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PubMed2003

A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions.

We demonstrate successful, simultaneous polymerase chain reaction (PCR) amplification of up to 300 000 discrete reactions in a novel platform, the PicoTiterPlate. In addition to elevated throughput, the PicoTiterPlate based amplifications (PTPCR) can be performed in extremely small volumes: individual reactions volumes are as low as 39.5 pL, with a total 15.3 microL reaction volume for the entire PicoTiterPlate. The bulk PTPCR product can be recovered and assayed with real-time PCR, or discrete PTPCR products can be driven to solid supports, enabling downstream applications such as translation/transcription or sequencing.

Base Sequence↗
PubMed2000

Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135.

A novel 135 kDa centrosomal component (Cep135) was identified by immunoscreening of a mammalian expression library with monoclonal antibodies raised against clam centrosomes. It is predicted to be a highly coiled-coil protein with an extensive alpha-helix, suggesting that Cep135 is a structural component of the centrosome. To evaluate how the protein is arranged in the centrosomal structure, we overexpressed Cep135 polypeptides in CHO cells by transient transfection. HA- or GFP-tagged full (amino acids 1-1144) as well as truncated (#10, 29-1144; Delta3, 29-812) polypeptides become localized at the centrosome and induce cytoplasmic dots of various size and number in CHO cells. Centrosomes are associated with massive approximately 7 nm filaments and dense particles organized in a whorl-like arrangement in which parallel-oriented dense lines appear with a regular approximately 7 nm periodicity. The same filamentous aggregates are also detected in cytoplasmic dots, indicating that overexpressed Cep135 can assemble into elaborate higher-ordered structures in and outside the centrosome. Sf9 cells infected with baculovirus containing Cep135 sequences induce filamentous polymers which are distinctive from the whorl seen in CHO cells; #10 forms highly packed spheroids, but the Delta3-containing structure looks loose. Both structures show an internal repeating unit of dense and less dense stripes. Although the distance between the outer end of two adjacent dense lines is similar between two types of polymers ( approximately 120 nm), the dense stripe of Delta3 polymers ( approximately 40 nm) is wider than #10 ( approximately 30 nm). The light band of Delta3 ( approximately 40 nm) is thus narrower than #10 ( approximately 60 nm). Since thin fibers are frequently seen to extend from one dense line to the next, the coiled-coil rod of Cep135 may span the light band. These results suggest that overexpressed Cep135 assemble into distinctive polymers in a domain-specific manner.

Animals↗
PubMed2002

Expression profiling following traumatic brain injury: a review.

Traumatic brain injury (TBI) elicits a complex sequence of putative autodestructive and neuroprotective cellular cascades. It is hypothesized that the genomic responses of cells in the injured brain serve as the basis for these cascades. Traditional methods for analyzing differential gene expression following brain trauma demonstrate that immediate early genes, cytokines, transcription factors, and neurotrophic factors can all participate in the brain's active and directed response to injury, and may do so concurrently. It is this complexity and multiplicity of interrelated molecular mechanisms that has demanded new methods for comprehensive and parallel evaluation of putative as well as novel gene targets. Recent advances in DNA microarray technology have enabled the simultaneous evaluation of thousands of genes and the subsequent generation of massive amounts of biological data relevant to CNS injury. This emerging technology can serve to further current knowledge regarding recognized molecular cascades as well as to identify novel molecular mechanisms that occur throughout the post-traumatic period. The elucidation of the complex alterations in gene expression underlying the pathological sequelae following TBI is of central importance in the design of future therapeutic agents.

Animals↗
PubMed2026

Promoter identity shapes splicing outcomes and fidelity.

Gene expression is a complex process subject to regulation at multiple functionally interconnected levels. One prominent example is the crosstalk between transcription and splicing regulation. Past work has shown that transcription can influence splicing in multiple ways, but a systematic investigation of this complex interplay is lacking. Here we employ massively parallel reporter assays of large combinatorial promoter-splice site libraries to dissect how promoter identity and transcription dynamics affect alternative splicing in human cells. We find that promoter identity, rather than expression level, exerts strong and highly context-specific effects on cassette exon inclusion, exceeding the effect of pharmacological inhibitors of transcription initiation or elongation. Groups of exons display coordinated promoter-dependent splicing behavior, and we identified predictive sequence and structural features underlying this sensitivity. Promoter and gene architecture also shape isoform diversity by modulating cryptic splice site usage. These findings present promoters as central regulators of splicing outcomes and fidelity.

Humans↗
PubMedDate not supplied

Inducible expression of cytokinin biosynthesis in Agrobacterium tumefaciens by plant phenolics.

Nopaline strains of Agrobacterium tumefaciens contain a gene, tzs, that encodes a cytokinin biosynthetic prenyl transferase. The gene is located adjacent to the Ti plasmid virulence region and is constitutively expressed at low levels. As a result, bacteria containing tzs secrete low levels of zeatin into the medium. We find zeatin secretion to be induced more than 100-fold by acetosyringone, one of a number of naturally occurring phenolics produced by plants in response to wounding. Induction was very sensitive to the pH of the medium (optimum pH 5.5) and was due to massive overexpression of tzs-encoded cytokinin prenyl transferase activity. The relative ability of members of a set of phenols to induce tzs expression was examined and found to be parallel to that reported for activation of other virulence genes. A series of molecular cloning experiments established that virA and virG, two genes known to be essential to the virulence induction process, were necessary and sufficient for phenolic-induced tzs expression. Sequences present in the promoter region of tzs were found to be similar to those present in genes regulated by bacterial two-component positive regulatory systems.

Acetophenones↗
PubMed2001

High-throughput microarray technologies: from genomics to clinics.

BACKGROUND: Despite continuous research efforts in the past decades, there are still cancers where no effective treatment is available, such as advanced kidney cancer or hormone-refractory prostate cancer. A better understanding of the molecular mechanisms of cancer development and progression is the basis for the development of new diagnostic and therapeutic strategies. Current developments in genomics have a dramatic impact on the whole field of research. The sequence of the entire human genome will soon be fully sequenced and provide the 'book of life' as a basis for the understanding of human disease. METHODS AND RESULTS: New technologies have emerged to translate the human genome sequence into gene function and improved diagnostics or treatment modalities. New technologies such as microarrays are not only important for fundamental research, but will also be useful for diagnostic, prognostic or therapeutic purposes in individual patients. DNA microarrays make it possible to analyze the mRNA expression of thousands of genes simultaneously. The resulting comprehensive gene expression surveys lead to the identification of new genes and pathways with importance in cancer development and progression, or as targets for new therapies. The validation and prioritization of genes emerging from genome screening analyses in large series of clinical tumors has become a new bottleneck in research. Therefore, we have recently developed the tissue microarray (TMA) technology to efficiently test the clinical relevance of candidate genes. TMAs are microscope slides containing samples from hundreds of individual tumor specimens. They can be used for large-scale, massively parallel in situ analysis of genetic alterations on a DNA, RNA and protein level using in situ hybridization or immunohistochemistry on hundreds of tumor specimens at a time. Microarray technologies are already increasingly being used in urologic research, and will also have a strong impact on clinical urology. CONCLUSIONS: DNA microarrays and TMAs provide a powerful approach to identify large numbers of new candidate genes, and rapidly validate their clinical impact in large series of human tumors. These technologies will soon lead to a better molecular understanding of urologic tumors, and accelerate the identification of new prognostic markers or therapeutic targets.

Clinical Medicine↗
PubMed2026

Massively parallel characterization and predictive modelling of neuronal regulatory variation.

Disease-associated variants reside frequently in noncoding cis-regulatory elements (CREs), yet their functional consequences remain poorly understood. We performed a large-scale lentiMPRA in human excitatory neurons, quantifying the impact of >46,000 naturally occurring variants across >27,000 candidate CREs near 524 disease-associated genes. These data improved regulatory variant effect predictions beyond state-of-the-art models. Significant allelic effects occurred at comparable rates across common, rare, and singleton variants, demonstrating that, within MPRA-measurable effects, population frequency carries limited information about per-variant regulatory impact. Variant effect detectability and magnitude were governed primarily by baseline activity of the enclosing regulatory element and local sequence context. Regulatory effects were distributed across numerous transcription factors rather than concentrated in master regulators, consistent with a combinatorial enhancer architecture. We establish a large-scale functional variant catalog and provide a complementary benchmark and resource for developing and evaluating models of noncoding regulatory variation.

Journal Article↗
PubMed2003

Insights into nucleic acid conformational dynamics from massively parallel stochastic simulations.

The helical hairpin is one of the most ubiquitous and elementary secondary structural motifs in nucleic acids, capable of serving functional roles and participating in long-range tertiary contacts. Yet the self-assembly of these structures has not been well-characterized at the atomic level. With this in mind, the dynamics of nucleic acid hairpin formation and disruption have been studied using a novel computational tool: large-scale, parallel, atomistic molecular dynamics simulation employing an inhomogeneous distributed computer consisting of more than 40,000 processors. Using multiple methodologies, over 500 micro s of atomistic simulation time has been collected for a large ensemble of hairpins (sequence 5'-GGGC[GCAA]GCCU-3'), allowing characterization of rare events not previously observable in simulation. From uncoupled ensemble dynamics simulations in unperturbed folding conditions, we report on 1), competing pathways between the folded and unfolded regions of the conformational space; 2), observed nonnative stacking and basepairing traps; and 3), a helix unwinding-rewinding mode that is differentiated from the unfolding and folding dynamics. A heterogeneous transition state ensemble is characterized structurally through calculations of conformer-specific folding probabilities and a multiplexed replica exchange stochastic dynamics algorithm is used to derive an approximate folding landscape. A comparison between the observed folding mechanism and that of a peptide beta-hairpin analog suggests that although native topology defines the character of the folding landscape, the statistical weighting of potential folding pathways is determined by the chemical nature of the polymer.

Computer Simulation↗
PubMed2004

Massively parallel networks for edge localization and contour integration--adaptable relaxation approach.

The paper describes a new adaptive neural network for edge-based pattern extraction problems. In the model, each neuron represents an edge with continuous state variables describing its location and orientation. The post-synaptic distribution is dependent on the state variables. Each neuron adjusts its state to increase its membrane potential, which results in highly adaptive dynamics of the synaptic weight distribution. The network allocates multiple neurons with different orientation modes for each edge. The strategy allows accurate modeling of multi-modal distributions at key-points such as corners and junctions. As a result, the network delineates edges at sub-pixel accuracy while preserving key-points. It is also capable of processing a sequence of images and following moving objects. The network is extended for contour integration and key-point detection tasks. The paper presents experiments conducted on both synthetic and non-synthetic data to demonstrate the effectiveness of the technique.

Artificial Intelligence↗
PubMedDate not supplied

Induction of retinal degeneration in a crab by light and okadaic acid in vitro: comparison with the Drosophila light-dependent retinal degeneration mutant w rdgBKS222.

Retinae of the crab Leptograpsus which had been maintained on a 12-h light/12-h dark cycle were cultured in vitro and exposed to 1 microM okadaic acid (OKA) at 0.75 h before light onset. Control retinae were subjected to the same routine and sampled at the same times without OKA treatment. At the concentration used, OKA totally inhibits types 1 and 2A protein phosphatases, minimally inhibits type 2B, and does not affect type 2C. 1 microM OKA provoked a diminution of rhabdom diameter measured at the level of the photoreceptor nuclei in the dark, some ommatidial cartridges being stripped of rhabdomeral microvilli altogether. After 1-h illumination (225-320 lux), further reduction of rhabdom diameter was modest in control retinae but precipitate in those treated with OKA. After 2 h, control rhabdom diameters showed a further, not significant, decline, but OKA had induced a resynthesis of massive structures with the light-microscopic appearance of rhabdoms. Electron microscopy revealed that they were heterogeneous and of the following kinds: (1) a minority of rhabdoms with normally disposed but distorted microvilli; (2) rhabdoms in the throes of events that parody normal assembly; and (3) rhabdomal volumes occupied by saccular organelles or by pleats or ruffles of irregular architecture. The cytoplasm of all such receptors was packed with free and bound ribosomes and endomembranes. The sequence of events parallels that seen during light-induced degeneration of photoreceptors of the Drosophila mutant w rdgBKS222. Preliminary experiments show that a protein kinase activator SC-9 mimics many of these effects in the dark in the presence of 1 microM OKA. As a working hypothesis, it is proposed that light activates protein kinases via diacylglycerols generated by the phototransduction cascade, and that in both crab retinas challenged with OKA and retinas of rdg BKS222 activation of a nuclear regulatory protein by hyperphosphorylation provokes a runaway transcription whose selectivity and extent remain to be determined.

Animals↗
PubMed1980

Kinetics of erythroid precursors in mice infected with the anemic or the polycythemic strain of Friend leukemia virus.

The kinetics of both erythroid burst-forming and colony-forming units (BFU-E, CFU-E) and myelomonocytic precursors [myelomacrophage colony-forming unit (CFU-C)] have been evaluated in tibial marrow, peripheral blood, and spleen of DBA/2 mice at time intervals after inoculation of either the anemic (FLV-A) or the polycythemic (FLV-P) strain of Friend leukemia virus. Either one of the viruses induced, at 7-10 days after infection, a massive increase in the number of BFU-Es in peripheral blood, in parallel with their depletion in tibial marrow and increase in spleen. A comparable increase in the blood BFU-E number was observed in splenectomized FLV-infected mice. These results indicate a marrow-spleen migration of BFU-Es. In spleen, the increase of the BFU-E number was associated with an increase in the CFU-E pool. In tibial marrow, a sequence of expansion/depletion waves occurred reciprocally at the level of BFU-E and CFU-E. The cycling of BFU-E([(3)H]thymidine in vitro suicide index) in marrow, blood, and spleen was enhanced, whereas that of CFU-E and CFU-C showed little or no modification. These kinetic data suggest that the main target cell of FLV may be the BFU-E or a closely related element. In plates without added erythropoietin (but containing it in fetal calf serum), expression of CFU-E from FLV-P-treated animals was maximal; that of CFU-E from FLV-A-injected mice was either virtually absent or only slight in marrow or spleen, respectively. BFU-E growth always was fully dependent upon erythropoietin addition. Control studies in FLV-infected resistant mice and in susceptible mice given diluted or heat-inactivated virus provide convincing evidence that the phenomena described are induced by FLV.

Animals↗
PubMed1997

Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta.

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene.

Animals↗
PubMed2000

Thoughts on the cerebellum as a model for cerebral cortical development and evolution.

This chapter explores the prospect of using the cerebellar cortex as a model for the development and evolution of the cerebral neocortex. At first, this would seem a nearly fruitless task given the readily apparent structural and functional differences between the two cortices. Cerebellum and cerebrum perform different associative tasks, the cellular 'circuit diagram' of the two structures is different, even the developmental sequences that give rise to the two structures differ markedly. Yet there are similarities between the structures at the conceptual level that are difficult to ignore. Both structures have a relatively simple modular circuitry and achieve their complexity by an increase in either the size or number of the modules. Both have massive commisures connecting the left and right halves of the structure. For the cortex this commisure is the obvious corpus callosum; the cerebellar commisure is made up of parallel fibres of the granule cells that pass freely across the midline. As they are thin and unmyelinated, the number of these crossing fibres may well exceed the number of the callosal axons by a significant amount. By far the most obvious similarity between cortex and cerebellum, however, is that they are both topologically sheet-like in structure. They are broad and wide in the two-dimensional plane of the pial membrane with a relatively modest thickness in the radial dimension. The question for this chapter then is whether these similarities, in particular the sheet-like organization are coincidental or indicative of larger themes that play deeper roles in the development and function of these two seemingly disparate brain regions.

Animals↗
PubMed2000

Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta.

Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line derived from rat pancreatic beta-cells, before and after challenge with 30 and 1,000 pg/ml of recombinant human IL-1beta. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we found that 146 full-length genes and a large number of expressed sequence tags were differentially regulated 3-fold or more. Most of the differentially regulated transcripts have not previously been described to be regulated by IL-1beta in beta-cells. We have analysed the expression data and sorted the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune- and stress-related pathways as well as in insulin secretion and vesicle trafficking, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated.

Animals↗
PubMed2001

Kinetics and mechanisms of hepatic acute phase response to subtotal partial hepatectomy and cultural impact on environmental hepatic end-stage liver injury in the homeless.

Intoxication and liver damage induced by carbon tetrachloride (CCl(4)), aflatoxin B1, diabetes, and subtotal partial hepatectomy (PH(90)) in rats in which approximately 90% of the total hepatic tissue mass is surgically removed produces an acute-phase response (APR) whose initial stage prior to regression closely mimics the APRs associated with the life-threatening hepatic failure seen in the homeless. Rats treated by PH(90)were either healthy, CCl(4)-intoxicated, diabetic, or alflatoxin B1 (AFB1) intoxicated to the point of 75% liver insufficiency. It is well documented that high rates of mortality following PH(90)in aseptic rats could be minimized by supplementing drinking water with 20% glucose, organic components of L-15 medium and housing animals in cages maintained at 33-35;C. Aseptic rats showed a mild 20-30% decrease in APR proteins during the first 4-5 days following PH(90), while a maximal APR was noted 9-12 days post PH(90)and lasted for ~30 days when it returned to values close to those of healthy controls. This delay in hepatic APR of the remnant caudate lobe favoured replacement of lost basophilic clumps and ribosomes. The newly synthesized ribosomes of the nascent hepatocytes quantitatively maintained the APR signals of the injured caudate hepatocytes, and biosynthesized and released a typical spectrum of APR proteins. We suggest that massively injured liver has decoded an already stored and irreversible DNA-biochemical sequence of events in which priority is given to recovery of lost tissues by delaying an APR response to injury. In PH(90)of diabetic and CCl(4)-intoxicated rats, the hepatic dual functions of regeneration and APR processes associated with intoxication-initiated catabolic signals, created a heavy metabolic burden on the remnant caudate lobe leading to higher rates of mortality. APR of healthy rats to AFB1 parallels that of alpha-amanitin-induced intoxication. Similarly, within shorter time scale proportional to the severity of surgery, livers undergoing 75% partially hepatectomy (PH(75)) delayed both the onset and regression of APR. We are therefore led to believe that approaches other than liver transplantation should be considered as viable alternatives in the treatment of various acute and chronic liver diseases to avoid rejection and retransplantation. Scarcity of cadaveric liver has forced the medical community to investigate xenotransplantation with its unknown risks. Concomitantly, it is suggested that in view of the incalculable risks of indifference, the homeless must receive much improved medical care as we have found that two-dimensional immunoelectrophoretic assay of their serum is indicative of acute and chronic liver injury. The scientific and moral interrelationships of related matters are illuminated.

Acute-Phase Reaction↗
PubMed2002

[DNA chip and SNP].

Single nucleotide polymorphism (SNP) is high-density genetic marker that could be utilized to dissect genetic traits that influence susceptibility to common diseases and responsiveness to drugs. Requirement for massive SNP analysis has enhanced development of various SNP genotyping technologies. DNA chip is one of such methods, which detects SNPs in parallel on an array of surface bound oligonucleotides. Recent application of DNA chip for high-throughput SNP analysis as well as clinical diagnosis will be presented.

Genetic Markers↗
PubMed2001

Temporal sequence and kinetics of proinflammatory and anti-inflammatory cytokine secretion induced by toxic shock syndrome toxin 1 in human peripheral blood mononuclear cells.

The staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1) induces massive cytokine production, which is believed to be the key factor in the pathogenesis of TSS. The temporal sequence and kinetics of both proinflammatory and anti-inflammatory cytokines induced by TSST-1 in human peripheral blood mononuclear cells were investigated. A panel of loss-of-function single-amino-acid-substitution mutants of TSST-1, previously demonstrated to be defective in either major histocompatibility complex (MHC) class II binding (G31R) or T-cell receptor (TCR) interaction (H135A, S14N), was studied in parallel to further elucidate the mechanisms of cytokine secretion. Wild-type recombinant (WT r) TSST-1 induced a biphasic pattern of cytokine secretion: an early phase with rapid release of proinflammatory cytokines (especially gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha [TNF-alpha]) within 3 to 4 h poststimulation, and a later phase with more gradual production of both proinflammatory (IL-1beta, IL-12, and TNF-beta) and anti-inflammatory (IL-6, IL-10) cytokines within 16 to 72 h poststimulation. G31R, which is defective in MHC class II binding, induced a cytokine profile similar to that of WT rTSST-1, except that secretion of the early-phase proinflammatory cytokines was delayed and production of IL-1beta and IL-12 was markedly reduced. In contrast, mutant toxins defective in TCR interaction either demonstrated complete absence of any cytokine secretion during the entire observation period (H135A) or resulted in complete abolishment of IL-2 and other early-phase proinflammatory cytokines, while secretion of IL-10 appeared unaffected (S14N). Neither WT rTSST-1 nor the mutant toxins induced IL-4 or transforming growth factor beta. Our data indicate that effective TCR interaction is critical for the induction of the early-phase proinflammatory cytokine response, thus underscoring the importance of T-cell signaling in TSS.

Adult↗
PubMed2002

Sequences of elongation factors-1 alpha and -1 gamma and stimulation by juvenile hormone in Locusta migratoria.

Two cDNAs encoding the alpha and gamma subunits of translation elongation factor-1 (EF-1) have been cloned and sequenced from the African migratory locust, Locusta migratoria. Southern blotting and real-time PCR analyses indicated that these sequences represent single copy genes. Comparison with sequences from other species indicated greater conservation for EF-1 alpha than for EF-1 gamma. The developmental profiles for EF-1 alpha and -1 gamma mRNA expression in the fat body paralleled reported changes in the hemolymph juvenile hormone (JH) titer in the fifth instar and were elevated during early reproductive maturation in the female adult. In maturing adults, there was a greater accumulation of EF-1 alpha and -1 gamma transcripts in females than in males. The levels of both transcripts were greatly increased by an enriched diet, previously shown to elevate JH titers and accelerate vitellogenin production. Treating JH-deprived adult females with the JH analog, methoprene, resulted in more than doubling of transcript levels of both genes, supporting the hypothesis that JH could stimulate the accumulation of LmEF-1 alpha and -1 gamma transcripts. We suggest that production of elongation factors, increased by JH, may contribute to the massive protein synthesis required for egg production.

Amino Acid Sequence↗