bluuebunny/crossref_metadata_2025 huggingface dataset:
Seasonal succession and long-term stability of pelagic community in a productive reservoir
Authors: Matveev V. F. , Matveeva L. K.
Published: November 2005

In Lake Hume, a reservoir located in an active agricultural zone of the Murray River catchment, Australia, time series for the abundances of phytoplankton and zooplankton taxa, monitored from 1991 through to 1996, were stationary (without trends), and plankton taxonomic composition did not change. This indicated ecosystem resilience to strong fluctuations in reservoir water level, and to other potential agricultural impacts, for example eutrophication and pollution. Although biological stressors such as introduced fish and invertebrate predators are known to affect planktonic communities and reduce biodiversity in lakes, high densities of planktivorous stages of alien European perch (Perca fluviatilis) and the presence of carp (Cyprinus carpio) did not translate into non-stationary time series or declining trends for plankton in Lake Hume. However, the seasonal successions observed in the reservoir in different years did not conform well to the Plankton Ecology Group (PEG) model. Significant deviations of the Lake Hume successional pattern from the PEG model included maxima for phytoplankton abundance being in winter and the presence of a clear water phase without large zooplankton grazers. The instability of the water level in Lake Hume probably causes the dynamics of most planktonic populations to be less predictable, but did not initiate the declining trends that have been observed in some other Australian reservoirs. Both the PEG model and the present study suggest that hydrology is one of the major drivers of seasonal succession.
Utility and efficiency of multi-mesh gill nets and trammel nets for sampling assemblages and populations of estuarine fish
Authors: Gray Charles A. , Jones Martine V. , Rotherham Douglas , Broadhurst Matt K. , Johnson Daniel D. , Barnes Lachlan M.
Published: November 2005

Two replicate multi-mesh gill and trammel nets, each comprising five 30 m long panels made from different-sized mesh (38, 54, 70, 90 and 100 mm stretched mesh openings) were fished in a south-east Australian barrier estuary over seven nights to evaluate their potential as sampling gears for fishery-independent surveys of estuarine fish assemblages. There were no differences in composition and structure of assemblages, mean abundance, or diversity of catches between the two types of net. The composition and structure of catches differed between mesh sizes, with the panels made from 38 and 54 mm mesh retaining significantly more fish and species than the larger-sized meshes. The two smallest mesh sizes were important for capturing sub-adults and juveniles of some species. Based on a greater precision of catch per unit effort (CPUE) estimates, less sampling effort and greater ease of use, the multi-mesh gill net was a better sampling unit than the trammel net for assessments of estuarine fish populations.
Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR
Authors: Bourne D. G. , Blakeley R. L. , Riddles P. , Jones G. J.
Published: November 2005

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.
Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types
Authors: Walisser Jacqueline A. , Glover Edward , Pande Kalyan , Liss Adam L. , Bradfield Christopher A.
Published: November 2005

The aryl hydrocarbon receptor (AHR) plays a role in three areas of biology that include the adaptive metabolism of xenobiotics, the toxic responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-
p
-dioxin (dioxin), and vascular remodeling of the developing embryo. To test the hypothesis that receptor signaling in different cell types is responsible for these aspects of AHR biology, we generated a conditional
Ahr
allele where exon 2 is flanked by
loxP
sites. Through the use of
Cre
-lox technology, we then investigated the role of AHR signaling in hepatocytes or endothelial cells in mediating prototypical endpoints of adaptive, toxic, or developmental signaling. Using this model, we provide evidence that AHR signaling in endothelial/hematopoietic cells is necessary for developmental closure of the ductus venosus, whereas AHR signaling in hepatocytes is necessary to generate adaptive and toxic responses of the liver in response to dioxin exposure. Taken together, these data illustrate the importance of cell-specific receptor signaling for the generation of distinct AHR-dependent physiological outcomes.
Identifying the conserved network of cis-regulatory sites of a eukaryotic genome
Authors: Wang Ting , Stormo Gary D.
Published: November 2005

A major focus of genome research has been to decipher the cis-regulatory code that governs complex transcriptional regulation. We report a computational approach for identifying conserved regulatory motifs of an organism directly from whole genome sequences of several related species without reliance on additional information. We first construct phylogenetic profiles for each promoter, then use a
blast
-like algorithm to efficiently search through the entire profile space of all of the promoters in the genome to identify conserved motifs and the promoters that contain them. Statistical significance is estimated by modified Karlin–Altschul statistics. We applied this approach to the analysis of 3,524
Saccharomyces cerevisiae
promoters and identified a highly organized regulatory network involving 3,315 promoters and 296 motifs. This network includes nearly all of the currently known motifs and covers >90% of known transcription factor binding sites. Most of the predicted coregulated gene clusters in the network have additional supporting evidence. Theoretical analysis suggests that our algorithm should be applicable to much larger genomes, such as the human genome, without reaching its statistical limitation.
Activation-coupled inactivation in the bacterial potassium channel KcsA
Authors: Gao Lizhi , Mi Xianqiang , Paajanen Vesa , Wang Kun , Fan Zheng
Published: November 2005

X-ray structures of the bacterial K
+
channel KcsA have led to unparalleled progress in our understanding of ion channel structures. The KcsA channel has therefore been a prototypic model used to study the structural basis of ion channel function, including the gating mechanism. This channel was previously found to close at near-neutral intracellular pH (pH
i
) and to open at acidic pH
i
. Here, we report the presence of a previously unknown channel inactivation process that occurs after the KcsA channel is activated. In our experiments, mammalian cells transfected with a codon-optimized synthetic gene encoding the KcsA protein expressed K
+
-selective channels that activated in response to a decrease in pH
i
. Using patch-clamp and rapid solution exchange techniques, we observed that the KcsA channels inactivated within hundreds of milliseconds after channel activation. At all tested pHs, inactivation always accompanied activation, and it was profoundly accelerated in the same pH range at which activation increased steeply. Recovery from inactivation was observed, and its extent depended on the pH
i
and the amount of time that the channel was inactive. KcsA channel inactivation can be described by a kinetic model in which pH
i
controls inactivation through pH-dependent activation. This heretofore-undocumented inactivation process increases the complexity of KcsA channel function, but it also offers a potential model for studying the structural correspondence of ion channel inactivation.
Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity
Authors: Li Xiao-Dong , Sun Lijun , Seth Rashu B. , Pineda Gabriel , Chen Zhijian J.
Published: November 2005

Hepatitis C virus (HCV) is a global epidemic manifested mainly by chronic infection. One strategy that HCV employs to establish chronic infection is to use the viral Ser protease NS3/4A to cleave some unknown cellular targets involved in innate immunity. Here we show that the target of NS3/4A is the mitochondrial antiviral signaling protein, MAVS, that activates NF-κB and IFN regulatory factor 3 to induce type-I interferons. NS3/4A cleaves MAVS at Cys-508, resulting in the dislocation of the N-terminal fragment of MAVS from the mitochondria. Remarkably, a point mutation of MAVS at Cys-508 renders MAVS resistant to cleavage by NS3/4A, thus maintaining the ability of MAVS to induce interferons in HCV replicon cells. NS3/4A binds to and colocalizes with MAVS in the mitochondrial membrane, and it can cleave MAVS directly
in vitro
. These results provide an example of host–pathogen interaction in which the virus evades innate immunity by dislodging a pivotal antiviral protein from the mitochondria and suggest that blocking the cleavage of MAVS by NS3/4A may be applied to the prevention and treatment of HCV.
Light-switching excimer probes for rapid protein monitoring in complex biological fluids
Authors: Yang Chaoyong James , Jockusch Steffen , Vicens Marie , Turro Nicholas J. , Tan Weihong
Published: November 2005

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from ≈400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (≈40 ns) than that of the background (≈5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.
The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8
Authors: Hou Haitong , Subramanian Kanagaraj , LaGrassa Tracy J. , Markgraf Daniel , Dietrich Lars E. P. , Urban Jörg , Decker Nadine , Ungermann Christian
Published: November 2005

Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3/Ynl326c is required for efficient localization of Vac8 to vacuoles
in vivo
, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuole-associated Vac8 appears to be palmitoylated in a
pfa3
mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate/palmitate (as in Vac8) but also one that is targeted by a myristate/basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole.
Crosstalk in G protein-coupled receptors: Changes at the transmembrane homodimer interface determine activation
Authors: Guo Wen , Shi Lei , Filizola Marta , Weinstein Harel , Javitch Jonathan A.
Published: November 2005

Functional crosstalk between G protein-coupled receptors in a homo- or heterodimeric assembly likely involves conformational changes at the dimer interface, but the nature of this interface is not yet established, and the dynamic changes have not yet been identified. We have mapped the homodimer interface in the dopamine D2 receptor over the entire length of the fourth transmembrane segment (TM4) by crosslinking of substituted cysteines. Their susceptibilities to crosslinking are differentially altered by the presence of agonists and inverse agonists. The TM4 dimer interface in the inverse agonist-bound conformation is consistent with the dimer of the inactive form of rhodopsin modeled with constraints from atomic force microscopy. Crosslinking of a different set of cysteines in TM4 was slowed by inverse agonists and accelerated in the presence of agonists; crosslinking of the latter set locks the receptor in an active state. Thus, a conformational change at the TM4 dimer interface is part of the receptor activation mechanism.
Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease
Authors: Dodge James C. , Clarke Jennifer , Song Antonius , Bu Jie , Yang Wendy , Taksir Tatyana V. , Griffiths Denise , Zhao Michael A. , Schuchman Edward H. , Cheng Seng H. , O'Riordan Catherine R. , Shihabuddin Lamya S. , Passini Marco A. , Stewart Gregory R.
Published: November 2005

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.
Outpatient treatment with corticosteroids and antibiotics for acalculous cholecystitis in chronic granulomatous disease
Authors: ROJO PABLO , RUIZ‐CONTRERAS JESUS , GONZALEZ‐TOME MARIBEL , SERRANO CARMELO , MARIN MIGUEL A.
Published: November 2005

We report the outpatient management of acalculous colecistitis in an 18‐y‐old male with X‐linked chronic granulomatous disease. The patient complained of abdominal pain and the initial ultrasound showed a gallbladder with a thickened wall.

Conclusion: In chronic granulomatous disease, pain from a thickened gallbladder disappears after oral treatment with glucocorticoids and antibiotics.
Iron supplements reduce erythrocyte copper‐zinc superoxide dismutase activity in term, breastfed infants
Authors: DOMELLÖF MAGNUS , DEWEY KATHRYN G. , COHEN ROBERTA J. , LÖNNERDAL BO , HERNELL OLLE
Published: November 2005

Aim: To investigate whether iron supplements compromise copper status in infants.

Methods: 214 healthy, term, breastfed Swedish and Honduran infants were randomized to (1) iron supplements (1 mg/kg/d) from 4–9 mo of age, (2) iron supplements from 6–9 mo, or (3) placebo. Blood samples were obtained at 4, 6, and 9 mo and analyzed for plasma copper (p‐Cu) and, at 9 mo, for copper/zinc‐dependent superoxide dismutase (CuZn‐SOD) activity.

Results: P‐Cu increased with infant age. At 9 mo, Honduran infants had significantly higher p‐Cu (1.40 ± 0.29 vs 1.09 ± 0.22 mg/1, p < 0.001) and CuZn‐SOD activity (1.09 ± 0.29 vs 0.93 ± 0.21 U/mg Hb, p < 0.001) than Swedish infants. Infants receiving iron supplements from 4–9 mo had significantly lower CuZn‐SOD at 9 mo of age (0.95 ± 0.27 vs 1.08 ± 0.24 U/mg Hb,p = 0.023) than those receiving placebo.

Conclusion: There is a physiologic increase in p‐Cu during the first 9 mo of life. Differences in copper status between Swedish and Honduran infants may be due to genetic or nutritional differences. Iron supplementation decreases CuZn‐SOD activity, probably due to a negative effect on copper status. Possible clinical implications remain to be elucidated.
The global obesity epidemic: Snacking and obesity may start with free meals during infant feeding
Authors: ERLANSON‐ALBERTSSON CHARLOTTE , ZETTERSTRÖM ROLF
Published: November 2005

Feeding is vital for survival. The brain has strong hunger and reward mechanisms that ensure optimal food intake for adequate nutrition. The drive for feeding is particularly strong in humans whose large brains require large energy support. This starts immediately after birth; the newborn child being able to taste sucrose and suck the sweet and fat from its mother's milk. At present, mothers are generally advised to breastfeed children as often as they like, which may be up to 15 times a day. At the same time, childhood obesity is rapidly developing. One reason for the rapidly increasing prevalence of childhood obesity may be overfeeding with snack food.

Conclusion: We hypothesize that non‐rule breastfeeding favours the development of snacking throughout the day during childhood, a habit which in turn favours the development of obesity.
When newborn infants are bound to die
Authors: SAUGSTAD OLA DIDRIK
Published: November 2005

Withholding and withdrawing of intensive care of severely ill newborns may occasionally be needed when there is severe injury and extremely poor prognosis. In such cases, strict guidelines and procedures should be followed. In this issue of Acta Paediatrica, Arlettaz et al. describe such guidelines and report practice from the University Hospital in Zürich. To ensure an unbiased discussion, it is important that those who make decisions regularly receive input from external sources regarding indications of viability. This may be the case if, for instance, a unit has decided that the limit of viability is < 26 wk, and many children with good life expectancy would in such units be denied intensive care. Does a liberal view on withholding or withdrawing treatment lead to poorer overall outcome results even for babies above the threshold of viability specified by the institution?

Conclusion: Strict procedures should be followed in the case of withholding or withdrawing intensive care of severely ill newborn infants. In every institution, the indications for ending life should be discussed on a regular basis, preferably with external input. However, euthanasia of newborn infants is not compatible with Western cultural traditions and should be abandoned.
LC‐PUFA content in human milk: Is it always optimal?
Authors: AGOSTONI CARLO
Published: November 2005

The content of long‐chain polyunsaturated fatty acids (LC‐PUFAs) in human milk has been connected with infant growth and developmental indices. The LC‐PUFA content of human milk usually reflects the dietary habits of mothers, so questions have been raised regarding the possibility of enriching maternal diet with LC‐PUFAs during lactation (or even before) in order to improve infant outcome. Nevertheless, environmental and genetic factors have independent roles in affecting both maternal milk composition and infant development.

Conclusion: Diet‐related differences in the LC‐PUFA composition of human milk are under active investigation for their possible contribution to infant development, but environment‐ and gene‐related differences in both human milk composition and maternal diet should be considered in evaluating the adaptive mechanisms of infants and the effects of specific LC‐PUFA dietary supplementations.
New insights into juvenile parotitis
Authors: KOLHO KAIJA‐LEENA , SAARINEN RIITTA , PAJU ANNUKKA , STENMAN JACOB , STENMAN ULF‐HÅKAN , PITKÄRANTA ANNE
Published: November 2005

Aim: We enquired about the possibility of a familial trend in juvenile parotitis and evaluated the role of SPINK1 mutations in juvenile parotitis.

Methods: The clinical records of all children admitted to the Helsinki University Hospital during 1995 to May 2003 because of swelling in the parotid gland were reviewed. A questionnaire on possible recurrences and on familial cases was mailed. As disturbances in trypsin inhibition might be involved in the pathogenesis, we assessed the SPINK1 gene encoding for Kazal‐type trypsin inhibitor in voluntary patients. The study group comprised 133 children (boys 82 girls 51) with juvenile parotitis. The median age at presentation of first symptoms was 6.0 y (range 1–19 y).

Results: Recurrent symptoms in the parotid gland were common (57%), and 29% of the children (38/133) had suffered from four or more episodes. A young age at the first episode of symptoms increased the likelihood of recurrences (p < 0.0001). Familial cases of parotid swelling were common (22%; response rate 67%). A total of 47 patients (35%) agreed to testing for SPINK1 status. Four children had a major mutation (N34S or P55S), corresponding to an 8.5% (4/47) prevalence, but this was not different from the controls (5%).

Conclusion: It is likely that inherited factors are involved in the manifestation of juvenile parotitis in a subset of patients. It is tempting to speculate that disturbed proteolytic balance may play a role in the development of symptoms.
Clinical features of nosocomial infections by extended‐spectrum β‐lactamase‐producing Enterobacteriaceae in neonatal intensive care units
Authors: CHIU SHUN , HUANG YHU‐CHERLNG , LIEN REY‐IN , CHOU YI‐HONG , LIN TZOU‐YIEN
Published: November 2005

Aim: To determine the risk factors for the acquisition of nosocomial extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae infection in infants hospitalized in neonatal intensive care units (NICUs) and to evaluate the therapeutic outcome of these infants.

Methods: We retrospectively reviewed the medical records of infants with nosocomial ESBL‐producing Enterobacteriaceae infection hospitalized in NICUs at Chang Gung Children's Hospital in 2001. The clinical features of these infants were compared with a cohort of non‐ESBL‐producing Enterobacteriaceae‐infected infants during the same period. The therapeutic outcome of the infants in the ESBL group was analysed.

Results: Seventy infants were included in this study. Thirty‐one infants with 34 isolates were identified in the ESBL group and 39 infants with 42 isolates in the non‐ESBL group. Of the parameters analysed, including gestational age, birthweight, length of hospital stay before onset, the number of antibiotics used, the duration of third‐generation cephalosporin usage and the number of patients receiving a third‐generation cephalosporin prior to the onset of infection, no significant difference was found between the two groups. The infection‐contributed case fatality rate was 3.0% (1 of 33) in the ESBL group, not significantly different from that in the non‐ESBL group (1 of 41, 2.4%). Of the 31 patients in the ESBL group, 18 were treated with a carbapenem as definitive therapy while 13 were treated with a non‐carbapenem antibiotic regimen. No significant difference was noted in terms of mortality rate between the two subgroups.

Conclusion: The outcome of the infants hospitalized in the NICU with ESBL‐producing enterobacterial infections was not indispensably grave, even when treated with a non‐carbapenem antibiotic regimen. The risk factors for the acquisition of ESBL‐producing enterobacterial infections in these infants were not identified in this series.
Epidermal growth factor receptor and proliferating cell nuclear antigen expression in urine ThinPrep specimens
Authors: Politi E. N. , Lazaris A. C. , Lambropoulou S. , Alexopoulou D. , Kyriakidou V. , Koutselini H.
Published: November 2005

Objective:  To evaluate proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR) expression in urine ThinPrep (TP) specimens, to compare these findings with clinical and histological features and to determine whether these immunomarkers are predictive of clinical stage.

Patients and Methods:  The TP processed urine samples and the corresponding tissue sections from 42 patients with newly diagnosed bladder cancer (18 non‐muscle invasive and 24 muscle invasive) were included in our study. Urine was collected for cytological evaluation before transurethral resection. Tumour grade and clinical stage were assessed from the transurethral resection specimens. The EGFR and PCNA expression was obtained by an automated immunostainer.

Results:  There was a remarkable concordance in the expression of both antibodies in TP smears and tissue sections. No significant association was detected for any of the immunomarkers examined with regard to tumour grade. The EGFR expression as well as grade of malignancy were significantly associated with stage of disease (P = 0.0001). PCNA was not found to be a significant predictor of stage (P = 0.210).

Conclusion:  Our data suggest that the evaluation of grade of malignancy and EGFR immunopositivity can be considered as reliable predictors of disease stage in urine TP specimens.
Reconstitution of γ‐secretase by truncated presenilin (PS) fragments revealed that PS C‐terminal transmembrane domain is critical for formation of γ‐secretase complex
Authors: Shiraishi Hirohisa , Marutani Toshihiro , Wang Hua‐Qin , Maeda Yasuhiro , Kurono Yukihisa , Takashima Akihiko , Araki Wataru , Nishimura Masaki , Yanagisawa Katsuhiko , Komano Hiroto
Published: November 2005

The presenilin (PS) complex, including PS, nicastrin (NCT), APH‐1 and PEN‐2, is essential for γ‐secretase activity. Previously, the PS C‐terminal tail was shown to be essential for γ‐secretase activity. Here, to further understand the precise mechanism underlying the activation of γ‐secretase regulated by PS cofactors, we focused on the role of the PS1 C‐terminal region including transmembrane domain (TM) 8 in γ‐secretase activity. For this purpose, we co‐expressed C‐terminally truncated PS1 (PS1ΔC) completely lacking γ‐secretase activity and the PS1 C‐terminal short fragment in PS‐null cells, because the successful reconstitution of γ‐secretase activity in PS‐null cells by the co‐expression of PS1ΔC and the PS1 C‐terminal short fragment would allow us to investigate the role of the PS1 C‐terminal region in γ‐secretase activity. We found that the exogenous expression of the PS1 C‐terminal short fragment with NCT and APH‐1 completely rescued a defect of the γ‐secretase activity of PS1ΔC in PS‐null cells. With this reconstitution system, we demonstrate that both TM8 and the PS1 C‐terminal seven‐amino‐acid‐residue tail are involved in the formation of the active γ‐secretase complex via the assembly of PS1 with NCT and APH‐1.
Direct binding of TReP‐132 with TdT results in reduction of TdT activity
Authors: Fujisaki Seiichiro , Sato Asami , Toyomoto Tsuguri , Hayano Takahide , Sugai Maki , Kubota Takashi , Koiwai Osamu
Published: November 2005

N regions at the junction of V, D and J DNA segments are synthesized with large protein complexes including terminal deoxynucleotidyltransferase (TdT) during V(D)J recombination in B‐ or T‐cells. TdT directly binds to TdIF1, TdIF2, PCNA and the Ku70/86 heterodimer. Using a yeast two‐hybrid system, we isolated a cDNA clone encoding the gene for TReP‐132, which is involved in P450scc gene expression in steroid‐hormone‐producing cells or lymphoid cells. Interaction between TReP‐132 and TdIF1 was confirmed by pull‐down assay and immunoprecipitation assay using specific antibodies against TReP‐132 both in vitro and in vivo. TdT also directly bound to TReP‐132 through its confined N‐terminal region. Furthermore, the co‐expression of TdIF1 and TReP‐132 or TdT and TReP‐132 in COS7 cells showed that these proteins are co‐localized within the nucleus. TReP‐132 reduces TdT activity to 2.5% of its maximum value in the in vitro assay system using double‐stranded DNA with a 3′ protrusion as a primer. These findings suggest that TdT synthesizes N region under a negative control of TReP‐132 during V(D)J recombination.
Visualization of the interaction between archaeal DNA polymerase and uracil‐containing DNA by atomic force microscopy
Authors: Asami Yasuo , Murakami Masahiro , Shimizu Masatomi , Pisani Francesca M. , Hayata Isamu , Nohmi Takehiko
Published: November 2005

Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B‐family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil‐containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil‐containing single‐stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double‐stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil‐containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis.
Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells
Authors: Huang F.‐M. , Chen Y.‐J. , Chou M.‐Y. , Chang Y.‐C.
Published: November 2005

Aim  To investigate the tissue type plasminogen activator (t‐PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3‐kinase (PI3K) inhibitor LY294002.

Methodology  The supernatants of P. endodontalis were used to evaluate t‐PA activity in human pulp cells using casein zymography and enzyme‐linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t‐PA activity.

Results  The main casein secreted by human pulp cells migrated at 70 kDa and represented t‐PA. Secretion of t‐PA was found to be stimulated with P. endodontalis during 2‐day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t‐PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t‐PA production (P > 0.05).

Conclusions  Porphyromonas endodontalis enhances t‐PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t‐PA.
Calcium hydroxide dressings using different preparation and application modes: density and dissolution by simulated tissue pressure
Authors: Peters C. I. , Koka R. S. , Highsmith S. , Peters O. A.
Published: November 2005

Aim  To study the effect of different apical shapes in prepared simulated root canals on the application of a commercially prepared calcium hydroxide paste by a syringe or lentulo spiral.

Methodology  Three different types of root canal preparation were performed in 90 simulated canals: group A to an apical size 20 and a 0.10 taper using hand and rotary instruments, group B to an apical size 30 and a 0.08 taper using GT rotary instruments and group C to an apical size 40 and a 0.04 taper using ProFile 0.04 instruments. The insertion of calcium hydroxide [Ca(OH)2] paste was accomplished using either a lentulo spiral or a syringe. After 1 week of simulated fluid pressure applied to the apical end of the canal using physiological saline solution, the solution was evaluated for released Ca(OH)2. The specimens were weighed initially, after preparation, after insertion of Ca(OH)2 paste, after temporization with Cavit and after 1 week of simulated fluid pressure. Digital radiographs of the filled canals were taken and canal areas in mm2, gray values of the Ca(OH)2 dressings, total area of voids in mm2, as well as location of voids in the apical, middle or coronal thirds of the root canals were measured. Analyses of variance, with Scheffe's post‐hoc tests, as well as chi‐square tests were performed.

Results  Canals in group C had significantly fewer (P < 0.01) radiographic voids than canals in groups A and B. Using a lentulo spiral resulted in significantly (P < 0.05) fewer voids compared with the injection technique. More voids were detected coronally compared with middle and apical root canal thirds (P < 0.05).

Conclusions  Canal shape and method of application had an impact on the amount and radiodensity of calcium hydroxide dressings in simulated root canals. Canals prepared to an apical size 40 and a taper of 0.04 had the least number of voids; Ca(OH)2 was placed with significantly fewer voids using a lentulo spiral compared with the injection technique.
Pleistophora infestation in fathead minnows, Pimephales promelas (Rafinesque)
Authors: Ruehl‐Fehlert C , Bomke C , Dorgerloh M , Palazzi X , Rosenbruch M
Published: November 2005

Pleistophora infestation was observed in adult fathead minnows, Pimephales promelas, held under laboratory conditions. Fish were clinically healthy, and presented no gross findings at necropsy. Histopathology revealed parasitic stages only in the ovaries. Spores within sporophorous vesicles were mainly encountered in late vitellogenic oocytes and were ultrastructurally identified as a microsporidian parasite. Heavily parasitized oocytes underwent degeneration followed by the release of spores into the ovarian interstitium. Degenerating oocytes and interstitial spores caused ovarian inflammation. Male fish showed no parasites in the testes. Parasitic infestation was compared with body length, body weight, gonadal weight, gonadosomatic index and plasma vitellogenin levels, and revealed no statistically significant differences between non‐parasitized and parasitized females. The isolated holding conditions of the fish and the presence of parasitic stages in the ovaries suggested that an infestation with Pleistophora ovariaeSummerfelt, 1964 was more probable than that with Pleistophora mirandellae (Vaney & Conte, 1901).
Mortality patterns in infectious salmon anaemia virus outbreaks in New Brunswick, Canada
Authors: Hammell K L , Dohoo I R
Published: November 2005

Mortality levels attributed to infectious salmon anaemia viral (ISAV) infections were examined at the net pen and site level in the 1996 smolt year class in three areas of New Brunswick, Canada. The year class in this region was the first known to have potential exposure to ISAV beginning at the time of seawater transfer. There was considerable variability in mortality patterns among net pen groups of fish. Net pen outbreak definitions were based on at least seven high mortality days in which there were at least 100 per 100 000 fish per day or >5% cumulative mortality for the study period. There were 106 net pen outbreaks in a study population consisting of 218 net pens. Although the number of new cases decreased as water temperature decreased, overall mortality levels at the study sites did not decrease noticeably. The median peak daily mortality rate during outbreaks was 492 per 100 000 fish per day, with 10% of cases experiencing >5200 mortalities per 100 000 fish per day. The median duration of outbreaks in net pens for which the fish were not slaughtered during the outbreak was 33 days and the median total loss in those outbreaks was 6600 per 100 000 fish.
Risk factors associated with mortalities attributed to infectious salmon anaemia virus in New Brunswick, Canada
Authors: Hammell K L , Dohoo I R
Published: November 2005

Outbreaks of unexplained mortalities attributed to infectious salmon anaemia (ISA) were examined in the 1996 year class of Atlantic salmon in three regions of New Brunswick, Canada. A total of 218 net pens at 14 sites deemed to have been exposed to ISA virus (ISAV) were surveyed for mortality records and management, environmental and host characteristics. Based on definitions of mortality patterns, clinical ISA disease outbreaks occurred in 106 net pens. There were eight sites in which >50% of net pens experienced ISA outbreaks during the study period. Factors related to their potential role in transmission of virus to new sites or new net pens at the same site were identified as sea lice vectors, divers visiting multiple sites, sites belonging to companies with more than one site, exposure to other year classes at the site, and proximity to other infected net pens. Host resistance factors associated with greater risk of outbreaks were identified as larger groupings, general health following smolt transfer, stressful husbandry procedures during growout, and health or productivity during colder water periods. Despite very close proximity between sites, modification of these management factors would probably influence the severity of mortalities caused by ISAV.
Comparative phylogeographic summary statistics for testing simultaneous vicariance
Authors: HICKERSON M. J. , DOLMAN G. , MORITZ C.
Published: November 2005

Testing for simultaneous vicariance across comparative phylogeographic data sets is a notoriously difficult problem hindered by mutational variance, the coalescent variance, and variability across pairs of sister taxa in parameters that affect genetic divergence. We simulate vicariance to characterize the behaviour of several commonly used summary statistics across a range of divergence times, and to characterize this behaviour in comparative phylogeographic datasets having multiple taxon‐pairs. We found Tajima'sDto be relatively uncorrelated with other summary statistics across divergence times, and using simple hypothesis testing of simultaneous vicariance given variable population sizes, we counter‐intuitively found that the variance across taxon pairs in Nei and Li's net nucleotide divergence (πnet), a common measure of population divergence, is often inferior to using the variance in Tajima'sDacross taxon pairs as a test statistic to distinguish ancient simultaneous vicariance from variable vicariance histories. The opposite and more intuitive pattern is found for testing more recent simultaneous vicariance, and overall we found that depending on the timing of vicariance, one of these two test statistics can achieve high statistical power for rejecting simultaneous vicariance, given a reasonable number of intron loci (> 5 loci, 400 bp) and a range of conditions. These results suggest that components of these two composite summary statistics should be used in future simulation‐based methods which can simultaneously use a pool of summary statistics to test comparative the phylogeographic hypotheses we consider here.
Morphological and Immunohistochemical Study of the Zebrafish (Danio rerio) Oral Cavity
Authors: Abbate F. , Germanà G. P. , Montalbano G. , Germanà A. , Ciriaco E.
Published: November 2005

The oral cavity in vertebrates plays a very important role in food intake mechanics and also contains chemical sensors for food and environmental water. Comparative studies revealed morphological and functional inter‐specific differences that can be correlated to evolutionary events and might represent the adaptation to environmental conditions present in different habitats (Iwasaki, 2002). Particularly these events are strictly dependent on the food intake mechanisms determining the success of the adaptation of vertebrates to their environment and therefore their survival (Roth and Wake, 1989). Since data about the morphology of the zebrafish oral cavity are scarce the aim of the present research was to study it by light, scanning and transmission electron microscopy and immunohistochemistry for some growth factor receptors, i.e. trkA and trkB. The zebrafish was chosen because as a vertebrate it has valuable features as a model for study of vertebrate development (Withfield, 2002). Adult zebrafishes were anesthesized with MS222 and the samples were taken and routinely processed for light and electron microscopy and for indirect peroxidase immunohistochemistry. The topographical and ultrastructural study demonstrated the presence of three different zones. The outer zone is characterized orally by the presence of a papilla and by an epithelium with numerous taste buds that are chemosensory endorgans consisting of modified epithelial cells. Their function is to sample potential food and selecting it according to its edibility (Hansen et al., 2002). Both in upper and lower intermediate zones, the presence of internal valves with a semi‐lunar shape was evident, probably able to regulate the mouth closure. The most internal lower zone corresponds to the tongue that in zebrafish is only a thickening of the pavement mucosa with a stratified epithelium. Immunohistochemistry demonstrated both in upper and lower zones the presence of numerous taste buds that were immunoreactive to TrkA and TrkB‐like neurotrophin receptors, confirming the role of these receptors in taste buds sensory cells (Germanà et al., 2004). The present results give a contribution to the morphological knowledge of the zebrafish oral cavity, an animal model commonly used for developmental studies of vertebrates.
Perineuronal Nets in the Central Nervous System
Authors: Sayed R. , Mubarak W. , Ohtsuka A. , Atoji Y. , Murakami T.
Published: November 2005

One hundred and twenty years ago, Camillo Golgi described reticular or fenestrated sheaths of extracellular matrix (ECM), enwrapping the cell bodies, axon initial segments and proximal dendrites of certain pyramidal and non‐pyramidal neurons in the adult mammalian central nervous system. Such structures, currently termed perineuronal nets (PNs), are preferentially associated with GABAergic, parvalbumin‐containing fast‐spiking inhibitory types of cells, and mostly expressing the Kv3.1 subunit of voltage‐gated potassium channels. Although some neuroanatomists validated the existence of the PNs on selected neurons in the adult brain, there was a lack of agreement concerning their origin, composition and function. Recent data suggest that PNs result from the visualization of ECM molecules that are confined to the space interposed between glial processes and the nerve cells that they outline. The substance confined to these spaces could be visualized selectively by antibodies directed to glycoproteins, proteoglycans, markers for hyaluronan, some plant lectins recognizing N‐acetylgalactosamine or by monoclonal antibodies directed to epitopes on unknown molecules. The PNs appear only after birth, which they could serve as recognition molecules between certain neurons and their surrounding cells. Other putative roles include stabilization of synapses, maintenance of cellular relationships in the adult brain, concentration of growth factors around certain neurons, generation of a polyanionic ion‐buffering microenvironment, as well as prevention of extracellular‐space occlusion and the formation of certain link with the intracellular cytoskeleton.
Surface Ultrastructure of Ultimobranchial Epithelium in the Thyroid Gland of Donkey (Equus asinus)
Authors: Sayed R. , Mubarak W. , Saleh A. M.
Published: November 2005

Surface ultrastructural observation of the thyroid gland in donkey revealed the coexistence of certain unusual follicles or cysts, the ultimobranchial (UB) follicles, which exhibited large diameters and less regular outlines. In addition, several small follicles, cell nests or solid cellular masses were also demonstrated in association with these structures. Surface of most UB follicular cells was almost hexagonal in shape, studded with sparse or dense pleomorphic microvilli, which were much denser at the cellular borders. Each follicular cell bore single cilium that projecting over the cell surface. Interestingly, bleb‐like apocrine protrusion of the apical cytoplasm of some follicular cells was frequently demonstrated. These apocrine blebs (or aposomes) were varied in shape, size and showed smooth or irregular surface. In addition to such aposomes, deep to shallow circumscribed or eroded areas of the apical cell surface was frequently demonstrated. These findings indicate that the UB follicular cells in donkeys, similar to those of camels, exhibit an apocrine activity alongside with the usual merocrine mode of secretion.