摘要:

AbstractThe 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 ge

ISSN:0360-4012    

DOI:10.1002/jnr.490280402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe ionic mechanism of the effect of extracellularly ejected recombinant human tumor necrosis factor‐alpha (rhTNF‐α) on the membrane of identified neurons R9 and R10 ofAplysia kurodaiwas investigatedwith conventional voltage‐clamp, micropressure ejection, and ion substitution techniques. Micropressureejected rhTNF caused a marked hyperpolarization in the undamped neuron. Clamping the same neuron at its resting potential level (–60 mV) and reejecting rhTNF‐α with the same doseproduced a slow outward current [Io(TNF)] associated with a decrease in input membrane conductance. Io(TNF) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io}(TNF) was approximately +10 mV. Ion substitution and pharmacological experiments suggest that I}o(TNF) in identified neurons R9 and R10 ofA. kurodaiis due to a decreased Na+conductance but not due to an activation of the Na+‐K+Pump. Our results demonstrate that the immunomodulator TNF can act directly on the nervous system as well as on the

ISSN:0360-4012    

DOI:10.1002/jnr.490280403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractTo elucidate the relationship between amyloid fibril formation in Alzheimer disease (AD) and the primary structure of the β‐amyloid protein (β‐AP), we investigated the ability of peptides sharing sequences with β‐AP to form fibrils in vitro and to recognize anti‐β‐amyloid antisera. The peptides, which were synthesized using a FMOC solid phase procedure and purified by HPLC, consisted of residues 6–25 from the putativeaqueous domain, residues 22–35, which overlaps the putative aqueous and transmembrane domains, and residues 1–38 and 1–40 representing nearly the full length of β‐AP. Electron microscopy of negative‐stained or thin‐sectioned preparations revealed that the peptides assembled into fibrils having different morphologies, some of which resembled in situ AD amyloid. Peptide 6–25 fibrils had diameters of 50–80 å and occasionally showed a central groove suggestive of constituent filaments. Cross sections of the fibril showed a penta‐ or hexameric arrangement ofglobular subunits with diameters of 25–30 å. Peptide 22–35 fibrils were helical, with a pitch of 1,100 å and a width of 120 A at its greatest and 50–60 å at its narrowest. The fibrils formed by peptides 1–38 and 1–40 were 70–90 å in diameter. When the peptide assemblies were singly oriented by sedimentation or doubly oriented in a magnetic field, their X‐ray diffraction patterns all showed reflections typical of a cross‐β pleated sheet conformation. The patterns differed mainly in their small‐angle equatorial intensity, which arises from the packing of fibrils having different widths. Antiserum raised to either native amyloid or to synthetic peptide β‐(l–28) was highly reactive in an inhibition‐ELISA assay to β‐(6–25) and β‐(1–38), but not to β‐(22–35), and immunostained β‐(1–40) on Western blots. These studies show that the β‐(6–25), β‐(l–38) and β‐(

ISSN:0360-4012    

DOI:10.1002/jnr.490280404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe cell line PC12, derived from a rat pheochromocytoma, has served as a model for studies on the mechanism of action of nerve growth factor, as well as for the exploration of neuronal differentiation in general. When treated with nanomolar concentrations of nerve growth factor, these neoplasticchromaffin‐like cells stop dividing and acquire, for all intents and purposes, the phenotype of mature sympathetic neurons. This phenotype is characterized by the extensive outgrowtb of electrically excitable neurites, the ability to form functional synapses, and the acquisition of a number of biochemical markers.Treatment of PC12 cells with retro viral vectors encoding the K‐ras, the N‐ras, or the v‐src oncogenes also produces a marked morphological differentiation very similar to that seen upon treatment with nerve growth factor. Treated cells stop dividing and develop an extensive network of neurites. It has recently been shown that PC12 cells differentiated with v‐src, while resembling, morphologically, those treated with nerve growth factor, differ substantially in the biochemical characteristics normally associated with nerve growth factor‐induced differentiation.Cells infected with K‐ras also develop a neurite network similar to that seen after treatment with nerve growth factor. In addition, such cells develop tetanus toxin‐binding sites and saxitoxin‐binding sites, as do cells treated with nerve growth factor. Decreases in the binding of epidermal growth factor and in the activity of calpain also occur and these, as well, are characteristic of nerve growth factor‐treated cells. But the adhesive properties of cells infected with K‐ras are different than those of nerve growth factor‐treated cells, and the former do not show an increase in the NILE glycoprotein. Finally, K‐252a, an inhibitor of the actions of nerve growth factor on PC 12 cells, has no effect on the neurite outgrowth produced by infection with K‐ras. Thus, many of the key markers of nerve growth factor‐induced differentiation of PC 12 cells also appear upon differentiation with K‐ras, but there are, nevertheless, some crucial differences in the proper

ISSN:0360-4012    

DOI:10.1002/jnr.490280405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractPrevious studies from this laboratory have shown that C‐6 rat glioma cells (2B clone) exhibit specific phenotypic characteristics depending on passage in culture and that these populations respond differentially to addition of various exogenous compounds to the medium. Early passage (90% of the phospholipid was taken into the cells within the first hour of incubation. We compared the PAF effects with that of dibutyryl cyclic AMP (dBcAMP) and RO20‐1724, a phosphodiesterase inhibitor. Cells from the early passage responded to both dBcAMP and RO20‐1724 treatments with a significant increase in GS activity whereas cells from the late passage showed no significant change, confirming earlier reports from this laboratory. These findings indicate that the response of C‐6 glioma cells to PAF (at least in the late passage) is not mediated via cyclic AMP. We suggest that in early passage cells PAF promotes expression of the astrocytic phenotype and in late passage cells PAF mediates a gl

ISSN:0360-4012    

DOI:10.1002/jnr.490280406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractResponses of rat embryonic septal cells to reconstituted basement membrane, laminin, and laminin A chain‐derived synthetic peptides were studied in culture. Dissociated fetal E16/17 septal cells were grown for three days on differently coated plastic substrata. Reconstituted basement membrane (Ma‐trigel), laminin, and a 19‐amino acid synthetic peptide CSRARKQAASIKVAVSADR‐NH2(PA22‐2) from the laminin A chain sequence mediated cell‐substratum adhesion and promoted neurite outgrowth. In contrast, cells did not attach to or form processes on uncoated plastic or on plastic substrata coated with synthetic, laminin‐derived control peptides. Polyethylenimine (PEI) supported the adhesion and survival of fetal septal cells; however, when lami‐nin was added to the medium during cell plating or 18 hr afterward, a dose‐dependent increase was observed in neurite outgrowth of cells attached to this substratum. Cells grown for 6 days on PEI in the presence of laminin showed a determined increase in the number of cholinergic neurons as marked by acetylcholinesterase staining. These data suggest that the subpopulation of cholinergic septal neurons present in the septal cells studied here were also responding to laminin. The results of this in vitro study suggest potential uses for basement membrane, laminin, or synthetic peptides, sucha as PA22‐2, in fetal septal grafts to enhance regeneration in the damaged septo

ISSN:0360-4012    

DOI:10.1002/jnr.490280407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe presence of cytochrome P‐45011βin 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‐45011βimmunoreactivity is selectively localized to the tracts of myelinated fibers throughout the brain. The specificity of immunohistochemical stainings with P‐45011βantibodies was established by control tests including nonimmune rabbit immunoglobulin Gs and P‐45011βantibodies absorbed with purified antigen.Western immunoblots of homogenates from different brain areas with P‐45011βantibodies, 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‐45011βand 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‐45011βbands 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‐45011βwas 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‐45011βin 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 tract

ISSN:0360-4012    

DOI:10.1002/jnr.490280408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe 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

ISSN:0360-4012    

DOI:10.1002/jnr.490280409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractAntibodies against the mitochondrial enzyme glutaminase (EC 3.5.1.2), have been used in previous immunocytochemical studies to help identify glutamatereleasing neurons among all glutamate‐containing neurons. The studies were based on the idea that glutaminase is enriched within the releasable “transmitter” pools of glutamate. However, evidence is also available to suggest that the enzyme does not occur exclusively within glutamate‐releasing neurons. Thus we sought to determine whether glutaminase was immunocytochemically detectable within presynaptic terminals forming asymmetric (putatively excitatory) synapses or, alternatively, occurs in association with mitochondria throughout the cell. For this purpose, we examined the cellular and subcellular distribution of glutaminase‐ immunoreactivity in neocortical (vi‐sual and somatosensory) areas known to contain glutamatergicperikarya. This localization was compared with the distribution in striatal(caudateputamen and nucleus accumbens) regions recognized to contain high densities of glutamatergic terminals out fewer, if any, glutamatergic perikarya. Glutaminase‐immunoreactive perikarya were numerous within the infragranular, lamine of neocortex (∼1 per 1,000 μm2tissue area) but sparse within me caudate‐putamen nuclei and accumbens nuclei, (

ISSN:0360-4012    

DOI:10.1002/jnr.490280410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA low concentration of transition metal ions Co2+and Ni2+increases the inward current density in neurons from the land snailHelix aspersa. The currents were measured using a single electrode voltage‐clamp/internal perfusion method under conditions in which the external Na+was replaced by Tris+the predominant external current carrying cation was Ca2+and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was addedexternally to block K+carrent. In presence of Co2+(3 mM) Ni2+Ca2+currents were stimulated normally by voltage‐dependent activation of Ca2+channels. There was a 5‐10% decrease in the rate of rise of the inward current The principal effect of Co2+and Ni2+current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage‐dependent or Ca2+‐dependent outward current and/or an inhibition of Ca2+channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+efflux through the voltage activated or Ca2+‐activated nonspecification channels. Co2+is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normalHelixRinger solution showed that the Ca2+‐dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co2+‐dependent increase in inward (depolarizing) current seen in voltage‐

ISSN:0360-4012    

DOI:10.1002/jnr.490280411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY