bluuebunny/crossref_metadata_2025 huggingface dataset:
Parkin is recruited selectively to impaired mitochondria and promotes their autophagy
Authors: Narendra Derek , Tanaka Atsushi , Suen Der-Fen , Youle Richard J.
Published: December 2008

Loss-of-function mutations in Park2, the gene coding for the ubiquitin ligase Parkin, are a significant cause of early onset Parkinson's disease. Although the role of Parkin in neuron maintenance is unknown, recent work has linked Parkin to the regulation of mitochondria. Its loss is associated with swollen mitochondria and muscle degeneration in Drosophila melanogaster, as well as mitochondrial dysfunction and increased susceptibility to mitochondrial toxins in other species. Here, we show that Parkin is selectively recruited to dysfunctional mitochondria with low membrane potential in mammalian cells. After recruitment, Parkin mediates the engulfment of mitochondria by autophagosomes and the selective elimination of impaired mitochondria. These results show that Parkin promotes autophagy of damaged mitochondria and implicate a failure to eliminate dysfunctional mitochondria in the pathogenesis of Parkinson's disease.
Active Tuberculosis in a Healthcare Worker: Are You Ready?
Authors: Fraser Thomas G. , Kowalczyk Joan , Schmitt Steven , Bernstein Jill , Terpeluk Paul , Tomford J. Walton , Gordon Steven M.
Published: December 2008

We describe a healthcare worker with a previously positive tuberculin skin test result who developed active tuberculosis. An investigation revealed 280 potential contacts, 3 of whom had positive tuberculin skin test results. Our experience demonstrates the potential benefits of therapy for latent tuberculosis infection as a component of a tuberculosis-control program.
Personal Healthcare Worker (HCW) and Work-Site Characteristics That Affect HCWs' Use of Respiratory-Infection Control Measures in Ambulatory Healthcare Settings
Authors: Turnberg Wayne , Daniell William , Simpson Terri , Van Buren Jude , Seixas Noah , Lipkin Edward , Duchin Jeffery
Published: December 2008

Objectives.

To identify healthcare worker (HCW) and work-site characteristics associated with HCWs' reported use of recommended respiratory-infection control practices in primary and emergency care settings.

Design.

A cross-sectional study using a self-administered questionnaire for HCWs during the summer and fall of 2005.

Setting.

Primary and emergency care clinics at 5 medical centers in King County, Seattle, Washington.

Results.

Nurse professionals who reported receiving training (odds ratio [OR], 2.5 [confidence interval {CI}, 1.1–5.9]; P = .029), instructional feedback from supervisors (OR, 3.0 [CI, 1.5–5.9]; P = .002), and management support for implementing safe work practices had a higher odds of also reporting adherence to recommended respiratory precautions, compared with nurses who did not. Training was the only important determinant for adherence to respiratory precaution measures among medical practitioners (OR, 5.5 [CI, 1.2–25.8]; P = .031). The reported rate of adherence to hand hygiene practices was higher among nurse professionals who were male (OR, 2.2 [CI, 1.0–4.9]; P = .045), had infants, small children, or older adults living at home (OR, 2.2 [CI, 1.2–3.9]; P = .007), reported cleanliness and orderliness of the establishment where they worked (OR, 2.0 [CI, 1.1–3.5]; P = .019), had received respiratory-infection control training (OR, 3.2 [CI, 1.8–6.0]; P<.001), and reported fears about catching a dangerous respiratory infection at work (OR, 2.3 [CI, 1.2–4.5]; P= .011).

Conclusion.

A number of HCW and work-site characteristics associated with HCWs' use of recommended respiratory-infection control measures have been identified. These potentially influential characteristics should be considered as targets or guides for further investigation, which should include the evaluation of intervention strategies.
Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication
Authors: Yi Xia , de Vries Hilda I. , Siudeja Katarzyna , Rana Anil , Lemstra Willy , Brunsting Jeanette F. , Kok Rob M. , Smulders Yvo M. , Schaefer Matthias , Dijk Freark , Shang Yongfeng , Eggen Bart J.L. , Kampinga Harm H. , Sibon Ody C.M.
Published: December 2008

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.
The Extracellular Domain of Lrp5/6 Inhibits Noncanonical Wnt Signaling In Vivo
Authors: Bryja Vitezslav , Andersson Emma R. , Schambony Alexandra , Esner Milan , Bryjová Lenka , Biris Kristin K. , Hall Anita C. , Kraft Bianca , Cajanek Lukas , Yamaguchi Terry P. , Buckingham Margaret , Arenas Ernest
Published: December 2008

Lrp5/6 are crucial coreceptors for Wnt/β-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/β-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6−/− mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.
Slow Growth Induces Heat-Shock Resistance in Normal and Respiratory-deficient Yeast
Authors: Lu Charles , Brauer Matthew J. , Botstein David
Published: December 2008

Yeast cells respond to a variety of environmental stresses, including heat shock and growth limitation. There is considerable overlap in these responses both from the point of view of gene expression patterns and cross-protection for survival. We performed experiments in which cells growing at different steady-state growth rates in chemostats were subjected to a short heat pulse. Gene expression patterns allowed us to partition genes whose expression responds to heat shock into subsets of genes that also respond to slow growth rate and those that do not. We found also that the degree of induction and repression of genes that respond to stress is generally weaker in respiratory deficient mutants, suggesting a role for increased respiratory activity in the apparent stress response to slow growth. Consistent with our gene expression results in wild-type cells, we found that cells growing more slowly are cross-protected for heat shock, i.e., better able to survive a lethal heat challenge. Surprisingly, however, we found no difference in cross-protection between respiratory-deficient and wild-type cells, suggesting induction of heat resistance at low growth rates is independent of respiratory activity, even though many of the changes in gene expression are not.
Reference alignment of SNP microarray signals for copy number analysis of tumors
Authors: Pounds Stan , Cheng Cheng , Mullighan Charles , Raimondi Susana C. , Shurtleff Sheila , Downing James R.
Published: December 2008

A new procedure to align single nucleotide polymorphism (SNP) microarray signals for copy number analysis is proposed. For each individual array, this reference alignment procedure (RAP) uses a set of selected markers as internal references to direct the signal alignment. RAP aligns the signals so that each array has a similar signal distribution among its reference markers. An accompanying reference selection algorithm (RSA) uses genotype calls and initial signal intensities to choose two-copy markers as the internal references for each array. After RSA and RAP are applied, each array has a similar distribution of signals of two-copy markers so that across-array signal comparisons are biologically meaningful. An upper bound for a statistical metric of signal misalignment is derived and provides a theoretical basis to choose RSA-RAP over other alignment procedures for copy number analysis of cancers. In our study of acute lymphoblastic leukemia, RSA-RAP gives copy number analysis results that show substantially better concordance with cytogenetics than do two other alignment procedures.

Availability: Documented R code is freely available from www.stjuderesearch.org/depts/biostats/refnorm.

Contact:  stanley.pounds@stjude.org

Supplementary information:  Supplementary data are available at Bioinformatics online.
Australia’s deep-water reserve network: implications of false homogeneity for classifying abiotic surrogates of biodiversity
Authors: Williams Alan , Bax Nicholas J. , Kloser Rudy J. , Althaus Franziska , Barker Bruce , Keith Gordon
Published: December 2008

Williams, A., Bax, N. J., Kloser, R. J., Althaus, F., Barker, B., and Keith G. 2009. Australia’s deep-water reserve network: implications of false homogeneity for classifying abiotic surrogates of biodiversity. – ICES Journal of Marine Science, 66: 214–224. Australia’s southeast network of deep-water marine reserves, declared in July 2007, was designed using a hierarchy that represented the distribution of marine biodiversity as a nested set of bioregions. In this hierarchy, geomorphic units, individual or aggregations of seabed geomorphic features, are the finest scale used in the design process. We evaluated the interaction between two hierarchical levels (depth and geomorphic features), using video survey data on seamounts and submarine canyons. False within-class homogeneity indicated that depth, size, complexity, configuration, and anthropogenic impact need to be added as modifiers to allow geomorphic features to act as surrogates for biodiversity distribution. A consequence of using unmodified geomorphic surrogates, and of not correctly nesting geomorphic features within depth, is the diminished recognition of the importance and comparative rarity of megafaunal biodiversity of the continental margin (<1500-m depths). We call this area the zone of importance, because it is where targeted marine impacts coincide with the greatest megafaunal biodiversity. Refining the geomorphic classification is desirable for future biodiversity characterization, but an alternative approach is to define patterns in biodiversity and abiotic variables jointly, and to utilize finer scale information and provide a classification that preserves the maximum information of both datasets.
Host Range, Purification, and Genetic Variability in Sweet potato chlorotic fleck virus
Authors: Aritua V. , Barg E. , Adipala E. , Gibson R. W. , Lesemann D. E. , Vetten H. J.
Published: December 2008

Sweet potato chlorotic fleck virus (SPCFV) has recently been classified as a putative new member of the genus Carlavirus (family Flexiviridae) on the basis of its molecular properties. In this study, SPCFV was characterized in terms of host range, physical and biological characteristics, and genetic variability. In addition to sweet potato, SPCFV infected some plant species in the families Convolvulaceae, Chenopodiaceae, and Solanaceae. Limited numbers of virus particles were observed in the assimilation parenchyma cells of infected plant tissues; some cells had a distorted and enlarged endoplasmic reticulum though without any cytoplasmic and amorphous inclusions. The normal length of SPCFV particles was determined to be approximately 800 nm. In enzyme-linked immunosorbent assays, polyclonal antibodies raised against purified SPCFV virions were able to detect the virus in infected sweet potato and indicator plant tissues. In immunoelectron microscopy, SPCFV particles were all strongly decorated when reacted with homologous antiserum. Comparison of the 3′ terminal part of the genome of a range of geographically diverse isolates revealed a high level of genetic diversity. The amino acid sequence identity in the coat protein and the nucleic acid binding protein ranged from 89 to 99.7% and from 75.9 to 99.2%, respectively. Phylogenetic analysis of both proteins showed a geographically associated clustering into two genogroups.
The PDGF signaling pathway controls multiple steroid-producing lineages
Authors: Schmahl Jennifer , Rizzolo Kamran , Soriano Philippe
Published: December 2008

The platelet-derived growth factor (PDGF) signaling pathway regulates numerous lineages of mesenchymal cell origin during development and in the adult. The transcriptional targets of this pathway have been shown to be required in several PDGF-dependent processes, but the roles of these targets in specific tissues is just beginning to be identified. In this study, we show that five different PDGF target genes are essential for male and/or female fertility. Mutations in each of these five different genes lead to defects in the steroid-producing cells in the testis and/or ovary and altered hormone production, suggesting that the PDGF pathway controls steroidogenesis through these genes in both sexes. Furthermore, conditional mutations of both PDGF receptors revealed a requirement in steroid-producing cells in multiple organs, including the testis, ovary, and adrenal cortex. Therefore, PDGF signaling may constitute a common mechanism in the control of multiple steroidogenic lineages.
Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines
Authors: Link Andrew J. , LaBaer Joshua
Published: December 2008

INTRODUCTION

The Nucleic Acid Programmable Protein Array (NAPPA) approach for producing protein microarrays uses cell-free extracts to transcribe and translate cDNAs encoding target proteins directly onto glass slides. Following array preparation, the identification of protein interactions using NAPPA can be accomplished by either of two general schemas. The first is to probe an expressed NAPPA slide with a purified protein of interest (the query protein) and look for interactors. Signals of these interactions can be detected either by directly labeling the query protein or by using a labeled antibody either to the protein itself or to a tag on the protein. This approach works well when there is access to the purified protein, and it has the advantage that users can test query protein binding with and without post-translational modifications or under a variety of binding conditions. The second schema entails coexpressing the query protein on the NAPPA slide at the same time that all the target proteins are expressed. This article describes the advantages of using a coexpressed query protein and provides advice on choosing a suitable epitope tag.
Whole-Mount In Situ Hybridization in Amphimedon
Authors: Larroux Claire , Fahey Bryony , Adamska Maja , Richards Gemma S. , Gauthier Marie , Green Kathryn , Lovas Erica , Degnan Bernard M.
Published: December 2008

INTRODUCTION

Developmental gene expression is analyzed predominantly via whole-mount in situ hybridization using digoxigenin-labeled RNA probes. This protocol describes how to perform this procedure in Amphimedon queenslandica, including fixation, hybridization, and sectioning of embryonic, larval, and post-larval juvenile stages.
Cultures of Cerebellar Granule Neurons
Authors: Bilimoria Parizad M. , Bonni Azad
Published: December 2008

INTRODUCTION

Primary cultures of granule neurons from the post-natal rat cerebellum provide an excellent model system for molecular and cell biological studies of neuronal development and function. The cerebellar cortex, with its highly organized structure and few neuronal subtypes, offers a well-characterized neural circuitry. Many fundamental insights into the processes of neuronal apoptosis, migration, and differentiation in the mammalian central nervous system have come from investigating granule neurons in vitro. Granule neurons are the most abundant type of neurons in the brain. In addition to the sheer volume of granule neurons, the homogeneity of the population and the fact that they can be transfected with ease render them ideal for elucidating the molecular basis of neuronal development. This protocol for isolating granule neurons from post-natal rats is relatively straightforward and quick, making use of standard enzymatic and mechanical dissociation methods. In a serum-based medium containing an inhibitor of mitosis, cerebellar granule neurons can be maintained with high purity. Axons and dendrites can be clearly distinguished on the basis of morphology and markers. For even greater versatility, this protocol for culturing granule neurons can be combined with knockout or transgenic technologies, or used in cerebellar slice overlay assays.
Chicken Erythrocyte Histone Octamer Preparation
Authors: Peterson Craig L. , Hansen Jeffrey C.
Published: December 2008

INTRODUCTION

Core histones can be purified from a variety of cell sources, including Drosophila embryos, HeLa tissue culture cells, calf thymus, or chicken erythrocytes. Chick erythrocytes are an excellent source of cellular histones: Large quantities of source material are readily obtainable, the purified histones have low levels of post-translational modifications, and linker histones can also be purified from the same cell sample. Also, avian histones have an amino acid sequence identical to that of human histones. Histone stocks can be stored successfully for more than a year at 4°C and for several years at −20°C. With this protocol, 200 mL of blood usually yields in excess of 50 mg of purified histone octamers. Additional optional procedures are also presented for the purification of H1 and H5 linker histones, as well as for the preparation of H3/H4 tetramers and H2A/H2B dimers.
Analysis of theNicotiana tabacumStigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species
Authors: Quiapim Andréa C. , Brito Michael S. , Bernardes Luciano A.S. , daSilva Idalete , Malavazi Iran , DePaoli Henrique C. , Molfetta-Machado Jeanne B. , Giuliatti Silvana , Goldman Gustavo H. , Goldman Maria Helena S.
Published: December 2008

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.
The t(X;7)(q22;q34) in paediatric T‐cell acute lymphoblastic leukaemia results in overexpression of the insulin receptor substrate 4 gene through illegitimate recombination with the T‐cell receptor beta locus
Authors: Karrman Kristina , Kjeldsen Eigil , Lassen Carin , Isaksson Margareth , Davidsson Josef , Andersson Anna , Hasle Henrik , Fioretos Thoas , Johansson Bertil
Published: December 2008

The t(X;7)(q22;q34), a translocation not previously reported in a neoplastic disorder, was identified and molecularly characterised in a paediatric T‐cell acute lymphoblastic leukaemia (T‐ALL), subsequently shown also to harbour a deletion of 6q, a STIL/TAL1 fusion and an activating NOTCH1 mutation. The t(X;7) was further investigated using fluorescence in situ hybridisation (FISH), real‐time quantitative polymerase chain reaction (RQ‐PCR) and Western blot analyses. FISH revealed a breakpoint at the T‐cell receptor beta locus at 7q34 and mapped the corresponding breakpoint to Xq22.3. The latter region contains only two known genes, namely insulin receptor substrate 4 (IRS4) and collagen, type IV, alpha 5 (COL4A5), the expressions of which were analysed by the use of RQ‐PCR. COL4A5 was not differentially expressed in the t(X;7)‐positive sample compared to five T‐ALL controls. However, a marked, 1000‐fold overexpression of IRS4 was identified. Western blot analysis with a monoclonal antibody against IRS4 showed overexpression also at the protein level. Considering that forced expression of several members of the IRS family has been shown to result in increased cell proliferation, for example in haematopoietic cells, we hypothesise that the IRS4 up‐regulation in T‐ALL is pathogenetically important as a mitogenic stimulus.
Epstein‐Barr virus and cytomegalovirus are differentially associated with numbers of cytokine‐producing cells and early atopy
Authors: Nilsson C. , Larsson Sigfrinius A.‐K. , Montgomery S. M. , Sverremark‐Ekström E. , Linde A. , Lilja G. , Blomberg M. T.
Published: December 2008

Background
We have previously shown that Epstein‐Barr virus (EBV) seropositivity, at 2 years of age, was inversely related to IgE‐sensitization and that this effect was enhanced when EBV is combined with cytomegalovirus (CMV) seropositivity. We hypothesize that early exposure to EBV or CMV will affect the cytokine balance in the individual.

Objective
The aim of this study was to relate the cytokine profile in peripheral blood mononuclear cells (PBMC) to the EBV and CMV serostatus and IgE‐sensitization in children at 2 years of age.

Methods
Seventy‐five children were followed prospectively from birth until 2 years of age. Their EBV and CMV serostatus was correlated to the numbers of IFN‐γ, IL‐4, IL‐10 and IL‐12‐producing PBMC following PHA stimulation in vitro. Skin prick tests and allergen‐specific IgE antibodies were used to assess IgE‐sensitization.

Results
In the study cohort, there was an inverse association between EBV seropositivity and IgE‐sensitization but not with CMV seropositivity. Following linear regression analysis, we did not detect any statistically significant associations between children with IgG antibodies against EBV at 2 years of age and the investigated cytokines. However, there was a non‐significant tendency to a positive association between high numbers of all individual cytokine‐producing cells and EBV seropositivity. Children who were CMV seropositive had significantly higher numbers of IFN‐γ and lower numbers of IL‐4‐producing cells compared with CMV negative children. There was a significant, positive association between the number of IL‐4‐producing cells and IgE‐sensitization.

Conclusion
Taken together our results indicate that infections with EBV and CMV in different ways will interact with the immune system and may protect children from developing early atopy.
CD26/dipeptidyl peptidase 4-deficiency alters thymic emigration patterns and leukcocyte subsets in F344-rats age-dependently
Authors: Klemann C , Schade J , Pabst R , Leitner S , Stiller J , Von Hörsten S , Stephan M
Published: December 2008

As CD26 (dipeptidyl peptidase 4/DPP4) rapidly truncates incretins N-terminally, including glucagon-like peptide-1, DPP4-inhibitors have been developed for treatment of diabetes type 2. To some extent this is surprising, as CD26/DPP4 is also deeply involved in immune regulation. Long-term pharmacological studies are hampered by off-target inhibition of DPP4-homologues. Therefore, we studied the effects of genetic CD26/DPP4-deficiency by investigating blood, spleen and thymus leucocyte subpopulations of wild-type and CD26-deficient F344-rats at different ages. In young animals at 1 and 3 months of age, there were no differences in leucocyte subsets, while in older animals the T cell composition was changed significantly. From the age of 6 months onwards, reduced numbers of recent thymic emigrants and memory T cells, and consequently an increased amount of naive T cells were observed in CD26-deficient rats. In addition, the architecture of the thymus was altered, as observed by a reduced density of lymphocytes in the medulla. Furthermore, the number of proliferating cells in the thymus was decreased in CD26-deficient rats at a higher age. Moreover, CD26-deficiency resulted in markedly reduced numbers of B cells in later life. Additionally, an age- but not CD26-dependent increase of regulatory T cells and a decrease of natural killer cell numbers were detected in the blood and spleen. Our findings indicate an important role of CD26 in maintaining lymphocyte composition, memory T cell generation and thymic emigration patterns during immunosenescence, with possible implications for using DPP4-inhibitors.
FAMILIAL BENIGN HYPERCALCAEMIA: HYPERCALCIURIA AND HYPOCALCIURIA IN AFFECTED MEMBERS OF A SMALL KINDRED
Authors: PASIEKA JANICE L. , ANDERSEN MEL A. , HANLEY DAVID A.
Published: December 2008

Familial benign hypercalcaemia has also been termed familial hypocalciuric hypercalcaemia because a major feature of this condition has been a relative hypocalciuria in relation to the hypercalcaemia, such that the calcium to creatinine clearance ratio is less than 0.016. The following is a report of a small kindred of patients with familial benign hypercalcaemia in which two of the affected members have frank hypercalciuria.
FACTORS INFLUENCING URINARY 6‐SULPHATOXYMELATONIN, A MAJOR MELATONIN METABOLITE, IN NORMAL HUMAN SUBJECTS
Authors: BOJKOWSKI C. J. , ARENDT J.
Published: December 2008

6‐Sulphatoxymelatonin (aMT6) has been measured, by a direct radioimmunoassay, in urine from 130 normal volunteers aged 2–80 years. Its relationship to a number of physiological parameters has been assessed. Total urinary excretion of aMT6s did not vary in a group of 40 children aged 2–20 years (24 boys and 16 girls) except when expressed as a function of body weight. In this case, total aMT6s excretion over 24 h decreased as a function of age. In 90 adult volunteers (44 men and 46 women) aged 20–80 years, there was an age‐related decline in total 24 h aMT6s excretion with significantly lower values in elderly subjects. In this same adult group no relationships were found between total aMT6s excretion and body weight or height. No sex differences were found either in the 2–20 years or the 20–80 years groups. Pineal calcification was assessed by lateral skull X‐ray in 26 adult volunteers (17 men and 9 women) aged 20–50 years. No significant differences in aMT6s excretion were found as a function of pineal calcification. In 16 of these subjects plasma melatonin and aMT6s also showed no relationship to pineal calcification. These studies confirm the usefulness of aMT6s as an index of melatonin secretion in normal volunteers.
INSULIN SECRETION, INSULIN SENSITIVITY AND GLUCOSE‐MEDIATED GLUCOSE DISPOSAL IN THYROTOXICOSIS: A MINIMAL MODEL ANALYSIS
Authors: PESTELL RICHARD , ALFORD FRANK , RAMOS ROMULUS , SAWYER STEVEN , BEST JAMES , WARD GLENN
Published: December 2008

In order to evaluate simultaneously in thyrotoxic subjects the relative contributions of insulin secretion, insulin‐sensitivity (SI) and glucose‐mediated (SG) glucose disposal to overall glucose tolerance, seven non‐obese patients with thyrotoxicosis were studied by the minimal model analysis of the frequently sampled intravenous glucose tolerance test, before and > 1 month after being rendered euthyroid, and compared with eight healthy control subjects. Basal glucose, C‐peptide and glucagon levels were similar in all groups but, in the toxic and euthyroid states, basal insulin levels were significantly elevated compared to the control group (11.2 ±2‐0 and 7.9± 1‐1 vs 5.1 ±0.6 μU/ml, mean±SE, P<0.02). FFA levels were raised in the thyrotoxic subjects prior to treatment (0.95 ±0.11 vs 0.68 ± 0.08 and 054 ± 008 mmol/1, P <0.0). Glucose tolerance (Kg) was reduced in the thyrotoxic subjects compared to the euthyroid state (1.16 ± 0.12 vs 1.44 ± 0.13 per min, P <0.025) and control group (1.44± 1.0 per min, 0.05 <P<0.1). First phase (ψ1) and second phase (ψ2) insulin release were both significantly elevated in the thyrotoxic and euthyroid states compared to the control group (ψ1), 7.10± 1.88 and 5.29± 1.03 vs 1.72 ± 0.17 μU/mg/minx 10‐2 P<0.01; ψ2 18.64 ± 3.14 and 16.74 ± 4.48 vs 9.23 ± 0.74 μU/mg/minx 10‐2 respectively, P<0.02). SG was similar in all groups but SI was significantly reduced in the thyrotoxic subjects compared to the control group (2.24 ± 0.62 vs 5.92 ± 1.50/minμU/ml × 104, P<0.02) and rose post‐treatment in the euthyroid subjects (4.23 ± 1.75/minμU/ml × 104). In the thyrotoxic subjects before and after treatment, log SI correlated negatively with basal FFA levels (r=‐0.57, P<0.05) and with ψ2 (r= ‐0.58, P<0.05). The fractional clearance rate of insulin was unaltered by the thyrotoxic state. It is concluded that in thyrotoxicosis the impairment of Kg is due to reduced insulin sensitivity in the presences of enchanced insulin secretion, but glucose‐medicated glucose disposal is unaltered by the toxic state.
THE PREVALENCE OF POLYCYSTIC OVARIES IN PATIENTS WITH CONGENITAL ADRENAL HYPERPLASIA AND THEIR CLOSE RELATIVES
Authors: HAGUE WILLIAM M. , ADAMS JUDITH , RODDA CHRISTINE , BROOK CHARLES G. D. , DE BRUYN ROSE , GRANT DAVID B. , JACOBS HOWARD S.
Published: December 2008

Seventy‐seven female patients (36 adults and 41 children aged under 16 years) with congenital adrenal hyperplasia (CAH) were assessed using pelvic ultrasound, as well as with standard endocrine tests and HLA typing. Forty‐six close female relatives were also tested for ovarian morphology using ultrasound after assessment of their heterozygous state using HLA typing. The association of CAH with ultrasonically detected polycystic ovaries (PCO) was confirmed in 30/36(83%) adult patients, 4/10 (40%) postpubertal girls and 1/31 (3%) pre and peripubertal girls: in all, 35/46 (76%) postmenarcheal patients. Six out of nine (67%>) pre‐menopausal mothers of patients with PCO, and 8/10 (80%) sisters of patients with PCO also had PCO. The proportions of both CAH patients and heterozygote subjects with PCO were significantly greater than that found in a normal population (P<00.001). The finding, however, of two homozygous non‐CAH‐affected adult sisters with PCO and, conversely, of 10 heterozygous adult relatives and of 12 postmenarcheal CAH patients with normal ovaries, indicates that the ovarian morphological change may be independent of the adrenal lesion.
DECREASED HDL CHOLESTEROL IN SUBCLINICAL HYPOTHYROIDISM: THE EFFECT OF L‐THYROXINE THERAPY
Authors: CARON Ph. , CALAZEL C. , PARRA H. J. , HOFF M. , LOUVET J. P.
Published: December 2008

Thyroid function and lipid tests were measured in 29 premenopausal women with subclinical hypothyroidism. This group was compared with 41 euthyroid women matched for age and metabolic parameters. In basal condition there was no difference in thyroid hormone levels between the two groups except for TSH concentration (P<0.01). Total cholesterol, triglycerides and apolipoprotein (apo A1, A2, B) of women with subclinical hypothyroidism were not different from controls. HDL cholesterol was significantly decreased in subclinical hypothyroidism compared to the controls (P<0.01). With thyroxine therapy, normalization of serum TSH was associated with (1) no significant change in total cholesterol and triglycerides, (2) an increase of HDL cholesterol (P<0.01) and apoprotein Al (P<0.05) levels. Total cholesterol/HDL cholesterol ratio was increased in subclinical hypothyroidism (P<0.01). During L‐thyroxine therapy this ratio returned to normal value. Decreased HDL cholesterol concentration might cause coronary heart disease reported in women with subclinical hypothyroidism.
CIRCULATING BIOACTIVE FOLLICLE STIMULATING HORMONE AND IMMUNOREACTIVE INHIBIN LEVELS DURING THE NORMAL HUMAN MENSTRUAL CYCLE
Authors: REDDI K. , WICKINGS E. J. , McNEILLY A. S. , BAIRD D. T. , HILLIER S. G.
Published: December 2008

To examine the relationship between circulating levels of bioactive FSH (B‐FSH) and immunoactive inhibin and oestradiol we studied five women during ovulatory cycles. Daily blood samples were collected from each subject during one menstrual cycle. B‐FSH was measured using a modified, highly sensitive in‐vitro rat granulosa cell bioassay. The inclusion of IGF‐1 (10 μg/1) and transferrin (50 mg/I) in the assay system enhanced granulosa ceil responsiveness to FSH and resulted in increased assay sensitivity. Inhibin was measured by a heterologous radioimmunoassay (RIA) using an antibody raised against 31 kDa bovine inhibin. Bioactive FSH (B‐FSH) levels were closely correlated to those of immunoactive FSH (I‐FSH, r = 0.79, P<0001) throughout the cycle. Peak levels of B‐FSH were observed during the early follicular phase (day –13, 44.7 ± 9.6 IU/1, mean ± SEM) and during the midcycle surge (35.2 ± 6.2 IU/1); lowest levels occurring during the luteal phase (nadir 3.9 ± 0.27 IU/1). Plasma oestradiol levels increased significantly during the follicular phase (P<0.001) to a peak on day – 1 and were negatively correlated with B‐FSH during the late follicular phase (day –8 to – 1; r= –0.45. P<002). There was no change in the concentration of inhibin (range 55.3–72.3 U/1) during the follicular phase until day –2 after which an increase to a midcycle peak of 139± 10.6 U/1 was observed. No correlation was observed between inhibin and B‐FSH during the follicular phase. A second increase in the concentration of inhibin was seen during the luteal phase; peak levels occurred by day 6 (311 ±25.8 U/1), remained elevated until day 12, and were negatively correlated with B‐FSH (r=–0.53, P<0001). No correlation was observed between oestradiol and inhibin or B‐FSH during the luteal phase. We conclude that (1)
Egg size affects larval performance in a coleopteran parasitoid
Authors: BOIVIN GUY , GAUVIN MARIE‐JOSÉE
Published: December 2008

Abstract 1. Optimal progeny size models assume that the more eggs a female produces, the lower the amount of resource allocated per egg. As egg size generally correlates with the fitness of the emerging immature, this trade‐off can be expressed as a choice between the production of numerous low quality or fewer high quality progeny.

2. The first‐instar larvae of the coleopteran parasitoid Aleochara bilineata have to search for and parasitise dipteran pupae. The present study found a positive correlation between egg size and larval weight, but not between egg size and development time. Larger first‐instar larvae survived longer, were more active, and found and parasitised their host more rapidly.

3. Female A. bilineata may invest smaller larvae in conditions of high host density and low intraspecific competition, but investing fewer, larger larvae would bring them more fitness when hosts are scarce and competition high.
Accumulation of heterocyclic nitrogen in humified organic matter: a 15N‐NMR study of lowland rice soils
Authors: Mahieu N. , Olk D. C. , Randall E. W.
Published: December 2008

Recent intensification of cropping and the attendant longer submergence of the soil for lowland rice in tropical Asia appear to have altered the nature of the soil organic matter, and perhaps also nutrient cycling. To identify the dominant forms of organic nitrogen in the soils we extracted the labile mobile humic acid (MHA) and the more recalcitrant calcium humate (CaHA) fractions from soils under several long‐term field experiments in the Philippines and analysed them by 15N‐nuclear magnetic resonance spectroscopy. Amide N dominated the spectra of all humic acid (HA) samples (60–80% of total peak area). Its proportion of total spectral area increased with increasing intensity of cropping and length of time during which the soil was flooded and was greater in the MHA fraction than in the CaHA fraction. Simultaneously the spectral proportion of free amino N and other chemical shift regions decreased slightly with increasing length of submergence. Heterocyclic N was detected at modest proportions (7–22%) and was more prevalent in more humified samples, especially in the CaHA of aerated soils. Correlations of spectral proportions of heterocyclic N with other properties of the HA, reported elsewhere, were highly significant. Correlations were positive with visible light absorption (r= 0.86) and concentration of free radicals (r= 0.85), both of which are indices of humification, and negative with concentration of H (r= −0.86), a negative index of humification. Correlations of spectral proportions of amide N with these properties were also highly significant but in each case of opposite sign to that of heterocyclic N. Proportions of heterocyclic N declined with increasing duration of submergence. The results suggest that (i) 15N‐NMR can reproducibly measure some portion of heterocyclic N, (ii) formation of heterocyclic N is associated solely with gradual humification occurring over many years, and (iii) the abundant phenols in the submerged rice soils did not promote formation of heterocyclic N, and hence some other process is responsible for a substantial decrease in the availability of native N associated with intensive rice cropping.
Depth‐related variations in organic matter at the molecular level in a loamy soil: reference data for a long‐term experiment devoted to the carbon sequestration research field
Authors: Celerier J. , Rodier C. , Favetta P. , Lemee L. , Ambles A.
Published: December 2008

Three lipid fractions, namely the freely extractable fraction and those associated with humin and humic acid fractions, were obtained from the loamy soil of a carefully maintained long‐term experiment located on Deffend ORE field, Poitiers, France. The analyses showed differences in molecular distribution, suggesting different sources and diagenetic states of the source material. Despite a major input of plant material to the soil organic matter, intensive bacterial activity was suspected. Most distributions suggested an increase in the microbial/terrestrial lipid ratio from the free to humin to humic fractions. Molecular evidence of fungal activity, especially in the top layer, was also found in the distributions of n‐alkanes and n‐alkanoic acids. In the surface horizon A1 alkanes were the major compounds, followed by n‐alkanoic acids and sterols. The degraded horizons, poorer in organic matter, i.e. the A2 and B horizons, were dominated by long‐chain (>C20) n‐alkanoic acids with a strong even‐over‐odd predominance and C24 and C26n‐alkanols. Sterols had been removed from these horizons through degradation. A comparison of humic acid and humin composition on the basis of Py(methylation)‐GC‐MS showed that the two fractions produce partly similar pyrolysis products. Most prominent were molecules from plant and microbial carbohydrates, lignin building blocks and linear aliphatic (carboxylic acids, esters) and nitrogen compounds. The investigation showed that while low‐molecular‐weight soil lipids were highly dominated by compounds derived from the overlying vegetation, pyrolysis data from the corresponding high‐molecular‐weight fractions reflected the incorporation of microbial biomarkers into the humic‐type fractions.
Mass flowering oilseed rape improves early colony growth but not sexual reproduction of bumblebees
Authors: Westphal C. , Steffan‐Dewenter I. , Tscharntke T.
Published: December 2008

Pollination is a vital ecosystem service, which is endangered by the ongoing declines of pollinators. These declines also affect bumblebees (Bombus spp.), which are important generalist pollinators in agricultural landscapes. Most studies focussing on the conservation of bumblebees have investigated the effects of local flower‐rich habitats on bumblebee density and diversity. However, bumblebee densities do not necessarily correlate with the colonies’ reproductive success (i.e. the presence or absence of males and/or queens).

We analysed the effects of landscape‐wide availability of mass flowering oilseed rape Brassica napus on the growth and sexual reproduction of Bombus terrestris colonies. Thirty‐two young colonies were established and monitored in different resource environments represented by 16 landscapes (circular study areas with 3000 m radius) with large or small amounts of oilseed rape. As an indicator of colony growth, we used weight gain, which was strongly correlated with the numbers of brood cells in the colonies.

The colonies gained significantly more weight in study areas with large amounts of oilseed rape particularly during early colony stages.

Despite early weight gain, the colonies in study areas with large amounts of oilseed rape did not reproduce more successfully. The frequencies of colonies that produced males and/or queens did not differ between the two resource environments.

Synthesis and applications. Early mass flowering oilseed rape has a beneficial effect on colony growth, which however, does not translate into a greater likelihood of colonies producing sexual offspring. This may be due to food plant scarcity later in the colony cycle. Conservation measures should enhance food plant availability in agricultural landscapes, particularly during the most critical phases of the colony cycle: the colony establishment in spring and the reproductive phase in mid‐ to late summer.
Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA
Authors: Xing Guangxin , Kirouac Kevin , Shin Yoon Jung , Bell Stephen D. , Ling Hong
Published: December 2008

DNA polymerases are co‐ordinated by sliding clamps (PCNA/β‐clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full‐length Y‐family polymerase, Dpo4, in complex with heterodimeric PCNA1–PCNA2 at 2.05 Å resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo‐ or DNA‐bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C‐terminal peptide of Dpo4 becomes structured with a 310 helix and dominates the specific binding. The Y‐family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation‐dependent protein–protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding‐coupled multiple conformations of a Y‐family polymerase facilitate its recruitment and co‐ordination on the sliding clamp. The conformational flexibility would turn the error‐prone Y‐family polymerase off when more efficient high‐fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.
Extracellular signalling, translational control, two repressors and an activator all contribute to the regulation of methylenomycin production inStreptomyces coelicolor
Authors: O'Rourke Sean , Wietzorrek Andreas , Fowler Kay , Corre Christophe , Challis Greg L. , Chater Keith F.
Published: December 2008

Bioinformatic analysis of the plasmid‐linked gene cluster associated with biosynthesis of methylenomycin (Mm) suggested that part of the cluster directs synthesis of a gamma‐butyrolactone‐like autoregulator. Autoregulator activity could be extracted from culture fluids, but differed from gamma‐butyrolactones in being alkali resistant. The activity has recently been shown to comprise a series of novel autoregulator molecules, the methylenomycin furans (termed MMF). MMF autoregulator activity is shown to account for the ability of certain Mm non‐producing mutants to act as ‘secretors’ in cosynthesis with other ‘convertor’ mutants. Three genes implicated in MMF biosynthesis are flanked by two regulatory genes, which are related to genes for gamma‐butyrolactone‐binding proteins. Genetic evidence suggests that these two genes encode components of a hetero‐oligomeric repressor of MMF and Mm biosynthesis. The Mm biosynthetic genes themselves depend on the activator genemmyB, which appears to be repressed by the putative MmyR/MmfR complex until enough MMF accumulates to release repression. The presence of TTA codons inmmyBand the main MMF biosynthetic gene causes Mm production to be dependent on the pleiotropically actingbldAgene, which encodes the tRNA for the rarely used UUA codon.
Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis
Authors: Jakubowski Simon J. , Kerr Jennifer E. , Garza Isaac , Krishnamoorthy Vidhya , Bayliss Richard , Waksman Gabriel , Christie Peter J.
Published: December 2008

Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB‐encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N‐terminal cytoplasmic region, (ii) transmembrane (TM) α‐helix, (iii) proline‐rich region (PRR) and (iv) C‐terminal β‐barrel domain. Mutations conferring a transfer‐ and pilus‐minus (Tra‐, Pil‐) phenotype included PRR deletion and β‐barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7–VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra+, Pil‐) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra+ mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of α‐helical projections extending from the β‐barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its β‐barrel domain with the VirB7–VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus.