bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractTo 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

AbstractThree 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

AbstractNeurotensin 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

AbstractPeripheral 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

AbstractBy 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

AbstractPeptides 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

AbstractImmunohistochemical 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

AbstractBy 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

AbstractThe 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

AbstractFura‐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

AbstractA 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

AbstractCell 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

AbstractPermeability‐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.
Mitotic potential of adult rat oligodendrocytes in culture
Authors: Vick R. S. , DeVries G. H. 
Published: January 2005

AbstractThe proliferation of adult oligodendrocytes (OLGs) was examined in response to membrane bound and soluble mitogens. OLGs were isolated according to Vick, et al., J Neurosci Res 25:524–534, 1990, and co‐cultured with dorsal root ganglia (DRGs). Less than 5% of the total cells incorporated a 48 hr pulse of 3H thymidine during the first 4 days of co‐culture. From day 4 to day 6 there was a dramatic increase in proliferation which reached a plateau at 40% and gradually decreased to 25% from days 10 to 20 of co‐culture. Axolemma‐enriched fractions (AEF) were weak mitogens for OLGs (less than 5% proliferation after 7 days of stimulation), however, heparin extracts of AEF were five‐fold more mitogenic than the AEF from which they were derived. Basic and acidic fibroblast growth factor were effective mitogens for the adult OLGs (labelling indices of 28% and 12%, respectively) provided that the cells were treated for 7 days with the growth factor and that the cells had been in culture for at least 14 days. Other soluble growth factors (IL‐2 and PDGF) gave no mitotic response. We conclude that adult OLGs are mitotically responsive to mitogens provided that (1) the adult OLG has been cultured for sufficient time (14 days, acidic or basic fibroblast growth factor); (2) the axonal mitogen is allowed to interact with the OLG for a sufficient time (neuritic mitogen); and (3) the axonal mitogen is presented to the OLG in an activated form (AEF‐heparin extract). © 1992 Wiley‐Liss, Inc.