bluuebunny/crossref_metadata_2025 huggingface dataset:
The active site of lysosomal sphingomyelinase: Evidence for the involvement of hydrophobic and ionic groups
Authors: Callahan J. W. , Jones C. S. , Davidson D. J. , Shankaran P.
Published: January 2005

The natural substrate for sphingomyelinase contains hydrophobic and polar moieties. In this study, we have employed pH rate studies and examined hydrophobic compounds and phosphorylated esters for their effect on sphingomyelinase activity in an attempt to determine some of the kinetic properties of this enzyme. Sphingomyelinase, purified from human placentae, undergoes noncompetitive inhibition by octylglucoside and Nonidet P‐40, two nonionic detergents containing terminal octyl groups. The effect of these detergents at the hydrophobic binding site is somewhat different from that of Triton X‐100, which contains an isooctyl terminal group, and this may serve to identify a structural basis for the effects.

Sphingomyelinase activity is also modulated by several nucleotides. Inhibition by 5′‐adenosine monophosphate (5′‐AMP) is also noncompetitive. Other nucleotide monophosphates (such as 5′‐uridine monophosphate (5′‐UMP), 5′‐cytidine monophosphate (5′‐CMP), 2′‐adenosine monophosphate (2′‐AMP), and 3′‐adenosine monophosphate (3′‐AMP) and phosphorylated intermediates (such as phosphorylcholine, phosphorylethanolamine and hexose phosphates) have a lower inhibitory effect. The data suggest that the inhibition by 5′‐AMP involves the combined effect of the phosphate group and the purine ring, structural requirements which may also be satisfied by bis(4‐methylumbelliferyl)phosphate, a synthetic enzyme substrate.

Studies of pH rate indicate that the maximal velocity for the hydrolysis of sphingomyelin is independent of pH over the range 3.5–6.2 while the Km value shows a pH dependence. The Km value is lowest from pH 4.0–5.2 and rises at pH values outside this range. The log Vmax/Km and pKm relationships, when plotted as a function of pH, have been used to identify the dissociation constants for the binding of sphingomyelin by the enzyme. These occur at pK values of 4.1 and 5.5. The activity of sphingomyelinase is also reduced when the enzyme is photooxidized in the presence of methylene blue or rose bengal and carbamylated by diethylpyrocarbonate (DEPC).

These results are interpreted to show that (1). the ezyme contains a hydrophobic binding site which involves linear aliphatic moieties containing at least eight carbon atoms; (2) two ionic groups are involved in formation of the enzyme substrate complex, one of which is presumed to be the carboxylate group of aspartate or glutamate (represented by pK 4.1) and the second may be the protonated imidazolium group of histidine (represeted by pK 5.5); and 3) since the maximal velocity shows no pH dependence, the interactions involving the hydrophobic and ionic groups affect only the binding of te substrate to the enzyme and formation of the enzyme‐substrate complex.
Measurement of prostaglandin biosynthetic capacity in discrete areas of the rabbit hippocampal formation
Authors: Lysz T. W. , Needleman P. , Ferrendelli J. A.
Published: January 2005

Assay conditions sensitive enough to measure prostaglandin (PG) biosynthetic capacity in discrete brain areas have been developed. Serial sections of the rabbit hippocampal formation were dissected from the brain, frozen, and 16 m̈m sections were cut and freeze dried. The assay was performed with 40—200 m̈g dry weight of tissue in a 30 m̈l incubation volume containing high specific activity arachidonic acid. The whole hippocampal formation slice or its subdivisions (subiculum, dentate gyrus, and hippocampus) when assayed individually demonstrated the ability to produce approximately 30—50 pmol/mg dry weight PGF2α, PGE2, and lesser amounts of PGD2 (in a ratio of 1:0.8:0.3). A modest yet significant increase in PGE2 production was measured in the dentate gyrus when compared to PGE2 formation in the subiculum or hippocampus. An intact PGE2 isomerase was confirmed in the hippocampal formation slice by incubations with PGH2 endoperoxide. These results demonstrate that quantitative histochemical techniques can be used to measure PG metabolism in microscopic regions of brain.
Influence of the visual cortex on responses of retinal ganglion cells in the rat
Authors: Molotchnikoff S. , Tremblay F.
Published: January 2005

The objective of the present investigation was to answer the following question: Does the visual cortex affect the neuronal firing of retinal ganglion cells in the rat? To test this hypothesis, the visual cortex was inactivated by a reversible cryoblockade. Action potentials of a ganglion cell were recorded from its axon at the optic tract level prior to, during, and following cortical blockade. The results indicated that indeed the visual cortex influenced the retinal output since its inactivation led to a modification of the firing pattern evoked in response to a flash of light. In most cases the modification was an increase of the bursting pattern of the evoked discharges. By contrast cooling nonvisual areas failed to modify ganglion cells' discharge. A comparison between cortico‐geniculate and cortico‐retinal feedback loops seems to suggest that the first path is involved mostly with the spatial organization of center‐surround receptive fields, whereas the second path is associated with temporal aspects of the retinal responses in the rat.
Intracellular ion changes of astrocytes in response to extracellular potassium
Authors: Walz W. , Hertz L.
Published: January 2005

Intracellular changes of K+, Na+, and Cl− were investigated by the aid of radiotracers in primary cultures of astrocytes when extracellular K+ was (1) increased from 3 to 12 mM and subsequently again decreased to 3 mM; and (2) increased from 5.4 to 54 mM with subsequent decrease to 5.4 mM. In both situations the K+ content increased by 50% within seconds, and it doubled within 1‐2 min. The increase must be carrier mediated, because keeping the K x Cl product (Donnan equilibrium) constant did not lower the K+ accumulation rates. The Na+ content decreased when K+ was increased to 12 mM, but the decrease corresponded only to 10% of the accumulated K+. When K+ was increased to 54 mM, the Na+ content increased transiently. Cl− increased by about 15‐25% of the accumulated K+. Return of extracellular K+ to original levels evoked a very fast K+ release, reversing all ion changes. The Na+ content increased transiently during the release process. For an interpretation of these observations, it is necessary to postulate endogenous production of an anion and of H+, which in turn is partly exchanged with Na+.
Neurons cultured from developing rat brain attach and spread preferentially to laminin
Authors: Liesi Päivi , Dahl D. , Vaheri A.
Published: January 2005

Neurons, mechanically dissociated from newborn rat brain and identified by immunostaining for neurofilaments, attached preferentially to laminin‐coated coverslips without need of an underlying glial monolayer. The most extensive neurite outgrowth was seen when 20–30 μg/ml of laminin was used to coat the coverslips. Higher concentrations of laminin (> 30 μg/ml) supported single neurons to spread on the coverslips. Fibronectin coating of the coverslips allowed glial cells to attach more rapidly than on uncoated surfaces, but it did not support neuronal spreading or neurite outgrowth. Spreading of neurons and neurite outgrowth were completely inhibited by preincubation of laminin‐coated coverslips with laminin antibodies but were unaffected by fibronectin antibodies. These results indicate that laminin is an attachment and spreading factor for central neurons in culture and suggest the presence of a laminin receptor on the neuronal cell surfaces.
Reversal potential of the end‐plate currents and potassium feedback at the neuromuscular synapse
Authors: Matyushkin D. P. , Krivoj I. I. , Shabunova I. A.
Published: January 2005

The experiments were carried out at the neuromuscular junction of the frog. Erev of epc was estimated by two methods—extrapolation and interpolation. In K+‐ enriched solution Erev obtained by linear extrapolation from high‐mp region changed according to the Takeuchi equation. The behavior of Erev obtained by interpolation was better described by the Goldman‐Hodgkin‐Katz equation. Under the change of the nerve stimulation frequency from 0.5 to 50/sec, the positive shift of Erev estimated by both methods was observed. This indicates K+ accumulation in active synaptic segments during activity. The elimination of the potassium current of epc by clamping of mp at the − 130 mV level resulted in the lowering of facilitation under paired nerve stimulation (20 msec interval) and in the decrease of the rate of facilitation under 50/sec frequency stimulation. The data support the presence of antidromic K+ action in the neuromuscular synapse.
Formation of desTyr dynorphins 5–17 by a purified cytosolic aminopeptidase of rat brain
Authors: Berg M. J. , Marks N.
Published: January 2005

An aminopeptidase purified to homogeneity from cytosol of rat brain cleaved dynorphins having 5–17 residues and selected proenkephalins at the Tyr–Gly bond only to release Tyr and the desTyr fragments. The enzyme protein consisted of a single polypeptide chain of Mr 103,000 and was inhibited by puromycin, bestatin, and chelating reagents to yield Ki in the micromolar range. Hydrolysis of Leu‐2‐naphthylamide was inhibited by Dyn 1–5 competitively (Ki, 18 μM); the Km for Dyn 1–5, the best substrate of the series, was 63.8 μM (Kcat/Km ratio 580 mM−1 min−1). Rates of N‐tyrosyl release decreased with peptide size; the presence of Arg in position 6 led to 50% loss for Dyn 1–6, and the C‐terminal extensions of Dyn 1‐13 or 1–17 to a 98% loss in activity as compared to the pentapeptide. Rapid degradation of small peptides is consistent with a paracrine (neurotransmitter) role as compared to the postulated precursor or exocrine roles for the dynorphins with 13 residues or more.
Effects of dopamine on photopic L‐type s‐potentials in the catfish retina
Authors: Hida E. , Negishi K. , Naka K.‐I.
Published: January 2005

Photopic L‐type responses were recorded from the soma and the axon terminal of horizontal cells of the catfish (Ictalurus punctatus) retina in eye‐cup preparations. The responses were produced by a spot of light with 100‐μm diameter (intensity, 10 μ/ cm2), which was flashed or steadily illuminated and swept along a 6‐mm length over the retinal surface at a speed of 0.95 mm/sec. In some experiments, a traveling random bar stimulus was used instead of the sweeping spot. While recording the responses, dopamine (DA) was applied in a jet form via a nebulizer over the retinal preparation or as superfusate to the eye‐cup preparation. DA increased the response amplitude by about 50% and markedly narrowed the spatial profile of the responses from the soma but not from the axon terminal. These DA effects were observed in both normal retinas and those from which DA cells had been deprived by prior intraocular injection of 6‐hydroxydopamine. Deprival of DA cells from the retina resulted in a slightly wider spatial profile of the soma and axon terminal responses than that in normal retinas. The results indicate that the spatial properties of photopic L‐type responses are modulated by DA at the soma level of horizontal cells.
The influence of systemic factors on acrylamide‐induced changes in brain, nerve, and other tissues
Authors: Lapin E. P. , Maker H. S. , Weissbarth S. , Weiss C. , Lehrer G. M.
Published: January 2005

In order to define the locus of acrylamide neurotoxicity, the effects of chronic intoxication (total dose 500 mg/kg) on cholinergic synthesis and transport, the Schwann cell‐myelin complex, lysosomal activity, and several metabolic pathways were determined in rat sciatic nerve, spinal cord, and brain. No changes were found in hematological measures or in the levels of clinically important blood enzymes, indicating no major damage to other organs. The activities of choline acetyltransferase (ChAT), 2′,3′‐cyclic nucleotide phosphohydrolase, beta‐glucuronidase, and lactate dehydrogenase were unaffected in acrylamide paralyzed animals, but creatine kinase (CK) decreased in sciatic nerve, muscle, and brain, particularly in animals dying of the intoxication. CK blood and the CK isoenzyme patterns in blood were unchanged. The synthesis of protein in brain and spinal cord (measured in vivo) were decreased in rats exposed to high‐dose acrylamide. However, in brain and cord, CK decreased only after animals became systemically ill and suffered weight loss, with the lowest activities in those animals sick enough to die. The degree of stress to which the animals had been subjected was indicated by enlargement of the adrenal glands and decreased sulfolipid synthesis in the adrenals. Rats exposed to 25 mg/kg/day acrylamide to a total dose of 250 mg/kg developed leg weakness but not paralysis or weight loss and had a 25% decrease in CK only in the distal sciatic nerve. Because of the apparently stress‐related or agonal loss of CK, no specific effect of acrylamide on the enzyme could be definitely demonstrated. Neither could the changes in protein synthesis be attributed solely to a direct effect of the toxin. These results illustrate the difficulties encountered in interpreting intoxication studies that produce systemic illness and support the suggestion that CK activity may be a useful marker of the severity and duration of the agonal state in studies of postmortem human brain.
Detection of a cell surface antigen found on rat peripheral nervous system neurons and multiple glia: Astrocytes, oligodendrocytes, and Schwann cells
Authors: Akeson R. , Warren S. L.
Published: January 2005

A cell surface component has been identified that is found on cultured rat dorsal root ganglion neurons and Schwann cells and also cultured brain astrocytes and oligodendrocytes. This component was detected with a monoclonal antibody originally generated to the NG108 (N18 mouse neuroblastoma × C6 rat astrocytoma) hybrid cell line. The antibody, designated B2C11, binds to cultured peripheral nervous system cells: intact dorsal root ganglion and trigeminal neurons and cultured dorsal root ganglion and sciatic nerve Schwann cells. The binding of B2C11 to dorsal root ganglion neurons in vivo was confirmed by immunofluorescence analysis of cryostat sections. However, cultured embryonic rat central neurons showed no detectable binding of B2C11. Cultured brain cells containing glial fibrillary acidic protein (astrocytes) and also oligodendrocytes cultured from corpus collosum did bind B2C11 on their surfaces. B2C11 immunoprecipitation of detergent‐solubilized membrane proteins from both lactoperoxidase iodinated C6 and PC12 rat pheochromocytoma cells indicated a single band with an apparent molecular weight of 21,000–23,000. Analysis of B2C11 binding to particulate protein preparations from adult rat organs showed highest specific activity in dorsal root ganglia. Other neural tissues had substantial binding. Some nonneural tissues (lung, kidney, and small intestine) expressed significant antigen levels, whereas others (heart, liver, and skeletal muscle) had a B2C11 antigen‐specific activity less than 5% of that of dorsal root ganglia. Thus the B2C11 antigen is enriched in neural tissues, where it is found on the surfaces of a unique set of neuronal and glial cells.
Acquisition of submandibular gland nerve growth factor (SMG‐NGF) responsiveness to thyroxine administration in neonatal mice
Authors: Lakshmanan J. , Beri U. , Perheentupa J. , Grueters A. , Kim H. , Macaso T. , Fisher D. A.
Published: January 2005

The growth of the submandibular gland (SMG) was studied in newborn mice from birth to 15 days of age. Progressive changes in wet weight were observed to accompany changes in biochemical constituents such as RNA, protein, and lipid. Thyroxine (T4) administration from days 0–6 produced changes in SMG growth and SMG accumulation of RNA, protein, and lipid components relative to control pups treated with a similar volume of vehicle. This hormone regimen produced no measurable changes in SMG nerve growth factor (SMG‐NGF) concentration. T4 responsiveness also was studied from days 0–15. Three patterns of T4 injection (from days 0–6, 7–14, and 0–14) were found to elicit a differential response in the three biochemical constituents measured, but treatment from days 7–14 and 0–14 elicited precocious increments in SMG‐NGF concentrations on day 15. The effect of T4 injection from birth was more effective in augmenting SMG‐NGF concentration than hormone treatment initiated from days 7–14. A persistent T4 effect on SMG‐NGF also was observed on day 21 following hormone treatment from days 7–14 or 0–14. In summary, the acquisition of SMG‐NGF responsiveness to T4 appears to develop during the neonatal period. The administration of T4 during this period also precociously stimulates the mechanisms that govern the normal ontogeny of SMG‐NGF at the time of weaning.
Inhibition of the chicken retinal UDP‐GalNAc:GM3, N‐acetylgalactosaminyltransferase by blood serum and by pineal gland extracts
Authors: Quiroga S. , Caputto B. L. , Caputto R.
Published: January 2005

The effects of the pineal gland extract and blood serum on the activity of the UDP‐GalNAc:GM3, N‐acetylgalactosaminyltransferase (GM3:GalNAc‐T) from chicken retina were studied. Both preparations have inhibitory capability on the enzyme activity. Two types of inhibitory capabilities were found: one is heat labile and decomposes UDP‐GalNAc and another is heat stable.

When the pineal gland extracts were prepared from light‐exposed chickens, the inhibitory capability increased with respect to the extracts from dark‐maintained animals; vice versa, blood serum from dark‐maintained animals had higher heat‐labile and heat‐stable capabilities than that from light‐exposed chickens.

In in vitro experiments, no difference was found in the inhibitory capability of blood serum extracts from pinealectomized animals compared to control animals. In vivo labeling experiments with pinealectomized animals in either light or dark showed similar differences in the labeling of the optic tectum gangliosides as the normal animals.
Biological activity of preformed cholera toxin‐ganglioside GM1 complex
Authors: Fiani M. L. , Macioce P. , Gallina A. , Tomasi M.
Published: January 2005

Synthetic and natural amphiphiles, octyl glucoside, Nonidet P40, sodium dodecyl sulfate (SDS), gangliosides GM1 and GD1a, interact with cholera toxin (CLT) and with its active region (protomer A). The formation of CLT‐amphiphile complex leads to inhibition of ADP‐ribosyltransferase activity, a characteristic of protomer A elicited after thiol‐reagents treatment. In all cases the interaction produces the maximum inhibitory effect above the critical micellar concentration of amphiphiles, although monomers of SDS show inhibition activity as well. The gangliosides appear to be capable of altering bilayer organization of membrane, similar to synthetic detergents.

When CLT‐ganglioside complexes were incubated with cell culture medium containing 10% fetal calf serum (FCS) the ADP‐ribosyltransferase activity was completely restored both in cholera toxin and in protomer A. Some protein of FCS, which is avid of gangliosides, seems to be responsible for reversibility of inhibition. The results indicate that the active site of protomer A may be located in a hydrophobic pocket of the toxin structure.

Furthermore, CLT was bound to reconstituted Sendai virus envelopes (RSVEs), containing a small amount of GM1. The RSVEs are made of membranous vesicles, capable of binding and fusing with host cell membrane. The incubation for 1 lhr of RSVE bearing CLT with Friend's erythroleukemic cells produced the stimulation of adenylate cyclase. This stimulation appears to be due to the translocation of the active subunit of CLT in the inner half of plasma membrane.
GM1 micelles modify the transport properties of the lonophore gramicidin D in artificial planar bilayers
Authors: Gambale F. , Marchetti C. , Usai C. , Robello M. , Gorio A.
Published: January 2005

We have analyzed the effects induced in different phospholipid planar bilayers by monosialoganglioside micelles containing the inonophore gramicidin D. The membrane conductance increases after the addition of GM1 micelles at various ionophore/ganglioside ratios. We believe this fact may be ascribed to gramicidin molecules that incorporate into the bilayer together with gangliosides.

In the presence of micelles the mean lifetime and the amplitude of the gramicidin single channel did not present relevant modifications when dioleoylphosphatidylcholine or phosphatidylserine were used to form the bilayer. Calcium proved to trigger the interaction between phosphatidylethanolamine membranes and GM1 micelles containing gramicidin. In this case the ionic pore presents a longer lifetime and a lower amplitude with respect to pure gramicidin.

We suggest that different properties developed by gramicidin may depend on structural organization of gangliosides when incorporated into the phospholipid bilayer.
Interaction of ganglioside GM1 and myelin basic protein studied by carbon‐13 and proton nuclear magnetic resonance spectroscopy
Authors: Ong R. L. , Yu R. K.
Published: January 2005

The interaction of the myelin basic protein (MBP) and the major endogenous ganglioside GM1 in myelin of the central nervous system has been investigated using both 500‐MHz 1H and 67.89 MHz 13C NMR. Titration of MBP by GM1 resulted in 13C NMR signal shifts for the Ile and His residues of MBP at a GM1/MBP mole ratio of one or less. The carbohydrate head group of GM1 was also found to be perturbed. 1H NMR results obtained in a similar manner demonstrated the perturbation of His and Phe residues. At a GM1/MBP mole ratio of 0.5, small perturbation of Trp #116 was observed, and at mole ratios of two and beyond significant involvement of Phe residues and methylated Arg #107 was found. Met #167 was more perturbed than Met #20; hence, more extensive interaction of the lipid is occurring with the C‐terminus of the protein than with the N‐terminus. No resonances from GM1 bound to MBP at mole ratios of up to one appeared in the spectra. However, as the GM1/MBP mole ratio was increased to eight or greater a major conformational change of MBP was detected. An upfield shift of the GM1 midchain methylene resonance was observed for the GM1/MBP complex. This observation provides strong evidence that the state of GM1 interacting with MBP is different from that of GM1 micelles. The number of saturable GM1 binding sites on MBP is estimated to be four. The data also favor a rapid exchange between bound GM1 and GM1 micelles. Interaction of MBP with the oligosaccharide derived from GM1 was found to be weaker than with GM1. Based on our data, a model for the interaction can be proposed: the first GM1 molecule is bound to the protein molecule through its head group and hydrocarbon chains, followed by the formation of a GM1/MBP complex with a concomitant conformational change of MBP as more GM1 is added.
Endogenous material in brain inhibiting [3H]nicotine and [3H]acetylcholine binding
Authors: Sershen H. , Reith M. E. A. , Hashim A. , Lajtha A.
Published: January 2005

The supernatant obtained from mouse brain homogenates contains material that inhibits the saturable binding of [3H]nicotine in mouse cerebral cortex. This inhibitory material was further purified by heat denaturation, ultrafiltration through an Amicon PM‐10 membrane filter, and gel chromatography on Sephadex G‐10. The material inhibited the binding of [3H]acetylcholine with the same potency as it did that of [3H]nicotine. It also had some affinity for the sites that specifically bind [3H]D‐Ala, D‐Leu enkephalin, but had much lower affinity for the binding sites for [3H]quinuclidinyl benzilate (QNB), [3H]spiroperidol, [3H]naloxone, or [3H]imipramine. Acid hydrolysis destroyed the activity. These preliminiary results suggest the presence in brain of “nicotinelike” substances, one of which may be the endogenous ligand for the sites that specifically bind [3H]nicotine.
Estimation of the apparent affinity of the striatal dopamine receptors for the radioligand [3H]spiperone([3H]spiroperidol)
Authors: Hruska R. E.
Published: January 2005

The characteristics of the receptor population labeled by the ligand [3H]spiperone were determined by several experimental procedures. Varying the assay volumes, and hence both the receptor and ligand concentrations, did not alter the specificity for the dopamine (DA) receptor. The density of binding sites estimated from saturation analyses varied little (6%) over a greater than 50‐fold tissue concentration range. In contrast, variation in tissue concentration did alter the apparent affinity of [3H]spiperone for the DA receptors more than 16‐fold, as determined from saturation analyses. This was most marked at large receptor or tissue concentrations. The standard correction for depletion of the free ligand by that bound to the receptors reduced the range to threefold or 200%. Two separate measurements of the DA receptor affinity, using the rates of association and dissociation and the shift in Ki for DA, gave affinity measurements in the same range as that obtained using low concentrations of receptors in saturation analyses. Therefore, three separate estimates for the affinity of [3H]spiperone binding to the striatal DA receptors agreed that it is probably in the range of 10–15 pM, and single saturation experiments reach this level when the tissue concentration is very low, ie, less than 15 μg protein per 1 ml assay volume.
Membrane specializations of neuritic growth cones in vivo: A quantitative IMP analysis
Authors: Small R.K.
Published: January 2005

The internal structure of the membranes of axonal and Schwann cell growth processes was examined by freeze‐fracture in the growing olfactory nerve, a simple in vivo system consisting of a homogeneous neuronal population. Excision of the mature nerve of adult bullfrogs provides well‐synchronized primary neuritic outgrowth that is highly enriched in growth cones at its distalmost segment. The extreme uniformity of olfactory axons in terms of their diameter and their intramembrane particle (IMP) composition permits clear identification of the cellular origin of the growth cone structures observed in replicas. In vivo, growth cones of the olfactory nerve appear as irregularly shaped enlargements of the distal tip; filopodia are only infrequently exposed by the fracture plane. Axonal and Schwann cell growth cones are distinguished by (1) the larger size of the Schwann cell growth cone and the smaller diameter of its attached processes, and (2) the distinct differences in IMP composition of Schwann cell and axonal growth cones and cell processes. Schwann cell growth cones display a uniformly high IMP density on their P‐face leaflet, with the exception of circumscribed moundlike protrusions that are relatively free of IMPs. In contrast, axonal growth cones display sharp regional variation in IMP density on their P‐face: broad regions almost devoid of IMPs are interspersed with zones of high IMP density. Cytotic profiles occur within high IMP density zones located, most often, at the base of the axonal growth cone. A comparison of IMP size histograms of both high and low‐density regions of axonal growth cones and that of the neighboring distal shaft of the axon indicates a strict partitioning of membrane components between these two regions. The IMP profile of the axonal growth cone, notable for its relative enrichment in large‐diameter particles, suggests that IMP components of the growth cone are delivered to the distal tip by a mechanism that is distinct from the lateral diffusion process described for particles of the growing axon's shaft [cf. Small and Pfenninger, 1984]. The IMP profile of the concave P‐face leaflet of the internal vesicles found clustered at the base of the growth cone is more similar in composition to the profile of the neuronal shaft than that of the growth cone.
Growing tips of embryonic cerebellar axons in vivo
Authors: Mason C.A.
Published: January 2005

Few studies have focused on the transformation of growth cones to mature synaptic arbors. To study these events in developing axons in vivo, we have labeled growing cerebellar axons with horseradish peroxidase (HRP) in postnatal stages [Mason and Gregory, 1984]. This report will provide the first data on embryonic cerebellar axons, and will ask whether growth cones differ in tracts and in target tissue and what features characterize axons that enter the cerebellum in fetal periods.

During the earliest embryonic (E) periods examined (E16–19), axons in tracts have enlarged growth cones with lamellopodia and short filopodia that contain small and large vesicles. In contrast, axons within the cerebellar anlage from E16‐postnatal day (P) 5 have fine calibers with a minimum of branching, and have small tapered growing tips. If synaptic contacts are made by such growing tips, there is little concomitant change in their shape. As target cells form layers and as their dendrites extend (P5–P7), growing tips and synaptic boutons differentiate according to the type of synaptic arrangement in which they engage. Enlarged, irregular expansions of growing tips correspond to synaptic contacts with multiple dendritic partners and are filled with large and small clear vesicles. Filopodia arising from such swellings, like the small undifferentiated growing tips of the type seen on embryonic axons, contain a mixture of vesicle types but make simple synapses with single profiles. Many axons make both kinds of synaptic structures, especially during the period when maturing axons give rise to long filopodia.

Thus, growing tips have immature forms long after synaptogenesis begins, and use filopodial structures to elaborate synaptic arrangements. This analysis should elucidate the changes in growth cone form and cytology that reflect cell‐cell interactions during synaptogenesis.
Regulation of growth cone morphology by nerve growth factor: A comparative study by scanning electron microscopy
Authors: Connolly J.L. , Seeley P.J. , Greene L.A.
Published: January 2005

The object of this study was to document and analyze local regulation by nerve growth factor (NGF) of neuronal growth cone properties and to explore the possible diversity of this effect in various NGF‐responsive preparations. In particular, scanning electron microscopy was used to characterize the morphology of neuronal growth cones in cultures of dissociated chick embryo dorsal root ganglia (DRG) under conditions of continuous NGF exposure, withdrawal of NGF for 5–6 hr, and restoration of NGF for various times. Comparison was made with similarly manipulated cultures of dissociated newborn rat sympathetic ganglia and neurite‐bearing PC12 pheochromocytoma cells. The growth cones of most of the continuously NGF‐treated DRG neurons (cultured on poly‐L‐lysine or collagencoated glass coverslips) had relatively compact central flattened areas and numerous prominent filopodia. Withdrawal of NGF resulted in a marked spreading of the central growth cone area so that the average maximum width of this structure increased by about threefold as compared to nondeprived cultures. The mean number and lengths of filopodia were unaffected. Restoration of NGF brought about, over a time course of tens of minutes, a return of the original type of growth cone morphology. Rather different responses were observed for the sympathetic neuron and PC12 cultures. Here, surface ruffles, only rarely seen in the chick cultures, were a major feature of the growth cones, whereas filopodia, though present, were less prominent. Removal of NGF led to loss of ruffles and to rounding up of the growth cones; NGF readdition elicited a rapid (< 30 sec) reinitiation of ruffling and a more gradual (over tens of minutes) respreading of growth cones. These findings illustrate not only that NGF can regulate growth cone properties, but also that there is a diversity as to how this is manifested. Possible mechanisms and biological roles for this regulation are discussed.
Single channel activity differs in growing and nongrowing growth cones of isolated identified neurons of Helisoma
Authors: Cohan C.S. , Haydon P.G. , Kater S.B.
Published: January 2005

When plated in cell culture, Helisoma neurons extend new neurites with large growth cones. These growth cones progress from actively growing organelles to stable structures over a period of several days. The ability to distinguish morphologically between these growth states has provided the opportunity to test whether changes in growth status are correlated with changes in the ionic properties of the growth cone membrane. This investigation has begun to study these changes by examining single channel ionic currents using patch clamp recording techniques and has demonstrated the presence of at least one ion channel having a conductance of 70 pS in the membrane of Helisoma growth cones. Recordings from cell‐attached patches show that this channel is normally active in growing growth cones, whereas channel activity is absent in recordings from stable growth cones. The absence of channel activity in growth cones that have reached the stable state is not due to the loss of channels from these growth cones. Rather, the channel is present in an inactivated state. The activity of this channel is not dependent on the presence of Ca, as channel activity persists when Ca is removed from the solution that bathes the internal surface of the membrane. These data demonstrate a correlation between channel activity and the growth state of neuronal growth cones.
An analysis of peripheral neuronal survival factors present in muscle
Authors: Bonyhady R.E. , Hendry I.A. , Hill C.E. , Watters D.J.
Published: January 2005

Extracts of bovine heart, rat heart, and rat skeletal muscle were chromatographically separated and the fractions tested for their ability to maintain the survival of dissociated sympathetic, parasympathetic, and sensory neurones. Bovine heart contained at least five, rat heart at least six, and rat skeletal muscle at least four active components differing in their physicochemical properties and their target selectivity. Bovine and rat heart appeared to contain at least two components in common. The differences in active components found in the three tissues are consistent with a complex and perhaps tissue‐specific system regulating neuronal survival.
Synthesis of arachidonoyl coenzyme a and docosahexaenoyl coenzyme a in synaptic plasma membranes of cerebrum and microsomes of cerebrum, cerebellum, and brain stem of rat brain
Authors: Reddy T.S. , Bazan N.G.
Published: January 2005

Synthesis of arachidonoyl CoA and docosahexaenoyl CoA in homogenates and microsomes from cerebrum, cerebellum, and brain stem and in synaptic plasma membranes from cerebrum of control rats and rats undergoing bicuculline‐induced status epilepticus were studied. Arachidonoyl CoA synthesis was 3–5 times higher than docosahexaenoyl CoA in homogenates and microsomes. The synaptic plasma membranes showed only 1.5‐ to 2.5‐fold higher activity. The presence of Triton X‐100 (0.1%) in the incubation medium did not alter the activity of arachidonoyl CoA synthesis but did increase the synthesis of docosahexaenoyl CoA in homogenates, microsomes, and especially in synaptic plasma membranes. The synthesis of these polyenoic fatty acyl CoAs were 4–6 times higher in microsomes than in homogenates. Synaptic plasma membranes exhibited about the same amount of activity as homogenates in the synthesis of docosahexaenoyl CoA, but only half the activity of the latter in arachidonoyl CoA synthesis. The synthesis of arachidonoyl CoA and docosahexaenoyl CoA in cerebral homogenates and microsomes was higher than that of cerebellum and brain stem.

The apparent Km values for labeled arachidonic acid (17 μM) and docosahexaenoic acid (12 μM) in synaptic plasma membranes were lower than the values for microsomes isolated from different brain regions. The Vmax values were also 4–10 times lower. Microsomes from different regions did not differ in their apparent Km values, but did show variations in apparent Vmax values. Cerebellar microsomes showed lower Vmax values than the other two regions. The presence of Triton X‐100 caused a significant decrease in the apparent Km values with little change in the Vmax values. Bicuculline‐induced seizures did not alter the kinetic properties of arachidonoyl CoA and docosahexaenoyl CoA synthesis, except there was a significant decrease in the apparent Km and Vmax values for cerebellar microsomal docosahexaenoyl CoA synthesis. In conclusion, there were marked differences in the activation of polyenoic fatty acids in different parts of the brain and in subcellular fractions. Although bicuculline‐induced convulsions accumulate free polyenoic fatty acids in the brain, no changes were detected when the fatty activation was assayed with exogenous cofactors, except in cerebellum.
Functional role of the potassium‐induced stimulation of oxygen uptake in brain slices studied with cesium as a probe
Authors: Hertz L. , Kjeldsen C. S.
Published: January 2005

The potassium‐induced stimulation of oxygen consumption in brain slices has a threshold value of 15–20 mM potassium, and it reaches its maximum at 35–50 mM. Although this phenomenon now has been known for almost 50 years, its physiological role remains undetermined. One reason for this may be that the high concentration of potassium that are required for this response also have many other consequences, e.g., a depolarization of the cells, and that the different effects to some extent may mask each other. For this reason this investigation studied the effects of cesium, which evokes a maximal stimulation of oxygen consumption already at 15 mM. Like potassium, concentrations of cesium that stimulate oxygen consumption also lead to an enhanced swelling. Unlike potassium, the sodium content is affected very little by these concentrations of cesium, whereas cesium and chloride contents are increased. On this basis it is concluded that the cesium‐induced stimulation of oxygen uptake is a metabolic manifestation of an active uptake of cesium and chloride, which secondarily leads to an uptake of water, i.e., the cesium‐induced swelling. Analogously, it is suggested that the potassium‐induced stimulation of oxygen uptake represents an active accumulation of potassium and chloride.
Deacylation of myelin proteolipid protein in organic solvents
Authors: Bizzozero O. A. , Dominguez F. , Pasquini J. M. , Soto E. F.
Published: January 2005

A procedure has been developed for the deacylation of the hydrophobic, myelin proteolipid apoprotein using hydroxylamine in an alkaline organic solvent medium. Complete removal of covalently bound fatty acids was obtained after 4 hr of treatment. After deacylation, no changes could be detected in the electrophoretic pattern or in the number of free sulfhydryl groups. The deacylated apoprotein remains soluble in chloroform‐methanol mixtures and is suitable for further physicochemical characterization.
Tuning the power spectrum of physiological finger tremor frequency with flickering light
Authors: Isokawa‐Akesson M. , Komisaruk B. R.
Published: January 2005

Fast Fourier Transform analyses were performed on finger tremor movements at 0.2‐Hz intervals from 0.4 to 40 Hz in 10 human subjects, under a flickering light condition of 4–15 Hz and an unstimulated control condition. Under the control condition, the power spectrum showed an essentially normal curve distribution, except for an early frequency component in the histogram. In contrast, when the flickering light stimulus was presented, the power of specific frequency components at 8–1 Hz was strongly enhanced. This effect was induced exclusively at a frequency of 8, 9, or 11 Hz of flickering light, and this flickering frequency producing the enhancement effect differed from subject to subject. There existed a significant correlation between the frequencies of flicker and tremor at the tuned frequency. These findings demonstrate that a specific frequency of flickering light can intensify a specific frequency of physiological finger tremor, and that different individuals exhibit different optimal “tuning” frequencies.
Development and elimination of presynaptic elements on polylysine‐coated beads implanted in neonatal rat cerebellum
Authors: Burry R. W. , Hayes D. M.
Published: January 2005

Polylysine‐coated sepharose beads were implanted in the cerebellum of neonatal rats and examined at 3 hr, 3 days, 14 days, and 21 days after surgery. Previous studies at 5 or 8 days after implantation showed that axons formed neuronal swellings that appeared to be presynaptic elements, with the bead surface in the position of a postsynaptic element. Results reported here show that no beads at 3 hr had presynaptic elements, whereas the number of beads with presynaptic elements increased to high levels at 3 and 7 days but dropped to low values at 14 and 21 days after implantation. Presynaptic elements were seen on beads regard less of their distance from cerebellar tissue except at 3 hr, when no axons were seen in the implant, indicating that axons first grew into the implant and then formed presynaptic elements. The morphological measurements of presynaptic elements on beads at 3 to 7 days after implantation showed increases in area and number of synaptic vesicles, which then decreased at 14 and 21 days after implantation.

These results show that axons can grow into implants of polylysine‐coated beads and form presynaptic elements that do not survive with increased time after implantation. The survival of presynaptic elements on beads can be used as a model for investigations into regeneration of axons and presynaptic elements in the injured brain.
Growth patterns of glial cells dissociated from newbron and aged mouse brain with cell passage
Authors: Vernadakis A. , Davies D. , Sakellaridis N. , Mangoura D.
Published: January 2005

Glial cell cultures derived from newbron and aged (18‐month‐old) mouse cerebral hemispheres and maintained up to cell passage 11 were characterized immunocytochemically by using glial fibrillary acidic protein (GFA), and biochemically by using glutamine synthetase (GS), for astrocytes, and 2′,3′ cyclic nucleotide 3′ phosphohydrolase (CNP) for oligodendrocytes. We report here the changes occurring during passages 5–1. GS and CNP activities did not significantly changes with cell passage in cultures from newbron mouse. In cultures derived from aged mouse, CNP activity did not change signigicantly whereas GS activity increased severalfod. A characteristic finging in higher cell passages (passage #7) was the loss of GFA‐positive stained cells and the appearance of multinucleated cells. We interpret these changes in culture to represent possible signs of cellular senescence.
μ‐type calcium‐activated neutral protease in the rat peripheral nerve
Authors: Kamakura K. , Ishiura S. , Sugita H.
Published: January 2005

Previously we reported results of an incubation experiment with neurofilaments that supported the existence of a μ‐type of Ca2+‐activated neutral protease in the rat peripheral nerve; it was active with μM order Ca2+ (μ‐CANP). This time, we partially purified the μ‐CANP from a crude CANP fraction of rat peripheral nerve by using a DE52 column and a Phenyl‐Sepharose column followed again by DE52 column chromatography. The presence of μ‐CANP was verified by an immunoblotting technique. The μ‐CANP degraded the neurofilament triplet as previously reported; i.e., among the neurofilament triplet, the 160K component was most sensitive, and in the order of the 160K, 68K, and 200K components, respectively.
Metabolism of choline in brain of the aged CBF‐1 mouse
Authors: Saito M. , Kindel G. , Karczmar A. G. , Rosenberg A.
Published: January 2005

In order to quantify the changes that occur in the cholinergic central nervous system with aging, we have compared acetylcholine (Ach) formation in brain cortex slice preparations from 2‐year‐old aged CBF‐1 mouse brains and compared the findings with those in 2–4‐month‐old young adult mouse brain slices. Incorporation of exogenous radioactively labelled choline (31 nM [3H] choline) into acetyl choline in incubated brain slices was linear with time for 90 min. Percentage of total choline label distributed into Ach remained constant from 5 min after starting the incubation to 90 min. In contrast, distribution of label into intracellular free choline (Ch) and phosphorylcholine (Pch) changed continuously over this period suggesting that the Ch pool for Ach synthesis in brain cortex is different from that for Pch synthesis. Incorporation of radioactivity into Ach was not influenced by administration of 10 μM eserine, showing that the increment of radioactivity in Ach reflects rate of Ach formation, independently from degradation by acetylcholine esterases. Under our experimental conditions, slices from cortices of aged 24‐month‐old mouse brain showed a significantly greater (27%) incorporation of radioactivity into intracellular Ach than those from young, 2–4‐month‐old, brain cortices. Inhibitors of Arch release, 1 mM ATP or GABA, had no effect. Since concentration of radioactive precursor in the incubation medium was very low (31 nM), the Ch pool for Ach synthesis in slices was labelled without measurably changing the size of the endogenous pool. These data suggest a compensatory acceleration of Ach synthesis or else a smaller precursor pool specific for Ach synthesis into which labelled Ch migrated in aged brain. We also measured in detail the incorporation of [3H]CH into choline phospholipids: phosphatidyl choline, lysophosphatidyl choline, and sphingomyelin of young and old mouse brain. We could not detect any difference between young and old mouse brain cortical slices, suggesting that the difference in Ach synthesis that we have observed may reflect a specific alteration with aging in the metabolic mechanism for Ach synthesis.
Effect of selective kainate lesions on the release of glutamate and aspartate from chick retina
Authors: López‐Colomé A. M. , Somohano F.
Published: January 2005

In order to contribute evidence leading to establishing the excitatory pathways in the vertebrate retina, we selectively lesioned chick retinas by intraocular injection of 6, 60, 120, and 200 nmol of kainate, which selectively damages OFF‐bipolars, amacrines, horizontals, and ON‐bipolars, and measured the K+‐stimulated, Ca++‐dependent release of L‐(3H)‐glutamate and L‐(3H)‐aspartate. We also measured (3H)‐GABA release as a marker for horizontal cells and a population of amacrines, as well as (14C)‐glycine release as a tracer of a different subpopulation of amacrines. All four amino acids were released from control retinas by a depolarizing K+ concentration in a Ca++‐dependent fashion. GABA and glycine, however, showed an additional Ca++‐independent component of release. Lesion induced by 6 nmol of kainate decreased by 50% the release of glutamate and by 20% that of aspartate; glycine release was reduced 40% while GABA release was unaffected. Injection of 60 nmol of kainate reduced glutamate release a further 20% and significantly decreased GABA (50%) and glycine (75%) release; aspartate release remained unmodified; 120 nmol of kainate caused a further 30% reduction in aspartate and GABA release. Neither compound was significantly released after treatment with 200 nmol of kainate. These results seem to suggest that while OFF‐bipolars could release glutamate as transmitter, aspartate is released from a different cell population which is less sensitive to kainate, probably ON‐bipolars.