The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.

InterProScan is a tool that scans given protein sequences against the protein signatures of the InterPro member databases, currently--PROSITE, PRINTS, Pfam, ProDom and SMART. The number of signature databases and their associated scanning tools as well as the further refinement procedures make the problem complex. InterProScan is designed to be a scalable and extensible system with a robust internal architecture.

BACKGROUND

The Perl-based InterProScan implementation is available from the EBI ftp server (ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/) and the SRS-basedInterProScan is available upon request. We provide the public web interface (http://www.ebi.ac.uk/interpro/scan.html) as well as email submission server (interproscan@ebi.ac.uk).

We have developed ClueGO, an easy to use Cytoscape plug-in that strongly improves biological interpretation of large lists of genes. ClueGO integrates Gene Ontology (GO) terms as well as KEGG/BioCarta pathways and creates a functionally organized GO/pathway term network. It can analyze one or compare two lists of genes and comprehensively visualizes functionally grouped terms. A one-click update option allows ClueGO to automatically download the most recent GO/KEGG release at any time. ClueGO provides an intuitive representation of the analysis results and can be optionally used in conjunction with the GOlorize plug-in.

The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein-ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.

Of the ions involved in the intricate workings of the heart, calcium is considered perhaps the most important. It is crucial to the very process that enables the chambers of the heart to contract and relax, a process called excitation-contraction coupling. It is important to understand in quantitative detail exactly how calcium is moved around the various organelles of the myocyte in order to bring about excitation-contraction coupling if we are to understand the basic physiology of heart function. Furthermore, spatial microdomains within the cell are important in localizing the molecular players that orchestrate cardiac function.

Hepcidin is a peptide hormone secreted by the liver in response to iron loading and inflammation. Decreased hepcidin leads to tissue iron overload, whereas hepcidin overproduction leads to hypoferremia and the anemia of inflammation. Ferroportin is an iron exporter present on the surface of absorptive enterocytes, macrophages, hepatocytes, and placental cells. Here we report that hepcidin bound to ferroportin in tissue culture cells. After binding, ferroportin was internalized and degraded, leading to decreased export of cellular iron. The posttranslational regulation of ferroportin by hepcidin may thus complete a homeostatic loop: Iron regulates the secretion of hepcidin, which in turn controls the concentration of ferroportin on the cell surface.

Metastasis causes most cancer deaths, yet this process remains one of the most enigmatic aspects of the disease. Building on new mechanistic insights emerging from recent research, we offer our perspective on the metastatic process and reflect on possible paths of future exploration. We suggest that metastasis can be portrayed as a two-phase process: The first phase involves the physical translocation of a cancer cell to a distant organ, whereas the second encompasses the ability of the cancer cell to develop into a metastatic lesion at that distant site. Although much remains to be learned about the second phase, we feel that an understanding of the first phase is now within sight, due in part to a better understanding of how cancer cell behavior can be modified by a cell-biological program called the epithelial-to-mesenchymal transition.

BACKGROUND

The relative effectiveness of second-generation (atypical) antipsychotic drugs as compared with that of older agents has been incompletely addressed, though newer agents are currently used far more commonly. We compared a first-generation antipsychotic, perphenazine, with several newer drugs in a double-blind study.

METHODS

A total of 1493 patients with schizophrenia were recruited at 57 U.S. sites and randomly assigned to receive olanzapine (7.5 to 30 mg per day), perphenazine (8 to 32 mg per day), quetiapine (200 to 800 mg per day), or risperidone (1.5 to 6.0 mg per day) for up to 18 months. Ziprasidone (40 to 160 mg per day) was included after its approval by the Food and Drug Administration. The primary aim was to delineate differences in the overall effectiveness of these five treatments.

RESULTS

Overall, 74 percent of patients discontinued the study medication before 18 months (1061 of the 1432 patients who received at least one dose): 64 percent of those assigned to olanzapine, 75 percent of those assigned to perphenazine, 82 percent of those assigned to quetiapine, 74 percent of those assigned to risperidone, and 79 percent of those assigned to ziprasidone. The time to the discontinuation of treatment for any cause was significantly longer in the olanzapine group than in the quetiapine (P<0.001) or risperidone (P=0.002) group, but not in the perphenazine (P=0.021) or ziprasidone (P=0.028) group. The times to discontinuation because of intolerable side effects were similar among the groups, but the rates differed (P=0.04); olanzapine was associated with more discontinuation for weight gain or metabolic effects, and perphenazine was associated with more discontinuation for extrapyramidal effects.

CONCLUSIONS

The majority of patients in each group discontinued their assigned treatment owing to inefficacy or intolerable side effects or for other reasons. Olanzapine was the most effective in terms of the rates of discontinuation, and the efficacy of the conventional antipsychotic agent perphenazine appeared similar to that of quetiapine, risperidone, and ziprasidone. Olanzapine was associated with greater weight gain and increases in measures of glucose and lipid metabolism.

Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming and laborious. Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids.

Receiver operating characteristic (ROC) curves are used to describe and compare the performance of diagnostic technology and diagnostic algorithms. This paper refines the statistical comparison of the areas under two ROC curves derived from the same set of patients by taking into account the correlation between the areas that is induced by the paired nature of the data. The correspondence between the area under an ROC curve and the Wilcoxon statistic is used and underlying Gaussian distributions (binormal) are assumed to provide a table that converts the observed correlations in paired ratings of images into a correlation between the two ROC areas. This between-area correlation can be used to reduce the standard error (uncertainty) about the observed difference in areas. This correction for pairing, analogous to that used in the paired t-test, can produce a considerable increase in the statistical sensitivity (power) of the comparison. For studies involving multiple readers, this method provides a measure of a component of the sampling variation that is otherwise difficult to obtain.

The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.

While the tumor suppressor functions of p53 have long been recognized, the contribution of p53 to numerous other aspects of disease and normal life is only now being appreciated. This burgeoning range of responses to p53 is reflected by an increasing variety of mechanisms through which p53 can function, although the ability to activate transcription remains key to p53's modus operandi. Control of p53's transcriptional activity is crucial for determining which p53 response is activated, a decision we must understand if we are to exploit efficiently the next generation of drugs that selectively activate or inhibit p53.

OBJECTIVE

To construct growth curves for school-aged children and adolescents that accord with the WHO Child Growth Standards for preschool children and the body mass index (BMI) cut-offs for adults.

METHODS

Data from the 1977 National Center for Health Statistics (NCHS)/WHO growth reference (1-24 years) were merged with data from the under-fives growth standards' cross-sectional sample (18-71 months) to smooth the transition between the two samples. State-of-the-art statistical methods used to construct the WHO Child Growth Standards (0-5 years), i.e. the Box-Cox power exponential (BCPE) method with appropriate diagnostic tools for the selection of best models, were applied to this combined sample.

RESULTS

The merged data sets resulted in a smooth transition at 5 years for height-for-age, weight-for-age and BMI-for-age. For BMI-for-age across all centiles the magnitude of the difference between the two curves at age 5 years is mostly 0.0 kg/m(2) to 0.1 kg/m(2). At 19 years, the new BMI values at +1 standard deviation (SD) are 25.4 kg/m(2) for boys and 25.0 kg/m(2) for girls. These values are equivalent to the overweight cut-off for adults >> or = 25.0 kg/m(2)). Similarly, the +2 SD value (29.7 kg/m(2) for both sexes) compares closely with the cut-off for obesity >> or = 30.0 kg/m(2)).

CONCLUSIONS

The new curves are closely aligned with the WHO Child Growth Standards at 5 years, and the recommended adult cut-offs for overweight and obesity at 19 years. They fill the gap in growth curves and provide an appropriate reference for the 5 to 19 years age group.

OBJECTIVE

To compare the clinical features, natural history, and outcomes for women with "triple-negative" breast cancer with women with other types of breast cancer.

METHODS

We studied a cohort of 1,601 patients with breast cancer, diagnosed between January 1987 and December 1997 at Women's College Hospital in Toronto. Triple-negative breast cancers were defined as those that were estrogen receptor negative, progesterone receptor negative, and HER2neu negative. The prognostic significance of triple-negative breast cancer was explored.

RESULTS

The median follow-up time of the 1,601 women was 8.1 years. One hundred and eighty of 1,601 patients (11.2%) had triple-negative breast cancer. Compared with other women with breast cancer, those with triple-negative breast cancer had an increased likelihood of distant recurrence (hazard ratio, 2.6; 95% confidence interval, 2.0-3.5; P < 0.0001) and death (hazard ratio, 3.2; 95% confidence interval, 2.3-4.5; P < 0.001) within 5 years of diagnosis but not thereafter. The pattern of recurrence was also qualitatively different; among the triple-negative group, the risk of distant recurrence peaked at approximately 3 years and declined rapidly thereafter. Among the "other" group, the recurrence risk seemed to be constant over the period of follow-up.

CONCLUSIONS

Triple-negative breast cancers have a more aggressive clinical course than other forms of breast cancer, but the adverse effect is transient.

The dementia with Lewy bodies (DLB) Consortium has revised criteria for the clinical and pathologic diagnosis of DLB incorporating new information about the core clinical features and suggesting improved methods to assess them. REM sleep behavior disorder, severe neuroleptic sensitivity, and reduced striatal dopamine transporter activity on functional neuroimaging are given greater diagnostic weighting as features suggestive of a DLB diagnosis. The 1-year rule distinguishing between DLB and Parkinson disease with dementia may be difficult to apply in clinical settings and in such cases the term most appropriate to each individual patient should be used. Generic terms such as Lewy body (LB) disease are often helpful. The authors propose a new scheme for the pathologic assessment of LBs and Lewy neurites (LN) using alpha-synuclein immunohistochemistry and semiquantitative grading of lesion density, with the pattern of regional involvement being more important than total LB count. The new criteria take into account both Lewy-related and Alzheimer disease (AD)-type pathology to allocate a probability that these are associated with the clinical DLB syndrome. Finally, the authors suggest patient management guidelines including the need for accurate diagnosis, a target symptom approach, and use of appropriate outcome measures. There is limited evidence about specific interventions but available data suggest only a partial response of motor symptoms to levodopa: severe sensitivity to typical and atypical antipsychotics in approximately 50%, and improvements in attention, visual hallucinations, and sleep disorders with cholinesterase inhibitors.

We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.

LTP and LTD, the long-term potentiation and depression of excitatory synaptic transmission, are widespread phenomena expressed at possibly every excitatory synapse in the mammalian brain. It is now clear that "LTP" and "LTD" are not unitary phenomena. Their mechanisms vary depending on the synapses and circuits in which they operate. Here we review those forms of LTP and LTD for which mechanisms have been most firmly established. Examples are provided that show how these mechanisms can contribute to experience-dependent modifications of brain function.

Synaptic transmission is a dynamic process. Postsynaptic responses wax and wane as presynaptic activity evolves. This prominent characteristic of chemical synaptic transmission is a crucial determinant of the response properties of synapses and, in turn, of the stimulus properties selected by neural networks and of the patterns of activity generated by those networks. This review focuses on synaptic changes that result from prior activity in the synapse under study, and is restricted to short-term effects that last for at most a few minutes. Forms of synaptic enhancement, such as facilitation, augmentation, and post-tetanic potentiation, are usually attributed to effects of a residual elevation in presynaptic [Ca(2+)]i, acting on one or more molecular targets that appear to be distinct from the secretory trigger responsible for fast exocytosis and phasic release of transmitter to single action potentials. We discuss the evidence for this hypothesis, and the origins of the different kinetic phases of synaptic enhancement, as well as the interpretation of statistical changes in transmitter release and roles played by other factors such as alterations in presynaptic Ca(2+) influx or postsynaptic levels of [Ca(2+)]i. Synaptic depression dominates enhancement at many synapses. Depression is usually attributed to depletion of some pool of readily releasable vesicles, and various forms of the depletion model are discussed. Depression can also arise from feedback activation of presynaptic receptors and from postsynaptic processes such as receptor desensitization. In addition, glial-neuronal interactions can contribute to short-term synaptic plasticity. Finally, we summarize the recent literature on putative molecular players in synaptic plasticity and the effects of genetic manipulations and other modulatory influences.

A new lineage of effector CD4+ T cells characterized by production of interleukin (IL)-17, the T-helper-17 (T(H)17) lineage, was recently described based on developmental and functional features distinct from those of classical T(H)1 and T(H)2 lineages. Like T(H)1 and T(H)2, T(H)17 cells almost certainly evolved to provide adaptive immunity tailored to specific classes of pathogens, such as extracellular bacteria. Aberrant T(H)17 responses have been implicated in a growing list of autoimmune disorders. T(H)17 development has been linked to IL-23, an IL-12 cytokine family member that shares with IL-12 a common subunit, IL-12p40 (ref. 8). The IL-23 and IL-12 receptors also share a subunit, IL-12Rbeta1, that pairs with unique, inducible components, IL-23R and IL-12Rbeta2, to confer receptor responsiveness. Here we identify transforming growth factor-beta (TGF-beta) as a cytokine critical for commitment to T(H)17 development. TGF-beta acts to upregulate IL-23R expression, thereby conferring responsiveness to IL-23. Although dispensable for the development of IL-17-producing T cells in vitro and in vivo, IL-23 is required for host protection against a bacterial pathogen, Citrobacter rodentium. The action of TGF-beta on naive T cells is antagonized by interferon-gamma and IL-4, thus providing a mechanism for divergence of the T(H)1, T(H)2 and T(H)17 lineages.

Two small temporal RNAs (stRNAs), lin-4 and let-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs (miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed with let-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply that, as a class, miRNAs have broad regulatory functions in animals.

Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.