bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear envelope defects cause stem cell dysfunction in premature-aging mice
Authors: Espada Jesús , Varela Ignacio , Flores Ignacio , Ugalde Alejandro P. , Cadiñanos Juan , Pendás Alberto M. , Stewart Colin L. , Tryggvason Karl , Blasco María A. , Freije José M.P. , López-Otín Carlos
Published: April 2008

Nuclear lamina alterations occur in physiological aging and in premature aging syndromes. Because aging is also associated with abnormal stem cell homeostasis, we hypothesize that nuclear envelope alterations could have an important impact on stem cell compartments. To evaluate this hypothesis, we examined the number and functional competence of stem cells in Zmpste24-null progeroid mice, which exhibit nuclear lamina defects. We show that Zmpste24 deficiency causes an alteration in the number and proliferative capacity of epidermal stem cells. These changes are associated with an aberrant nuclear architecture of bulge cells and an increase in apoptosis of their supporting cells in the hair bulb region. These alterations are rescued in Zmpste24−/−Lmna+/− mutant mice, which do not manifest progeroid symptoms. We also report that molecular signaling pathways implicated in the regulation of stem cell behavior, such as Wnt and microphthalmia transcription factor, are altered in Zmpste24−/− mice. These findings establish a link between age-related nuclear envelope defects and stem cell dysfunction.
CXCR4 is required for the quiescence of primitive hematopoietic cells
Authors: Nie Yuchun , Han Yoon-Chi , Zou Yong-Rui
Published: April 2008

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4−/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.
WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1–CUL4–ROC1 ligase
Authors: Hu Jian , Zacharek Sima , He Yizhou Joseph , Lee Hyun , Shumway Stuart , Duronio Robert J. , Xiong Yue
Published: April 2008

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either the TSC1 or TSC2 genes. TSC1 and TSC2 proteins form a functionally interdependent dimeric complex. Phosphorylation of either TSC subunit by different kinases regulates the function of TSC and represents a major mechanism to integrate various signals into a centralized cell growth pathway. The majority of disease-associated mutations targeting either TSC1 or TSC2 results in a substantial decrease in protein level, suggesting that protein turnover also plays a critical role in TSC regulation. Here we report that TSC2 protein binds to FBW5, a DDB1-binding WD40 (DWD) protein, and is recruited by FBW5 to the DDB1–CUL4–ROC1 E3 ubiquitin ligase. Overexpression of FBW5 or CUL4A promotes TSC2 protein degradation, and this is abrogated by the coexpression of TSC1. Conversely, depletion of FBW5, DDB1, or CUL4A/B stabilizes TSC2. Ddb1 or Cul4 mutations in Drosophila result in Gigas/TSC2 protein accumulation and cause growth defects that can be partially rescued by Gigas/Tsc2 reduction. These results indicate that FBW5–DDB1–CUL4–ROC1 is an E3 ubiquitin ligase regulating TSC2 protein stability and TSC complex turnover.
The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast
Authors: Easlon Erin , Tsang Felicia , Skinner Craig , Wang Chen , Lin Su-Ju
Published: April 2008

Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate–aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1Δaat1Δ double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate–aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.
Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin
Authors: Dechat Thomas , Pfleghaar Katrin , Sengupta Kaushik , Shimi Takeshi , Shumaker Dale K. , Solimando Liliana , Goldman Robert D.
Published: April 2008

Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remarkably large number of disease-causing mutations in the human lamin A gene. This review provides an up-to-date overview of the functions of nuclear lamins, emphasizing their roles in epigenetics, chromatin organization, DNA replication, transcription, and DNA repair. In addition, we discuss recent evidence supporting the importance of lamins in viral infections.
A topoisomerase II-dependent mechanism for resetting replicons at the S–M-phase transition
Authors: Cuvier Olivier , Stanojcic Slavica , Lemaitre Jean-Marc , Mechali Marcel
Published: April 2008

Topoisomerase II (topo II) is required for chromosome segregation and for reprogramming replicons. Here, we show that topo II couples DNA replication termination with the clearing of replication complexes for resetting replicons at mitosis. Topo II inhibition impairs completion of DNA replication, accounting for replication protein A (RPA) stabilization onto ssDNA. Topo II inhibition does not affect the caffeine-sensitive ORC1 degradation found upon origin firing, but it impairs the cdk-dependent degradation/chromatin dissociation of an ORC1/2 reservoir at mitosis. Our results show that ORC1 degradation is rescued by Pin1 depletion and that this topo II-dependent clearing of ORC1/2 from chromatin involves the APC.
Xenopus Paraxial Protocadherin regulates morphogenesis by antagonizing Sprouty
Authors: Wang Yingqun , Janicki Patricia , Köster Isabelle , Berger Corinna D. , Wenzl Christian , Großhans Jörg , Steinbeisser Herbert
Published: April 2008

Xenopus Paraxial Protocadherin (xPAPC) has signaling functions that are essential for convergent extension (CE) movements and tissue separation during gastrulation. PAPC modulates components of the planar cell polarity (PCP) pathway, but it is not clear how PAPC is connected to β-catenin-independent Wnt-signaling. By yeast two-hybrid screen, we found that the intracellular domain of PAPC interacts with Sprouty (Spry), an inhibitor of CE movements. Upon binding to PAPC, Spry function is inhibited and PCP signaling is enhanced. Our data indicate that PAPC promotes gastrulation movements by sequestration of Spry and reveal a novel mechanism by which protocadherins modulate β-catenin-independent Wnt-signaling.
A new way to explore the world of extracellular protein interactions
Authors: Sanderson Christopher M.
Published: April 2008

Eukaryotic genomes encode large numbers of proteins that are either secreted or have exposed extracellular domains. It is highly likely that these proteins facilitate many important biological processes: however, as yet, most remain uncharacterized. Progress in this area of research has been impaired by the lack of a robust screening system that can be used to investigate interactions between large numbers of different extracellular proteins. In this issue, Bushell et al. introduce AVEXIS (avidity-basedextracellularinteractionscreen), a high-throughput screening procedure, which can be used to identify even weak extracellular protein interactions with extremely high confidence. This assay represents an important development in the field of network biology. By combining data from the AVEXIS system with data produced by classical or variant yeast two-hybrid methods, it will be possible to assemble binary protein interaction networks that connect extracellular and intracellular processes. This information will dramatically increase our ability to understand a wide range of physiological processes and facilitate the development of better therapeutic strategies.
Sectioning of Brain Tissues
Authors: Paul Carol Ann , Beltz Barbara , Berger-Sweeney Joanne
Published: April 2008

INTRODUCTION

A fixed brain, although harder than the brain in its natural state, is still not hard enough to cut into very thin slices. To make uniform, thin sections, the brain must be frozen. A wide variety of sectioning devices are available, each with their own requirements. With a microtome, for example, a very sharp knife is drawn manually across a brain that has been frozen to a chilled platform using dry ice. After each cut, the knife is lowered mechanically by a preset amount, giving consistent sections of a required thickness. A drawback of this instrument is that the temperature of the platform can fluctuate, thus causing changes in the quality of the sections. Other instruments, such as a cryostat, maintain the platform-mounted brain and the knife inside an enclosed refrigerated compartment. The temperature is kept at -20°C, and electronics are used to control the speed of the knife and the thickness of the sections. Some models can be set up to make a series of sections automatically.
Perfusion of Brain Tissues with Fixative
Authors: Paul Carol Ann , Beltz Barbara , Berger-Sweeney Joanne
Published: April 2008

INTRODUCTION

In many experiments it is necessary to section the brain to determine the location of a treatment (lesion or electrode) or to look at the histology of the brain using various staining techniques. Because the texture of the brain is so soft (often likened to soft cheese), it must be “fixed” before it can be removed from the skull. A fixative is a chemical that cross-links the molecules of the tissue, rendering it hard and preserving the tissue. This protocol describes a method for perfusing the brain with fixative (specifically, it describes how to perfuse a rat brain; slight modifications may be needed for different animals). A relatively simple gravity feed and the pumping mechanism of the heart is used to get fixative into the brain. A cannula is placed in the heart, or directly in the ascending aorta, of a deeply anesthetized animal. Blood is flushed out with saline first, and then with a fixative. The choice of fixative is often important if a specific staining technique is to be used, especially in immunocytochemistry, because the fixative can interfere with the staining sensitivity.
Transduction of Primary Hematopoietic Cells by Retroviral Vectors
Authors: Cornetta Kenneth , Pollok Karen E. , Miller A. Dusty
Published: April 2008

INTRODUCTION

Noncycling cells are relatively resistant to transduction with retroviral vectors. Because most immortalized cell lines are actively proliferating, this is not an issue. However, for many primary cells, especially quiescent populations such as primitive progenitor and stem cells, the gene-transfer rate can be particularly low. This protocol describes transduction of primary hematopoietic cells. Two interventions are combined to maximize gene transfer in hematopoietic progenitor cells: (1) cytokines and other growth factors are used to stimulate cell cycling in hematopoietic cells, and (2) matrix proteins such as fibronectin are used to mediate colocalization of target cells and vector.
Scanning Electron Microscopy (SEM) of Plant Tissues
Authors: Bomblies Kirsten , Shukla Vipula , Graham Charles
Published: April 2008

INTRODUCTION

Scanning electron microscopy (SEM) is used to produce detailed images of surface structures. This article details the steps required to prepare a sample for SEM. In brief, the sample is fixed, dehydrated, subjected to critical-point drying, mounted, and coated with an electrically conducting material such as gold or palladium.
Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU): Figure 1.
Authors: Salinger Andrew P. , Justice Monica J.
Published: April 2008

INTRODUCTION

This protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 × 10−3 in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed.
Desorption Electrospray Ionization (DESI) Analysis of Intact Proteins/Oligopeptides
Authors: Takats Zoltan , Wiseman Justin M. , Ifa Demian R. , Cooks R. Graham
Published: April 2008

INTRODUCTION

Desorption electrospray ionization (DESI) is amenable to the study of intact proteins in complex mixtures, including blood or other biological media. Intact proteins can be desorbed and ionized from the surface under gentle (soft) conditions to produce compact conformations of the protein. A procedure for DESI analysis of intact proteins and oligopeptides using mass spectrometry (MS) is described here. DESI-MS is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary depending on the application.
In Situ Desorption Electrospray Ionization (DESI) Analysis of Tissue Sections
Authors: Takats Zoltan , Wiseman Justin M. , Ifa Demian R. , Cooks R. Graham
Published: April 2008

INTRODUCTION

Desorption electrospray ionization (DESI) allows in situ analysis of biological tissues. The analysis of less abundant protein constituents within a tissue sample often requires the removal of lipid species prior to analysis, similar to the situation with matrix-assisted laser desorption/ionization (MALDI). After removal of lipid constituents, the tissue can be treated with protease to degrade proteins present in the tissue. The tryptic products can be investigated directly from the tissue using DESI. The spectra obtained feature ions of tryptic fragments from abundant proteins present in the tissue sample. The digestion is usually not complete; hence, the presence of missed cleavage sites is typical in the peptides detected. The signal is more stable for longer times than in the case of deposited samples, so the recording of mass spectrometry (MS)/MS data is simple in this case. DESI-MS is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol for DESI-MS analysis of tissue sections presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary depending on the application.
Retroviral Vectors for Gene Transfer
Authors: Cornetta Kenneth , Pollok Karen E. , Miller A. Dusty
Published: April 2008

INTRODUCTION

Retroviral vectors from the γ-retrovirus genus were the first retroviral vectors to be developed. They have been called oncoretroviral vectors or simple retroviral vectors because of their derivation from oncogenic retroviruses having a simple gag-pol-env genome structure. Later additions to the retroviral vector family include the lentiviral and foamy viral vectors derived from more complex retroviruses that contain multiple accessory genes in addition to the standard gag-pol-env genes. This article describes the advantages and disadvantages of retroviral vectors for gene therapy. It also discusses the issues that must be considered in designing retroviral vectors and in choosing retroviral packaging cell lines.
The Maximum Likelihood Approach for Phylogenetic Prediction: Figure 1.
Authors: Mount David W.
Published: April 2008

INTRODUCTION

Maximum likelihood (ML) methods are especially useful for phylogenetic prediction when there is considerable variation among the sequences in the multiple sequence alignment (msa) to be analyzed. ML methods start with a simple model, in this case a model of rates of evolutionary change in nucleic acid or protein sequences and tree models that represent a pattern of evolutionary change, and then adjust the model until there is a best fit to the observed data. For phylogenetic analysis, the observed data are the observed sequence variations found within the columns of an msa. The ML method is similar to the maximum parsimony method in that the analysis is performed on each column of an msa.
Development of thermotolerance requires interaction between polymerase‐β and heat shock proteins
Authors: Takahashi Akihisa , Yamakawa Nobuhiro , Mori Eiichiro , Ohnishi Ken , Yokota Shin‐ichi , Sugo Noriyuki , Aratani Yasuaki , Koyama Hideki , Ohnishi Takeo
Published: April 2008

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat‐induced DNA double‐strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat‐labile repair proteins such as DNA polymerase‐β (Polβ). Here the authors show that thermotolerance was partially suppressed in Polβ−/– mouse embryonic fibroblasts (MEF) when compared to the wild‐type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat‐induced γH2AX was suppressed in the thermotolerant cells. These results suggest that Polβ at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat‐induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat‐labile proteins besides Polβ. The present novel findings provide strong evidence that Polβ functions as a critical element involved in thermotolerance and exerts an important role in heat‐induced DSB. (Cancer Sci 2008; 99: 973–978)
Identification of SMURF1 as a possible target for 7q21.3‐22.1 amplification detected in a pancreatic cancer cell line by in‐house array‐based comparative genomic hybridization
Authors: Suzuki Ayako , Shibata Tatsuhiro , Shimada Yutaka , Murakami Yoshinori , Horii Akira , Shiratori Keiko , Hirohashi Setsuo , Inazawa Johji , Imoto Issei
Published: April 2008

Pancreatic cancer (PC) cell lines provide a useful starting point for the discovery and functional analysis of genes driving the genesis and progression of this lethal cancer. To increase our understanding of the gene copy number changes in pancreatic carcinomas and to identify key amplification and deletion targets, we applied genome‐wide array‐based comparative genomic hybridization using in‐house array (MCG Cancer Array‐800) to 24 PC cell lines. Overall, the analyses revealed high genomic complexity, with several copy number changes detected in each line. Homozygous deletions (log2ratio < –2) of eight genes (clones) were seen in 14 of the 24 cell lines, whereas high‐level amplifications (log2ratio > 2) of 10 genes (clones) were detected in seven lines. Among them, we focused on high‐level amplification at 7q22.1, because target genes for this alteration remain unknown. Through precise mapping of the altered region by fluorescence in situ hybridization, determination of the expression status of genes located within those regions, and functional analysis using knockdown of the gene expression or the ectopic overexpression approach in PC cell lines, as well as immunohistochemical analyses of candidates in primary tumors of PC, we successfully identified SMURF1 as having the greatest potential as a 7q21.3‐22.1 amplification target. SMURF1 may work as a growth‐promoting gene in PC through overexpression and might be a good candidate as a therapeutic target. Our results suggest that array‐based comparative genomic hybridization analysis combined with further genetic and functional examinations is a useful approach for identifying novel tumor‐associated genes involved in the pathogenesis of this lethal disease. (Cancer Sci 2008; 99: 986–994)
Induction of multinucleated cells and apoptosis in the PC‐3 prostate cancer cell line by low concentrations of polyethylene glycol 1000
Authors: Fukuta Katsuhiro , Kohri Kenjiro , Fukuda Hirokazu , Watanabe Masatoshi , Sugimura Takashi , Nakagama Hitoshi
Published: April 2008

Polyethylene glycol (PEG) has been reported to inhibit the development of colonic lesions in carcinogen‐treated rats when administered orally. However, the precise mechanism for the chemopreventive activity of PEG remains largely elusive. Based on a characteristic feature of PEG as a ‘fusogen’, we investigated its potential as a chemotherapeutic agent through the induction of multinucleated cell formation and apoptosis induction in PC‐3 prostate cancer cells. When PC‐3 cells were treated with 0.5 and 1.0% PEG 1000, multinucleated cells were induced at a frequency of 8.4 and 13%, respectively, 36 h after PEG treatment under high cell density (1 × 106 cells in 100 µL PEG solution) in vitro. Although abnormality of cell cycle progression was not evident in PEG‐treated PC‐3 cells, multinucleated cells substantially disappeared at around 38 h due to apoptosis. In contrast, no apparent growth suppression was observed when PC‐3 cells were exposed to up to 1.0% PEG at a much lower cell density, namely under ordinary culture conditions. Furthermore, injection of 0.5% PEG solution in vivo into PC‐3 xenografts implanted in BALB/c‐nu/nu male mice significantly suppressed tumor growth compared to phosphate‐buffered saline injection. Multinucleated TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL)‐positive cells were observed inside the PEG‐injected tumors. PEG was here demonstrated to have anticell proliferation and antitumor effects via induction of apoptosis, possibly by cell fusion. PEG injection therapy could therefore be adopted as an alternative chemotherapeutic strategy for localized prostate cancers, including those that become refractory to androgen‐deprivation therapy. (Cancer Sci 2008; 99: 1055–1062)
Downregulation of Tie2 gene by a novel antitumor sulfolipid, 3′‐sulfoquinovosyl‐1′‐monoacylglycerol, targeting angiogenesis
Authors: Mori Yoko , Sahara Hiroeki , Matsumoto Kayo , Takahashi Nobuaki , Yamazaki Takayuki , Ohta Keisuke , Aoki Satoko , Miura Masahiko , Sugawara Fumio , Sakaguchi Kengo , Sato Noriyuki
Published: April 2008

We previously reported that 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG) was effective in suppressing the growth of solid tumors due to hemorrhagic necrosis in vivo. In the present study, we investigated the antiangiogenic effect of SQMG. In vivo assessment of antitumor assays showed that some tumor cell lines, but not others, were sensitive to SQMG. Microscopic study suggested that in SQMG‐sensitive tumors, but not SQMG‐resistant tumors, angiogenesis was reduced. We next investigated gene expression relating to angiogenesis in tumor tissues by quantitative real‐time polymerase chain reaction. Consequently, although vascular endothelial growth factor gene expression was not detected with significant differences among the cases, significant downregulation of Tie2 gene expression was observed in all SQMG‐sensitive tumors as compared with controls, but not in SQMG‐resistant tumors. These data suggested that the antitumor effects of SQMG could be attributed to antiangiogenic effects, possibly via the downregulation of Tie2 gene expression in SQMG‐sensitive tumors. (Cancer Sci 2008; 99: 1063–1070)
Alteration of p53‐binding protein 1 expression during skin carcinogenesis: Association with genomic instability
Authors: Naruke Yuki , Nakashima Masahiro , Suzuki Keiji , Matsuu‐Matsuyama Mutsumi , Shichijo Kazuko , Kondo Hisayoshi , Sekine Ichiro
Published: April 2008

Epidermal cells are the first cells to be exposed to environmental genotoxic agents such as ultraviolet and ionizing radiations, which induce DNA double strand breaks (DSB) and activate DNA damage response (DDR) to maintain genomic integrity. Defective DDR can result in genomic instability (GIN) which is considered to be a central aspect of any carcinogenic process. P53‐binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DSB and rapidly form nuclear foci, the presence of 53BP1 nuclear foci can be considered as a cytological marker for endogenous DSB reflecting GIN. The levels of GIN were analyzed by immunofluorescence studies of 53BP1 in 56 skin tumors that included 20 seborrheic keratosis, eight actinic keratosis, nine Bowen's disease, nine squamous cell carcinoma, and 10 basal cell carcinoma. This study demonstrated a number of nuclear 53BP1 foci in human skin tumorigenesis, suggesting a constitutive activation of DDR in skin cancer cells. Because actinic keratosis showed a high DDR type of 53BP1 immunoreactivity, GIN seems to be induced at the precancerous stage. Furthermore, invasive cancers exhibited a high level of intense, abnormal 53BP1 nuclear staining with nuclear accumulation of p53, suggesting a disruption of DDR leading to a high level of GIN in cancer cells. The results of this study suggest that GIN has a crucial role in the progression of skin carcinogenesis. The detection of 53BP1 expression by immunofluorescence can be a useful histological marker to estimate the malignant potential of human skin tumors. (Cancer Sci 2008; 99: 946–951)
Effects of imatinib (Glivec) on the pharmacokinetics of metoprolol, a CYP2D6 substrate, in Chinese patients with chronic myelogenous leukaemia
Authors: Wang Yanfeng , Zhou Li , Dutreix Catherine , Leroy Elisabeth , Yin Qi , Sethuraman Venkat , Riviere Gilles‐Jacques , Yin Ophelia Q. P. , Schran Horst , Shen Zhi‐Xiang
Published: April 2008

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Imatinib, a tyrosine kinase inhibitor, exhibits a competitive inhibition on the CYP450 2D6 isozyme with a Ki value of 7.5 μm. However, the clinical significance of the inhibition and its relevance to 2D6 polymorphisms have not been evaluated. The pharmacokinetics of imatinib have been well studied in Caucasians, but not in a Chinese population.

• Metoprolol, a CYP2D6 substrate, has different clearances among patients with different CYP2D6 genotypes. It is often used as a CYP2D6 probe substrate for clinical drug–drug interaction studies.

WHAT THIS STUDY ADDS

• Co‐administration of imatinib at 400 mg twice daily increased the plasma AUC of metoprolol by approximately 23% in 20 Chinese patients with chronic myeloid leukaemia (CML), about 17% increase in CYP2D6 intermediate metabolizers (IMs) (n = 6), 24% in extensive metabolizers (EMs) (n = 13), and 28% for the subject with unknown 2D6 status (n = 1) suggesting that imatinib has a weak to moderate inhibition on CYP2D6 in vivo.

• The clearance of imatinib in Chinese patients with CML showed no difference between CYP2D6 IMs and EMs, and no major difference from Caucasian patients with CML based on data reported in the literature.

AIMS

To investigate the effect of imatinib on the pharmacokinetics of a CYP2D6 substrate, metoprolol, in patients with chronic myeloid leukaemia (CML). The pharmacokinetics of imatinib were also studied in these patients.

METHODS

Patients (n = 20) received a single oral dose of metoprolol 100 mg on day 1 after an overnight fast. On days 2–10, imatinib 400 mg was administered twice daily. On day 8, another 100 mg dose of metoprolol was administered 1 h after the morning dose of imatinib 400 mg. Blood samples for metoprolol and α‐hydroxymetoprolol measurement were taken on study days 1 and 8, and on day 8 for imatinib.

RESULTS

Of the 20 patients enrolled, six patients (30%) were CYP2D6 intermediate metabolizers (IMs), 13 (65%) extensive metabolizers (EMs), and the CYP2D6 status in one patient was unknown. In the presence of 400 mg twice daily imatinib, the mean metoprolol AUC was increased by 17% in IMs (from 1190 to 1390 ng ml−1 h), and 24% in EMs (from 660 to 818 ng ml−1 h). Patients classified as CYP2D6 IMs had an approximately 1.8‐fold higher plasma metoprolol exposure than those classified as EMs. The oral clearance of imatinib was 11.0 ± 2.0 l h−1 and 11.8 ± 4.1 l h−1 for CYP2D6 IMs and EMs, respectively.

CONCLUSIONS

Co‐administration of a high dose of imatinib resulted in a small or moderate increase in metoprolol plasma exposure in all patients regardless of CYP2D6 status. The clearance of imatinib showed no difference between CYP2D6 IMs and EMs.
Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved
Authors: Lam H‐Y. , Van Hoffen E. , Michelsen A. , Guikers K. , Van Der Tas C. H. W. , Bruijnzeel‐Koomen C. A. F. M. , Knulst A. C.
Published: April 2008

Background
Studies on cow's milk allergy (CMA) in adults are scarce. Little is known about the clinical symptoms, eliciting doses (ED), and allergens involved.

Objective
The aim of this study was to analyse the clinical symptoms, ED and allergen recognition in adult CMA patients, compared with cow's milk (CM)‐sensitized, but tolerant controls.

Methods
Adult CMA patients were evaluated by standardized questionnaires (n=30), skin prick tests (SPTs) and specific IgE for CM allergens (n=18), and a double‐blind placebo‐controlled food challenge (DBPCFC, n=10). A control group (n=25) of CM‐sensitized, but tolerant adults was included.

Results
The majority of CMA patients (20/30, 67%) reported severe symptoms. In all patients participating in DBPCFC, CMA was confirmed. ED for subjective symptoms (0.3–300 mg CM protein) were significantly lower than that for objective symptoms (300–9000 mg CM protein). The severity of CMA by history and ED was not correlated with SPT or IgE. Patients had higher SPT reactivity than controls for CM, α‐lactalbumin and β‐lactoglobulin (P=0.002, P=0.014 and P=0.004) but not for casein. Specific IgE to CM tended to be higher (P=0.068) and IgE to casein was higher in patients than that in controls (P=0.016). No difference was observed for IgE to α‐lactalbumin and β‐lactoglobulin.

Conclusion
Adult CMA is severe in nature. ED are low, starting from 0.3 mg CM protein. Patients with CMA recognize the same major allergens (casein and whey proteins) as controls, but display a stronger SPT and IgE reactivity.
IL‐13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells
Authors: Chibana K. , Trudeau J. B. , Mustovitch A. T. , Hu H. , Zhao J. , Balzar S. , Chu H. W. , Wenzel S. E.
Published: April 2008

Background
Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T‐helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells.

Objectives
We hypothesized that primary human airway epithelial cells in air–liquid interface (ALI) culture would increase iNOS expression and activity in response to IL‐13, while decreasing arginase expression.

Methods
iNOS and arginase mRNA (real‐time PCR) and protein expression (Western blot and immunofluorescence) as well as iNOS activity (nitrite levels) were measured in ALI epithelial cells cultured from bronchial brushings of normal and asthmatic subjects following IL‐13 stimulation.

Results
IL‐13 up‐regulated iNOS mRNA primarily at a transcriptional level in epithelial cells. iNOS protein and activity also increased, arginase1 protein expression decreased while arginase 2 expression did not change. The changes in iNOS protein correlated strongly with changes in nitrites, and inclusion of arginase (1 or 2) did not substantially change the relationship. Interestingly, iNOS mRNA and protein were not correlated.

Conclusions
These results contrast with many previous results to confirm that Th2 stimuli enhance iNOS expression and activity. While arginase 1 protein decreases in response to IL‐13, neither arginase appears to substantially impact nitrite levels in this system.
Panbiogeographical analysis of the genus Bomarea (Alstroemeriaceae)
Authors: Alzate Fernando , Quijano‐Abril Mario Alberto , Morrone Juan J.
Published: April 2008

Aim  A panbiogeographical analysis of the genus Bomarea was undertaken in order to determine generalized tracks and biogeographical nodes, and to evaluate the current distribution of the genus based on the available tectonic information and the biogeographical regionalization of Latin America.

Location  The Neotropical region from northern Mexico to northern Argentina, and the Nearctic and Andean regions.

Method  A total of 2205 records of 101 species were analysed, representing 95% of the species assigned to Bomarea. Localities were represented on maps and their individual tracks were drawn. Based on their comparison, generalized tracks were detected and mapped. Nodes were identified in the areas where different generalized tracks were superimposed.

Results  Five generalized tracks were recovered. One is located in the Eastern Central America and Western Panamanian Isthmus provinces (Caribbean subregion, Neotropical region), which was supported by three species of Central American distribution. The four remaining generalized tracks were located in South America, in the North Andean Paramo, Cauca and Puna biogeographical provinces. These tracks were supported by species of Bomarea with an Andean distribution. Biogeographical nodes were established in the Central Andean region of Colombia, central Ecuador and central Peru.

Main conclusions  The nodes obtained for Bomarea support a hybrid origin for the Andean region, which presents diverse components from both northern and southern South America. Likewise, the track recovered between Colombia and Ecuador includes Andean and Neotropical areas, providing further support for this hypothesis. The nodes obtained are coherent with vicariant elements evident for Bomarea. Species of three clades proposed for Bomarea are distributed in specific generalized tracks.
Tertiary relict trees in a Mediterranean climate: abiotic constraints on the persistence of Prunus lusitanica at the eroding edge of its range
Authors: Pulido Fernando , Valladares Fernando , Calleja Juan Antonio , Moreno Gerardo , González‐Bornay Guillermo
Published: April 2008

Aim  To investigate the ecophysiological traits allowing persistence of a subtropical relict tree (Prunus lusitanica L.) under a dry Mediterranean climate at the eroding edge of its range.

Location  A glasshouse for the study under controlled conditions and two marginal populations located in riparian forests of central Spain and exposed to summer drought, in contrast to subtropical populations that grow in mountain cloud forests.

Methods  Two experiments were conducted to assess tolerance to the abiotic conditions found in riparian habitats. In a glasshouse experiment, gas‐exchange and light‐use parameters were measured to evaluate seedling responses to a factorial combination of irradiance (60%, 10%, 2% and 0.5% full sun) and moisture (well watered control and drought stress). In a parallel field experiment, irradiance and soil moisture were measured as predictors of seedling survival at two sites in which half the seedlings were subjected to a threefold increase in summer precipitation by adding water every 10 days.

Results  Soil moisture strongly determined survival both in the glasshouse and in the two field sites. In the field, periodic addition of water failed to increase survival. Water‐use efficiency (WUE) increased with drought. Seedlings did not tolerate deep shade (2–0.5%) and their performance and survival were better when exposed to moderate (10%) or high (60%) irradiance. The effect of water stress on seedling performance was stronger at moderate irradiance, moderate at high irradiance and negligible at very low light. Seedling size (height and number of leaves) attained after 1 month of experimental treatments had a positive effect on survival at the end of the summer, hence greater survival was not achieved at the expense of vertical growth.

Main conclusions  While studies in Macaronesia have shown that P. lusitanica occupies a wide range of moisture conditions along mountain slopes, it behaves as an obligate riparian species in dry peripheral populations. Intolerance to deep shade and tolerance to moderate and high irradiance allow the species to grow in small and moist gaps, or in treeless river sections. Despite the species’ low range filling in marginal, drought‐prone regions, long‐term persistence might have been achieved through shifts to riparian habitats serving as local refuges.
The effects of dietary modification as denned by NACNE on the eating habits of 28 people
Authors: Bradley Anna , Theobald Alison
Published: April 2008

Twenty‐eight members of the general public, with little nutritional knowledge and no previous commitment to dietary change, completed 7‐day‐weighed dietary diaries. They then attempted to change their diets, where appropriate, in line with current nutritional guidelines.

The panel achieved changes in diet that were consistent with the recent recommendations but several problems arose. In particular, although fat levels were reduced, energy levels fell and fibre goals were not reached; in addition, certain of the initial vitamin and mineral intakes gave cause for concern. These results emphasize the importance of giving advice not only about foods that might be reduced in the diet, but also about food that might be used as alternatives. They also illustrate the difficulty of persuading people to make radical changes to their eating habits even when given personal advice from a nutritionist.

The panel was small and not necessarily representative, but the results illustrate the need for more dietary analysis studies. It is not enough simply to formulate appropriate guidelines from a nutritional point of view; it is also important to ensure that the public understands them and that people make sensible decisions about their diet.
Calcium intake in the elderly: validation of a dietary questionnaire
Authors: Nelson Michael , Hague Gillian F. , Cooper Cyrus , Bunker Valda W.
Published: April 2008

Epidemiological studies of calcium and osteoporosis have been hampered by the lack of a suitable tool for assessing calcium intake. This report describes a new frequency and amount questionnaire for measuring present and past calcium intake in the elderly. The validity of the questionnaire was tested against two commonly used standards of dietary assessment, five‐day duplicate diets and seven‐day weighed dietary inventories. The resulting correlation coefficients were, respectively, r= 0.76 and r= 0.69, while that for repeatability was r= 0.84. Furthermore, the questionnaire categorized subjects into thirds of the distribution of intake with almost no gross misclassification. It is suggested that the present findings may be extended to the majority of normal, healthy elderly subjects, implying wide application for the questionnaire in the assessment of calcium intake in the elderly.
Calcium intake in normotensive and hypertensive pregnant women
Authors: Manocha S. , Gupta M. C. , Aggarwal N.
Published: April 2008

The present study was undertaken to investigate the dietary intakes of protein, fat, CHO, total calories and calcium in normotensive pregnant females and patients with pregnancy induced hypertension. A significantly higher intake of protein and calcium was found in normotensive females. The relationship also held true when the dietary intake of a subset of 13 hypertensive women was compared with normotensive women matched for age, parity and socio‐economic status.
Toxoplasmosis with hemophagocytic syndrome after bone marrow transplantation: diagnosis at autopsy
Authors: Duband S. , Cornillon J. , Tavernier E. , Dumollard J.‐M. , Guyotat D. , Péoc'h M.
Published: April 2008

Abstract: Toxoplasmosis is a rare but well recognized opportunistic infection that can occur after allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Besides encephalitis, other common presentations of Toxoplasma gondii infection are interstitial pneumonitis and myocarditis. Because of its non‐specific clinical and biological signs and its lethal outcome, toxoplasmosis is often misdiagnosed and only revealed at autopsy. We report a case of a postmortem diagnosis of disseminated toxoplasmosis associated with hemophagocytic syndrome, which underlines the value of necropsy in cases of death after transplantation. We also discuss clinical presentations and risk factors that lead to toxoplasmosis in allo‐HSCT recipients.
Effects of Exercise on the Morphology of the Myenteric Neurons of the Duodenum of Wistar Rats during the Ageing Process
Authors: De Britto Mari R. , Clebis N. K. , Gagliardo K. M. , Guimarães J. P. , Stabille S. R. , De Mello Germano R. , De Souza R. R.
Published: April 2008

This study aimed to evaluate the effects of regular physical activity on the morphology of the myenteric plexus of the duodenum in rats during the ageing process. To this end, 45 Wistar rats were divided into three groups: C (sedentary – 6 months old), S (sedentary – 12 months old) and T (trained – 12 months old). The animals of group S were given with a physical activity programme consisting of a 10‐min‐treadmill workout once a week. The animals of group T were submitted to the physical activity programme five times a week. Their duodenums were collected and submitted to the techniques of nicotinamide adenine dinucleotide (NADH)‐diaphorase enzyme histochemistry for whole‐mount preparations and transmission electron microscopy. No differences in the constitution of the myenteric plexuses were found when the sedentary and trained groups were compared with the control group. The ultrastructural features were similar for the three groups. However, it was verified that the physical activity of the trained animals resulted in a similar myenteric neuron morphology to that of the adult animals (6 months old), thereby confirming its beneficial effect, as the sedentary animals had larger alterations in the collagen fibrils and the basal membrane that occur through ageing. The quantitative analysis showed that the NADH‐diaphorase positive neurons decreased with ageing and increased with physical activity (P > 0.05). No significant alteration (P > 0.05) in the neuronal profile area of the NADH‐diaphorase positive neurons has been observed with ageing.
Portal‐systemic shunt encephalopathy presenting with diffuse cerebral white matter lesion: An autopsy case
Authors: Kimura Noriyuki , Kumamoto Toshihide , Hanaoka Takuya , Nakamura Kenichiro , Hazama Yusuke , Arakawa Ryuki
Published: April 2008

We report herein an autopsy case of portal‐systemic encephalopathy (PSE) presenting with diffuse tissue rarefaction in the cerebral deep white matter. Clinically, the patient showed recurrent episodes of unconsciousness, abnormal behavior and urinary incontinence, as well as flapping tremor. Cognitive impairment and peripheral neuropathy developed following recurrent episodes. Although conventional arterial portography revealed a small portal‐systemic collateral vessel of a left gastro‐renal venous shunt, abdominal CT and liver biopsy showed no evidence of liver cirrhosis and serum ammonia level showed a mild increase. T2‐weighted MRI demonstrated symmetrical signal hyperintensities in the deep white matter. Neuropathological findings showed Alzheimer type II astrocytes in the deep layers of the cerebral cortices and severe tissue rarefaction with no or slight reactive astrocytosis in the subcortical and deep white matter. These white matter changes have been reported infrequently in patients with PSE. The present case suggests that chronic PSE without liver cirrhosis may develop diffuse white matter lesions.
Disentangling interactions between adaptive divergence and gene flow when ecology drives diversification
Authors: Räsänen Katja , Hendry Andrew P.
Published: April 2008

Adaptive diversification is driven by selection in ecologically different environments. In absence of geographical barriers to dispersal, this adaptive divergence (AD) may be constrained by gene flow (GF). And yet the reverse may also be true, with AD constraining GF (i.e. ‘ecological speciation’). Both of these causal effects have frequently been inferred from the presence of negative correlations between AD and GF in nature – yet the bi‐directional causality warrants caution in such inferences. We discuss how the ability of correlative studies to infer causation might be improved through the simultaneous measurement of multiple ecological and evolutionary variables. On the one hand, inferences about the causal role of GF can be made by examining correlations between AD and the potential for dispersal. On the other hand, inferences about the causal role of AD can be made by examining correlations between GF and environmental differences. Experimental manipulations of dispersal and environmental differences are a particularly promising approach for inferring causation. At present, the best studies find strong evidence that GF constrains AD and some studies also find the reverse. Improvements in empirical approaches promise to eventually allow general inferences about the relative strength of different causal interactions during adaptive diversification.