bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamate receptor agonists increase the expression of Fos, Fra, and AP‐1 DNA binding activity in the mammalian brain
Authors: Sonnenberg J. L. , Mitchelmore C. , Macgregor‐Leon P. F. , Hempstead J. , Morgan J. I. , Curran T.
Published: January 2005

Administration of convulsant doses of Metrazole (pentylenetetrazol) and picrotoxin, as well as maximal electroshock, results in a rapid but transient increase in c‐fos mRNA in mouse brain. Elevation of c‐fos mRNA is followed by the accumulation and subsequent disappearance of Fos, the protein encoded by c‐fos. In addition, immunoblots reveal the induction of two additional proteins that are antigenically related to Fos (Fra, Fos‐related antigens). Fos and the various Fra appear and disappear in a staggered manner over an 8 hour period, such that at longer times after stimulation the brain contains no Fos but relatively large amounts of Fra (the latter being designated here by their apparent molecular weights, Fra‐46K and Fra‐35K). Previous studies have established that Fos, as well as several Fra, contribute to transcription factor AP‐1 nucleoprotein complexes along with Jun, the product of the jun proto‐oncogene. The appearance in brain of Fos and Fra coincides with a protracted increase in total AP‐1 DNA binding activity, indicating that all the Fos‐like proteins can participate in AP‐1 complexes. Furthermore, the molecular composition of these complexes alters with time after stimulation. The induction of c‐fos by Metrazole is blocked or attenuated by known anticonvulsants such as diazepam and valproate as well as the N‐methyl‐D‐aspartate (NMDA) receptor antagonists, 2‐amino‐5‐phosphonovaleric acid (APV) and MK‐801. This suggests that fos induction might involve stimulation of a glutamate receptor. This conclusion was strengthened by the observations that two glutamate receptor agonists, kainic acid and NMDA, induced c‐fos expression. The induction of c‐fos by NMDA had a similar kinetic and pharmacological profile to that of Metrazole. Kainic acid, in contrast, caused a protracted induction of c‐fos not seen in any of the other paradigms. Therefore, it is likely that both the kainate and NMDA forms of the glutamate receptor may participate independently in the induction of c‐fos in the rodent nervous system.
Evolution of the neuron‐specific alternative splicing product of the c‐src proto‐oncogene
Authors: Raulf F. , Robertson S. M. , Schartl M.
Published: January 2005

The observation of a slower migrating form of pp60c‐src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of the c‐src gene (Martinez et al.: Science 237:411–415, 1987; Levy et al.: Mol Cell Biol 7:4142–4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini‐exon, gives rise to six amino acid residues not found in the non‐neuronal (termed fibroblastic) form of pp60c‐src.

We have addressed the question of the evolutionary origin of the c‐src neuronal insert and its functional significance regarding neural‐specific expression of the c‐src gene. To this end we have investigated whether the c‐src gene of a lower vertebrate (the teleost fish Xiphophorus) gives rise to a neural‐specific transcript in an analogous manner. We could show that the fish c‐src gene does encode for a “fibroblastic” and a “neuronal” form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini‐exon. The miniexon is also 18 basepairs long and we could demonstrate directly that this exon lies within the intron separating exons 3 and 4.

For comparative purposes we have examined whether the fish c‐yes gene, the member of the src gene family most closely related to c‐src, also encodes a neural tissue‐specific transcript. No evidence for a second transcript form in brain was obtained. This result suggests that the mini‐exon arose within the c‐src gene lineage sometime between the src/yes gene duplication event and the divergence of the evolutionary lineage giving rise to the teleost fish. Published genomic sequence of src‐related genes in Drosophila and our own results with Hydra demonstrate no intron in these species at the analogous location, consistent with first appearance of this mini‐exon sometime between 550 and 400 million years ago.
Induction of c‐fos and TIS genes in cultured rat astrocytes by neurotransmitters
Authors: Arenander A. T. , de Vellis J. , Herschman H. R.
Published: January 2005

The interaction of neurotransmitters with their specific receptors initiates a cascade of intracellular biochemical events which lead to induction of specific genes. Included in this cascade is the rapid and transient induction of a family of primary early response genes we term TIS genes (Lim et al.: Oncogene 1:263–270, 1987). Expression of six TIS genes, including c‐fos, was examined in secondary cultures of rat neocortical astrocytes exposed to muscarinic and adrenergic agonists and antagonists to study the early genomic responses which accompany neurotransmitter‐induced alteration of glial morphology and physiology. Carbachol induced accumulation of mRNA for c‐fos and the other TIS genes. Carbachol‐meditated induction of TIS mRNA expression was sensitive to atropine blockade and was potentiated by lithium. Norepinephrine (NE), isoproterenol, or phenylephrine also induced TIS mRNA accumulation. In order to determine which second‐messenger pathways mediate NE induction of TIS gene expression, the influences of the beta(B) antagonist propranolol (PR), the alpha 1(A1) antagonist prazosin (PZ), and the alpha 2(A2) antagonist yohimbine (YB) were examined. The induction of TIS1 mRNA by NE was partially blocked by PR or PZ alone, and completely abolished by both antagonists in combination. YB had no effect on TIS1 mRNA expression. These results suggest that NE induces TIS1 mRNA through both B‐ and A1‐adrenergic, but not A2, pathways. The lack of effect of inhibitors of phospholipase A2 and cyclooxygenase suggests that the A1 component is mediated through a protein kinase C pathway. The induction of transient gene expression by neurotransmitters may mediate the secondary genomic responses and phenotypic changes occurring in astrocytes in response to alterations in neuronal neurotransmitter release.
Rapid growth of regenerating axons across the segments of sciatic nerve devoid of schwann cells
Authors: Sketelj J. , Bresjanac M. , Popović M.
Published: January 2005

The characteristic response of Schwann cells (SC) accompanies peripheral nerve injury and regeneration. To elucidate their role, the question of whether or not regenerating axons can elongate across the segments of a peripheral nerve devoid of SC was investigated. Rat sciatic nerve was crushed so that the continuity of SC basal laminae was not interrupted. A segment about 15 mm long distal to the crush was either repeatedly frozen/thawed to eliminate SC or scalded by moist heat which, in addition, denatured the proteins in the SC basal laminae, too. Both sensory and motor axons grew rapidly across the frozen/thawed segment of the nerve. Their rate of elongation was reduced by only 30% in comparison to control crushed nerves. SC were not present along the path of growing axons adhering tightly to the bare SC basal laminae. The rate of elongation of regenerating sensory and motor axons in scalded nerve segments was eight times lower than in control crushed nerves. SC were present in that part of the scalded region that had been invaded by the regenerating axons but no further distally.

These results suggest that acellular basal laminae of SC provide very good, although not optimal, conditions for elongation of regenerating sensory and motor axons. If biochemical integrity of the basal lamina is destroyed, the regenerating axons must be accompanied or preceded by viable SC, and axon elongation rate is significantly reduced.
Release from live choroid plexus of apical fragments and electrophoretic characterization of their synthetic products
Authors: Gudeman D. M. , Brightman M. W. , Merisko E. M. , Merril C. R.
Published: January 2005

Protein synthesis and secretion by the choroid plexus (CP) has been implicated as a major source of certain proteins in cerebrospinal fluid (CSF), such as transthyretin. The suggestion that proteins are elaborated from CP through apocrine secretion has been borne out by the presence of newly labeled proteins in apical protrusions from CP (Agnew et al.: Cell and Tissue Research 208:261–281, 1980a). When the protrusions (aposomes) separate from the cells, they continue to incorporate labeled amino acids (Gudeman et al.: Tissue and Cell 19:101–109, 1987). In the present work the formation of aposomes in live CP explants indicated that these spheroids were not the result of fixation. Aposomes were also identified within rat CSF by immunohistochemistry with monoclonal directed against aposomes as well as with anti‐transthyretin serum. The protein product of aposomes was characterized by 2‐dimensional SDS‐PAGE and compared to the protein products of whole CP tissue. Paradoxically, transthyretin, a heavily labeled protein in the tissue, was virtually undetected in the aposome synthetic profile. However, four other proteins were expressed in relatively equivalent amounts by the aposomes. The presence of mRNA in aposomes was detected with a poly dT probe, and the presence of actin was revealed by phalloidin staining of aposomes. These studies provide a more comprehensive definition of aposomes, but the functions of their secreted proteins remains to be determined.
Brain mitochondrial swelling induced by arachidonic acid and other long chain free fatty acids
Authors: Hillered L. , Chan P. H.
Published: January 2005

Polyunsaturated fatty acids (PUFAs), arachidonic acid in particular, are well known, potent inducers of edema in the brain, while monounsaturated and saturated long chain fatty acids do not possess this quality. This investigation has compared the ability of some free fatty acids (FFAs), known to be released during cerebral ischemia, to induce brain mitochondrial swelling in vitro. The PUFAs tested, especially arachidonic acid (20:4), were more potent in causing swelling than saturated or monounsaturated ones, as measured by the decrease in light absorbance of the mitochondrial suspension. This finding is in line with the unique potency of 20:4 to induce brain edema. Incubation of brain mitochondria with 20:4 for 20 min caused a dose‐dependent swelling. ATP‐MgCl2 both prevented and reversed this swelling, while binding of the 20:4 by the addition of bovine serum albumin could only prevent but not reverse the swelling. The contraction of the swollen mitochondria appeared to be mediated by a mechanism dependent upon high‐energy phosphates, potentiated by MgCl2.

The concentration of 20:4 required to induce swelling was about 20 times higher than the concentration required to induce inhibition of mitochondrial respiratory function (L Hillered and P H Chan: J Neurosci Res 19:94–100, 1988a). Morover, reversal of the swelling occurred without recovery of respiratory function. These results suggest that swelling is a phenomenon of minor importance as an indicator of brain mitochondrial dysfunction, at least when induced by 20:4 in vitro.
Autoradiographic localization of [3H]dextromethorphan in guinea pig brain: Allosteric enhancement by ropizine
Authors: Canoll P. D. , Smith P. R. , Gottesman S. , Musacchio J. M.
Published: January 2005

Dextromethorphan (DM) is an antitussive with anticonvulsant activity that binds to high‐ and low‐affinity sites in guinea pig brain homogenates. We examined the autoradiographic localization of [3H]DM using the anticonvulsant ropizine, an allosteric modifier that decreases the dissociation rate of [3H]DM. Competition studies demonstrated that the binding to brain sections was identical to that of brain homogenates [Craviso and Musacchio: Mol Pharmacol 23:629–640, 1983b]. Computer‐assisted quantitative analysis of the autoradiographic images demonstrated that [3H]DM binds to discrete structures throughout the brain, but with higher density in the midbrain, pons, and medulla oblongata. The most intense labeling was observed in the rhabdoid, dorsal raphe, median raphe, caudal linear raphe nuclei, and cranial motor nerve nuclei. The central gray showed moderate to high‐density labeling throughout its entire rostro‐caudal extent, with very high binding in the dorsal tegmental nucleus and the locus coeruleus. Moderate and high binding was also seen in several hypothalamic structures. Distinct bands of moderate binding were seen in the pyramidal cell layer of the piriform cortex, the retrosplenial cortex, the granular cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus, and the Purkinje cell layer of the cerebellum. The striking similarity between the binding distribution of [3H]DM and sigma ligands, plus competition studies in brain homogenate, support the hypothesis that DM and sigma ligands share a common highaffinity binding site [Musacchio et al: Mol Pharmacol 35:1–5, 1989]. The distribution of [3H]DM binding provides possible anatomical substrates for both the antitussive and anticonvulsant actions of DM.
Intravenous injection of [1‐14C]arachidonate to examine regional brain lipid metabolism in unanesthetized rats
Authors: DeGeorge J. J. , Noronha J. G. , Bell J. , Robinson P. , Rapoport S. I.
Published: January 2005

We examined the metabolic disposition and brain distribution of an unsaturated fatty acid, [1‐14C]arachidonate, between 5 and 240 min following its intravenous bolus injection in unanesthetized adult rats. Injected [1‐14C]arachidonate was cleared rapidly from plasma, with less than 10% remaining by 2 min. Total brain radioactivity, 0.2% of the injected dose, was near maximal by 5 min, reached a peak by 15 min, then slowly declined. Radioactivity in brain lipids constituted greater than 82% of the total brain radioactivity at all times. Radioactivity in aqueoussoluble metabolites was greatest at 5 min (13% of total) and declined to 5% by 240 min. Protein pelletassociated radioactivity gradually rose to a peak of 7% by 120 min. Within the lipid fraction, more than 92% of radioactivity was in glycerolipids, with greater than 81% in phospholipids. Radioactivity in inositol phosphoglyceride was maximal at 5 min (47% of phospholipid radioactivity); and declined to 34% by 20 min, whereas radioactivity in choline phosphoglyceride peaked at 15 min (41% of phospholipid radioactivity) and was constant thereafter. In contrast, radioactivity in ethanolamine phosphoglycerides increased from 7 to 17% during the course of the experiment. Quantitative autoradiography of brain sections indicated incorporation of [1‐14C]arachidonate into gray‐matter regions was 1.5‐ to threefold that into white‐matter regions. The data were analyzed in terms of a model for brain fatty acid uptake from plasma. Estimates of unidirectional transfer constants, k, for [1‐14]arachidonate from plasma to brain regions with an intact blood‐brain barrier ranged from 0.0005 to 0.0015 ml · sec−1 · g−1 and were correlated with those for [9,10‐3H]palmitate. The results indicate that brain phospholipid metabolism in awake animals can be examined regionally and quantitatively using intravenous injection of [1‐14C]arachidonate combined with quantitative autoradiography and biochemical analysis.
Multiple and novel specificities of monoclonal antibodies O1, O4, and R‐mAb used in the analysis of oligodendrocyte development
Authors: Bansal R. , Warrington A. E. , Gard A. L. , Ranscht B. , Pfeiffer S. E.
Published: January 2005

Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage‐specific manner, 01, 04 (Sommer and Schachner, 1981), and R‐mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. 01 reacted with galactocerebroside, monogalactosyl‐diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R‐mAb reacted with galactocerebroside, monogalactosyl‐diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R‐mAb with sulfatide was nearly equal to that with galactocerebroside. 04 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with 04 that had not yet begun to express the 01 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in 01‐positive cells. Therefore, 04 stains, in addition to sulfatide and seminolipid, an unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing 04+ oligodendrocyte progenitors stained slightly earlier with R‐mAb than with 01, and thus R‐mAb transiently stained a larger population of oligodendrocytes than did 01. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that 04, R‐mAb, and 01 have multiple overlapping specificities, including previously unrecognized cross‐reactions.
Expression of heat shock genes (hsp70) in the mammalian brain: Distinguishing constitutively expressed and hyperthermia‐inducible mRNA species
Authors: Brown I. R. , Rush S. J.
Published: January 2005

The mammalian genome contains both constitutively expressed and heat‐shock‐inducible members of the hsp70 gene family. Riboprobes derived from members of these two classes of heat shock genes were utilized in Northern blot studies to analyze brain mRNA isolated from control rabbits and rats and from animals subjected to hyperthermic treatment. Ariboprobe derived from a constitutively expresed hsp70 gene detected a 2.5 kilobase (kb) mRNA species in control rat brain. These brain mRNAs showed little change in abundance in animals which were subjected to hyperthermic treatment. A riboprobe derived from a heat‐shock‐inducible hsp70 gene detected an abundant 2.7 kb brain transcript in hyperthermic rabbits which was not apparent in control animals. A time course study revealed that the induction of this mRNA species was transient and paralleled the rise and fall in body temperature. Peak induction was observed at 1 hr. The level of this message had greatly decreased by 5 hr and only trace levels were present at 10 and 24 hr. In the rat brain the induced hsp70 mRNA species was slightly larger than that observed in rabbit (2.9 kb vs. 2.7 kb). The riboprobe which detected the hyperthermia‐inducible mRNA species was highly specific and did not cross react to the constitutively expressed mRNA species under the conditions employed in the Northern blot studies.
Neonatal hyperthyroidism in the rat produces an increase in the activity of microperoxisomal marker enzymes coincident with biochemical signs of accelerated myelination
Authors: Adamo A. M. , Aloise P. A. , Soto E. F. , Pasquini J. M.
Published: January 2005

The effect of neonatal hyperthyroidism produced by injection of tri‐iodothyronine (T3) on myelination and on the microperoxisomal population of the brain was studied in young rats. Data on the lipid composition of myelin show that myelinogenesis starts earlier in treated animals. In coincidence with the early appearance of myelin, there is an increase in the population of brain microperoxisomes, indicated by the increase in the activity of two enzymes that have been shown to be located in these organelles: catalase and acyl CoA‐dihydroxyacetone phosphate acyl transferase. Double‐label experiments using (1,2,3‐3H) and (2‐3H) glycerol to study the synthesis of glycerophospholipids throug the dihydroxyacetone phosphate (DHAP) pathway give further support to the above‐mentioned findings and suggest that there is an active participation of microperoxisomes in the synthesis of myelin lipids during the period of myelin formation.
Rat dopamine β‐hydroxylase: Molecular cloning and characterization of the cDNA and regulation of the mRNA by reserpine
Authors: McMahon A. , Geertman R. , Sabban Esther L.
Published: January 2005

A number of cDNA clones for rat dopamine β‐hydroxylase (DBH) were isolated from a rat pheochromocytoma tumor cDNA library. The 2445 nucleotide sequence revealed a single open reading frame of 1860 nucleotides and a 3′ untranslated region containing two polyadenylation addition signals. The cDNA coded for a 620 amino acid protein of 69,883 daltons. Six potential glycosylation sites and one potential phosphorylation site were identified. Amino acid residues likely to be involved in the active site of DBH and in copper ligand binding were identified. The N‐terminal 42 amino acids appeared to constitute a typical but unusually long signal sequence. Hydropathy analysis indicated that this N‐terminal region contained the only extensive hydrophobic domain and thus constituted the only obvious potential membrane attachment site. Northern analysis detected two mRNA species of 2.5 and 2.7 kb. The relative abundance of the 2.7 vs. 2.5 kb mRNAs was differentially regulated in PC12 cells and adrenals upon treatment with reserpine.
Mapping of the Alz 50 epitope in microtubule‐associated proteins tau
Authors: Ksiezak‐Reding H. , Chien C.‐H. , Lee V. M.‐Y. , Yen Shu‐Hui
Published: January 2005

Alz 50 and seven other monoclonal antibodies have been shown to react with both tau and Alzheimer brain proteins of molecular mass 60–70 kDa. The location of some of the epitopes of these antibodies (Alz 50, Tau‐2, NP14, Ab 636.7) on the tau molecule is unknown, whereas those of others (Tau 60, Tau 14, Tau‐1, Tau 46) have recently been demonstrated in fetal human tau at amino acid residues 60–72, 83–120, 131–140, and 315–352. To determine the location of the unknown epitopes, human tau was digested with chymotrypsin and trypsin, and the bovine microtubule fraction was incubated with chymotrypsin. Comparison of the immunoblots of chymotryptic digested tau with those of untreated preparations showed that the Alz 50 epitope was more sensitive than other tau epitopes to proteolysis. Cleavage of a 3–4 kDa polypeptide from the periphery of tau was sufficient to remove the Alz 50 epitope, but not the epitopes of Tau 46 (C‐end) or Tau 60 (N‐end). The distribution of the Alz 50 epitope in endogenously degraded, chymotrypsin or trypsin digested tau fragments was different from that of the Tau 46 epitope known to be located within 38 residues from the C‐terminus of the tau molecule. Based on these observations Alz 50 epitope was considered to be located within 3–4 kDa of the N‐terminus of tau. A comparison of immunoblots of different tau‐reactive antibodies showed similarities between Tau 60 and Tau‐2, and between Tau 14, Tau‐1, NP14, and Ab 636.7. Microtubule fractions, after chymotryptic digestion, retained small fragments (<29 kDa) reactive with all tau antibodies except Alz 50 and Tau 60. The two smallest fragments (<20–22 kDa), which were bound to digested microtubules and presumably contained microtubule binding domains, were recognized by Ab 636.7 and Tau 46. Among the eight tau‐reactive antibodies, the epitopes of Tau‐1, Ab 636.7, NP14, and Tau 46 were conserved in tau from human, bovine, rat, and mouse. The epitopes of the other four antibodies were more species specific. These results support our conclusion that the Alz 50 epitope is located at the N‐terminus of tau, the Tau‐2 epitope is located between Tau 60 and Tau 14 epitopes, and the Ab 636.7 and NP14 epitopes are in the C‐half of tau molecule closer than Tau‐1 epitope to the C‐terminus.
Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro
Authors: Murphy M. , Drago J. , Bartlett P. F.
Published: January 2005

We have developed an in vitro culture system to study the regulation of proliferation and differentiation of neural precursor cells contained within the neuroepithelium of embruonic day 10 mice. Adnumber of soluble growth factoes have been tested for their ability to regulate these early events and, of these factors, we have found that the fibroblast growth factors [FGFs] can directly stimulate the proliferation and survival of the neuroepithelial cells. At least 50% of the neuroepithekuak cekks divide in the presence of FGF whereas in the absence of FGF all of the cells die within 6 days of cultural. At higher concentrations of FGF, the cells change from being nonadherent round cells in tight clusters into a more flattened cell type which adheres to the substratum. This morphological change is accompanied by the expression of both neurofilament and GFAP, which are definitive markersof the two major cell types in the central nervous system: neurons and glia. In addition a neuroepithelial cell line, which does not rely on FGF for survival or proliferation, expresses both of these markers in response to FGF. These results indicate that FGF is stimulating the differentation of the neuroepithelial cells into mature neurons and glia.
Multiple sclerosis and HLA class II susceptibility and resistance genes
Authors: Haegert D. G. , Michaud M. , Schwab C. , Francis G. S.
Published: January 2005

Probes to the HLA class II genes DRβ, DQβ, and DQα were used to study DNA from unrelated Caucasian multiple sclerosis (MS) patients by sequential restriction fragment length polymorphism (RFLP) analysis in Taq1 restriction enzyme digests. Comparison of 104 patients and 108 controls, who were not matched for DR type, has identified for the first time a linked series of allele‐specific RFLPs or allogenotypes which form an extended haplotype that is preferentially associated with MS. These allogenotypes include DRw15 or DR2(15);DQβlb, which corresponds at the DNA level to the DQwl (DQw6) serotype; a DQA1 allogenotype termed DQα1b; and a 2.2kb DX (DQA2) allogenotype termed DXαU (DQA2U).

The role of HLA class II genes in susceptibility to MS was found to be complex. First, 23 of 104 MS patients showed DR‐DQ linkages which were not observed in our control population. We suggest these anomalous associations may be important in the pathogenesis of MS. Second, homozygosity of a 2.0 KB DX (DQA2) gene, termed DXαL (DQA2L), Showed a strong negative association with MS. DXαL (DQA2L) is in strong linkage disequilobrium with DR1, 5(w11)7, and a subset of DR4, all of which also showed a negative association with MS, Since DXαL (DQA2L) does not code for any known product, DR1, 5(11), 4, and 7 become candidates for disease resistance genes. Third, in EcoR1 and EcoRV digests of DNA from both controls and patients homozygous for DQβ1b a number of different RFLP patterns were identified and these RFLPs patterns were identified and these RFLPs were associated with either relapsing‐remitting or progressive MS. This suggests there may be HLA sequence differences between individuals bearing a particular class II allele and these may correlate with the clinical course of MS.
Effect of chronic lead exposure on biopterin metabolism in the rat neostriatum
Authors: Martínez‐Fong Daniel , Gutiérrez M. E. , Aceves J.
Published: January 2005

Chronic (3 months) lead exposure increased the quinonoid dihydropteridine reductase activity and the tetrahydrobiopterin content but did not affect the GTP‐cyclohydrolase activity and the dihydrobiopterin content. These results suggest that lead intoxication may enhance dopamine metabolism in neostriatum by increasing the content of tetrahydrobiopterin, the regulating cofactor of tyrosine hydroxylase activity.
Expression of macrophage colony‐stimulating factor gene in the mouse brain during development
Authors: Théry C. , Hétier E. , Evrard C. , Mallat M.
Published: January 2005

We have investigated the expression of macrophagecolony stimulating factor (M‐CSF) gene in mouse brain during development. Northern blot analysis of cerebral RNA evidenced a 4.5‐kb M‐CSF transcript from day 14 of gestation until 2 weeks after birth. The cell type responsible for this transcription was studied using in vitro cell cultures. The 4.5‐kb M‐CSF transcript was found both in astrocyte primary cultures and in immortalized astrocytic cell lines. M‐CSF mRNA was also detected in lipopolysaccharide‐stimulated brain macrophage cultures. These results suggest that M‐CSF is involved in the outgrowth of microglia during ontogenesis.
Cultured microglial cells have a distinct pattern of membrane channels different from peritoneal macrophages
Authors: Kettenmann H. , Hoppe D. , Gottmann K. , Banati R. , Kreutzberg G.
Published: January 2005

Microglia are the source of the resident macrophages of the brain and thus belong to one of the most reactive cell types in cerebral tissue. They are attributed to have an important role in a number of pathological conditions, such as multiple sclerosis, viral infections like AIDS, and in lethal or sublethal injuries of neurons where the blood‐brain barrier is left intact (Streit et al., 1988; McGeer et al., 1988., Gendelman et al.,1989). Microglia share a number of macrophage characteristics but so far lack a distinguishing positive marker. In this study it is shown that microglia are distinguished from other macrophages by a unique pattern of ion channels.

We compared membrane currents of microglial cells with those from peritoneal macrophages cultured under identical conditions. Although in macrophages a delayed outward K+ current was previously described (Randriamampita and Trautmann,1987), microglial cells lacked any specific outward current. Instead, these cells were characterized by large inwardly rectifying currents, activated by hyperpolarizing voltage steps. The reversal potential in different K+ gradients and the sensitivity of the current to Ba2+, TEA, and 4‐AP indicates that this current is K+ selective. In single‐channel recordings, a 30 pS K+ selective channel similar to the classical inward rectifier K+ channel was observed. Thus, the expression of membrane channels served not only to distinguish microglia from other cells inside and outside the brain, e.g., blood macrophages, but also suggests a unique functional state of this cell population.
Subtractive cDNA cloning of RC3, a rodent cortex‐enriched mRNA encoding a novel 78 residue protein
Authors: Watson J. B. , Battenberg E. F. , Wong K. K. , Bloom F. E. , Sutcliffe J. G.
Published: January 2005

A rodent cortex‐enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex‐minus‐cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNA encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine‐rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen‐like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.
Solubilization of A1 adenosine receptor from pig brain: Characterization and evidence of the role of the cell membrane on the coexistence of high‐ and low‐affinity states
Authors: Casadó V. , Cantí C. , Mallol J. , Canela E. I. , Lluis C. , Franco R.
Published: January 2005

The present solubilization strategy recognizes the important role of detergent cocktails in the solubilization and subsequent stability of adenosine A1, receptors from pig brain cortical membranes. The 3‐[3‐(cholamidopropyl) dimethylammonio]‐1‐propane‐sulfonate‐digitonin mixture produced the extraction of up to 52% of the receptor with an enrichment of 1.2‐fold with respect to crude membranes. The binding activity of the soluble extract was very stable even in the absence of glycerol. In crude membranes the existence of high‐ and low‐affinity states was detected, but in the soluble extract and in the detergent‐treated membranes only the high‐ affinity state was detected. Association‐dissociation curves showed that in crude membranes no interconversion between high‐ and low‐affinity sites is produced by the association of the ligand [3H]R‐N6 ‐phenylisopro‐pyladenosine. These results suggest that the high‐ and the low‐affinity states are different conformations induced by the structure of the membrane. The modulation of the binding activity by (Gpp(NH)p)5′ ‐guanylylimidodiphosphate and Mg2+ was studied. In crude membranes Gpp(NH)p shifted the high‐affinity state to the low‐affinity state, whereas the contrary occurred when Mg2+ was used. The effect of both Mg2+ and Gpp(NH)p was also assayed with the soluble extact and with the detergent‐treated membranes. In addition to a decrease of the overall binding capacity, Gpp(NH)p promoted a conversion to all low‐affinity states in the detergent‐treated membranes or to all very‐low‐affinity sites in the soluble extract. Mg2+ and Gpp(NH)p counteracted their effects in intact membranes, whereas Mg2+ could not reverse the uncoupling effect of Gpp(NH)p with solubilized or detergent‐treated membranes. Thus, it is suggested that Mg2+ acts at sites other than guanine‐nucleotide sites. If high‐affinity states correspond to receptor/G protein complexes and low‐affinity states correspond to the uncoupled receptor, we should conclude that Mg2+, as well as the loss of membrane integrity, favours the interaction of A1 receptor molecule with G protein.
Po mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin
Authors: Brunden K. R. , Brown D. T.
Published: January 2005

Schwann cells of the peripheral nervous system depend on the presence of both axons and basal lamina to achieve a myelinating phenotype. Furthermore, removal of axonal influence results in the cessation of myelination and down‐regulation of myelin protein expression by Schwann cells. Here we examine whether both axons and basal lamina are required by Schwann cells for the expression of mRNA encoding the major myelin glycoprotein, Po. Cultures of Schwann cells and neurons obtained from dorsal root ganglia of 15 day embryonic rat pups were grown for up to 20 days in vitro under conditions that either allowed or prohibited basal lamina and myelin formation. These cultures were assayed for the expression of Po. mRNA by using an S1 nuclease‐protection assay. After 20 days in vitro, the cultures that did not assemble basal lamina and that were incapable of myelin formation expressed Po. mRNA at a level which was comparable to that seen in identically aged, myelinating cultures. Both the myelinating and nonmyelinating cultures demonstrated an appreciable increase in Po mRNA when compared to the starting embryonic dorsal root ganglia Schwann cells. The latter had a low, but detectable, level of mRNA for this myelin glycoprotein. The cultures that were devoid of basal lamina and myelin showed a clear increase in Po mRNA by 11–15 days in culture. This increase in expression depended on the presence of neurons/neurites, since Schwann cells which were grown in neuron‐depleted cultures expressed little, if any, Po mRNA. In contrast to the levels of Po. mRNA, the nonmyelinating cultures had a significantly lower amount of Po. glycoprotein than did the cultures which assemble myelin. This suggests that the nonmyelinating Schwann cells regulate the level of this glycoprotein at the translational and/or the posttranslational level. The data presented here suggest that myelin protein mRNA expression and myelin assembly by Schwann cells are separable events, with the former depending on one or more neuronal/axonal factors.
Phosphorylation of the glial fibrillary acidic protein
Authors: Noetzel M. J.
Published: January 2005

The glial fibrillary acidic protein (GFAP) was found to be phosphorylated in vivo after intracerebral injection of [32P]‐orthophosphate, in brain Slices, and in a cell free system. The phosphorylated proteins were separated by two‐dimensional gel electrophoresis and then transferred to nitrocellulose sheets. Two isoelectric variants of GFAP were immunochemically identified by monoclonal antibodies. Autoradiography demonstrated that only the more acidic isoelectric variant of GFAP was phosphorylated. Phosphoamino acid analysis revealed that under all conditions GFAP was phosphorylated at serine and threonine residues. Incubation of brain slices with[32P]‐orthophosphate and the protein kinase C activator phorbol 12‐myristate 13‐acetate or forskolin, an activator of cyclic AMP‐dependent protein kinase, stimulated phosphorylation of GFAP. Likewise phosphorylation of GFAP was also accentuated by calcium/phosphatidylserine/diolein and by exogenous cyclic AMP‐dependent kinase in a cell free system. These findings announce that protein kinase C and cyclic‐AMP dependent kinase may play physiologic roles in the in situ phosphorylation of GFAP. When isolated cytoskeletal preparations were incubated with [γ‐32P] ATP, GFAP was phosphorylated in vitro by two additional protein kinases, a Ca++/calmodulin‐depen‐dent kinase and an effector‐independent kinase. The results of these investigations strongly suggest that phosphorylation of GFAP appears to be regulated by multiple second messenger pathways.
Recombinant peripheral myelin protein Po confers both adhesion and neurite outgrowth‐promoting properties
Authors: Schneider‐Schaulies J. , von Brunn A. , Schachner M.
Published: January 2005

To probe into the functional properties of the major peripheral myelin cell surface glycoprotein Po, its ability to confer adhesion and neurite outgrowth‐promoting properties was studied in cell culture. To this aim, Po was expressed as integral membrane glyco protein at the surface of CV‐1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin‐like extracellular domain of Po (Po‐ED) was expressed as soluble protein in a bacterial expression system and used as substrate coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth‐promoting assays. The adhesion of Po‐expressing CV‐1 cells to Po‐ED substrate was specifically inhibitable by polyclonal Po antibodies (54% ± 6%). In addition, the specific interaction between Po molecules could be reduced (49% ± 8%) by adding soluble Po‐ED to the culture medium, demonstrating that the homophilic interaction between recombinant Po molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig‐like domain. Substrate‐coated Po‐ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 μm. This neurite outgrowth was specifically inhibitable by soluble Po (74% ± 14%) and Po antibodies (65% ± 9%). These observations indicate that Po is capable of displaying two different types of functional roles in the myelination process of peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interaction may indicate its role in the self‐recognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.
Phagocytosis of peripheral nerve myelin in vitro: Effect of antibody
Authors: Smith M. E. , Sadler R. H. , Dyer C. , Benjamins J. A. , Yu A. C. H.
Published: January 2005

We have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C‐labeled PNS increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole Po and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to al most the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self‐associate to form large clumps which were bound to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin. The presence of high amounts of glycoprotein in PNS myelin may account for this difference in binding to macrophages, either through self‐adhesion of the PNS myelin particles, or by the presence of carbohydrate receptors in macrophages. These results may define a role for antibody in myelin destruction in cell‐mediated demyelinative diseases, and may be relevant to the differences seen in vivo between the rates of demyelination of peripheral and central nerves.
Differential regulation of peripherin and neurofilament gene expression in regenerating rat DRG neurons
Authors: Wong J. , Oblinger M. M.
Published: January 2005

Quantitative in situ hybridization and RNA blotting methods were used to define the time course and magnitude of changes in expression of mRNAs encoding peripherin and the neurofilament (NF) triplet proteins in rat dorsal root ganglion (DRG) neurons during axonal regeneration. mRNA levels in adult rat L4 and L5 DRGs were examined in autoradiograms after in situ hybridization with specific 35S‐labeled cDNA probes 1–56 days following unilateral crush lesions of the sciatic nerve. The results of quantitative analyses indicated that peripherin mRNA levels were significantly increased in large‐sized (>1000 μm2) DRG neurons at 7, 14, and 28 days after axotomy while the mRNA levels for each of the NF triplet proteins were significantly decreased at these same time points. The mRNA levels of the low (NF‐L) and middle (NF‐M) sized NF subunits were significantly decreased as early as 1 day postaxotomy but the mRNA level of the large NF subunit (NF‐H) did not change until 7 days after axotomy. The maximal reduction in NF mRNA levels was observed at 14 days postaxotomy when NF‐L mRNA levels were only 35% of those in large‐sized, normal control neurons. Recovery toward normal levels of both NF and peripherin mRNAs was observed at 8 weeks postaxotomy. RNA blot analyses with total RNA obtained from DRGs at different postaxotomy times confirmed that NF‐L mRNA levels were reduced in the DRG during the first 4 weeks after axotomy but, interestingly, failed to detect an increase in peripherin mRNA levels. This difference concerning peripherin mRNA levels in axotomized preparations obtained by RNA blotting vs. in situ hybridization was attributed to the fact that RNA blots utilized total DRG RNA which includes mRNAs from both small and large‐sized DRG neurons. A recent in situ hybridization study showed that the small‐sized DRG neurons which contain the majority of the peripherin mRNA in the DRG do not increase their peripherin mRNA levels 14 days after axotomy (Oblinger et al., 1989b). This may mask any change in the large neuron response when total RNA is examined. Overall, the results of this study demonstrate (1) that type III (peripherin) and type IV (NF) intermediate filament genes are regulated differently during axonal regeneration, and (2) that the three NF genes are down‐regulated in a fairly coordinate manner during regeneration. These data suggest that an important component of the regeneration program is the alteration of the composition of the IF component of the cytoskeleton. A cytoskeleton with fewer Type IV IFs and more Type III IFs may be more compatible with the specific structural and functional requirements of regenerating neurons.
6‐[18F]Fluoro‐L‐DOPA probes dopamine turnover rates in central dopaminergic structures
Authors: Barrio J. R. , Huang S. C. , Melega W. P. , Yu D. C. , Hoffman J. M. , Schneider J. S. , Satyamurthy N. , Mazziotta J. C. , Phelps M. E.
Published: January 2005

6‐[18F]Fluoro‐L‐DOPA (FDOPA) cerebral kinetics and metabolism were correlated in normal primates (Macaca nemestrina) and primates with 1‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) induced unilateral Parkinsonism. Application of a tracer kinetic model to positron emission tomography (PET) data indicated that the model allows reliable estimation of FDOPA blood brain barrier transport, decarboxylation and release of stored 6‐[18F]fluorodopamine (FDA) radioactivity in normal striatum (k4 = 0.005/min, turnover half‐time ≥ 2 hr), in agreement with biochemical data. PET scans of MPTP treated monkeys revealed 40‐50% reduction in total striatal activity in comparison with pre‐MPTP scans. Monkey brain biochemical analysis revealed that the reduction in activity was mainly due to a decrease in FDA and its metabolites, 6[18F]fluorohomovanillic (FHVA) and 6‐[18F]fluoro‐3, 4‐dihydroxyphenylacetic acid (FDOPAC). The remaining activity in tissue was 3‐0‐methyl‐6‐[18F]fluoro‐L‐DOPA (3‐OMFD) of peripheral origin. The (FHVA + FDOPAc)/FDA ratio was 1:2 in normal putamen and ≥6:1 in the lesioned putamen, indicative of a dramatic increase in turnover of FDA. Both kinetic and biochemical data indicate that FDOPA labels a slow turnover rate pool of dopamine in rat and primate brain. This turnover rate for stored dopamine (DA) is accelerated with dopaminergic cell losses (e.g., MPTP‐induccd Parkinsonism).
Reverse‐phase high‐performance liquid chromatography of nerve growth factor receptor‐like proteins identified with monoclonal antibodies
Authors: Shan D.‐E. , Beck C. E. , Werrbach‐Perez K. , Perez‐Polo J. R.
Published: January 2005

Human neuroblastoma SK‐N‐SH‐SY5Y (SY5Y) and rat pheochromocytoma PC12 cells are model cell lines used in the study of nerve growth factor (NGF) effect. The effects of NGF are initiated by binding to cell surface receptors (NGFR). The amino acid sequence for NGFR has been deduced based on the identification of a single gene for NGFR. However, there are two kinds of NGF binding activities and several reported molecular weights of NGFR. We report here on the demonstration of NGFR‐like proteins from PC12 and SY5Y cells by sequential lectin chromatography, reverse‐phase HPLC, and SDS‐PAGE analysis of immunoprecipitates obtained with NGFR‐specific monoclonal antibodies. For both human and rodent NGFR, there was a tendency for the higher molecular‐ weight species of NGFR‐like proteins to be eluted in more hydrophobic fractions. Also, the expression of different species of NGFR could be modified by treatment with retinoic acid (RA). These results are consistent with the hypothesis that the different molecular species of NGFR may result from the generation of a truncated form of NGFR, the presence of sugar residues on the NGFR protein, dimer formation between NGFR, or the association of NGFR with a receptor‐ associated protein.