bluuebunny/crossref_metadata_2025 huggingface dataset:
Commissural size in neonatal rats: Effects of sex and prenatal alcohol exposure
Authors: Zimmerberg Betty , Scalzi Lisabeth V.
Published: April 2003

Sex differences have been reported in the size of the adult corpus callosum in both humans and rodents. This experiment investigated whether sex and/or different prenatal treatment conditions would influence commissural size at birth. Male and female 3‐day‐old Long Evans rats were selected from one of three prenatal treatment histories: prenatal alcohol‐exposed (35% ethanol‐derived calories, 35% EDC), nutritional control (0% ethanol‐derived calories, 0% EDC) or standard control (lab chow). Midline sagittal areas of the corpus callosum and the anterior commissure were determined for these subjects. Male control subjects had significantly larger callosal areas than females. Prenatal alcohol exposure significantly abolished this sexual dimorphism, with 35% EDC males having a significantly smaller callosal area than males from both control groups. This effect was independent of prenatal treatment differences in body or brain size. There were no significant sex differences in the midline sagittal area of the anterior commissure, nor were there any apparent effects of prenatal treatment on this measure. These results indicate that sex differences in the size of the corpus callosum are present at birth. Since a difference in myelination cannot account for this difference in area, there may be a sex difference in the number of fibers or in the average fiber size. Additionally, the effects of prenatal alcohol exposure on male, but not female, offspring suggest that this alcohol‐related birth defect is hormonally mediated.
The ontogeny of [3H]5‐hydroxytryptamine binding in the lamb: Effects of in vivo thyroidectomy
Authors: Richards Gail E. , Lakoski Joan M. , Black Eric W. , Halpern Rebekah E. , Ball Karen , Gluckman Peter D.
Published: April 2003

The effects of congenital hypothyroidism in the late gestation ovine fetus include changes in serotonin concentrations in specific brain areas. To investigate possible ontogenic patterns of changes in 5‐HT receptor function, we studied the binding characteristics of [3H]5‐HT in the midbrain, hypothalamus and cerebral cortex in the late gestation ovine fetus and young lamb. We compared the binding characteristics of control fetuses to those of thyroidectomized fetuses, with or without thyroxine replacement therapy.

In each of the areas examined, age‐dependent changes in the receptor density (Bmax) for [3H]5‐HT was observed. In cerebral cortex, Bmax was constant from 120 days gestation through the early neonatal period and increased significantly only at 25–30 days after birth. In hypothalamus, [3H]5‐HT binding density decreased late in gestation (140–145 days) with a return after birth to values comparable to those at 120–125 and 130–135 days gestation. The midbrain also exhibited a significant age‐dependent pattern of altered receptor density with a decrease in the 130–135 and 140–145 day gestational age groups compared to both younger and older lambs. In contrast, the affinity constant (Kd) for [3H]5‐HT did not change over the ages evaluated in cerebral cortex or hypothalamus. In the midbrain. however, there was a significant increase in Kd at 1–5 days after birth compared to all other age groups. The ability of fetal thyroidectomy, with or without thyroxine replacement therapy, to alter patterns of [3H]5‐HT binding was also tested. In the hypothyroid animals, examined at 130–135 days gestation, both the cerebral cortex and midbrain receptor affinities (Kd) were the same as control animals and animals of the same gestational age which had undergone throidectomy followed 10 days later by thyroxine treatment. In contrast, the receptor density (Bmax) for [3H]5‐HT was increased significantly in the midbrain of both thyroidectomy animals and animals which had thyroidectomy plus thyroxine treatment.

These data demonstrate marked patterns of ontogenic change in characteristics of [3H]5‐HT binding, particularly in the midbrain region of this species. In addition, these studies support the hypothesis that changes in 5‐HT receptor binding which result from hypothyroidism may be an index of thyroiddependent brain maturation.
Changes in acetylcholine receptor distribution and binding properties at the neuromuscular junction during aging
Authors: Smith Dean O. , Williams Kevin D. , Emmerling Mark
Published: April 2003

Junctional and extrajunctional acetylcholine receptors were characterized in diaphragm muscle obtained from mature adult and aged rats. Rhodamine‐conjugated α‐bungarotoxin was used to visualize receptor localization. At this level of resolution, there were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. Specific binding characteristics were assayed by measuring 125I‐α‐bungarotoxin binding. Maximal binding to intact junctional and extrajunctional tissue samples was greater in the older rats. The association rate constant in minced tissue decreased in the older animals. Retardation of the initial rate of toxin binding by d‐tubocurarine was described by a two‐component nonlinear Hofstee plot; values of Ki were about the same for both age groups, but there was a significant shift towards the low‐affinity values in the aged rats. Miniature end‐plate currents (m.e.p.c.s) were recorded under voltage‐clamp conditions before and after AChE inhibition. When AChE activity was inhibited m.e.p.c. amplitudes and decay time‐constants increased in both age groups. The magnitude of these increases was larger in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACh‐induced membrane noise. Lipid composition was assayed in whole muscle and isolated sarcolemma. Muscle cholesterol concentration rose 15–20 percent, but phospholipid concentrations were maintained. However, neither cholesterol, phospholipid levels, nor membrane fluidity changed significantly with age in isolated sarcolemmal membrane fractions.

These data indicate that the numbers of junctional and extrajunctional receptors increase with age. In the junctional region, this is quite likely due to an expanded field of receptors and not an increased density. This is associated with an increased fraction of receptors with lower binding affinity during aging. These changes apparently are not caused by major changes in membrane fluidity or lipid composition.
Cell‐type specific effects of N‐methyl‐d‐aspartate on biochemical differentiation of subcortical neurons in culture
Authors: Patel Ambrish J. , Hunt Anthony , Sanfeliu Coral
Published: April 2003

The possible involvement of N‐methyl‐d‐aspartate (NMDA) receptors in the biochemical differentiation of cultured neurons derived from the medial frontal part of the forebrain containing the septum‐diagonal band region was studied in terms of the activities of enzymes important in the synthesis of neurotransmitter compounds. The activity of choline acetyltransferase (ChAT) was used as a marker for cholinergic neurons, glutamate decarboxylase (GAD) for GABAergic neurons and phosphate‐activated glutaminase (GLNase) and aspartate aminotransferase (ASP‐AT) for glutamatergic neruons, while lactate dehydrogenase (LDH) was included as an ubiquitous enzyme. The exposure of cultures to a depolarizing concentration of K+ (40 mM) for the last 3 days (i.e. between 2 and 5 days in vitro) significantly enhanced the expression of ChAT, GAD and GLNase activities, but high K+ caused little alteration in the activities of ASP‐AT and LDH. On the other hand, treatment with NMDA markedly elevated the specific activities of GAD and GLNase only, and the compound had no significant effects on the activities of ChAT, ASP‐AT and LDH enzymes. The enhancements of the specific activities of GAD and GLNase were completely blocked by the NMDA receptor antagonist, 2‐amino‐5‐phosphonovaleric acid, and by the NMDA receptor‐linked Ca2+ ion channel blocker, MK‐801. On the basis of the present findings it is concluded that, (a) contrary to an earlier proposal, ASP‐AT does not appear to be a good marker for the glutamatergic neurons, (b) the failure of the subcortical cholinergic neurons to respond by an increase in ChAT activity to NMDA may indicate that these nerve cells lack NMDA subtype excitatory amino acid receptors, and (c) as the septal GABAergic input in the hippocampus is involved in the modulation of long‐term potentiation, the presence of NMDA receptors on these neurons would now suggest that NMDA receptors are linked to both the initiation and the modulation of hippocampal plasticity in the mammalian brain.
Appearance and localization of dystrophin in normal human fetal muscle
Authors: Prelle A. , Chianese L. , Moggio M. , Gallanti A. , Sciacco M. , Checcarelli N. , Comi G. , Scarpini E. , Bonilla E. , Scarlato G.
Published: April 2003

We studied the localization of dystrophin in normal human fetal muscle by immunohistochemistry. Our results show the appearance of dystrophin at week 11 and a progressive organization of the protein along membrane in the following weeks of gestation. At week 22 almost all fibers show a clear membrane immunostaining. Concomitant analysis of muscle fiber‐type composition reveals no correlation between progressive appearance of dystrophin and muscle fiber‐type differentiation. Our findings suggest that synthesis and localization of dystrophin in developing human skeletal muscle is time‐related and probably independent of neuronal influences.
Expression of c‐myc and c‐fos oncogenes in different rat brain regions during postnatal development
Authors: Parma Diana L. , Benasayag Silvia J. , Szijan Irene
Published: April 2003

We have studied the expression of c‐myc and c‐fos proto‐oncogenes in various areas of the central nervous system during postnatal development, c‐myc mRNA levels increased during the first 5 days and then decreased over the next 15 days in all nervous regions studied, c‐fos mRNA levels changed in a different way in four brain areas. While in cerebral cortex and cerebellum there was a sharp decrease during the first 10 days, in white matter and hypothalamus c‐fos transcript levels remained high during the same period, decreasing at a later stage.

Changes in oncogenes mRNA levels are related to various developmental events, such as neurite growth, myelination and cell proliferation. The dissimilar patterns of c‐myc and c‐fos expression suggests that they play different functions in CNS maturation, c‐myc mRNA levels are temporally related to active neurite growth and to cell proliferation. Changes in c‐fos mRNA correlate in time with early developmental processes and also with those occurring at later stages, such as myelination.
Elevated potassium prevents neuronal death but inhibits network formation in neocortical cultures
Authors: Baker R.E. , Ruijter J.M. , Bingmann D.
Published: April 2003

Chronic depolarization is inimical to neuronal growth and synaptogenesis so that spontaneous action potential generation appears to be required for the normal cytomorphological maturation of neocortical networks. The efficacy of 25 mM K in suppressing spontaneous bioelectric activity was monitored by extra‐ and intracellular recording from the explants. Intracellular recording from individual neurons showed that membrane potentials were reduced to ca −30 mV in potassium cultures but rapidly repolarized to ca −50 mV when returned to normal growth medium. Though action potentials could be readily evoked from these explants, spontaneous discharges and postsynaptic potentials were absent from potassium‐treated cultures. Both spontaneous bioelectric activity and postsynaptic potentials returned to the cultures by 5 days after returning the explants to normal growth medium. Extracellular recordings also showed that the explants were bioelectrically silent in the presence of 25 mM K or 25 mM K plus tetrodotoxin. In contrast to tetrodotoxin alone, bioelectric activity was absent when the cultures (with or without tetrodotoxin) were returned to normal growth medium. The explants gradually began to evince spontaneous bioelectric activity between 3 and 5 days after being returned to normal growth medium. Massive cell death induced by chronic exposure to tetrodotoxin was totally prevented by concomitant addition of 25 mM potassium, though these explants were significantly thinner than controls due to a large decrease in neuropil.

We conclude that chronic depolarization of neonatal cortical explants by potassium results in a delayed return of spontaneous bioelectric discharges. Chronic depolarization results in a retardation of network formation in these explants apparently due to a lack of neurite and/or synapse formation. These effects are reversible with function returning to the explants over a period of several days.
Some observations on the localization of hyaluronic acid in adult, newborn and embryonal rat brain
Authors: Bignami A. , Asher R.
Published: April 2003

Hyaluronic acid was localized in acetone‐fixed cryostat sections of brain and spinal cord obtained from adult, newborn and embryonal rat. The sections were incubated with glial hyaluronatebinding protein (GHAP) of human origin and the protein was visualized by indirect immunofluorescence with monoclonal antibodies raised to human GHAP and not staining rat brain by immunofluorescence. GHAP is a brain extracellular matrix (ECM) glycoprotein, approximately 60,000 molecular weight, which is structurally related to the HA‐binding region of cartilage ECM proteins. The distribution of hyaluronate in adult brain white matter and cerebellar cortex was similar to that previously reported for GHAP. In both cases, the reaction product formed a mesh surrounding myelinated axons and granule cells. Hyaluronate was also found in parts of the brain that did not contain GHAP. A finely reticulated mesh was observed in the neuropil between cell bodies in cerebral cortex and basal ganglia. Scattered cortical neurons were surrounded by a rim of reactive material. Perineural staining was the rule rather than the exception in spinal cord anterior horn motoneurons, inferior olivary nucleus, large bulbar reticular neurons and dentate nucleus of cerebellum. The only part of the brain which appeared relatively free of hyaluronate was the molecular layer of the cerebellum. In newborn and embryonal rat, the densely packed cell bodies in cerebral gray matter, periventricular germinal layer and external granular layer of cerebellum were surrounded by hyaluronate. Small droplets of hyaluronate were observed inbetween the cylindrical epithelial cells lining the neural tube in 11 day embryos. Non‐myelinated fiber tracts and the molecular layer of the developing cerebellum were relatively unstained. No hyaluronate was detected in the ependyma lining the cerebral ventricles and the central canal of the spinal cord.
Effects of pentylenetetrazol‐induced seizures on dopamine and norepinephrine levels and on glucose utilization in various brain regions of the developing rat
Authors: Hamdi Ghéziel El , Boutroy Marie‐Jeanne , Nehlig Astrid
Published: April 2003

Levels of dopamine and norepinephrine were measured in seven brain areas after 60 min of sustained seizure activity induced by intraperitoneal repetitive timed administrations of pentylenetetrazol in rats at 10, 14, 17 and 21 days of postnatal life. The tissue levels of norepinephrine were markedly reduced in the majority of brain structures, except for striatum at 10 and 14 days. Conversely, dopamine concentrations increased in many areas and at various ages, except in cerebral cortex at 10 and 14 days and in midbrain between 14 and 21 days. PTZ seizures induced marked increases over control levels in the rates of glucose utilization, measured by the quantitative autoradiographic [14C]2‐deoxyglucose method, in all dopamine‐ and norepinephrine‐innervated areas studied at 10 and 14 days, except in cerebellar cortex at both ages and in frontal cortex and anteroventral thalamus at 14 days. At 17 and 21 days, glucose utilization remained increased over control levels in some areas, mainly in catecholaminergic cell groupings such as substantia nigra, ventral tegmental area and locus coeruleus, but was significantly reduced in cortex, caudate nucleus and thalamus, and similar to control rates in other regions.

The present results suggest that pentylenetetrazol‐induced seizures lead to a simultaneous increase in functional activity of norepinephrine neurons and an inhibition of dopaminergic‐mediated neurons. They also confirm the maturation of connections, of metabolic activity and of neurotransmitter interaction within the brain, occurring mainly during the third week of postnatal life, paralleled by an increased selective vulnerability of some regions to this kind of insult.
Neuritogenesis on collagen substrates. Involvement of integrin‐like matrix receptors in retinal fibre outgrowth on collagen
Authors: Carri Néstor G. , Rubin Kristofer , Gullberg Donald , Ebendal Ted
Published: April 2003

Extracellular matrix molecules such as laminin, fibronectin and collagen promote neurite out‐growth in vitro. We have investigated the capacity of hydrated gels of collagen types I–III and monomeric collagen types I–VI on plastic surfaces to support neuritogenesis. The attachment and survival of explants from the day 6 chick embryo were studied and neurite outgrowth measured as mean elongation rate and maximal neurite length. Collagen types I and III, both as three‐dimensional gels or as native monomers supported neuritogenesis equal to or better than laminin. Collagen type V also supported neurite out‐growth although less effectively. Collagen types II, IV and VI, as well as denatured collagens of all types tested, did not support outgrowth. The monoclonal anti‐β1 integrin antibody (CSAT), as well as rabbit polyclonal antibodies directed to the integrin β1‐chain, effectively inhibited neurite outgrowth on permissive collagenous substrata, indicating that collagen‐binding integrins were involved in the neuritogenesis. These β1‐integrins were independent of Arg‐Gly‐Asp (RGD) since neurite formation proceeded in the presence of synthetic RGD‐containing peptides. Fluorescence immunohistochemistry revealed the presence of the integrin β1‐chain on the outgrowing neurites. The results suggest a possible function of collagen and collagen‐binding integrins in the development of the visual system.
Development of the cholinergic fibres innervating the cerebral cortex of the rat
Authors: Kiss Jozsef , Patel Ambrish J.
Published: April 2003

The ontogeny of innervation of the cholinergic fibres from the basal forebrain into the cingulate, frontal, parietal and piriform cortices of the rat has been examined using a modified histochemical method of acetylcholinesterase (AChE). The method produced crisp fibre staining with enhanced visibility and a clear back‐ground, and a pattern of the distribution of these fibres was comparable to that achieved by choline acetyltransferase (ChAT) immunocytochemistry. In the rat, the AChE‐stained fibres developed progressively from the deep cortical white matter towards the cortex itself. In general, a few AChE‐positive fibres were seen in the subcortical white matter and the cingulum bundle, entering into the cerebral cortex by about 5 postnatal days. The number of these AChE‐positive processes increased dramatically during the following two weeks. Thereafter, the general appearance of the overall pattern of distribution of the AChE fibres changed little, but the staining density became gradually more intense and by about 28 days after birth it was virtually indistinguishable from that in the adult. The onset and the development of the AChE‐positive fibre network varied considerably between individual cortical regions, and indicated, in general, an anterior to posterior gradient. Within the dispersed AChE fibre network in the cerebral cortex, three bands of relatively enriched cholinergic processes, namely the deep cortical, mid‐cortical and superficial layers, developed in an ‘inside‐out’ fashion. The exact position of some of these AChE‐rich bands varied from one cortical region to another and during development. A striking correlation during ontogeny was observed in the cerebral cortex between the changing patterns of AChE fibre network and the activity of ChAT, the enzyme synthesizing acetylcholine. The present findings can also provide an important anatomical baseline for future studies related to the factors controlling the expression of ChAT activity and the development of cholinergic neurotransmitter system in the rat.
Glial fibrillary acidic protein and its encoding mRNA exhibit mosaic expression in a glioblastoma multiform cell line of clonal origin
Authors: Bocchini V. , Beccari T. , Arcuri C. , Bruyere L. , Fages C. , Tardy M.
Published: April 2003

The expression of two astroglial differentiation markers, vimentin and glial fibrillary acidic protein, was investigated in a previously established human glioma cell line of clonal origin (GL15). Vimentin immunolabelling was homogeneously expressed in all cells. Glial fibrillary acidic protein and its encoding message, investigated by immunocytochemistry and in situ hybridization, showed a mosaic‐like expression. Only 30% of the cell population expressed glial fibrillary acidic protein and its mRNA. Western and Northern blots performed for both markers confirmed the presence of both proteins and messages, and their level was correlated with the observed antigenic and molecular probe labelling. The overall antigenic pattern suggests that GL‐15 cells do not belong to the O‐2 A progenitor cell lineage and may arise from a clonal expansion of astrocyte precursors.
The pH of jimpy glia is increased: Intracellular measurements using fluorescent laser cytometry
Authors: Knapp Pamela E. , Booth Charles S. , Skoff Robert P.
Published: April 2003

The jimpy mutation lies in the gene which codes for myelin proteolipid protein, and the brains and spinal cords of jimpy mice contain little myelin and no measurable proteolipid protein. It has been thought that the mutation affected only the myelin forming oligodendroglial cells, but there is now considerable evidence that astroglia are also a target of the mutation since jimpy astrocytes exhibit a prominent gliosis along with defects in metabolism and proliferation. Because cell proliferation is associated with an increase in intracellular pH, we investigated whether the pH of jimpy glia was abnormal. Using a pH sensitive fluorescent dye and a laser cytometry system we measured the intracellualr pH of individual cells in cultures derived from both jimpy and normal brains. The relative pH of flat astrocytes in jimpy cultures was higher than in normal cultures by an average of 0.24 pH units, and these increased values were evident 2–3 days after plating. At this in vitro age the cultures contain only a few oligodendrocytes, none of which express detectable proteolipid protein. The pH of the process‐bearing cell population, which contains the oligodendrocytes as well as some astrocytes and presumptive glial precursors, was also increased but not until 7 days in culture.

The finding that a mutation in the myelin proteolipid protein gene can alter the normal pH of astrocytes is quite unexpected since, as far as is known, astrocytes do not make proteolipid protein. These results and others discussed in this paper support the hypothesis that either proteolipid protein itself, or some other product of the gene, may have an important role in central nervous system development.
Microglia‐derived plasminogen enhances neurite outgrowth from explant cultures of rat brain
Authors: Nagata Koichi , Nakajima Kazuyuki , Takemoto Nagisa , Saito Hiroshi , Kohsaka Shinichi
Published: April 2003

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett.308, 179–182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum‐free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16‐day‐old rat brain by using 125I‐Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high‐ and low‐affinity binding sites on the neurons. The estimated dissociation constants of high‐ and low‐affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia‐derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.
Relationship between ischemia and ischemic neuronal necrosis to astrocyte expression of glial fibrillary acidic protein
Authors: Petito Carol K. , Halaby Issam A.
Published: April 2003

It is not entirely clear whether the proliferative changes in astrocytes following cerebral ischemia are in response to neuronal injury or are secondary to the direct effects of ischemia on the astrocytes. Therefore, the following study examined the relationship between post‐ischemic astrocytosis with the extent of neuronal necrosis and the severity of the ischemia. Astrocyte reactivity was assessed by alterations in glial fibrillary acidic protein (GFAP), using immunohistochemistry and evaluation by optical density analysis. Cerebral ischemia was produced in rats by temporary occlusion of the carotid and vertebral arteris for 2,10 and 30 min. This results in damage to the CA1 neurons after a characteristic delay of several days, the duration of which is inversely proportional to the severity of the ischemia. CA3 neurons are resistant to the ischemia and do not suffer permanent injury. The results showed that GFAP immunoreactivity significantly increased in the CA1 region after all three ischemic intervals but the rise of GFAP in the CA3 area reached significance only after 30 min of ischemia. The peak and duration of the GFAP increases thus correlated with the extent and the maturation of the neuronal necrosis. This suggests that with mild injury (2 and 10 min ischemia), post‐ischemic astrocytosis is closely related to its neuronal environment rather than to the ischemic insult itself. Furthermore, the results showed an initial decrease in and delay of the subsequent GFAP rise. This initial decline in GFAP and the delay in its rise have both been identified in other models of brain injury and may be related in part to transient astrocyte swelling as well as to a finite interval required for sufficient increases in transcriptional activity to affect a noticeable rise in GFAP immunoreactivity.
Age‐dependent changes in the proliferative response of S‐100 protein‐positive glial cells to injury in the rat brain
Authors: Janeczko Krzysztof
Published: April 2003

A mechanical injury was inflicted to the left cerebral hemisphere in rats of four age groups: newborns, 6, 14 and 30 days old. The injury was followed by [3H]thymidine injections at different time intervals. Brain sections were immunostained for S‐100 protein and subjected to autoradiography. During microscopic observations of the injury region, locations and numbers of the autoradiographically labeled astrocytes expressing S‐100 protein were recorded. On the basis of the observations, injury‐induced changes in the total number of proliferating astrocytes, as well as in their distribution, were analysed quantitatively.

In rats injured neonatally, as well as those injured on postnatal days 6 and 14, the reactive increase in the number of proliferating astrocytes began on the first post‐traumatic day. In 30‐day‐old rats the increase was slower and appeared on day 2.

The maximal increase in the astrocyte proliferative activity occurred in 6‐day‐old rats as early as day 1 after injury and was about eight times higher than that recorded in newborns, and nearly twice as high as that recorded in brains of 30‐day‐old rats.

The results suggest that the intensity of astrocyte proliferative response to injury cannot be regarded as simply being proportional to the developmental progress of the brain tissue. Rather, these results indicate that changes in glial proliferative responses to injury follow a developmental time course, with a peak around the end of the first postnatal week.
Characterization of N‐cadherin messenger RNA and polypeptide expression in rat
Authors: Linnemann Dorte , Gaardsvoll Henrik , Dalseg Anne‐Marie , Zhernosekov Dmitri , Lundgren Torben , Edvardsen Klaus , Bock Elisabeth
Published: April 2003

The cell adhesion molecule N‐cadherin is a member of the cadherin gene superfamily. The protein is involved in morphogenetic processes, including neurite extension. In this study, N‐cadherin mRNA and polypeptide expression were investigated in rat brain, liver, muscle, heart, kidney and lung during postnatal development and aging. Six synthetic oligonucleotide probes covering different parts of mouse N‐cadherin cDNA all hybridized to 5.2,4.3–4.4 and 3.5 kb mRNAs in rat tissues. The mRNA pattern differed between tissues and, furthermore, the amount of N‐cadherin mRNA and polypeptides in brain, liver and heart was higher than in muscle, kidney and lung. N‐cadherin expression decreased slightly during early postnatal development in all tissues, whereas no changes in N‐cadherin expression were observed during aging.

Antibodies against a fusion protein containing the transmembrane and cytoplasmic sequence of chick N‐cadherin were produced. These antibodies, termed anti‐N‐cad‐cyt, were compared to the R‐156 antibodies which recognize the 24 C‐terminal amino acids of N‐cadherin and which have been shown to react with a broad spectrum of cadherins. Using these two antibodies, it was shown that the 130 kDa N‐cadherin polypeptide was subject to calcium‐dependent cleavage of the cytoplasmic domain. Conversely, in the absence of calcium the polypeptide was cleaved extracellularly, producing two C‐terminal fragments of 85 and 95 kDa. A 122 kDa polypeptide was recognized by both antibodies and may be either an alternatively spliced form of N‐cadherin or a closely related cadherin.
Presynaptic ecto‐ and postsynaptic endo‐calcium‐adenosine‐triphosphatases in synaptosomes: Doubts about biochemical interpretation of localization
Authors: Kittel A. , Bácsy E.
Published: April 2003

Ecto‐ and endo‐Ca‐adenosine‐triphosphatase (ATPase) activity was identified as electrondense lead or cerium phosphate precipitate in the rat cortical synaptosomes by transmission electron microscopy and enzyme histochemistry. The formation of the deposit was dependent on the presence of ATP (the substrate), Ca (activator) and levamisole, quercetin or ouabain (inhibitors of different phosphatases and ATPases). Reaction products were found at the external surface of the presynaptic membrane, both surfaces of the postsynaptic membrane, in the synaptic cleft and in the free mitochondrial membranes. In the presence of ATP and the three inhibitors together, the quantity of the precipitate decreased markedly, but we still found some deposit on the external surface of the presynaptic membrane (this activity is probably due to the so‐called ecto‐ATPase) and on the internal surface of the postsynaptic one (endo‐ATPase). The distinction between ecto‐ and endo‐ATPases in biochemical fractions solely upon biochemical differential measurements must be interpreted with caution.
The potential role of nerve growth factor, brain‐derived neurotrophic factor and neurotrophin‐3 in avian cochlear and vestibular ganglia development
Authors: Bernd P. , Zhang D. , Yao L. , Rozenberg I.
Published: April 2003

The role of the nerve growth factor family of neurotrophins in the development of cochlear and vestibular ganglia is unclear. In order to predict the potential importance of nerve growth factor, brain‐derived neurotrophic factor or neurotrophin‐3, we examined the expression of neurotrophin mRNA and full‐length neurotrophin receptor mRNA by in‐situ hybridization and reverse transcription‐polymerase chain reaction, as well as whether high affinity 125I‐nerve growth factor binding was present, in cochlear and vestibular ganglia of the quail at several stages of development (stages 26, 31 and 36). Nerve growth factor, brain‐derived neurotrophic factor and neurotrophin‐3 mRNA was detected at all ages examined, suggesting that these neurotrophins may serve an autocrine or paracrine function, especially prior to target contact. In addition, we found full‐length trkA and trkC mRNA was expressed, the products of which are the functional neuronal receptors for nerve growth factor and neurotrophin‐3, respectively. Although full‐length trkA mRNA was found, physiologically important high affinity 125I‐nerve growth factor binding was not detected. Since nerve growth factor's effects on survival and neurite outgrowth are mediated through high affinity binding, nerve growth factor may serve an as yet unidentified role in this system. Full‐length trkB mRNA, the product of which is the functional neuronal receptor for brain‐derived neurotrophic factor, was not detected using reverse transcription‐polymerase chain reaction, however, truncated (non‐catalytic) trkB was present, at least in cochlear ganglia at stage 31. It is not known what function may be subserved by these truncated receptors.
Rapid response genes as markers of cellular signaling during cortical histogenesis: Their potential in understanding mental retardation
Authors: Kaufmann W.E. , Yamagata K. , Andreasson K.I. , Worley P.F.
Published: April 2003

Aberrations of dendritic morphology are seen in most forms of mental retardation (MR). Normal cortical development is dependent on neural activity that modulates developmental processes such as dendritic differentiation. Indeed, many of the classical histological correlates of MR are reproduced in models that alter activity during development. To explore the hypothesis that MR results from aberrant activity signals during development, it would be useful to have histochemical markers that are sensitive to neural activity. Recent studies indicate that certain immediate early genes (IEGs) are normally expressed at relatively high levels in cortical neurons during postnatal development and are rapidly regulated by natural activity. We have begun to assess the possible use of IEG markers to study MR by examining the pre‐ and postnatal developmental time course of a panel of known IEG transcription factors as well as a set of novel IEGs identified in our laboratory. One of these recently characterized clones encodes a novel, mitogen‐inducible cyclo‐oxygenase that is expressed during a critical period for dendritic formation and is regulated by N‐methyl‐d‐aspartate‐dependent synaptic activity and by environmental stimuli. These data suggest a role for prostaglandin signaling in postnatal cortical development. Other partially characterized novel IEGs are expressed in a cell‐specific fashion in the cortical plate. Application of these histochemical markers to the study of MR pathogenesis in animal models is discussed.
Neuropathology of twitcher mice: examination by histochemistry, immunohistochemistry, lectin histochemistry and fourier transform infrared microspectroscopy
Authors: Levine Steven M. , Wetzel David L. , Eilert Arnold J.
Published: April 2003

The twitcher mouse is an authentic animal model of globoid cell leukodystrophy, which is a genetic disease that affects the lysosomal enzyme galactocerebroside β‐galactosidase. This enzyme deficiency causes one of its substrates, galactosylsphingosine (psychosine), to accumulate in myelin‐forming cells, which eventually results in their death. In the central nervous system, the death of oligodendrocytes is thought to cause a series of secondary pathological changes. In this study, several techniques were utilized to examine the neuropathology of two different brain regions in the twitcher mouse—the hindbrain and the cerebrum. Neuropathological changes were as follows: (1) demyelination was detected in the hindbrain but not in the cerebrum, (2) a high density of periodic acid‐Schiff‐positive cells were detected in the hindbrain and to a lesser extent in the cerebrum, (3) astrocyte gliosis was pronounced in both the hindbrain and cerebrum, and (4) macrophages were abundant in both the hindbrain and the cerebrum. We found that Periodic acid‐Schiff‐positive cells, astrocyte gliosis and macrophage infiltration were present in white and gray matter regions of the cerebrum, while they were generally absent from the granule and molecular layers of the cerebellum. In addition to these studies, we utilized the technique of Fourier transform infrared (FT‐IR) microspectroscopy to identify the in situ distribution of psychosine in the brains of twitcher mice. Evidence was obtained that indicates a large accumulation of psychosine in the hindbrain, and to a lesser extent in the white matter of the cerebrum in the twitcher mouse, but not the normal mouse. There was no evidence for the accumulation of psychosine in the molecular layer of the cerebellum from the twitcher or normal mouse. Our conclusions are as follows: (1) pathology is more advanced in the hindbrain compared to the cerebrum, which is likely due to the hindbrain becoming myelinated prior to the cerebrum, (2) demyelination is not necessary for the development of secondary pathological changes, (3) pathology is not limited to white matter in the cerebrum, (4) pathology is not present in all brain regions, i.e. the granule and molecular layers of the cerebellum are devoid of pathological changes, and (5) psychosine accumulates in both the cerebrum and hindbrain, but not in the molecular layer of the cerebellum in the twitcher mouse. This study demonstrates that FT‐IR microspectroscopy can be used to correlate chemical changes to histopathological changes in brains from twitcher mice, which suggests that FT‐IR microspectroscopy may be a useful tool for studies examining other brain diseases.
Disabled‐2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells
Authors: Tseng Ching-Ping , Huang Chien-Ling , Huang Ching-Hui , Cheng Ju-Chien , Stern Arnold , Tseng Chin-Hsiao , Chiu Daniel Tsun-Yee
Published: April 2003

Previous studies have shown that Disabled‐2 (DAB2) is up‐regulated during megakaryocytic differentiation of human K562 cells. To delineate the consequences of DAB2 induction, a DNA vector‐based small interfering RNA (siRNA) was designed to intervene in DAB2 expression. We found that DAB2 siRNA specifically inhibited DAB2 induction, resulting in the modulation of cell–cell adhesion and mitogen‐activated protein kinase (MAPK) phosphorylation. The morphological changes and β3 integrin expression associated with megakaryocytic differentiation were not affected. Since the MAPK pathway has been shown to involve DAB2 induction [Tseng et al., Biochem. Biophys. Res. Commun. 285 (2001) 129–135], our results suggest a reciprocal regulation between DAB2 and MAPK in the differentiation of K562 cells. In addition, we have demonstrated for the first time that DAB2 siRNA is a valuable tool for unveiling the biological consequences of DAB2 expression.
C/EBP homologous protein (CHOP) up‐regulates IL‐6 transcription by trapping negative regulating NF‐IL6 isoform
Authors: Hattori Takayuki , Ohoka Nobumichi , Hayashi Hidetoshi , Onozaki Kikuo
Published: April 2003

Interleukin‐6 (IL‐6) production is up‐regulated by several stimuli through the activation of transcription factors. We have previously demonstrated that CCAAT/enhancer binding protein homologous protein (CHOP) positively regulates IL‐6 production at the transcriptional level in the human melanoma cell line A375. In this study, we provide evidence that CHOP up‐regulates the IL‐6 transcription without binding to the IL‐6 promoter. CHOP dimerized more preferentially with an inhibitory isoform of nuclear factor for IL‐6 expression (LIP (liver‐enriched inhibitory protein)) than with a positively acting isoform (LAP, liver‐enriched activator protein). These results indicate that CHOP plays an important role in IL‐6 production without binding to its promoter, probably by trapping protein(s) such as LIP, which would otherwise inhibit IL‐6 transcription.
The fully conserved Asp residue in conserved sequence region I of the α‐amylase family is crucial for the catalytic site architecture and activity
Authors: Leemhuis Hans , Rozeboom Henriëtte J. , Dijkstra Bauke W. , Dijkhuizen Lubbert
Published: April 2003

The α‐amylase family is a large group of starch processing enzymes [Svensson, B. (1994) Plant Mol. Biol. 25, 141–157]. It is characterized by four short sequence motifs that contain the seven fully conserved amino acid residues in this family: two catalytic carboxylic acid residues and four substrate binding residues. The seventh conserved residue (Asp135) has no direct interactions with either substrates or products, but it is hydrogen‐bonded to Arg227, which does bind the substrate in the catalytic site. Using cyclodextrin glycosyltransferase as an example, this paper provides for the first time definite biochemical and structural evidence that Asp135 is required for the proper conformation of several catalytic site residues and therefore for activity.
Comparison of the anti‐apoptotic effects of Bcr‐Abl, Bcl‐2 and Bcl‐xL following diverse apoptogenic stimuli
Authors: Brumatti Gabriela , Weinlich Ricardo , Chehab Cristina F , Yon Monica , Amarante-Mendes Gustavo P
Published: April 2003

Ectopic expression of Bcr‐Abl, Bcl‐2 or Bcl‐xL in HL‐60 cells conferred resistance to apoptosis against a variety of death‐inducing agents. Bcr‐Abl‐mediated interference with mitochondrial events was confirmed by the analysis of the loss of mitochondrial transmembrane potential and cytochrome c release. HL‐60.Bcr‐Abl cells were extremely resistant to all apoptogenic stimuli tested, even in circumstances where HL‐60.Bcl‐2 or HL‐60.Bcl‐xL cells were only partially protected from apoptosis. The levels of Mcl‐1, Bax, Bid, Akt, c‐IAP‐1, c‐IAP‐2, XIAP and c‐FLIP were compared in all HL‐60 lines. Our findings show that Bcr‐Abl is a more powerful anti‐apoptotic molecule than Bcl‐2 or Bcl‐xL.
Enhanced plasma ghrelin levels in rats with streptozotocin‐induced diabetes
Authors: Masaoka Tatsuhiro , Suzuki Hidekazu , Hosoda Hiroshi , Ota Takayuki , Minegishi Yuriko , Nagata Hiroshi , Kangawa Kenji , Ishii Hiromasa
Published: April 2003

Ghrelin is a novel gastrointestinal peptide that stimulates growth hormone secretion, food intake, and body weight gain. Increased ghrelin secretion has been reported in such negative energy states as starvation and low body weight. We investigated the dynamics of ghrelin in rats with streptozotocin‐induced diabetes, because they present reduced body weight and hyperphagia. The plasma ghrelin levels and gastric preproghrelin mRNA expression levels of the diabetic rats increased significantly and their gastric ghrelin levels decreased significantly. Negative energy balance may enhance preproghrelin mRNA expression and ghrelin secretion into the bloodstream.
Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases
Authors: Pitson Stuart M. , Voragen Alphons G.J. , Beldman Gerrit
Published: April 2003

The stereochemical course of hydrolysis catalyzed by various enzymes acting on arabinofuranosyl linkages has been determined. 1H‐NMR analysis of the action of endo‐(1 → 5)‐α‐l‐arabinanases from Aspergillus niger and Aspergillus aculeatus showed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. niger enzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of α‐l‐arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatum and Bacillus subtilis were investigated using both 1H‐NMR and high performance anion exchange chromatography to follow glycosyl transfer reactions to methanol. In all cases these enzymes catalyzed the reaction with retention of configuration, and therefore probably operate via double displacement hydrolytic mechanisms. From the results with arabinofuranosidase A and B from A. niger we predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1 → 4)‐β‐d‐arabinoxylan arabinohydrolases from A. awamori, Trichoderma reesei and Bifidobacterium adolescentis only enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.
Ribonuclease A mutant His119Asn: the role of histidine in catalysis
Authors: Panov Konstantin I. , Kolbanovskaya Elena Yu. , Okorokov Andrei L. , Panova Tatiana B. , Terwisscha van Scheltinga Anke C. , Karpeisky Marat Ya. , Beintema Jaap J.
Published: April 2003

Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His12, His119, and Lys41) are known to be involved in catalysis. Mutation of His119 to asparagine was generated to study the role of His119 in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His119 of the wild‐type enzyme must be protonated in an enzyme‐substrate productive complex. Thus our results indicate that a contribution of protonated His119 into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His119 participates directly in a transition state stabilization via hydrogen bonding.
Expression of activin A in human keratinocytes at early stages of cultivation
Authors: Seishima Mariko , Nojiri Mari , Akiyama Tomoko , Seishima Mitsuru , Noma Akio , Etoh Yuzuru , Kitajima Yasuo
Published: April 2003

Activins are members of the TGF‐β superfamily and are classified into 3 types: activin A, which consists of a homodimer of β
A, activin B, which consists of a homodimer of β
B, and activin AB, which consists of a heterodimer of β
A
β
B. We studied the expression of activin mRNAs by RT‐PCR in normal human epidermis, cultured keratinocytes, and DJM‐1 cells (a squamous cell carcinoma line). We could detect only activin A mRNA (β
A) in normal human epidermis. In cultured keratinocyte and DJM‐1 cells, activin β
A mRNA was observed at 4 h but not at 96 h after plating. Activin A activity was detected in the conditioned medium of DJM‐1 cells within 48 h. In addition, although follistatin mRNA was not observed in human epidermis in situ, it was transiently expressed in cultured cells at 4 h after plating. These findings suggest that the expression of these molecules in keratinocytes is associated with cell proliferation. In an in vitro tissue injury model, activin A was observed at the wound edge, where cell migration and proliferation may be activated. In DJM‐1 cells cultured for 92 h, β
A mRNA was observed 4 h after injury treatment. These findings suggest that activin A acts as a potent inducer of proliferation in vitro, at least in keratinocytes.