bluuebunny/crossref_metadata_2025 huggingface dataset:
To best measure cell proliferation in samples from the intestine
Authors: Alferez D. , Goodlad R. A.
Published: March 2007

Abstract.  Arrangement of the intestinal cell lining, as it is, into distinct anatomically defined zones where proliferation is confined to the crypts, makes it an ideal tissue to study growth control mechanisms. While many methods have been used to quantify cell proliferation in the gut, several of them have severe limitations and others (although potentially better) have been misused and misinterpreted. Here, correct use and interpretation of labelling studies will be described as will a well established alternative method that provides equivalent results for one‐sixth of the effort.
Immortalization of human urothelial cells by human papillomavirus type 16 E6 and E7 genes in a defined serum‐free system
Authors: Carmean N. , Kosman J. W. , Leaf E. M. , Hudson A. E. , Opheim K. E. , Bassuk J. A.
Published: March 2007

Abstract.  Normal human epithelial cell cultures exhibit a limited (although different between tissues) lifespan in vitro. In previous studies, urothelial cell cultures were immortalized using retroviral transformation with human papillomavirus type 16 E6 and E7 genes, in undefined culture systems containing serum or bovine pituitary extract. Objective: Due to the variability of results in such systems, we instead developed a procedure for the immortalization of urothelial cells using a defined, serum‐free culture system. Method and results: Immortalization through retroviral transformation with human papillomavirus type 16 E6 and E7 was successful, and transformation of urothelial cells conferred an extended over normal lifespan and restored telomerase activity. Transformed cells retained typical morphology and exhibited a similar growth rate, cytokeratin immunoreactivity pattern, and response to growth factors as observed in untransformed cells. Karyotype analysis revealed a gradual accumulation of genetic mutations that are consistent with previously reported mutations in epithelial cells transformed with human papillomavirus type 16 E6 and E7. Conclusion: The ability to extend the in vitro lifespan of cells holds the potential to reduce the continuous need for tissue samples and to enable complete investigations with one cell line.
Extracts from plastrum testudinis promote proliferation of rat bone‐marrow‐derived mesenchymal stem cells
Authors: Chen D. F. , Zeng H. P. , Du S. H. , Li H. , Zhou J. H. , Li Y. W. , Wang T. T. , Hua Z. C.
Published: March 2007

Abstract.  Objectives: The purpose of this study is to identify active components of PT involved in promoting proliferation of MSCs and to investigate its mechanism. PT was extracted with petroleum ether, ethyl acetate, ethanol and water respectively. Materials and methods: Evidence provided by MTT, HE stain, BrdUrd, PCNA immunoreactivity and cell cycle indicated that Plastrum Testudinis Extracted with ethyl acetate (PTE) is the only active components responsible for increasing MSCs proliferation. Results: This finding leads us to identify the chemical component of PTE. Steroid, fatty acids and their esters components in PTE were determined by GC‐MS and HPLC. The mechanism of PTE action may be associated with the up‐regulation of BMP4. Conclusions: Our findings give novel insights into the promoting effects of Plastrum Testudinis on proliferation of MSCs and help to identify the chemical component and to clarify the mechanism of its pharmacological activities.
Pollen‐induced airway inflammation, hyper‐responsiveness and apoptosis in a murine model of allergy
Authors: Conejero L. , Higaki Y. , Baeza M. L. , Fernández M. , Varela‐Nieto I. , Zubeldia J. M.
Published: March 2007

Background
Previous studies indicate that murine models are useful tools for studying the allergic diseases, including certain aspects of bronchial asthma such as cellular tissue inflammation and pulmonary function.

Objective
To develop an experimental model of allergic lung inflammation based on a relevant human allergen, olive pollen, and to establish the immunological, cellular and functional airway features of the allergic response in this model.

Methods
Induction of systemic allergic response was achieved by the subcutaneous administration of Olea europaea extract in BALB/c mice. Olea‐specific Igs (IgG1, IgG2a and IgE) and cytokines from splenocyte cultures IL‐4, IL‐5 IL‐10, IL‐13 and IFN‐γ were measured. Allergic airway response was generated by transnasal instillation of the allergens. Airway responsiveness was monitored by non‐invasive methacholine inhalation challenge. Lungs were paraffin embedded and histologically analysed. Apoptosis was studied by the TUNEL technique in the lung tissue and through cell cycle analysis by flow cytometry in splenocytes.

Results
Our results demonstrate that Olea‐sensitized mice develop a specific allergic antibody (IgG1 and IgE) and cytokine (IL‐4, IL‐5, IL‐10 and IL‐13) response. After transnasal Olea instillation, they show inflammatory infiltration of lung tissue, mucus secretion and non‐specific hyper‐responsiveness in the airway. Concomitantly, differences in the rate of apoptosis are observed in the lung cells as well as a significant reduction of spontaneous apoptosis in the splenocytes of allergic mice.

Conclusion
We present a novel animal model of olive pollen‐allergic disease. This model presents traits associated with human allergic asthma and could be an interesting tool in the study of underlying molecular mechanisms and in exploring the therapeutic approaches to this disease.
Exhaled air temperature in asthma: methods and relationship with markers of disease
Authors: Piacentini G. L. , Peroni D. , Crestani E. , Zardini F. , Bodini A. , Costella S. , Boner A. L.
Published: March 2007

Background
Exhaled breath temperature has been proposed as a surrogate marker for the evaluation of airway inflammation in asthmatic patients.

Objective
The aim of the present study was to extend the investigation of exhaled air temperature as a means for the evaluation of airway inflammation using a professionally developed instrument.

Methods
Fifty‐seven children, 41 allergic mild asthmatics and 16 healthy controls have been evaluated. They underwent exhaled air temperature and lung function measurement. The asthmatic children also underwent exhaled nitric oxide measurement, and hypertonic saline sputum induction for the evaluation of eosinophil (EOS) percentage.

Results
The level of exhaled temperature was significantly higher in asthmatics than in controls, being 30.18±0.14°C vs. 27.47±0.24°C (P<0.001). In asthmatic children, a positive relationship was observed between exhaled air temperature and both exhaled nitric oxide (r=0.39; P=0.01) and EOS percentage in samples from induced sputum (ρ=0.53; P=0.04).

Conclusion
The data from the present study support the hypotheses that exhaled breath temperature is related to the degree of airway inflammation in asthma.
Perinatal maternal application of Lactobacillus rhamnosus GG suppresses allergic airway inflammation in mouse offspring
Authors: Blümer N. , Sel S. , Virna S. , Patrascan C. C. , Zimmermann S. , Herz U. , Renz H. , Garn H.
Published: March 2007

Background
Clinical studies indicate that maternal exposure to probiotic bacteria may protect from the development of allergic disease later in life.

Objective
The purpose of this study was to analyse the effects of a perinatal Lactobacillus rhamnosus GG (LGG) supplementation on the development of allergic disorders in offspring.

Methods
Female BALB/c mice received intragastric LGG every other day before conception, during pregnancy and lactation (perinatal supplementation group) or before conception and during pregnancy only (prenatal supplementation group). Cytokine expression of placental tissues was examined. Offspring of LGG‐supplemented and sham‐exposed mothers were sensitized to Ovalbumin (OVA), followed by aerosol allergen challenges. Development of experimental asthma was assessed by bronchoalveolar lavage analysis, lung histology and lung function measurement. Cytokine production of splenic mononuclear cells was analysed following in vitro stimulation.

Results
Intestinal colonization with LGG was observed in mother mice only, but not in the offspring. However, a reduced expression of TNF‐α, IFN‐γ, IL‐5 as well as IL‐10 was observed in mice derived from perinatally LGG‐supplemented mothers, whereas IL‐13 and IL‐4 expression remained unchanged. Moreover, in offspring of prenatally or perinatally LGG‐supplemented mothers allergic airway and peribronchial inflammation as well as goblet cell hyperplasia were significantly reduced as compared with mice derived from non‐supplemented mothers. In contrast, airway hyperresponsiveness to methacholine was not affected. Exposure to LGG during pregnancy only shifted the placental cytokine expression pattern with a markedly increased TNF‐α level.

Conclusion
Our data suggest that LGG may exert beneficial effects on the development of experimental allergic asthma, when applied in a very early phase of life. Immunological effects are, at least in parts, mediated via the placenta, probably by induction of pro‐inflammatory cell signals.
Increased expression of HLA‐DR and CD86 in nasal epithelial cells in allergic rhinitics: antigen presentation to T cells and up‐regulation by diesel exhaust particles
Authors: Takizawa R. , Pawankar R. , Yamagishi S. , Takenaka H. , Yagi T.
Published: March 2007

Background
A proportion of nasal epithelial cells (NEC) in patients with allergic rhinitis (AR) are known to express the major histocompatibility complex Class II molecule (HLA‐DR).

Objective
We hypothesized that NEC may play a role in antigen presentation to T cells. To elucidate the possible role of NEC in antigen presentation, we examined the expression of HLA‐DR, CD80 and CD86 in NEC, their regulation by cytokines and the capacity of NEC to induce antigen‐specific proliferation of T cells.

Methods
We examined the expression of HLA‐DR, CD80 and CD86 in nasal epithelial scrapings of patients with seasonal allergic rhinitis (SAR) to Japanese cedar pollen pre‐season and in‐season, by immunohistochemistry. Next, we examined the effect of IL‐1β, TNF‐α, (IFN‐γ), IL‐4 α, IL‐13 and diesel exhaust particles (DEP) on the HLA‐DR, CD80 and CD86 expression in cultured nasal epithelial cells (CNEC), by flow cytometry. Further, we analysed the capacity of mite antigen (Der f II)‐pulsed mitomycin‐C‐treated CNEC to induce proliferation of autologous T cells from patients with perennial allergic rhinitis.

Results
NEC constitutively expressed HLA‐DR and CD86, but not CD80. The expression of HLA‐DR and CD86 in NEC was significantly increased in‐season, in patients with SAR as compared with that of pre‐season. While IFN‐γ up‐regulated the expression of HLA‐DR, IL‐1β and TNF‐α up‐regulated the expression of CD86 in CNEC. Furthermore, in the presence of mite antigen, CNEC induced the proliferation of autologous peripheral blood T lymphocytes. Anti‐CD86 and anti‐HLA‐DR monoclonal antibody but not anti‐CD80 inhibited the epithelial cell‐induced T cell proliferation. Stimulation with a combination of DEP and mite antigen significantly up‐regulated HLA‐DR and CD86 expression in CNEC.

Conclusions
These studies suggest that NEC in patients with AR may play a role in antigen presentation through the enhanced expression of HLA‐DR and CD86. Furthermore, these results suggest the possibility that DEP may enhance the antigen‐presenting function of CNEC.
Eulittoral macroinvertebrate communities of lowland lakes: discrimination among trophic states
Authors: BRAUNS MARIO , GARCIA XAVIER‐FRANÇOIS , PUSCH MARTIN T. , WALZ NORBERT
Published: March 2007

1. Nutrient inputs from urban and agricultural land use often result in shifts in species composition of pelagic and profundal invertebrate communities. Here, we test if nutrient enrichment affects the composition of eulittoral macroinvertebrate communities, and, if so, if macroinvertebrate communities of five different habitat types reflect differences in trophic state.

2. Macroinvertebrate community composition of 36 lakes was significantly correlated with total phosphorus (TP) concentration, the proportion of coarse woody debris (CWD) and root habitats and the proportion of grassland.

3. However, macroinvertebrate communities of five major habitat types from eight lakes were more dissimilar among habitats than among trophic states. Community composition of reed and stone habitats was significantly correlated with wind exposure but not TP concentration, while macroinvertebrate composition of sand habitats was related to TP concentration and coarse sediments. In CWD and root habitats, both TP concentration and a predominance of invasive species covaried, which made it difficult to relate the observed compositional differences to either trophic state or to the effects of competition between native and invasive species.

4. Trophic state influenced the composition of eulittoral macroinvertebrate communities but to a lesser extent than has been previously reported for profundal habitats. Moreover, the effects of trophic state were nested within habitat type and were partially superseded by biotic interactions and small‐scaled habitat complexity. Although eulittoral macroinvertebrate communities were not strong indicators of the trophic state of lowland lakes, they may be used to assess other anthropogenic impacts on lakeshores.
The fission yeast DASH complex is essential for satisfying the spindle assembly checkpoint induced by defects in the inner‐kinetochore proteins
Authors: Kobayashi Yasuyo , Saitoh Shigeaki , Ogiyama Yuki , Soejima Saeko , Takahashi Kohta
Published: March 2007

Spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance system for chromosome missegregation. We isolated fission yeast Hos2, a component of the Dam1/DASH complex, as a multicopy suppressor of temperature‐sensitive (ts) growth of nnf1‐495 mutant that exhibits the minichromosome instability (mis) phenotype, producing lethal aneuploids without prominent mitotic delay. It remains elusive why SAC is satisfied in mis mutants despite the occurrence of missegregation. We found that Hos2 binds to the inner‐kinetochore regions in both prometaphase and metaphase. Hos2 is essential for kinetochore localization of Dis1, a microtubule (MT) associated Dis1/XMAP215/TOG family protein that is required for proper MT dynamics. Cells lacking DASH exhibit cold‐sensitive (cs) growth with the defective in sister‐chromatid disjoining (dis) phenotype, which is characterized by hyper‐condensed sister‐chromatid pairs and elongated spindle MTs. Although DASH‐deficient cells are viable at high temperatures, DASH‐deletion transforms all the inner‐kinetochore mis mutants so far tested into a constitutively active state of SAC, leading to the dis phenotype. We also discovered that Hos2 over‐expression commonly suppresses growth retardation in a variety of inner‐kinetochore mutants. These genetic interactions highlight the DASH‐action(s) in satisfying SAC when aneuploids are formed during mitosis in the inner‐kinetochore‐defective mis mutants.
A requirement of FancL and FancD2 monoubiquitination in DNA repair
Authors: Seki Sohsuke , Ohzeki Mioko , Uchida Akiko , Hirano Seiki , Matsushita Nobuko , Kitao Hiroyuki , Oda Tsukasa , Yamashita Takayuki , Kashihara Naoki , Tsubahara Akio , Takata Minoru , Ishiai Masamichi
Published: March 2007

The rare hereditary disorder Fanconi anemia (FA) can be caused by mutations in components of the FA core complex (FancA/B/C/E/F/G/L/M), a key regulator FancD2, the breast cancer susceptibility protein BRCA2/FancD1, or the newly identified FancJ/BRIP1 helicase. By performing yeast two‐hybrid (Y2H) screens using N‐terminal chicken (ch) FancD2 as a bait, we have identified chFancL, the likely ubiquitin E3 ligase subunit of the FA core complex. We also found that ectopically expressed FancD2 and FancL co‐immunoprecipitated in 293T cells, and this interaction was dependent on the PHD domain of FancL. FANCL‐disrupted chicken DT40 cells displayed defects in both FancD2 monoubiquitination and focus formation. Importantly, cell lines lacking the FANCL or FANCD2 genes, or carrying a “knock‐in” mutation of the FancD2 monoubiquitination site (where the Lys 563 residue is changed to Arg), displayed quantitatively identical defects in the repair of I‐SceI‐induced chromosomal breaks by homologous recombination (HR). These data establish the role of FANCL and FancD2 monoubiquitination in HR repair.
Cytoplasmic splicing of tRNA in Saccharomyces cerevisiae
Authors: Yoshihisa Tohru , Ohshima Chié , Yunoki‐Esaki Kaori , Endo Toshiya
Published: March 2007

The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre‐tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature‐sensitive (ts) sen2 (HA‐sen2‐42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA‐sen2‐42 cells accumulated large amounts of pre‐tRNAs in the cytoplasm at a restrictive temperature, but the pre‐tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse‐chase/hybrid‐precipitation techniques, we showed that the pre‐tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre‐tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm.
Epigenetic regulation of Nanog gene in embryonic stem and trophoblast stem cells
Authors: Hattori Naoko , Imao Yuko , Nishino Koichiro , Hattori Naka , Ohgane Jun , Yagi Shintaro , Tanaka Satoshi , Shiota Kunio
Published: March 2007

The Nanog and Oct‐4 genes are essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that DNA methylation and chromatin remodeling underlie the cell type‐specific mechanism of Oct‐4 gene expression. In the present study, we found that there is a tissue‐dependent and differentially methylated region (T‐DMR) in the Nanog up‐stream region. The T‐DMR is hypomethylated in ES cells, but is heavily methylated in trophoblast stem (TS) cells and NIH/3T3 cells, in which the Nanog gene is repressed. Furthermore, in vitro methylation of T‐DMR suppressed Nanog promoter activity in reporter assay. Chromatin immunoprecipitation assay revealed that histone H3 and H4 are highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus in ES cells. Conversely, histone deacetylation and H3‐K4 demethylation occurred in TS cells. Importantly, in TS cells, hypermethylation of H3‐K9 and ‐K27 is found only at the Nanog locus, not the Oct‐4 locus, indicating that the combination of histone modifications associated with the Nanog gene is distinct from that of the Oct‐4 gene. In conclusion, the Nanog gene is regulated by epigenetic mechanisms involving DNA methylation and histone modifications.
Regulation of platelet‐derived growth factor‐induced Ras signaling by poliovirus receptor Necl‐5 and negative growth regulator Sprouty2
Authors: Kajita Mihoko , Ikeda Wataru , Tamaru Yoshiyuki , Takai Yoshimi
Published: March 2007

Necl‐5, known as a poliovirus receptor and up‐regulated in many cancer cells, enhances platelet‐derived growth factor (PDGF)‐induced activation of Ras‐Raf‐MEK‐ERK signaling, but not PDGF‐induced tyrosine phosphorylation of PDGF receptor, resulting in facilitation of cell proliferation. Here, we showed that Necl‐5 interacted with Sprouty2, known to be a negative regulator of growth factor‐induced signaling, and reduced the inhibitory effect of Sprouty2 on PDGF‐induced Ras signaling. Necl‐5 was reported to be down‐regulated by its trans‐interaction with nectin‐3 upon cell–cell contact, initiating cooperative cell–cell adhesion with cadherin. This down‐regulation of Necl‐5 caused tyrosine phosphorylation of Sprouty2 by c‐Src, which was activated by PDGF receptor in response to PDGF, and inhibited PDGF‐induced Ras signaling. Thus, Necl‐5 and Sprouty2 cooperatively regulate PDGF‐induced Ras signaling. The roles of Necl‐5 and Sprouty2 in contact inhibition for cell proliferation are also discussed.
Effects of habitat on GPS collar performance: using data screening to reduce location error
Authors: LEWIS JESSE S. , RACHLOW JANET L. , GARTON EDWARD O. , VIERLING LEE A.
Published: March 2007

Global positioning system (GPS) technology enables researchers to evaluate wildlife movements, space use and resource selection in detail for extended periods of time. Two types of errors, missed location fixes and location error, are inherent to GPS telemetry and can bias location data sets. Habitat characteristics can influence both types of errors, but no studies have reported how continuous ranges of canopy cover and terrain simultaneously affect location error at different positional dilution of precision (PDOP) and signal quality levels. This information can assist in developing a protocol for removing large location errors from GPS data sets.

The objectives of this study were to quantify how canopy cover and terrain affected GPS collar performance within a mountainous region of northern Idaho, USA, and evaluate different data‐screening options for GPS location data sets from stationary test collars and free‐ranging black bears Ursus americanus.

The fix rate for test collars was very high in all habitats (mean 99·5%, SE 0·14, range 97·9–100%) and was not related to canopy cover or terrain obstruction. However, habitat variables strongly influenced location error, PDOP values and proportion of three‐dimensional (3‐D) fixes. The 95% circular error probable (CEP) equalled 106·8 m for locations at all test sites, and varied substantially with canopy cover, terrain obstruction and signal quality categories, ranging from 14·3 m to 557·0 m. Location errors for two‐dimensional (2‐D) fixes were more variable at higher PDOP values and were significantly larger compared with 3‐D fixes.

Data screening increased the accuracy of test collar location data sets by removing large location errors that were associated with high PDOP values. Data‐screening options that focused on screening 2‐D locations were most effective in reducing location error and retaining the greatest number of locations. For black bear data sets, the four data screening options resulted in data reduction ranging from 8% to 35%.

Synthesis and applications. We have demonstrated how location data can be analysed and screened based on 2‐D and 3‐D fixes in relation to PDOP values to eliminate locations with potentially large location errors. This information can be applied to GPS location data for individual animals to increase data accuracy for analyses.
Organizing learning materials through hierarchical topic maps: an illustration through Chinese herb medication
Authors: Shih B.‐J. , Shih J.‐L. , Chen R.‐L.
Published: March 2007

Abstract This research aims to use hierarchical topic maps to compile digital learning material and to discuss its design and application possibilities. The system renders tremendous original assets and then embeds a self‐organizing map (SOM) in the material database to produce topical learning materials, as in this case, an illustration through Chinese herb medication. It helps to demonstrate robust professional information as well as knowledge structures, and provides a customized and interactive learning dynamic to support both progressive and constructive learning styles.

The paper first gives a detailed procedural description of the material construction, explains how topic map techniques were applied, and observes the implications and potentials of the technology to education.

Both the technical and educational evaluations of using SOM topic maps in compilation of learning materials have resulted in positive feedback. SOM allows users to review the complete databank in structural hierarchical order, which provides comprehensive understanding of the entire set of learning materials, and also brings opportunities to users to discover knowledge related to their study area.
The Neuropeptide Pituitary Adenylate Cyclase‐Activating Polypeptide Exerts Anti‐Apoptotic and Differentiating Effects during Neurogenesis: Focus on Cerebellar Granule Neurones and Embryonic Stem Cells
Authors: Falluel‐Morel A. , Chafai M. , Vaudry D. , Basille M. , Cazillis M. , Aubert N. , Louiset E. , DeJouffrey S. , Le Bigot J. F. , Fournier A. , Gressens P. , Rostène W. , Vaudry H. , Gonzalez B. J.
Published: March 2007

Pituitary adenylate cyclase‐activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its hypophysiotrophic activity. It has subsequently been shown that PACAP and its receptors are widely distributed in the central nervous system of adult mammals, indicating that PACAP may act as a neurotransmitter and/or neuromodulator. It has also been found that PACAP and its receptors are expressed in germinative neuroepithelia, suggesting that PACAP could be involved in neurogenesis. There is now compelling evidence that PACAP exerts neurotrophic activities in the developing cerebellum and in embryonic stem (ES) cells. In particular, the presence of PACAP receptors has been demonstrated in the granule layer of the immature cerebellar cortex, and PACAP has been shown to promote survival, inhibit migration and activate neurite outgrowth of granule cell precursors. In cerebellar neuroblasts, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through activation of the MAPkinase extracellular regulated kinase. ES cells and embryoid bodies (EB) also express PACAP receptors and PACAP facilitates neuronal orientation and induces the appearance of an electrophysiological activity. Taken together, the anti‐apoptotic and pro‐differentiating effects of PACAP characterised in cerebellar neuroblasts as well as ES and EB cells indicate that PACAP acts not only as a neurohormone and a neurotransmitter, but also as a growth factor.
Influence of thermal expansion mismatch and fatigue loading on phase changes in porcelain veneered Y‐TZP zirconia discs
Authors: DE KLER M. , DE JAGER N. , MEEGDES M. , VAN DER ZEL J. M.
Published: March 2007

Summary  There is a rapidly growing interest for the use of Y‐TZP zirconia as core material in veneered all‐ceramic prostheses. It was hypothesized that a mismatch in coefficient of thermal expansion between the veneering porcelain and the Y‐TZP zirconia core of these prostheses causes transformation of the tetragonal to the monoclinic structure in Y‐TZP zirconia at the interface boundary when exposed to fatigue loading, resulting in fracture at the interface boundary. Y‐TZP zirconia discs were veneered with three porcelains differing in coefficient of thermal expansion. Finite element analysis was used to investigate the stress distribution in the bi‐layered discs because of the mismatch in thermal expansion. Two of these three groups were fatigued with the veneering ceramic in tension. X‐ray diffraction was used to measure the intensity of monoclinic and tetragonal zirconia phase present at the zirconia core surface after sintering, airborne abrasion, veneering, and fatigue loading. It was found that the sintered tetragonal structure was converted to monoclinic up to a depth of 27 μm after airborne abrasion, and reversed back to tetragonal after porcelain veneering with porcelain. Fatigue loading of veneered discs did not, even with the highest possible thermal mismatch stress, cause any conversion from tetragonal to monoclinic phase.
Traumatic axonal damage in the brain can be detected using β‐APP immunohistochemistry within 35 min after head injury to human adults
Authors: Hortobágyi T. , Wise S. , Hunt N. , Cary N. , Djurovic V. , Fegan‐Earl A. , Shorrock K. , Rouse D. , Al‐Sarraj S.
Published: March 2007

Immunohistochemistry staining for β‐amyloid precursor protein (β‐APP) is a sensitive method to detect early axonal damage in traumatic brain injury, which was previously estimated to be of minimum 60–90 min after head injury. We present seven cases of well‐documented posttraumatic survival of 35–60 min where β‐APP detects early axonal damage. Cases were selected from routine work where documentation about survival is judged to be accurate. These are divided into three groups: group 1: severe head injury (n = 7) with documented survival between 35 and 60 min. Group 2: severe head injury (n = 4) with documented survival of less than 30 min. Group 3: cases (n = 4) where death was not due to head injury but survival is documented between 45 and 109 min. The brains were fixed in formalin for 4 weeks and six regions (frontal lobe with anterior corpus callosum, parietal lobe with deep white matter, basal ganglia with posterior limb of internal capsule, cerebellum with white matter and middle cerebellar peduncle and pons with basis pontis and superior cerebellar peduncle) were sampled. All blocks were stained for haematoxylin and eosin and β‐APP and selected ones for CD68, using antigen retrieval method. In group 1 sections revealed β‐APP immunoreactivity in forms of small globules and granules and occasionally as thin and short filaments. These were detected in the pons, corpus callosum, internal capsule and cerebral white matter, with some variation in localization and intensity. In groups 2 and 3 all the sections were negative for β‐APP staining. None of the cases showed evidence of severe brain swelling, increased intracranial pressure, ischaemia or infection. Using the antigen retrieval method, β‐APP immunohistochemistry can detect axonal damage within 35 min after severe head injury. These results may have an implication in the consideration of minimal survival time after traumatic head injury in medico‐legal practice.
Pathogenesis and molecular targeted therapy of spinal and bulbar muscular atrophy
Authors: Adachi H. , Waza M. , Katsuno M. , Tanaka F. , Doyu M. , Sobue G.
Published: March 2007

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is a motor neurone disease characterized by muscle atrophy, weakness, contraction fasciculations and bulbar involvement. SBMA mainly affects males, while females are usually asymptomatic. SBMA is caused by expansion of a polyglutamine (polyQ)‐encoding CAG trinucleotide repeat in the androgen receptor (AR) gene. AR belongs to the heat shock protein 90 (Hsp90) client protein family. The histopathologic hallmarks of SBMA are diffuse nuclear accumulation and nuclear inclusions of the mutant AR with expanded polyQ in residual motor neurones in the brainstem and spinal cord as well as in some other visceral organs. There is increasing evidence that the ligand of AR and molecular chaperones play a crucial role in the pathogenesis of SBMA. The success of androgen deprivation therapy in SBMA mouse models has been translated into clinical trials. In addition, elucidation of its pathophysiology using animal models has led to the development of disease‐modifying drugs, that is, Hsp90 inhibitor and Hsp inducer, which inhibit the pathogenic process of neuronal degeneration. SBMA is a slowly progressive disease by nature. The degree of nuclear accumulation of mutant AR in scrotal skin epithelial cells was correlated with that in spinal motor neurones in autopsy specimens; therefore, the results of scrotal skin biopsy may be used to assess the efficacy of therapeutic trials. Clinical and pathological parameters that reflect the pathogenic process of SBMA should be extensively investigated.
IgE Anti‐Borrelia burgdorferi Components (p18, p31, p34, p41, p45, p60) and Increased Blood CD8+CD60+ T Cells in Children with Lyme Disease
Authors: Bluth M. H. , Robin J. , Ruditsky M. , Norowitz K. B. , Chice S. , Pytlak E. , Nowakowski M. , Durkin H. G. , Smith‐Norowitz T. A.
Published: March 2007

Immunoglobulin (Ig) E may provide immunity against Borrelia burgdorferi infection (Lyme disease) in children which lasts throughout adulthood. We investigated the presence and persistence of IgE anti‐B. burgdorferi antibodies (Abs) in paediatric patients infected with Lyme disease over time. Serum immunoglobulin levels, presence of IgG and IgE anti‐B. burgdorferi components, and distributions of blood T, B and natural killer lymphocyte subsets were studied in B. burgdorferi‐infected and ‐uninfected children (nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay, Western blot, flow cytometry). Total serum IgM, IgG, IgE and IgA levels, and distributions of blood lymphocytes (CD4+, CD8+, CD19+) of both groups, excluding CD8+CD60+ T cells, were within normal ranges. However, infected, but not uninfected children made IgG anti‐B. burgdorferi proteins p18, p31, p34, p41, p45, but not IgG anti‐p60, and IgE anti‐B. burgdorferi proteins p31, p34, p41, p45, p60, but not IgE anti‐p18. These proteins were also detected in an infected child 1 year post‐infection. Interestingly, CD8+CD60+ T‐cell numbers were significantly increased (fourfold) in infected, compared with uninfected, patients (P = 0.001). These results demonstrate that specific IgE anti‐B. burgdorferi Abs are generated and persist in children with Lyme disease and that CD8+CD60+ T cells may play an important role in these responses.
Mesenchymal Stem Cells Stimulate Antibody Secretion in Human B Cells
Authors: Rasmusson I. , Le Blanc K. , Sundberg B. , Ringdén O.
Published: March 2007

Mesenchymal stem cells (MSC) have immunomodulatory effects and inhibit T‐cell responses to alloantigens and mitogens in vitro and in vivo. We wanted to examine the effect of MSC on human B cells. MSC stimulated IgG production, measured in an enzyme‐linked immunospot (ELIspot) assay in blood and spleen lymphocytes. MSC only induced a low proliferation. When a semipermeable membrane separated MSC and mononuclear cells, the IgG production was stimulated in unfractionated lymphocytes. In contrast, enriched B cells required cell contact with MSC to produce IgG. Co‐cultures of MSC and lymphocytes increased IFN‐γ production. MSC produce IL‐6, and addition of MSC to spleen cells dramatically increased IL‐6 levels. After lymphocyte stimulation with lipopolysaccharide (LPS), cytomegalovirus or varicella zoster virus, MSC either stimulated or inhibited IgG response, depending on the level of stimulation by LPS or the viral antigens. Similar results were obtained for enriched B cells. To conclude, MSC stimulate B‐cell antibody secretion. The IgG secretion by activated B cells may be stimulated or inhibited by the addition of MSC, depending on the level of stimulation.
A leucine‐rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana
Authors: Shahollari Bationa , Vadassery Jyothilakshmi , Varma Ajit , Oelmüller Ralf
Published: March 2007

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single‐copy gene DMI‐1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica‐insensitive‐2; pii‐2), and a corresponding insertion line, are impaired in a leucine‐rich repeat protein (At1g13230). The protein pii‐2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X‐100‐insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine‐rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii‐2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica‐induced growth promotion and enhanced seed production in Arabidopsis thaliana.
A method for cellular localization of gene expression via quantitative in situ hybridization in plants
Authors: Küpper Hendrik , Seib Laura Ort , Sivaguru Mayandi , Hoekenga Owen A. , Kochian Leon V.
Published: March 2007

A quantitative in situ hybridization technique (quantitative whole‐mount in situ hybridization, QISH) for plants is described. It employs direct hybridization of fluorescently labelled gene‐specific oligonucleotides in large tissue pieces combined with optical sectioning. It dramatically increases the throughput compared with conventional antibody‐ and microtome‐based in situ mRNA hybridization methods, while simultaneously eliminating artefact‐prone preparation steps that prevent reliable quantification in conventional methods. The key feature of this technique is the quantification of gene expression using housekeeping genes (cytosolic GAPDH and 18S RNA) as internal standards. This feature enables a correction of varying cytoplasm/vacuole ratios in different cell types, as well as tissue optical effects and non‐specific signals. The quantitative nature of the technique allows for analysis of gene expression in response to different environmental conditions, as well as tissue‐ and age‐dependent differences in gene expression patterns. In addition to testing tissue permeabilization, structural preservation, specificity, linearity and tissue optical effects, we verified the reliability of the technique with three Arabidopsis thaliana genes of known function and distribution. These were the rbcL gene for ribulose 1,5‐bisphosphate carboxylase, the developmentally related gene SCARECROW (AtSCR) and PHOT‐1, a photoreceptor kinase. As expected, rbcL mRNA was found in all photosynthetic cells, while SCR mRNA was detected mainly in bundle sheath cells and PHOT‐1 was found predominantly in epidermal and cortical cells of the apical hook of light‐grown seedlings. As an application example, QISH was used to measure transcript abundance for a zinc transporter from the ZIP family of transporters in the Zn/Cd hyperaccumulator model plant, Thlaspi caerulescens, and the related non‐accumulator Thlaspi arvense. This showed that QISH can be used to compare differences in mRNA levels between cell types, plant growth conditions and plant species. Messenger RNA for the zinc transporter gene ZNT1 was abundant in photosynthetic cells, but not in the epidermal storage cells where metal hyperaccumulation in T. caerulescens occurs. This indicates that ZNT1 does not directly participate in metal hyperaccumulation within the leaf. Growing T. caerulescens with high zinc levels strongly reduced ZNT1 transcript abundance in the spongy mesophyll cells, but less in the other cell types. In T. arvense, ZNT1 mRNA levels were generally much lower, and were furthermore drastically reduced by growth at increased zinc levels, confirming earlier reports regarding ZNT1 regulation in these two Thlaspi species.
SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans‐acting endogenous siRNA
Authors: Hernandez‐Pinzon Inmaculada , Yelina Nataliya E. , Schwach Frank , Studholme David J. , Baulcombe David , Dalmay Tamas
Published: March 2007

Post‐transcriptional gene silencing (PTGS) is a sequence‐specific RNA degradation process conserved in fungi, plants and animals. The trigger of the mechanism is double‐stranded RNA derived from transgenic or endogenous loci and formed by intra‐ or inter‐molecular interactions of single‐stranded RNAs or the action of RNA‐dependent RNA polymerases (RDRs). Double‐stranded RNA from various sources is processed by one of the four Dicer‐like (DCL) proteins in Arabidopsis, and the resulting short RNAs enter into at least four different pathways, one of which involves the production of trans‐acting short interfering RNAs (tasiRNAs). We report here a novel gene (SDE5) that is required for transgene silencing and the production of tasiRNAs. Mutation in SDE5 also results in hyper‐susceptibility to cucumber mosaic virus but not turnip mosaic virus. However, like RDR6, SDE5 is not involved in inverted repeat‐induced transgene silencing or the biogenesis of microRNAs and 24 nt siRNAs produced by DCL3. Based on these results, we propose that SDE5 acts together with RDR6 in generating double‐stranded RNA from specific single‐stranded RNAs. As the sequence of SDE5 has sequence features shared by TAP, a human mRNA export factor, we propose that its role could be in the transport of RNA molecules that are converted into a double‐stranded form by RDR6.
Offsets for land clearing: No net loss or the tail wagging the dog?
Authors: Gibbons Philip , Lindenmayer David B.
Published: March 2007

Summary  Offsets (also known as mitigation banks, compensatory habitat, set‐asides) is a policy instrument recently introduced in several States in Australia to permit some land clearing while striving for no net loss in the extent and condition of native vegetation overall. Offsetting is criticized with respect to the amount of gain required to compensate for losses from clearing, the equivalence of losses and gains, the time lag between losses and gains and a poor record of compliance. Despite these criticisms, we conclude that offsetting is a useful policy instrument while governments continue to permit some clearing of native vegetation. However, offsets will only contribute to no net loss if (i) clearing is restricted to vegetation that is simplified enough so that its functions can be restored elsewhere with confidence or clearing is restricted to vegetation that is unlikely to persist and is not practicable to restore irrespective of clearing; (ii) any temporary loss in habitat between clearing and the maturation of an offset, or differences between the habitat lost from clearing and gained through an offset, does not represent significant risk to a species, population or ecosystem process; (iii) there will be gains of sufficient magnitude on the offset site to compensate for losses from clearing; (iv) best practice adaptive management is applied to offsets; (v) offsets are in place for at least the same duration as the impacts from clearing; and (vi) there is adequate compliance. Land clearing with offsets outside these parameters is inconsistent with ‘no net loss’.
Potential microbial and chemical hazards to waterbirds at the Western Treatment Plant
Authors: Hamilton Andrew J.
Published: March 2007

Summary  The Western Treatment Plant, a major sewage treatment plant west of Melbourne, Australia, is widely regarded as a significant conservation site for waterbirds. But experiences from various parts of the world suggest that sewage can also be hazardous to waterbirds, and has probably been responsible for mass‐kill events. The intent of this contribution is to raise awareness about the potential for adverse impacts of sewage treatment plants on waterbirds, and to stimulate debate on the issue, with the ultimate objective of developing appropriate management strategies to mitigate the risk of mass kills.
Landholder participation in native vegetation management in irrigation areas
Authors: Mendham Emily , Millar Joanne , Curtis Allan
Published: March 2007

Summary  Landholder adoption of conservation practices has been extensively researched in dryland areas, but there has been less research into the adoption of biodiversity conservation practices in irrigation areas. The Murray Catchment Management Authority (MCMA) and Murray Irrigation Limited (MIL) in south‐west New South Wales offer landholders monetary incentives to enhance native vegetation management. However, uptake of the incentives and recommended practices has been slower in the irrigation areas than in dryland areas serviced by the MCMA. We explored landholder participation in native vegetation management in the MIL area using semistructured interviews with landholders and extension staff. Our findings suggest there are important differences between irrigation and dryland contexts. Landholders in irrigation districts face higher opportunity costs and are more likely to mistrust government intentions. Other constraints to adoption include lack of financial resources, restricted time to carry out works and a high turnover of extension staff. Landholders adopted recommended vegetation management practices because those practices matched their values and goals. The importance of regular contact with a positive and enthusiastic extension officer was also evident. These findings have important implications for conservation policies and programs. Extension should move beyond arousing interest and include substantial follow‐up contact to maintain landholder motivation and confidence, as well as provide recognition for past landholder efforts. Incentive programs should be designed to provide wider landholder support with different entry points allowing participants to adapt, learn and build confidence. Incentives should address the constraints experienced by landholders, particularly at times of peak workloads and during drought. Work targets set for field staff can be too high and result in program staff focusing on accomplishing on‐ground work, rather than developing irrigators’ longer‐term commitment to vegetation conservation.
Schizophrenia housing and supportive relationships
Authors: Browne Graeme , Courtney Mary
Published: March 2007

ABSTRACT:  Schizophrenia can be a very disabling illness that affects between 0.5% and 1% of the population. This illness has a great personal impact on the individual sufferer, their family and friends. In addition, it makes significant demands on health services and the community in general. This paper reviews the literature on housing and supportive relationships for people with schizophrenia. The literature reports that people’s experience of their schizophrenia is that it not only causes symptoms, but often impacts on their ability to maintain the basic resources in life. These resources include the ability to maintain reasonable quality housing, which seems to further impact negatively on their illness and their ability to maintain supportive social relationships. People with schizophrenia (and people in general) rely on their social relationships and family to maintain their mental health. The loss of social relationships and inability to maintain quality housing seem to be related – if people cannot maintain quality housing, they find it difficult to maintain supportive social relationships.