, the synaptic elements and the cell processes of the neuropil. The neuronal surface of the soma and of the proximal dendrites were positive, but the pre‐ and postsynaptic membranes and clefts were negative.
Antibodies to the retina N‐acetylgalactosaminylphosphotransferase inhibit neurite outgrowth
Authors: Gayá‐González L. , Balsamo J. , Swaminathan N. , Lilien J.
Published: January 2005

The neural retina N‐acetylgalactosaminylphospho‐transferase (GalNAcPTase) is a cell surface molecule (Balsamo and Lilien, 1980, 1983; Balsamo et al., 1986a) that is tightly associated with, and glycosy‐lates, the calcium‐dependent, cell‐cell adhesion molecule, N‐cadherin (Balsamo and Lilien, 1990). N‐cad‐herin has been implicated in neuronal attachment and neurite outgrowth when at the surface of cels (Bixby et al., 1987, 1988; Matsunaga et al., 1988; Neugebauer et al., 1988; Tomaselli et al., 1988). The intimate association of the GalNAcPTase and N‐cadherin prompted us to test the possibility that the GalNAcPTase is also involved in the process of neurite outgrowth. We tested the effect of one polyclonal and two monoclonal anti‐GalNAcPTase antibodies in cultures of chick neural retina cells extending neurites on substrates requiring N‐cadherin, beta inte‐grin receptors, or the chicken homologue of L1, G4. The length and number of neurites produced were dramatically reduced on all of these substrates by the polyclonal and one of the monoclonal anti‐GalNAcPTase antibodies. The second monoclonal antibody bound to the cell surface but was not inhibitory, indicating that it reacts with a different epitope. The mechanism through which the retina cell surface GalNAcPTase may modulate neurite outgrowth on many substrates is discussed.
Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor
Authors: Deloulme J. C. , Baudier J. , Sensenbrenner M.
Published: January 2005

A primary culture system of nearly pure neuronal cells from 14‐day‐old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum‐free medium, and bo‐rated polylysine‐coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron‐specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non‐motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth‐associated protein GAP‐43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined, by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bro‐modeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron‐specific enolase. Re‐combinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor β1 (TFG‐β1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE‐pos‐itive neurons, probably belonging to the non‐motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE‐positive.
Acidic fibroblast growth factor (aFGF) is expressed in the neuronal and glial spinal cord cells of adult mice
Authors: Tourbah A. , Oliver L. , Jeanny J. C. , Gumpel M.
Published: January 2005

Fibroblast growth factors (FGFs) are known to be synthesized in the central nervous system (CNS) and to act on CNS cells in vitro, but less is known about their synthesis, expression, and role in vivo. In this work, using specific anti‐acidic fibroblast growth factor (aFGF) antibodies, we have shown for the first time., by immunohistochemistry, that aFGF is expressed in spinal cord cells of young adult normal mice. This expression is predominant in the cell nucleus. Using immunohistochemical double staining procedures, we identified the cell type expressing aFGF as neurons, astrocytes, and oligodendrocytes, : but for each type, cells were not all positively immunostained.
Expression of the neurofilament protein NF‐H in L cells
Authors: Soifer D. , Nicoletti V. , Cabane K. , Mack K. , Poulos B.
Published: January 2005

We have inserted a Not1‐Sal1 fragment of the mouse gene coding for the neurofilament protein NF‐H behind the dexamethasone‐inducible transcription promoter of MMTV in a vector derived from pMAMneo (Clonetech Labs). This construct, which includes all four exons of the NF‐H gene, was amplified and in corporated into liposomes for transfection of L cells. Transfectants were selected in G418‐containing medium and cloned. Clones were grown in serum‐containing medium and screened for expression of the NF‐H mRNA by extraction of total RNA, generation of cDNAs by reverse transcription, and amplification of a 900‐base portion of the NF‐H cDNA by PCR. Positive clones were detected by the presence of a band of the correct size on agarose gels. This was confirmed by Southern blotting of the gels probed with a 185‐base segment of the amplified region. Immunofluorescent analysis of two positive clones, C33 and C34, showed that C33 cells grown in serum‐containing medium or in serum‐free medium in the presence of dexamethasone have a network of SMI32 (Sternberger/Meyer Inc.– monoclonal antibody against a nonphosphorylated epitope on NF‐H)‐positive filaments with the same distribution as filaments stained with antibodies to vimentin, while C34 cells do not react with antibodies against neurofilament proteins. Neither clone reacted with antibodies against highly phosphorylated NF‐H (SMI31).
Dynamics of mammalian high‐molecular‐weight neurofilament subunit phosphorylation in cultured rat sympathetic neurons
Authors: Clark E. A. , Lee V. M.‐Y.
Published: January 2005

To better understand the function(s) of the multiphosphorylation repeat (MPR) of the high molecular weight neurofilament (NF) subunit (NF‐H), we sought to determine how phosphorylation within this region is regulated in cultured rat sympathetic neurons. To do this, monoclonal antibodies specific to phosphorylated or nonphosphorylated tandem repeats of the ammo acid sequence Lys‐Ser‐Pro‐Ala‐Glu‐Ala found within the MPR were identified and used to determined the extent of phosphorylation, the time course of phosphorylation, and the rate of turnover of phosphate groups within the NF‐H MPR in cultured sympathetic neurons. We showed that (1) the synthesis and phosphorylation of NF‐H occurs in these neurons cultured for 1 or 4 weeks; (2) the conversion from poorly to more highly phosphorylated variants of NF‐H occurs slowly in cultured neurons; and (3) the turnover of phosphate groups on both poorly and highly phosphorylated variants of NF‐H occurs more rapidly than the turnover of NF‐H itself. In addition, we showed that the 200‐kD highly phosphorylated NF‐H can contain at least five consecutive nonphosphorylated tandem repeats as well as phosphorylated tandem repeats, whereas we were unable to detect consecutively phosphorylated tandem repeats in the 160‐kD form of NF‐H.

These findings allow us to propose a mechanism whereby NF‐H is initially phosphorylated singly at sites distributed throughout the MPR. This “poorly phosphorylated NF‐H,” which is the predominant form in immature neurons, is then converted by phosphorylation at additional sites within the MPR to the more “highly phosphorylated” species of NF‐H typically found in mature neurons. These latter phosphorylation events generate NF‐H variants in which tandem repeats within the MPR have been phosphorylated.
Differences in the abilities of human tau isoforms to promote microtubule assembly
Authors: Scott C. W. , Blowers D. P. , Barth P. T. , Lo M. M. S. , Salama A. I. , Caputo C. B.
Published: January 2005

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, τ3 (tau containing three micro‐tubule‐binding domains), τ4 (tau containing four microtubule‐binding domains) and τ4L (tau containing four microtubule binding domains plus a 58‐amino‐acid insert near the N‐terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4–0.45 μM. However, τ4 induced microtubule formation at a rate five‐ to tenfold faster than either τ3 or τ4L. The rate of microtubule elongation seen with τ4 was twofold greater than with τ3 or τ4L, suggesting that the faster rate of microtubule assembly seen with τ4 was due, at least in part, to faster elongation. τ4 induced a greater number of microtubules to form at steady state than did τ3 or τ4L. The microtubules generated with each tau isoform had similar steady‐state length distributions and were equally susceptible to cold‐induced disassembly. These results indicate that the additional microtubule‐binding domain in τ4 enhances microtubule assembly, while the 58‐amino‐acid insert negates the stimulatory effect of the fourth microtubule‐binding domain.
Vesicular and cytoplasmic localization of neurotensin‐like immunoreactivity (NTLI) in neurons postsynaptic to terminals containing NTLI and/or tyrosine hydroxylase in the rat central nucleus of the amygdala
Authors: Bayer V. E. , Towle A. C. , Pickel V. M.
Published: January 2005

Neurotensin and catecholamines in the central nucleus of the amygdala (CNA) have both been implicated in the integration of autonomic responses to stress. We examined whether there might be a cellular substrate for interactions involving these putative neurotransmitters in the CNA. Sections of acrolein‐fixed rat brain were processed either (1) for the ultrastructural localization of a rat antiserum against neurotensin using the peroxidase–antiperoxidase (PAP) method, or (2) for the dual localization of rat neurotensin antiserum and rabbit antiserum against the catecholamine‐synthesizing enzyme, tyrosine hydroxylase (TH), using the PAP method and immunoautoradiography. The rat polyclonal antiserum against neurotensin was shown in immunoblots to recognize neuromedin N and Lys‐Arg‐neurotensin (LANT‐6) in addition to neurotensin. In single and dual labeling studies, the neurotensin‐like immuno reactivity (NTLI) was detected in perikarya and processes. The NTLI was localized predominantly to dense core vesicles in one group of perikarya and dendrites, while a second group had labeling both in dense core vesicles and more diffusely throughout the cytoplasm. Terminals also showed NTLI, particularly in association with dense core vesicles. The labeled terminals formed primarily symmetric junctions with both cell bodies and dendrites. In the dual labeling study, perikarya contained only NTLI while terminals contained TH and/or NTLI. Terminals containing TH or NTLI separately innervated cell bodies and dendrites displaying NTLI, and formed separate or convergent inputs onto unlabeled neuronal targets. Terminals colocalizing both TH and NTLI formed junctions only on unlabeled dendrites. These findings show that in the rat CNA two populations of neurons differ with respect to their distribution of NTLI, and that the output from neurons containing NTLI is modulated by direct synaptic input from terminals containing neurotensin and/or catecholamines. Release of neurotensin and catecholamines, most likely dopamine, from the same or separate terminals on common targets, in the CNA may account for certain similarities in their stress‐related functions.
Peripheral benzodiazepine induces morphological changes and proliferation of mitochondria in glioma cells
Authors: Shiraishi T. , Black K. L. , Ikezaki K. , Becker D. P.
Published: January 2005

Peripheral benzodiazepine (PBD) receptors are localized on the mitochondrial membrane and are highly expressed in brain tumors compared to normal brain. To elucidate the biological role of the PBD receptor on mitochondria, we examined the effect of PBDs on mitochondrial morphology in C6 and T98G glioma cells using rhodamine 123 and quantitative electron microscopy. In cells incubated in serum‐free medium alone, mitochondria were distributed in a filamentous pattern throughout the cytoplasm. By contrast, the mitochondria aggregated in the perinuclear region in PK11195 or Ro5‐4864 (10 nM) treated cells. Quantitative electron micrography revealed a 250% increased in the number of mitochondria with elongated cristae and a fivefold increase in dividing mitochondria in PK11195‐treated cells compared with cells incubated in serum‐free medium alone. PBD treatment also resulted in vacuolation within the matrix and mitochondrial swelling. These data suggest that PBDs influence mitochondrial morphology and induce mitochondrial replication in cultured glioma cells.
Immunohistochemical evidence for the coexistence of substance P, thyrotropin‐releasing hormone, GABA, methionin‐enkephalin, and leucin‐enkephalin in the serotonergic neurons of the caudal raphe nuclei: A dual labeling in the rat
Authors: Kachidian P. , Poulat P. , Marlier L. , Privat A.
Published: January 2005

By means of dual immunohistochemical labeling on the same brain section examined with a light microscope, the present study reports the presence with serotonin (5‐hydroxytryptamine; 5‐HT) of gamma‐aminobutyric acid (GABA), substance P (SP), thyro‐tropin‐releasing hormone (TRH), leucin‐enkephalin (LEU‐enk), or methionin‐enkephalin (MET‐enk), within the same neuron in the nuclei raphe magnus, raphe obscurus, and raphe pallidus of the rat. On the one hand, peptides or GABA are detected with specific rabbit antibodies by indirect peroxidase labeling using peroxidase‐conjugated Fab fragments, and on the other, 5‐HT is detected with a rabbit antibody against the BSA‐serotonin conjugate by radio‐immunocytochemistry using [125I]‐labeled protein A. The possible coexistence of TRH and SP in these neurons is also investigated by using peroxidase labeling and radio‐immunocytochemical detection, respectively.

In the whole caudal raphe nuclei the proportion of each coexisting peptide with 5‐HT appears in decreasing order as: TRH > SP > MET‐enk # LEU‐enk > GABA. In all instances the level of coexistence differs considerably in B1–B2 vs. B3 cell groups. No SP/TRH dually labeled cells have ever been found in any of the serotonergic nuclei of the caudal raphe.

Given the evidence that these raphe nuclei project possibly to the spinal cord, these data constitute an anatomical substrate for the several distinct physiological functions presumably subserved by 5‐HT in the cord, namely the modulation of nociception, motor, and autonomic functions.
Characterization of a panel of neurofilament antibodies recognizing N‐terminal epitopes
Authors: Kaplan M. P. , Chin S. S. M. , Macioce P. , Srinawasan J. , Hashim G. , Liem R. K. H.
Published: January 2005

Peptides corresponding to sequences from the ammoterminal “head” regions of the low, middle, and high molecular weight neurofilament proteins (NF‐L, NF‐M, and NF‐H) were synthesized by a modification of the Merrifield solid‐phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS‐1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters.
A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains
Authors: Connor J. R. , Menzies S. L. , St. Martin S. M. , Mufson E. J.
Published: January 2005

Immunohistochemical and histochemical staining were performed on Alzheimer's diseased brain tissue obtained at autopsy. The iron‐regulatory proteins transferrin and ferritin as well as iron are, in general, found predominantly in oligodendrocytes similar to that previously reported for normal brain tissue. However, in the vicinity of senile plaques, the staining pattern is altered for both proteins and iron. Transferrin is homogenously distributed around the senile plaques and is apparently extracellular. In addition, transferrin is found in astrocytes in the cerebral cortical white matter of the Alzheimer's tissue rather than its normal distribution in oligodendrocytes. A robust ferritin immunoreaction accompanies senile plaques and many blood vessels in the Alzheimer's brain tissue. Although many ferritin‐positive oligodendrocytes are present in the Alzheimer's tissue, most of the ferritin‐containing cells associated with senile plaques and blood vessels are microglia. Iron can also be demonstrated in the senile plaques. The iron reaction product is observed both diffusely in proximity of the plaques and in cells associated with the plaques. These data strongly suggest a disruption in brain iron homeostasis in Alzheimer's disease as demonstrated by alterations in the normal cellular distribution of iron and the proteins responsible for iron regulation. These data will contribute to understanding both the potential for oxidative damage and the potential for metal neurotoxicity in Alzheimer's disease.
Muscimol and N,N‐dimethylmuscimol: From a GABA agonist to a glycine antagonist
Authors: Aprison M. H. , Lipkowitz K. B.
Published: January 2005

By using molecular modeling methods, a molecular mechanism was identified which can explain how the incorporation of two methyl groups in place of two hydrogen atoms on the terminal nitrogen atom of muscimol can not only convert this potent agonist at GABAnergic receptors to an inactive molecule at these receptors, but also can convert this new derivative to an antagonist of glycine at glycinergic receptors. This insight into the molecular mechanism operative in the conversion of physiological function provides a basis for understanding how a single molecule may be able to act at both the GABA‐ and glycine‐inhibitory receptors.
Characteristics of large neutral amino acid‐induced release of preloaded L‐glutamine from rat cerebral capillaries in vitro: Effects of ammonia, hepatic encephalopathy, and γ‐glutamyl transpeptidase inhibitors
Authors: Hilgier W. , Puka M. , Albrecht J.
Published: January 2005

The release of newly loaded L‐[14C]glutamine (L‐Gln) from rat cerebral cortical capillaries was stimulated by L‐transport system substrates: tryptophan (TRY), leucine (leu), and nonlabeled L‐Gln, respectively, by 32, 50, and 40% above the basal release resulting from superfusion with standard Krebs–Henseliet buffer. However, no stimulation was observed upon treatment with D‐Gln or L‐glutamate (L‐Glu), which are not the L‐system substrates, or with ammonium chloride. The stimulatory effect of TRY was temperature dependent but sodium independent, and was abolished in the presence of a sulfhydryl reagent N‐ethylmaleimide (NEM). The results support the view that the L‐Gln‐stimulated uptake of large neutral amino acids (LNAA) across the blood–brain barrier involves the L‐system mediated Gln‐LNAA exchange. The TRY‐stimulated Gln release was enhanced in vitro by simultaneous addition of ammonium chloride, and in capillaries derived from rats with acute hepatic encephalopathy (HE). These results confirm the role of Gln‐LNAA exchange in the excessive accumulation of LNAA in brain observed in a variety of hyperammonemic conditions.

Superfusion of L‐Gln‐loaded capillaries in a buffer containing γ‐glutamyl transpeptidase (GGT) inhibitors, serine borate (SB) or 6‐diazo‐5‐oxo‐L‐norleucine (DON), increased the basal L‐Gln release and made it irresponsive to subsequent treatment with TRY. However, the basal release was also increased by superfusion with serine alone or Leu, and this treatment abolished the subsequent effect of TRY as well. Moreover, DON stimulated L‐Gln release from capillaries superfused in a standard way, and the effects of DON and TRY were additive. Hence, in the present conditions, SB and DON acted as L‐system substrates rather than as GGT inhibitors. Taken together, the results do not support the concept that GGT mediates the Gln‐LNAA exchange. © 1992 Wiley‐Liss, Inc.
Advances in the use of the fluorescent probe fura‐2 for the estimation of intrasynatposomal calcium
Authors: Yates S. L. , Fluhler E. N. , Lippiello P. M.
Published: January 2005

Fura‐2 has been used to measure intracellular Ca2+ with great success in a variety of cell and subcellular preparations, including synaptosomes. There is however, a great deal of variability in the reported estimates of resting intrasynaptosomal Ca2+ ([Ca2+]i). Fura‐2 AM is highly lipophilic and passes readily across the plasma membrane into the cytoplasm, where it is de‐esterified and trapped. The lipophilicity of fura‐2, however, promotes the formation of micelles in aqueous media, which may impede the passage of the probe across cell membranes. Our results suggest that some of the variability in the reported [Ca2+]i estimates may be related to fura‐2 de‐esterification and loading efficiencies. The use of the nonionic detergent pluronic F‐127 is recommended to prevent the formation of fura‐2 micelles. The use of a detergent is not always an acceptable practice, however, especially in studies in which detergent–lipid interactions may influence membrane parameters. We found that fatty acid free bovine serum albumin (BSA) (0.25%) greatly increases the intrasynaptosomal concentration of the probe, resulting in a significant increase in the signal‐to‐noise (S/N) ratio. The mechanism appears to be independent of effects of BSA on synaptosomal integrity and directly related to the prevention of fura‐2 micelle formation, as evidenced by light spectroscopic scattering measurements. Thus, BSA appears to keep the probe in a form that crosses the synaptic plasma membrane more readily. The effectiveness of BSA in improving the loading of fura‐2 into synaptosomes was comparable to the detergent pluronic F‐127, making it possible to measure [Ca2+]i without compromising membrane integrity. © 1992 Wiley‐Liss, Inc.
Morphological differentiation and proteoglycan synthesis regulate Alzheimer amyloid precursor protein processing in PC‐12 and human astrocyte cultures
Authors: Baskin F. , Rosenberg R. N. , Davis R. M.
Published: January 2005

A morphologically differentiated strain of rat pheochromocytoma (PC‐12H) metabolically labeled with [35S]methionine and incubated with a phorbol ester displayed reduced 140‐kDa and increased 15 kDa bands relative to cells incubated without phorbol ester after immunoprecipitation with antisera elicited by the C‐terminal peptide of the Alzheimer amyloid precursor protein (APP). These bands correspond to glycosylated full length APP and a C‐terminal fragment previously reported by Anderson et al. (Neurosci. Lett. 120:126–128, 1991) to result from a cleavage within the amyloidotic A4 region of APP, which releases a 120 kDa extracellular fragment. The 15 kDa fragment, not immunoprecipitated with an antisera elicited by the N‐terminal portion of A4 amyloid, is nonamyloidogenic. Incubation of these cells with p‐nitrophenylxyloside, known to inhibit proteoglycan formation, also increased this nonamyloidogenic cleavage of APP.

In contrast to these results, an undifferentiated low passage PC‐12‐L strain constitutively displayed rapid nonamyloidogenic APP cleavage. Incubation of PC‐12‐L with phorbol ester did not affect the relative abundance of 140 or 15 kDa bands. Growth of PC‐12‐L with 7 S NGF or dibutyryl cAMP resulted in increased morphological differentiation and deceased APP cleavage which was now phorbol‐inducible. Similar analyses of dividing and senescent human astrocytes and normal and F‐AD fibroblasts indicate 5‐fold lower rates of mid‐A4 APP cleavage. Phorbol esters decreased the 140 kDa APP band without affecting the intensity of the 15 kDa band in these cells. Phorbol esters similarly reduces the 140 kDa band in 1 of 5 human neuroblastomas without affecting a constitutive level of 15 kDa APP which was 10‐fold lower than that seen in PC‐12‐L.

These data indicate that (1) rapid mid‐A4 APP cleavage occurs in additional cell types including CNS astrocytes; (2) nonamyloidogenic APP cleavage is inhibited in constitutively or chemically differentiated PC‐12 cells; (3) phorbol ester inductions of mid‐A4 APP cleavage product accumulations and reduced accumulations of products of APP maturation are not coupled in all cell types; and (4) inhibition of proteoglycan formation increases this APP cleavage. These results may be relevant to alterations in APP processing in AD‐affected brain regions. © 1992 Wiley‐Liss, Inc.
Cell proliferation and protooncogene induction in oligodendroglial progenitors
Authors: Bhat N. R. , Hauser K. F. , Kindy M. S.
Published: January 2005

Cell proliferation and the expression of the protoon‐cogenes c‐fos and c‐jun have been examined in the primary cultures of oligodendroglial (OL) progenitor cells in response to phorbol 12‐myristate 13‐acetate (PMA), serum, insulin, insulin‐like growth factor‐I (IGF‐I), platelet‐derived growth factor (PDGF), and fibroblast growth factor (FGF). Combined [3H]thymidine autoradiography and immunocytochemistry was used to asses the mitogenic response of O4 (an oligodendrocyte‐specific marker)‐positive OL progenitors. In addition, the rate of DNA synthesis was measured by the incorporation of [3H]thymidine into acid‐precipitable material. It was found that all of the agents tested stimulated DNA synthesis in OL progenitors and induced a rapid increase in c‐fos and c‐jun protooncogene expression. The induction of c‐fos gene expression and DNA synthesis in response to PMA was completely blocked by 1‐(5‐isoquinolinyl‐sulfonyl)‐2‐methylpiperazine (H‐7), a potent inhibitor of protein kinase C (PKC), thereby suggesting a role for PKC in the control of c‐fos expression and cell proliferation in OL progenitors. © 1992 Wiley‐Liss, Inc.
Enhanced permeabilities of cationized‐bovine serum albumins at the blood‐nerve and blood‐brain barriers in awake rats
Authors: Wadhwani Kishena C. , Shimon‐Hophy M. , Rapoport S. I.
Published: January 2005

Permeability‐surface area products (PAs) of the blood‐nerve barrier (BNB) and blood‐brain barrier (BBB) to 125I‐labeled native bovine serum albumin (nBSA, pI ∼︁4), and to 2 cationized albumins (cBSA) of differing pI (pI ∼︁ 8 and 11), were quantitatively determined in awake rats, using an i.v. bolus injection technique. Mean PAs of the BNB and BBB to 125I‐nBSA, after a circulation time of up to 120 min, were (0.17 ± 0.23) and (0.09 ± 0.05) × 10−5 ml/s.g. wet wt, respectively (n =; 12 rats), and were not significantly different from 0 (P > 0.05). Mean PAs of the BNB and BBB to 125I‐cBSA (pI ∼︁ 8), after circulation time of 12 min, were (1.9 ± 0.1) and (1.7 ± 0.1) × 10−5 ml/s.g wet wt, respectively (n =; 8). Significant greater PAs, at both the BNB and BBB to 125I‐cBSA (pI ∼︁ 11) [(8.2 ± 1.8) and (3.0 ± 0.6) × 10−5 ml/s.g wet wt, respectively (n =; 12)], than both PA's of nBSA and cBSA (pI ∼︁ 8) were found. The accumulation of 125I‐cBSA in epi‐perineurial tissues also was higher than that of 125I‐nBSA, and was related to the degree of cationization. Our results indicate that, as at the BBB, the transfer of cationized serum albumin is enhanced over that of native albumin at the BNB of the mammalian peripheral nerve. © 1992 Wiley‐Liss, Inc.