bluuebunny/crossref_metadata_2025 huggingface dataset:
Abscisic acid activates acid invertases in developing grape berry
Authors: Pan Qiu‐Hong , Li Mei‐Jun , Peng Chang‐Cao , Zhang Na , Zou Xun , Zou Ke‐Qin , Wang Xiu‐Ling , Yu Xiang‐Chun , Wang Xiao‐Fang , Zhang Da‐Peng
Published: September 2005

Acid invertases play a key role in sugar metabolism, and the plant hormone abscisic acid (ABA) enhances sugar accumulation in crop sink organs, but information about the relationship between ABA and acid invertases has been limited. The present experiments were done with both in vivo pre‐incubation of the grape (Vitis vinifera × V. labrusca L.) berry tissues in ABA‐containing medium and in vivo infiltration of ABA into the intact berries. The results show that ABA activates both the soluble and cell wall‐bound acid invertases during fruit development by enhancing their activities and amounts as assessed by immunoblotting or enzyme‐linked immunosorbent assay. This activation was pH, time course and ABA dose dependent. The serine/threonine protein kinase inhibitors K252a, staurosporine and H7 and acid phosphatase increased the activation of ABA‐induced acid invertase, but the tyrosine protein kinase inhibitor quercetin strongly suppressed the ABA‐induced effects, suggesting that a complex reversible protein phosphorylation is involved in the ABA‐induced activation of acid invertases. The effects of the protein kinase inhibitors were dependent on the in vivo state of the tissues but independent of the expression of acid invertases. Two ABA analogues, (–)‐ABA and trans‐ABA, had no effect on acid invertases, showing that the ABA‐induced activation of acid invertases is specific to the physiologically active form of ABA. These data suggest that ABA may be involved in fruit development by activating acid invertases.
Assessment of lovastatin application as tool in probing cytokinin‐mediated cell cycle regulation
Authors: Hartig Katja , Beck Erwin
Published: September 2005

Lovastatin, a potent inhibitor of the mevalonate pathway, has been used in plant cell cycle studies to eliminate the cytosolic cytokinin biosynthesis. However, several implications can blur the results, as cytokinins may be alternatively formed from isopentenylpyrophosphate produced by the plastid 1‐deoxy‐xylulose 5‐phosphate pathway and because the endogenous cytokinin levels oscillate considerably in the course of a cell cycle. In the work presented here, short‐ and long‐term effects of lovastatin on suspension‐ cultured Nicotiana tabacum (L.) BY‐2 cells were differentiated. The short‐term experiments revealed a fast action of lovastatin, resulting in a significantly, though not completely, decreased content of endogenous cytokinins that became visible already after 10 min and was most pronounced after 30 min. But the impact of lovastatin on cell cycle progression depended also on the phase of the cell cycle at which it was administered. Lowering of the cytokinin level during the early S phase, when the endogenous cytokinin levels increased, delayed the S/G2 transition, whereas the same treatment in the late S phase, when the cellular cytokinin concentrations had already started to decrease, promoted it. Incubation periods longer than 48 h resulted in about 50% loss of viable of the cells and also in a reduced capability of division of the survivors. These cells later on resumed cell division. A second treatment with lovastatin of that culture again killed about 50% of the cells, but the surviving cells showed faster re‐growth. In conclusion, lovastatin appears as a useful inhibitor of cytokinin biosynthesis in short‐term studies, but its use in long‐term experiments may create complex effects and therefore requires substantial caution.
Partitioning of respiratory electrons in the dark in leaves of transgenic tobacco with modified levels of alternative oxidase
Authors: Guy Robert D. , Vanlerberghe Greg C.
Published: September 2005

This study examined tobacco (Nicotiana tabacum cv. Petit Havana SR1) leaf respiration in the dark, utilizing both wild‐type plants and transgenic plants with increased or decreased levels of alternative oxidase (AOX) protein. AOX represents a non‐energy‐conserving branch in mitochondrial electron transport. Inhibitor studies showed that the maximum possible flux of electrons to AOX (AOX capacity) correlated with the level of AOX protein present in the different plant lines. A comparison of the plants using online 18O isotope discrimination was done to determine whether AOX protein level would impact the actual steady‐state partitioning of electrons to AOX (AOX engagement). Under a range of pretreatment and measurement conditions, there was little if any effect of AOX protein level on the degree of engagement. This suggests that the metabolic conditions inherent to a particular growth condition and/or the biochemical regulatory properties of AOX itself are the critical factors that control partitioning. Interestingly, we found that measurement temperature and water status are parameters that may have some influence over AOX engagement.
Induction of Ym1/2 in mouse bone marrow‐derived mast cells by IL‐4 and identification of Ym1/2 in connective tissue type‐like mast cells derived from bone marrow cells cultured with IL‐4 and stem cell factor
Authors: Lee Eunkyung , Yook Jumin , Haa Kyungmi , Chang Hyeun Wook
Published: September 2005

Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR‐selected cDNA subtraction to establish the profile of induced genes in the connective tissue (CT) type‐like mast cells derived from bone marrow cells cultured in the presence of IL‐4 and stem cell factor. Two hundred and fifty cDNA clones were obtained from the CT type‐like mast cells by PCR‐selected cDNA subtraction. Among them, Ym1/2, a chitinase‐like protein, is one of the most abundantly induced genes. Ym1 is produced by activated macrophages in a parasitic infection, whereas its isotype, Ym2, is highly upregulated in allergic lung disease. In order to differentiate which isotype is expressed in bone marrow cells, specific primers for bone marrow‐derived mast cells (BMMC), and CT type‐like mast cells were used for RT‐PCR. The results showed that Ym1 was constitutively expressed in bone marrow cells and gradually decreased in the presence of IL‐3, whereas Ym2 was induced only in the presence of IL‐4. CT type‐like mast cells from bone marrow cells expressed Ym1 throughout the culture period and Ym2 was induced only by the addition of IL‐4 into BMMC, indicating that IL‐4 is essential for the expression of Ym1/2 genes.
Tissue localization and frequency of antigen‐specific effector CD4+ T cells determines the development of allergic airway inflammation
Authors: Harris Nicola L , Holloway John , Fitzharris Penny , McDonald Michael , Camberis Mali , Fazekas De St Groth Barbara , Ronchese Franca , Le Gros Graham
Published: September 2005

Previous activation of effector Th2 cells is central to the development of allergic inflammatory responses. We have observed that priming of allergen‐specific Th2 cells in C57BL/6 or B10.A mice with allergen delivered via the i.p. or s.c. routes results in very different outcomes following subsequent airway exposure to the same allergen. Systemic allergen immunization (via the i.p. route) resulted in the formation of a lung‐resident population of allergen‐specific T cells, and mice developed severe allergic airway inflammation in response to inhaled allergen. The localization of cells to the lung did not require the presence of antigen at this site, but reflected a large pool of circulating activated allergen‐specific T cells. In contrast, localized immunization (via the s.c. route) resulted in a small T‐cell response restricted to the draining lymph node, and mice were not responsive to inhaled allergen. These data indicate that prior sensitization to an allergen alone was not sufficient for the induction of allergic inflammation; rather, responsiveness was largely determined by precursor frequency and tissue localization of the allergen‐specific effector Th2 cells.
Polymorphisms of CARD15/NOD2 and CD14 genes in New Zealand Crohn's disease patients
Authors: Leung Euphemia , Hong Jiwon , Fraser Alan G , Merriman Tony R , Vishnu Prakash , Abbott William GH , Krissansen Geoffrey W
Published: September 2005

Polymorphisms in the CARD15/NOD2 gene, which encodes a cytosolic protein involved in bacterial recognition, are associated with development of Crohn's disease (CD). Other potential susceptibility genes such as CD14 may compound the risk of developing CD. We examined the frequency of the three major CARD15 risk alleles (3020insC/L1007fsinsC, G908R and R702W), and a functional polymorphism (–159C/T) in the promoter of the CD14 gene in 185 CD patients in New Zealand and 187 ethnically matched controls. The frequencies of the 3020insC (8.1 vs 0.8%, P < 0.0001), G908R (3.5 vs 2.4%, P = 0.37) and R702W (7.3 vs 5.1%, P = 0.21) alleles in CD patients and controls, respectively, were similar to those described in Australia, and the ancestral countries of Scotland, Ireland and the UK. Only the 3020insC polymorphism was found to be a significant risk factor for CD in our New Zealand cohort (odds ratio = 10.91 [95% confidence intervals 3.30–36.08]; P < 0.0001 for heterozygotes), but not a single patient was homozygous for the 3020insC polymorphism. The T allele (51 vs 50%, P = 0.77) and TT genotype (26 vs 24%, P = 0.84) frequencies of the –159C/T CD14 gene promoter polymorphism did not significantly differ between CD patients and controls. In summary, our findings provide evidence that the CARD15 3020insC risk allele influences disease susceptibility in a small proportion (<17%) of New Zealand CD patients, whereas there was no evidence that the CD14 –159C/T polymorphism is associated with CD.
Production and purification of human indoleamine 2,3‐dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO
Authors: Webster Nicole L , Wee Janet , Uren Sandra J , Boyle William , Sandrin Mauro S
Published: September 2005

The human indoleamine 2,3‐dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa‐histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme‐containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti‐HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN‐γ‐stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.
ATLAS OF IMMUNOLOGY, 2nd edition
Authors: Vinuesa Carola G
Published: September 2005

Immunology and Cell Biology (2005) 83, 584; doi:10.1111/j.1440‐1711.2005.01376.x
Methods in Molecular Biology, Volume 295: IMMUNOCHEMICAL PROTOCOLS, 3rd edition
Authors: Grollo Lara , Chua Brendon , Jackson David C
Published: September 2005

Immunology and Cell Biology (2005) 83, 584–585; doi:10.1111/j.1440‐1711.2005.01377.x
Role of oxidative stress in ERK and p38 MAPK activation induced by the chemical sensitizer DNFB in a fetal skin dendritic cell line
Authors: Matos Teresa J , Duarte Carlos B , Gonçalo Margarida , Lopes M Celeste
Published: September 2005

The intracellular mechanisms involved in the early phase of dendritic cell (DC) activation upon contact with chemical sensitizers are not well known. The strong skin sensitizer 2,4‐dinitrofluorobenzene (DNFB) was shown to induce the activation of mitogen‐activated protein kinases (MAPK) in DC. In the present study, we investigated a putative role for oxidative stress in DNFB‐induced MAPK activation and upregulation of the costimulatory molecule CD40. In a DC line generated from fetal mouse skin, DNFB induced a significant increase in protein oxidation, measured by the formation of carbonyl groups, while it had almost no effect on lipid peroxidation. The antioxidants glutathione and vitamin E, which inhibit protein and lipid oxidation, respectively, were used to assess the role of oxidative stress in DNFB‐induced MAPK activation. Glutathione, but not vitamin E, inhibited DNFB‐induced p38 MAPK and ERK1/2 phosphorylation, whereas none of the antioxidants interfered significantly with the DNFB‐induced upregulation of CD40 protein levels. Taken together, these results indicate that DNFB activates p38 MAPK and ERK1/2 via production of reactive oxygen species, and that protein oxidation plays an important role in MAPK activation.
Methods in Molecular Biology, Volume 302: HANDBOOK OF ELISPOT: METHODS AND PROTOCOLS
Authors: Thomson Scott
Published: September 2005

Immunology and Cell Biology (2005) 83, 586; doi:10.1111/j.1440‐1711.2005.01381.x
IFN‐γ enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis
Authors: Denis Michel , Wedlock D Neil , Buddle Bryce M
Published: September 2005

We sought to determine the impact of bovine IFN‐γ on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF‐α, IL‐1β and IL‐12 upon infection with bacille Calmette−Guérin (BCG). Prior pulsing of cells with IFN‐γ significantly enhanced the release of NO and IL‐12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro‐inflammatory mediators, compared to levels released upon BCG infection. IFN‐γ treatment of macrophages enhanced the release of pro‐inflammatory mediators, but did not modify bacterial replication in M. bovis‐infected macrophages. Treatment of macrophages with a combination of IFN‐γ and LPS led to a reduction in bacterial replication. Infected cells treated with IFN‐γ/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL‐10 and neutralizing anti‐TNF‐α). We conclude that virulent M. bovis is a major determinant of release of pro‐inflammatory cytokines by macrophages. IFN‐γ amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF‐α release.
SonoCT improves on conventional ultrasound in the visualization of the pancreatic and bile duct: A pilot study
Authors: RICKES STEFFEN , BÖHM JANETT , MALFERTHEINER PETER
Published: September 2005

Background and Aims:  In sonographic diagnostic investigation, evaluation of the ductus Wirsungianus and the ductus hepatocholedochus plays an important role. The present prospective study sought to establish whether SonoCT can improve on conventional B‐mode sonography in the visualization of the pancreatic and bile duct.

Subjects and Methods:  In the pilot study, ductus Wirsungianus and ductus hepatocholedochus of 14 healthy volunteers were assessed by an experienced and an inexperienced examiner, respectively, using both traditional B‐mode sonography and SonoCT. Assessment was based on a predetermined set of criteria.

Results:  Both examiners were able to determine duct structures significantly more effectively using SonoCT. Compared with traditional B‐mode sonography, the use of SonoCT also led to significant improvements in terms of delineation and clarity of representation in both ductus Wirsungianus and ductus hepatocholedochus.

Conclusion:  SonoCT yields better results than conventional B‐mode sonography in terms of delineation, visualization of borders and artefact‐free representation of both ductus Wirsungianus and ductus hepatocholedochus. Appropriate training and experience of the examiner, however, remain key prerequisites for success in evaluating pathological changes of the ducts. Future studies will be needed to determine whether certain diseases can be diagnosed more readily using SonoCT rather than conventional sonographic technique.
Xanthine oxidase‐derived free radicals directly activate rat pancreatic stellate cells
Authors: TANIOKA HIROAKI , MIZUSHIMA TAKAAKI , SHIRAHIGE AKINORI , MATSUSHITA KOKI , OCHI KOJI , ICHIMURA MITSUKO , MATSUMURA NAOKI , SHINJI TOSHIYUKI , TANIMOTO MITSUNE , KOIDE NORIO
Published: September 2005

Background and Aim:  Free radicals are reported to be associated with fibrosis in the pancreas. It is generally accepted that pancreatic stellate cells (PSC) play an important role in pancreatic fibrosis. However, the exact role of free radicals in activation of PSC has not been fully elucidated. In the present study, using a superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC) with cultured PSC, we investigated how free radicals act on the activation of PSC.

Methods:  PSC were isolated from male Wister rats. Cultured rat PSC were incubated with DDC for 48 h. Intracellular SOD activity and lipid peroxidation were examined in DDC‐treated PSC. Activation of PSC was examined by determining the expression of α‐smooth muscle actin (α‐SMA) by immunocytochemistry. The number of PSC using a hemocytometer, type I collagen secretion with ELISA and matrix metalloproteinases (MMP) activities with gelatin zymography were also examined. Secretion of transforming growth factor‐β1 (TGF‐β1) was evaluated by ELISA. The effects of the allopurinol, a xanthine oxidase (XOD) inhibitor, on PSC were also examined.

Results:  DDC decreased SOD activity and increased lipid peroxidation products in PSC. DDC activated PSC, increasing the number of α‐SMA positive cells, enhancing secretion of type I collagen and MMP, inhibiting PSC proliferation. Secretion of TGF‐β1, which is known to activate PSC, was increased by DDC treatment. These alterations were prevented by allopurinol.

Conclusion:  These results suggest that free radicals generated by XOD might directly activate PSC.
Is a limited lymphadenectomy targeting obturator nodes alone an adequate procedure for Japanese men undergoing radical prostatectomy?
Authors: MIYAKE HIDEAKI , SAKAI IORI , HARADA KEN‐ICHI , HARA ISAO , ETO HIROSHI
Published: September 2005

Abstract

Background: The objective of the present study was to investigate the significance of pelvic lymphadenectomy during radical prostatectomy in Japanese men with prostate cancer.

Methods: A total of 178 consecutive patients who underwent radical prostatectomy and standard pelvic lymphadenectomy targeting the external iliac nodes and obturator fossa for clinically localized prostate cancer were studied. The median observation period of this series was 18 months (range: 3–36 months).

Results: Lymph node metastases were detected in 13 patients; that is, positive nodes were located in the external iliac nodes alone in seven patients, the obturator fossa alone in four patients, and both external iliac nodes and obturator fossa in two patients. Of these 13 patients, all of the seven with more than one positive node demonstrated biochemical recurrence, whereas five of the six with single node involvement remained without signs of biochemical recurrence. Furthermore, a single positive node was located in the external iliac region in five of the six patients. When a group at high‐risk for lymph node metastasis was defined as those meeting more than two of the following three criteria: (i) pretreatment serum prostate specific antigen value ≥ 20 ng/mL; (ii) biopsy Gleason sum ≥ 8; or (iii) percentage of positive biopsy core ≥ 50%, the incidence of lymph node metastasis was 24.5% in the high‐risk group and 0.8% in the low‐risk group.

Conclusions: These findings suggest that limited dissection of the obturator node alone may not be sufficient for Japanese men undergoing radical prostatectomy; therefore, we recommend performing standard pelvic lymphadenectomy targeting both the external iliac nodes and the obturator fossa for patients at high‐risk of lymph node involvement.
Effect of above‐ground plant species on soil microbial community structure and its impact on suppression of Rhizoctonia solani AG3
Authors: Garbeva P. , Postma J. , Van Veen J. A. , Van Elsas J. D.
Published: September 2005

The extent of soil microbial diversity is seen to be critical to the maintenance of soil health and quality. Different agricultural practices are able to affect soil microbial diversity and thus the level of suppressiveness of plant diseases. In a 4‐year field experiment, we investigated the microbial diversity of soil under different agricultural regimes. We studied permanent grassland, grassland turned into arable land, long‐term arable land and arable land turned into grassland. The diversity of microbial communities was described by using cultivation‐based and cultivation‐independent methods. Both types of methods revealed differences in the diversities of soil microbial communities between different treatments. The treatments with higher above‐ground biodiversity generally maintained higher levels of microbial diversity. Moreover, a positive correlation between suppression of Rhizoctonia solani AG3 and microbial diversity was observed. Permanent (species‐rich) grassland and grassland turned into maize stimulated higher microbial diversities and higher levels of suppressiveness of R. solani AG3 compared with the long‐term arable land. Effects of agricultural practices on Bacillus and Pseudomonas communities were also observed and clear correlations between the levels of suppressiveness and the diversities of these bacterial groups were found. This study highlighted the importance of agricultural management regime for soil microbial community structure and diversity as well as the level of soil suppressiveness.
Selective enrichment and molecular characterization of a previously unculturedNitrospira‐like bacterium from activated sludge
Authors: Spieck Eva , Hartwig Christine , McCormack Iris , Maixner Frank , Wagner Michael , Lipski André , Daims Holger
Published: September 2005

Previously uncultured nitrite‐oxidizing bacteria affiliated to the genusNitrospirahave for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of theNitrospira‐like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features ofNitrospira‐like bacteria and includes the selective repression of coexistingNitrobactercells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml−1. The enrichment process was monitored by electron microscopy, fluorescencein situhybridization (FISH) with rRNA‐targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novelNitrospiraspecies closely related to unculturedNitrospira‐like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘CandidatusNitrospira defluvii’.
The ‘pH optimum anomaly’ of intracellular enzymes of Ferroplasma acidiphilum
Authors: Golyshina Olga V. , Golyshin Peter N. , Timmis Kenneth N. , Ferrer Manuel
Published: September 2005

A wide range of microorganisms, the so‐called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0–3), whereas intracellular enzymes would be optimally active at the near‐neutral pH of the cytoplasm (4.6–7.0). The genes of several intracellular or cell‐bound enzymes, a carboxylesterase and three α‐glucosidases, from Ferroplasma acidiphilum, a cell wall‐lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaα‐glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7–4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This ‘pH optimum anomaly’ suggests the existence of yet‐undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed.
The Daily Life of the Garden‐Variety Neurotic: Reactivity, Stressor Exposure, Mood Spillover, and Maladaptive Coping
Authors: Suls Jerry , Martin René
Published: September 2005

AbstractThis article describes a series of studies using the daily process paradigm to describe and understand the affective dynamics of people who experience frequent and intense bouts of a wide range of negative emotions. In several studies, community residents reported on problem occurrence and affect several times a day or at the end of the day. We found reliable evidence that persons who scored high (vs. low) in Neuroticism reported more daily problems, tended to react with more severe emotions, experienced more mood spillover from prior occasions, and exhibited stronger reactions to recurring problems (the “neurotic cascade”). The susceptibility of neurotics to stress seems to extend to all types of problems while certain other dimensions of personality (e.g., Agreeableness) are associated with hyperreactivity to particular kinds of problems. The research demonstrates how daily process research can provide insight about classic problems in the field of individual differences.
Increased risk of early virological failure in non‐European HIV‐1‐infected patients in a Dutch cohort on highly active antiretroviral therapy
Authors: Van Den Berg JB , Hak E , Vervoort SCJM , Hoepelman IM , Boucher CAB , Schuurman R , Schneider MME
Published: September 2005

Objective
To compare early and late responses to highly active antiretroviral therapy (HAART) in European and non‐European HIV‐1 infected patients in a Dutch cohort.

Methods
We retrospectively analysed the response to HAART of 216 previously treatment‐naive HIV‐1‐infected patients using the University Medical Centre Utrecht HIV database. African (n=51), Asian (n=7), and Central/South American (n=6) patients were classified as non‐European, and others as European (n=152). Early failure was defined as a viral load that remained above 400 HIV‐1 RNA copies/mL after 6 months of treatment with HAART. Late‐phase failure was determined in patients who were successfully treated in the early phase and was defined as two consecutive viral load measurements above 400 copies/mL, a new AIDS‐defining event or death.

Results
In the early phase, four of 152 (2.6%) European and eight of 64 (12.5%) non‐European patients failed HAART. A significant increased risk of virological failure in the early phase of treatment was observed for non‐Europeans as compared to Europeans (odds ratio 4.6; 95% confidence interval 1.1–20.2). Low serum drug levels in the absence of resistant virus were often seen at the time of early failure. No difference in late‐phase failure was observed between the two groups (adjusted hazard ratio 0.6; 95% confidence interval 0.3–1.2).

Conclusions
Non‐European patients had a 4.6 times higher risk of virological failure than their European counterparts in the first 6 months of treatment with HAART. This failure seemed to be associated with low serum drug levels at the time of failure. However, if HAART was successful in the early phase, response rates in the late phase were similar for Europeans and non‐Europeans.
HIV‐1 subtypes in Spain: a retrospective analysis from 1995 to 2003
Authors: Lospitao E , Álvarez A , Soriano V , Holguín A
Published: September 2005

Objective

To perform a retrospective analysis of all HIV‐1 non‐B variants circulating in Spain from 1995 to 2003 and extend their virological characterization.

Methods

Samples from a total of 396 HIV‐infected subjects with epidemiological suspicion of being infected with non‐B clades were analysed during the study period. Subtyping was carried out on the protease (PR), reverse transcriptase (RT) and envelope (env) genes.

Results

PR sequences belonging to non‐B subtypes were recognized in 43.2% of cases (23A, 13C, 6D, 3F, 118G, 3H, 4J and 1U). Subtype G and AG recombinants were the most frequent variants (69%), and were found most often in subjects from West and Central Africa. Up to 70% of pol (PR, RT) sequences belonging to subtype G harboured env sequences belonging to clade A (55%), B (13.8%) or K (3.4%). Nearly half were mosaic GA viruses, and a few were CRF14_BG viruses. Up to 14 new recombinant viruses, which could not be assigned to previously described circulating recombinant forms (CRFs), were found.

Conclusions

There is great diversity in the HIV‐1 variants and recombinant viruses circulating in Spain. Non‐B sequences may be underestimated if only the env region is examined in phylogenetic analyses. Drug resistance testing provides the advantage of pol subtyping, and its additional use for this purpose should be encouraged.
Combining Predictors for Classification Using the Area under the Receiver Operating Characteristic Curve
Authors: Pepe Margaret Sullivan , Cai Tianxi , Longton Gary
Published: September 2005

SummaryNo single biomarker for cancer is considered adequately sensitive and specific for cancer screening. It is expected that the results of multiple markers will need to be combined in order to yield adequately accurate classification. Typically, the objective function that is optimized for combining markers is the likelihood function. In this article, we consider an alternative objective function—the area under the empirical receiver operating characteristic curve (AUC). We note that it yields consistent estimates of parameters in a generalized linear model for the risk score but does not require specifying the link function. Like logistic regression, it yields consistent estimation with case–control or cohort data. Simulation studies suggest that AUC‐based classification scores have performance comparable with logistic likelihood‐based scores when the logistic regression model holds. Analysis of data from a proteomics biomarker study shows that performance can be far superior to logistic regression derived scores when the logistic regression model does not hold. Model fitting by maximizing the AUC rather than the likelihood should be considered when the goal is to derive a marker combination score for classification or prediction.
A Hypothesis Test for the End of a Common Source Outbreak
Authors: Brookmeyer Ron , You Xiaojun
Published: September 2005

SummaryThe objective of this article is to develop a hypothesis‐testing procedure to determine whether a common source outbreak has ended. We consider the case when neither the calendar date of exposure to the pathogen nor the exact incubation period distribution is known. The hypothesis‐testing procedure is based on the spacings between ordered calendar dates of disease onset of the cases. A simulation study was performed to evaluate the robustness of the methods to various models for the incubation period of infectious diseases. We investigated the impact of multiple testing on the overalloutbreak‐wisetype I error probability. We derive expressions for the outbreak‐wise type I error probability and show that multiple testing has minimal effect on inflating that error probability. The results are discussed in the context of the 2001 U.S. anthrax outbreak.
cDNA cloning and characterization of a novel calmodulin‐like protein from pearl oyster Pinctada fucata
Authors: Li Shuo , Xie Liping , Ma Zhuojun , Zhang Rongqing
Published: September 2005

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full‐length cDNA encoding a novel calmodulin‐like protein (CaLP) with a long C‐terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca2+‐binding domains, is highly heat‐stable and has a potentially high affinity for calcium. CaLP also displays typical Ca2+‐dependent electrophoretic shift, Ca2+‐binding activity and significant Ca2+‐induced conformational changes. Ca2+‐dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.
A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells
Authors: Mitsui Shinichi , Okui Akira , Kominami Katsuya , Konishi Eiichi , Uemura Hidetoshi , Yamaguchi Nozomi
Published: September 2005

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, γ‐tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT‐PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)‐His‐Glu‐Lys‐methylcoumaryl amidide (MCA) and t‐butyloxycarbonyl (Boc)‐Gln‐Ala‐Arg‐MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.
Revisiting the 13C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence
Authors: Kleijn Roelco J. , van Winden Wouter A. , van Gulik Walter M. , Heijnen Joseph J.
Published: September 2005

The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping‐pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping‐pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C2) and dihydroxyacetone moiety (C3) fragments producing and consuming half‐reactions. It is shown that a stoichiometric model based upon these half‐reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C2 and C3 fragment pools in the pentose phosphate pathway and can lead to different label distributions for 13C‐tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published 13C‐based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several micro‐organisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the 13C‐label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the half‐reaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.
Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells
Authors: Vago Riccardo , Marsden Catherine J. , Lord J. Michael , Ippoliti Rodolfo , Flavell David J. , Flavell Sopsamorn‐U , Ceriotti Aldo , Fabbrini M. Serena
Published: September 2005

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER‐associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL‐like C‐terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome‐inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome‐inactivating protein that can enter cells by receptor‐mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi‐independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin‐based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi‐mediated retrograde transport to reach their retrotranslocation site.
Wound conditioning of a deep tissue defect including exposed bone after tumour excision using PROMOGRAN* Matrix, a protease‐modulating matrix
Authors: Tausche Anne‐Kathrin , Sebastian Günther
Published: September 2005

A case study reporting on the successful treatment of a patient affected by a basal cell carcinoma is submitted. Because the carcinoma had infiltrated deeply, a wide excision was necessary, including the removal of bone tissue. The deep tissue defect was treated with PROMOGRAN* Matrix, a protease‐modulating matrix, to promote granulation and ensure that the skin graft do survive and heal successfully. In this case study, a rapid development of granulation tissue on the exposed surface of the bone was observed. The benefits of the dressing enabled a successful split‐thickness skin grafting to be carried out which gave very good aesthetic and functional results.
A critical review of irrigation techniques in acute wounds
Authors: S Chatterjee Justin
Published: September 2005

Irrigation is a fundamental concept to the care of acute wounds and therefore of prime importance in all surgical specialties. A significant amount of research has been done in search of the most effective irrigation technique over the last three decades. There is evidence to show that irrigation is beneficial in the management of acute traumatic and elective wounds, but exactly which techniques and irrigation pressures needed for optimal outcome are still undetermined. A significant number of the studies to date lack the rigid scientific design needed in a climate of evidence‐based medicine. There are substantial methodological flaws and a lack of standardisation between many studies hindering the ability to draw scientific conclusion and compare different studies. There are few randomised, controlled studies on human wounds, and extrapolation from animal studies must be done with caution. Investigation into different irrigation pressures for different levels of contamination is lacking. This would possibly produce a more logical approach for clinically managing acute wounds rather than a blanket treatment for all acute wounds irrespective of the level of contamination. Further research in this direction with greater attention to scientific methodology and design is needed.
A liquid film‐forming acrylate for peri‐wound protection: a systematic review and meta‐analysis (3M™ Cavilon™ no‐sting barrier film)
Authors: Schuren Jan , Becker Anja , Gary Sibbald R
Published: September 2005

To undertake a systematic review of all reliable evaluations of the clinical performance and cost‐effectiveness of a film‐forming liquid acrylate in the protection of the chronic ulcer peri‐wound skin. Results from searches were scrutinised by two reviewers to identify possible randomised controlled trials and full reports of these were obtained. Details of eligible studies were extracted and summarised independently by two reviewers using a data extraction sheet. Meta‐analysis was used to combine the results of trials where the interventions and outcome measures were sufficiently similar. A total of nine eligible studies were identified. The review reveals that a liquid‐film forming acrylate (Cavilon no‐sting barrier film, 3M, Minneapolis, MN, USA) is a safe and effective skin barrier to protect the peri‐wound area of chronic ulcers. There is no difference between the protective properties of different barrier methods that are used to protect the peri‐wound skin around chronic ulcers. Compared with no treatment or a placebo, this liquid film‐forming acrylate has a significant impact on the integrity of the peri‐wound skin. In addition, it has significant benefits in terms of pain control and patient comfort, and its use may reduce nursing time.
Brachytherapy source characterization for improved dose calculations using primary and scatter dose separation
Authors: Russell Kellie R. , Carlsson Tedgren Åsa K. , Ahnesjö Anders
Published: September 2005

In brachytherapy, tissue heterogeneities, source shielding, and finite patient/phantom extensions affect both the primary and scatter dose distributions. The primary dose is, due to the short range of secondary electrons, dependent only on the distribution of material located on the ray line between the source and dose deposition site. The scatter dose depends on both the direct irradiation pattern and the distribution of material in a large volume surrounding the point of interest, i.e., a much larger volume must be included in calculations to integrate many small dose contributions. It is therefore of interest to consider different methods for the primary and the scatter dose calculation to improve calculation accuracy with limited computer resources. The algorithms in present clinical use ignore these effects causing systematic dose errors in brachytherapy treatment planning. In this work we review a primary and scatter dose separation formalism (PSS) for brachytherapy source characterization to support separate calculation of the primary and scatter dose contributions. We show how the resulting source characterization data can be used to drive more accurate dose calculations using collapsed cone superposition for scatter dose calculations. Two types of source characterization data paths are used: a direct Monte Carlo simulation in water phantoms with subsequent parameterization of the results, and an alternative data path built on processing of AAPM TG43 formatted data to provide similar parameter sets. The latter path is motivated of the large amounts of data already existing in the TG43 format. We demonstrate the PSS methods using both data paths for a clinical source. Results are shown for two geometries: a finite but homogeneous water phantom, and a half‐slab consisting of water and air. The dose distributions are compared to results from full Monte Carlo simulations and we show significant improvement in scatter dose calculations when the collapsed‐cone kernel‐superposition algorithm is used compared to traditional table based calculations. The PSS source characterization method uses exponential fit functions derived from one‐dimensional transport theory to describe both the primary and scatter dose contributions. We present data for the PSS characterization method to different , , and brachytherapy sources. We also show how TG43 formatted data can be derived from our data to serve traditional treatment planning systems, as to enable for a gradual transfer to algorithms that provides improved modeling of heterogeneities in brachytherapy treatment planning.
A technique for on‐board CT reconstruction using both kilovoltage and megavoltage beam projections for 3D treatment verification
Authors: Yin Fang‐Fang , Guan Huaiqun , Lu Wenkai
Published: September 2005

The technologies with kilovoltage (kV) and megavoltage (MV) imaging in the treatment room are now available for image‐guided radiation therapy to improve patient setup and target localization accuracy. However, development of strategies to efficiently and effectively implement these technologies for patient treatment remains challenging. This study proposed an aggregated technique for on‐board CT reconstruction using combination of kV and MV beam projections to improve the data acquisition efficiency and image quality. These projections were acquired in the treatment room at the patient treatment position with a new kV imaging device installed on the accelerator gantry, orthogonal to the existing MV portal imaging device. The projection images for a head phantom and a contrast phantom were acquired using both the On‐Board Imager™ kV imaging device and the MV portal imager mounted orthogonally on the gantry of a Varian Clinac™ 21EX linear accelerator. MV projections were converted into kV information prior to the aggregated CT reconstruction. The multilevel scheme algebraic‐reconstruction technique was used to reconstruct CT images involving either full, truncated, or a combination of both full and truncated projections. An adaptive reconstruction method was also applied, based on the limited numbers of kV projections and truncated MV projections, to enhance the anatomical information around the treatment volume and to minimize the radiation dose. The effects of the total number of projections, the combination of kV and MV projections, and the beam truncation of MV projections on the details of reconstructed kV/MV CT images were also investigated.