bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphocytic choriomeningitis virus infection in a province of Spain: Analysis of sera from the general population and wild rodents
Authors: Lledó Lourdes , Gegúndez María Isabel , Saz José Vicente , Bahamontes Noemí , Beltrán María
Published: April 2003

Lymphocytic choriomeningitis virus (LCMV) is a rodent‐borne virus belonging to the family Arenaviridae, genus Arenavirus, which causes a wide spectrum of human disease. However, data on LCMV infection in Spain is scant. To investigate whether this virus causes infection in Spain, 400 serum samples from the general population (191 males, 209 females) and 100 from wild rodents were studied by immunofluorescence assay (IFA) using L‐929 cells infected with LCMV. The study was performed in the “Community of Madrid,” a region with both rural and urban areas in different ecological settings. Of the 400 human serum samples tested, antibodies against LCMV were detected in 7 (1.7%). No statistical differences in prevalence were found with respect to either age or rural or urban residence, but differences were seen with respect to sex. Nine (9%) of the rodent serum samples were positive. These results confirm the occurrence of LCMV infections in Man and rodents in Spain. J. Med. Virol. 70: 273–275, 2003. © 2003 Wiley‐Liss, Inc.
Cellular and humoral immune responses to measles in immune adults re‐immunized with measles vaccine
Authors: Wong‐Chew Rosa Maria , Beeler Judy A. , Audet Susette , Santos José Ignacio
Published: April 2003

The objective of this study was to characterize the kinetics of the cellular and humoral immune responses elicited by measles vaccine given to previously immune adults. The cellular and humoral immune responses to measles were measured in seven healthy adults, before vaccination and at 1, 2, 3, and 4 weeks and 3 months after vaccination, using measles‐specific T‐cell proliferation and plaque reduction neutralization assays. All study subjects had detectable measles antibodies, but only six (85%) showed protective titers, defined as >1:120, before immunization. However measles‐specific T‐cell proliferation was not detectable before vaccination in any of the subjects. The six subjects with protective titers showed a positive stimulation index (SI) of >3.0 within the first 4 weeks after vaccination, an SI of 5 at the 4th week, and an SI of 3 at 3 months after vaccination. The subject with a low antibody titer (1:99) before vaccination developed a high SI at 3 months after vaccination. This subject was the only participant whose neutralizing antibody titers increased more than 4‐fold by 3 months after vaccination. No significant increases in geometric mean titers were detected in the other six subjects during the follow‐up period. These data suggest that high measles antibody titers interfere with the humoral response in subjects who receive a booster immunization, whereas the cellular response is boosted at least transiently, after revaccination. J. Med. Virol. 70: 276–280, 2003. © 2003 Wiley‐Liss, Inc.
Seroprevalence of human metapneumovirus in Japan
Authors: Ebihara Takashi , Endo Rika , Kikuta Hideaki , Ishiguro Nobuhisa , Yoshioka Mikio , Ma Xiaoming , Kobayashi Kunihiko
Published: April 2003

A new human pneumovirus, provisionally designated human metapneumovirus, was discovered by Dutch researchers. We examined 142 serum samples from the general population aged from 1 month to 35 years in Japan for human metapneumovirus antibody by indirect immunofluorescence assays using human metapneumovirus‐infected monkey kidney cells. The overall prevalence of human metapneumovirus infection was 72.5%. The seropositive rate was lowest in the age group of 6 months to 1 year and gradually increased with age. All of the children had been exposed to human metapneumovirus by the age of 10 years. The results show that human metapneumovirus is circulating in the Japanese population and is a ubiquitous virus acquired early in life. J. Med. Virol. 70: 281–283, 2003. © 2003 Wiley‐Liss, Inc.
Identification of a new genotype H wild‐type mumps virus strain and its molecular relatedness to other virulent and attenuated strains
Authors: Amexis Georgios , Rubin Steven , Chatterjee Nando , Carbone Kathryn , Chumakov Kostantin
Published: April 2003

A single clinical isolate of mumps virus designated 88‐1961 was obtained from a patient hospitalized with a clinical history of upper respiratory tract infection, parotitis, severe headache, fever and lymphadenopathy. We have sequenced the full‐length genome of 88‐1961 and compared it against all available full‐length sequences of mumps virus. Based upon its nucleotide sequence of the SH gene 88‐1961 was identified as a genotype H mumps strain. The overall extent of nucleotide and amino acid differences between each individual gene and protein of 88‐1961 and the full‐length mumps samples showed that the missense to silent ratios were unevenly distributed. Upon evaluation of the consensus sequence of 88‐1961, four positions were found to be clearly heterogeneous at the nucleotide level (NP 315C/T, NP 318C/T, F 271A/C, and HN 855C/T). Sequence analysis revealed that the amino acid sequences for the NP, M, and the L protein were the most conserved, whereas the SH protein exhibited the highest variability among the compared mumps genotypes A, B, and G. No identifying molecular patterns in the non‐coding (intergenic) or coding regions of 88‐1961 were found when we compared it against relatively virulent (Urabe AM9 B, Glouc1/UK96, 87‐1004 and 87‐1005) and non‐virulent mumps strains (Jeryl Lynn and all Urabe Am9 A substrains). J. Med. Virol. 70: 284–286, 2003. © 2003 Wiley‐Liss, Inc.
Hemagglutinin‐neuraminidase sequence and phylogenetic analyses of mumps virus isolates from a vaccinated population in Singapore
Authors: Lim C.S. , Chan K.P. , Goh K.T. , Chow Vincent T.K.
Published: April 2003

During 1999–2000, a sustained mumps outbreak in the highly vaccinated population in Singapore was attributed to vaccine failure associated with the Rubini vaccine strain. To explain this phenomenon, the complete nucleotide and amino acid sequences of the hemagglutinin‐neuraminidase (HN) gene of eight mumps virus isolates from patients with parotitis in Singapore were determined and compared with those of known vaccine strains. Phylogenetic trees constructed on the basis of HN nucleotide and amino acid sequences showed that the Singapore mumps virus isolates were more closely related to the Urabe strain and belonged to a different cluster from the Rubini and Jeryl‐Lynn strains. The Rubini vaccine showed only ∼93% nucleotide and ∼96% amino acid sequence similarity to Urabe and Singapore isolates. Compared with the vaccine strains, six of the eight isolates lacked the extracellular glycosylation site at residues 400–402. Other significant amino acid disparities (e.g., at residue 354) may also affect the antigenic properties of the HN protein. These findings suggest that the evolution and adaptation of the currently circulating mumps virus strains in the community has led to the emergence of genetically distinct viral strains. The low vaccine efficacy of the Rubini strain represents a major reason for the recent mumps resurgence and failure of mumps immunization in Singapore. J. Med. Virol. 70: 287–292, 2003. © 2003 Wiley‐Liss, Inc.
VP1 pseudocapsids, but not a glutathione‐S‐transferase VP1 fusion protein, prevent polyomavirus infection in a T‐cell immune deficient experimental mouse model
Authors: Vlastos Andrea , Andreasson Kalle , Tegerstedt Karin , Holländerová Dana , Heidari Shirin , Forstová Jitka , Ramqvist Torbjörn , Dalianis Tina
Published: April 2003

The ability to vaccinate against polyomavirus infection in a T‐cell deficient as well as a normal immune context was studied using polyomavirus major capsid protein (VP1) pseudocapsids (VP1‐ps) or a glutathione‐S‐transferase‐VP1 (GST‐VP1) fusion protein. VP1‐ps (1 or 10 μg) were administered subcutaneously, alone or together with Freund's complete and incomplete adjuvant, to CD4−/−8−/− T‐cell deficient or normal C57Bl/6 mice on four occasions. Alternatively, CD4−/−8−/− and normal mice were inoculated with either GST‐VP1 or Py‐VP1‐ps (5 μg). Following immunisation, antibody titres were tested by ELISA to VP1‐ps or GST‐VP1 or by haemagglutination inhibition (HAI). Mice were then infected with polyomavirus. Three weeks post‐infection, the mice were killed and examined for the presence of polyomavirus DNA by PCR. Viral DNA was not detected in CD4−/−8−/− mice immunised with either VP1‐ps alone or in combination with Freund's complete and incomplete adjuvant, or in any of the normal mice immunised with VP1‐ps or GST‐VP1. However, viral DNA was detected in 2/5 of the CD4−/−8−/− mice immunised with GST‐VP1 and in non‐immunised controls. Greater antibody titres were observed to VP1‐ps than to GST‐VP1 in CD4−/−8−/− mice after VP1‐ps compared to GST‐VP1 immunisation and antibody responses were better in normal than in immune‐deficient mice. Only immunisation with VP1‐ps resulted in haemagglutination inhibition. Complete protection against polyomavirus infection in the T‐cell deficient context was obtained with VP1‐ps, but not with GST‐VP1, immunisation using the present vaccination protocol. J. Med. Virol. 70: 293–300, 2003. © 2003 Wiley‐Liss, Inc.
Immune responses to the nonglycosylated ectodomain of respiratory syncytial virus attachment glycoprotein mediate pulmonary eosinophilia in inbred strains of mice with different MHC haplotypes
Authors: Hancock Gerald E. , Tebbey Paul W. , Scheuer Catherine A. , Pryharski Karin S. , Heers Kristen M. , LaPierre Natisha A.
Published: April 2003

Development of subunit vaccines against respiratory syncytial virus (RSV) for naive human infants is hindered by concerns that immunization with the fusion or attachment (G) proteins will elicit polarized Type 2 T cell responses and cause immunopotentiation upon subsequent natural infection. We investigated the regions of G protein responsible for inducing a Type 2 T cell phenotype in inbred mice of different MHC haplotype toward development of vaccines with improved safety. As demonstrated by IL‐5‐dependent pulmonary eosinophilia after challenge and serum anti‐G protein IgG1 to IgG2 ratios, highly purified native G protein sensitized all strains for a Type 2 T cell phenotype. Stimulation of G protein‐primed splenocytes with synthetic overlapping peptides indicated that the nonglycosylated ectodomain was primarily responsible. Respectively the recall responses of BALB/c (H2d), C57BL/6 (H‐2b), SJL (H‐2s), and C3H/HeJ (H‐2k) mice were directed against epitopes within peptides spanning amino acids 184–198 (pep184–198), 168–181 (pep168–181) or 171–185 (pep171–185), 176–190 (pep176–190), and 104–118 (pep104–118) or 159–173 (pep159–173). Injection of pep184–198conjugated to KLH (pep184–198‐KLH) primed H2d[BALB/c, B6.C‐H2d/bBy], but not H‐2b[C57Bl/6, C.B10‐H2b/LiMcd] mice for pulmonary eosinophilia. Sensitization with a peptide‐KLH conjugate encompassing amino acids 149–200 (pep149–200‐KLH) further confirmed that Type 2 T cell responses in BALB/c, C57BL/6 and SJL, but not C3H/HeJ mice were induced by the nonglycosylated ectodomain of G protein. These data are important for design of safe and efficacious subunit and attenuated vaccines for RSV. J. Med. Virol. 70: 301–308, 2003. © 2003 Wiley‐Liss, Inc.
Moderate local and systemic respiratory syncytial virus‐specific T‐cell responses upon mild or subclinical RSV infection
Authors: de Waal L. , Koopman L.P. , van Benten I.J. , Brandenburg A.H. , Mulder P.G.H. , de Swart R.L. , Fokkens W.J. , Neijens H.J. , Osterhaus A.D.M.E.
Published: April 2003

Respiratory syncytial virus (RSV) infections are a major cause of severe respiratory disease in infants. It has been shown that there is an increased frequency of childhood wheezing in ex‐bronchiolitic preteen children. This was postulated to be mediated by a vigorous virus‐specific Th2 response influencing the further development of the immune system. Little is known about the possible role of the immune response to clinically mild RSV infections in this respect. We have studied the RSV‐specific cellular immune response in infants with a laboratory‐confirmed RSV upper respiratory tract infection (URTI; n = 13, mean age 12 months, range 2–22 months) in comparison with infants with non‐RSV mediated URTI (n = 9, mean age 9.3 months, range 4–18 months) or infants with severe RSV bronchiolitis (n = 11, mean age 2.3 months, range 1–6 months). RSV‐specific cytokine‐producing cells were enumerated using the ELISPOT method in peripheral blood mononuclear cells and nasal brush T‐cells, collected during the acute and convalescent phase of the infection. Mixed Th1 (IFN‐γ) and Th2 (IL‐4 and IL‐13) responses were detected in all three groups. Frequencies of RSV‐specific T‐cells were lower in both URTI groups than in the RSV bronchiolitis group, and not significantly different between the RSV URTI and the non‐RSV URTI group. The absence of vigorous virus‐specific Th2 responses upon mild RSV infection does not support the hypothesis that these infections influence the development of the immune system and that they predispose for the development of atopic disease. J. Med. Virol. 70: 309–318, 2003. © 2003 Wiley‐Liss, Inc.
Detection of rhinovirus RNA in middle turbinate of patients with common colds by in situ hybridization
Authors: Pitkäranta Anne , Puhakka Tuomo , Mäkelä Mika J. , Ruuskanen Olli , Carpen Olli , Vaheri Antti
Published: April 2003

Human rhinovirus 14 RNA was determined by in situ hybridization from middle turbinate biopsies in 32 patients with diagnosed common colds and in five control individuals. Twenty‐two (69%) biopsies from common colds patients but none of the five control biopsies showed reactivity for human rhinovirus 14 antisense probe. The signal was detected both in the respiratory epithelium and in mucosal inflammatory cells. In situ hybridization of the middle turbinate tissue yielded more positive results than RT‐PCR (47%) or virus culture (34%) assayed from nasopharyngeal aspirates, but no statistical significant differences were observed (P = 0.265, P = 0.425, respectively). The results indicated that in situ hybridization procedure was slightly more sensitive than PCR assays and classical culture for the detection of human rhinovirus infection of upper respiratory tract. However, in situ hybridization procedure appeared to be an interesting methodology to investigate the physiopathology of respiratory tract infection by rhinoviruses. J. Med. Virol. 70: 319–323, 2003. © 2003 Wiley‐Liss, Inc.
Distribution of human group a rotavirus VP7 and VP4 types circulating in Seoul, Korea between 1998 and 2000
Authors: Song Mi‐Ok , Kim Ki‐Jung , Chung Sang‐In , Lim Inseok , Kang Shien‐Young , An Chang‐Nam , Kim Wonyong
Published: April 2003

Three hundred forty‐eight fecal specimens collected from young children with acute diarrhea in Seoul, Korea between January 1998 and February 2000 were examined for G and P types. Of these, 205 samples (59%) were confirmed as group A rotavirus by ELISA for the detection of VP6 antigen. Confirmed rotavirus isolates were characterized using G serotyping ELISA and RT‐PCR methodologies for G and P genotyping of the outer capsid proteins VP7 and VP4, respectively. Serotyping of the outer capsid protein, VP7, revealed G4 as the dominant circulating serotype (41%) followed by G1 (28%) and quite a high incidence of mixed infection (14%). Genotyping of the VP4 protein was carried out on 55 of the rotavirus isolates with the dominant type being P[8] (46%). Of interest were a number of unusual G and P type combinations detected in Korea for the first time, especially the P[4] genotype associated with non‐G2 serotypes. There were also a number of P[6] isolates identified including one G2P[6] isolate. J. Med. Virol. 70: 324–328, 2003. © 2003 Wiley‐Liss, Inc.
Live varicella vaccine polarizes the mucosal adjuvant action of cholera toxin or its B subunit on specific Th1‐type helper T cells with a single nasal coadministration in mice
Authors: Sasaki Keiko , Kato Michio , Takahashi Tsuyoshi , Ochi Sadayuki , Ichinose Yoshio , Shiraki Kimiyasu , Asano Yoshizo , Iwanaga Masaaki , Tsuji Takao
Published: April 2003

This study was undertaken to determine whether the specific Th1‐ or Th2‐cell response to varicella‐zoster virus was induced predominantly by a mucosal adjuvant, cholera toxin, in mice. A commercially available live varicella vaccine (Oka strain) and cholera toxin or its B subunit were administered simultaneously via the nasal route. Delayed‐type hypersensitivity to the Oka vaccine was induced, but the systemic neutralizing antibody response was low. The delayed‐type hypersensitivity evoked after a single administration was relatively higher than that on administration three times. When spleen cells from mice immunized once with the vaccine and cholera toxin or its B subunit were restimulated with the live vaccine in vitro, there was greater thymidine uptake and production of interleukin‐ 2 (IL‐2) than controls, but only a low level of IL‐4 production. The production of IL‐2 induced by the B subunit of cholera toxin was less than that by cholera toxin and a mutant of Escherichia coli enterotoxin on co‐immunization with the vaccine in mice. Cholera toxin and its B subunit have been reported to induce predominantly a specific Th2‐type T‐cell response to various antigens. However, the Oka vaccine is an antigen that polarizes the activation of specific Th1/Th2‐type T cells by cholera toxin or its B subunit to the Th1‐type side. Cholera toxin and its B subunit are thus useful mucosal adjuvants for inducing cellular immunity to the Oka vaccine similar to Escherichia coli enterotoxin. J. Med. Virol. 70: 329–335, 2003. © 2003 Wiley‐Liss, Inc.
Adenosine triphosphate induces proliferation of human neural stem cells: Role of calcium and p70 ribosomal protein S6 kinase
Authors: Ryu Jae K. , Choi Hyun B. , Hatori Kozo , Heisel Rochelle L. , Pelech Steven L. , McLarnon James G. , Kim Seung U.
Published: April 2003

Human neural stem cells (NSCs) grown in culture responded to extracellularly applied adenosine triphosphate (ATP), and the rate of proliferation increased as shown by immunocytochemical and RT‐PCR analysis. Activation of P2 purinoceptors by ATP is coupled to the release of intracellular calcium ([Ca2+]i) from thapsigargin‐sensitive intracellular stores. ATP‐induced proliferation was blocked by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+‐ATPase. Neither EGTA, a calcium chelator, nor caffeine had any effect on ATP‐induced [Ca2+]i increases. Multiblot kinase analysis, by which activation of 24 different kinases could be determined, showed that application of ATP to NSCs predominantly activated p70 ribosomal protein S6 kinase (p70 S6 kinase). As well, rapamycin, a p70 S6 kinase inhibitor, blocked the ATP‐mediated proliferative response in NSCs. After outlining a role for p70 S6 kinase in ATP‐mediated NSC proliferation, we examined the possibility that phosphatidylinositol 3‐kinase (PI3‐kinase) acts upstream of p70 S6 kinase. The application of wortmannin, a PI3‐kinase inhibitor, decreased both ATP‐mediated p70 S6 kinase activation and NSC proliferation. From these results, we conclude that ATP application to NSCs induces release of Ca2+ from intracellular Ca2+ stores and that this increase in intracellular Ca2+ in turn promotes NSC proliferation. The increase in NSC proliferation observed following ATP application can also be mediated by PI3‐kinase‐dependent p70 S6 kinase activation. © 2003 Wiley‐Liss, Inc.
Pituitary adenylate cyclase‐activating polypeptide prevents C2‐ceramide‐induced apoptosis of cerebellar granule cells
Authors: Vaudry David , Falluel‐Morel Anthony , Basille Magali , Pamantung Tommy F. , Fontaine Marc , Fournier Alain , Vaudry Hubert , Gonzalez Bruno J.
Published: April 2003

The sphingolipid metabolites, ceramides, are critical mediators of the cellular stress response and play an important role in the control of programmed cell death. In particular, ceramides have been shown to induce apoptosis of cerebellar granule cells. We show that pituitary adenylate cyclase‐activating polypeptide (PACAP) prevents C2‐ceramide‐induced apoptosis. The neuroprotective effect of PACAP was dose‐dependent and blocked by its antagonist, PACAP6‐38, whereas the PACAP‐related peptide VIP was inactive. The effect of PACAP on cell survival was mimicked by dibutyryl‐cAMP (dbcAMP) and forskolin and prevented by the MEK inhibitor U0126, indicating that both the adenylyl‐cyclase and MAP‐kinase pathways contribute to the neuroprotective action of the peptide. C2‐ceramide and PACAP induced opposite effects on phosphorylated forms of ERK and JNK without affecting the total amounts of ERK and JNK, suggesting that a balance between these two MAP‐kinases is critical for the cell survival/death decision. The effect of PACAP on ERK phosphorylation was blocked by U0126, but was not affected by H89 or chelerythrine indicating that PACAP activates ERK through a PKA‐ and PKC‐independent mechanism. C2‐ceramide induced a time‐dependent activation of caspase‐3, enhanced the amount of cleaved caspase‐3 and stimulated the DNA fragmentation process, while PACAP strongly inhibited the C2‐ceramide‐induced activation of caspase‐3, reduced the expression of cleaved caspase‐3 and blocked DNA fragmentation. Taken together, the present results show that C2‐ceramide induces apoptosis of cerebellar granule cells through a mechanism involving activation of caspase‐3. Our data also demonstrate that PACAP is a potent inhibitor of C2‐ceramide‐induced apoptosis. © 2003 Wiley‐Liss, Inc.
Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre‐degenerated peripheral nerves
Authors: Marcol Wieslaw , Kotulska Katarzyna , Święch‐Sabuda Ewa , Larysz‐Brysz Magdalena , Golka Beata , Górka Dariusz , Lewin‐Kowalik Joanna
Published: April 2003

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non‐pre‐degenerated or 7‐day‐pre‐degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain‐derived neurotrophic factor (BDNF) or fibrin only. We examined DiI‐labeled motoneurons, toluidine blue‐labeled myelinated fibers in the mid‐part of the chambers, and AChE‐positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7‐day‐pre‐degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF. © 2003 Wiley‐Liss, Inc.
Cellular migration in the postnatal rat cerebellar cortex: Confocal‐infrared microscopy and the rapid Golgi method
Authors: Liesi Päivi , Akinshola Emmanuel , Matsuba Kenji , Lange Kurt , Morest Kent
Published: April 2003

Confocal laser microscopy of DiI‐labeled slices of postnatal rat cerebellum (postnatal Day 4–10; P4–10) was compared to infrared microscopy and the rapid Golgi method (P0–14) to investigate postnatal migration of granule neurons. Vertical migration of the granule neurons occurred already at birth (P0). Surprisingly, mossy fibers often reached the external granule cell layer and were in close contact with the external granule cells. These mossy fibers may play a role in initiating granule cell migration. At this age, cell bodies of the immature neurons were attached to the external basal lamina by a process and extended down toward the presumptive internal granule cell layer. At P14, some granule cells remained attached to the surface, although their cell bodies exhibited the typical morphology of mature granule neurons and were located deep in the internal granule cell layer. These cells extended their endfeet‐like processes all the way to the surface of the brain. These results indicate that the vertical pathways of granule cell migration form early and persist throughout the period of granule cell migration. Confocal infrared microscopy of DiI‐labeled sections and the rapid Golgi method also allowed demonstration of tangentially migrating neurons that made one or more turns on the way to the internal granule cell layer. The rapid Golgi method confirmed that many Bergmann glial processes end at the level of the tangentially migrating granule cells whereas others project to the surface. These observations show that migratory granule cells take several different routes to their final destination, which cannot be explained by so‐called radial glial guidance. The only mode of migration in evidence is consistent with process elongation and translocation of the nucleus within the preformed processes. © 2003 Wiley‐Liss, Inc.
Differential connexin expression, gap junction intercellular coupling, and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons
Authors: Boucher Sherri , Bennett Steffany A.L.
Published: April 2003

Connexin‐mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT‐G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell‐replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT‐G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT‐G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye‐uptake studies. Dye coupling was inhibited by the gap junction blocker 18α‐glycyrrhetinic acid. Hemichannel activity was inhibited by the dual‐specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells. © 2003 Wiley‐Liss, Inc.
Arachidonic acid activates an open rectifier potassium channel in cultured rat cortical astrocytes
Authors: Ferroni Stefano , Valente Pierluigi , Caprini Marco , Nobile Mario , Schubert Peter , Rapisarda Carmela
Published: April 2003

A pathophysiological increase in free arachidonic acid (AA) is thought to regulate the channel‐mediated astrocytic swelling occurring in several brain injuries. We report that in cultured rat type‐1 cortical astrocytes, exposure to 10 μM AA activates an open rectifier K+ channel, which exhibits many similarities with TREK/TRAAK members of the two‐pore‐domain K+ channel family KCNK. Patch‐clamp experiments showed that the current developed with a long latency and was preceded by a depression of the previously described outward rectifier K+ conductance. Pharmacologic studies indicate that the K+ open rectifier was differentially sensitive to classic K+‐channel blockers (quinine, quinidine, tetraethylammonium, and barium) and was inhibited potently by gadolinium ions. The activation of this K+ current occurred independently of the AA metabolism as pharmacologic inhibition of the lipoxygenase, cyclooxygenase, and cytochrome P450 epoxygenase signaling cascades did not alter the AA effect. Moreover, neither the neutralization of the NADPH‐oxidase pathway nor scavenging intracellular free radicals modified the AA response. Finally, the AA‐induced K+ current was unaffected by protein kinase C inhibitors. The activation mechanism of the K+ open rectifier was through an extracellular interaction of AA with the plasma membrane. RT‐PCR analysis revealed that the AA‐induced K+ conductance was mediated likely by TREK‐2 channels. Collectively, the results demonstrate that in cultured cortical astrocytes, pathological levels of AA directly activate an open rectifier K+ channel, which may play a role in the control of K+ homeostasis under pathophysiological conditions. © 2003 Wiley‐Liss, Inc.
Involvement of calcium/calmodulin‐dependent protein kinase II in the induction of mPer1
Authors: Nomura Kazumi , Takeuchi Yusuke , Yamaguchi Shun , Okamura Hitoshi , Fukunaga Kohji
Published: April 2003

Recent studies suggest that CaM kinase II is involved in light‐induced phase delays and induction of Per1 and Per2 genes in the hamster suprachiasmatic nucleus (SCN) (Yokota et al.,2001). We focused on intracellular mechanisms of the CaM kinase II‐induced mPer1 gene expression. Immunoblotting and immunohistochemical analyses with isoform‐specific antibodies against different isoforms of CaM kinase II and CaM kinase IV showed abundant expression of the δ isoform of CaM kinase II without significant expression of CaM kinase IV in the lateral ventral region of the rat SCN. We next defined the CaM kinase II‐responsive region on the mPer1 promoter using a luciferase reporter gene assay. Transfection of the constitutively‐active CaM kinase IIδ greatly increased mPer1 promoter activity in NG108‐15 cells and increased activity slightly but significantly in NB2A and C6 glioma cells. Similarly, transfection of a constitutively‐active MEKK, an upstream kinase of mitogen‐activated protein kinase (MAPK), greatly increased promoter activity in NB2A cells. Deletion and mutation analyses of the mPer1 promoter revealed that a 5′‐GAGGGG‐3′ sequence motif near exon 1B, in which several zinc finger proteins seem to bind, was essential for the CaM kinase II‐induced activation of the mPer1 promoter. These results suggest that CaM kinase IIδ but not CaM kinase IV plays an essential role for mPer1 expression through the 5′‐GAGGGG‐3′ motif on the mPer1 promoter. © 2003 Wiley‐Liss, Inc.
Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei, and apoptosis
Authors: Bresgen N. , Karlhuber G. , Krizbai I. , Bauer H. , Bauer H.C. , Eckl P.M.
Published: April 2003

There is evidence accumulating that brain microvasculature is involved critically in oxidative stress‐mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system. © 2003 Wiley‐Liss, Inc.
Postnatal dendritic morphogenesis of cerebellar basket and stellate cells in vitro
Authors: Spatkowski Gabriele , Schilling Karl
Published: April 2003

Inhibitory interneurons in the molecular layer of the cerebellar cortex play an essential role in cerebellar physiology by providing feed‐forward inhibition to efferent Purkinje cells. Morphologic characteristics have been utilized to classify these cells as either basket cells or stellate cells. Conflicting evidence exists as to whether these cells are of distinct lineage and develop by employing discrete genetic programs, or whether their characteristic morphologic differences result from external cues that they encounter only after they have settled in their final territory in the molecular layer. We used primary dissociated cerebellar cultures established from early postnatal mice to study dendritogenesis of basket/stellate cells, identified by immunostaining for parvalbumin, under experimentally controlled conditions. We find that the radial axonal orientation of stem dendrites is non‐random, suggesting a cell‐intrinsic component defining this morphologic trait. In contrast, the expanse and complexity of basket/stellate cell dendrites is modulated by the granule cell derived neurotrophin, BDNF. BDNF‐induced morphogenetic effects decline with ongoing development. Overall, our data do not provide evidence for a distinct lineage or genetic makeup of cerebellar molecular layer inhibitory interneurons. © 2003 Wiley‐Liss, Inc.
Tropomyosin isoforms from the γ gene differing at the C‐terminus are spatially and developmentally regulated in the brain
Authors: Vrhovski Bernadette , Schevzov Galina , Dingle Sharon , Lessard James L. , Gunning Peter , Weinberger Ron P.
Published: April 2003

Tropomyosin is an actin‐binding protein responsible for stabilizing the actin microfilament system in the cytoskeleton of nonmuscle cells and is involved in processes such as growth, differentiation, and polarity of neuronal cells. From the γ gene, at least 11 different isoforms have been described, with three different C‐terminal exons used (9a, 9c, 9d). The precise roles that the different isoforms play are unknown. To examine the localization and hence determine the function of these isoforms in developing mouse, specific antibodies to exons 9a and 9c were made. These were used with previously developed 9d and N‐terminal 1b antibodies on Western blots and immunohistochemical analysis of mouse brains. We were able to show that all three C‐termini are used in the brain. 9c isoforms are highly enriched in brain and neural cells, and we also detected significant amounts of 9a‐containing isoforms in brain. γ gene activity is relatively constant in the brain, but the choice of C‐terminus is developmentally regulated. A more detailed study of the brain revealed regional expression differences. The hippocampus, cerebellum, and cortex were analyzed in depth and revealed that different isoforms could be sorted into different neuronal compartments, which change with development for 9d. Furthermore, a comparison with a homologous exon to 9c from the α‐tropomyosin gene showed that expression from these exons is related to the maturational state of the neuron, even though both are sorted differently intracellularly. These data suggest that the large numbers of tropomyosin isoforms are likely to have specific roles in microfilament dynamics and neural cell function. © 2003 Wiley‐Liss, Inc.
Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord
Authors: Wu Sufan , Suzuki Yoshihisa , Ejiri Yoko , Noda Toru , Bai Hongliang , Kitada Masaaki , Kataoka Kazuya , Ohta Masayoshi , Chou Hirotomi , Ide Chizuka
Published: April 2003

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord‐derived neurosphere cells by either cell mixing or making monolayered‐feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered‐feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting. © 2003 Wiley‐Liss, Inc.
Preservation of hematopoietic properties in transplanted bone marrow cells in the brain
Authors: Ono Kenji , Yoshihara Ken , Suzuki Hiromi , Tanaka Kenji F. , Takii Takemasa , Onozaki Kikuo , Sawada Makoto
Published: April 2003

Recent studies have described the possible transdifferentiation of bone marrow cells (BMC) into neurons and glia when they migrate to the brain. However, we have reported that some immature BMC migrating into the brain parenchyma after bone marrow transplantation express early hematopoietic markers but not neural or glial markers. The present study further characterizes transplanted BMC that migrate to the brain. Double immunolabeling confirmed that BMC migrating to the brain expressed hematopoietic but not neural markers, such as nestin, microtubule‐associated protein‐2 and glial fibrillary acidic protein, even 4 and 18 weeks after bone marrow transplantation. BMC that expressed green fluorescent protein also expressed hematopoietic but not neural markers when cultured with mixed brain cells according to double immunolabeling and single‐cell dissection using a laser. Analysis of the DNA content indicated that most of the migrated BMC were arrested at the G0/G1 phase, and aneuploidy or tetraploidy was undetectable. Thus, BMC that migrate to the brain probably have preserved hematopoietic properties under physiological conditions. © 2003 Wiley‐Liss, Inc.
A peptide fragment of ependymin neurotrophic factor uses protein kinase C and the mitogen‐activated protein kinase pathway to activate c‐Jun N‐terminal kinase and a functional AP‐1 containing c‐Jun and c‐Fos proteins in mouse NB2a cells
Authors: Adams David S. , Hasson Brendan , Boyer‐Boiteau Anne , El‐Khishin Adam , Shashoua Victor E.
Published: April 2003

Ependymin (EPN) is a goldfish brain neurotrophic factor previously shown to function in a variety of cellular events related to long‐term memory formation and neuronal regeneration. CMX‐8933, an 8‐amino‐acid synthetic peptide fragment of EPN, was designed for aiding an investigation of the biological properties of this glycoprotein. We reported from previous studies that treatment of mouse neuroblastoma (NB2a) cultures with CMX‐8933 promotes activation of transcription factor AP‐1, a characteristic previously associated with the following full‐length neurotrophic factors: nerve growth factor, neurotropin‐3, and brain‐derived neurotrophic factor. The CMX‐8933‐activated AP‐1 specifically bound an AP‐1 consensus probe and appeared to contain c‐Jun and c‐Fos protein components in antibody supershift experiments. Because AP‐1 influences a variety of positive and negative cellular processes, determined in part by its exact protein composition and mechanism of activation, we extended these initial AP‐1 observations in the current study to confirm the identity of the CMX‐8933‐activated c‐Jun and c‐Fos components. CMX‐8933 increases the enzymatic activity of c‐Jun N‐terminal kinase (JNK), increases the phosphorylation of JNK and c‐Jun proteins, and increases the cellular titers of c‐Jun and c‐Fos mRNAs. Furthermore, the AP‐1 activated by CMX‐8933 is functional, insofar as it transactivates both synthetic and natural AP‐1‐dependent reporter plasmids. Inhibition studies indicate that activation of the 8933‐induced AP‐1 occurs via the mitogen‐activated protein kinase pathway. These data are in agreement with the recently proposed model for the conversion of short‐ to long‐term synaptic plasticity and memory, in which a JNK‐activated transcription factor AP‐1, containing c‐Jun and c‐Fos components, functions at the top of a hierarchy of transcription factors known to regulate long‐term neural plasticity. © 2003 Wiley‐Liss, Inc.
c‐Src protein tyrosine kinase activity is required for muscarinic receptor‐mediated DNA synthesis and neurogenesis via ERK1/2 and c‐AMP‐responsive element‐binding protein signaling in neural precursor cells
Authors: Zhao Wei‐Qin , Alkon Daniel L. , Ma Wu
Published: April 2003

The G protein‐coupled muscarinic acetylcholine receptor (mAChR) isoforms have been identified in neural stem/progenitor (or precursor) cells. In previous studies, activation of these receptors induced elevations in intracellular Ca2+ signals and mitogen‐activated protein (MAP) kinase activity that led to enhanced DNA synthesis along with neurogenesis in neural precursor cells. Here we report that the nonreceptor protein tyrosine kinase c‐src activity is required for the muscarinic receptor‐activated MAP kinase and cAMP‐responsive element‐binding protein (CREB). Stimulation of neural precursor cells dissociated from embryonic day 13 rat cortical neuroepithelium with the muscarinic receptor agonist carbachol (CCh) induced phosphorylations of c‐src that were detected by antibodies raised against phospho‐Tyr416 (Ptyr416), phospho‐Tyr527 (Ptyr527), and phospho‐Tyr215 (Ptyr215) of the kinase. Although an increase in Ptyr416 suggested direct activation of c‐src, Ptyr215 may serve as an alternative mechanism underlying activation of c‐src without dephosphorylation of Ptyr‐527. Both extracellular signal‐regulated kinase (Erk1/2) and CREB were significantly activated after CCh treatment indicated by increases in phosphorylation of these two proteins. The c‐Src inhibitor PP1 abolished the CCh‐induced activation of Erk1/2 and CREB in a dose‐dependent manner. Moreover, CCh stimulated expression of the neuronal specific marker MAP2, which was inhibited by PP1. Cell proliferation assays and immunocytochemistry revealed that PP1 inhibited the CCh‐induced DNA synthesis and MAP2+ production. These results suggest that c‐src activity is essential for the muscarinic receptor‐mediated proliferation and neurogenesis in neural precursor cells via Erk1/2 and CREB signaling pathways. © 2003 Wiley‐Liss, Inc.
Role of radial fibers in controlling the onset of myelination
Authors: Nakahara Jin , Takemura Masaaki , Gomi Hiroshi , Tsunematsu Ken‐ichiro , Itohara Shigeyoshi , Asou Hiroaki , Ogawa Masaharu , Aiso Sadakazu , Tan‐Takeuchi Kyoko
Published: April 2003

Recent in vitro study showed that astrocytes induce oligodendrocyte processes to adhere to axons. However, the role of astrocytes in myelination in vivo remains unknown. We have, therefore, conducted a study to clarify the possible involvement of astrocytes during the initial myelination process. In newborn mice, the expression of glial fibrillary acidic protein (GFAP), a marker for astrocytes, was restricted to a few fibrous architectures in the subventricular zone (SVZ), but we did not observe any GFAP‐positive astrocytes. Prior to the onset of myelination, GFAP became transiently expressed in the cells with radial fibers elongating from the SVZ to the pia of cerebral cortex, and myelin‐associated glycoprotein (MAG)‐positive premyelinating oligodendrocytes appeared as neighbors to them, with the processes attaching to radial fibers, but not to axons. These GFAP‐positive “radial” cells lost their fibrous architecture and became typical GFAP‐positive astrocytes at about 10 days postnatally, when myelination set in, indicating that the disappearance of radial fibers coordinates with the initiation of myelination. From these results, we propose that premyelinating oligodendrocytes are in contact with radial fibers rather than axons and that the cytoarchitectural transformation of radial fibers into astrocytes is involved substantially in controlling the onset of initial myelination. Our proposal was further confirmed by GFAP‐deficient mice, in which the disappearance of these radial fibers and the initiation of myelination were delayed in parallel. Our findings together suggest that myelination in vivo is in concert with astrocytic differentiation, involving radial fibers therein, rather than being a mere axon–oligodendrocyte interaction. © 2003 Wiley‐Liss, Inc.
Adjusting urban bias in the regional mean surface temperature series of South Korea, 1968–99
Authors: Choi Youngeun , Jung Hyun‐Sook , Nam Kyung‐Yeub , Kwon Won‐Tae
Published: April 2003

The purpose of this paper is to produce a higher quality regional surface temperature series by removing urban biases in South Korean surface temperatures using statistical procedures. Monthly mean temperatures for 16 stations were obtained for a period of 32 years (1968–99). Each station is defined as an urban station or a rural station. Urban (rural) stations are defined as those that have population densities greater (less) than 1000 persons per squared kilometer in 1995. Ten urban stations and six rural stations are identified. Again, urban stations are subdivided into two groups according to whether their population totals exceed one million to examine magnitude changes of urban biases with the size of urban areas. Estimates of urban bias magnitude are calculated by averaging the difference between each urban station and every rural station. Estimates of mean urban bias magnitude (T̄u–r) are calculated by averaging the yearly urban bias estimates. Estimates of the urban trend (ΔTu–r) are obtained by differencing period means (by doubling the differences obtained between yearly estimates averaged over two 16 year periods, 1968–83 and 1984–99). For annual or seasonal mean temperature Ti, the adjusted temperature T′i is determined. As all estimates of T̄u–r are greater than zero, it suggests that temperatures in urban stations are warmer than those in rural stations. Estimates of the annual mean magnitude of urban bias range from 0.35 °C for smaller urban stations to 0.50 °C for large urban stations. Also, all estimates of ΔTu–r are positive, indicating an increasing trend in the urban bias time series. Seasonal variations are found in T̄u–r and ΔTu–r. After adjusting the urban bias, an increasing trend in surface temperature series is still evident. Copyright © 2003 Royal Meteorological Society