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Large-scale analysis of the yeast proteome by multidimensional protein identification technology.
We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.
Evaluation of an optimized system for random amplified polymorphic DNA (RAPD)-analysis for genotypic mapping of Candida albicans strains.
A simple, rapid, and cost-effective protocol has been developed for a PCR-based molecular typing method for Candida albicans, which includes the use of a commercially available medium (Chelex 100 Resin) for DNA extraction and a single set of two arbitrarily chosen oligonucleotide (10 nt length) primers for random amplified DNA(RAPD)-analysis. The optimized parameters for the amplification components and conditions for the selected primer combination have been determined to avoid artifactual variation (absence/presence of bands) in RAPD banding patterns in repeated assays. The optimized RAPD-assay consistently generated DNA-patterns of 33 genetically unrelated C. albicans isolates that contained ten polymorphic markers in the non-artifactual banding patterns. The intralaboratory reproducibility of RAPD patterns was efficient and consistent provided the optimized amplification conditions were rigidly controlled. Interlaboratory reproducibility was tempered by slight variations in time of cyclers of different thermocyclers. In comparison, the RAPD assay was almost equal to restriction enzyme analysis (REA) (Eco RI digested chromosomal DNA) in discrimination, and the RAPD assay was able to group isolates of C. albicans that were untypable by REA. The protocol outlined for an optimized RAPD-assay of C. albicans has the potential to be widely useful epidemiological screening tool that can be easily applied in the clinical laboratory.
High-speed interlaced spin-echo magnetic resonance imaging.
A new method is introduced for increasing the efficiency in multislice single spin-echo MRI. The method interlaces the excitation and measurement of different slices, resulting in an effective use of the echo delay time between RF excitation and reception. Under certain conditions, the method allows for scan time reduction compared to standard single spin-echo MRI, in particular for long echo times. The technique is demonstrated in examples of brain scans, indicating that a substantial increase is scan speed can be achieved without loss in image signal-to-noise ratio or contrast. Potential applications include perfusion imaging using T(2)-contrast agents, as well as BOLD-based functional imaging. Magn Reson Med 43:905-908, 2000. Published 2000 Wiley-Liss, Inc.
Genomics of human dysmorphogenesis.
A survey of Mendelian Inheritance in Man emphasizes the large Mendelian contribution to human dysmorphogenesis and contrasts single gene conditions with chromosomal disorders. There were 1761 conditions that involved altered morphogenesis (49% of disease entries), including 1040 multiple defect syndromes and 721 inherited single birth defects. Premature death (36-57%), mental retardation (20-59%), and growth retardation (37-59%) are more frequent in autosomal recessive or X-linked syndromes, while predisposition to tumorigenesis was more common in dominant (16%) than recessive (3.4%) syndromes. Comparison of the Mendelian conditions with 100 chromosomal disorders showed a strikingly similar spectrum of malformation, with skeletal, craniofacial, eye, epidermal, and neuromuscular systems being most frequently affected. Chromosomal syndromes average 10.6 systems affected per disorder, in contrast to 3.55 for Mendelian syndromes, and pleiotropy does correlate weakly with aneuploid segment length. Genomic understanding of these relationships is still primitive, with 74 of 1609 (4.6%) autosomal conditions and 43 of 152 (29%) X-linked conditions mapped to specific chromosomal regions. The societal toll of human dysmorphogenesis and the evident progress with X-linked disorders provide a powerful rationale for the Human Genome Project.
Evaluation of algorithms used for cross-species proteome characterisation.
The ability to effectively search databases for the identification of protein spots from two-dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N- and C-terminal microsequence, amino acid composition, and peptide-mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty-four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7-100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non-sequence-dependent software in proteome analysis. The data obtained also predicted that 'sequence tagging' of peptide fingerprints would need to be accompanied by at least 11-20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.
Brain activity associated with painfully hot stimuli applied to the upper limb: a meta-analysis.
The capacity of pain to alert against potential injury or focus attention on damaged tissue is enhanced by the intrinsically aversive nature of the experience. Finding methods to relieve pain will ultimately be facilitated by deeper understanding of the processes that contribute to the experience, and functional brain imaging has contributed substantially toward that end. An impressive body of literature has identified a distributed network of pain-related activity in the brain that is subject to considerable modulation by different stimulus parameters, contextual factors, and clinical conditions. The fundamental substrates of the pain network are yet to be distilled from the highly variable results of studies published thus far. Qualitative reviews of the pain-imaging literature have been contributory, but lack the greater surety of quantitative methods. We employ the activation likelihood estimation (ALE) meta-analytic technique to establish the most consistent activations among studies reporting brain responses subsequent to the application of noxious heat. A network of pain-related activity was replicated for stimuli to either upper limb that included two discernible regions of the mid-anterior cingulate cortex, bilateral thalami, insula, and opercula cortices, posterior parietal cortex, premotor cortex, supplementary motor area, and cerebellum. The findings of the meta-analysis resonate with other streams of information that continue to enhance our understanding of pain in the brain. The results also point toward new areas of research that may be fruitful for the exploration of central pain processing.
A comparison of label-based review and ALE meta-analysis in the Stroop task.
Meta-analysis is an important tool for interpreting results of functional neuroimaging studies and is highly influential in predicting and testing new outcomes. Although traditional label-based review can be used to search for agreement across multiple studies, a new function-location meta-analysis technique called activation likelihood estimation (ALE) offers great improvements over conventional methods. In ALE, reported foci are modeled as Gaussian functions and pooled to create a statistical whole-brain image. ALE meta-analysis and the label-based review were used to investigate the Stroop task in normal subjects, a paradigm known for its effect of producing conflict and response inhibition due to subjects' tendency to perform word reading as opposed to color naming. Both methods yielded similar activation patterns that were dominated by response in the anterior cingulate and the inferior frontal gyrus. ALE showed greater involvement of the anterior cingulate as compared to that in the label-based technique; however, this was likely due to the increased spatial level of distinction allowed with the ALE method. With ALE, further analysis of the anterior cingulate revealed evidence for somatotopic mapping within the rostral and caudal cingulate zones, an issue that has been the source of some conflict in previous reviews of the anterior cingulate cortex.
Prostate cancer chemoprevention by silibinin: bench to bedside.
Prostate cancer (PCA) is the most invasive malignancy and second leading cause of cancer deaths in American males. One approach to reduce PCA incidence, growth and metastasis is prevention and intervention targeted towards mitogenic and survival signaling and cell-cycle regulation. This approach is based on the rationale that overexpression of receptor tyrosine kinases (RTKs) and/or non-receptor tyrosine kinases leads to persistent autocrine stimulation of malignant cells for deregulated cell-cycle progression and uncontrolled growth. PCA progression has also been associated with transition from a paracrine to an autocrine relationship between receptors and growth ligands as this malignancy progresses to an advanced androgen-independent aggressive stage. Together, these studies suggest that targeting RTK-mediated signaling pathways along with cell-cycle regulators could be a practical and translational approach for PCA prevention and intervention. Here, we provide evidence that a naturally occurring nontoxic flavanoid, silibinin, targets the epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R) and NF-kappaB (nuclear factor-kappa B) pathways in PCA. Furthermore, it modulates cell-cycle regulators, including cyclin-dependent kinases (CDKs), Cip/Kip and cyclins for its anticancer efficacy against PCA. Silibinin inhibits growth of PCA cells from human, mouse, and rat origins, and also suppresses human prostate tumor xenograft growth in nude mice. Silibinin also inhibits PCA growth in the transgenic adenocarcinoma of mouse prostate (TRAMP) mouse model. Now, silibinin has been entered into phase I/II clinical trials in human PCA patients where preliminary observations were suggestive of its further study in a larger base of the patient population.
Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi Liston (Culicidae).
A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the 3H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.
Guidelines for nomenclature of genetically determined biochemical variants in the house mouse, Mus musculus.
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[(1)H MR spectroscopy. Methods and applications in diagnosis and assessment of surgical and conservative treatment strategies in epilepsies].
Techniques and methods of clinical (1)H-MR spectroscopy are described in this manuscript. The role of (1)H-MRS in the multimodal focus analysis of temporal lobe epilepsy (TLE) is illustrated with special respect to focus lateralization and differentiation between mesial and lateral (neocortical) TLE. Additionally the application of (1)H-MRS for evaluating postoperative outcome and monitoring conservative antiepileptic treatment schedules is summarized.
Validation of mRNA/EST-based gene predictions in human Xp11.4 revealed differences to the organization of the orthologous mouse locus.
Careful manual annotation of the human reference sequence provides a solid basis for the identification of disease-associated genes. Toward this end, we focused on a medically relevant 2.6-Mb region of the human chromosome Xp11.4 between markers DXS9851 and DXS9751 and identified 16 transcription units according to the Vertebrate Genome Annotation (Vega) rules. In order to validate these annotations, we performed a comprehensive RT-PCR expression analysis and a human-mouse comparison. This revealed, despite the high overall genomic conservation of the region, remarkable differences of the gene content between human and mouse. Whereas 12 of 16 annotations were confirmed by RT-PCR in human tissues, for only seven genes mouse orthologs could be identified and found to be expressed. This indicates that a comprehensive and experimentally supported annotation effort of the human genome simultaneously highlights regions with striking differences in gene organization to other species and may indicate evolutionary events specific to the human lineage demanding further functional analyses.
Mapping of gene-specific markers on the genetic map of chickpea (Cicer arietinum L.).
With the exception of the fact that it is made up of eight different chromosomes, the physical organization of the 738-Mb genome of the important legume crop chickpea (Cicer arietinum L.) is unknown. In an attempt to increase our knowledge of the basic structure of this genome, we determined the map positions of a series of genes involved in plant defence responses (DR) by genetic linkage analysis. Exploiting the sequence data available in GenBank, we selected genes known to be induced in chickpea and other plants by pathogen attack. Gene-specific primers were designed based on conserved regions, and used to detect the corresponding gene sequences in a segregating population derived from an interspecific cross between Cicer arietinum and C. reticulatum. Forty-seven gene-specific markers were integrated into an existing map based on STMS, AFLP, DAF and other anonymous markers. The potential of this approach is discussed.
Analysis of bacteriophage T7 early RNAs and proteins on slab gels.
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A new approach to optical imaging applied to rat barrel cortex.
Several groups have described using intrinsic optical imaging to form images of activity patterns in the cortex. Because the signal is small, the general approach has been to use expensive camera equipment with a high dynamic range to make these measurements. However, by using signal averaging to compensate for lower dynamic range, images can be obtained using equipment already available in many laboratories. This modified technique has been used for imaging activity in 'barrel' cortex of the rat. A map of the representation of a single whisker as determined by the imaging technique corresponded well with a similar map made using standard electrophysiology. A map of several whiskers was made by overlaying images of single-whisker representations. The details of the images differ from those previously described. Possible mechanisms for the signal are discussed.
DNA typing of the D1S8 (MS32) locus by rapid detection minisatellite variant repeat (MVR) mapping using polymerase chain reaction (PCR) assay.
The typing of the D1S8 (MS32) locus using the minisatellite variant repeat (MVR) polymerase chain reaction (PCR) method was performed by visualising amplified DNA stained with ethidium bromide. The results from rapid detection MVR-PCR were compared with those from the original MVR-PCR using Southern blot hybridisation with a 32P-labelled probe. With genomic DNA extracted from blood samples of 40 healthy unrelated Japanese individuals, the first 41 codes, on average, were correctly determined by rapid detection MVR-PCR without band intensity information, compared with at least 60 codes typed by the original MVR-PCR. The rapid detection MVR-PCR method was applied to bloodstains to simulate forensic samples. On average, 39 code positions could be determined from DNA extracted from 3-month-old bloodstains of six persons. Rapid detection MVR-PCR is more convenient than the original MVR-PCR, furnishes much information with regard to personal identification, and should be applicable to forensic fields.
Integrating human brain maps.
Perception, action, cognition, and emotion can now be mapped in the brain by a growing family of techniques. Positron emission tomography, functional magnetic resonance imaging, event-related electrical potentials, event-related magnetic fields, and other non-invasive imaging techniques are rapidly evolving and providing an increasingly rich literature on the functional organization of the human brain. Although no two techniques map identical physiological processes or physical parameters, replications of functionally specific maps by different techniques indicate sufficient common ground for multimodality integration. The process of integration is multi-tiered. Recent advances in integration range from simple image fusion, to model-based synthetic analyses, to collective databases for neural-system modeling. Spatially, temporally, physiologically, and cognitively accurate computational models of the neural systems of human behavior are the ultimate objective of functional brain mapping. This objective will be reached only through integrating the diversity of modern brain-mapping methods.