bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurons from substantia nigra increase the efficacy and potency of second messenger arising from striatal astroglia dopamine receptor
Authors: Hansson Elisabeth , Rönnbäck Lars
Published: January 2005

Dopamine (D1) receptors were demonstrated to be present on astroglial cells from striatum in primary culture. In a cocultivation system, the astrocytes were influenced by neurons from one of their natural projection areas (substantia nigra) to increase the efficacy and potency of second messenger (cyclic AMP) from the dopamine receptor. This provides evidence for a heterogeneity among astroglia from the various brain regions with respect to the expression of receptors.
Astroglial differentiation in the opossum superior colliculus
Authors: Barradas Penha C. , Cavalcante Leny A. , Mendez‐Otero Rosalia , Vieira Alex M.
Published: January 2005

Glial markers, namely, vimentin, glial fibrillary acidic protein (GFAP), and glycogen, as well as accumulation of axon‐borne horseradish peroxidase (HRP), were used to visualize radial glial cells in the developing opossum superior colliculus (SC) and to follow changes in young astrocytes of the superficial layers. Vimentin, GFAP, and glycogen are relatively abundant in elements of the median ventricular formation (MVF), which persists at least as late as weaning time, i.e., postconception day 103, postnatal day 90 (PND90). Radial profiles and end‐feet in the remaining collicular sectors (main radial system, MRS) are also vimentin‐positive but show little or no glycogen or anti‐GFAP staining. The numeric density of MRS profiles is very high at the final stages of neuronal migration (PND12) but falls to vestigial numbers by PND 56–60. Antivimentin staining and filling of MRS profiles by axon‐borne HRP disappear in parallel. Before total regression of MRS profiles, young astrocytes of the superficial gray layer exhibit a transiently high GFAP expression that is not found in those of the subjacent layers. The results suggest that (1) radial glia at or near the collicular midline are well equipped for a mechanical supportive role, and their abundant glycogen accumulation may reflect their eventual transformation in cells with high glycolytic metabolism, including tanycytes; (2) in most collicular sectors, some radial glia cells persist for long periods after cessation of neuronal migration and may interact with afferent fibers coursing through the superficial neuropil; (3) radially oriented astrocytes of the superficial gray layer exhibit a transiently high GFAP expression that is temporally correlated with late transformations of the retinocollicular projections.
Uptake, metabolism, and release of [3H]‐histamine by glial cells in primary cultures of chicken cerebral hemispheres
Authors: Huszti Zsuzsanna , Rimanóczy Ágnes , Juhász Anna , Magyar Kálmán
Published: January 2005

Labelled histamine was taken up into cultured glial cells of chick embryonic brain by a system with high affinity for histamine and diffusion. The active uptake, occurring at low concentrations of the amine, was Na+ dependent and gave an apparent Km of 0.24 μM and a Vmax of 0.31 pmol X mg protein−1 X min−1. The uptake was completely blocked by desmethylimipramine (Ki = 2.5 μM) and partially by the histamine agonists and histamine‐N‐methyltransferase blockers 4‐methylhistamine and 2‐methylhistamine (I30 values obtained were 2 μM and 5 μM). Other psychoactive drugs were either ineffective (imipramine) or they showed moderate inhibitory effects (amitryptyline and cocaine). Ouabain (100 μM) inhibited uptake by ∼50%. Diffusion occurred at high concentrations of the amine, was insensitive to extracellular Na+, and was proportional to histamine concentration up to 1 mM.

[3H]‐Histamine, taken up into the cells, was metabolized and/or released. The spontaneous efflux of the radioactivity measured after 10 min of exposure to [3H]‐histamine (when most of it was still unmetabolized), was moderately Ca++ dependent, accelerated by both reduced concentrations of extracellular Na+ and enhanced concentrations of K+ and inhibited by desmethylimipramine. After prolonged (60 min) incubation, histamine metabolites detected in the cells presented 78% of the chromatogram radioactivity and consisted of Nτ‐methylhistamine and Nτ‐methylimidazole acetic acid.

These results indicate that at low nM concentrations, histamine is taken up and metabolized by (and released from) glial cells by an Na+‐dependent system, and the intracellular metabolism seems to serve an increased uptake of the amine.
Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system
Authors: D'Amelio Fernando , Eng Lawrence F. , Gibbs Michael A.
Published: January 2005

Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.
Extracellular potassium influences DNA and protein syntheses and glial fibrillary acidic protein expression in cultured glial cells
Authors: Canady Karen S. , Ali‐Osman Francis , Rubel Edwin W.
Published: January 2005

Previous reports of increases in glial cell number and expression of glial fibrillary acidic protein (GFAP) in stimulated brain regions or epileptic tissue have implicated a role for increases in extracellular potassium concentration ([K+]o) in glial reactions. We examined the effects of altered [K+]o on DNA and protein syntheses and GFAP expression of cultured glial cells isolated from the posthatch chick brain stem. [K+]o was varied by adding both KCl and NaCl to K+, NaCl‐free medium to achieve final [K+] of 1–50 mM. DNA and protein syntheses were measured by incorporation of 3H‐thymidine and 3H‐leucine, respectively, into acid‐insoluble material. GFAP expression was measured by a dot‐immunoblotting assay. DNA synthesis in glial cells cultured in high (5–50 mM) K+o was 45–60% less than that of cells cultured in low (1–3 mM) K+o. Protein synthesis per cell was increased 34–44% in cells cultured in high K+ as compared to those cultured in low K+. GFAP expression was inversely related to [K+]o over the 1–10 mM range. Compared to the baseline of 3 mM K+o, GFAP per cell was increased 65% at 1 mM and decreased 45% at 10 mM. These data suggest that increases in glial cell number and GEAP immunoreactivity found in sites of increased neuronal activity and in pathological tissues may not be caused solely by persistent increases in [K+]o. Instead, these results suggest that neuronal activity, through the release of K+, may have an inhibitory influence on glial proliferation and GFAP expression. In light of work by others implying a relationship between GFAP immunoreactivity and rigidity of astroglial processes together with the data presented here, we suggest that the elevated [K+]o accompanying neuronal activity, by inhibiting GFAP expression, may facilitate the morphological plasticity of glial cells. Conversely, conditions of low [K+]o may contribute to rigidity of astrocytic processes.
Stretch‐activated channels in human retinal muller cells
Authors: Puro Donald G.
Published: January 2005

The cell‐attached and excised patch configurations of the patch clamp technique were used to study stretch‐activated ion channels in Muller glial cells that were obtained from postmortem adult human retinas and were maintained in culture. A stretch‐activated channel permeable to monovalent and divalent cations was found. Ion channels of this type have not been demonstrated previously in glial cells, though indirect evidence has suggested that such stretch‐activated channels may help mediate a compensatory response of glia to swelling. Consistent with a possible role for volume regulation is the finding that the activation of calcium‐permeable stretch‐sensitive channels is associated with an increase in the activity of calcium‐activated potassium channels. Activation of potassium channels to produce an efflux of potassium with a subsequent loss of anions and cell water could be an effective mechanism to decrease glial cell volume.
Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. II. Differences in cultures prepared from different brain regions
Authors: Amundson R. H. , Goderie S. K. , Kimelberg H. K.
Published: January 2005

Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4‐day‐old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na+‐dependent, [3H] L‐glutamate, and fluoxetine‐sensitive [3H] 5‐HT uptake varied markedly. The relative order of uptake for [3H] 5‐HT was MS (mesencephalon) > CC (cerebral cortex) > BG + DI (basal ganglia + diencephelon) > HP (hippocampus) > BS (brain stem) > CB (cerebellum). For [3H] L‐glutamate the order was HP > CC > BG + DI > MS = BS > CB. For [3H] 5‐HT this essentially corresponds to the reported order of binding in situ of the [3H] 5‐HT‐specific uptake ligand [3H] citalopram. For [3H] L‐glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double‐label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65–98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra‐ and inter‐regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo. © 1992 Wiley‐Liss, Inc.
Intracellular pH regulation in single cultured astrocytes from rat forebrain
Authors: Boyarsky Gregory , Ransom Bruce , Schlue Wolf‐R. , Davis Marion B. E. , Boron Walter F.
Published: January 2005

We used the fluorescent pH‐sensitive dye 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) to monitor intracellular pH (pHi) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO2/HCO3− ‐free medium buffered to pH 7.40 with HEPES at 37°C, the cells had a mean pHi of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO3− caused the steady‐state pHi to increase by an average of 0.35, suggesting the presence of a HCO3− ‐dependent acid‐extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3−‐free (HEPES‐buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pHi to fall to a value substantially below the initial one. pHi spontaneously recovered from this acid load, stabilizing at a value ∼ 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cells bathed in HEPES‐buffered solutions, removing extracellular Na+ caused pHi to decrease rapidly by 0.5. Returning the Na+ caused pHi to increase rapidly, indicating the presence of an Na+‐dependent/HCO3−‐independent acid‐extrusion mechanism; the final pHi after returning Na+ was ∼ 0.08 higher than the initial value. This pHi recovery elicited by returning Na+ was not substantially affected by 50 μM ethylisopropylamiloride (EIPA), but was speeded up by 50 μM 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS). Increasing [K+]− from 5 to 25 mM caused pHi to increase reversibly by ∼ 0.2 in nominally CO2/HCO3−‐free solutions, and by ∼ 0.1 in CO2/HCO3−‐containing solutions, although the initial pHi was ∼ 0.17 higher in the presence of CO2/HCO3‐. These results suggest the presence of a depolarization‐induced alkalinization. Our results suggest the presence of both HCO3− dependent and ‐independent acid‐base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pHi is sensitive to changes in either membrane voltage or [K+]0 per se. © 1993 Wiley‐Liss, Inc.
A standardized interview for the elderly (GMS): Reliability studies comparing the Dutch language version with the original
Authors: Hooijer C. , Jonker C. , Dewey M. E. , Van Tilburg W. , Copeland J. R. M.
Published: January 2005

A Dutch translation of the Geriatric Mental State Schedule was prepared and used. Here we report the replication of previous reliability (inter‐rater and retest) studies and present the first figures for the reliability of factor scores. The results have proved valuable for solving some (apparent) discrepancies between previous studies. Overall reliability results were shown to be satisfactory. Using the GMS scores Dutch patient groups appeared to be similar to the US‐UK project patient samples. Preliminary results of a small follow‐up study are provided. The total set of results makes it plausible that Dutch findings can be generalized and the use of the GMS in Holland can be extended.
Jim who? Recall of national leaders by elderly people in three countries
Authors: Ames David , Ashby Deborah , Flicker Leon , Snowdon John , West Christopher , Weyerer Siegfried
Published: January 2005

The Organic Brain Syndrome scale (OBS) of the Brief Assessment Scale (BAS) was administered to 1202 elderly subjects in London, Mannheim, Sydney and Melbourne. Subjects' knowledge of current and last national leaders was assessed, and two methods of scoring the OBS were compared. Taking into account the degree of cognitive impairment found using seven items of the OBS were compared. Taking into account the degree of cognitive impairment found using seven items of the OBS other than the national leader item, knowledge of current leader was much more consistent across countries than was knowledge of last leader. When subjects were allocated an error point for failing to know either current or last leader, dementia prevalence measured by the OBS was consistently 5–6% less than that found when they were allocated an error point for failing to know both leaders. Future studies using the BAS should publish prevalence data for both scoring methods in order to permit comparison between studies.
Nosocomial transmission of HCV in the liver unit of a tertiary care center
Authors: Forns Xavier , Martínez-Bauer Eva , Feliu Anna , García-Retortillo Montserrat , Martín Marta , Gay Eugeni , Navasa Miquel , Sánchez-Tapias Jose María , Bruguera Miquel , Rodés Juan
Published: January 2005

Despite its medical and legal implications, there are no prospective studies analyzing the incidence and mechanisms involved in the nosocomial transmission of hepatitis C virus (HCV) in liver units. This study prospectively investigates the nosocomial transmission of HCV in the liver unit of a tertiary care center from August 2000 to October 2002. The median prevalence of HCV infection among hospitalized patients was 50%. Anti-HCV–negative patients admitted to the liver unit during the study period were prospectively followed, and serum markers of HCV infection were repeated 6 months after discharge. All known risk factors for HCV transmission (including the physical allocation of HCV-infected and noninfected patients during hospitalization) were recorded. Complete follow-up data were available in 1,301 (84.5%) of 1,540 patients. Six patients (0.46%) acquired HCV infection (annual incidence: 0.27/100 admissions). Phylogenetic analyses of recovered HCV sequences identified the source of infection as an HCV-infected roommate (3 cases) and a patient receiving care by the same nurse team (1 case). The most relevant risk factors associated with HCV acquisition were duration of hospitalization (>10 days; OR, 35; 95% CI, 1.96-622) and hospitalization with an HCV-infected roommate (>5 days; OR, 12; 95% CI, 1.39-103). In fact, HCV infection occurred in 1.7% of the 357 patients hospitalized longer than 10 days.
In conclusion,

HCV nosocomial infection appears to occur via patient-to-patient transmission in liver units, particularly in individuals who require long hospitalizations. Continuous reinforcement of universal prevention measures and, when possible, isolation of patients at higher risk might further reduce nosocomial HCV transmission. (Hepatology 2005;41:115–122.)
The role of cell cycle on polyplex‐mediated gene transfer into a retinal pigment epithelial cell line
Authors: Männistö Marjo , Rönkkö Seppo , Mättö Mikko , Honkakoski Paavo , Hyttinen Mika , Pelkonen Jukka , Urtti Arto
Published: January 2005

Background

Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non‐viral gene delivery into a RPE cell line (D407).

Methods

D407 (human) cells were transfected with cationic DNA complexes. Cells were synchronized into different phases of the cell cycle and transfected using poly‐L‐lysine (PLL) or polyethyleneimine (PEI) carriers for 3 h. The effects of different reporters (β‐galactosidase, luciferase) or promoters (CMV, SV40, tk, PDE‐β) on gene expression were evaluated 43 h later. Cellular uptake of ethidium monoazide/DNA complexes with PLL or PEI was determined by flow cytometry. Fluorescent DNA and the complexes were localized with a confocal microscope. The role of cell cycle in transcription was evaluated by stable luciferase‐expressing cells.

Results

PLL showed lower transfection levels than PEI in synchronized cells and only slight dependence on cell cycle. PEI showed minimal efficiency at G1 phase and maximum level at S phase. All promoters and reporter genes showed dependence on cell cycle. Cellular uptake of polyplexes was highest at S phase (80–90%) and lowest at G1 phase (5–30%). Confocal microscopy showed minor differences of free DNA between groups in the nucleus, where it was largely carrier‐bound. Cell cycle effects on luciferase expression were clear in stable cell line

Conclusions

Transfection by polyplexes in the RPE cell line is influenced by cellular uptake and transcription, and both processes are cell‐cycle‐dependent. The results have implications in retinal gene therapy. Copyright © 2004 John Wiley & Sons, Ltd.
The use of microchemical techniques for the identification of new transmitter molecules in neurons
Authors: Giacobini E.
Published: January 2005

The author reviews available microtechniques and results on cellular analysis of single neurons emphasizing the identification of putative neurotransmitter molecules.

Highly sensitive microquantitative methods for identification and assay of known and putative neurotransmitters have been developed within the last 5 years. Several of the methods reported in the article fulfill the requirements of both specificity and sensitivity for exploring and measuring the level of transmitter molecules in single neurons.

Some of the available techniques, mostly those which associate GC or TLC with MS, have been able to unambiguously demonstrate the presence of putative neurotransmitter molecules, or at least of substances which are strongly suspected of fulfilling a functional role in the nervous system.
Topographical distribution of acetylcholinesterase and butyrylcholinesterase in the cerebral hemisphere of the garden lizard, calotes versicolor
Authors: Sethi J. S.
Published: January 2005

Differential patterns of distribution of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are observed in the cerebral nuclei and fiber tracts of Calotes versicolor. The majority of the nuclei reveal a positive reaction for AChE varying from moderate to intense. In contrast, almost all the nuclei are found negative to BChE, except for the area of the nucleus commissurae anterioris and of the paleostratum where the positive activity is due to BChE positive fibers. The medial forebrain bundle demonstrates intense activity for AChE, while the lateral forebrain bundle and commissures present a negative picture. Interestingly, all the fiber bundles and commissures show an intense reaction for BChE. The nature of nuclei and fiber tracts in relation to the differential patterns of AChE and BChE is discussed.
A model of GABA transport by cortical synaptosomes from the long‐evans rat
Authors: Wheeler D. D. , Hollingsworth R. G.
Published: January 2005

The initial velocity of uptake of GABA by cortical synaptosomes from the Long‐Evans rat has been measured as a function of both sodium and GABA concentration. These data were then fitted to the rate equation for each of several possible models, and the models giving least error were identified. Although one cannot unequivocally distinguish between two of the models on the basis of the goodness of fit, deductions based on the fits of subsets of the models point to one of the two models. The major predictions from this model are as follows: (1) Uptake of GABA is totally sodium‐dependent. (2) Although plots of 1/v versus 1/[Na]2 are nonlinear, the coupling ratio for transport (Na/GABA) is 2. (3) For transport to take place, the order of combination with carrier must be Na, Na, GABA. (4) Maximal velocity will occur only at infinite Na and GABA concentrations. (5) There is a sigmoidal relationship between apparent maximal velocity (Va) and [Na]. (6) Kt, the [GABA] that gives a velocity equal to Va/2, rises and then falls as [Na] is increased from zero, passing through a maximum at 33.52 mM [Na]. (7) The relationship between initial velocity and [Na] is sigmoidal. (8) Jm, rate of uptake with infinite [Na], is hyperbolically related to [GABA]; Jm approaches Vmax as [GABA] becomes very large. (9) KNa, the [Na] giving a velocity equal to Jm/2, declines rapidly from 10−7M to 10−5M GABA, but is essentially constant at 10−4M and above. (10) One GABA molecule is translocated per carrier molecule.

The model is consistent with observations in the literature concerning the effects of sodium on the kinetics of transport.
Cerebral amino acid levels and uptake in rats after portocaval anastomosis : I. Regional studies in vitro
Authors: Zanchin Giorgio , Rigotti Paolo , Bettineschi Fulvio , Vassanelli Paolo , Battistin Leontino
Published: January 2005

Uptake of various amino acids was studied in slices from brain regions of rats four weeks after portocaval anastomosis.

No differences of the inulin compartment were observed between control and experimental animals.

After 60‐minutes incubation, uptake showed an overall pattern of diminution. This was more evident for some amino acids: valine, methionine, and lysine exhibited a lowering of about 30%, which was fairly uniform in the four tested regions; others showed a regional decrease – alanine in pons‐medulla, phenylalanine in cerebellum, histidine and GABA in mesodiencephalon.

This decrease did not seem to be related to transport classes. The restricted entry of amino acids into brain cells in portocaval encephalopathy is somewhat difficult to explain; a decreased rate of protein synthesis may be of some importance, but other factors, such as a “carrier” impairment, effects on release and on amino acid metabolism, may also be involved.
Effects of anesthetics on sulfate transport in the red cell
Authors: Makriyannis Alexandros , Fesik Stephen W.
Published: January 2005

We have measured the effect of anesthetics on sulfate transport in the human erythrocyte and have found that a wide variety of chemical classes of anesthetics, including the anesthetic steroid alphaxalone, inhibit sulfate transport in human erythrocytes at concentrations paralleling the concentrations that block nerve conduction. These experiments suggest that anesthetics act through a general mechanism and that the sulfate transport system in the red blood cell is a good model for studying the mechanism of action of anesthetics.
Separation of radiolabelled protein from brain and spinal cord of spinal hemisected rats on SDS polyacrylamide slab gels
Authors: Wells Michael R.
Published: January 2005

After hemisection of the spinal cord in rats, alterations in amino acid incorporation into protein have been observed in both brain and spinal cord. Proteins mediating these changes have been studied in the present experiment. Male, Long‐Evans hooded rats were given either a laminectomy and dura incision (sham) at spinal segment T2, a left spinal cord hemisection, or no operative procedures. One hour prior to utilization at 1, 3 and 14 days postoperation, animals were injected subcutaneously with 200 μCi of [3H]‐L‐lysine and 200 μCi of [3H]‐L‐amino acid mixture. Samples were prepared for electrophoresis and proteins separated on linear gradient SDS polyacrylamide slab gels (7.5–20% acrylamide). The Gel slabs were cut into slices and processed for scintillation counting. In right somatomotor cortex, there was evidence for a general stimulation of amino acid incorporation at one day postoperation in animals which received surgery. At other postoperation intervals, significant increases in proteins in the regions of 90–150,000, 50–65,000 and 35–45,000 molecular weight (MW) were observed. In spinal cord, significant increases in proteins in the regions of 55–65,000, 30–45,000 and 15,000 MW were observed over postoperation time in all animals receiving surgery compared to normals. A large, significant increase in the radioactivity of proteins in the region of 18–20,000 MW were observed in the spinal cord lesion site of spinal hemisected animals alone at three days postoperation. These results indicate that increases in amino acid incorporation into the brain and spinal cord of laminectomized and spinal hemisected animals observed previously are the result of both specific and nonspecific changes in protein incorporation of precursors.
The fate of tritium‐labeled reserpine in the chick embryo: A preliminary report
Authors: Dahl Anne‐Lise , Giacobini E. , Serra G. , Manara L.
Published: January 2005

After a single administration of 3H‐reserpine, 10 μgm, 2.5 μCi, into the yolk of fertilized chicken eggs, gross radioactivity (reserpine plus degradation products) and the unchanged labeled drug, separated by thin layer chromatography, were assayed on the 8th and 19th day of incubation. Nanogram per gram concentrations of unchanged 3H‐reserpine were recovered at both times. The 3H‐reserpine recovered in the whole egg on the 8th day of incubation accounted for up to 20% of the administered dose. On the 19th day of incubation, brain showed the largest share (28%) of 3H‐reserpine as a percentage of gross radioactivity of all organs investigated. Reserpine underwent considerable biodegradation in the developing chick embryo, but the concentrations of unchanged drug recovered in embryonal tissues throughout development were of the same order as those persisting in target organs of rats given a pharmacological dose of reserpine.
Role of glycoproteins in the uptake of L‐ 14C ‐ leucine by a synaptosomal fraction
Authors: Rossowska Magdalena
Published: January 2005

The present study deals with the effect of modification of membrane glycoproteins on the accumulation of amino acids by isolated synaptosomal fractions, measured by the uptake of 14C‐leucine. Superficial sugar receptors were modified by concanavalin A, neuraminidase, and neuraminidase followed by concanavalin A binding. It is demonstrated that neuraminidase treatment followed by concanavalin A binding results in significant diminution of 14C‐leucine uptake. A similar effect was observed in experimental hypoxia followed by concanavalin A binding to isolated synaptosomal fractions.

In this case, however, (Na+K+) –ATPase activity remained unchanged. These results seem to indicate that 14C−leucine uptake was dependent on integrity of glycoprotein structure of synaptosomal membranes.
Neuron‐specific enolase levels in primary cultures of neurons
Authors: Schengrund C.‐L. , Marangos P. J.
Published: January 2005

Primary neuronal cell cultures, derived by centrifugal elutriation of cells dissociated from embryonic rat cerebra, have been analyzed for their content of neuron‐specific enolase (NSE) and non‐neuronal enolase (NNE). Both immunocytochemical staining and radioimmunoassay establish that NSE is present in these cells. Furthermore, the observed increase in NSE and decrease in the NNE/NSE ratio with time in culture parallels that observed in vivo. It has recently been shown that during in vivo neurogenesis and maturation a “switch over” occurs from NNE to NSE which correlates with neuronal differentiation. The presence of a neuron‐specific protein in these primary neuronal cultures and the changes observed during growth further support their use as a model system for the study of nervous tissue differentiation.
Sodium dependence of GABA transport in rat hypothalamic synaptosomes
Authors: Wheeler D. D.
Published: January 2005

The sodium dependence of γ‐aminobutyric acid (GABA) uptake has been studied in rat hypothalamic synaptosomes, and the results compared to previous studies in cortical synaptosomes. Initial velocity of GABA uptake was measured as a function of both GABA and sodium concentration, and these data were fitted to the rate equation for each of several plausible models. The minimal best‐fit model was found to be identical to that for cortical synaptosomes. However, the constants which quantitate the model were found to differ for hypothalamus and cortex. As a result, uptake at any combination of [Na] and [G] in hypothalamic synaptosomes is approximately double that in cortical synaptosomes. The rate equation for the minimal best‐fit model was utilized to define and compute certain parameters which are useful in comparing transport mechanisms (Vmax, Va, Kt, Jm, and KNa). In all cases, differences were found between hypothalamus and cortex.
Spatial distribution of catecholaminergic cells in the fish retina
Authors: Negishi K. , Drujan B. D. , Laufer M.
Published: January 2005

The cell density, distribution pattern, and morphology of catecholaminergic (CA) cells have been studied by fluorescence microscopy of retinal flatmounted preparations from various species of fresh water, estuary, and marine fishes, and the toad. In all fish retinas examined the CA‐cell density (cells/mm2) is lowest in the central region surrounding the optic disc, slightly higher in the intermediate region, and highest in the periphery. The size of CA‐cells is smaller the higher their density. Following administration of L‐Dopa, dompamine, or noradrenaline, the density of CA‐cells approximately doubled, due to the appearance of small fluorescent cells. CA‐cells are arranged in rows along radial lines which fan out from the optic disc. In large cells of the central and intermediate regions three to five processes arise from the soma and extend and ramify irregularly in the inner plexiform layer, while in small cells from the intermediate and peripheral regions two processes arise from opposite poles and extend regularly in a direction perpendicular to the rows of cells and parallel to the retinal margin. In the retina of the marine fish Holocentrus sp CA‐cells are fewer in number compared to other fish studied and their processes extend without any regular pattern. In the toad their size and density are homogenous throughout the retina, and their processes show a regular arrangement.
S‐180 cells secrete nerve growth factor protein similar to 7S‐nerve growth factor
Authors: Barklis E. , Perez‐Polo J. R.
Published: January 2005

The nerve growth factor protein (NGF) has been identified by biological assay and rocket immunoelectrophoresis in media conditioned by monolayers of mouse S‐180 sarcoma cells, a transformed cell line of salivary gland origin. By utilization of a purification procedure designed to enrich for acid‐dissociable, high‐molecular‐weight complexes similar to the 7S‐NGF isolated from male mouse submaxillary gland, it has been determined that a significant portion of mouse sarcoma NGF is initially present as a complex at neutral pH with a molecualr weight indistinguishable from that of 7S‐NGF. At pH 4.0 the mouse sarcoma NGF complex dissociates, and the active subunit can be isolated as a species with a molecular weight of less than 40,000. The induced dissociation at pH 4.0 of the mouse sarcoma NGF high‐molecular‐weight complex, as well as the complex's behavior in isoelectric focusing and sucrose gradient sedimentation is consistent with the hypothesis that 7S‐NGF is packaged as a subunit containing protein intracellularly prior to secretion into the extracellular space. Moreover, the stability of the mouse sarcoma NGF complex at dilute concentrations is similar to that reported for the purified 7S‐NGF complex.
Ontogenesis of neuropeptide degrading enzymes in the mouse brain
Authors: Faivre‐Bauman A. , Knisatschek H. , Tixier‐Vidal A. , Bauer K.
Published: January 2005

The ontogenesis of neuropeptide degrading enzymes was studied in the mouse brain, from the 10th gestational day up to adulthood. Two activities were followed: the pyroglutamate aminopeptidase and the post‐proline cleaving enzyme, using either TRH or specific fluorogenic peptides as substrates. In the hypothalamus as well as in cerebral hemispheres, the specific activities of both enzymes was highest on the 13th fetal day and decline thereafter until the 20–22nd post‐natal day, with a plateau around birth. In contrast, a classical peptidase, the leucyl‐arylamidase increased only in fetal life, and reached the adult level before birth.
Monoclonal antibodies against mouse nerve growth factor produced by somatic cell hybrids
Authors: Businaro Rita , Butler Richard H. , Rubenstein Allan E. , Revoltella Roberto P.
Published: January 2005

Spleen leukocytes from rats and mice immunized against mouse 2.5S nerve growth factor (NGF) and peripheral blood leukocytes from rabbits hyperimmunized against the same antigen were fused with the mouse plasmacytoma P3 × 63Ag8. Hybridomas were screened by immunological assays (microcomplement fixation test and solid phase radioimmunoassay) for production of antibodies that reacted with NGF. Significant variations were seen between culture fluids from different hybrid cells. In addition, most but not all hybridoma antibodies that reacted immunologically with NGF prevented neurite outgrowth from 8‐day chick embryo sensory ganglia explants after binding to NGF. These results suggest that the hybridoma antibodies produced by the different clones react with different antigenic sites on the NGF molecule.
Localization of kainic acid‐sensitive cells in mammalian retina
Authors: Hampton Cynthia K. , Garcia Charles , Redburn Dianna A.
Published: January 2005

Short‐term (15 minutes) in vitro exposure to kainic acid (KA), a rigid structural analog of L‐glutamic acid (Glu), caused two morphologically distinct neuronal lesions in retinas of several species. In rabbit retina, one type of lesion was characterized by rapid swelling after exposure to low concentrations of KA (10−4 M). This lesion was observed in elements of both plexiform layers and, more specifically, in cell bodies and neurites of horizontal cells that contact cones. A few cell bodies from the amacrine cell layer showed some limited swelling. The swelling was completely blocked when sodium was removed from the incubation medium. The second type of lesion was generally seen after longer exposures of after exposure to higher concentrations of KA and was evidenced by degeneration of neurons in the amacrine and ganglion cell layers. One exception was noted in that a few cells from the ganglion cell layer degenerated even under low exposure conditions. The second type of lesion was not blocked by removal of sodium ions. Photoreceptor cells appeared resistant to all effects of KA. The results suggest that a correlation may exist between certain KA‐induced lesions of the retina and putative glutamoreceptive neurons. At the same time, the two types of retinal lesions produced by KA are morphologically and chemically differentiable and may be useful in elucidating the differences between specific, Glu‐related toxicity and nonspecific toxicity of KA.
The role of cAMP in the induction of ornithine decarboxylase by nerve and epidermal growth factors
Authors: Lacorbiere Monique , Schubert David
Published: January 2005

Nerve growth factor (NGF), epidermal growth factor (EGF), adenosine 3′,5′‐cyclic monophosphate (cAMP), and cholera toxin all increase the specific activity of ornithine decarboxylase (ODC) in the PC12 nerve‐like cell line. A phosphodiesterase inhibitor augments the effect of NGF on the induction of ODC in these cells, while it is only additive with the EGF response. These data suggest that cAMP has an intermediary role in the induction of ODC by NGF and that cAMP is not involved in the EGF response.
In vivo structure of frog sciatic nerve myelin membranes: An x‐ray diffraction study at 13Å resolution
Authors: Padróoan Raúual , Mateu L.
Published: January 2005

The x‐ray diffraction pattern of myelin membranes from a frog sciatic nerve was recorded in vivo at 13 äAR resolution using a new experimental technique recently developed in our laboratory. The electron density profile was determined for the myelin membranes first in vivo and then, after elimination of the intraneural blood microcirculation, by transection of the nerve. The small structural differences observed between these conditions were statistically nonsignificant (at 95% confidence level). In addition, our results show that the stacking disorder that is present in the myelin membranes of freshly dissected frog sciatics is also characteristic of the myelin sheath in vivo.
Synthesis of lipids in mouse brain cell cultures during development
Authors: Siegrist Hans Peter , Bologa‐Sandru Liane , Burkart Thomas , Wiesmann Ulrich , Hofmann Karel , Herschkowitz Norbert
Published: January 2005

Several metabolic activities in dissociated cultures of newborn mouse brain were compared to the situation in vivo. The developmental activity pattern of cerebroside‐sulfotransferase, cyclic nucleotide phosphohydrolase, and β‐hydroxy‐β‐methyl glutaryl‐coenzyme A‐reductase and the synthesis and deposition of sulfatide and cholesterol in culture were estimated. The enzyme activity patterns in vivo and in culture are the same. Since the cultures show very little myelin formation, the parallel increase of enzyme activities necessary for myelination in vivo and in culture suggest the existence of intrinsic factors regulating the biochemical differentiation. In addition, the formation of the products, determined in culture, follows the patterns of the enzyme activities. Dissociated brain cell cultures are therefore a valid model for the study of biochemical parameters related to the synthesis of brain lipids during development.
Effect of denervation on cyclic nucleotide metabolism in different types of skeletal muscle of the rat
Authors: McLane Jerry A. , Held Irene R.
Published: January 2005

Slow‐twitch soleus and fast‐twitch extensor digitorum longus muscles of the rat were denervated unilaterally by sciatic nerve section at mid‐thigh level. Activities of adenylate cyclase, guanylate cyclase, low Km and high Km cyclic AMP phosphodiesterase, and cyclic GMP phosphodiesterase were compared on the same, freshly prepared homogenates of denervated and shamoperated contralateral muscles one, two, three, or five days after surgery. As an early consequence of denervation, cyclic AMP metabolism was differentially affected in these different types of skeletal muscle. The adenylate cyclase activity of soleus muscle increased significantly by the second day following denervation and continued to rise through the fifth day, while this enzyme did not increase in denervated extensor digitorum longus even by the fifth day. The high Km cyclic AMP phosphodiesterase was already increased by day one in the denervated soleus, but not until the fifth day in the denervated extensor digitorum longus. Parallel increases beginning the first day were observed for the low Km cyclic AMP phosphodiesterase in both muscles. Since the activity of cytosolic cyclic AMP‐dependent protein kinase of soleus muscle was also increased two days following denervation, the changes in cyclic AMP synthetic and degradative enzymes apparently result in a rise in intracellular cyclic AMP concentration. Alterations of the cyclic GMP enzymes following denervation were similar in the soleus and extensor digitorum longus, but were delayed relative to the increases in activity in the cyclic AMP enzymes.
Cathechol‐O‐methyltransferase activity in cultured human skin fibroblasts from controls and patients with dystonia musculorum deformans
Authors: Breakefield Xandra O. , Braverman Muriel , Riker Donald Kay , Giller Earl L.
Published: January 2005

Fibroblasts provide a source of living cells that can be obtained easily from humans and used to evaluate inherited differences in the activities of enzymes important in neurotransmitter and drug metabolism. Here, we describe biochemical characteristics of catechol‐O‐methyltransferase (COMT, EC 2.1.1.6) activity in homogenates of cultured human skin fibroblasts. Many properties of the enzyme, including apparent affinity for dihydroxybenzoic acid and Sadenosyl methionine, optimal pH and (Mg++), and inhibition by Ca++, are similar to those reported in lysates of human erythrocytes. Culture and assay conditions have been established for optimal and reproducible measurement of COMT activity in individual fibroblast lines. In 16 control lines, COMT activity ranged from 115 to 263 pmol/min/mg protein with a mean of 181 pmol/min/mg protein. Enzyme activity did not vary with the age or sex of the donor. The COMT activities in fibroblasts from eight patients with dystonia musculorum deformans, an inherited movement disorder of unknown etiology, were not significantly different from controls. Monoamine oxidase (MAO, EC 1.4.3.4) type A activity was measured in 12 lines from patients with dystonia, and values did not differ significantly from age‐ and sex‐matched controls. We conclude that inherited variation in activity of these two catabolic enzymes is not sufficient to explain alterations in monoamine metabolism described in this disorder.
Membrane properties of a human neuroblastoma II: Effects of differentiation
Authors: Kuramoto T. , Werrbach‐Perez K. , Perez‐Polo J. R. , Haber B.
Published: January 2005

The SK‐N‐SH cell line is a human neuroblastoma which when grown under standard culture conditions remains relatively undifferentiated. The undifferentiated SK‐N‐SH cells are relatively inexcitable: they show only partial active responses to injections of current pulses and lack the depolarizing component of the action potential generating mechanism [Kuramoto et al, 1977]. In this study we report on the membrane properties of two subclones of the SK‐N‐SH, the SK‐N‐SH‐IN (referred to as IN) and the SK‐N‐SH‐Sy5Y (referred to as 5Y) which exhibit extensive morphological differentiation when grown with 1mM dibutyryl cAMP. Fully differentiated IN and 5Y cells have higher resting membrane potentials, in the range of −50 to −80 mV, and higher input resistance and time constants than do the undifferentiated SK‐N‐SH parental cell line. After 1 week in culture the differentiated IN and 5Y cells exhibit spike potentials in response to injection of current to the cell body. The presence of the Na+‐dependent depolarization was verified directly by the use of tetrodotoxin (TTX) and indirectly in experiments where veratridine (0.1 mM) markedly enhanced the influx of 22Na+. Taken together, the data indicate that generation of action potentials in these human neuroblastoma cells is to a large extent a function of morphological differentiation.
Covalent interaction of [3H]‐dopamine with rat brain proteins in vivo and with the dopamine‐reuptake site of nerve endings in vitro
Authors: Davies B. D. , Abood L. G.
Published: January 2005

Isolated nerve endings have been demonstrated to undergo saturable, covalent interactions with [3H]‐dopamine under physiological conditions; and the reaction is greatly accelerated by flash photolysis with ultraviolet light. With intact nerve endings, under conditions where dopamine reuptake occurs, benztropine and cocaine (inhibitors of dopamine reuptake), but not atropine or haloperidol (a postsynaptic antagonist), prevent the reaction. The reaction also occurs in vivo following the intraventricular administration of [3H]‐dopamine, the reaction being greatest with mitochondria, followed by the nerve ending and myelin. With the use of sodium dodecylsulfate–gel electrophoresis, a number of proteins of varying molecular weight were labeled, and the pattern of labeling was similar in vitro and in vivo. One protein, with a MW of about 60,000 was labeled to an exceptionally high degree. A number of protein bands showed decreased radiolabeling in the presence of benztropine, a finding which suggests that they may be associated with the reuptake site. Both the addition of ascorbic acid and unlabeled dopamine inhibited the reactivity of [3H]‐dopamine, and the effects were concentration dependent. In the absence of photolysis, the reaction of [3H]‐dopamine to synaptic membranes attained saturation within 10 min, but with photolysis the reaction continued at a constant rate even after 20 min. The results are discussed in relation to the use of [3]‐dopamine as a photoaffinity label of the dopamine reuptake site and in relation to the nature of the reactions with and without photolysis.
Cerebral RNA synthesis in experimental hepatogenic encephalopathy
Authors: Albrecht Jan
Published: January 2005

Cerebral slices from rats subjected either to chronic (2‐ to 6‐month) administration of carbon tetrachloride or to prolonged (21‐day) portocaval shunting showed an increased above control ability to incorporate [3H]uridine in vitro into the RNA of the large cell nuclei, mostly derived from astrocytes but not the small oligodendroglial cell nuclei. This result is interpreted to indicate that brain reacts to extended periods of hepatogenic insult or chronic omission of hepatic circulation with a stimulation of RNA synthesis in the astrocytes. The possible relation of this phenomenon to the characteristic astrocytes response accompanying hepatogenic encephalopathy is discussed.
Physical properties of bovine white matter proteolipid apoprotein–sodium dodecyl sulfate complexes
Authors: Sakura J. David
Published: January 2005

The interaction of bovine white matter proteolipid apoproteins with sodium dodecyl sulfate (SDS) was studied. Equilibrium dialysis binding measurements show that the apoprotein binds approximately 1.5 gm SDS per gram protein at high ionic strength (T/2 =0.17). At low ionic strength (T/2 = 0.01) the protein binds only 0.90 gm per gram protein. The Stokes radius of the proteolipid protein is 40 Å. Based on the circular dichroism spectrum, the apoprotein contains less than 20% α‐helix structure in either aqueous or 0.1% sodium dodecyl sulfate solution. Analytical SDS‐PAGE (polyacrylamide gel electrophoresis) revealed two polypeptides. The major proteolipid protein migrated with an apparent molecular weight of 26,000 daltons. The minor proteolipid protein exhibited a molecular weight of 22,200 daltons. The apoprotein showed a marked tendency to aggregate on 8 M urea‐SDS‐polyacrylamide gels or when heated at 100° in excess detergent. These results suggest that the proteolipid proteins have hydrophobic regions that are not embedded within the detergent micelle but are exposed to the solvent. These regions may serve as sites for intermolecular aggregation.